%0$503"5
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%NVUEDELOBTENTIONDU
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$ÏLIVRÏPAR
Université Toulouse III Paul Sabatier (UT3 Paul Sabatier)
$ISCIPLINEOUSPÏCIALITÏ
Interactions plantes-microorganismes
0RÏSENTÏEETSOUTENUEPAR
REY
Thomas
LE mercredi 20 février 2013
4ITRE
Mise en évidence et caractérisation d'interconnexions moléculaires entre
symbiose et résistance chez la légumineuse
modèle Medicago truncatula
%COLEDOCTORALE
Sciences Ecologiques, Vétérinaires, Agronomiques
et Bioingénieries (SEVAB)
5NITÏDERECHERCHE
UMR5546 CNRS/UPS Laboratoire de Recherche en Sciences Végétales
$IRECTEURSDE4HÒSE
Professeur Christophe Jacquet
2APPORTEURS
Dr Florian Frugier, Directeur de recherche, Gif sur Yvette
Dr Maïté Vicre-Gibouin, Maître
de Conférences, Rouen
MEMBRESDUJURY:
Pr Guillaume Bécard, Toulouse (Président)
Dr Florian Frugier, Directeur de recherche, Gif sur Yvette
Dr Patrick Saindrenan, Chargé de recherche, Orsay
Dr Maïté Vicre-Gibouin, Maître de Conférences, Rouen
Pr Christophe Jacquet, Toulouse
Remerciements
Les" travaux" présentés" dans" ce" manuscrit" rendent" compte" des" cinq" années" passées" au" LRSV." Mon"
travail" n’aurait" pu" être" mené" sans" le" concours" de" collaborateurs" à" l’étranger," en" France" et"
particulièrement" au" LIPM" de" Toulouse," qui" nous" ont" fourni" matériels" biologiques" et" outils."
Je"remercie" toutes" les" personnes" côtoyées" au" cours" de" mon" doctorat," une" aventure" avant" tout"
humaine."
Je"souhaite"en"premier"lieu"à"remercier"Maïté&Vicré+Gibouin,"Florian&Frugier,"Patrick&Saindrenan"et"
Guillaume&Bécard"qui"ont"pris"le"temps"d’examiner"ce"manuscrit."Je"tiens"à"exprimer"ma"gratitude"à"
l’équipe" Effecteurs" Microbiens" et" Immunité" " Végétale" pour" son" accueil." Je" pense" avant" tout" à"
Christophe" Jacquet& qui" m’a" tout" de" suite" accordé" sa" confiance" et" offert" une" grande" liberté" dans"
la"conduite"de"mes"travaux,"tout"en"étant"présent"pour"les"jalons"importants"telle"que"la"préparation"
de" chaque" communication" orale" ou" écrite." Je" pense" aussi" à" Arnaud& Bottin" qui" a" particulièrement"
œuvré" pour" l’obtention" du" projet" ANR" à" l’origine" de" mon" projet" de" thèse" et" son" étudiant" Amaury&
Nars& qui& a" joué" un" rôle" déterminant" dans" mes" travaux" ." Je" tiens" à" remercier" Bernard& Dumas,"
directeur"du"groupe,"pour"son"impitoyable"clairvoyance"lorsqu’il"s’agit"de"synthétiser"des"données"et"
de"fixer"un"cap."Je"garde"une"pensée"particulière"pour"Marie+Thérèse&Esquerré+Tugayé"sans"qui"je"ne"
me"serais"peutPêtre"jamais"tourné"vers"l’étude"des"interactions"plantesPmicroorganismes."Je"remercie"
aussi"tous"les"autres"membres"de"l’équipe,"dans"le"désordre":"Claude&Laffitte,"Olivier"André,"Sophie&
Vergnes,"Yacine&Badis,"Valérie&Jaulneau,""Donia&Cheriet,"Ilham&Baddredine,""Elodie&Gaulin,"Salona&
Amatya,"Elodie&Belmas,"Laurent&Camborde,&Mathieu&Larroque,&&Diana&Ramirez,"Thomas&Martinez"et"
Maxime&Bonhomme&pour"leur"aide"scientifique"mais"surtout"pour"le"plaisir"que"j’ai"eu"à"travailler"à"
leur"côté"au"jour"le"jour."Parmi"tous"ces"collègues,"je"tiens"à"faire"une"mention"spéciale"pour" Marc&
Cazaux,& thésard" de" Chrisophe" en" 2008& qui" a" été" le" premier" à" me" former" et& Mathilde& Rech,&
ma"première"stagiaire,"qui"a"contribué"à"certains"résultats"présentés"ici."
Je" tiens" à" exprimer" ma" reconnaissance" à" l’ensemble" des" personnels" des" services" communs" du"
laboratoire"que"ce"soit"le"service"culture"avec"Jean+Louis&Luc,&&Paticia&Panégos&et&Patrick&Bermudes"
pour" les" nombreuses" plantes" qu’ils" m’ont" aidé" à" entretenir," le" service" stérilisation" avec" David&
Laimant" et" Annabelle& Dupas& pour" les" hectolitres" de" milieu" qu’ils" ont" vus" passer" et" le" secrétariat,"
Nicole& Saccol," Catherine& Deprey& et& Michèle& Escassut,& pour" leur" assistance" dans" les" tâches"
administratives.&Je"remercie"aussi"la"plateforme"de"microscopie,"Cécile&Pouzet,&Alain&Jauneau,&Yves&
Martinez&et&Aurélie&Leru&pour"leur"aide,"leurs"conseils"et"le"temps"qu’ils"ont"pris"pour"me"former"à"
diverses" techniques" mais" aussi" le" service" commun" séquençage" notamment" Nathalie& Ladouce," et"
1"
"
Francis& Carbonne," pour" les" nombreuses" analyses" qu’ils" ont" lancées" pour" moi." Je" remercie" aussi"
l’ensemble" des" personnels" de" la" plateforme" génomique," toujours" disponible" pour" répondre" à"
mes"questions"et"régler"mes"problèmes"et"tout"particulièrement"Frédéric&Martins&qui"m’a"formé"et"
permis"de"réaliser"moiPmême"quelques"dizaines"de"milliers"de"points"en"qPCR.""
Je"tiens"à"remercier"les"nombreuses"personnes"avec"qui"j’ai"pu"collaborer,"tout"d’abord"dans"le"cadre"
du" projet" ANR" Sympasignal" dirigé" par" Jean& Jacques& Bono.& Un" grand" merci" à& Clare& Gough,& Julie&
Cullimore,& & Judith& Fliegmann,& Benoît& Lefebvre& et& Delphine& Pitorre& pour" les" discussions" que"
nous"avons" eues" autour" des" LysMPRLKs" et." Je" tiens" aussi" à" remercier" Andreas& Niebel& qui" s’est"
pleinement" investi" dans" notre" étude" du" rôle" de" MtNF%YA%1" dans" l’immunité" en" obtenant" avec"
Christophe"un"financement"de"la"FR3450"pour"la"réalisation"de"transcriptomes."A"ce"titre,"je"remercie"
aussi"l’URGV"d’Evry"pour"la"réalisation"des"puces,"Marie+Françoise&Jardinaud&pour"son"aide"précieuse"
dans" l’analyse" de" ces" données" et" Philippe& Laporte" avec" qui" nous" avons" souvent" travaillé" à" quatre"
mains" et" dans" la" bonne" humeur." En" dehors" de" ces" deux" projets," j’ai" eu" la" chance" d’être" associé" à"
l’étude"de"la"perception"des"petits"chitooligosaccharides"chez"Medicago0truncatula"par"David&Barker"
à"qui"je"tiens"à"adresser"mes"remerciements"ainsi"qu’à"l’ensemble"des"auteurs"de"cette"publication"et"
Alain&Puppo"pour"sa"participation"à"mes"deux"comités"de"thèse."
Ayant" enseigné" à" l’IUT" de" génie" biologique" d’Auch," je" tiens" à" remercier" Othmane& Merah& qui" m’a"
encadré"et"l’ensemble"des"personnels"qui"ont"assuré"la"préparation"des"enseignements."Je"tiens&enfin"
à"remercier"les"personnels"non"permanents"du"laboratoire."Ce"fut"un"plaisir"de"vous"représenter"en"
2010," mention" spéciale" à" Marion& Molès," Daniel& Da& Silva," Christophe& Lachaud," Coline& Balzergue,&
Audrey&Courtial,&Carole&Bassa,&&Cyril&Jourda,"Louis&Jérome&Leba,&Nicolas&Denancé"et"Nicolas&Amelot.&
Bien" que" cela" ne" soit" point" détaillé" dans" ce" qui" va" suivre," j’ai" eu" aussi" à" étudier" des" interactions"
improbables" entre" des" écotypes" naturels" de" scientifiques" maximisant" les" variabilités"
comportementales." Qu’ils" soient" de" SaintPSauveur," Francoalgerojamaïcains," Landais" à" ne" pas"
confondre" avec" les" Béarnais," Tarnais," Castrais," Blablablais," Portugais," Bretons," Gersois," ou"
Toulousains," ils" ont" tous" su" cohabiter" malgré" des" manies" variées…" Cleptomanie," mitainomanie,"
snipomanie," procrastinomanie," muffinomanie," volubilomanie," musculomanie," prestidigitatomanie,"
strangulomanie," nicotinomanie," yackomanie," phenolomanie," caféiomanie," consuelomanie,"
mandibulomanie," ouéééomanie," répétomanie," jeunomanie," cernomanie," râlomanie," manies"
des"grands"et"gros"yeux"(de"paires),"veinomanie"frontale,"soufflomanie"nasale."Merci"à"tous"pour"tous"
ces"sujets"d’investigations"!"
Enfin" je" tiens" à" adresser" mes" derniers" et" plus" profonds" remerciements" à" ma" famille," mes" amis" et"
Marie,"mon"épouse,"pour"leur"soutien"au"quotidien."
2"
"
Résumé
"
La" rhizosphère," définie" comme" l’espace" du" sol" qui" entoure" les" racines," héberge" de" nombreux"
microorganismes" à" l’origine" d’une" grande" variété" d’interactions" avec" la" plante." Au" cours" des" dix""
dernières"années","un"grand"nombre"de"gènes"végétaux"impliqués"dans"le"dialogue"moléculaire"qui"
s’établit"dans"le"cadre"des"différentes"symbioses"ou"lors"de"la"réponse"de"la"plante"aux"parasites"ont"
été" identifiés." Les" résultats" de" ces" travaux" ont" notamment" révélé" l’existence" de" fortes" similitudes"
entre" les" signaux" et" les" récepteurs" impliqués" dans" les" deux" types" d’interactions." Toutefois,"
l’identification"de"gènes"essentiels"à"la"fois"aux"réponses"immunitaires"et"mutualistes"n’a"pas"encore"
été"décrite,"en"raison"notamment"de"l’incapacité"de"la"plante"modèle"Arabidopsis0thaliana"à"établir"
des" interactions" symbiotiques." Pour" étudier" l’existence" d’interconnexions" moléculaires" entre"
symbioses"et"résistances"aux"parasites,"la"légumineuse"modèle"Medicago0truncatula"a"été"utlisée."En"
exploitant" des" mutants" symbiotiques" et" deux" pathosystèmes" développés" avec" cette" plante," les"
résultats" décrits" dans" ce" travail" relatent" la" découverte" de" l’implication" de" plusieurs" gènes" avec" un"
rôle"clé"dans"la"mise"en"place"de"la"symbiose"et"l‘immunité"de"la"plante."L’implication"du"récepteur"
putatif"aux"facteurs"NOD"NFP"et"celle"du"facteur"de"transcription"NF%YA1,0marqueur"de"la"symbiose"
fixatrice"d’azote"ont"été"particulièrement"étudiées"après"inoculation"d’Aphanomyces0euteiches0(Ae),"
un"oomycète"racinaire."Des"approches"de"phénotypage"complémentaires"(notations"des"symptômes,"
cytologie,"détection"moléculaire"du"parasite)"ont"montré"que"NFP"est"impliqué"dans"la"résistance"de"
la"plante"tandis"que"NF%YA1"est"associé"à"la"sensibilité"de"cellePci."Le"comportement"des"mutants"nfp"
visPàPvis"de"Colletotrichum0trifolii,0un"champignon"parasite,"s’est"révélé"similaire"à"celui"observé"visPàP
vis"d’Ae,0permettant"de"généraliser"son"rôle"face"aux"organismes"filamenteux"pathogènes."En"ce"qui"
concerne" NF%YA1," il" est" apparu" que" sa" mutation" implique" aussi" un" meilleur" développement" des"
racines" latérales." Son" expression" a" d’ailleurs" pu" être" détectée" dans" les" primordia" de" racines,"
suggérant" qu’il" possède" un" rôle" régulateur" de" ces" organes." Des" analyses" transcriptomiques"
comparant" les" mutants" de" ces" deux" gènes" et" la" plante" sauvage" inoculée" par" Ae0 ont" révélé" des"
modifications" non" seulement" sur" l’expression" de" gènes" dans" la" signalisation" et" la" mise" en" place" de"
réponses" immunitaires" mais" aussi" dans" des" fonctions" associées" à" des" processus" de" dynamique"
cellulaire."Ainsi"l’analyse"détaillée"du"rôle"de"NFP"et"NF%YA1,"ainsi"que"l’initiation"de"plusieurs"autres"
travaux" sur" d’autres" mutants" symbiotiques" suggèrent" un" nombre" d’interconnexions" moléculaires"
beaucoup"plus"important"qu’imaginé"initialement"entre"symbiose"et"immunité."
"
3"
"
Abstract
"
Rhizophere" can" be" defined" as"the" soil" area" surrounding" the" roots." It" contains" important" amount" of"
microbes"that"initiate"a"wide"variety"of"interactions"with"plants."During"the"last"decades,"numerous"
plant" genes" involved" in" the" molecular" dialog" leading" either" to" symbiotic" or" immune" responses" has"
been" discovered." These" studies" unravelled" strong" homologies" between" signals" and" their" cognate"
receptors"involved"in"both"types"of"interactions."Nonetheless,"whether"some"genes"can"act"in"both"
kind"of"interaction"is"not"an"addressed"question"yet,""notably"beacause"the"model"plant"Arabidopsis0
thaliana" cannot" establish" any" kind" of" symbiosis." To" investigate" potential" molecular" crossPtalks"
between" symbiosis" and" disease" resistance," the" model" legume" plant" Medicago0 truncatula" has" been"
used."Taking"advantage"of"symbiotic"mutants"and"two"pathosystems"developped"with"this"plant,"the"
results" described" in" this" work" highlight" roles" in" immune" responses" of" several" symbiotic" actors."
Implication"of"the"putative"Nod"factor"receptor"NFP0and"of"the"transcription"factor"NF%YA1,0marker"of"
nodule" development" maintenance," has" been" particularly" studied" upon" inoculation" of" the" root"
oomycete" Aphanomyces0 euteiches0 (Ae)." Complementary" phenotyping" approaches" (symptoms"
notations," cytology," molecular" detection" of" parasite)" showed" the" involvement" of" NFP" in" plant"
immunity"whereas"NF%YA1"appears"as"a"susceptibility"gene."nfp"mutants"are"also"more"susceptible"to""
Colletotrichum0trifolii,0a"parasitic"fungus."This"result"indicates"that"the"role"in"resistance"of"this"gene"
can"be"generalised"to"filamentous"microbes."In"parallel,"we"showed"that"NF%YA1"disruption"lead"also"
to"a"better"root"development"upon"oomycete"invasion"and"its"expression"was"localised"in"lateral"root"
primordia" through" histological" observations," suggesting" a" regulation" role" for" this" gene" in" root"
architecture." Transcriptomic" analyses" comparing" mutants" and" wild" type" plants" inoculated" with" Ae"
were" generated" and" revealed" expression" alterations" of" genes" involved" in" signaling" and" immune"
responses"but"also"associated"to"cell"dynamic"processes."Hence,"detailed"analyses"of"NFP"and"NF%YA10
roles" along" with" others" studies" with" symbiotic" genes" and" Ae" initiated" in" this" work" suggest" an"
unexpected" number" of" molecular" crosstalks" " between" symbiosis" and" immunity" than" previously"
thought."
"
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4"
"
Abréviations
"
ABA":"Abscisic"Acid"
ABI&:"ABA"insensitive"
ADN":"Acide"désoxyribonucléique"
ANOVA":"Analysis"of"variance"
ANR&:"Agence"nationale"de"la"recherche"
ARF":"Auxin"Response"Factor"
ARN":"Acide"ribonucléique"
BAK1":"BRI1"associated"kinase"
BR":"Brassinolide"
BRI1":"Brassinolide"receptor"insensitive"1"
CC&:"Coil"Coiled"
CCAMK:"Calcium"calmoduline"dependent"kinase"
CDPK":"Calcium"dependent"protein"kinase"
CEBiP":"Chitin"elicitor"binding"protein"
CERK1":"Chitin"elicitor"receptor"kinase"1"
CK":"Cytokinine"
CLE":"Clavata"3"surrounding"embryo"
CMA":"Corn"meal"agar"
CRE1":"Cytokinin"receptor"1"
COI1&:"Coronatine"insensitive"1"
COs":"Chitooligosaccharides"
CSP":""Common"symbiotic"pathway"
DAMP":"Danger"associated"molecular"pattern"
DAPG":"2,4Pdiacétylphloroglucinol"
5"
"
DMI":"Does"not"make"infection"
EFD":""ERF"for"differentitiation"
EFR":"Elongation"factor"receptor"
EGF":"Epidermal"growth"factor"
ERF":"ET"responsive"factor"
EMS":"EthymethylPsulfonate"
EST":"Expressed"sequence"tag"
EIN":"ET"insensitive"
ERN&:"ERF"for"nodulation"
ET":""Ethylene"
ETI":"Effector"triggered"immunity"
FLS2":"Flagellin"sensing"2"
GA&:"Gibberrelic"acid"
GID1":"Gibberrelin"insensitive"dwarf"
GWAS":"Genome"wide"association"study"
HPAEC+PAD:"HighPPerformance"AnionPExchange"Chromatography"with"Pulsed"Amperometric"
Detection"
HR":"Hypersensitive"response"
IAA":"Indole"acetic"acid"
ISR":"Induced"systemic"resistance"
JA":"Jasmonic"acid"
LATD":"Lateral"deficiency"
LCOs":""LipoCOs"
LecRK":"Lectin"receptor"kinase"
LIN":"lumpy"infection"
LRR":"Leucine"Rich"Repeat"
LYK":"LysMPRLK"
LYP:"LysM"protein"
6"
"
LYR":"LysMPRLK"related"
LysM":"Lysin"motif"
MAMP":"Microbe"associated"molecular"pattern"
MAPK":"Mitogen"associated"protein"kinase"
MISSP7":"Mycorrhizal"induced"small"secreted"protein"7"
NAG":"NPacetylglucosamine"
NBS":"Nucleotide"binding"site"
NCR":"Nodule"cystein"rich"
NFP&:""Nod"factor"perception"
NFR":"Nod"factor"receptor"
NF+Y&:"Nuclear"factor"Y"box"
NIL":""Near"isogenic"line"
NIN&:"Nodule"inception"
NIP":"Numerous"infection"and"polyphenolics"
NPR":""Not"PR"
NSP":""Nod"signaling"pathway"
PAMP":"Pathogen"associated"molecular"pattern"
RT+PCR":""Reverse"transcription"polymerisation"chain"reaction"
pdf1.2&:"Protein"defensin"1.2"
PEPR&:"Pep"receptor"
PGPF":"Plant"growth"promoting"fungus"
PGPR":"Plant"growth"promoting"rhizobacteria"
PGPF&:"Plant"growth"promoting"fungus"
PIF":"Phytochrome"interacting"factor"
PR&:"Pathogenesis"related"
PRR":"Pattern"recognition"receptor"
PTI":""PAMP"triggered"immunity"
7"
"
PUB":"Protein"ubiquitin"
QTL&:"Quantitative"trait"locus"
RBoH":Respiratory"burst"homologue"
RIP1":"Rhizobium"induced"peroxydase"1"
RLK":"Receptor"like"kinase"
RLP":"Receptor"like"protein"
ROS":"Reactive"oxygen"species"
RPG":"Rhizobium"directed"polar"growth"
SA":"Salicylic"acid"
SAMP":"Symbiotic"associated"molecular"pattern"
SAR":"Systemic"acquired"resistance"
SNP":"Single"Nucleotide"Polymorphism"
SP7":""Secreted"protein"7"
SUNN":"Super"numeric"nodule"
SYMREM":"Symbiotic"remorin"
SYMRK":"Symbiotic"receptor"kinase"
TILLING":"Targeting"induced"local"lesion"in"genome"
TIR&:"Toll"interleukine"receptor"
VLCFA":"Very"long"chain"fatty"acid"
WGA+FITC":"Wheat"germ"agglutinin"fluorescein"isothiocyanate"
"
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8"
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Sommaire
Remerciements0.........................................................................................................................01"
Résumé0.....................................................................................................................................03"
Abstract0.....................................................................................................................................04"
Abréviations0..............................................................................................................................05"
Sommaire0..................................................................................................................................09"
Introduction0............................................................................................................................014"
I." Racines&et&interactions&biotiques&.........................................................................................&14"
A." Diversité"des"interactions"plantes"–"microbes"....................................................................................."17"
1." Les"associations"mutualistes"............................................................................................................"17"
a." La"symbiose"mycorhizienne"à"arbuscules"...................................................................................."17"
b." Les"symbioses"fixatrices"d’azote".................................................................................................."19"
2." Les"associations"parasitaires"............................................................................................................"20"
a." Les"interactions"plantesPchampignons"........................................................................................"22"
a." Les"interactions"plantesPoomycètes"............................................................................................"23"
b." Les"interactions"plantesPbactéries"..............................................................................................."23"
c." Les"interactions"plantesPnématodes"............................................................................................"23"
B." Antagonismes"et"synergies"entre"interactions"biotiques"....................................................................."24"
1." Mycorhization"et"autres"interactions"biotiques"..............................................................................."24"
a." La"mycorhization"et"la"symbiose"fixatrice"d’azote"......................................................................."24"
b." La"mycorhization"et"les"maladies"racinaires"................................................................................"25"
2." Interactions"entre"bactéries"bénéfiques"et"parasites"......................................................................"25"
a." La"symbiose"fixatrice"d’azote"......................................................................................................."25"
b." Les"bactéries"bénéfiques":"Plant"Growth"Promoting"Rhizobacteria"(PGPR)"................................"25"
3." Interactions"entre"parasites"............................................................................................................."26"
II." Mécanismes&moléculaires&et&cellulaires&de&réponses&des&plantes&au&microbiome&................&27"
A." Le"dialogue"moléculaire"entre"plantes"et"microbes":"signaux"et"récepteurs"........................................"27"
1." Molécules"présentes"dans"les"exsudats"racinaires"perçues"par"le"microbe"....................................."27"
a." Exsudats"végétaux"influençant"de"manière"globale"le"microbiome"............................................"27"
b." Les"strigolactones"........................................................................................................................"28"
c." Les"flavonoïdes."............................................................................................................................"28"
2." Signaux"et"molécules"perçus"par"la"plante"......................................................................................."28"
a." MAMPs"et"DAMPs"induisant"la"résistance"basale"et"PRRs"associés"............................................."30"
b." Perception"des"signaux"mutualistes":"les"SAMPs"et"leurs"récepteurs".........................................."34"
c." Effecteurs"microbiens"et"leurs"détections"par"la"plante".............................................................."39"
B." La"signalisation"....................................................................................................................................."41"
1." Flux"ioniques,"ROS"et"cascades"de"phosphorylations"......................................................................."41"
a." Dans"les"réponses"immunitaires".................................................................................................."41"
b." Dans"les"réponses"mutualistes"...................................................................................................."43"
2." Modification"de"la"balance"hormonale"............................................................................................"45"
a." Dans"les"réponses"immunitaires".................................................................................................."46"
9"
"
b." Dans"les"réponses"mutualistes"...................................................................................................."48"
3." Facteurs"de"transcription"et"régulations"par"le"protéasome"............................................................"50"
a." Dans"les"réponses"immunitaires".................................................................................................."50"
b." Dans"les"réponses"mutualistes"...................................................................................................."53"
C." Réponses"des"plantes"aux"microorganismes"........................................................................................"54"
1." Réponses"cellulaires"et"tissulaires"de"la"plante"................................................................................"54"
a." Réarrangements"membranaires"et"cellulaires":"signalisation"et"colonisation"............................."54"
b." Modifications"de"la"paroi"végétale"et"exocytose"........................................................................."55"
c." Modifications"de"l’architecture"de"la"racine"................................................................................"57"
d." Mort"cellulaire":"réponse"hypersensible"et"autophagie"..............................................................."58"
2." Réponses"moléculaires"et"biochimiques"de"la"plante"aux"microorganismes"..................................."59"
a." Reprogrammations"transcriptionnelles"......................................................................................."59"
b." Réponses"biochimiques"de"la"plante"..........................................................................................."61"
III." Medicago)truncatula,&légumineuse&modèle&pour&les&interactions&&biotiques&racinaires&........&63"
A." Ressources"génétiques,"génomiques"et"transcriptomiques"disponibles"pour"Medicago0truncatula".."64"
B." Medicago0truncatula,"modèle"pour"la"comparaison"des"associations"mutualistes"et"parasites".........."65"
1." Exploitation"de"deux"pathosystèmes"modèles"pour"évaluer"les"connexions"entre"symbiose"et"
immunité".................................................................................................................................................."66"
a." Aphanomyces"euteiches".............................................................................................................."66"
b." Colletotrichum"trifolii".................................................................................................................."70"
IV." Objectifs&de&la&thèse&............................................................................................................&73"
Résultats0et0Discussion0............................................................................................................076"
Chapitre&I.&Criblage&d’une&collection&de&mutants&symbiotiques&..................................................&76"
A." Phénotype"des"mutants"de"la"signalisation"symbiotique"....................................................................."79"
B." Niveau"de"résistance"des"mutants"de"l’organogénèse"symbiotique"et"des"mutants"hormonaux"........"81"
C." Développement"des"racines"des"mutants"au"cours"de"l’invasion"par"Aphanomyces0euteiches"..........."83"
1." Mutants"de"la"signalisation"symbiotique"........................................................................................."83"
2." Mutants"hormonaux"et"d’organogénèse"nodulaire"........................................................................."84"
D." Cytologie"de"l’invasion"par"Aphanomyces0euteiches"des"mutants"symbiotiques"................................"86"
E." Conclusion"du"chapitre"........................................................................................................................."89"
Chapitre&II.&Caractérisation&du&rôle&de&NFP)dans&l’immunité&......................................................&90"
A." Article"..................................................................................................................................................."90"
B." Analyses"transcriptomiques"complémentaires".................................................................................."102"
C." Vers"l’identification"de"signaux"d’Aphanomyces0euteiches"perçus"par"NFP"......................................."104"
1." Préparation"de"MAMPs"issus"d’Aphanomyces0euteiches"..............................................................."105"
2." Analyse"des"réponses"de"Medicago0truncatula"aux""filtrats"de"culture"d’Aphanomyces0euteiches106"
a." Les"espèces"réactives"de"l’oxygène"............................................................................................"106"
b." Les"marqueurs"transcriptionnels"..............................................................................................."109"
D." Conclusion"du"chapitre"......................................................................................................................"111"
Chapitre&III.&&Caractérisation&du&rôle&de&NF2YA1&dans&la&résistance&à&Ae&...................................&112"
Introduction":""Les"facteurs"de"transcription"NF%Y"chez"les"plantes"..........................................................."112"
A." Article"................................................................................................................................................."114"
B." Conclusion"du"chapitre"......................................................................................................................."136"
Matériels0et0Méthodes0..........................................................................................................0138"
I." Matériel&Biologique&...........................................................................................................&138"
10"
"
A." Microorganismes"pathogènes":"souche,"culture"et"inoculation"........................................................."138"
1." Aphanomyces0euteiches"................................................................................................................."138"
a." Souche"et"culture"......................................................................................................................."138"
b." Inoculations"et"notations"de"symptômes".................................................................................."139"
2." Colletotrichum0trifolii"....................................................................................................................."140"
a." Souche"et"culture"......................................................................................................................."140"
b." Inoculations"et"notations"de"symptômes".................................................................................."141"
B." Medicago0truncatula".........................................................................................................................."141"
1." Origine"des"lignées"sauvages"et"mutantes"....................................................................................."141"
2." Germination"..................................................................................................................................."142"
3." Transformation"par"Agrobacterium0rhizogenes"............................................................................."142"
4." Traitements"MAMPs"......................................................................................................................"143"
II." Méthodes&..........................................................................................................................&144"
A." Cytologie"............................................................................................................................................"144"
1." Préparation"de"coupes"fines".........................................................................................................."144"
2." Microscopie"à"épifluorescence"......................................................................................................"144"
3." Coloration"glucuronidase"..............................................................................................................."144"
B." Biologie"moléculaire"..........................................................................................................................."145"
1." RTPqPCR".........................................................................................................................................."145"
a." Extraction"des"ARNs"..................................................................................................................."145"
b." Reverse"transcription"................................................................................................................."146"
c." qPCR"..........................................................................................................................................."146"
d." Biomark"(qPCR"haut"debit)"........................................................................................................"147"
2." Analyse"de"données"transcriptomiques"........................................................................................."147"
Conclusions0générales0et0perspectives0..................................................................................0150"
Annexes0.................................................................................................................................0156"
I." Participation&à&divers&articles&.............................................................................................&156"
A." Etude"de"la"perception"de"chitosaccharides"de"petits"degrés"de"polymérisation"chez"Medicago0
truncatula"..................................................................................................................................................."156"
B." Mise"au"point"du"bioessai"d’élicitation"de"Medicago0truncatula"......................................................."168"
C." Analyse"des"phénotypes"non"symbiotiques"contrôlés"par"CRE1"........................................................"183"
II." Participations&&en&congrès&et&colloques&..............................................................................&185"
A." Oomycetes"Molecular"and"Genetic"Network"....................................................................................."185"
B." International"Congress"of"Legume"Genetic"and"Genomic".................................................................."186"
C." Model"Legume"Congress"...................................................................................................................."187"
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Introduction
13"
"
Introduction
"
Le" cadre" général" de" cette" thèse" concerne" la" façon" dont" les" plantes" perçoivent" et" répondent" à" leur"
environnement" biotique," en" parvenant" à" s’adapter" aux" types" de" microorganismes" qu’elles"
rencontrent." C’est" pourquoi" l’introduction" bibliographique" de" ce" travail" est" axée" d’une" part," sur" la"
description" de" la" diversité" des" interactions" biotiques" racinaires" et" d’autre" part," sur" les" mécanismes"
moléculaires" qui" expliquent" comment" la" plante" peut" faire" le" «"tri"»" entre" les" microorganismes"
bénéfiques"et"les"autres."Pour"l’étude"de"ces"premiers"microbes,"les"plantes"modèles"de"la"famille"des"
légumineuses" constituent" un" outil" de" choix" puisqu’elles" ont" la" faculté" de" s’associer" à" des"
champignons" et" des" bactéries" mutualistes" contrairement" à" Arabidopsis0 thaliana," modèle" végétal"
majeur" qui" a" été" largement" utilisé" pour" l’étude" des" interactions" pathogènes." En" utilisant" les"
connaissances"générées"sur"chacune"de"ces"plantes"modèles"pour"mieux"comprendre"les"deux"types"
d’interactions,"un"des"objectifs"de"cette"introduction"est"de"souligner"les"similitudes"entre"la"réponse"
immunitaire" et" celle" qui" existe" lors" de" la" mise" en" place" des" symbioses" bénéfiques" chez" les"
légumineuses." Cette" synthèse" permettra" de" resituer" le" cadre" des" travaux" réalisés" au" cours" de" ma"
thèse." CeuxPci" ont" étudié" des" connections" moléculaires" pouvant" exister" chez" Medicago0 truncatula"
entre" les" mécanismes" immunitaires" et" symbiotiques." Ce" travail" a" été" favorisé," notamment," par"
l’utilisation"d’un"pathosystème"modèle"entre"cette"plante"et"l’oomycète"Aphanomyces0euteiches0qui"
sera"présenté"en"fin"d’introduction."Chacun"de"ces"partenaires"possède"en"effet"des"caractéristisques"
génétiques" et" biologiques" qui" ont" permis" par" la" suite" d’identifier" des" gènes" préalablement"
découverts" dans" le" cadre" de" la" symbiose" et" qui" jouent" aussi" un" rôle" majeur" dans" la" réponse" de" la"
plante"aux"parasites."
"
I.
Racines et interactions biotiques
Un" seul" gramme" de" sol" peut" renfermer" des" centaines" " de" milliards" de" bactéries" appartenant" à" des""
dizaines"de"milliers"d’espèces"et"des"centaines"de"mètres"d’hyphes"de"microbes"filamenteux"(van"der"
Heijden" et" al.," 2008)." Les" racines," compartiments" clés" de" l’ancrage" au" sol" et" de" la" nutrition" des"
plantes,"sont"ainsi"en"contact"avec"une"flore"microbiologique"dense"et"variée"(van"der"Heijden"et"al.,"
2008)." Ces" microorganismes" telluriques" appartiennent" à" des" " règnes" " divers," champignons,"
oomycètes," bactéries," nématodes" ou" virus" et" présentent" des" modes" de" trophisme" variés" qui"
14"
"
déterminent" notamment" les" répercussions" positives" ou" négatives" à" l’égard" des" plantes" qu’ils"
rencontrent"(Bulgarelli"et"al.,"2012";"Lundberg"et"al.,"2012)."
Une" grande" majorité" de" ces" microbes" n’interagissent" pas" directement" avec" les" plantes" mais" les"
influencent"de"part"leur"proximité,"l’utilisation"des"ressources"voisines"et"l’influence"qu’ils"ont"sur"des"
microorganismes"capables"de"s’associer"aux"plantes":"on"parle"alors"de"mode"de"vie"commensal"pour"
cette" catégorie" de" microbes." CeuxPci" peuvent" néanmoins" avoir" un" effet" positif" sur" la" nutrition" de"
cellesPci" en" minéralisant" et" solubilisant" des" composés" organiques" " (acides" nucléiques," protéines,"
sucres" de" parois" fongiques)" qui" deviennent" autant" de" sources" d’azote" " et" de" carbone" essentielles"
pour" la" croissance" et" le" développement" de" la" plante." Ces" commensaux" favorisent" aussi" d’autres"
microbes" à" même" d’aider" la" nutrition" végétale" et" qui" sont" alors" dits" bénéfiques." Par" exemple," des"
bactéries"fixatrices"d’azote"vivant"à"l’état"libre"dans"le"sol"peuvent"aider"la"nutrition"azotée"tout"en"
s’opposant" à" la" colonisation" de" la" rhizosphère" par" les" microbes" délétères," en" limitant" l’accessibilité"
aux" nutriments" ou" en" sécrétant" des" enzymes" lytiques" et" des" composés" antimicrobiens" qui"
empêcheront" leur" croissance." Ces" mêmes" bactéries," en" étant" identifiées" comme" non" soi," stimulent"
l’immunité" végétale" et" conduisent" au" développement" de" sols" dits" suppressifs" qui" empêchent"
l’émergence"de"maladies"(Newton"et"al.,"2010";"Berendsen"et"al.,"2012)(Figure"1)."L’ensemble"de"ces"
microorganismes" favorisant" le" développement" des" plantes" est" communément" nommé"«"Plant"
Growth" Promoting" Rhizobacteria"»" PGPR" ou" «"Plant" Growth" Promoting" Fungus"»" PGPF." Outre" leur"
participation" à" l’enrichissement" " du" sol" en" nutriments" disponibles" pour" les" plantes," les" microbes"
jouent"aussi"un"rôle"tampon"dans"leur"utilisation":"en"séquestrant"ces"composés"dans"les"périodes"où"
les" plantes" sont" faiblements" actives," ils" évitent" ainsi" leur" lessivage" par" la" pluie." Dans" une" moindre"
mesure,""la"libération"d’acides"organiques"et"la"solubilisation"de"formes"précipitées"de"phosphate"par"
des"bactéries"peuvent""aussi"favoriser"le"développement"des"plantes"(van"der"Heijden"et"al.,"2008)."A"
l’inverse," il" peut" aussi" exister" d’autres" microbes" dits" délétères" qui" entrent" indirectement" en"
compétition"avec"les"plantes"pour"acquérir"les&"nutriments"disponibles"dans"le"sol"en"les"assimilant"
directement" ou" en" les" transformant" en" formes" non" assimilables" pour" les" plantes." Ces" derniers"
peuvent"aussi"interférer"avec"la"croissance"de"microorganismes"commensaux"ou"bénéfiques"(van"der"
Heijden"et"al.,"2008";"Berendsen"et"al.,"2012)."Bien"sûr,"les"plantes"aussi,"au"travers"de"la"sécrétion"de"
leurs"exsudats"racinaires"dans"la"rhizosphère,"façonnent"cette"flore"microbiologique"en"stimulant"ou"
en" inhibant" le" développement" de" certains" de" ces" microbes." Dans" leur" ensemble," ces" interactions"
entre" plantes" et" microorganismes" ont" un" impact" prépondérant" sur" la" plasticité" phénotypique" des"
hôtes" végétaux" qui" se" répercute" alors" sur" la" constitution" des" écosystèmes" naturels" et" sur" le"
rendement"des"plantes"de"grande"culture"(Berg"and"Smalla,"2009";"PartidaPMartínez"and"Heil,"2011";"
Bakker"et"al.,"2012)."Même"si"les"interactions""directes"entre"plantes"et"microorganismes""sont"bien"
15"
"
plus" rares" que" celles" décrites" ciPdessus," elles" possèdent" un" impact" prépondérant" sur" l’hôte" végétal"
qui" sera" illustré" dans" cette" première" partie." On" s’intéressera" aux" différents" types" d’interactions" et"
aux"effets"croisés"qu’elles"exercent"les"unes"sur"les"autres"d’un"point"de"vue"cellulaire"et"cytologique."
"
Figure"1":"Interactions"dans"la"rhizosphère.""
Les" plantes" sont" capables" d’influencer" la" composition" et" l’activation" de" leur" microbiome" rhizosphérique" en" exsudant" des"
composés"qui"stimulent"(flèche"verte)"ou"inhibent"(flèche"rouge)"la"croissance"des"microorganismes."Vice"versa,"un"grand"
panel" d’agents" pathogènes" telluriques" est" capable" d’affecter" la" santé" végétale." Avant" l’infection," ces" microbes" délétères"
sont" en" compétition" avec" beaucoup" d’autres" microbes" dans" la" rhizosphère" pour" les" nutriments" et" l’espace." Dans" cette"
bataille" pour" les" ressources," les" microbes" mutualistes" limitent" le" succès" du" parasite" par" la" production" de" composés"
biostatiques," la" consommation" de" micronutriments" ou" la" stimulation" du" système" immunitaire" végétal." La" plupart" des"
microbes" n’affecteront" " ni" la" plante," ni" le" parasite" de" manière" directe" car" ils" occupent" différentes" niches" " écologiques"
(commensaux)."Cependant,"ils"sont"susceptibles"d’interagir"avec"les"autres"microorganismes"à"des"degrés"variés""au"travers"
de" réseaux" complexes." Abréviation" :" ISR," Induced" Systemic" Resistance." Berendsen,& R.L.& et& al." (2012)." The" rhizosphere"
microbiome"and"plant"health."Trends0in0plant0science"
"
"
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16"
"
"
A. Diversité des interactions plantes – microbes
"
Si" les" interactions" plantePmicroorganisme" indirectes" décrites" ciPdessus" sont& très" répandues," les"
associations" dites" symbiotiques" (au" sens" de" vivre" ensemble)," qu’elles" soient" mutualistes" ou"
parasitaires,"constituent"des"cas"plus"rares"qui"ont"nécessité"une"importante"coPévolution"des"deux"
partenaires."Dans"le"premier"cas,"plante"et"microbe"se"sont""acceptés"et"ont"trouvé"un"modus"vivendi"
où"les"bénéfices"sont"réciproques."La"deuxième"situation"a"conduit"à"une"course"aux"armements"où"
chacun"essaie"de"contourner"les"stratégies"mises"en"place"par"l’autre."
1. Les associations mutualistes
On"distingue"classiquement"deux"principaux"types"d’associations"mutualistes"chez"les"plantes."Celles"
avec"des"mycorhizes"et"celles"avec"des"bactéries"endosymbiotiques"qui"sont"fixatrices"d’azote."Parmi"
les" mycorhizes," les" ectomycorhizes" s’associent" à" la" plante" en" formant" un" manchon" de" mycélium"
autour"des"cellules"du"rhizoderme,""permettant"des"échanges"de"nutriments."Ces"champignons"sont"
également" capables" de" vivre" à" l’état" libre" dans" le" sol" (Martin" et" al.," 2008";" VeneaultPFourrey" and"
Martin,"2011)."Par"opposition,"les"endomycorhizes"sont"des"biotrophes"obligatoires"qui"différencient"
au"sein"de"l’hôte"des"sites"d’échanges"intracellulaires"appelés"arbuscules"(Parniske,"2008)(Figure"2)."Il"
est" très" probable" que" cette" symbiose" ait" été" un" préPrequis" déterminant" pour& l’apparition" d’autres"
endosymbioses" qui" impliquent" des" bactéries" du" genre" Rhizobia" et" Frankia." Ces" dernières" fixent"
l’azote" atmosphérique" in0 planta" dans" des" organes" plus" ou" moins" spécialisés" (Gough" and" Cullimore,"
2011)."Chez"les"légumineuses,"ce"deuxième"type"d’endosymbiose"aboutit"à"la"formation"d’un"nouvel"
organe"sur"les"racines"de"l’hôte"appelé"nodosité."Dans"cette"structure,"la"bactérie"se"différencie"afin"
d’accomplir"les"fonctions"requises"pour"les"échanges"de"nutriments"avec"la"plante."
a. La symbiose mycorhizienne à arbuscules
Les" symbioses" mycorhiziennes" reposent" sur" des" échanges" dans" lesquels" la" plante" donne""
principalement" du" glycogène" et" du" triacylglycerol," et" en" retour," le" champignon" fournit" la" plante" en"
eau"et"phosphate"assimilable"(Parniske,"2008)."Dans"le"cas"des"endomycorhizes"qui"concernent"80%"
des" espèces" terrestres," il" a" été" montré" que" des" composés" exsudés" par" la" plante" stimulent" " le"
branchement"du"champignon"(Buee"et"al.,"2000)"."Parallèlement,"on"observe"la"sécrétion"de"signaux"
symbiotiques" par" le" mycélium" qui" reconfigure" l’architecture" racinaire" et" stimule" la" mycorhization"
(Oláh"et"al.,"2005";"Maillet"et"al.,"2011)."Ces"événements""permettent"l’adhésion"du"champignon"à"la"
surface" du" rhizoderme" et" la" formation" d’appareils" de" préPpénétration" au" sein" de" la" plante" qui" fait"
17"
"
suite" au" contact" de" l’hyphopode" différencié" par" le" mycelium." Cet" appareil" de" préPpénétration"
permettra" le" passage" du" " mycélium" à" travers" les" cellules" corticales." Sa" formation" est" liée" à" un"
déplacement"du"noyau"et"des"organites"requis"pour"l’adressage"de"protéines"et"métabolites"vers"le"
milieu" extracellulaire" ou" apoplastique." Plusieurs" noyaux" issus" de" cellules" situées" dans" différentes"
assises"cellulaires"se"synchronisent"pour"permettre"une"synthèse"de0novo"et"polarisée"de"paroi"et"de"
membrane" aboutissant" à" la" formation" d’un" conduit" qui" sera" emprunté" par" le" champignon"
mycorhizien." Ainsi" guidés," les" hyphes" se" développent" entre" les" assises" cellulaires" de" la" plante"
jusqu’au"cortex"interne"où"se"différencie"une"importante"surface"d’échanges"appelée"arbuscule"issue"
du" champignon" (Novero" et" al.," 2002" ;" Genre" et" al.," 2005";" Genre" et" al.," 2009)." L’ensemble" de" ces"
processus" est" sous" le" contrôle" de" la" plante" qui" régule" les" échanges" en" fonction" de" ses" propres"
besoins," comme" l’illustre" l’inhibition" de" cette" symbiose" par" un" apport" suffisant" de" phosphate"
assimilable"(Breuillin"et"al.,"2010)."
"
Figure"2":"Etapes"du"développement"de"la"symbiose"mycorhizienne"à"arbuscules."
"Les" racines" exsudent" des" strigolactones" qui" induisent" " la" germination" des" spores" et" le" branchement" du" mycélium" et"
accroissent"l’activité"physiologique"des"spores"et"des"hyphes."Les"strigolactones"induisent"aussi"la"germination"des"graines"
de"plantes"parasites"telles"que"les"Striga.0Le"champignon"produit"des"facteurs"de"mycorhize"(Facteurs"Myc)"qui"sont"définis"
par" leur" capacité" à" induire" des" oscillations" calciques" dans" les" cellules" rhizodermiques" et" à" activer" les" gènes" relatifs" au"
mutualisme."Les"champignons"mycorhiziens"à"arbuscules""forment""des"apporessoria"particuliers"appelés"hyphopodes"qui"
par"définition"se"développent"à"partir"d’hyphes"matures"et"non"germinatifs."En"conséquence"de"ces"stimulations"chimiques"
et"physiques"séquentielles,"les"cellules"végétales"produisent"un"appareil"de"préPpénétration"(PPA)."Par"la"suite,"un"hyphe"se"
développant"à"partir"de"l’hyphopode"entre"dans"le"PPA"qui"guide"le"champignon"au"travers"des"cellules"végétales"du"cortex."
A" cet" endroit," " le" champignon" se" ramifie" et" croît" latéralement" le" long" de" l’axe" de" la" racine." Ces" hyphes" induisent" le"
développement" " de" structures" ressemblant" au" PPA" dans" le" cortex" interne" avant" d’entrer" et" de" se" développer" à" nouveau"
dans" les" cellules" de" l’hôte." Des" vésicules" qui" sont" supposées" jouer" un" rôle" de" stockage" chez" le" champignon" sont" parfois"
formées" dans" l’apoplasme" (non" montré)." En" fin" de" cycle," de" nouvelles" spores" sont" générées" hors" de" la" racine," à"
l’extrémité"principale" d’hyphes" isolés." Parniske,& M." (2008)." Arbuscular" mycorrhiza:" the" mother" of" plant" root"
endosymbioses."Nature0reviews.0Microbiology"6:"763P75."
"
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b. Les symbioses fixatrices d’azote
Les"symbioses"fixatrices"d’azote"sont"apparues"bien"plus"tardivement"au"sein"du"règne"végétal,"ce"qui"
explique" qu’elles" ne" touchent" qu’un" groupe" plus" restreint" de" plantes":" les" légumineuses" avec" les"
Rhizobia" et" les" plantes" actinorhiziennes" avec" Ies" Frankia0 (Parniske," 20080;0 Markmann" and" Parniske,"
2009";"Markmann"et"al.,"2008)."La"symbiose"rhizobienne"est"la"plus"documentée"(Jones"et"al.,"2007)."
Dans"ce"cas"de"figure,"des"signaux"de"reconnaissance"mutuelle"sont"émis"par"les"deux"partenaires"(cf."
partie"suivante)"et"aboutissent"à"la"prolifération"des"bactéries"dans"une"poche"formée"à"l’extrémité"
de"poils"absorbants"qui"se"recourbent"(Gough"and"Cullimore,"2011)."Sous"l’impulsion"de"ces"échanges"
de" signaux," des" mécanismes" d’accommodation" intracellulaire" se" mettent" en" place," entraînant" la"
formation"d’un"cordon"infectieux"rappelant"l’appareil"de"préPpénétration"de"l’endomycorhize."Cette"
fois," c’est" la" formation" d’un" conduit" à" l’intérieur" du" poil" absorbant" qui" achemine" les" bactéries" à"
travers" les" tissus" végétaux." Là" encore," des" mouvements" nucléaires" sont" associés" à" la" formation" de"
cette"invagination"pariétale"et"cela,"de"cellule"en"cellule,"jusqu’au"site"de"libération"des"symbiontes."
En"parallèle,"un"primordium"de"nodosité,"initie"sa"formation"puis"est"rejoint"par"le"cordon"infectieux"
qui" libère" en" son" sein" les" bactéries" (Oldroyd" and" Downie," 2008)." Selon" la" légumineuse," il" se" forme"
alors"des"nodosités"à"croissance"indéterminée"(par"exemple"Medicago0truncatula"ou"Pisum0sativum)"
ou"à"croissance"déterminée"(Lotus0japonicus,0Glycine0max)."La"biologie"des"Rhizobia"capables"de"vivre"
à"l’état"libre"dans"le"sol"est"drastiquement"modifiée"pour"favoriser"la"fixation"de"l’azote"(Oldroyd"et"
al.," 2010";" Popp" and" Ott," 2011)(Figure" 3)" et" l’on" observe" une" différenciation" des" bactéries" en"
bactéroïdes" qui" est" irréversible" et" constitue" un" état" de" dépendance" totale" de" la" bactérie" envers" la"
plante" qui," théoriquement," ne" maintient" l’interaction" qu’avec" les" meilleurs" fixateurs." De" manière"
similaire"à"ce"qui"se"produit"pour"l’endomycorhize,"c’est"cette"fois"le"statut"de"la"nutrition"azotée"de"
la"plante"qui"régule"son"aptitude"à"s’investir"dans"la"symbiose"(Ruffel"et"al.,"2008)."Le"détail"de"ces"
mécanismes"au"niveau"moléculaire"sera"abordé"dans"la"partie"II."
19"
"
"
Figure"3":"Développement"à"croissance"déterminée"et"indéterminée"de"nodosités"et"gènes"impliqués"dans"les"différentes"
étapes"de"leur"formation."
La" perception" des" facteurs" Nod" induit" des" divisions" cellulaires" périclines" dans" le" péricycle," suivies" d’une" prolifération"
cellulaire" pendant" l’infection." Le" développement" du" primordium" nodulaire" est" accompagné" de" celui" d’un" méristème"
persistant" menant" à" l’apparition" de" zones" dans" le" nodule" indéterminé" comprenant" le" méristème" (ZI)," la" zone" d’infection"
(ZII)," l’interzone" (ZIIPIII)," la" zone" de" fixation" (III)" et" de" sénescence" (IV)." Les" nodules" déterminés" dérivent" de" divisions"
cellulaires"dans"le"cortex"externe"où"l’activité"méristèmatique"est"perdue"dans"les"nodosités"matures.""Nombres"de"mutants"
chez"Medicago0truncatula""(indéterminée)"et"Lotus0japonicus"(déterminée)"affectés"dans"la"perception"des"facteurs"Nod"ont"
été"identifiés."Les"phases"précoces"de"l’infection"situées"en"amont"de"la"formation"du"primordium"nodulaire"ont"aussi"pu"
être"ordonnées,"dans"la"mesure"où"différents"stades"phénotypiques"ont"pu"être"associés"au"rôle"des"récepteurs"et"à"la"voie"
de"signalisation"commune"à"la"mycorhization."Les"mutants"et"approches"de"RNAi"associés"sont"regroupés"dans"la"catégorie"
«""absence"d’initiation"de"cordons"infectieux"»."Popp,&C.&and&Ott,&T."(2011)."Regulation"of"signal"transduction"and"bacterial"
infection"during"root"nodule"symbiosis."Current0opinion0in0plant0biology"14:"458P467"
"
2. Les associations parasitaires
Les" microorganismes" parasites" racinaires" de" plantes" peuvent" être" des" bactéries," des" champignons,"
des" oomycètes" ou" encore" des" nématodes." Les" virus" responsables" de" maladies" généralisées" à"
l’ensemble"de"la"plante"ne"seront"pas"traités"ici."La"compréhension"de"l’immunité"végétale"nécessite"
de"s’arrêter"sur"les"modes"de"vie"adoptés"par"les"microorganismes"phytopathogènes."CeuxPci"peuvent"
déployer"un"panel""varié"de"comportements"envers"l’hôte."Soit"ils"se"développent"exclusivement"sur"
des"tissus"vivants":"on"parle"alors"de"biotrophie"qui"peut"être"obligatoire"ou"facultative"à"la"survie"du"
microbe," " soit" ils" se" développent" sur" des" tissus" morts" et" on" parle" alors" de" nécrotrophie." Certains"
20"
"
microorganismes" pathogènes" ont" un" mode" de" vie" en" deux" phases," passant" de" la" biotrophie" à" la"
nécrotrophie" :" ils" sont" alors" considérés" comme" hémibiotrophes." " Il" est" à" noter" que" des" similitudes"
existent" au" niveau" de" l’accommodation" intracellulaire" du" mycelium" entre" le" développement" de" la"
biotrophie" obligatoire" des" champignons" endomycorhiziens" et" celle" de" certains" champignons"
biotrophes"(Parniske,"2000)."Dans"ces"deux"types"d’interactions","le"mycélium"du"partenaire"fongique"
se"développe"en"effet"de"manière"intercellulaire"et"non"dommageable"pour"les"tissus"de"l’hôte."Par"la"
suite,"il"différencie"des"organes"d’échanges,"appelés""arbuscules"dans"le"cas"mutualiste"et"haustorium"
dans" le" parasitisme," qui" permettent" les" échanges" entre" les" deux" organismes." Selon" le" type"
d’agresseur,"les"plantes"mettent"en"place"des"mécanismes"de"défenses"adaptés""(Panstruga,"2003";"
van" Kan," 2006";" Glazebrook," 2005";" Spoel" et" al.," 2007" ;" Kliebenstein" and" Rowe," 2008";" Mengiste,"
2012)(Figure"4).""
"
Figure"4":"DAMPs,"PAMPs,"facteurs"de"virulence"et"réponses"immunitaires"à"des"parasites"de"modes"de"vie"variés."
(a)"Schéma"simplifié"montrant"la"reconnaissance"de"DAMPs"et"PAMPs"par"des"PRRs"et"l’activation"de"réponses"immunitaires"
basales."(b)"Les"parasites"nécrotrophes"à"spectre"d’hôte"large"produisent"divers"PAMPs"et"induisent"la"libération"de"DAMPs"
qui" activent" les" réponses" immunitaires" végétales" tandis" que" leurs" facteurs" de" virulence" ou" effecteurs" suppriment" ces"
réponses." (c)" Les" parasites" nécrotrophes" à" hôte" spécifique" produisent" des" toxines" qui" sont" des" acteurs" majeurs" de" leur"
virulence."En"retour,"les"plantes"ont"des"enzymes"de"détoxification"ou"possèdent"des"allèles"insensibles"à"ces"toxines."Les"
toxines" sont" aussi," sans" doute," des" cibles" directes" dont" la" manipulation" est" reconnue" par" des" protéines" de" résistance"
menant"à"l’ETI.""Les"gènes"LOV1"d’Arabidopsis,"TSN1"du"blé"et"PC"du"sorgho"codent"pour"des"protéines"de"résistance"qui"
contrôlent" la" " sensibilité" aux" toxines" sans" pour" autant" interagir" directement" avec" cellesPci." (d)" Schéma" simplifié" de" l’ETI"
communément" associé" " aux" parasites" hémibiotrophes" ou" biotrophes." Quelle" que" soit" leur" spécificité," les" toxines" sont"
présumées"cibler"des"protéines"de"l’hôte"pour"promouvoir"la"sensibilité."Dans"tous"les"cas"de"figure"et"pour"simplifier,"seuls"
les" facteurs" majeurs" sont" soulignés." Les" flèches" hachurées" indiquent" les" preuves" exéprimentales" limitées" ou" inexistantes."
Abréviations":"CWDE,"cell"wall"degrading"enzymes";""NEPs","necrosis"and"ethylene"inducing"proteins";""TTI,"Toxin"Triggered"
Immunity.0Mengiste,&T."(2012)."Plant"Immunity"to"Necrotrophs."Annual0review0of0phytopathology:"1P28."
21"
"
Pour"répondre"à"l’attaque"des"microorganismes,"les"plantes"ont"développé"différentes"stratégies"qui"
vont"se"traduire"par"des"phénotypes"de"résistance"différents."On"distingue"d’abord"la"résistance"non"
hôte" qui" se" traduit" le" plus" souvent" par" l’absence" de" symptôme" (Heath," 2000";" Nürnberger," 2005" " ;"
Lipka" et" al.," 2008" ;" Zellerhoff" et" al.," 2010)." " Elle" est" liée" à" des" mécansimes" de" défense" préformée"
et/ou"à"l’activation"de"la"résistance"(ou"PTI"cf"II)."Le"deuxième"niveau"est"la"résistance"racePcultivar"
(ou"ETI"cf"II)."CellePci"est"spécifique,"qualitative"et"se"traduit"en"général"par"le"déclenchement"d’une"
réponse" hypersensible." Cette" dernière" se" traduit" par" le" «"suicide"»" du" tissu" végétal" infecté" qui"
empêche"le"parasite"de"s’implanter"dans"des"tissus"propices"à"son"développement","un"peu"à"l’image"
d’une"stratégie"de"terre"brûlée."Le"dernier"niveau"de"résistance"concerne"les"réponses"quantitatives,"
par"opposition"aux"autres"formes"de"résistances."Dans"ce"type"de"résistance,"le"parasite"est"capable"
d’effectuer" son" cycle" biologique" complet" mais" sa" présence" est" associée" à" des" symptômes" moins"
intenses"ou"retardés"lorsqu’on"les"compare"avec"ceux"observés"chez"une"lignée"sensible."Ce"dernier"
type"de"résistance"constitue"encore"un"champ"de"recherche"très"ouvert"car"l’étude"des"mécanismes"
impliqués" a" été" longtemps" marginalisée" au" profit" de" celle" sur" les" résistances" qualitatives." Une"
caractérisitique" centrale" des" réponses" de" défenses" observées" dans" ce" type" de" résistance" est" leur"
aspect" multivarié" qui" implique" de" vastes" réseaux" de" gènes" et" limite" ainsi" les" risques" de"
contournement"de"la"résistance"par"le"parasite"(Poland"et"al.,"2009)."Dans"les"paragraphes"suivants"
sont" illustrés" quelques" exemples" d’interactions" décrites" dans" la" littérature" scientifique" qui" mettent"
en"jeu"différents"types"de"parasites"racinaires."
a. Les interactions plantes-champignons
Parmi"les"maladies"fongiques"les"plus"dommageables"(Dean"et"al.,"2012),"Fusarium0oxysporum"est"la"
première"maladie"racinaire"(Repp"et"al.,"2009)."Ce"champignon"possède"un"large"spectre"d’hôtes"qu’il"
colonise" de" manière" semblable." Après" adhésion" à" la" surface" de" la" racine," il" atteint" le" cortex," sans"
former" de" structure" de" pénétration." Une" fois" dans" le" cortex," les" hyphes" sont" directements" dirigés"
vers"le"cylindre"central"où"ils"colonisent"les"vaisseaux"de"xylème."C’est"à"ce"stade"de"développement"
de" la" maladie" que" les" symptômes" visibles" apparaissent." Ils" se" caractérisent" par" un" flétrissement"
complet" des" parties" aériennes" et" une" coloration" foncée" des" tissus" vasculaires" qui" conduisent" à" la"
mort" de" la" plante." Un" autre" champignon," Colletotrichum0 graminicola," initialement" décrit" comme"
pathogène"foliaire"hémibiotrophe"chez"le"maïs,"est"également"capable"d’attaquer"les"racines"de"son"
hôte." Cette" fois," le" mycélium" différencie" des" hyphopodes" pour" traverser" le" rhizoderme" puis" se"
développe" principalement" de" manière" intercellulaire" et" biotrophe," colonisant" de" manière" éparse"
l’intérieur" des"cellules" tuées," sans" jamais" former" d’haustoria." Ainsi," ce" champignon" présente" deux"
modes"d’infection"distincts"selon"le"tissu"infecté"qui"lui"permettent"tous"deux"d’effectuer"son"cycle"de"
reproduction" (Sukno" et" al.," 2008)." Des" observations" similaires" ont" également" été" obtenues" en"
22"
"
inoculant"Magnaporthe0oryzae0sur"les"racines"du"riz":"dans"ce"cas,"il"y"a"utilisation"d’hyphopodes"pour"
entrer"dans"la"racine"(Marcel"et"al.,"2010)."Des"travaux"menés"dans"notre"équipe"de"recherche"ont"
conduit" à" la" caractérisation" d’un" comportement" similaire" pour" Colletotrichum0 trifolii," agent" de"
l’anthracnose" de" la" luzerne" chez" Medicago0 truncatula" ":" ces" données" seront" reprises" plus" en" détail"
ultérieurement"dans"ce"rapport"(Cazaux,"2009)."
a. Les interactions plantes-oomycètes
Les" oomycetes" sont" des" organismes" filamenteux" distincts" des" champignons." Ils" sont" à" l’origine" des"
maladies"végétales"parmi"les"plus"importantes"(Hein"et"al.,"2009";"Bozkurt"et"al.,"2012)."Phytophtora0
sojae"et"Phytophtora0parasitica"sont"responsables"respectivement"de"la"pourriture"racinaire"du"soja"
et"de"la"pourriture"des"racines"et"du"collet"chez"un"large"spectre"d’hôtes"(Attard"et"al.,"2010";"Kebdani"
et" al.," 2010";" Han" et" al.," 2011)." Ces" deux" parasites" sont" considérés" comme" hémibiotrophes" dans" la"
mesure"où"une""phase"de"croissance"biotrophe,"caractérisée"par"un"développement"intercellulaire"du"
mycelium"et"le"développement"d’haustoria"est"suivie"par"une"phase"de"croissance"dans"des"cellules"
tuées."Au"final,"ces"deux"oomycètes"provoquent"tout"d’abord"des"brunissements"racinaires"puis"des"
flétrissements" aériens" affectant" la" survie" de" l’hôte." Le" parasite" Aphanomyces0 euteiches," modèle"
d’étude"au"sein"du"groupe,"sera"présenté"ultérieurement."
b. Les interactions plantes-bactéries
De" toutes" les" bactéries" pathogènes" racinaires" de" plantes," Ralstonia0 solanacearum" cause" les" plus"
grands"dommages,"notamment"en"raison"de"son"important"nombre"d’hôtes"potentiels"(Mansfield"et"
al.," 2012)." Cette" bactérie" colonise" rapidement" le" cortex" sans" se" multiplier," après" avoir" pénétré" la"
racine" par" des" blessures." Elle" atteint" le" cylindre"central" et" l’intérieur" du" xylème" où" elle" se" multiplie"
abondamment"(Digonnet"et"al.,"2012)."C’est"cette"dernière"étape"de"l’invasion"qui,"en"empêchant"le"
transport"d’eau"vers"les"parties"aériennes"de"la"plante,"induit"un"flétrissement"des"parties"aériennes"
et" la" mort" de" la" plante" notamment" chez" Medicago0 truncatula" (Vailleau" et" al.," 2007"" ;" Turner" et" al.,"
2009)."
c. Les interactions plantes-nématodes
Bien" que" les" nématodes" ne" soient" pas" des" microorganismes" stricto0 sensus,0 ils" font" partie" des"
pathogènes"racinaires"les"plus"répandus"(Gheysen"and"Mitchum,"2011)."Les"réponses"qu’ils"induisent"
chez" la" plante" ressemblent" parfois" à" celles" observées" avec" d’autres" microorganismes" pathogènes"
reposant" d’ailleurs" sur" les" mêmes" types" de" déterminismes" moléculaires" (Tarek" and" Thomas," 2012)."
Dans" le" cas" des" nématodes" racinaires," l’entrée" dans" la" plante" se" fait" par" le" méristème" puis" ils"
s’installent"dans"l’endoderme"et"utilisent"un"stylet"pour"se"nourrir"et"accéder"à"l’intérieur"de"la"stèle."
Ici," ils" établissent" avec" la" plante" une" interaction" biotrophe" basée" sur" la" formation" d’une" cellule"
23"
"
nourricière"sous"la"manipulation"de"protéines"et"de"signaux"sécrétés"par"le"parasite."Ainsi"les"cellules"
végétales"forment"des"syncitia,"c’estPàPdire"des"cellules"fusionnées"aboutissant"à"la"formation"d’une"
grande"cellule"plurinuclée":"on"parle"alors"de"nématodes"à"kyste."Parmi"ceuxPci,"le"nématode"du"soja"
Heterodera0glycines"entraîne"la"maladie"qui"provoque"les"pertes"économiques"les"plus"importantes."
D’autres" " nématodes" à" spectre" d’hôtes" très" large," tel" que" Melidogine0 incognita," induisent" la"
formation" de" cellules" géantes":" on" parle" alors" des" nématodes" à" galles." Dans" ce" dernier" cas," les"
déterminismes" moléculaires" conduisant" à" la" formation" de" ces" cellules" nourricières" reposent" sur"
certaines" fonctions," aussi" impliquées" dans" le" développement" des" nodosités" fixatrices" d’azote" qui"
seront"explicités"en"partie"II"(Damiani"et"al.,"2012).""
"
B. Antagonismes et synergies entre interactions
biotiques
"
Même"si,"pour"des"besoins"de"recherche"fondamentale,"l’étude"des"pathosystèmes"racinaires"met"en"
jeu"une"espèce"de"microorganisme"et"la"plante"dont"on"souhaite"évaluer"les"réactions"en"présence"de"
ce" microbe," il" est" clair" que," dans" le" sol," ces" mêmes" plantes" doivent" en" réalité" faire" face"
simultanément" à" des" individus" ou" des" populations" de" plusieurs" dizaines" ou" centaines" d’espèces"
microbiennes."En"dépit"de"leur"appartenance"à"des"règnes"différents,"de"leurs"modes"de"vie"variés"et"
de" leurs" effets" contrastés" sur" les" plantes," les" différents" microorganismes" vivant" " dans" le" sol"
influencent" la" mise" en" place" et" le" devenir" des" autres" interactions" plantePmicroorganismes." Ce"
phénomène" peut" s’expliquer" par" des" effets" de" compétition" ou" de" synergie" entre" les" microbes" euxP
mêmes" ou" bien" par" la" stimulation" du" système" immunitaire" de" la" plante" qui" participera" alors" à" la"
régulation" des" différentes" interactions." Bien" que" les" interactions" multiples" au" sein" d’une" même"
plante"soient"délicates"à"étudier"et"que"peu"de"rapports"soient"disponibles","cette"section"a"pour"but"
de" faire" un" tour" d’horizon" des" connaissances" établies" sur" les" interférences" existant" entre" les"
différentes"interactions.""
"
1. Mycorhization et autres interactions biotiques
a.
La mycorhization et la symbiose fixatrice d’azote
La" mycorhization," en" améliorant" la" nutrition" phosphatée" de" la" plante," favorise" son" métabolisme"
énergétique"et"sa"croissance."Ainsi"les"plantes"sont"davantage"à"même"d’entrer"en"interaction"avec"
24"
"
les"bactéries"fixatrices"d’azote"puisque"le"système"racinaire"est"plus"étendu"et"la"plante"possède"une"
plus" grande" capacité" à" fournir" en" énergie" les" bactéroïdes" (Miransari," 2011)." Des" essais" de" coP
inoculation"des"deux"symbiontes"aboutissent"à"une"meilleure"croissance"des"plantes"testées"dès"lors"
que"le"sol"est"pauvre"en"l’un"et"l’autre"des"nutriments"fournis"par"ces"interactions"(Wang"et"al.,"2012)."
b. La mycorhization et les maladies racinaires
Les" champignons" mycorhiziens" sont" capables" d’entrer" en" compétition" avec" les" parasites" pour" les"
nutriments"du"sol,"d’occuper"les"sites"d’infection"au"sein"de"la"plante"potentiellement"utilisable"par"
les" parasites," d’améliorer" la" santé" végétale" au" travers" de" la" nutrition" et" d’activer" des" réponses" de"
défense"(AzcónPAguilar"and"Barea,"1997";"Slezack"et"al.,"2000";"Pozo"and"AzcónPAguilar,"2007";Jung"et"
al.,"2012)."Un"exemple"d’effet"positif"de"la"mycorhization"visPàPvis"de"la"résistance"aux"parasites"du"
sol" est" la" tolérance" accrue" du" pois" à" son" parasite" Aphanomyces0 euteiches" Bødker" et" al.," 2002." Par"
ailleurs,"en"mobilisant"des"réponses"hormonales"de"la"plante,"les"champignons"mycorhiziens"peuvent"
aussi"améliorer"par"des"voies"systémiques"l’immunité"aérienne"des"plantes"(Jung"et"al.,"2012).""
"
2. Interactions entre bactéries bénéfiques et parasites
a. La symbiose fixatrice d’azote
Certaines" souches" de" Rhizobium," à" même" de" noduler" la" plante" Medicago0 truncatula," peuvent"
posséder" une" activité" inhibitrice" visPàPvis" de" la" croissance" d’agents" pathogènes" de" ce" même" hôte."
Cette"interaction"négative"a"été"montrée"visPàPvis"de""Phoma0medicaginis,0un"parasite"nécrotrophe."
Ainsi,"des"plantes"en"interaction"avec"ses"Rhizobia"présentent"une"diminution"des"symptômes"liés"au"
développement" de" cette" maladie" (Djébali" et" al.," 2011)." Inversement," l’activation" de" l’immunité"
végétale" par" la" présence" de" bactéries" pathogènes" a" un" effet" négatif" sur" la" formation" de" nodules"
(LopezPGomez"et"al.,"2012)."
b. Les bactéries bénéfiques : Plant Growth Promoting Rhizobacteria (PGPR)
Les"interactions"entre"les"microorganismes"pathogènes"et"le"grand"nombre"de"microbes,"notamment"
des"bactéries"(mais"aussi"des"champignons)"qui"n’interagissent""pas""directement"avec"les"plantes,"se"
traduisent" en" général" par" une" réduction" de" l’efficacité" des" premiers" à" coloniser" la" plante." Certains"
microorganismes" libèrent" des" composés" qui" influencent" et" favorisent" le" développement" végétal" en"
rendant" les" plantes" plus" résistantes" aux" agressions" (Jäderlund" et" al.," 2008)." Par" ailleurs," la" forte"
présence"de"divers"microorganismes"vivant"dans"le"sol"entraîne"une"compétition"pour"les"nutriments,"
à"même"de"limiter"le"développement"des"microorganismes"parasites."En"effet,"bien"que"ces"derniers"
soient"capables"d’être"saprophytes,"ils"s’avèrent"moins"efficaces"que"les"microorganismes"du"sol"et"
25"
"
leur"développement"est"ainsi"inhibé"(Berendsen"et"al.,"2012)."Ces"effets"de"suppression"peuvent"être"
généraux"ou"spécifiques"d’une"maladie"donnée."Ils"reposent"sur"la"production"par"des"bactéries"de"
genre" Pseudomonas" de" toxines" tels" que" le" 2,4Pdiacetylphloroglucinol" (DAPG)," la" pyrrolnitrin," la"
pyoluteorin," les" phenazines," des" lipopeptides" cycliques" ou" encore" du" cyanide" d’hydrogène" " qui"
affectent" des" fonctions" clés" de" la" biologie" des" parasites" (respiration," sporulation…)" (Raaijmakers" et"
al.," 2008)." En" ce" qui" concerne" l’établissement" des" symbioses," le" même" type" de" pression" peut"
s’appliquer" mais" il" faut," de" plus," considérer" que" la" forte" présence" de" microbes" libres" minéralisant"
l’azote"et"le"phosphate"peuvent"diminuer"l’acceptation"des"plantes"visPàPvis"de"la"symbiose"fixatrice"
d’azote"et"la"mycorhization"(van"der"Heijden"et"al.,"2008).""
"
3. Interactions entre parasites
Comme" illustrée" par" les" complexes" de" pourriture" racinaire," la" colonisation" d’une" plante" par" un"
parasite,"en"participant"à"son"affaiblissement,"permet"à"d’autres"microorganismes"du"sol"de"coloniser"
les"tissus"végétaux"alors"qu’ils"ne"seraient"pas"à"même"d’infecter"la"plante"de"manière"isolée."De"tels"
complexes"reposent"fréquemment"sur"l’infection"des"racines"par"des"champignons"de"type"Fusarium"
qui"induisent"par"la"suite"la"colonisation"par"des"champignons"tels"que"Botryodiplodia0theobromae"et"
Armillaria0spp."à"titre"d’exemple"chez"le"manioc"(Bandyopadhyay"et"al.,"2006)."
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II.
Mécanismes moléculaires et
cellulaires de réponses des plantes
au microbiome
"
Face"à"la"complexité"du"microbiome"et"à"la"large"influence"qu’il"peut"avoir"sur"la"biologie"des"plantes,"
cellesPci" ont" développé" au" cours" de" l’évolution" des" réponses" adaptées" à" chaque" situation."
Schématiquement,""l’établissement"ou"le"rejet"des"interactions"se"déroule"en"quatre"grandes"étapes."
Il" y" a," d’abord," l’émission" de" signaux" dans" la" rhizosphère" de" la" part" de" chaque" partenaire," puis" la"
perception"de"ces"signaux":"cellePci"entraîne"l’activation"de"différentes"voies"de"signalisation"chez"la"
plante" qui" conduisent" à" un" vaste" panel" de" réponses" lui" permettant" de" s’adapter" au" mieux" au"
microorganisme" rencontré." La" dernière" décennie" a" vu" notre" connaissance" de" ces" événements"
s’étendre" considérablement," notamment" du" point" de" vue" moléculaire," soulignant" de" façon"
étonnante,"l’existence"d’acteurs"moléculaires"très"similaires""lors"de"la"mise"en"place"des"mécanismes"
impliqués"dans"l’établissement"des"symbioses"et"dans"l’activation"de"l’immunité"végétale."Ces"deux"
aspects" seront" traités" en" parallèle" dans" le" cadre" des" trois" grandes" étapes" de" la" mise" en" place" des"
interactions":""la"reconnaissance,"la"signalisation"et"la"réponse."
A. Le dialogue moléculaire entre plantes et
microbes : signaux et récepteurs
"
Il"s’agit"ici"de"rappeler"quels"sont"les"molécules"issues"des"plantes"ou"les"composés"produits"par"les"
microbes"qui"vont"servir"au"dialogue"entre"les"partenaires"des"différentes"interactions."
1. Molécules présentes dans les exsudats racinaires perçues par le microbe
L’un" des" rôles" majeurs" des" sécrétions" végétales" dans" le" sol" est" de" favoriser" l’importation" de"
nutriments"qui"sont"sous"forme"non"assimilable"dans"le"milieu."Néanmoins,"certains"de"ces"exsudats"
sont"aussi"importants"pour"l’attraction"et"le"développement"des"microorganismes"du"sol."
a. Exsudats végétaux influençant de manière globale le microbiome
De" façon" générale," les" plantes" libèrent" dans" le" sol" plus" de" 100"000" composés" de" petits" poids"
moléculaires."On"y"trouve"une"grande"diversité"de"sucres,"d’acides"aminés,"de"métabolites,"d’ions,"et"
27"
"
des" mucilages" constitués" de" polysaccharides" qui" peuvent" être" utilisés" par" les" différents" organismes"
(Harsh"et"al.,"2010)."A"contrario,"des"molécules"toxiques"(phytoalexines,"phénylpropanoïdes,"indoles,"
terpènes,"isoflavonoïdes,"espèces"réactives"de"l’oxygène"ou"enzymes)"participent""à"la"protection"de"
la"rhizosphère"contre"une"invasion"par"des"organismes"compétiteurs"ou"parasites"(Bais"et"al.,"2004";"
Bais"et"al.,"2006";"Bakker"et"al.,"2012)."Ainsi"l’ensemble"de"ces"éléments"sécrétés"contribue"à"équilibre"
physicoPchimique"qui"permet"aux"microorganismes"adaptés"de"coloniser"la"rhizosphère."
"
b. Les strigolactones
Ces"petites"molécules"font"partie"des"métabolites"sécrétés"par"les"plantes"qui"stimulent"et"attirent"les"
microorganismes"symbiotiques."Ce"sont"des"terpènes"connus"initialement"pour"activer"la"germination"
de"plantes"parasites"du"genre"Striga0(Matusova"et"al.,"2005)."Plus"récemment,"leur"rôle"positif"sur"la"
germination" des" spores" de" champignon" mycorhizien" et" le" développement" du" réseau" mycélien" a" pu"
être" établi." Des" effets" sur" la" croissance" de" champignons" parasites" ont" également" été" documentés,"
révélant"que"ces"métabolites"peuvent"jouer"des"rôles"stimulateurs"ou"inhibiteurs"de"croissance"d’un"
parasite" à" l’autre" (Dor" et" al.," 2011)." Leur" labilité" dans" le" sol" entraîne" l’existence" d’un" gradient" de"
concentration" depuis" le" point" de" leur" sécrétion" jusqu’au" reste" de" la" rhizosphère," permettant" ainsi"
d’attirer" le" symbiote" dans" la" bonne" direction" (Akiyama" et" al.," 2005)." Par" ailleurs," ces" molécules" se"
sont"même"vu"attribuer"une"troisième"fonction":"elles"ont"un"rôle"hormonal"et"régulent"négativement"
le" branchement" des" parties" aériennes" des" plantes" " (Umehara" et" al.," 2008" ;" GomezPRoldan" et" al.,"
2008)."Plus"récemment,"de"nouvelles"avancées"ont""également"montré"que"les"strigolactones"sont"à"
même"d’influencer"l’architecture"racinaire"des"plantes"(Kapulnik"et"al.,"2011";"Delaux"et"al.,"2012)"et"
de"favoriser"la"formation"de"nodosités"fixatrices"d’azote"(Soto"et"al.,"2010";"Foo"and"Davies,"2011";"Liu"
et"al.,"2011)."
c. Les flavonoïdes.
Avant" les" strigolactones," les" flavonoïdes" ont" été" identifiés" comme" activateurs" du" programme"
symbiotique"chez"les"bactéries"du"genre"Rhizobium0(Long,"2001)."Ce"composé"a"pu"être"caractérisé"à"
partir" d’exsudats" de" graines" germées" de" luzerne." Ainsi," cette" famille" de" métabolites" secondaires,"
aussi"connue""pour"l’activité"antimicrobienne"de"certains"de"ses"membres,"peut"également"jouer"des"
rôles"chimioPattractifs."Inversement,"des"composés"de"la"même"famille,"la"daidzeine"et"la"genisteine,"
euxPmêmes" induits" chez" le" soja" par" la" présence" de" bactéries" symbiotiques," sont" aussi" capables"
d’attirer"les"zoospores"de"l’oomycète"pathogène"Phytophtora0sojae0(Morris"et"al.,"1998)."
"
2. Signaux et molécules perçus par la plante
28"
"
Les" microorganismes" produisent" aussi" dans" la" rhizosphère" des" composés" susceptibles" d’être"
reconnus" par" la" plante" et" ceuxPci" serviront" alors" de" signaux" pour" induire" les" mécanismes"
immunitaires"ou"la"mise"en"place"de"la"symbiose."Chaque"microorganisme"peut"délivrer"une"grande"
diversité"de"signaux.""En"dépit"de"cette"observation,"des"travaux"récents"ont"montré"que,"de"façon"
surprenante," les" réponses" mutualistes" ou" immunitaires" reposent" en" partie" sur" la" perception" de"
signaux" structuralement" apparentés." La" découverte" des" différents" acteurs" moléculaires" qui"
conduisent" à" la" compatibilité" ou" l’incompatibilité" d’une" interaction" plantePmicroorganisme" donne"
lieu"à"un"modèle"en"zigPzag"de"l’immunité"végétale"(Jones"and"Dangl,"2006)."Nous"verrons"aussi"que"
ce"modèle"peut,"dans"certaines"mesures,"se"transposer"aussi"aux"interactions"mutualistes."
Le" modèle" (Figure" 5)" se" décompose" en" au" moins" trois" phases" qui" illustrent" notamment" la" lutte" coP
évolutive"que"se"livrent"plantes"et"parasites."La"perception"de"signaux"bien"conservés"appelés"PAMPs"
(Pathogen"Associated"Molecular"Pattern)"chez"le"parasite"qui"peuvent"être"généralisés"au"terme"de"
MAMP" chez" les" autres" microorganismes," entraîne" le" déclenchement" d’une" résistance" basale," non"
spécifique,"la"PTI"(PAMP"triggered"Immunity,"PTI)."Chez"les"symbiotes,"il"a"été"mis"en"évidence"que"
des"altérations"subtiles"de"la"séquence"des"MAMPs""peuvent"leur"permettre"d’échapper"aux"défenses"
de"la"plante"ou"bien"même"favorisent"leur"acceptation"(Felix"et"al.,"1999)."""
La" deuxième" phase" du" modèle" en" zigPzag" repose" sur" l’utilisation" par" les" microbes" pathogènes" de"
contrePmesures" pour" bloquer" ou" contourner" cette" PTI" et" permettre" ainsi" l’établissement" de" la"
maladie."Ce"rôle"est"joué"par"des"molécules"injectées"dans"la"plante"appelées"effecteurs"microbiens."
La"troisième"phase"du"modèle"correspond"à"une"remontée"du"niveau"de"résistance"de"la"plante"grâce"
à" l’utilisation" de" récepteurs" capables" de" neutraliser" les" effecteurs" microbiens." Suite" à" cette"
reconnaissance"spécifique,"directe"ou"indirecte,"la"plante"met"en"place"une"résistance"généralement"
spécifique" du" parasite," longtemps" caractérisée" sous" le" terme" de" résistance" gène" pour" gène" et"
maintenant"appelée"ETI"(Effector"Triggered"Immunity)"dans"ce"modèle."Le"terme"de"zigPzag"concerne"
l’alternance"des"baisses"et"des"remontées"du"niveau"de"résistance"au"fur"et"à"mesure"de"la"mise"en"
place"évolutive"des"contrePmesures"d’attaque"du"parasite"et"de"défense"de"la"plante."De"plus"en"plus"
d’études" récentes" montrent" que" ce" modèle" en" zigPzag" semble" pouvoir" s’appliquer" aussi" aux"
interactions"plantesPsymbiotes."Les"paragraphes"suivants"reprennent"chacune"des"phases"du"modèle"
en"les"illustrant"avec"différents"exemples"d’interactions"(Figure"5).""
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29"
"
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Figure"5":"Un"modèle"en"zigPzag"illustrant"de"manière"évolutive"et"quantitative"le"système"immunitaire"végétal."
Dans"ce"schéma,"l’amplitude"maximale"de"la"résistance"ou"de"la"sensibilité"à"la"maladie"est"proportionnelle"à"PTIPETS+ETI."
Dans" la" phase" 1," les" plantes" détectent" les" MAMPs" (losange" rouge)" via" les" PRRs" pour" déclencher" la" PTI." En" phase" 2," le"
parasite" adapté" délivre" des" effecteurs" qui" interfèrent" avec" la" PTI" et" induisent" l’ETS" permettant" la" nutrition" et" la"
dissémination"du"parasite."En"phase"3,"un"effecteur"(en"rouge)"est"reconnu"par"une"protéine"NBPLRR"qui"active"l’ETI,"une"
version"amplifiée"de"la"PTI"qui"peut"culminer"en"une"réponse"de"type"HR."En"phase"4,"les"isolats"d’agents"pathogènes"ayant"
perdu" ou" acquis" certains" effecteurs" (en" bleu)" par" transfert" horizontal," sont" sélectionnés" et" peuvent" supprimer" l’ETI." La"
sélection"favorise"alors"de"nouveaux"allèles"de"NBPLRR"à"même"de"reconnaître"un"des"nouveaux"effecteurs"acquis"résultant"
à"nouveau"en"une"ETI.&Jones,&J.D.G.&and&Dangl,&J.L."(2006)."The"plant"immune"system."Nature"444:"323P9."
"
a. MAMPs et DAMPs induisant la résistance basale et PRRs associés
Les" MAMPs" sont" des" signatures" ou" motifs" moléculaires," conservés" chez" les" microorganismes" et"
absents" chez" les" plantes" qui" les" reconnaissent" comme" non" soi" et" provoquent" des" réponses"
immunitaires." Les" motifs" sont" souvent" présents" dans" des" molécules" structurales" de" surface" du"
parasite" qui" peuvent" être" de" nature" protéique," saccharidique," lipidique" ou" mixte" (Boller" and" Felix,"
2009)."De"nombreux"exemples"ont"été"décrits"pour"chacun"de"ces"types"de"MAMPs"dans"différentes"
espèces"de"parasites."Les"MAMPs"peptidiques"connus"sont"issus"de"protéines"cruciales"à"la"biologie"
des" organismes" qui" les" synthétisent." Chez" les" bactéries," on" peut" citer" flg22" issu" de" la" fagelline" ou"
elf18"issu"du"facteur"d’élongation"(Zhang"and""Zhou,"2010)"mais"aussi"ax21"impliqué"dans"le"quorum"
sensing."Chez"les"champignons,"le"motif"TKLGE"(Rotblat"et"al.,"2002)"issu"d’une"xylanase"fongique"ou"
pep13" issu" d’une" transglutaminase" d’oomycète" (Schwessinger" and" Zipfel," 2008)" ont" aussi" été"
identifiés" (Figure" 6)." Les" microbes" libèrent" aussi" des" MAMPs" saccharidiques." " Les"
chitooligosaccharides" (COs)" (Kombrink" et" al.," 2011)" et" les" hepta−! −glucanes" (Zipfel," 2008";" Zipfel,"
2009)"respectivement"libérés"par"les"champignons"et"oomycètes"ainsi"que"les"peptidoglycanes"et"les"
30"
"
lipopolysaccharides"bactériens"sont"pour"leur"part"issus"des"parois"de"ces"organismes"(Nicaise"et"al.,"
2009)."
Leur"reconnaissance"initie"la"PTI"qui"repose"sur"l’utilisation"de"récepteurs"membranaires"capables"de"
reconnaître" les" PAMPs":" ces" derniers" sont" appelés" Pattern" Recognition" Receptors" (PRR)." Ces" PRRs"
sont" en" général" constitués" par" une" partie" extracellulaire" qui" reconnaît" le" ligand," un" domaine"
transmembranaire" et" dans" la" majorité" des" cas," une" partie" intracellulaire" à" domaine" kinase" qui"
transmettra" le" signal." Pour" cette" raison," on" parle" de" Receptor" Like" Kinase" (RLK)," si" la" kinase" est"
absente," les" récepteurs" sont" des" RLP" (Receptor" Like" Protein)." On" peut" noter" que" la" caractérisation"
des" MAMPs" repose" sur" l’étude" de" leur" perception" qui" a," la" plupart" du" temps," été" menée" sur" les"
parties"aériennes"des"plantes."Néanmoins,"des"travaux"récents"suggèrent"que"les"mécanismes"de"ces"
reconnaissances"seraient"aussi"présents""au"niveau"racinaire"(Millet"et"al.,"2010)."
Certains"des"PRRs"à"ces"PAMPs"ont"été"identifiés"et"caractérisés"(Figure"6)."Une"première"classe"de"
récepteurs" est" constituée" par" des" protéines" membranaires" qui" possèdent" une" partie" extracellulaire""
qui" contient" des" répétitions" " de" domaines" riches" en" leucines" ou" LRR" (Leucine" Rich" Repeat)" qui"
interviennent" dans" la" spécificité" de" reconnaissance" des" molécules" détectées." " Les" représentants" de"
cette" classe" sont" AtFLS20 (Pour" Flageline" Sensitive)" qui" reconnaît" flg22" et" AtEFR0 (Elongation" Factor"
Receptor)0qui"perçoit"elf18"(Zipfel,"2008";"Zipfel,"2009";"Boller"and"Felix,"2009";"Monaghan"and"Zipfel,"
2012)." On" dénombre" plus" de" 600" LRRPRLK" chez" Arabidopsis0 thaliana," ainsi" il" est" très" probable" que"
davantage" de" membres" de" cette" famille" génique" jouent" des" rôles" dans" les" réponses" immunitaires."
D’autres" RLKs" sont" associés" à" des" domaines" de" type" EGF" (Epidermal" Growth" Factor)" tel" que" le"
récepteur" WAK1" impliqué" dans" la" perception" des" oligogalacturonates" libérés" à" partir" de" la" pectine"
pariétale"sous"l’effet"d’enzymes"de"dégradation"de"l’agresseur."En"effet,"la"dégradation"de"composés"
pariétaux" permet" la" libération" " de" Danger" Associated" Molecular" Patterns" (DAMPs)" reconnus" à" la"
manière" des" MAMPs." Dans" cet" exemple," le" récepteur" membranaire" lié" de" manière" covalente" à" la"
pectine" est" activé," dès" lors" que" l’intégrité" de" ce" constituant" de" la" paroi" est" atteinte," répondant" du"
coup" à" la" définition" de" PRR" de" DAMP" (Hématy" et" al.," 2009";" Li" et" al.," 2009";" Brutus" et" al.," 2010";"
Mengiste," 2012)." Bien" que" liant" des" ligands" distincts," ces" récepteurs" conduisent" à" des" réponses"
immunitaires" semblables." Ils" sont" aidés" en" cela" par" des" coPrécepteurs" communs" qui" participent"
notamment" à" la" première" étape" de" transduction" du" signal." Le" plus" connu" de" ces" coPrécepteurs" est"
aussi" un" LRRPRLK" qui" est" nommé" AtBAK1" (Pour" BRI1" (Brassinolide" insensitive" 1)" Associated" Kinase)."
Son"association"à"FLS2"et"EFR"a"pu"être"démontrée"mais"il"est"probablement"aussi"impliqué"dans"la"
signalisation" de" nombreux" autres" PAMPs" saccharididques" et" lipidiques" comme" les"
lipopolysaccharides" et" les" peptidoglycanes" ou" peptidiques" tels" que" INF1," HrpZ," csp22" et" les" DAMPs"
AtPeps" reconnus" par" les" LRRPRLKs" PEPRs" (Pep" Receptor)" (Roux" et" al.," 2011)." " Comme" son" nom"
31"
"
l’indique," AtBAK1" s’associe" également" à" BRI1" pour" la" signalisation" des" brassinolides." A" ce" titre," il"
constitue"sans"doute"un"acteur"clé"dans"la"régulation"du"comportement"de"la"plante"visPàPvis"de"deux"
processus"énergétiquement"coûteux":"la"défense"et"le"développement."
"
Figure"6":""Couples"PRRsPligands"connus."
Le" LRRPRLK" FLS2" reconnaît" la" flagelline" bactérienne" (ou" l’épitope" actif" flg22)" chez" Arabidopsis," la" tomate," Nicotiana0
benthamiana"et"le"riz."Le"LRRPRLK"EFR"qui"est"spécifique"des"Brasicaceae0reconnaît"le"facteur"d’élongation"bactérien"Tu""(ou"
bien"l’épitope"actif"elf18)"tandis"qu’un"proche"LRRPRLK"du"riz"nommé"XA21"lie"un"peptide"Ax21"(via"un"peptide"de"17"acides"
aminés)" associé" au" quorum" sensing" bactérien." Les" RLPs" à" domaines" LysM" LYM1" et" LYM3" sont" les" récepteurs" du"
peptidoglycane" (PGN)," tandis" que" CERK1" lie" ce" polymère" avec" une" faible" affinité." La" liaison" entre" un" PAMP" issu" d’une"
xylanase"fongique"et"les"récepteurs"LRRPRLPs"de"tomate"Eix1"et"Eix2"n’a"pas"encore"été"démontrée."Chez"le"riz,"le"LysMPRLP"
CEBiP"lie"la"chitine"et"interagit"avec"CERK1"(LysMPRLK)"pour"la"signalisation"intracellulaire"tandis"que"chez"Arabidopsis0CERK1"
est"le"récepteur"clé"de"la"chitine"(et"son"interaction"avec"CEBiP"n’est"pas"démontrée)."Chez"les"légumineuses,"une"protéine"
extracellulaire" de" liason" des" βPglucanes" (GBP)" reconnaît" l’heptaglucane" de" Phythophthora.0 Néanmoins," les" données"
décrivant"comment"cette"protéine"accomplit"sa"signalisation"intracellulaire"sont"encore"manquantes."Les"LRRPRLKs"PEPR1"et"
PEPR2" lient" les" peptides" endogènes" " AtPeps" et" le" RLK" WAK1" contenant" des" domaines" EGFPlike" est" un" récepteur" pour" les"
oligogalacturonides" (OGs)" dérivés" de" la" paroi." Monaghan,& J.& and& Zipfel,& C." (2012)." Plant" pattern" recognition" receptor"
complexes"at"the"plasma"membrane."Current0Opinion0in0Plant0Biology."
Une"autre"classe"de"PRR"qui"reconnaît"les"PAMPs"des"résidus"de"type"NPAcétylglucosamines"sont"Les"
récepteurs""dont"la"partie"extracytosolique"contient"des"domaines"Lysin"extracellulaires"LysM"(pour"
Lysine" Motif)." Ce" motif," identifié" initialement" chez" les" bactéries," est" particulièrement" présent" dans"
des" hydrolases" prokaryotes" où" il" améliore" l’affinité" pour" le" substrat" (Buist," Steen," Kok," &" Kuipers,"
2008)." Il" est" constitué" d’une" soixantaine" d’acides" aminés" dont" une" vingtaine" sont" plus" ou" moins"
conservés" et" séparés" par" des" séquences" variables" en" longueur" et" qui" renferment" des" sérines,"
thréonines,"asparagines"et"prolines"sans"ordre"particulier."Chez"les"plantes,"les"protéines"à"domaines"
LysM" peuvent" être" libres" dans" l’apoplasme," couplées" à" une" ancre" membranaire" ou" associées" à" un"
domaine"kinase,"les"LysMPRLK"étant"spécifiques"des"plantes."On"en"dénombre"cinq"chez"Arabidopsis"
dont"deux,"AtLYK1"aussi"appelés"AtCERK1"(Miya"et"al.,"2007)(pour"Chitin"Elicitor"Receptor"Kinase)"et"
AtLYK4" (Wan" et" al.," 2012)" impliqués" dans" la" résistance" envers" les" champignons" tel" qu’Alternaria0
brassicola0et"des"bactéries"comme"Pseudomonas0syringae(GimenezPIbanez"et"al.,"2009"";"Zeng"et"al.,"
2011";" AntolínPLlovera" et" al.," 2012)(Figure" 7)." De" ces" deux" récepteurs," le" premier" est" le" mieux"
caractérisé,"sans"doute"car"il"est"indispensable"à"la"perception"de"la"chitine""et"des"peptidoglycanes,"là"
où"le"second"n’apporte"qu’une"contribution"à"la"perception"de"ces"PAMPs."Ainsi"CERK1"est"capable"de"
32"
"
lier"de"manière"autonome"des"fragments"de"chitine"de"plus"de"huit"résidus,"ses"trois"domaines"lysM"
étant" importants" (Lizasa" et" al.," 2010)," ce" qui" provoque" la" phosphorylation" de" son" domaine" kinase"
(Petutschnig," Jones," Serazetdinova," Lipka," &" Lipka," 2010)." Une" homodimérisation" d’AtCERK1" en"
réponse"à"des"fragments"de"chitine"supérieurs"à"huit"résidus"de"NPacétylglucosamine"a"également"été"
identifiée"suite"à"la"liaison"du"chitosaccharide"au"cœur"du"domaine"lysM"central"qui"est"d’ailleurs"bien"
conservé"chez"plusieurs"espèces"végétales(Liu"et"al.,"2012)."En"revanche,"cellePci"ne"se"produit"pas"en"
présence"de"peptidoglycanes"où,"dans"ce"cas,"AtCERK1"pourrait"être"associé"à"AtBAK1"(Willmann"et"
al.," 2011";" Monaghan" and" Zipfel," 2012)." L’homodimérisation" d’AtCERK1" peut" être" empêchée" par" la"
présence" de" fragments" de" chitine" plus" courts" ce" qui" souligne" la" complexité" des" interactions"
moléculaires"mises"en"jeu"par"ces"types"de"récepteurs"visPàPvis"de"leurs"différents"ligands"(Liu"et"al.,"
2012)." Le" rôle" de" ces" récepteurs" LysMPRLK" dans" la" perception" de" chitooligosaccharides" et" de"
peptidoglycanes"dont"le"dénominateur"commun"est"la"présence"de"NPacetylglucosamine"montre"leur"
faculté"à"reconnaître"différents"ligands"apparentés."La"plasticité"de"ces"perceptions"est"aidée"par"des"
protéines"à"domaines"LysM"extracellulaires"dépourvus"de"domaines"kinases"tels"OsCEBiP"(Kaku"et"al.,"
2006)"pour"Chitin"Elicitor"Binding"Protein"chez"le"riz"et"AtLYM1"et"AtLYM3""(des"LYPs,"LysM"Protein)"
visPàPvis" des" peptidoglycanes" (Willmann" et" al.," 2011)." Chez" le" riz," des" protéines" analogues" à" ces"
dernières"reconnaissent"à"la"fois"les"peptidoglycanes"et"chitosaccharides,"permettant"ainsi"l’existence"
d’une" redondance" fonctionnelle" (Liu" et" al.," 2012)." Enfin," il" existe" d’autres" récepteurs" à" domaine"
kinase" interagissant" avec" des" sucres" :" les" lectinePRLKs." CellesPci" possèdent" des" domaines"
extracellulaires" variés" composés" notamment" de" feuillets" β" " susceptibles" de" reconnaître" différents"
enchaînements"de"sucres."Ce"type"de"récepteur""est"aussi"impliqué"dans"le"dialogue"moléculaire"avec"
des"parasites."Ainsi"LecRKPI.9,"en"étant"associé"au"contrôle"du"continuum"entre"membrane"plasmique"
et" paroi" végétale," participerait" à" la" perception" de" l’invasion" de" l’oomycète" Phythopthora0 brassicae"
(Bouwmeester"et"al.,"2011)."
33"
"
"
Figure"7:"Représentation"générale"des"rôles"des"récepteurs"à"domaines"LysM"de"plantes"dans"l’établissement"des"symbioses"
et"l’activation"de"la"MAMP"Triggered"Immunity."
La" perception" des" ligands" à" NPacetylglucosamine" que" sont" les" lipochitooligosaccharides" contrôle" les" interactions"
symbiotiques" mutualistes":" on" parle" de" facteur" Nod" et" Myc" selon" qu’il" s’agit" de" la" symbiose" fixatrice" d’azote" ou" de" la"
mycorhization."Les""réponses"immunitaires"sont"induites"par"la"perception"d’oligomères"de"chitine"ou"de"peptidoglycanes"
par" les" PRRs" à" LysM" qui" initient" la" MTI." Pour" interférer" avec" l’immunité" végétale," certains" champignons" sécrètent" des"
effecteurs"à"domaines"LysM"qui"masquent"ces"ligands,"supprimant"ainsi"la"mise"en"place"de"la"MTI."Gust,&A.&&et&al."(2012)."
Plant"LysM"proteins:"modules"mediating"symbiosis"and"immunity."Trends0in0plant0science:"1P8."
"
b. Perception des signaux mutualistes : les SAMPs et leurs récepteurs
Tout"comme"les"autres"champignons"et"bactéries"saprophytes"ou"pathogènes,"les"Rhizobia"fixatrices"
d’azote" et" les" champignons" mycorhiziens" arborent" des" polysaccharides" pariétaux" et" des" motifs"
peptidiques"susceptibles"de"déclencher"l’immunité"végétale."Une"hypothèse"permettant"d’expliquer"
comment" les" symbiotes" échappent" à" cette" réponse" est" leur" capacité" à" émettre" des" signaux"
particuliers" que" la" plante" pourrait" décoder" de" manière" parallèle" à" la" perception" des" MAMPs" afin"
d’infléchir" sa" réponse" vers" l’acceptation." Certains" de" ces" signaux" " SAMPs" (Symbiots" Associated"
Molecular" Pattern)(Zamioudis" and" Pieterse," 2012)" sont" étudiés" depuis" plus" de" vingt" ans" dans" la"
symbiose"fixatrice"d’azote"(facteurs"NOD)"et"des"analogues"ont"récemment"été"découverts"chez"les"
champignons" mycorhiziens":" ils" sont" pour" l’instant" dénommés" mycLCOs." Dans" " le" cas" des" bactéries,"
leur"synthèse"est"activée"par"la"perception"de"flavonoïdes."Il"s’agit"de"fragments"de"chitine"courts"de"
quatre"à"cinq"résidus"qui"possèdent"différents"types"de"substitutions,"sulfatations,"acétylations"mais"
aussi" des" groupements" lipidiques" qui" ont" donné" naissance" à" la" terminologie" de"
lipochitooligosaccharides."Ainsi"décorés,"ces"fragments"de"chitine,"qui"sont"en"outre"plus"courts"que"
ceux" issus" des" parois" fongiques" à" même" d’induire" la" défense," sont" à" l’origine" de" réponses" qui"
favorisent"les"symbioses"(Gough"and"Cullimore,"2011)(Figure"8)."
34"
"
"
Figure"8":"Structures"des"chitooligosaccharides"(CO)"et"lipoPchitooligosaccharides"(LCO)."
"A,"Structure"d’un"hexamère"linéaire"de"CO"montrant"une"orientation"alternée"des"résidus"de"NPAcetylglucosamine"liés"en"
β1P4."B,"Représentation"générique""d’un"LCO"symbiotique"montrant"les"sites"de"substitutions"chimiques."N"est"en"général"
égal" à" 1" ou" 2," résultant" en" des" tetramères" ou" pentamères" de" LCOs" substitués." C," " Structure" de" MycLCOs" sulfatés" ou" non"
sulfatés"de"Rhizophagus0irregularis0:0:"MycP"LCOPIV"(C16:0,"+/–S)"et"MycPLCOPIV"(C18:1∆9Z,"+/–S)."Notons"que"des"molécules"
similaires"mais"pentamèriques"ont"aussi"été"identifiées."D,"La"structure"des"facteurs"Nod"majeurs"de"Sinorhizobium0meliloti0
NodSmPIV(C16:2∆2E∆9Z," Ac," S)" et" Mesorhizobium0 loti0 NodMlPV" (C18:1∆11Z," Me," Cb," AcFuc).& Gough,& C.& and& Cullimore,& J."
(2011)." LipoPchitooligosaccharide" signaling" in" endosymbiotic" plantPmicrobe" interactions." Molecular0 plant%microbe0
interactions :0MPMI"24:"867P78."
La"recherche""des"mécanismes"de"perception"à"l’origine"des"symbioses"mutualistes"a"également"été"
entreprise" chez" des" plantes" légumineuses" telles" que" Medicago0 truncatula,0 Lotus0 japonicus0 (Hamel"
and"Beaudoin,"2010),0Pisum0sativum0(Limpens"et"al.,"2003)0ou"encore"la"non"légumineuse"Parasponia"
capable" d’interagir" avec" Rhizobium." Il" s’avère" que" des" LRRPRLKs" et" des" LysMPRLKs" sont" aussi"
impliqués" dans" la" mise" en" place" de" ces" associations." Les" légumineuses" telles" que" Medicago0
truncatula0(Arrighi"et"al.,"2006)"ou0Lotus0japonicus"(Lohmann"et"al.,"2010)"possèdent"une"vingtaine"de"
LysMPRLKs,"cette"expansion"de"la"famille"de"récepteurs"étant"potentiellement"liée"à"la"faculté"de"ces"
plantes" à" distinguer" chitooligosaccharides" et" lipochitooligosaccharides." Des" couples" de" LysMPRLKs,"
ont" pu" être" identifés" génétiquement," comme" " étant" requis" pour" promouvoir" les" réponses" aux"
35"
"
lipochitooligosaccharides" bactériens" Chez" Medicago0 truncatula" et" Lotus0 japonicus,0 il" s’agit"
respectivement"de"MtNFP"et"MtLYK3"et"de"LjNFR5"et"LjNFR1."Si"l’interaction"physique"entre"les"LysMP
RLKs"symbiotiques"n’a"pas"été"démontrée,"la"mutation"d’un"seul"des"deux"membres"bloque"tout"de"
même" le" processus" symbiotique." Ainsi" il" est" apparu" que" MtNFP" serait" un" récepteur" clé" de" la"
signalisation" précoce" et" un" acteur" important" de" la" colonisation" (Bensmihen" et" al.," 2011)" par" des"
souches" rhizobiennes" appropriées" tandis" qu’un" autre" récepteur" jouerait" un" rôle" de" reconnaissance"
plus"fin"des"facteurs"NOD"afin"de"ne"permettre"l’interaction"qu’avec"les"souches"bactériennes"les"plus"
favorables" à" l’hôte." Ce" récepteur" pourrait" être" MtLYK3" " qui" serait" spécifiquement" en" charge" du"
contrôle" de" l’infection" puisque" les" réponses" symbiotiques" sous" son" contrôle" ne" sont" pas" activables"
par" les" lipochitooligosaccharides" de" souches" rhizobiennes" pour" lesquelles" Medicago0 truncatula" est"
non"hôte"(Smit"et"al.,"2007)."MtNFP"appartient"au"sein"des"LysMPRLKs"à"la"catégorie"des"LYRs"(LysMP
RLK"Related)"et"non"des"LYKs"(LysMPRLK)."Cette"distinction"est"justifiée"par"la"présence"de"domaines"
kinases" incapables" de" s’autophosphoryler" chez" les" premiers." En" dépit" de" cette" impossibilité" à"
transmettre"de"manière"autonome"des"signaux,"MtNFP"a"un"rôle"central"dans"la"mise"en"place"de"la"
symbiose"et"on"peut"imaginer"que"l’association"avec"d’autres"LysMPRLKs"dont"le"domaine"kinase"est"
fonctionnel"soit"essentiel"à"son"action"(Gough"and"Cullimore,"2011)."Une"caractérisation"poussée"de"
ces"différents"domaines"a"permis"de"démontrer"chez"MtNFP"l’importance"du"domaine"lysM"central"
dans" la" reconnaissance" du" lipoochitooligosaccharide," soulignant" le" rôle" important" du" lipide" dans"
l’infection" rhizobienne" (Bensmihen" et" al.," 2011)." Les" nombreuses" glycosylations" des" domaines"
extracellulaires"ne"semblent"pas"cruciales"pour"ses"fonctions"biologiques."En"revanche,"les"cystéines"à"
l’origine"des"ponts"disulfures"extracellulaires"le"sont,"tout"comme"le"domaine"intracellulaire"bien"que"
ce"soit"une"kinase"inactive"dont"la"transphosporylation"n’est"pas"nécessaire"à"son"activité"(Lefebvre"et"
al.," 2012)." Les" récepteurs" aux" analogues" mycorhiziens" de" " ces" signaux" sont" toujours" non" identifiés"
chez" les" légumineuses." Cependant," l’identification" d’un" seul" et" même" LysMPRLK" " orthologue" de"
MtNFP" chez" Parasponia" (Op" den" Camp" et" al.," 2011)" requis" pour" les" deux" symbioses" suggère"
fortement" l’implication" de" ce" type" de" protéine" dans" la" perception" des" signaux" symbiotiques"
initiateurs" des" deux" types" de" symbiose." De" plus," le" traitement" d’un" mutant" " nfp" avec" des"
lipochitooligosaccharides" fongiques" aboutit" à" l’absence" totale" des" réponses" transcriptomiques"
observées"chez"le"sauvage,"confirmant"le"rôle"clé"de"ce"récepteur"dans"la"perception"initiale"de"ces"
signaux"(Czaja"et"al.,"2012),"en"dépit"de"la"faculté"des"champignons"mycorhiziens"à"interagir"avec"ce"
mutant." Enfin," la" forte" activation" transcriptionnelle" par" le" champignon" de" MtLYR1" pour" LysM"
receptor"like"kinase"Related"(Young"et"al.,"2011),"proche"homologue"du"récepteur"au"signal"bactérien"
MtNFP""suggère"que"ce"récepteur"pourrait"jouer"un"rôle"crucial"dans"la"symbiose"avec"le"champignon"
(Amor"et"al.,"2003";"Arrighi"et"al.,"2006";"Maillet"et"al.,"2011)."
36"
"
Outre"l’utilisation"d’outils"génétiques"pour"identifier"le"rôle"de"tels"récepteurs"dans"la"perception"des"
SAMPs," des" avancées" significatives" dans" l’étude" biochimique" de" la" reconnaissance" des" ligands" ont"
révélé"une"forme"de"versatilité"entre"les"ligands"reconnus"et"les"signaux"transmis"allant"de"la"défense"
à" la" symbiose." La" capacité" à" lier" les" lipochitooligosaccharides" des" LysMPRLKs" symbiotiques" de" Lotus0
japonicus" orthologues" de" NFP" et" LYK3," chez" Medicago0 truncatula" a" été" validée" expérimentalement"
très"récemment"(Broghammer"et"al.,"2012).""En"outre,"la"faculté"de"ces"récepteurs"à"reconnaître"des"
fragments"de"chitine"de"moins"de"cinq"résidus"suggère"qu’ils"puissent"aussi"être"acteurs"de"réponses"
immunitaires" et" qu’ils" ont" évolué" à" partir" des" récepteurs" initialement" nécessaires" à" l’activation" de"
l’immunité." L’activation" de" réponses" de" type" hypersensible" lorsque" NFR1" et" NFR5" " (Nod" factor"
Receptor)" sont" coexprimés" en" système" hétérologue" pourrait" constituer" une" réminiscence" de" cette"
évolution" (Madsen" et" al.," 2011)." Les" interconnexions" entre" symbiose" et" défense" sont" également"
renforcées" par" le" fait" que" les" lipochitooligosaccharides" (LCOs)" et" chitooligosaccharides" déclenchent"
chez"les"légumineuses"des"réponses"partiellement"identiques"(Nakagawa"et"al.,"2011)."De"plus,"une"
application" de" fortes" doses" de" lipochitooligosaccharide" aboutit" à" des" réponses" de" défense," sans"
doute" au" travers" de" la" saturation" aspécifique" de" récepteur(s)" de" la" chitine" par" le" squelette"
saccharidique"du"signal"symbiotique"(Duzan"et"al.,"2005)."En"dehors"de"la"reconnaissance"du"ligand"
proprement" dite," une" autre" explication" à" ce" recouvrement" peut" être" le" nombre" réduit" de"
substitutions" entre" les" domaines" kinases" activant" l’immunité" et" la" symbiose" chez" cette" famille" de"
récepteurs." L’analyse" phylogénétique" de" ces" domaines" kinases," révèle" que" l’on" peut" classer" ces"
protéines" selon" trois" groupes" dont" les" deux" plus" conséquents" mêlent" gènes" de" légumineuses" et" de"
non"légumineuses"(Figure"9)."A""titre"d’exemple,"le"changement"de"trois"acides"acides"aminés"dans"le"
domaine" kinase" de" CERK1" lui" permet," combiné" au" domaine" extracellulaire" de" NFR1," de"
complémenter"le"mutant"nfr1"(Nakagawa"et"al.,"2011)."Ainsi"les"légumineuses,"en"dépit"d’un"nombre"
important" de" LysMPRLKs," ne" parviennent" pas" à" discriminer" strictement" les" signaux" apparentés" que"
sont"les"chitooligosaccharides"et"les"lipochitooligosaccharides."
37"
"
Figure" 9" :" Arbre" phylogénétique" montrant" les" valeurs" de" bootstrap" des" domaines" kinases" des" LysMPRLK" de" Medicago0
truncatula0(16),"du"riz"(6)"d’Arabidopsis"(5)"et"de"NFR1"et"NFR5"de"Lotus0japonicus.00
Arrighi,& J.+F.& et& al." (2006)." The" Medicago" truncatula" lysin" [corrected]" motifPreceptorPlike" kinase" gene" family" includes" NFP"
and"new"nodulePexpressed"genes."Plant0physiology"142:"265P79.0
"En"marge"du"cas"des"saccharides"basés"sur"un"squelette"de"chitine,"d’autres"MAMPs"de"Rhizobia"sont"
aussi"sensiblement"modifiés"et"présentent"une"inhibition"de"leur"faculté"à"activer"la"défense."Ainsi"la"
flagelline" purifiée" de" Mesorhizobium0 meliloti" n’induit" pas" la" défense" (en" raison" d’une" modification"
dans" la" séquence" flg22" de" sa" flagelline)" (LopezPGomez" et" al.," 2012)." Par" ailleurs," des"
lipopolysaccharides," exopolysaccharides" et" glucanes" cycliques" libérés" par" plusieurs" souches"
rhizobiennes" se" révèlent" essentiels" au" développement" d’interactions" fructueuses" puisque" des"
bactéries" mutantes," incapables" de" synthétiser" ces" motifs" particuliers," déclenchent" les" réponses"
immunitaires"de"l’hôte"(Antuono"et"al.,"2005";"Staehelin"et"al.,"2006";"Jones"et"al.,"2008";"D’Antuono"et"
al.," 2008)." La" compétition" entre" ces" molécules" et" les" MAMPs" responsables" de" l’activation" des"
défenses" est" l’une" des" hypothèses" expliquant" le" mode" d’action" de" ces" molécules" (Mithöfer," 2002";"
Zamioudis" and" Pieterse," 2012)." Il" est" curieux" de" constater" que" les" symbiotes" aient" développé" un"
38"
"
arsenal" de" contrePmesures" sans" que" les" parasites" n’y" soient" parvenus." Il" existe" tout" de" même" un"
exemple" en" ce" sens" puisque" les" nématodes" sont" capables" de" déclencher" des" réponses" analogues" à"
celles" engendrées" par" les" lipochitooligosaccharides," en" empruntant," de" plus," la" même" voie" de"
perception"(Weerasinghe"et"al.,"2005)."Si"la"synthèse"de"ces"signaux"n’a"pas"été"démontrée,"depuis,"la"
capacité"de"ces"animaux"à"fabriquer"la"chitine"suggère"qu’ils"puissent"être"en"mesure"de"le"faire."De"
plus," les" recouvrements" dans" les" jeux" d’acteurs" moléculaires" impliqués" dans" la" formation" des"
nodosités"et"des"sites"nourriciers"de"nématodes"pourraient"trouver"leur"explication"dans"ce"composé"
issu"du"nématode"(Favery"et"al.,"2002";"Damiani"et"al.,"2012).""
Si" des" LysMPRLKs" sont" requis" pour" la" mise" en" place" des" symbioses," il" faut" également" noter" que" les"
événements"de"signalisation"nécessaires"aux"deux"interactions"convergent"rapidement"en"aval"vers"
un"LRRPRLK"appelé"DMI2"(Does"not""Make"Infection)"chez"Medicago0truncatula"et"SYMRK""(SYMbiotic"
Receptor"Kinase)"chez"les"espèces"capables"d’établir"la"symbiose"endomycorhizienne"(Gherbi"et"al.,"
2008";"Markmann"et"al.,"2008).""A"ce"titre,"ce"récepteur"marque"l’entrée"dans"la"voie"de"signalisation"
commune" CSP" (Common" Symbiotic" Pathway)." Il" faut" tout" de" même" noter" que" le" domaine"
extracelullaire" de" ce" récepteur" chez" les" légumineuses" exhibe," en" plus" des" LRRs," un" domaine"
extracellulaire"sans"homologie"connue,"suggérant"une"fonction"particulière"dans"la"symbiose"fixatrice"
d’azote."Pour"l’heure,"aucun"ligand"n’est"identifié."
Enfin,"parallèlement"à"ces"récepteurs"de"signalisation"symbiotique,"un"récepteur"lectine"MtLecRK1.1"
contribue"à"la"formation"de"nodosités"chez"Medicago0truncatula"puisque"sa"surexpression"augmente"
le" nombre" de" nodosités." " En" outre," une" autre" protéine" à" domaine" lectine" est" capable" de" lier" le"
lipochitooligosaccharide"bactérien,"ce"qui"active"l’activité"apyrase"à"laquelle"elle"est"associée,"jouant"
potentiellement" un" rôle" de" signalisation." L’expression" hétéroloque" de" cette" protéine" de" Medicago0
truncatula"chez"Lotus0japonicus"augmente"aussi"le"nombre"de"nodosités""développées"(De"Hoff"et"al.,"
2009)." " Enfin" un" récepteur" du" même" type" contrôlant" la" signalisation" symbiotique" commune" a"
également"été"identifié"chez"cette"dernière"plante"(Roberts"et"al.,"2012)."
c. Effecteurs microbiens et leurs détections par la plante
La"coévolution"permanente"entre"effecteurs"éteignant"la"PTI"et"gènes"de"résistance"à"ces"effecteurs"
visant"à"réactiver"les"défenses"via"l’ETI"a"longtemps"été"strictement"associée"aux"interactions"plantesP
pathogènes"(He"et"al.,"2007";"Boller"and"He,"2009";"Bozkurt"et"al.,"2012)."Parmi"les"multiples"exemples"
disponibles," nous" nous" focaliserons" sur" les" effecteurs" qui" affectent" des" fonctions" de" perception"
décrites" ciPdessus" afin" d’illustrer" le" zigPzag." Ces" protéines" peuvent" être" sécrétées" par" les"
microorganismes" dans" l’apoplasme" où" elles" interfèrent" notamment" avec" les" perception" de" MAMPs"
comme" c’est" notamment" le" cas" pour" des" protéines" à" domaine" LysM" capables" de" séquestrer" les"
39"
"
MAMPs" à" NPacétylglucosamine" (NAG)," à" titre" d’exemple" chez" le" champignon" Mycosphaerella0
graminicola"(Marshall"et"al.,"2011).""Les"effecteurs"peuvent"aussi"être"introduits"par"les"parasites"dans"
les" cellules" de" l’hôte" où" ils" vont" manipuler" les" événements" de" signalisation" menant" à" la" réponse"
immunitaire." Cela" est" illustré" par" Pseudomonas0 syringae" qui" injecte" AvrPtoB" via" son" système" de"
sécrétion"de"type"III"dans"les"cellules"de"l’hôte"pour"empêcher"la"signalisation"contrôlée"par" CERK10
qui"est"susceptible"de"répondre"au"peptidoglycane"du"parasite"(Zeng"et"al.,"2011";"GimenezPIbanez"et"
al.," 2009)." Les" oomycètes" possèdent" aussi" des" effecteurs," notamment" des" RXLR," du" nom" de" la"
séquence" en" acide" aminé" nécessaire" à" leur" endocytose" par" la" plante" (Kale" et" al.," 2010)," dont" un,""
AvrPio,""cible"LecRKPI.9""pour"faciliter"la"colonisation"par"Phytothphthora0brassicae"(Bouwmeester"et"
al.," 2011)." Les" bactéries" phytopathogènes" possèdent" des" dizaines" d’effecteurs" (Deslandes" &" Rivas,"
2012)"et"les"oomycètes"des"centaines"(Bozkurt"et"al.,"2012";"Stassen"and"Van"den"Ackerveken,"2011)"
tout"comme"les"champignons"(Stergiopoulos"&"De"Wit,"2009)"ou"les"nématodes"(Bellafiore"&"Briggs,"
2010)."Ainsi"ces"protéines"aux"fonctions"variées"peuvent"manipuler"diverses"fonctions"de"l’hôte"allant"
de"la"signalisation,""à"la"mise"en"place"des"réponses"de"défenses,"en"passant"par"le"développement"
végétal."Des"gènes"dits"de"résistance"ont"évolué"chez"les"plantes"afin"de"percevoir"la"présence"de"tels"
effecteurs"et"de"réactiver"les"réponses"immunitaires"appropriées":""on"en"dénombre"par"exemple"plus"
d’une" centaine" chez" Arabidopsis0 thaliana" (Jones" and" Dangl," 2006)." Ils" codent" pour" des" protéines" à"
domaines"Coil"Coiled"(CC)"ou"Toll"Interleukine"Receptor"(TIR)"capables"d’interagir"avec"leurs"cibles"qui"
peuvent" être" les" protéines" microbiennes" ou" plus" souvent" des" produits" de" l’activité" des" protéines"
microbiennes"dans"le"cadre"d’un"système"de"garde"employant"par"exemple"RIN4"(Jones"and"Dangl,"
2006)."Ces"domaines"sont"couplés"à"des"Nucleotide"Binding"Site"(NBS)"qui"vont"transmettre"le"signal"
d’une"manière"encore"méconnue."Ces"sites"NBS"sont"euxPmêmes"associés"à"des"LRRs"qui,"dans"ce"cas,"
jouent"un"rôle"de"régulateur"négatif,"évitant"l’activation"constitutive"de"défense."
En" parallèle," des" données" récentes" montrent" que" les" microorganismes" mutualistes" utilisent" aussi"
cette" stratégie" pour" coloniser" les" plantes." Ainsi," le" champignon" ectomycorhizien" Laccaria0 bicolor0
introduit"dans"les"cellules"végétales"une"protéine"MISSP7"(Mycorhizal"Induced"Small"Secreted"Protein"
7)" (Plett" et" al.," 2011)" par" des" mécanismes" d’endocytose" végétale" similaires" à" ceux" utilisés" par"
Phytophtora" (Kale" et" al.," 2010)." Une" fois" dans" l’hôte," cet" effecteur" est" adressé" au" noyau" où" il"
manipule" transcriptionnellement" la" cellule." Une" autre" protéine," découverte" cette" fois" chez" un"
champignon" endomycorhizien" et" nommée" SP7" (Secreted" Protein" 7)," est," elle" aussi," adressée" au"
noyau." Interagissant" directement" avec" un" facteur" de" transcription" de" Medicago0 truncatula," elle"
permet" la" croissance" du" champignon" in0 planta" mais" peut" aussi" favoriser" l’agression" par" " un"
champignon"pathogène"biotrophe,"Colletotrichum0trifolii0(Kloppholz"et"al.,"2011";"Sanders,"2011)."Si,"
dans"ces"cas,"le"symbiote"parvient"à"manipuler"son"hôte,"la"symbiose"rhizobienne"fournit"un"exemple"
40"
"
inverse" puisque" deux" récepteurs" typiques" de" l’ETI" de" type" TIRPNBSPLRR" (Toll" Interleukine" Receptor"
Nucleotide"Binding"SitePLRR)"ont"été"clonés"chez"le"soja"et"limitent"le"spectre"de"symbiotes"bactériens"
chez"cette"plante"(Yang"et"al.,"2010)."Cette"découverte"suggère"en"retour"que"ces"microbes"sécrètent"
in0 planta" des" effecteurs" facilitant" " l’acceptation" des" symbiotes" par" la" plante." Ils" ont" pu" être" mis" en"
évidence" chez" la" souche" Rhizobienne" NGR234" à" large" spectre" d’hôtes" dont" une" mutation" de" son"
système" de" sécrétion" de" type" III" empêche" l’interaction" avec" sa" centaine" d’hôtes" potentiels" en"
bloquant" la" sécrétion" d’une" vingtaine" de" protéines" (Deakin" and" Broughton," 2009" ;" Wenzel" et" al.,"
2010";"Zamioudis"and"Pieterse,"2012).""
"
B. La signalisation
"
Les" récepteurs" de" signaux" microbiens" sont" en" charge" d’une" transmission" rapide" de" l’information."
CellePci" se" matérialise" en" général" par" une" " autophosphorylation" du" récepteur" qui" entraîne"
consécutivement" des" phosphorylations" de" protéines" physiquement" proches" de" ce" dernier." Par"
exemple," AtFLS2" est" localisé" dans" les" «"lipid" rafts"»" directement" au" contact" de" coPrécepteurs," de"
pompes"ioniques"et"d’enzymes"qui"synthétisent"des"espèces"réactives"de"l’oxygène"Reactive"Oxygen"
Species,"(ROS)"(Keinath"et"al.,"2010)."Ainsi,"le"récepteur"peut"rapidement"interagir"de"manière"directe"
ou" indirecte" avec" des" protéines" contrôlant" des" messagers" secondaires" tels" que" des" cascades" de"
phosphorylation,"l’ion"calcium"ou"les"ROS."
1. Flux ioniques, ROS et cascades de phosphorylations
a. Dans les réponses immunitaires
La" première" minute" consécutive" à" la" perception" d’un" PAMP" voit" se" produire" une" dépolarisation"
membranaire" liée" à" des" flux" ioniques" massifs" qui" impliquent" notamment" un" influx" calcique"
cytoplasmique"(Jeworutzki"et"al.,"2010)."Cet"événement"active"des"voies"de"signalisation"reposant"sur"
des"cascades"de"phosphorylation"initiées"au"niveau"des"récepteurs"(Park"et"al.,"2012)"puis"impliquant"
des" senseurs" calciques" de" type" CDPK" (Calcium" Dependent" Protein" Kinase)" et" calmodulines" (Leba" et"
al.,"2012";"Du"et"al.,"2009)."Parallèlement,"et"de"manière"indépendante"du"calcium,"des"voies"MAPK"
Mitogen" Activated" Protein" Kinase" participent" de" manière" synergique" à" la" mise" en" œuvre" de" la"
réponse" (Boudsocq" et" al.," 2010";" Segonzac" and" Zipfel," 2011)(Figure" 10)." Le" calcium" est" en" revanche"
nécessaire"à"l’activation"de"l’activité"NADPH"oxydase,"source"de"ROS."Ces"enzymes"génèrent"en"effet"
.P
de" l’O2 ," une" espèce" réactive" de" l’oxygène" éphémère" qui" est" rapidement" convertie" en" péroxyde"
d’hydrogène," H2O2," qui" possède" des" propriétés" antimicrobiennes" et" qui" joue" aussi" un" rôle" dans"
41"
"
l’activation" de" réponses" de" défenses" diverses" (activation" systémique" de" l’immunité," réponse"
hypersensible,"métabolisme"secondaire,"lignifications"des"parois…)(Bolwell"and"Daudi,"2009)."L’oxyde"
.
nitrique"(NO )"est"une"autre"ROS"impliquée"dans"des"signalisations"immunitaires"où"des"réactions"de"
nitrosylation" de" substrats" activent" certaines" voies" associées" à" la" défense" (Leitner" et" al.," 2009)."
D’autres"messagers"secondaires"moins"caractérisés"existent"tels"que"les"lipides"membranaires"dont"le"
clivage"est"associé"à"la"réponse"hypersensible"(den"Hartog"et"al.,"2001";"Hartog"et"al.,"2003";"Vossen"et"
al.," 2010)." A" titre" d’exemple," la" dihydrosphingosine," en" plus" de" réguler" des" oscillations" calciques"
cytoplasmiques"et"aussi"nucléaires,"contrôle"la"production"de"ROS,"participant"ainsi"à"des"réponses"de"
défenses" non" associées" à" la" réponse" hypersensible"" (Lachaud" et" al.," 2011)." Enfin," les" protéines" G"
contrôlent" des" flux" ioniques" jouant" des" rôles" de" messagers" secondaires" en" aval" de" FLS2" et" EFR"
(Nicaise"et"al.,"2009)."
Figure"10":""Modèle"actuel"de"la"voie"de"signalisation"de"FLS2"chez"Arabidopsis.""
FLS2" interagit" constitutivement" avec" la" protéine" à" domaine" DENN" nommée" SCD1," BIK1" et" aussi" PBLs." BIK1" est" peutPêtre"
également"associée"à"BAK1"à"l’état"de"repos."Lors"de"la"liaison"de"flg22,"un"complexe"se"forme"entre"FLS2,"BAK1"et"BIK1"
instantanément."D’autres"SERKs,"tel"que"BKK1"pourraient"aussi"faire"partie"de"ce"complexe."Les"RLKs"FER"et"HERK"ainsi"que"
les"pompes"à"proton"AHA1"et"DET3"sont"présentes"dans"les"domaines"membranaires"résistant"aux"détergents""induits"par"
flg22" où" est" localisée" FLS2." Consécutivement" à" la" liaison" de" flg22," de" multiples" événements" de" phosphorylation" se"
produisent"rapidement"et"BIK1"est"alors"libérée"du"complexe."La"perception"de"flg22"conduit"à"l’activation"d’au"moins"deux"
cascades" de" MAPK," toutes" impliquées" dans" l’activation" de" l’expression" de" gènes" de" défense." La" reconnaissance" de" flg22"
déclenche"aussi"un"influx"calcique"qui"activerait"les"protéines"kinases"dépendantes"du"calcium"(CDPK)"et"la"NADPHPoxidase"
AtRbohD" requise" pour" la" production" de" ROS." De" manière" indépendante" et" synergique" les" CDPK" 4,5,6" et" 11" induisent"
l’expression"des"gènes"de"défense."Segonzac,&C.&and&Zipfel,&C."(2011)."Activation"of"plant"patternPrecognition"receptors"by"
bacteria."Current0opinion0in0microbiology"14:"54P61."
"
"
"
42"
"
b. Dans les réponses mutualistes
Des" mouvements" ioniques" sont" égalements" observés" en" réponse" aux" perceptions" mutualistes"
(HoldawayPclarke"et"al.,"2000)."L’importance"des"signaux"calciques"est"établie"depuis"plus"de"10"ans"
dans" la" symbiose" rhizobienne." Une" particularité" de" ceuxPci" est" l’observation" d’oscillations" calciques"
cytoplasmiques"dans"les"poils"absorbants"en"réponse"aux"lipochitoolisaccharides"dans"les"dizaines"de"
minutes" qui" suivent" le" traitement" (Ehrhardt" et" al.," 1996)." Ces" oscillations" sont" à" l’origine" de" la"
reprogrammation"symbiotique"(Miwa"et"al.,"2006)."L’analyse"d’une"sonde"rapportrice"des"oscillations"
calciques"nucléaires"révèle"que"ce"signal"y"est"aussi"présent."Ainsi"ces"oscillations,"que"l’on"nommera"
par"la"suite"«"spiking"»,"ont"aussi"été"caractérisées"au"niveau"nucléaire"(Kosuta"et"al.,"2008";"Sieberer"
et"al.,"2009)"que"ce"soit"en"réponse"aux"molécules"rhizobiennes"dans"le"poil"absorbant"ou"en"réponse"
aux"signaux"mycorhiziens"ou"au"contact"du"microorganisme"luiPmême""dans"les"cellules"épidermiques"
(Oldroyd"and"Downie,"2006";"Chabaud"et"al.,"2011";"Sieberer"et"al.,"2012)."Dans"toutes"les"interactions"
symbiotiques,"ce"spiking"dépend"des"gènes"de"la"voie"symbiotique"commune,"appelés"chez"Medicago0
truncatula" DMI1" et" DMI2." Le" premier" cité" code" pour" un" canal" ionique" situé" sur" la" membrane"
nucléaire" à" l’origine" du" contrôle" de" la" mobilisation" des" pools" calciques" renfermés" dans" l’enveloppe"
nucléaire"et""le"réticulum"endoplasmique"(Ané"et"al.,"2004";"Peiter"et"al.,"2007";"Capoen"et"al.,"2011)."
On"peut"noter"que"des"protéines"jouant"un"rôle"similaire"ont"aussi"été"étudiées"chez"Lotus0japonicus0
(Riely" et" al.," 2007";" Singh" and" Parniske," 2012)(Figure" 11)." Génétiquement" en" position" intermédiaire"
entre"le"spiking"DMI1/DMI2"dépendant""et"la"reprogrammation"transcriptionnelle,"DMI3"est"un"gène"
unique"chez"les"plantes"codant"une"«"Calcium"Calmoduline"dependent"Kinase"»"(CCAMK)."Située"dans"
le"noyau,"c’est"elle"qui"intègre"le"signal"calcique"(Mitra"et"al.,"2004)."Si"CCAMK"fait"partie"de"la"CSP,"et"
est"bien"conservée"des"légumineuses"au"non"légumineuses"tel"que"le"riz,"elle""possède"des"spécifités"
de"modes"d’action"dans"chacune"des"symbioses"qui"laissent"supposer"que"le"signal"calcique,"qui"est"
sensiblement" différent" entre" mycorhization" et" nodulation," puisse" être" discriminé" à" ce" niveau"
(Shimoda"et"al.,"2012)."Considérées"dans"leur"ensemble,"ces"données"mettent"en"lumière"la"grande"
importance" de" la" signalisation" calcique" dans" les" symbioses" mutualistes" et" illustrent" l’émergence" de"
mécanismes" originaux" en" comparaison" des" voies" impliquées" dans" l’immunité," fournissant" une" clé"
pour" comprendre" comment" les" plantes" adoptent" un" comportement" discriminant" visPàPvis" des"
différentes"interactions"(Singh"and"Parniske,"2012).""
43"
"
Figure"11":"Signalisation"de"la"perception"des"facteurs"Nod"dans"le"rhizoderme.""
La" perception" des" facteurs" Nod" implique" des" RLKs" contenant" des" motifs" LysM" et" LRR." Cette" perception" est" liée" à" des"
oscillations" calciques" nucléaires" et" des" changements" de" croissance" des" poils" absorbants," sans" doute" contrôlés" " par" un"
gradient"de"ROS"et"de"calcium"s’accroissant"vers"l’extrémité"des"poils."Un"second"messager"est"probablement"en"charge"de"
la" transmission" du" signal" de" la" membrane" plasmique" au" noyau." Un" canal" potassique" à" la" membrane" nucléaire" interne"
pourrait"être"la"cible"d’un"tel"messager"et"le"changement"de"concentration"en"potassium"conduirait"à"une"hyperpolarisation"
des"espaces"intermembranaires"nucléaires"qui"activerait"un"canal"calcique"voltage"dépendant."La"perception"des"oscillations"
calciques"requiert"une"calcium/calmodulinePdépendante"protéine"kinase"(CCaMK)"et"les"régulateurs"transcriptionnels"de"la"
voie"de"signalisation"de"la"nodulation"NSP1,"NSP2,"Ets2"et"ERN."Abréviations":"NFP/NFR5"Nod"Factor"perception/Nod"Factor"
Receptor;"DMI2/SYMRK"doesn’t"make"infection/symbiotic"receptor"kinase;"PLC/PLD"phospholipase"C/D,"NSP"Nod"signaling"
pathway," Ets2" Ethylene" triggered" susceptibility," ERN" ERF" required" for" nodulation," ENOD" ealy" nodulation" gene." Oldroyd,&
G.E.D.&and&Downie,&J.A."(2006)."Nuclear"calcium"changes"at"the"core"of"symbiosis"signalling."Current0opinion0in0plant0biology"
9:"351P7."
Cependant"le"calcium"ne"constitue"pas"l’unique"messager"secondaire"qui"contrôle"l’établissement"des"
endosymbioses." MtDMI2,0 situé" en" amont" de" cette" signalisation," interagit" directement" avec" une"
MAPK" (Chen" et" al.," 2012)" et" un" facteur" de" transcription" (Zhu" et" al.," 2008)" requis" pour" le" bon"
déroulement" de" la" nodulation." De" même," la" plupart" des" acteurs" connus" de" la" signalisation"
symbiotique"sont"susceptibles"d’être"phosphorylés"afin"d’être"mis"en"action"(Grimsrud"et"al.,"2010)."
D’autres" messagers" secondaires" importants" pour" la" nodulation" sont" les" espèces" réactives" de"
l’oxygène." Leur" régulation" apparaît" différente" entre" symbiose" et" immunité." Ainsi," les"
lipochitooligosaccharides"diminuent"la"libération"d’"H2O2" extracellulaire"et"empêchent"leur"induction"
par" des" éliciteurs" caractérisés" (Shaw" and" Long," 2003)." Au" niveau" interne," l’H2O2" est" consommé" par"
une" peroxydase" induite" par" les" signaux" rhizobiens," ce" qui" explique" une" diminution" de" sa" teneur"
(Ramu"et"al.,"2002";"Lohar"et"al.,"2007)."En"revanche,"sa"présence"est"augmentée"dans"le"bout"du"poil"
44"
"
absorbant" (Cárdenas" et" al.," 2008)," ce" qui" est" un" événement" clé" de" la" déformation" des" poils" pour"
l’accueil" du" symbiote." Dans" les" étapes" plus" avancées" de" l’interaction," une" NADPH" oxidase" est"
impliquée"dans"le"bon"fonctionnement"du"nodule"(Marino"et"al.,"2011)."Des"ROS"sont"aussi"observés"
au"contact"des"hyphes"mycorhiziens"et"dans"les"arbuscules"sénescents"(Fester"and"Hause,"2005)."Le"
NO" est" aussi" impliqué" dans" la" réponse" aux" symbiotes" puisqu’il" est" induit" par" la" perception" du"
champignon"(Calcagno"et"al.,"2011)"tandis"qu’il"est"un"régulateur"du"nombre"de"nodosités"(Cam"et"al.,"
2012)" et" de" la" fixation" d’azote" atmosphérique" par" Rhizobium" (Meilhoc" et" al.," 2011)." Enfin," La"
signalisation"basée"sur"des"phosphoinositides"est"aussi"impliquée"dans"la"signalisation"de"la"symbiose"
fixatrice" d’azote." CeuxPci," libérés" par" des" phospholipases" au" cours" de" la" signalisation"
lipochitooligosaccharidique,"permettent"les"événements"précoces"d’acceptation"au"niveau"des"poils"
absorbants." Les" protéines" G" en" hétérotrimères" sont" aussi" associées" à" ces" réponses," leur" activation"
pharmacologique"induisant"l’expression"de"nodulines"sous"le"contrôle"de"DMI30(Pingret"et"al.,"2004).""
"
2. Modification de la balance hormonale
L’étude" des" hormones" est" une" des" thématiques" de" recherche" les" plus" dynamiques" dans" la"
communauté" scientifique" végétale," tant" en" ce" qui" concerne" les" processus" développementaux""
(Santner"and"Estelle,"2009";"Durbak"et"al.,"2012)"que"ceux"liés"à"la"résistance"aux"stress,"notamment"
biotiques"(Pieterse"et"al.,"2009";"RobertPseilaniantz"et"al.,"2012";"Muday"et"al.,"2012)."S’il"apparaît"que"
les"hormones"jouent"des"rôles"similaires"pour"ce"qui"est"de"l’immunité"dans"les"parties"aériennes"et"
souterraines" (Millet" et" al.," 2010)," une" limitation" de" taille" se" situe" au" niveau" de" la" plante" modèle"
utilisée"pour"caractériser"ces"rôles."En"effet,"la"plupart"des"connaissances"proviennent"principalement""
de" l’étude" d’Arabidopsis0 thaliana," incapable" de" s’associer" à" Rhizobium" et" aux" champignons"
endomycorhiziens." Ainsi," on" peut" imaginer" que" la" dynamique" de" la" balance" hormonale" en" réponse"
aux" agents" pathogènes" soit" différente" chez" des" plantes" capables" d’établir" en" parallèle" des"
associations"mutualistes."En"ce"sens,"il"a"pu"être"démontré"que"la"résistance"à"un"parasite"fongique"
racinaire" dépend" de" l’éthylène" chez" Medicago0 truncatula" mais" pas" chez" Arabidopsis0 thaliana"
(Anderson"et"al.,"2010";"Anderson"and"Singh,"2011)."Ainsi"il"est"difficile"d’établir"des"relations"croisées"
entre"la"plupart"des"expériences"menées"pour"établir"le"rôle"des"hormones"dans"le"mutualisme"et"le"
parasitisme." Pour" ces" raisons," nous" considèrerons" d’abord" " d’un" point" de" vue" global" le"
fonctionnement" de" l’immunité" contrôlée" par" les" hormones" avant" d’entrer" dans" le" détail" des" rôles"
connus"pour"ces"métabolites"dans"les"symbioses.""
"
45"
"
a. Dans les réponses immunitaires
Les" principales" voies" de" signalisation" de" la" réponse" hormonale" visPàPvis" des" microorganismes""
parasites" sont," " d’une" part," celle" de" l’acide" salicylique" (SA";" Salicylic" Acid)" et," d’autre" part," celle"
associant"l’éthylène"(ET)"et"le"jasmonate"(JA";"Jasmonic"Acid)."Interagissant"de"manière"antagoniste,"
ces" deux" voies" constituent" les" leviers" principaux" activés" par" la" plante" pour" confiner" au" mieux" " le"
développement" du" parasite" (Pieterse" et" al.," 2012";" RobertPseilaniantz" et" al.," 2012" ;" Muday" et" al.,"
2012)(Figure" 12)." Ces" signalisations," lorsqu’elles" sont" affectées," conduisent" à"des" modifications"
importantes" de" la" constitution" du" microbiome" de" la" rhizopshère" d’Arabidopsis0 thaliana," preuve" de"
l’importance"de"ces"signaux"visPàPvis"des"microorganismes"telluriques"(Doornbos"et"al.,"2011)."Le"rôle"
de"cet"antagonisme"quant"à"la"réponse"aux"nématodes"et"aux"plantes"parasites"ne"sera"pas"détaillé"
ici,"même"s’il"apparaît"que"l’acide"salicylique""et"le"jasmonate"face"aux"premiers"et"l’acide"salicylique"
face" aux" dernières" soient" importants" pour" la" résistance" (Gutjahr" and" Paszkowski," 2009)."
"
Figure" 12a" :" Représentation" schématique" des" composantes" moléculaires" impliquées" dans" la" suppression" de" la" voie" de"
l’acide"jasmonique"par"l’acide"salicylique.Les"composantes"essentielles"à"ce"crosstalk"démontrées"à"l’aide"de"mutants"sont"
soulignées."Les"cadres"verts"montrent"les"composantes"pour"lesquelles"le"niveau"d’expression"dépend"de"l’acide"salicylique."
Les" lignes" pleines" indiquent" les" interactions" démontrées," les" lignes" hachurées" indiquent" des" interactions" supposées."
Pieterse,&C.M.J.&et&al."(2012)."Hormonal"Modulation"of"Plant"Immunity."Annual0review0of0cell0and0developmental0biology:"1P
33.Figure" 12b" :" Modulation" de" la" voie" de" signalisation" de" l’acide" jasmonique" par" l’éthylène," l’acide" abssicique" et" les"
gibérrellines.Les"agents"pathogènes"nécrotrophes"induisent"le"jasmonate"et"l’éthylène"tandis"que"les"insectes"induisent"le"
jasmonate" et" l’acide" abscisique." Les" branches" de" la" voie" de" l’acide" jasmonique" dépendant" de" l’éthylène" et" du" jasmonate"
sont" mutuellements" antagonistes." Les" lignes" pleines" indiquent" les" interactions" établies" et" les" hachurées," les" supposées."
Pieterse,&C.M.J.&et&al."(2012)."Hormonal"Modulation"of"Plant"Immunity."Annual0review0of0cell0and0developmental0biology:"1P
33."
46"
"
La"voie"de"l’acide"salicylique"contrôle"essentiellement"des"réponses"associées"à"la"résistance"face"aux"
agents" pathogènes" biotrophes." Elle" " active" des" batteries" de" fonctions" associées" à" la" défense," les"
Pathogenesis" related" " (PR)" protéines" et" notamment" PR1" marqueur" de" cette" voie." Elle" permet"
également"d’activer"l’immunité"dans"des"tissus"non"encore"atteints"par"le"parasite"en"jouant"un"rôle"
de"signal"via"un"transport"systémique":"on"parle"de"SAR"(Systemic"Acquired"Resistance)."Enfin,"elle"est"
aussi"actrice"associée"à"la"réponse"hypersensible,"particulièrement"efficace"contre"ce"type"de"parasite"
(Vlot"et"al.,"2009).""
La" voie" éthylènePjasmonate" est" le" plus" généralement" impliquée" dans" la" résistance" aux" parasites"
nécrotrophes." Elle" induit" la" synthèse" de" métabolites" secondaires" antimicrobiens," de" défensines""
comme"pdf1.2"("Proten"defensin";"protéines"toxiques"pour"le"microbe"de"petit"poid"moléculaire)"(van"
Loon"et"al.,"2006";"Browse,"2009";"Gutjahr"and"Paszkowski,"2009)."De"manière"parallèle"au"jasmonate,"
d’autres" molécules" appartenant" à" la" même" voie" des" oxylipines" sont" aussi" produites" au" cours" de"
l’infection" et" jouent" des" rôles" à" la" fois" antimicrobiens" et" dans" la" signalisation" des" défenses." Le"
jasmonate," en" inhibant" la" synthèse" d’acide" salicylique," favorise" la" synthèse" de" métabolites"
secondaires" tels" que" les" glucosinolates" chez" les" Brassicaceae" qui" sont" aussi" défavorables" à" la"
croissance"microbienne"(RobertPseilaniantz"et"al.,2012).""
Un" thème" de" recherche" émergeant" au" sein" de" l’étude" des" voies" hormonales" est" l’intégration" des"
hormones" généralement" associées" au" développement" dans" l’immunité" végétale" (Bari" and" Jones,"
2009";" RobertPseilaniantz" et" al.," 2012)(Figure" 13)." Ainsi," les" gibérellines" (GA";" Giberrelic" acid)"
favorisent"l’accumulation"de"ROS"et"celle"de"calcium"intracellulaire"qui"accroissent"la"quantité"d’acide"
salicylique" (Bari" and" Jones," 2009)" tandis" que" les" cytokinines" (CK)" joueraient" un" rôle" positif" dans"
l’activation" transcriptionnellele" dépendante" de" cette" hormone" (Choi" et" al.," 2011)." A" l’inverse," les"
brassinostéroïdes"(BR)"participent"à"la"résistance"aux"parasites"nécrotrophes"sous"la"dépendance"de"
l’acide"jasmonique""(Zhang"et"al.,"2009";"de"Vleesschauwer"et"al.,"2012)."L’auxine"(IAA";"Inole"Acetic"
Acid)" ," négativement" régulée" par" l’accumulation" d’acide" salicylique" peut" interagir" avec" la" voie"
ethylènePjasmonate," mais" aussi" au" travers" de" processus" développementaux" régulés" par" ces"
hormones" (Muday" et" al.," 2012";" Kazan" and" Manners," 2012)." Enfin" l’acide" abscissique" (ABA";" Absicic"
Acid)," identifié" " en" tant" qu’hormone" du" stress" abiotique," participe" aussi" à" la" défense" foliaire" en"
fermant"les"stomates,"points"d’entrée"préférentiels"de"certains"parasites."Cependant,"sous"certaines"
conditions" environnementales" défavorables," il" pourrait" affecter" l’immunité" et" favoriser"
l’établissement" de" la" maladie" selon" le" mode" de" vie" du" parasite" et" le" tissu" végétal" ciblé" (Ton" and"
MauchPMani," 2004";" Bari" and" Jones," 2009";" RobertPseilaniantz" et" al." 2012)." " A" l’image" de" l’acide"
salicylique"qui"contrôle"des"réponses"immunitaires"à"distance"dans"les"feuilles,"la"voie"du"éthylèneP
jasmonate"permet"une"réponse"systèmique"appellée"Induced"Systemic"Resistance"(ISR)"qui"est"initiée"
47"
"
au"niveau"des"racines""par"des"microorganismes"bénéfiques"en"général"et"conduit"à"une"amélioration"
de"la"résistance"de"la"plante,"y"compris"dans"les"parties"aériennes"(Okubara"&"Paulitz,"2005)."
"
Figure"13:"Un"modèle"simplifié"montrant"l‘implication"de"différentes"hormones"dans"la"régulation"positive"ou"négative"de"la"
résistance" à" divers" agents" pathogènes" biotrophes" ou" nécrotrophes." Bari,& R.& and& Jones,& J.D.G." (2009)." Role" of" plant"
hormones"in"plant"defence"responses."Plant0molecular0biology"69:"473P88."
b. Dans les réponses mutualistes
La" caractérisation" du" rôle" des" hormones" dans" les" interactions" mutualistes," et" donc" principalement"
chez"les"légumineuses,"a"débuté"de"manière"plus"tardive."Néanmoins,""des"résultats"obtenus"au"cours"
des" dernières" années" soulignent" l’importance" de" ces" signalisations" dans" l’établissement" des"
symbioses." C’est" particulièrement" vrai" dans" le" cas" de" la" nodulation," pendant" laquelle" les"
interconnexions"avec"les"hormones""associées"au"développement"sont"nombreuses."Les"cytokinines"
sont"en"effet"des"régulateurs"centraux"du"positionnement"des"primordia"nodulaires."Chez"Medicago0
truncatula,"le"récepteur"CRE1""permet"de"bloquer"le"transport"auxinique,""induisant"ainsi"la"formation"
du" méristème" nodulaire" dans" un" processus" compétitif" visPàPvis" de" la" formation" de" racines"
secondaires"(GonzalezPRizzo"et"al.,"2006";"Ding"and"Oldroyd,"2009";"Oldroyd"et"al.,"2010)."En"parallèle,"
la" voie" de" signalisation" CRE1" " (Cytokinine" Receptor" 1)" dépendante" interagit" directement" avec" des"
facteurs" de" transcription" attribués" à" la" signalisation" lipochitooligosaccharidique," NIN0 (Nodule"
Inception)0 ,0 ERN10 (Ethylene" responsive" transcription" factor" required" for" nodulation)0 et" NSP2" pour"
promouvoir" les" processus" d’organogénèse" en" plus" de" la" reprogrammation" transcriptionnelle"
provoquée"par"ces"signaux"(Plet"et"al.,"2011)."Enfin,"des"fonctions"relatives"à"l’acide"abscissique"sont"
nécessaires"à"l’émergence"de"nodules"chez"Medicago0truncatula0puisque"le"mutant"du"transporteur0
48"
"
LATD/NIP0 (Lateral" deficiency/numerous" infection" and" polyphenolics)0 peut" voir" ses" défauts" de"
formation"de"racines"latérales,"de"cordons"infectieux"et"de"développement"nodulaire"complémentés"
par"un"apport"exogène"d’acide"abscissique"(Veereshlingam"et"al.,"2004";"Bright"et"al.,"2005";"Liang"et"
al.," 2007";" Ding" et" al.," 2008)." D’autre" part," l’acide" abscissique" est" un" régulateur" négatif" de" la"
nodulation"où"elle"est"capable"d’interférer"avec"la"signalisation"des"cytokinines"et"la"perception"des"
lipochitooligosaccharides" (Ding" et" al.," 2008)." Enfin," c’est" aussi" un" régulateur" positif" de" la" formation"
des"arbuscules"chez"la"tomate"(HerreraPMedina"et"al.,"2007)."
En"ce"qui"concerne"l’antagonisme"acide"salicyliquePacide"jasmonique,"les"résultats"restent"difficiles"à"
obtenir,"en"raison"de"l’absence"de"mutants"de"ces"voies"de"signalisation"chez"les"légumineuses."Des"
travaux" révèlent" cependant" l’importance" de" ces" régulateurs" de" l’immunité" dans" les" interactions"
mutualistes."L’acide"salicylique""est"un"régulateur"négatif"de"la"croissance"des"Rhizobia"(Stacey"et"al.,"
2006)"et"pourrait"être"à"l’origine"de"réponses"de"types"hypersensibles"envers"les"cordons"infectieux"
de" Rhizobia" qui" n’aboutissent" pas" ." En" effet," des" plantes" de" Lotus0 japonicus," sousPaccumulant"
l’hormone," parviennent" à" augmenter" le" nombre" de" cordons" infectieux" et" de" nodules" développés"
(Stacey" et" al.," 2006)." Par" ailleurs," il" est" probable" que" la" perception" des" lipochitooligosaccharrides"
limite" l’activation" de" la" voie" acide" salicylique" chez" la" luzerne" (Gutjahr" and" Paszkowski," 2009)." Un"
résultat"concordant"a"été"observé"aussi"visPàPvis"de"la"mycorhization":"l’établissement"de"l’interaction"
entraîne" un" abaissement" de" la" quantité" de" cette" hormone" (Herrera" Medina," 2003" ;" Siciliano" et" al.,"
2007";" Jung" et" al.," 2012)"" et" des" lésions" nécrotiques" marquant" le" rejet" de" la" colonisation" ont" été"
observées"(Novero"et"al.,"2002";""Genre"et"al.,"2005";"Genre"et"al.,"2009).""
En"raison"de"la"grande"importance"de"l’acide"jasmonique"dans"l’immunité"végétale"(Browse,"2009),"
les" racines," lieux" de" contact" privilégié" avec" des" microorganismes," voient" leurs" réponses" fortement"
assujetties"au"comportement"de"cette"voie"visPàPvis"de"l’interaction"(Gutjahr"and"Paszkowski,"2009)."
Ainsi,"l’application"d’acide"jasmonique"ou"la"présence"de"blessures"répétées"sur"les"parties"aériennes"
des" plantes" améliorent" la" mycorhization," réduisent" les" symptômes" causés" par" le" parasite"
Aphanomyces0euteiches"mais"n’affectent"pas"la"nodulation"et"chez"Medicago0truncatula"(Landgraf"et"
al.,"2012)."Cependant,"un"traitement"de"MethylPJasmonate"directement"localisé"au"niveau"racinaire"a"
un"effet"négatif"sur"la"mycorhization"chez"la"tomate"(HerreraPMedina"et"al.,"2008)."Outre"l’effet"du"
jasmonate" sur" la" biologie" de" l’hôte," il" peut" aussi" modifier" le" comportement" du" partenaire." Il" est"
exsudé"dans"le"sol"où"il"est"capable"d’activer"les"gènes"nod"de"Rhizobia"(Rosas"et"al.,"1998";"Mabood"
et" al.," 2006)" tandis" qu’il" a" un" effet" négatif" sur" l’accumulation" de" nodosités" (Sun" et" al.," 2006)," les"
divisions"cellulaires"à"l’origine"du"méristème"nodulaire"(Gutjahr"and"Paszkowski,"2009),"la"perception"
des" lipochitooligosaccharides" et" le" calcium" spiking" (Sun" et" al.," 2006)." Enfin," la" colonisation" par"
Sinorhizobium" chez" Medicago0 truncatula" nécessite" une" répression" du" métabolisme" des" oxylipines"
49"
"
(Zhang"et"al.,"2012)."Parallèlement"à"l’acide"jasmonique,"l’éthylène"joue"un"rôle"synergique"et"bloque"
la" symbiose." Cette" information" a" été" démontrée" grâce" au" mutant" sickle" de" l’orthologue" d’EIN2""
(Ethylene"INsensitive)"qui"présente"un"accroissement"du"nombre"de"nodosités,"preuve"du"rôle"négatif"
de"cette"hormone"dans"le"contrôle"de"cette"association"(Penmetsa"et"al.,"2008)."Enfin,"on"peut"noter"
que" les" microorganismes" bénéfiques" du" sol" sont" fréquemment" initiateurs" de" l’ISR" (Zamioudis" &"
Pieterse,"2012)."
D’un" point" de" vue" écologique," l’aspect" fortement" intriqué" de" cet" antagonisme" entre" les" deux" voies"
hormonales," face" aux" symbiotes" et" aux" parasites," et" les" interconnexions" existantes" avec" le"
développement," participeraient" à" la" dynamique" de" la" réponse" végétale." Cette" plasticité" est" requise""
du"fait""d’un"environnement"biotique"sans"cesse"changeant"de"part"la"colonisation"de"la"rhizophère"
par"de"nouveaux"microbes"et"l’évolution"de"leur"comportement"au"cours"de"l’interaction."
"
3. Facteurs de transcription et régulations par le protéasome
a. Dans les réponses immunitaires
Les" événements" rapides" succédant" à" la" reconnaissance" de" MAMPs" aboutissent" à" l’activation" de"
régulateurs" transcriptionnels" chargés" de" reprogrammer" le" transcriptome" végétal." Cette"
reprogrammation" passe" par" des" modifications" post" traductionnelles" des" facteurs" de" transcription"
sous" l’impulsion" des" MAPKs," activées" en" amont" dans" les" cascades" de" signalisation" (Eulgem," 2005"
Nishimura" and" Dangl," 2010)," ou" par" la" dégradation" de" régulateurs" négatifs" au" travers" de" leur"
adressage"au"protéasome."
Un" mutant" d’Arabidopsis0 thaliana0 affecté" dans" l’ensemble" des" voies" hormonales" liées" à" la" défense"
est"incapable"d’assurer"la"signalisation"de"MAMPs"et"les"réponses"de"types"ETI"(Tsuda"et"al.,"2009)."
Ainsi," Il" est" impossible" de" considérer" séparément" ces" modules" de" régulations." La" plante" modèle"
Arabidopsis0 thaliana," au" travers" des" nombreux" pathosystèmes" et" réponses" transcriptomiques"
caractérisés"(Eulgem,"2005";"Nishimura"and"Dangl,"2010),"nous"confère"la"vision"la"plus"complète"des"
régulateurs" transcriptionnels" de" l’immunité." Il" s’agit" essentiellement" de" facteurs" de" types" WRKY"
(Pandey"and"Somssich,"2009)"mais"aussi"de"MYBs"ERFs,"whirly"et"bZIP"TGAs"(Eulgem,"2005";"Moore"et"
al.,"2011)."Ces"différents"types"de"régulateurs"appartiennent"à"de"vastes"familles"multigéniques"que"
l’on" retrouve" dans" l’activation" d’un" grand" panel" de" réponses" défensives" diverses." Les" réponses"
hormonales," essentielles" à" la" reprogrammation" transcriptionnelle," sont" elles" aussi" activées" par" des"
facteurs" de" transcription":" Les" TGAs" (facteurs" de" transcription" liant" des" cisPélément" TGA)" et" NPR1"
(pour"Not"PR1)"pour"l’acide"salicylique,"MYCs""pour"le"jasmonate,"ERFs"(Ethylene"responsive"factor)"et"
50"
"
pour"l’éthylène,"ARFs"(Auxine"responsive"factor)"pour"l’auxine,"PIFs"(Phytochrome"Interacting"factor)"
pour" les" giberrellines," ABI3" et" ABI5" " (Absicic" acid" insensitive)" pour" l’acide" abscissique" (Santner" and"
Estelle," 2009";" Pieterse" et" al.," 2009" ;" Kazan" and" Manners," 2012";" Pieterse" et" al.," 2012" ;" RobertP
seilaniantz"et"al.,"2012)"(Figure"14)."Certaines"voies"hormonales"étant"convergentes"dans"leurs"effets,"
il"n’est"pas"étonnant"de"noter"l’implication"de"régulateurs"dans"plusieurs"voies."Tous"ces"facteurs"de"
transcription" sont" régulés" par" des" protéines" susceptibles" de" les" faire" adresser" directement" ou"
d’adresser"leurs"régulateurs"positifs"ou"négatifs"au"protéasome"selon"que"la"signalisation"soit"allumée"
ou" éteinte":" Ainsi" les" NPR3" et" NPR4" inhibent" le" facteur" positif" NPR1" pour" l’acide" salicylique" (PelegP
Grossman" et" al.," 2009" ;" Fu" et" al.," 2012";" Wu" et" al.," 2012)," la" protéine" à" domaine" FPBox" COI1"
(coronatine" insensitive)" adresse" " les" répresseurs" JAZs" au" protéasome" permettant" au" facteur" de"
transcriptrion"MYC2"d’activer"la"voie"du"jasmonate,"EBF1"et"EBF2"répriment"(ethylene"binding"factor)"
le" stimulateur" d’ERFs" nommé" EIN3" (ethylene" insensitive)" pour" l’éthylène," TIR1" " supprime" les"
répresseurs" AUXPIAA" pour" l’auxine," GID1" (giberrellin" insensitive" dwarf)" supprime" les" régulateurs"
négatifs"DELLAs"pour"les"giberrellines."De"manière"intéressante,"les"DELLAs"peuvent"interagir"avec"les"
JAZs"et"affecter"la"régulation"de"la"voie"jasmonate,"balançant"les"processus"développementaux"et"les"
réponses" de" défense." Ces" différents" acteurs" sont" mis" en" action" ou" inhibés" par" leur" interaction"
physique"avec"l’hormone,"hormis"les"EBFs,"et"sont"à"ce"titre"considérés"comme"les"récepteurs"de"ces"
métabolites."
Figure"14:"Rôle"du"protéasome"dans"la"signalisation"des"hormones"végétales."
La"signalisation"hormonale"implique"généralement"des"changements"de"régulation"transcriptionnelle"via"des"modifications"
des"taux"d'ubiquitination"ou"de"transactivation"de"ses"régulateurs."Le"passage"de"l’état"inactif"à"l'état"actif"est"contrôlé"par"
la" dégradation" d'un" régulateur" négatif" (panneaux" a," b," c)" ou" la" suppression" de" la" dégradation" d'un" régulateur" positif"
(panneaux"c,"d,"e,"f)."(a)"Voie"à"l’état"inactif"de"l'auxine"(IAA),"de"l'acide"jasmonique"(JA),"et"de"l'acide"gibérrellique"(GA)."Les"
régulateurs" négatifs" respectifs" de" ces" trois" voies" (AUXPIAA," JAZ" et" DELLA)" lient" et" inactivent" les" régulateurs" positifs" (ARF,"
MYC2" et" PIF3/4)." ARF," MYC2" et" PIF3/4" sont" tous" des" facteurs" de" transcription." Un" deuxième" volet" inactivateur" est"
matérialisé"par"la"présence"de"Topless"(TPL)"et"NINJA."TPL"est"capable"de"se"lier"directement"aux"AUXPIAA,"tandis"que"TPL"
intervient"conjointement"avec"NINJA"pour"se"lier"aux"protéines"JAZ."Dans"les"deux"cas,"la"présence"de"TPL"et/ou"de"NINJA"
stabilise" le" complexe" dans" son" état" inactif." (b)" Voie" à" l’état" actif" de" l'IAA," du" JA," et" des" GAs." Suite" à" la" perception" des"
différentes"hormones,"les"régulateurs"négatifs"sont"dégradés,"libérant"le"régulateur"positif"qui"peut"activer"l’aval"de"la"voie"
de"signalisation."Les"récepteurs"à"l’IAA"et"au"JA"nommés"respectivement"TIR1"et"COI1"sont"des"protéines"à"domaine"FPbox."
L'hormone,"en"se"fixant"sur"son"récepteur,"facilite"l’interaction"entre"ce"dernier"et"le"régulateur"négatif"(AUXPIAA"ou"JAZ)"
déclenchant"la"dégradation"par"le"protéasome."La"perception"des"GAs"déclenche"un"changement"de"conformation"de"GID1"
qui"permet"la"reconnaissance"des"protéines"DELLA"par"SLY,"une"protéine"FPbox"contrôlant"leur"dégradation."(c)"Les"DELLAs"
régulent"en"outre,"de"manière"croisée,"la"signalisation"du"JA."L’augmentation"du"niveau"de"JA"entraîne"la"dégradation"des"
protéines"JAZs.""Les"faibles"teneurs"en"GAs"stabilisent"les"DELLAs"qui"sont"capables"de"se"lier"aux"protéines"JAZ."Cette"liaison"
ne"permet"pas"aux"protéines"JAZ"d'interagir"avec"MYC2"dont"l’activité"est"par"conséquent"maintenue."À"des"niveaux"élevés"
de" GAs," les" DELLAs" sont" dégradés" et" libèrent" les" JAZs" qui" inactivent" MYC2." À" de" faibles" niveaux" de" JA," COI1" n’induit" pas"
l'ubiquitination"des"JAZs":"la"voie"est"alors"éteinte."(d)"Lors"d’une"faible"abondance"d’ABA,"ABI5"est"inhibé"par"KEG"et"AFP."
Lors" d’une" élévation" de" la" quantité" d’ABA," KEG" est" ubiquitiné" puis" dégradé," libérant" le" facteur" de" transcription" ABI5" qui"
dans" le" même" temps," n’interagit" plus" avec" le" régulateur" négatif" AFP." (e)" Etat" inactif" de" la" voie" l'éthylène" (ET)." " L’ET" est"
perçue"par"des"récepteurs"liés"à"membrane"du"réticulum"endoplasmique."Les"récepteurs"ET"sont"des"régulateurs"négatifs"de"
la" voie" de" signalisation." EIN3" est" un" facteur" de" transcription" qui" régule" positivement" la" voie." En" l'absence" d'ET," EIN3" est"
ubiquitinée"et"dégradée"par"le"protéasome"via"les"protéines"FPbox"EBF1"et"EBF2."(f)""A"l'état"actif","le"récepteur"n'est"plus"en"
mesure"de"réprimer"la"voie"levant"la"répression"sur"EIN3."Robert+seilaniantz,&A.&et&al."(2012)."Hormone"Crosstalk"in"Plant"
Disease"and"Defense :"More"Than"Just"JASMONATEP"SALICYLATE"Antagonism."Annual0Review0of0Phytopathology."
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b. Dans les réponses mutualistes
Suite" à" l’identification" des" voies" de" signalisation" symbiotique," un" certain" nombre" de" facteurs" de"
transcription"participant"à"la"signalisation"ou"à"l’organogénèse"des"interactions"avec"Rhizobium"ont"
pu"être"identifiés."Les"premiers,""tels"que"NSP1,"NSP2"(Nod"signaling"pathway)"(Kaló"et"al.,"2005";"Smit"
et"al.,"2005";"Middleton"et"al.,"2007";"Hirsch"et"al.,"2009";"Cerri"et"al.,"2012)"ou"ERN1,"ERN2"et"ERN3"
(Marsh" et" al.," 2007";" Andriankaja" et" al.," 2007)" agissent" en" aval" de" DMI3" et" participent" à" la"
signalisation" liée" à" la" perception" des" lipochitooligosaccharides" en" régulant" des" nodulines" tel" que"
ENOD11" (Early" NODulin)." Récemment," un" facteur" de" transcription" nommé" RAM1" a" été" identifié"
comme" spécifiquement" requis" pour" la" symbiose" mycorhizienne." Induit" par" la" perception" des"
lipochitooligosaccharides" mycorhiziens," sa" mutation" affecte" la" colonisation" par" le" champignon" mais"
pas"les"processus"relatifs"à"la"symbiose"fixatrice"d’azote."RAM1"est"capable"d’interagir"physiquement"
avec" NSP2" mais" pas" NSP1," permettant" de" situer" le" point" de" divergence" de" la" voie" de" signalisation"
symbiotique"commune"à"son"niveau,"c'estPàPdire"en"aval"de"NSP2"(Gobbato"et"al.,"2012)."Il"a"pu"être"
démontré"que"RAM1"est"un"régulateur"direct"d’un"gène"RAM2"qui"est"requis"pour"la"colonisation"par"
les" champignons" mycorhiziens" mais" aussi" un" oomycète" racinaire":" ce" dernier" point" sera" traité" en"
détail"dans"le"chapitre"II"des"résultats""(Wang"et"al.,"2012)."
D’autres"facteurs"de"transcription"tels"que"NIN"(Nodue"Inception)"(Kaló"et"al.,"2005)"et"NFPYAP1"(pour"
Nuclear" factor" Y" box," préalablement" appelé" HAP2a)(Combier" et" al.," 2006";" Combier" et" al.," 2008)"
dépendent"de"la"signalisation"symbiotique"mais"sont"aussi"associés"à"l’organogénèse"nodulaire"tandis"
que"la"voie"cytokinine"via"CRE1"(cytokinine"receptor"1)"(GonzalezPRizzo"et"al.,"2006";"Plet"et"al.,"2011)"
contrôle"conjointement"avec"EFD"(ERF"required"for"differentiation)"un"régulateur"de"réponse"de"type"
A" nommé" RR4" (Response" Regulator)(Vernié" et" al.," 2008)" mais" aussi" de" manière" alternative" NSP1,"
NSP2"et"NIN."Les"NSPs"et"RAM1"appartiennent"à"la"famille"des"GRAS,"les"ERNs"et"EFD"sont"des"ERFs"
non" impliqués" dans" la" signalisation" éthylène" et" NIN" en" vertu" d’un" domaine" de" liaison" à" l’ADN" est"
probablement"un"facteur"de"transcription"même"si"son"fonctionnement"reste"mal"décrit"tout"comme"
celui"de"protéine"nucléaire"RPG"(Rhizobium"directed"Polar"Growth)"associée"à"l’infection"(Arrighi"et"
al.," 2008)." Ainsi," ces" facteurs" de" transcription" ne" semblent" pas" apparentés" à" ceux" de" l’immunité."
Certains"de"ces"gènes"sont"aussi"importants"pour"la"mycorhization,"notamment"les"NSPs"qui"sont"des"
régulateurs"de"la"biosynthèse"de"strigolactone"(Liu"et"al.,"2011";"Lauressergues"et"al.,"2012)."D’autres"
membres"de"la"famille"des"NFPY"sont"fortement"exprimés"dans"les"cellules"contenant"les"arbuscules"
(Hogekamp"et"al.,2011)."Il"en"est"de"même"pour"un"facteur"de"transcription"de"type"MYB"qui"contrôle"
l’architecture"racinaire"(Volpe"et"al.,"2012)."Enfin,"on"peut"noter"qu’ERF19"est"pertubé"par"l’effecteur"
mycorhizien"SP7"pour"promouvoir"la"colonisation"(Anderson"et"al.,"2010)."
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La"régulation"post"traductionelle"de"ces"facteurs"de"transcription"est"encore"mal"connue"et"aucune"
protéine"d’adressage"au"protéasome"ne"leur"a"été"pour"l’instant"associée."Néanmoins,"les"fonctions"
protéolytiques" semblent" importantes" dans" la" symbiose" rhizobienne." La" signalisation" précoce" des"
lipochitooligosaccharides" passe" par" la" phosphorylation" par" LYK3" d’une" E3Pligase" nommée" PUBP1""
(Protein"ubiquitin"ligase)"(Mbengue"et"al.,"2010)."Le"gène"LIN0(Lumpy"infection)"code"aussi"pour"une"
E3Pligase" qui" est" impliquée" dans" la" progression" du" cordon" infectieux" et" l’organogénèse" nodulaire,"
contrôlant" plus" particulièrement" la" colonisation" des" nodules" par" les" bactéries" (Kuppusamy" et" al.,"
2004";"Shimomura"et"al.,"2006";"Kiss"et"al.,"2009)."Enfin,"l’apparition"du"primordium"nodulaire"requiert"
la"réactivation"mitotique"de"cellules"endodermiques"qui"passe"par"la"dégradation"de"régulateurs"du"
cycle"cellulaire"("des"cyclines)"maintenant"la"quiescence""dans"l’endoderme"(Kondorosi"et"al.,"2005)."Si"
le" protéasome" n’est" pas" connu" dans" la" régulation" postPtraductionelle" de" facteurs" de" transcription,"
d’autres"mécanismes"faisant"notamment"intervenir"les"microARNs"ont"été"identifiés"(Combier"et"al.,"
2006";"Lauressergues"et"al.,"2012)."
"
C. Réponses des plantes aux microorganismes
"
Suite"aux"mécanismes"de"perception"et"de"signalisation"présentés"précédemment,"la"plante"met"en"
place"des"réponses"qui"ont"pour"but"de"contrôler"l’installation"des"microorganismes"symbiotiques"ou"
de" rejeter" les" parasites." " Ces" réponses" se" traduisent" par" l’apparition" de" nouvelles" structures"
cellulaires"et"la"production"de"nouvelles"protéines"et"métabolites"secondaires"et"par"la"modification""
de"l’architecture"racinaire."
1. Réponses cellulaires et tissulaires de la plante
a. Réarrangements membranaires et cellulaires : signalisation et colonisation
Les" PRRs" sont" capables" de" détecter" avec" rapidité" des" quantités" infimes" de" MAMPs" de" 10P6" à" 10P12"
molaires"selon"les"composés."Cela"présuppose"que"ces"récepteurs"puissent"rapidement"recruter"leurs"
partenaires"afin"d’assurer"les"événements"de"phosphorylation"et"transphosphorylation"à"l’origine"de"
la"signalisation"(Segonzac"and"Zipfel,"2011)."La"localisation"subcellulaire"de"ces"protéines,"en"présence"
ou"absence"de"leurs"ligands,"a"été"étudiée"ces"dernières"années."Ainsi,"certains"PRRs,"tel"qu’AtFLS2"
ou"MtLYK3,"se"voient"immobilisés""à"la"surface"de"la"cellule,"respectivement"en"présence"de"flg22"et"
de"bactéries"symbiotiques"(Ali"et"al.,"2007";"Haney"et"al.,"2011)."De"manière"frappante,"MtLYK3"est"
essentiellement" relocalisé" au" point" de" contact" avec" la" bactérie," au" bout" du" poil" absorbant" en"
présence"de"bactéries."AtFLS2"peut,"pour"sa"part,"être"recruté"ou"exclu"de"la"membrane"entourant""
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l’haustorium" de" deux" oomycètes" (Beck" et" al.," 2012)." Sa" localisation" dans" des" microdomaines"
lipidiques" riches" en" stérols" appelés" radeaux" lipidiques" ou" «"lipid" raft"»" pourrait" expliquer" les"
variations"dans"sa"distribution"en"présence"d’éliciteur"(Keinath"et"al.,"2010";"Urbanus"and"Ott,"2012)."
De"même,"MtLYK3"colocalise"avec"une"flotilline,"protéine"connue"pour"être"impliquée"dans"ces"sites"
membranaires" (Haney" and" Long," 2010)" et" interagit" physiquement" tout" comme" MtNFP" et" MtDMI20
avec0 MtSYMREM10 (Lefebvre" et" al.," 2010";" Popp" and" Ott," 2011),0 représentant" de" la" famille" des"
rémorines,""des"protéines"elles"aussi"associées"à"ces"microdomaines."La"présence"de"ces"récepteurs"
dans" des" domaines" membranaires" particuliers" avant" leur" activation" pourrait" aussi" être" liée" à" leur"
devenir" puisqu’AtFLS2" (Hong" et" al.," 2011)" ou" LjSYMRK" (Den" Herder" et" al.," 2012)" peuvent" être"
ubiquitinés" puis" endocytosés" pour" assurer" leur" fonction" de" signalisation" (Monaghan" and" Zipfel,"
2012)."MtLYK3,"quant"à"lui,"phosphoryle"une"E3"ligase"régulatrice"négative"de"la"nodulation"nommée"
PUB%1" (Mbengue" et" al.," 2010)." Par" ailleurs," les" rémorines" sont" susceptibles" d’interagir" avec" des"
composants" du" cytosquelette" (Raffaele" et" al.," 2007)," " ce" qui," dans" le" cas" de" la" symbiose," pourrait"
assurer" le" contrôle" des" étapes" d’infection." En" ce" sens," des" protéines" à" domaines" ankyrine"
susceptibles"d’interagir"avec"ces"éléments"sont"nécessaires"à"la"bonne"progression"des"appareils"de"
préPpénétration" et" des" cordons" infectieux." Chez" Medicago0 truncatula," le" membre" de" cette" famille"
impliqué" dans" les" symbioses" est" nommé" VAPYRIN0 (Pumplin" et" al.," 2010";0 Feddermann" et" al.," 2010";"
Murray"et"al.,"2011)."
b. Modifications de la paroi végétale et exocytose
La"plupart"des"microorganismes"pathogènes,"comme"les"symbiotes"mutualistes,"résident"in0planta"de"
manière" intercellulaire." Ainsi," les" cellules" végétales," afin" de" maîtriser" l’interaction," doivent" émettre"
dans" le" milieu" apoplastique" les" acteurs" du" dialogue" régulant" l’interaction." La" libération" des" sucres,"
métabolites" et" protéines" dans" cet" espace," repose" sur" un" mécanisme" clé" appelé" exocytose"
(Hückelhoven," 2007)(Figure" 15)." Dans" le" cas" de" la" pathogénie," ces" mécanismes" participent" au"
renforcement" de" la" paroi" en" libérant" des" oligomères" de" lignols" " et" des" protéines" riches" en" proline"
appellé" HRGP" Hydroxyproline" Rich" GlycoPProtein" qui," sous" l’action" de" peroxydases" et" de" leur"
catalyseur" l’H2O2" euxPmêmes" exocytés," sont" crosslinkés" et" polymérisés," assurant" une" résistance"
mécanique" accrue" face" à" l’envahisseur." Le" microorganisme," pour" sa" part," libère" des" batteries"
d’enzymes" lytiques" ciblant" les" principaux" constituants" de" la" paroi" végétale," polymères" relatifs" à" la"
cellulose"et"à"la"pectine,"afin"de"faciliter"l’accès"au"contenu"intracellulaire"de"l’hôte"et"d’obtenir"une"
source"d’énergie"saccharidique."Afin"de"limiter"les"dégâts"infligés"par"le"parasite,"les"cellules"libèrent"
également"dans"l’apoplasme"des"protéines"inhibitrices"de"ces"enzymes."Cette"résistance"mécanique"
peut"être"renforcée"par"des"appositions"de"papilles"de"callose."Ce"polymère"de"glucose"lié"de"manière"
originale""par"rapport"à"la"cellulose"est"résistant"aux"enzymes"fongiques"(Hématy"et"al.,"2009)."En"plus"
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de"participer"au"maintien"de"l’intégrité"pariétale,"l’exocytose"assure"également"la"mise"en"place"de"
mesures" actives" limitant" le" développement" du" microorganisme" pathogène." Ainsi," la" plupart" des"
fonctions"de"défense"énoncées"précédemment"et"affectant"la"biologie"du"microbe"sont"libérées"dans"
l’apoplasme"afin"d’affecter"le"parasite."
"
Figure" 15:" Intégration" des" mécanismes" biochimiques" et" moléculaires" associés" à" la" défense" pariétale." Abréviations:" CWA" :"
Cell"Wall"Apposition";"GLS":"Glucane"Synthase";""PM"Plasma"Membrane."Hückelhoven,&R."(2007)."Transport"and"secretion"in"
plantPmicrobe"interactions."Current0opinion0in0plant0biology"10:"573P9."
"Une" caractéristique" frappante" et" commune" de" l’exocytose," que" ce" soit" visPàPvis" de" parasites" ou" de"
symbiotes," est" qu’elle" requiert" une" réorganisation" des" organites" et" du" cytosquelette" de" la" cellule"
attaquée." " Ainsi" le" noyau," le" réticulum" et" le" Golgi" sont" polarisés" par" l’haustorium" de" l’agent"
pathogène" de" manière" semblable" aux" mouvements" nucléaires" anticipés" qui" se" produisent" au" cours"
de" la" formation" de" l’appareil" de" préPpénétration" mycorhizien" ou" de" la" formation" du" cordon"
d’infection" avec" Rhizobium." Dans" la" nodulation," l’exocytose" permet" l’assemblage" de" domaines"
membranaires"particuliers,"appelés"symbiosome,""facilitant"les"échanges"et"la"libération"des"peptides"
NCRs"nécessaires"à"la"différenciation"(Wang"and"Dong,"2011)."Ces"symbiosomes"sont"formés"à"l’aide"
de" processus" aussi" impliqués" dans" le" développement" des" arbuscules" (Ivanov" et" al.," 2012)."
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Récemment," un" marqueur" spécifique" à" la" paroi" du" cordon" d’infection" a" pu" être" indentifié":" il" s’agit"
d’une" pectate" lyase," soulignant" la" nécessité" d’un" assouplissement" des" parois" végétales" pour"
permettre" la" progression" des" structures" d’invasions" symbiotiques" (Xie" et" al.," 2011)." Ce" processus"
pourrait" être" contrebalancé" par" des" enzymes" et" protéines" capables" de" rigidifier" ces" mêmes" parois"
comme"la"peroxydase"RIP1"(Ramu"et"al.,"2002)"ou"la"protéine"ENOD110qui"interagit"avec"les"pectines"
et"est"induite"par"les"signaux"lipochitooligosaccharidiques"(BoissonPDernier"et"al.,"2005";"Maillet"et"al.,"
2011)."
c. Modifications de l’architecture de la racine
La" croissance" et" le" développement" des" racines" sont" essentiellement" régulés" par" la" balance" entre"
auxine" et" cytokinine" (De" Smet," 2012;" De" Smet" et" al.," 2006;" Ivanchenko" et" al.," 2008;" Kazan" and"
Manners,"2009";""Péret"et"al.,"2009)."En"présence"de"stress"biotiques,"l’architecture"racinaire"va"être"
modifiée" en" raison" d’une" modification" de" la" balance" entre" ces" deux" hormones" et" aussi" de"
l’intervention"d’hormones"supplémentaires"et"plus"particulièrement"de"l’éthylène"visPàPvis"des"stress"
biotiques" (Hodge" et" al.," 2009)." Les" microorganismes" mutualistes" modifient" indirectement"
l’architecture"racinaire"en"participant"à"la"nutrition"de"l’hôte"ou"directement"au"travers"des"signaux"
lipochitooligosaccharidiques" qui" induisent" la" formation" de" racines" latérales" (Maillet" et" al.," 2011)."
Outre"des"modifications"de"la"croissance"du"système"racinaire,"la"morphologie"des"tissus"euxPmêmes"
est"altérée."La"mycorhization"favorise"une"augmentation"du"diamètre"des"racines"afin"que"davantage"
de" mycélium" puisse" coloniser" le" site" d’interaction," tandis" que" l’élongation" est" ralentie." Les" poils"
absorbants,"structures"de"captage"direct"de"la"plante"dans"le"sol"voient"leur"présence"raréfiée,"sans"
doute"parce"qu’ils"sont"suppléés"de"manière"appropriée"par"l’activité"du"champignon"(Hodge"et"al.,"
2009)." Les" symbioses" fixatrices" d’azote" requièrent" toutes" l’émergence" d’organes" latéraux" plus" ou"
moins"spécialisés,"les"nodules"et"actinorhizes,"selon"qu’il"s’agisse"respectivement"d’interaction"avec"
des" bactéries" types" Rhizobia" ou" Frankia0 (Parniske," 20080;0 Markmann" et" al.," 20080 Markmann" and"
Parniske," 2009";" Gough" and" Cullimore," 2011)." Les" Plant" Growth" Promoting" Rhizobacteria" (PGPR)" ou"
Plant" Growth" Promoting" Fungus" (PGPF)," comme" leurs" noms" l’indiquent," favorisent" la" croissance" du"
système" racinaire" indirectement," par" une" amélioration" de" la" nutrition" ou," directement" via" la"
sécrétion" de" composés" influençant" les" régulations" hormonales" de" la" plante" (Yang" et" al.," 2009";"
Zamioudis"and"Pieterse,"2012).""
Les" champignons" délétères" limitent" la" plupart" du" temps" le" développement" racinaire" et" affectent" la"
qualité"de"la"nutrition."Cela"induit"de"multiples"émergences"de"racines"secondaires"peu"développées"
qui" limitent" le" transport" des" nutriments" captés" en" perturbant" l’organisation" de" la" vasculature" et" la"
poussée"racinaire"(Hodge"et"al.,"2009)."Les"nématodes""(Charon"et"al.,"1997";"Grunewald"et"al.,"2009)"
ou" les" bactéries" pathogènes" du" genre" Agrobacterium" (Tzfira" and" Citovsky," 2006)" sont" capables" de"
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manipuler" les" fonctions" développementales" des" cellules" racinaires" à" leur" profit." Des" signalisations"
reposant" sur" des" échanges" intercellulaires" et" systémiques" de" peptides" sont" aussi" des" régulateurs"
centraux"du"développement"végétal."Ces"peptides"appelés"CLEs"("CLavata"3"surrounding"Embryo)"ou"
CLEPlike" ont," en" premier" lieu," été" découverts" chez" un" nématode" parasite" qui" l’injectait" dans" l’hôte"
pour"manipuler"son"site"nourricier"(Betsuyaku"et"al.,"2011)."Par"la"suite,"des"peptides"CLEs"endogènes"
ont"été"identifiés"dans"le"contrôle"de"l’identité"méristèmatique"tant"au"niveau"aérien"que"racinaire"
(Wang" and" Fiers," 2010)," d’autres" encore" contrôlant" l’élongation" racinaire" (Meng" et" al.," 2012)" et" le"
développement"des"nodules"fixateurs"d’azote"(Batut"et"al.,"2011";"Mortier"et"al.,"2010";"Mortier"et"al.,"
2012)." Dans" ces" deux" derniers" cas," le" rôle" de" régulation" négative" du" nombre" de" nodules" par" le"
récepteur" SUNN" (SUper" Numeric" Nodule)" qui" code" un" LRRPRLK" de" type" CLAVATA0 a" pu" être"
précisé0(Schnabel"et"al.,"2005)."
d. Mort cellulaire : réponse hypersensible et autophagie
Lors" d’interactions" plantesPmicroorganismes," la" mort" programmée" de" cellules" végétales" au" contact"
d’un" microbe" constitue" parfois" une" étape" clé" des" processus" de" rejet" des" parasites" (Talbot" and"
Kershaw," 2009";" Mengiste," 2012)" ou" des" symbiotes" mutualistes" (Stacey" et" al.," 2006";" Genre" et" al.,"
2009)"mais"parfois"aussi"une"réponse"d’acceptation"tel"que"cela"est"illustré"chez"Arabidopsis0thaliana"
en"présence"de"Piriformospora0indica"qui"provoque"des"morts"cellulaires"pour"établir"une"relation"au"
final" mutualiste" (Deshmukh" et" al.," 2006";" Qiang" et" al.," 2012)." Ces" processus" reposent" sur" deux"
mécanismes" complémentaires" mis" en" œuvre" par" les" cellules" végétales," le" suicide" ou" réponse"
hypersensible"(HR)"(Kliebenstein"and"Rowe,"2008)"et"l’autophagie."Cette"dernière"vise"à"recycler"les"
contenus" cellulaires" de" la" cellule" morte." Si" la" réponse" hypersensible" est" décrite" depuis" plusieurs"
décennies"dans"l’étude"de"l’immunité"végétale,"le"rôle"de"l’autophagie"commence"seulement"à"être"
élucidé" (Hofius" et" al.," 2009" ;" Hofius" et" al.," 2011";" Liu" and" Bassham," 2012)." Elle" contrôle" différents"
processus"au"cours"de"l’interaction"avec"des"agents"pathogènes."VisPàPvis"des"biotrophes,"elle"limite"
l’extension"des"symptômes"nécrotiques"liés"à"la"réponse"hypersensible."Face"aux"lésions"provoquées"
par"les"microorganismes"nécrotrophes,"elle"permet"le"recyclage"des"ressources"de"cellules"mortes"par"
la"plante"au"détriment"du"parasite"mais"surtout"inhibe"l’extension"de"la"mort"cellulaire"(Eichmann"&"
Schäfer,"2012)."
"
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2. Réponses moléculaires et biochimiques de la plante aux
microorganismes
Les" reprogrammations" transcriptionnelleles" conduisent" les" plantes" " à" produire" des" métabolites,"
peptides" et" protéines" qui" manipulent" les" fonctions" microbiennes" participant" au" rejet" ou" à"
l’acceptation"du"microorganisme."
a. Reprogrammations transcriptionnelles
Les"dix"dernières"années"ont"vu"se"multiplier"les"analyses"transcriptomiques"chez"différentes"espèces"
végétales"dans"la"symbiose"mutualiste"comme"parasitaire,"que"ce"soit"au"cours"d’interactions"avec"les"
microorganismes"ou"de"traitements"avec"leurs"MAMPs.""
Dans" le" cas" de" la" pathogénie," de" nombreuses" études" chez" Arabidopsis0 thaliana" ont" souligné" le" fort"
recouvrement"entre"les"jeux"de"gènes"induits"et"réprimés"en"réponse"à"divers"microorganismes."Ces"
modifications" du" transcriptome" peuvent" représenter" jusqu’au" quart" des" gènes" de" l’hôte." La"
principale" distinction" entre" une" réponse" de" défense" menant" à" la" résistance" " et" une" réponse"
n’empêchant" pas" la" sensibilité" se" situe" dans" la" rapidité" et" le" maintien" de" la" régulation" des" gènes"
associés" à" la" réponse" immunitaire" (Eulgem," 2005)." L’établissement" progressif" de" bases" de" données"
transcriptomiques" chez" des" légumineuses" afin" d’étudier" les" symbioses" a" permis" de" souligner" des"
différences"visPàPvis"d’Arabidopsis0:"l’orthologue"phylogénétique"d’un"gène"de"la"crucifère"ne"possède"
pas" nécessairement" un" profil" d’expression" similaire" chez" les" légumineuses" (Benedito" et" al.," 2008" ;"
Libault" et" al.," 2010)." En" vue" de" comparer" les" réponses" transcriptomiques" entre" symbioses" et"
pathogénie,"je"me"suis"concentré"sur"les"légumineuses"modèles."Des"analyses"de"trancriptome"chez"
Medicago0 truncatula," en" réponse" à" deux" champignons" nécrotophes" à" large" spectre" d’hôte"
s’attaquant" aux" racines," ont" révélé" l’importance" de" la" voie" éthylènePjasmonate" dans" l’activation" du"
métabolisme"secondaire"et"des"PR"protéines."La"progression"de"la"colonisation"par"Phymatotrichopsis0
omnivora" s’associe" à" une" diminution" de" l’expression" de" la" défense" potentiellement" sous" la"
manipulation"du"parasite"(Uppalapati"et"al.,"2009)."Par"ailleurs,"chez"cette"même"plante,"la""résistance"
à"Macrophomina0phaseolina"emprunte"des"éléments"de"la"signalisation"auxinique"(Mah"et"al.,"2012).""
L’analyse" transcriptomique" de" la" mycorhization" a" longtemps" été" limitée" car" le" processus" de"
colonisation" par" le" champignon" mycorhizien" est" très" asynchrone," ce" qui" rend" les" études"
transcriptomiques"sur"des"tissus"racinaires"entiers"peu"représentatives"des"événements"nécessaires"à"
l’établissement" de" cette" symbiose." L’émergence" toute" récente" de" techniques" de" microdissection"
laser"combinée"à"une"analyse"transcriptomique"des"cellules"isolées"a"permis""une"grande"progression"
de" l’analyse" de" cette" association." Il" est" ainsi" apparu" que" les" cellules" les" plus" affectées" par" la"
mycorhization"sont"celles"qui"jouxtent"les"arbuscules,"ces"deux"types"de"cellules"présentant"une"forte"
59"
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altération" de" leur" fonction" de" transport" et" de" métabolisme" (Gaude" et" al.," 2011)." Deux" facteurs" de"
transcription" de" la" famille" NFPY" spécifiquement" exprimés" dans" les" arbuscules" ont" également" été"
identifiés"par"ce"type"d’approche"(Hogekamp"et"al.,"2011";"Guether"et"al.,"2009)."Ainsi,"cette"famille"
de" régulateurs" transcriptionnels" qui" agissent" en" hétérotrimères" semble" au" cœur" des" associations"
mutualistes"puisque"NFPYAP1"avait"auparavant"été"étudié"dans"le"développement"nodulaire."Enfin,"la"
comparaison"chez"le"riz"du"transcriptome"en"réponse"au"champignon"mycorhizien"et"à"deux"parasites"
fongiques"racinaires"a"révélé"l’existence"d’un"groupe"de"gènes"répondant"de"manière"similaire"à"tous"
les"microbes."De"telles"analyses"ont"aussi"été"déployées"pour"étudier"la"nodulation"et"ont"notamment"
révélé"que"la"reprogrammation"transcriptionnelle"ne"se"déroule"pas"seulement"dans"la"nodosité"mais"
aussi" dans" les" tissus" racinaires" qui" l’entourent" (Høgslund" et" al.," 2009)." L’étude" de" cinétiques" de"
transcriptomes"au"cours"du"développement"nodulaire"et"la"comparaison"avec"les"tissus"racinaires"ont"
permis"d’identifier"un"rôle"pour"le"jasmonate"dans"le"développement"du"nodule"(Moreau"et"al.,"2011)"
et"des"jeux"de"gènes"spécifiquement"exprimés"dans"cet"organe"(Benedito"et"al.,"2008)."Par"la"suite,"
ces" dernières" données" ont" été" comparées" à" la" réponse" des" feuilles" de" Medicago0 truncatula" à" des"
souches"de"Pseudomonas0syringae"déclenchant""des"réponses"hypersensibles"ou"la"maladie."Ainsi,"un"
groupe" de" gènes" impliqué" dans" les" sénescences" foliaires" est" aussi" associé" au" développement"
nodulaire"(Bozsó"et"al.,"2009)."Enfin,"des"comparaisons"de"cellules"en"cours"d’infection"par"Rhizobium"
et"des"cellules"nourricières"de"nématodes"ont"été"réalisées"par"microdissection"laser."Elles"ont"révélé"
l’aptitude" de" ces" parasites" à" provoquer" des" réponses" végétales" analogues" à" celles" induites" par" le"
symbiote,"notamment"au"niveau"du"métabolisme"primaire"et"hormonal"(Damiani"et"al.,"2012).""
Des" comparaisons" de" la" reprogrammation" transcriptionnelle" en" réponse" aux" chitooligosaccharides,"
lipochitooligosaccharides" et" la" flagelline" ont" été" entreprises" chez" Lotus0 japonicus." Elles" ont" révélé"
qu’approximativement" 50%" des" gènes" induits" par" les" lipochitooligosaccharides" le" sont" aussi" par" les"
MAMPs"chitiniques"et"peptidiques"selon"le"temps"après"traitement." Ainsi,"des"réponses"de"défense"
sont"induites"par"les"signaux"symbiotiques"et"inversement,"le"tout"sous"la"dépendance"des"récepteurs"
LysMPRLKs" symbiotiques" (Nakagawa" et" al.," 2011)." Très" récemment," suite" à" la" découverte" des"
lipochitooligosaccharides" mycorhiziens," une" comparaison" de" la" perception" de" ces" signaux" a" été"
conduite" chez" Medicago0 truncatula0 (Czaja" et" al.," 2012)." Il" a" ainsi" été" observé" que" la" dynamique" de"
perception"des"signaux"bactériens"et"fongiques"est"différente."Ainsi,"les"signaux"de"champignon"qu’ils"
soient"sulfatés"ou"non"ont"un"effet"rapide"et"transitoire"tandis"que"les"signaux"de"bactérie"mettent"
plus"de"temps"à"provoquer"des"réponses,"ces"dernières"étant"cependant"plus"soutenues"(Czaja"et"al.,"
2012)."Signaux"MycLCOs"et"NodLCOs"contrôlent"un"groupe"de"gènes"globalement"communs,"même"si"
des" subtilités" subsistent" en" dépit" du" faible" nombre" de" différences" entre" les" facteurs" NODs" et" les"
facteurs" MYCs" les" plus" proches" chimiquement." De" manière" surprenante," les" deux" signaux" MYCs"
60"
"
appliqués"séparement"ne"provoquent"pas"strictement"les"mêmes"réponses"que"les"MYCs"appliqués"
en" mélange," suggérant" une" régulation" très" fine" du" dialogue" au" travers" de" la" production" des" deux"
molécules."En"revanche,"l’étude"de"la"perception"de"ces"trois"signaux"chez"le"mutant"du"gène"NFP"a"
révélé"que"ce"LysMPRLK"est"indispensable"à"la"perception"de"tous"les"lipochitooligosaccharides,"une"
observation"intriguante"puisque"ce"mutant"est"tout"de"même"apte"à"la"mycorhization."On"peut"enfin"
noter"que"tous"ces"signaux"sont"intégrés"au"niveau"de"la"CSP"au"travers"de"DMI3"puisque"la"mutation"
de"ce"gène"entraîne"l’absence"de"toute"réponse"de"perception."
b. Réponses biochimiques de la plante
Les" réponses" biochimiques" des" plantes" aux" microorganismes" visent" à" bloquer" les" parasites" ou" à"
promouvoir"leurs"fonctions"mutualistes"selon"le"type"de"microbe"rencontré."Il"peut"s’agir"de"produits"
du"métabolisme,"de"peptides"ou"de"protéines."
Les" métabolites" associés" à" la" défense" sont" collectivement" nommés" phytoanticipines" puisqu’ils" sont"
accumulés" dans" les" tissus" végétaux" de" manière" non" toxique" et" se" voient" mobilisés" au" contact" du"
microorganisme,"au"point"de"pénétration"dans"les"tissus"hôtes."Ils"sont"considérés"à"ce"titre"comme"
des"moyens"de"défenses"préformés"(Bednarek"and"Osbourn,"2009";"Bednarek,"2012)."Comme"décrit"
antérieurement," la" sécrétion" de" flavonoïdes" et" de" strigolactones" attire" les" symbiotes." Les" premiers"
sont"tout"à"fait"centraux"aux"interactions"avec"les"microbes"puisque"des"voies"métaboliques"voisines"
telles"que"les"phénylpropanoïdes"et"les"isoflavonoïdes"synthétisent"des"phytoalexines"délétères"pour"
les" microbes." Dès" 2004," une" analyse" transcriptomique" a" illustré" la" convergence" des" métabolismes"
secondaires"dans"les"deux"types"d’interactions,"montrant"que"l’enzyme"en"charge"de"la"synthèse"de"
la" médicarpine," une" phytoalexine" de" type" isoflavonoïde" typique" des" légumineuses," est"
transcriptionnellement"induite"au"cours"du"développement"nodulaire"(Yahyaoui"et"al.,"2004).""
Des" peptides" végétaux" sont" aussi" mis" en" jeu" dans" les" deux" types" d’interaction." Les" défensines" sont"
des"petits"peptides"basiques"qui,"en"s’oligomèrisant,"forment"des"pores"dans"les"parois"microbiennes."
Une" de" leurs" particularités" est" qu’elles" sont" riches" en" cystéines" ce" qui" les" rend" semblables" à" des"
peptides" déployés" dans" les" réponses" immunitaires" animales" (Thomma" and" Cammue," 2002";""
Mergaert" et" al.," 2011)." Les" thionines" jouent" des" rôles" semblables" et" sont" aussi" riches" en" cystéines"
mais" sont" classifiées" séparement" en" raison" d’une" structure" tertiaire" distincte" (Sels," Mathys," De"
Coninck," Cammue," &" De" Bolle," 2008)." Curieusement," " d’autres" peptides" à" cystéines" sont" aussi"
impliqués"dans"la"maturation"des"nodules"et"sont"appelés"Nodule"Cystéine"Rich"(NCRs)"(Mergaert"et"
al." 2011";" Batut" et" al.," 2011)." CeuxPci" conduisent" notamment" les" bactéries" à" un" stade" de"
différenciation"irréversible,"se"caractérisant"par"des"endoréplications"et"une"augmentation"de"taille"
en"même"temps"que""leur"membrane"est"fortement"perméabilisée."
61"
"
Si" des" familles" de" métabolites" et" peptides" " apparentées" mais" distinctes" sont" impliquées" dans" les"
réponses" immunitaires" et" la" symbiose" rhizobienne," il" existe" des" batteries" de" protéines" végétales"
activées" dans" l’ensemble" des" interactions" biotiques." Originellement" identifiées" dans" le" cadre" de" la"
réponse" aux" microorganismes" pathogènes," elles" sont" appellées" Pathogenesis" Related" Proteins" (PR"
protéines)"(van"Loon"et"al.,"2006)(Figure"16)."
Figure"16:"van&Loon,&L.C.&et&al."(2006)."Significance"of"inducible"defensePrelated"proteins"in"infected"plants."Annual0review0of0
phytopathology"44:"135P62."
Réparties"dans"17"familles"selon"leurs"fonctions"antimicrobiennes,"elles"sont"fortement"corrélées"à"la"
régulation" de" la" balance" hormonale" et" donc" sujettes" aux" multiples" interconnections" soulignées"
précédemment"entre"réponses"mutualistes"et"immunitaires."On"peut"regrouper"ces"protéines"selon"
trois" grands" modes" d’actions." D’abord" les" glucanases" et" chitinases" qui" s’attaquent" à" la" paroi"
microbienne." Curieusement," certaines" sont" spécifiquement" induites" au" cours" de" la" symbiose"
rhizobienne"ou"mycorhizienne,"ce"qui"questionne"sur"les"rôles"qu’elles"pourraient"jouer"ici"(Salzer"et"
al.," 2000";" Salzer" et" al.," 2004)." Ensuite," les" inhibiteurs" de" protéases" et" de" polygalacturonases"
protègent"la"paroi"hôte"des"enzymes"de"dégradation"microbienne."CeuxPci"sont"particulièrement"mis"
en" jeu" face" aux" microorganismes" nécrotrophes" qui" assurent" leur" développement" en" dégradant" la"
paroi"végétale."Enfin,"les"systèmes"de"défenses"qui"rassemblent""des"activités"enzymatiques"toxiques"
pour" le" microorganisme" qui" sont" des" ribonucléases" et" protéinases," qui" affectent" l’expression" du"
62"
"
génome" microbien" ou" limitent" l’action" des" protéines" associées" à" l’adhésion" du" microbe" sur" l’hôte."
Ces" réponses" de" contrePattaque" incluent" aussi" les" enzymes" " associées" à" la" régulation" du" stress"
oxydant"qui"participe"au"renforcement"de"la"paroi"végétale."S’il"a"pu"être"démontré"que"l’inhibition"
de"certaines"de"ces"fonctions"conduit"à"un"déficit"de"résistance"à"des"parasites"(Samac"et"al.,"2011),"il"
apparaît" que" leur" activation" n’empêche" pas" la" mise" en" place" de" symbioses" mycorhiziennes," après"
quoi,"le"champignon"parvient"à"les"éteindre"(CamposPSoriano"et"al.,"2010).""
"
III. Medicago truncatula, légumineuse
modèle pour les interactions
biotiques racinaires
"
Les" légumineuses" constituent" la" deuxième" famille" de" plantes" la" plus" cultivée" (Graham" and" Vance,"
2003)."Leur"aptitude"unique"à"mettre"en"place"la"symbiose"fixatrice"d’azote"en"présence"des"bactéries"
du" genre" Rhizobium" et" à" être" mycorhizables" leur" donne" une" place" importante" dans" l’étude" des"
interactions" plantesPmicroorganismes" du" sol." Les" légumineuses" cultivées" ont" cependant" des"
caractéristiques" génétiques" (taille" et" complexité" du" génome)" et" biologiques" (difficulté" à" la"
transformation" par" Agrobacterium)" qui" les" ont" rendues" difficiles" à" étudier" et" à" améliorer." Pour"
contourner" ces" limitations," Lotus0 japonicus" et" Medicago0 truncatula0 ont" " été" sélectionnés" comme"
plantes" modèles" pour" représenter" respectivement" les" légumineuses" dont" les" nodosités" ont" une"
croissance"déterminée"(comme"le"soja"ou"le"haricot)""et"indéterminée"(comme"le"pois"ou"la"luzerne)"
(Popp" and" Ott," 2011)." La" capacité" de" Medicago" truncatula" à" former" des" nodosités" à" croissance"
indéterminée" fait" d’ailleurs" de" cette" plante" un" modèle" de" la" biologie" du" développement" végétal,"
notamment" pour" l’étude" des" fonctions" méristèmatiques" (Holmes" et" al.," 2008).Les" travaux" réalisés"
chez" ces" deux" plantes" ont" révélé" une" bonne" conservation" des" déterminismes" mutualistes." Compte"
tenu"de"leur"importance"agronomique"et"des"progrès"en"génomique,"le"soja"(Glycine0max)"ou"le"pois"
(Pisum0 sativum)" sont" eux" aussi" maintenant" bien" étudiés." Quoiqu’il" en" soit," les" différentes"
légumineuses"montrent"une"organisation"génomique"très"semblable"à"celle"des"plantes"modèles,"ce"
qui"souligne"l’intérêt"de"ces"dernières"pour"accélerer"l’amélioration"des"plantes"de"grande"culture,"en"
exploitant" la" synténie" existante" (Young" and" Bharti," 2012)." Plus" récemment," un" nouveau" modèle"
impliquant"la"non"légumineuse"Parasponia,"capable"d’interagir"avec"des"Rhizobia,0a"été"sélectionné"
63"
"
afin" de" mieux" comprendre" les" spécificités" des" symbioses" chez" les" différentes" espèces" concernées0
(Streng"et"al.,"2011";"Op"den"Camp"et"al.,"2011)."Cette"interaction"analogue"à"celle"qui"existe"chez"les"
légumineuses" est" une" occasion" d’observer" en" termes" évolutifs" comment" sont" mis" en" place" les"
différents" mécanismes" impliqués" dans" ces" symbioses." Les" légumineuses" étant" aussi" hôtes" de"
nombreux" parasites," il" est" donc" possible" de" comparer" chacune" des" interactions" et" d’étudier" les"
connexions" moléculaires" potentielles" dans" la" réponse" de" la" plante" visPàPvis" des" différents" types" de"
microorganismes."Pour"initier"ce"travail,"la"légumineuse"Medicago0truncatula"a"été"choisie"en"raison"
des"ressources"génétiques"et"génomiques"disponibles,"mais"également"suite"au"développement"et"à"
la"maîtrise"de"plusieurs"pathosystèmes"utilisés"en"routine"au"laboratoire."
"
A. Ressources génétiques, génomiques et
transcriptomiques disponibles pour Medicago
truncatula
Initialement"sélectionnée"au"début"des"années"1990"pour"étudier"les"déterminismes"génétiques"de"la"
symbiose" fixatrice" d’azote" (Barker" et" al.," 1990)," la" légumineuse" Medicago0 truncatula" a" rapidement"
été" utilisée" pour" l’étude" d’autres" processus" (Cook," 1999";" Rose," 2008)." Le" succès" de" son" utilisation"
repose" en" premier" lieu" sur" le" développement" de" nombreux" outils" génétiques" et" génomiques." La"
distribution"de"populations"naturelles"sur"le"pourtour"méditerranéen"est"à"l’origine"d’une"variabilité"
naturelle" qui" constitue" une" richesse" à" l’égard" de" la" résistance" à" des" stress" abiotiques" tels" que" la"
sécheresse"ou"la"salinité"(Larrainzar"et"al.,"2007";"Nunes"et"al.,"2008"";""Bianco"et"al.,"2009";"Gruber"et"
al.," 2009";" Badri" et" al.," 2010" ;" Arraouadi" et" al.," 2011)" mais" aussi" biotiques" (Vailleau" et" al.," 2007";"
AmelinePtorregrosa" et" al.," 2008" ;" Djébali" et" al.," 2009)." Le" développement" de" lignées" recombinantes"
(Recombinant"Inbred"Lines","RILs)"et"de"marqueurs"moléculaires"ont"permis"de"construire"des"cartes"
génétiques." Leur" exploitation" au" travers" d’analyses" génétiques" classiques" a" permis" d’identifier" de"
nombreux"QTLs"(Quantitative"Trait"Locus)"(cf"sections"suivantes)."Outre"la"génétique"conventionelle"
dite" «"forward"»," c'estPàPdire" du" phénotype" vers" la" fonction," des" approches" de" génétique" inverse,"
dites" «"reverse"»" allant" du" gène" mutagénisé" à" la" fonction" affectée," ont" aussi" été" déployées." Des"
collections"de"mutants"obtenus"par"mutagénèse"chimique"(Penmetsa"and"Cook,"2000";"Catoira"et"al.,"
2000)" ou" bombardement" de" neutrons" (Sagan" et" al.," 1995)" et" criblés" par" TILLING" (Targeted" Induced"
Local"Lesion"IN"Genome)"ont"été"obtenues"chez"la"lignée"de"référence"A17""(Tadege"et"al.,"2005)."Plus"
récemment,"une"banque"de"mutants"insertionnels"dérivée"de"la"lignée"R108"(Pislariu"et"al.,"2012)"a"
également" été" développée." Ainsi," des" mutants" des" cascades" de" signalisations" symbiotiques" ont" été"
isolés"dans"le"fond"génétique"A17."Fournissant"des"phénotypes"qualitatifs"d’absence"de"symbiose,"ils"
64"
"
ont"permis"de"conclure"sans"ambiguïté"sur"le"rôle"de"ces"acteurs"dans"la"symbiose"mutualiste."Il"s’agit"
de"NFP,0LYK3,0DMI1,DMI2,0DMI3,0NSP1,0NSP2."Les"mutants"d’organogénèses"présentent"souvent"des"
phénotypes" plus" discrets" et" quantitatifs." Pour" l’heure," PUB%1,0 LIN,0 RPG,0 NF%YA10 ,EFD,0 LATD,0 CRE1,0
EIN2,0 SUNN,0 " tous" impliqués" dans" la" nodulation," sont" issus" d’A17." VAPYRIN0 qui" contrôle" les"
événements" d’infection" dans" les" deux" symbioses" a," lui," été" " obtenu" dans" le" fonds" R108." Plus"
récemment," la" finalisation" de" la" séquence" du" génome" (Young" et" al.," 2011)" a" ouvert" la" porte" à" des"
approches" de" reséquençage" de" deux" cents" lignées" sauvages" qui," en" permettant" de" mieux"
appréhender" la" diversité" génétique" de" ce" modèle" et" de" multiplier" les" marqueurs" moléculaires,"
accélèrent" les" études" génétiques" basées" sur" la" «"Genome" Wide" Association" Mapping"»" (GWAS)"
http://medicagohapmap.org/"(Branca"et"al.,"2011)."
L’étude" de" la" transcriptomique" de" cette" plante" a" aussi" parfaitement" épousé" les" évolutions"
technologiques" successives." La" progression" de" la" qualité" des" bases" de" données" génomiques" et" de"
transcrits" (Expressed" Sequence" Tag)" (http://medicago.toulouse.inra.fr/Mt/EST/)" a" permis" de"
construire" des" puces" d’un" recouvrement" croissant" du" transcriptome" total" de" Medicago0 truncatula(0
Lamblin," 2003";" Küster" et" al.," 2004";" Becker" et" al.," 2005)." D’abord" restreintes" à" l’étude" de" la"
nodulation" et" de" la" mycorhization," les" études" réalisées" ont" été" étendues" au" développement" des"
graines,"à"différents"stress"et"à"des"analyses"organe"par"organe"(Benedito"et"al.,"2008";"De"Michele"et"
al.," 2009" ;" Gomez" et" al.," 2009" ;" He" et" al.," 2009)." La" connaissance" conjointe" du" génome" et" du"
transcriptome" de" cette" plante" ouvre" la" voie" à" des" approches" ambitieuses" de" protéomiques,"
notamment" sur" la" mécanistique" de" la" signalisation" chez" cette" plante" (Grimsrud" et" al.," 2010)." Enfin,"
tirant"toujours"profit"de"l’essor"des"technologies"de"séquençage"d’acides"nucléiques,"un"séquençage"
des"petits"ARNs"de"la"plante"dans"les"apex"racinaires"et"les"nodosités"a"permis"de"caractériser"certains"
de"ces"éléments"régulateurs"épigénétiques"(LelandaisPBrière"et"al.,"2009;"Khan"et"al.,"2011;"Bazin"et"
al.,"2012)(http://medicago.toulouse.inra.fr/Mt/RNA/MIRMED/LeARN/cgiPbin/learn.cgi)."
Medicago0 truncatula" est" aussi" hôte" pour" des" agents" pathogènes," notamment" les" bactéries"
Agrobacterium"capables"de"transférer"des"fragments"d’ADN"dans"le"génome"de"l’hôte,"ce"qui"permet"
de" générer" des" lignées" transgéniques." Elle" est" aussi" hôte" pour" certains" parasites" économiquement"
importants"et"spécifiques"des"légumineuses."
"
B. Medicago truncatula, modèle pour la comparaison
des associations mutualistes et parasites
"
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En"plus"des"caractéristiques"partagées"avec"Lotus0japonicus0:"cycle"reproductif"rapide,"abondance"de"
graines," transformation," diploïdie," petit" génome," Medicago0 truncatula" est" hôte" pour" un" grand"
nombre"de"microbes"parasites"d’importance"agronomique,"ce"qui"lui"donne"une"place"centrale"dans"
l’étude"des"relations"croisées"entre"parasitisme"et"mutualisme."
"
1. Exploitation de deux pathosystèmes modèles pour évaluer les
connexions entre symbiose et immunité
Medicago0truncatula"est"impliquée"dans"plusieurs"pathosystèmes"reposant"sur"des"déterminismes"à"
caractères" quantitatifs." De" nombreux" pathogènes," parmi" les" plus" dommageables" sur" les"
légumineuses"mais"aussi"à"spectre"d’hôtes"large,"sont"étudiés"au"travers"de"cette"plante,"que"ce"soit""
des" champignons" (Torregrosa" et" al.," 2004" ;" (AmelinePtorregrosa" et" al.," 2008);" Ellwood" et" al.," 2006" ;"
Moussart"et"al.,"2006";"Tivoli"et"al.,"2006";"Anderson"et"al.,"2010"";"Uppalapati"et"al.,"2010";"Mah"et"al.,"
2012";"Uppalapati"et"al.,"2012),"des"bactéries"""(Vailleau"et"al.,"2007"";"Turner"et"al.,"2009)"ou"encore"
des" insectes" (Gao" et" al.," 2007" ;" Gao" et" al.,2008" ;" Klingler" et" al.," 2009";" Gao" et" al.," 2010)" et" des"
oomycètes" (Samac" et" al.," 2011)." Ces" derniers" impliquent" des" réseaux" de" gènes" complexes" qui"
permettent," lors" de" l’étude" de" mutants," d’identifier" la" contribution" éventuelle" de" gènes" d’intérêt"
dans" la" réponse" immunitaire" globale." De" plus," certains" de" ces" pathosystèmes" ciblent" les" tissus"
racinaires," une" caractéristique" cruciale" pour" l’étude" des" interconnections" avec" la" symbiose" puisque"
nombre" des" gènes" symbiotiques" ne" sont" exprimés" que" dans" ces" tissus," rendant" inappropriée"
l’utilisation" d’agents" pathogènes" aériens" (Denny" Mellersh" and" Martin" Parniske," 2006)." Deux"
pathosystèmes"ont"été"retenus"dans"ce"travail":"les"paragraphes"suivants"présenteront"l’état"des"lieux"
sur"ces"derniers"et"justifieront"leur"utilisation"dans"le"cadre"de"notre"étude."
a. Aphanomyces euteiches
Les" oomycètes" tels" qu’Aphanomyces0 euteiches0 sont" des" microorganismes" filamenteux"
phylogénétiquement" très" distincts" des" champignons" (Baldauf," 2003)." Au" sein" de" cette" famille," ce"
parasite"appartient"au"clade"des"saprolegniales"où"il"est"le"seul"organisme"phytopathogène,"les"autres"
étant"des"saprophytes"ou"des"parasites"animaux."Ainsi,"cet"oomycète"est"significativement"différent"
des" espèces" de" Phytophtora0 sp.0 qui" appartiennent" aux" peronosporales" (DiéguezPUribeondo" et" al.,"
2009)et" sont" parasites" exclusifs" d’hôtes" végétaux." Le" spectre" d’hôte" d’Aphanomyces0 euteiches" est"
particulièrement"étendu"puisque"certaines"souches"peuvent"coloniser"le"pois,"la"fève,"le"pois"chiche,"
la"lentille,"le"lupin,"la"vesse,"ou"la"luzerne"pérenne"(Moussart"et"al.,"2008)(Figure"17).""
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Figure"17":""Arbre"phylogénétique"illustrant"les"relations"au"sein"des"Peronosporomycètes."Gaulin,&E&et&al.,"(2007)."Root"rot"
disease"of"legumes"caused"by"Aphanomyces"euteiches."Molecular0Plant0Pathology"8:"539P548."
Au"champ,"les"symptômes"se"manifestent"par"un"jaunissement"du"feuillage,"associé"à"une"diminution"
ou"une"absence"du"développement"des"gousses"et"un"brunissement"des"parties"racinaires""et,"parfois,"
du"collet.""Son"cycle"de"vie"se"caractérise"par"des"cycles"de"reproduction"sexuée"ou"asexuée"donnant"
respectivement" des" oospores" et" zoospores." Les" premières," formes" de" conservation," peuvent" rester"
inactives" dans" le" sol" plusieurs" années" avant" de" rencontrer" un" hôte" potentiel" (Gaulin" Elodie" et" al.,"
2007)."La" production" d’une" banque" " de" séquences" d’ADN" complémentaires" a" permis" de" mettre" en"
exergue"d’autres"originalités"de"sa"biologie"en"comparaison"notamment"aux"Phytophtora0sp.0(Gaulin"
et"al.,"2008)."Son"répertoire"d’effecteurs"classiques"de"types"RXLR"ou"Crinkle"paraît"plus"restreint,"en"
contrePpartie," certaines" voies" métaboliques" ou" fonctions" supplémentaires" se" sont" ajoutées" par"
rapport"à"Phytophthtora"telles"que"la"biosynthèse"de"stérols"ou"la"synthèse"de"certains"transporteurs."
Ceci" suggère" des" mécanismes" de" pathogénie" originaux" envers" ses" hôtes" (Madoui" et" al.," 2009)."
L’étude" de" la" paroi" de" cet" oomycète" a" révélé" une" étonnante" abondance" de" chitosaccharides" non"
cristallins,"et"donc"différents"de"la"chitine"classique,"là"où"les"parois"d’oomycètes"sont"le"plus"souvent"
purement"cellulosiques"(Badreddine"et"al.,"2008)."Ce"dernier"élément"fait"d’Aphanomyces0euteiches"
un"parasite"tout"à"fait"approprié"pour"étudier"les"liens"entre"immunité"et"mutualisme."L’étude"de"la"
maladie"causée"par"l’oomycète"chez"le"pois"a"révélé"que"l’initiation"de"la"colonisation"se"fait"dans"la"
zone" d’élongation" de" la" racine" en" croissance." Ainsi," apex" racinaire" et" zone" de" maturation"
apparaissent" protégés." L’accumulation" prévalente" d’un" composé" antimicrobien," la" pisatine,"
permettrait" dans" les" cellules" bordantes" (Gunawardena" &" Hawes," 2002)" et" de" l’apex" de" limiter" le"
développement"du"mycélium"(Cannesan"et"al.,"2011)."En"parallèle,""l’extrémité"des"racines"de"pois"est"
riche" en" protéines" à" arabinogalactanes" qui" possèdent" différentes" activités" sur" les" zoospores"
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d’A.euteiches."Dans"un"premier"temps,"cellesPci"exercent"un"pouvoir"chemoattractif"sur"les"zoospores"
puis" provoquent" leur" enkystement" et" empêchent" leur" germination," prévenant" ainsi" l’invasion"
(Cannesan"et"al.,"2012)."Ces"deux"éléments"supportent"le"rôle"des"cellules"bordantes"dont"la"quantité"
est" d’ailleurs" accrue" en" présence" du" parasite" dans" la" protection" visPàPvis" d’A.euteiches." Les"
déterminismes" génétiques" de" la" résistance" ont" été" étudiés" notamment" chez" le" pois" et" ont" permis"
d’identifier"trois"QTLs"majeurs"stables"et"consistants"dans"des"environnements"différents"(Wicker"and"
Rouxel,"2001";""PiletPNayel"et"al.,"2002";"Vandemark"and"Grünwald,"2004";""PiletPNayel"et"al.,"2005";"
McGee"et"al.,"2012)."Chez"Medicago0truncatula,"un"QTL"majeur"de"résistance""a"pu"être"identifié"sur"
le"haut"du"chromosome"III"à"l'issue"d'un"criblage"d'une"population"de"RILs"issues"des"lignées"DZA45.5"
et"F83005.5."Plus"récemment,"des"relations"épistatiques"entre"ce"locus"et"d'autres"QTLs"mineurs"ont"
pu" être" précisées" (Hamon" et" al.," 2010)." Par" ailleurs," un" QTL" a" été" identifié" " sur" le" haut" du"
chromosome" III," suite" à" l'analyse" de" RILs" issus" " également" d'un" croisement" avec" F83005.5" chez" la"
lignée"de"référence"A17"(Djébali"et"al.,"2009)(Figure"18).""
"
Figure" 18" :" Adaptée" de" Djébali,& N.& et& al." (2009)." Partial" resistance" of" Medicago0 truncatula" to" Aphanomyces0 euteiches0 is"
associated"with"protection"of"the"root"stele"and"is"controlled"by"a"major"QTL"rich"in"proteasomePrelated"genes."Molecular0
plant%microbe0interactions :0MPMI"22:"1043P55."
CeluiPci" contient" en" particulier" un" nombre" important" de" protéines" FPBox." Une" caractérisation"
cytologique" de" l'interaction" a" aussi" été" entreprise." Elle" a" révélé" que" l'oomycète" traverse" le"
rhizoderme" sans" former" de" structure" de" pénétration" puis" se" développe" de" manière" biotrophe" et"
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"
intercellulaire" dans" le" cortex." A" ce" stade," le" comportement" de" la" lignée" A17" et" celui" de" la" plante"
sensible"F83005.5"divergent."Une"production"de"composés"phénoliques"solubles"autour"du"cylindre"
central"et"la"division"de"cellules"pericycliques"couplées"à"la"formation"d'un"anneau"de"lignine"autour"
de" la" stèle" semblent" être" les" principaux" mécanismes" qui" empêchent" le" mycelium" d'accéder" à" la"
vasculature"chez"A17"mais"pas"F83"où"le"mycélium"entre"de"manière"intracellulaire"dans"cellePci."Des"
analyses" biochimiques" complémentaires" ont" révélé" un" rôle" important" joué" par" l'homéostasie" des"
ROS" dans" la" résistance" (Djébali" et" al.," 2011)." Il" est" apparu" que" ces" ROS" ne" sont" pas" accumulées" au"
niveau"racinaire"dans"des"proportions"toxiques"pour"le"microbe,"mais"sont"en"revanche"consommées"
par" des" activités" péroxydases" chez" A17" qui" participent" vraisemblablement" " à" la" polymérisation" des"
composés"phénoliques"insolubles"que"sont"les"lignines."Des"travaux"de"protéomique"ont"également"
été" entrepris." Les" premiers" d'entre" eux" ont" révélé" dès" 2005" que" des" protéines" FPBOX" similaires" à"
celles"renfermées"dans"le"QTL"sont"différentiellements"exprimées"entre"A17"et"F83005.5"(Colditz"et"
al.," 2005)." D'autre" part," les" auteurs" ont" constaté" que" l'application" d'acide" abscissique" renforce" la"
sensibilité"à"la"maladie"et"induit"l'expression"de"défenses"basales"tels"que"les"PR10"dont"l'expression"
est"corrélée"au"degré"de"colonisation"et"donc"de"sensibilité"(Colditz"et"al.,"2004)."Plus"récemment,"il"
est" apparu" qu'Aphanomyces0 euteiches" induit" les" enzymes" de" la" biosynthèse" de" l'acide" abscissique"
(Schenkluhn" et" al.," 2010)." Par" ailleurs," le" silencing" de" telles" PR10," en" induisant" de" manière"
compensatoire"des"PR5,"a"permis"d'accroître"la"tolérance"de"l'hôte"à"la"maladie"(Colditz"et"al.,"2007)."
La" mycorhization" permet" aussi" d'améliorer" la" résistance" à" Aphanomyces0 euteiches" (Slezack" et" al.,"
2000" ;" Colditz" et" al.," 2005" )," sans" doute" par" l'intermédiaire" de" modulation" de" la" signalisation"
jasmonate"qui"est"favorable"à"la"mycorhize,"et"n'affecte"pas"la"symbiose"fixatrice"d'azote"(Landgraf"et"
al.," 2012)." D'autres" analyses" protéomiques," comparant" les" inoculations" avec" des" symbiotes"
bactériens" et" fongiques" à" des" coPinoculations" avec" Aphanomyces0 euteiches," ont" démontré" un" effet"
protecteur"des"symbiotes"qui"conduit"à"une"induction"retardée"ou"diminuée"des"réponses"typiques"
de"la"défense."Forts"de"ces"approches"protéomiques"successives,"Colditz"et"ses"collègues"ont"identifié""
un"acteur"commun"à"ces"trois"interactions"racinaires"chez"Medicago0truncatula."Il"s'agit"d'une"petite"
GTPase" régulatrice" de" NADPH" oxydases" appellées" chez" les" plantes" RBOHs" (Respiratory" Bust"
Homologues)" à" l'origine" de" la" production" de" ROS" impliquée" dans" la" résistance" à" Aphanomyces0
euteiches" et" qui" est" aussi" un" régulateur" de" la" mycorhization" et" est" nécessaire" à" l'établissement" de""
nodosités"(Kiirika"et"al.,"2012)."
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b.
Colletotrichum trifolii
Les" Colletotrichum0 sp." sont" des" champignons" appartenant" au" groupe" des" deuteromycètes" qui"
constitue"un"large"groupe"d’espèces"connues"pour"s’attaquer"aux"parties"aériennes"de"nombreuses"
plantes" à" l’aide" de" modes" de" colonisation" allant" de" la" biotrophie" à" la" nécrotrophie" (Perfect" et" al.,"
1999";"O’Connell"et"al.,"2012)(Figure"19).""
"
Figure"19":"Phylogénie"et"mode"d’infection"de"Colletotrichum0higginsianum""et"Colletotrichum0graminicola.0(a)"Cladograme"
montrant" les" relations" entre" Colletotrichum" et" 13" autres" champignons" séquencés" utilisés" pour" comparaison" et" Rhizopus0
orizae0 en" hors" groupe." (b)" Processus" infectieux" de" Colletotrichum" higginsianum" et" symptômes" observés" sur" des"
brassicacées" et" Arabidopsis.0 (c)" Processus" infectieux" de" Colletotrichum0 graminicola0 0 et" symptômes" observés" sur" feuille" et"
tige"de"maïs."Abréviations:""SP"spore";"AP"appressorium";"PH"hyphe"primaire"biotrophe";"SH"hyphe"secondaire"nécrotrophe."
O’Connell,& R.J.& et& al." (2012)." Lifestyle" transitions" in" plant" pathogenic" Colletotrichum" fungi" deciphered" by" genome" and"
transcriptome"analyses."Nature0Genetics.""
Chez" les" légumineuses," Colletotrichum0 lindemuthianum" s’attaque" au" haricot" tandis" que"
Colletotrichum0trifolii"a"pour"hôte"la"luzerne"Medicago0sativa"ou"encore"Medicago0truncatula."Tous"
deux" sont" des" parasites" hémibiotrophes." L’étude" de" la" résistance" chez" la" légumineuse" modèle" a"
révélé" que" la" résistance" foliaire" de" la" lignée" A17" met" en" jeu" une" accumulation" de" composés"
phénoliques" solubles," la" déposition" de" papilles" de" callose" et" une" production" de" ROS" avant" de"
culminer"en"une"réponse"de"type"hypersensible"au"niveau"des"cellules"où"le"mycélium"différencie"ses"
appressoria." Chez" F83005.5," le" défaut" de" ces" réponses" entraîne" l’initiation" de" la" colonisation" et" la"
macération" des" tissus" aériens" (Torregrosa" et" al.," 2004)." Par" la" suite," des" analyses" génétiques"
(AmelinePtorregrosa"et"al.,"2008";"Yang"et"al.,"2008)"ont"permis"l’identification"d’un"acteur"classique"
de" la" résistance" de" type" gène" pour" gène," un" TIRPNBSPLRR" nommé" RCT1." Deux" lignées" quasiP
isogéniques" Near" Isogenic" Lines," NILs," différant" seulement" au" niveau" du" QTL" où" elles" portent"
respectivement" les" allèles" A17" ou" F83005.5," ont" été" étudiées" en" transcriptomique" au" cours" de"
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l’invasion" par" Colletotrichum0 trifolii" et" Colletotrichum0 lindemuthianum." Ainsi," des" processus" de"
signalisation" et" de" défense" régulés" de" manière" différentielle" entre" les" NILs" et" selon" le" type" de"
résistance"hôtePnon"hôte,"résistantPsensible"ont"été"observés":"ils"ont"montré"notamment"une"forte"
corrélation"entre"l’activation"du"métabolisme"secondaire"mais"aussi"d’une"PR10"et"la"résistance"non"
hôte" ou" incompatible" tandis" que" la" sensibilité" se" traduit" chez" les" porteurs" de" l’allèle" sensible"
F83005.5" par" une" réponse" tardive" (Jaulneau" et" al.," 2010)." Des" essais" d’inoculation" sur" organe"
racinaire"ont"révélé"l’aptitude"de"Colletotrichum0trifolii"à"attaquer"ces"tissus."Le"mutant"du"gène"DMI3"
présente" de" manière" intriguante" une" résistance" accrue" à" cette" colonisation" au" travers" de" morts" de"
cellules"épidermiques"au"contact"du"champignon"(Genre"et"al.,"2009)."Des"travaux"réalisés"au"sein"de"
l’équipe" et" encore" non" publiés" vont" plus" loin"":" ils" ont" révélé" qu’un" QTL," colocalisant" avec" celui"
impliqué" dans" la" résistance" aérienne," existe" et" explique" une" part" importante" des" phénotypes"
observés"chez"A17"et"F83005.5"toujours"respectivement"résistantes"et"sensibles,"soulignant"que"les"
déterminismes"de"la"résistance"pourraient"être"similaires"au"niveau"aérien"comme"racinaire."Dans"ce"
cas"de"figure,"Colletotrichum0trifolii"exhibe"un"comportement"différent"de"celui"qu’il"adopte"dans"les"
parties" aériennes." Chez" F83005.5," il" pénètre" l’épiderme" sans" appressorium" et" traverse" le" cortex" de"
manière" rapide" et" intercellulaire" pour" se" concentrer" dans" la" vasculature" et" former" des" aggrégats"
mycéliens" dans" l’endoderme." Dans" cet" hôte" sensible," son" développement" racinaire" est" aussi"
hémibiotrophe,"la"colonisation"entraînant"malgré"tout"la"mort"de"l’hôte"et"une"forte"macération"au"
bout"d’un"temps"plus"long"que"sur"les"parties"aériennes."A"l’inverse,"il"est"rapidement"arrêté"dans"les"
couches" superficielles" de" la" racine" chez" A17" (Figure" 20)." Par" rapport" à" ces" travaux," Colletotrichum0
trifolii"présente"l’intérêt"de"posséder"de"la"chitine"«"classique"»"dans"les"parois"de"son"mycélium"et"
est"donc"complémentaire"à"Aphanomyces0euteiches"pour"cet"aspectPlà."La"résistance"racinaire"d’A17"
facilite" en" plus" l’étude" du" rôle" des" gènes" symbiotiques" dans" la" résistance" en" recherchant," par"
exemple,"un"mutant"moins"résistant"que"le"sauvage."
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Figure"20":"Phénotypes"aériens"et"racinaires"de"la"résistance"à"Colletotrichum0trifolii0chez"A17"et"F83005.5."Torregrosa,&C.&et&
al."(2004)."Cytological,"genetic,"and"molecular"analysis"to"characterize"compatible"and"incompatible"interactions"between"
Medicago" truncatula" and" Colletotrichum" trifolii." Molecular0 plant%microbe0 interactions :0 MPMI" 17:" 909P20..Thèse& de& Marc&
Cazaux."
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IV. Objectifs de la thèse
"
L’objectif" de" cette" introduction" était" de" souligner" les" points" communs" associés" à" la" formation" des"
interactions" entre" plantes" et" microorganismes" mutualistes" et" pathogènes." Si" ces" deux" types" de"
symbioses" ont" été" intensément" étudiés" au" cours" des" dernières" décennies," les" mécanismes"
moléculaires"sousPjacents"commencent"à"peine"à"être"dévoilés."Ainsi,"l’objectif"de"ma"thèse"était"de"
tirer" profit" de" ces" nouvelles" connaissances" pour" évaluer" les" interconnections" moléculaires"
éventuelles"dans"le"contrôle"des"deux"types"d’interactions"biotiques."
Mon" travail" s’est" en" grande" partie" inscrit" dans" le" cadre" d’un" projet" ANR" (Agence" Nationale" de" la"
Recherche)" nommé" SYMPASIGNAL." CeluiPci" visait" à" comprendre" comment" les" plantes" telles" que"
Medicago0 truncatula" distinguent" des" signaux" présentant" des" similarités" structurales," les"
lipochitooligosaccharides" et" les" chitooligosaccharides," en" utilisant" des" jeux" de" récepteurs"
appartenant" à" la" même" famille," les" LysMPRLKs." Un" point" particulièrement" novateur" résidait" dans"
l’utilisation" du" parasite" racinaire" Aphanomyces0 euteiches," qui" possède" des" chitosaccharides" aux"
propriétés"chimiques"originales,"notamment"en"terme"de"solubilité"et"de"répartition"à"la"surface"du"
mycélium." Ainsi," la" caractérisation" biochimique" de" ces" composés" et" de" leur" perception" par" l’hôte"
constituaient" l’objectif" central" du" projet." A" titre" personnel," j’ai" étudié" l’implication" éventuelle" des"
voies" de" signalisation" symbiotique" dans" la" résistance" au" parasite" afin" d’obtenir" des" indications" sur"
l’importance"éventuelle"de"ces"voies"dans"la"perception"du"parasite"et"la"mise"en"place"de"la"défense."
En"ce"sens,"l’ensemble"des"mutants"disponibles"affectés"dans"la"signalisation"symbiotique"a"été"criblé"
au" niveau" phénotypique" et" cytologique." La" recherche" de" gènes" à" la" fois" acteurs" des" symbioses"
mutualistes"et"parasitaires"n’implique"pas"qu’ils"aient"le"même"degré"d’importance"dans"chacune"des"
associations." On" peut," en" effet," s’attendre" à" ce" que" les" parasites" capables" de" forcer" leur" passage"
jusqu’au"nutriment"de"l’hôte"soient"moins"affectés"par"l’absence"de"réponses"d’accommodation"de"la"
plante"qu’un"microbe"mutualiste"dépendant"strictement"de"certaines"réponses"végétales."De"même,"
telles" qu’elles" sont" décrites" à" ce" jour," les" réponses" immunitaires" reposant" sur" des" résistances" non"
hôtes" passives" ou" des" réponses" incompatibles" de" types" gène" pour" gène" hautement" coPévoluées"
dépendant"de"mécanisme"très"directs"et"spécialisés,"laissent"peu"de"place"à"d’éventuelles"relations"
croisées" visPàPvis" de" la" symbiose." La" résistance" quantitative," de" part" la" complexité" des" réseaux" de"
gènes" qu’elle" met" en" jeu," paraît" plus" propice" à" l’existence" de" liens" avec" les" déterminismes"
mutualistes."Ces"travaux"sont"à"l’origine"de"la"première"partie"des"résultats."
73"
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Ainsi," en" exploitant" à" la" fois" ce" pathosystème" et" les" mutants" symbiotiques" existants," le" premier"
chapitre"de"cette"thèse"met"en"évidence"des"réponses"à"Aphanomyces0euteiches"différentielles"chez"
certains" mutants" par" rapport" au" sauvage." Suite" à" ce" criblage," les" mutants" du" gène" NFP" et" NF%YA%1"
montrent" des" comportements" extrêmes" par" rapport" au" sauvage." ComptePtenu" de" leurs" rôles" clés"
dans" la" symbiose" rhizobienne" et" de" l’originalité" que" réprésentait" leur" implication" potentielle" dans"
l’immunité,"l’analyse"de"leurs"deux"mutants"a"été"approfondie"et"les"résultats"obtenus"sont"présentés"
dans"les"chapitres"II"et"III."
Grâce" à" l’attractivité" du" thème" de" recherche" présenté" dans" ce" travail," j’ai" eu" l’occasion" d’effectuer"
plusieurs"travaux"en"collaboration"avec"d’autres"chercheurs."Une"partie"des"résultats"obtenus"a"été"
valorisée"dans"des"articles"soumis"où"j’apparais"en"coPauteur."Ces"articles"sont"présentés"en"annexe"
de"ce"travail."
"
74"
"
Résultats et
Discussion
75"
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Résultats et Discussion
"
Chapitre I. Criblage d’une collection
de mutants symbiotiques
Depuis" plus" de" dix" ans," quelques" dizaines" de" gènes" intervenant" dans" la" symbiose" ont" été" identifiés"
chez"Medicago0truncatula"par"une"approche"de"génétique"inverse."Les"mutants"initiaux"proviennent"
d’une" population" mutagénéisée" par" EMS." Si" ces" mutations" sont" souvent" silencieuses," des" mutants"
nuls" de" type" codons" stop" précoces" ou" arborant" des" substitutions" d’acides" aminés" cruciaux" ont" pu"
être" obtenus" parmi" des" dizaines" de" milliers" d’individus." Plus" récemment," des" banques" de" mutants"
insertionnels"reposant"sur"des"transposons"ou"des"délétions"ont"permis"de"générer"des"mutants"nuls"
pour"peu"que"l’événement"se"soit"produit"dans"la"région"codante"des"gènes."Les"premiers"mutants"
symbiotiques" caractérisés" ont" été" obtenus" à" partir" d’une" collection" EMS" dérivée" du" parent" A17P
Jemalong" (lignée" de" référence" chez" Medicago0 truncatula)" tandis" que" les" plus" récents" sont" issus" de"
R108" (Medicago0 tricycla)," une" espèce" proche" de" la" première," qui" a" été" sélectionnée" pour" son"
aptitude"à"la"régénération"in0vitro."Les"deux"lignées"A17"et"R108,"comme"le"montrent"les"résultats"de"
ce"travail,"présentent"une"résistance"quantitative"à" Apahnomyces0euteiches."Ainsi,"en"se"basant"sur"
des" critères" quantitatifs," initialement" utilisés" pour" caractériser" la" résistance" partielle" d’A17" à"
Aphanomyces0 euteiches" (Djébali" et" al.," 2009)," nous" avons," dans" ce" premier" chapitre," comparé" le"
niveau" de" résistance" de" mutants" symbiotiques" à" celui" des" plantes" sauvages" correspondantes" pour"
identifier"les"gènes"en"interconnection"entre"la"symbiose"et"l’immunité"chez"notre"plante"modèle."
Ainsi,"les"mutants"affectés"dans"des"gènes"indispensables"à"la"symbiose"fixatrice"d’azote"(mais"aussi"
parfois"la"mycorhization)"ont"été"choisis"pour"évaluer"leur"rôle"potentiel"sur"le"niveau"de"résistance"à"
Aphanomyces0 euteiches." Ces" mutants" ont" été" séparés" en" deux" groupes," ceux" intervenant" dans" la"
signalisation" symbiotique" et" ceux" intervenant" dans" l’organogénèse" symbiotique" (cf." Introduction)."
Dans" le" premier" groupe," on" trouve" dmi1," dmi2," dmi3" qui" sont" indispensables" à" l’établissement" de"
toutes" les" symbioses" mutualistes" tandis" que" les" autres" sont" spécifiques" de" l’interaction" avec"
Rhizobium0 nfp,0 lyk3,0 nsp10 et0 nsp2." Il" faut" tout" de" même" noter" que" la" découverte" des"
lipochitooligosaccharides" fongiques" a" permis" de" mettre" en" évdence" une" implication" partielle" des"
NSPs"et"de"NFP"dans"l’endosymbiose"fongique"(Figure"1).""
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Figure"1":"Positionnement"des"réseaux"de"gènes"impliqués"dans"la"mise"en"place"des"événements"symbiotiques"d’un"point"
de"vue"génétique,"suite"à"l’analyse"des"phénotypes"de"ces"mutants."Actualisé"à"partir"de"Catoira&et&al.&2000."
Les" derniers" sont" tous" associés" à" des" dysfonctionnements" de" la" symbiose" fixatrice" d’azote" qui" se"
manifestent"par"des"malformations"des"nodosités,"à"l’exception"de" vapyrin"qui"affecte"pour"sa"part"
tous" les" mutualismes." L’approche" de" phénotypage" retenue" visait" à" identifier" rapidement" des"
phénotypes" de" mutants" significativement" différents" du" sauvage." La" grande" majorité" des" mutants" a"
été"testée"sur"au"moins"une"cinquantaine"de"plantes"au"cours"de"deux"à"trois"répétitions"et"beaucoup"
plus"dans"certains"cas"où"le"phénotypage"a"été"approfondi."Ceci"explique"les"écarts"qui"existent"quant"
au"nombre"de"plantes"phénotypées"pour"chaque"mutant"(Figure"2).""
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Figure"2":"Comptage"du"nombre""de"plantes"phénotypées"de"la"collection"de"mutants"symbiotiques.""Un"minimum"de"15"à"
20"plantes"inoculées"par"expérience"ont"été"phénotypées"sur"3"répétitions"(en"fonction"du"stock"de"graines"disponibles)."
Pour"améliorer"la"significativité"des"résultats,"3"à"4"répétitions"supplémentaires"ont"pu"être"rajoutées"sur"certains"mutants"
(notamment"ceux"qui"ont"été"étudiés"plus"en"détail"par"la"suite)."
Le"critère"prioritaire"pour"l’évaluation"du"niveau"de"résistance"est"le"pourcentage"de"brunissement"de"
l’hypocotyle":" celuiPci" s’est" révélé" être" le" plus" significatif" au" travers" des" analyses" réalisées"
antérieurement." De" plus," il" présente" l’avantage" d’être" à" caractère" continu" puisqu’il" repose" sur" la"
mesure"des"tissus"symptomatiques"de"chaque"individu,"permettant"ainsi"un"bon"niveau"de"précision"
quant"à"l’évaluation"de"la"résistance"du"mutant."Si"une"différence"semble"apparaître,"des"répétitions"
supplémentaires" sont" rajoutées" là" où" des" mutants" allèliques" sont" introduits." L’ensemble" de" ces"
mutants" est" issu" du" génotype" A17," parent" partiellement" résistant," à" l’exception" de" VAPYRIN" qui"
appartient"au"fonds"génétique"R108"dont"les"caractéristiques"génétiques"de"résistance"n’avaient"pas"
encore" été" étudiées" avant" ce" travail." Ainsi," la" plupart" des" mutants" contiennent" des" allèles" de" A17"
situés"dans"le"QTL"prAe1"de"résistance"partielle"à"Aphanomyces0euteiches."On"s’attend"donc"à"ce"que"
le"niveau"de"résistance"des"mutants"soit"assez"élevé."Cela"s’est"en"effet"traduit"par"un"faible"taux"de"
mortalité" des" mutants," rendant" ainsi" ce" critère" délicat" à" exploiter" en" raison" du" faible" niveau" de"
significativité." A" titre" de" contrôle," dans" toutes" les" expériences," la" lignée" A17" et" la" plante" sensible"
F83005.5" (Djébali" et" al.," 2009)" ont" été" introduites" dans" chaque" expérimentation" pour" vérifier" le"
niveau"d’agressivité"de"l’inoculum"d’Aphanomyces0euteiches0employé."Les"valeurs"des"répétitions"où"
ce"niveau"montre"une"trop"grande"variation"sur"ces"lignées"témoins"ont"été"éliminées."
"
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A. Phénotype des mutants de la signalisation
symbiotique
"
Un" total" de" neuf" mutants" de" signalisation" symbiotique" affectés" dans" sept" gènes" distincts" ont" été"
utilisés."Il"s’agit"de"simples"mutants"de"chaque"gène"pour""LYK3,"DMI1,"DMI2,"DM3,"NSP1"et"NSP2,"
d’un"couple"de"mutants"allèliques"de"NFP"nommés"nfp%1"et"nfp%2"et"d’un"double"mutant"obtenu"par"
croisement" de" nfp%1" et" lyk3." La" répartition" du" pourcentage" de" brunissement" de" l’ensemble" de" ces"
mutants"par"rapport"à"A17"(63%"de"brunissement""et"8%"de"mortalité"sur"228"plantes)"positionne"ce"
dernier" au" milieu" d’entre" eux" (Figure" 3a)." Les" mutants" dmi1" (64%" de" brunissement" et" 14%" de"
mortalité" à" n=43)," lyk30 (62%" de" brunissement" et" 5%" de" mortalité" à" n=57)" et" nsp1" (61%" de"
brunissement""et"4%"de"mortalité"à"n=57)"sont"indistinguables"du"sauvage"quant"au"premier"critère"
et" présente" des" taux" de" mortalité" qui" restent" proches" de" 10%." De" manière" frappante," toutes" les"
plantes" possédant" une" mutation" du" locus" NFP0 (nfp%1," 70%" brunissement" 18%" de" morts" sur"n=102;"
nfp%2,"69%"brunissement""28%"de"morts"sur"n=116,""et"nfp%1/lyk3"68%"brunissement"et"38%"de"morts"
sur"n=117)"sont"plus"sensibles"à"A.0euteiches"de"même"que"le"mutant"dmi20(67%"de"brunissements"et"
29%" de" morts" sur" n=114)" avec" des" niveaux" de" tissus" symptomatiques" avoisinant" les" 70%" et" un"
doublement"de"la"mortalité."A"l’inverse,"dmi30(58%"de"brunissement"et"11%"de"morts"sur"n=72),"et"
nsp2"(57%"de"brunissement"et"0"mort"sur"n=73)"sont"eux"plus"résistants,"présentant"un"brunissement"
en" deçà" de" celui" d’A17" et," pour" le" second," une" absence" de" plantes" mortes" (Figure" 3b)." Afin" de"
sélectionner"des"mutants"au"phénotype"significatif"pour"la"suite"de"nos"analyses,"une"ANOVA"a"été"
appliquée"à"nos"jeux"de"données."Elle"a"permis"la"détection"de"phénotype"hautement"significatif"pour"
l’ensemble" des" mutants" NFP," " tant" en" ce" qui" concerne" le" brunissement" (pvalue<0,001)" que" les"
plantes" mortes" (pvalue<0,001)." Les" moyennes" associées" à" une" résistance" accrue" ne" se" sont" pas"
révélées"significatives"sur"le"plan"statistique."A"l’issue"de"ce"criblage,"il"a"donc"été"décidé"de"focaliser"
le"travail"sur"les"mutants"NFP,"la"possibilité"d’utiliser"des"mutants"allèliques"renforçant"la"solidité"de"
l’implication"de"ce"gène"dans"la"résistance"à0A.0euteiches"(p<0,001"selon"les"deux"notations)."""
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Figure"3":"Notations"de"symptômes"sur"les"mutants"de"la"signalisation"symbiotiques."Pourcentage"de"tissus"présentant"du"
brunissement"14"jours"après"inoculation"(a)"et""pourcentage"de"plantes"mortes"à"21"jours"après"inoculation"(b)."
Ainsi," il" apparaît" de" manière" surprenante" que" certains" acteurs" de" la" signalisation" symbiotique"
pourraient" partiellement" altérer" la" résistance" de" A17," " alors" que," dans" la" symbiose," tous" ces" gènes"
favorisent" l’établissement" de" l’interaction." A" ce" stade," on" pourrait" donc" émettre" " l’hypothèse" que"
NFP"et"DMI2"puissent"participer"à"la"perception"de"parasites"en"vue"d’activer"la"défense,"tandis"que"
les"NSP2"et"DMI30auraient"plutôt"un"rôle"de"reprogrammation"transcriptionnelle"qui0irait"à"l’encontre"
de"la"réponse"immunitaire"expliquant"la"résistance"accrue"provoquée"par"leur"mutation."La"détection"
d’un" phénotype" distinct" entre" DMI2" et" DMI1" apparaît" intéressante" puisque," dans" la" symbiose," ces"
deux" gènes" sont" positionnés" de" manière" indissociée" en" amont" du" calcium" spiking" nécessaire" à" la"
nodulation"et"à"la"mycorrhization."Ainsi,"ce"test"avec"A.0euteiches"découple"pour"la"première"fois"le"
rôle" de" DMI1" et" DMI2" dans" une" interaction" plantePmicroorganisme," positionnant" DMI2" avec" NFP"
dans" une" voie" de" signalisation" commune" au" mutualisme" et" à" l’immunité" et" DMI1" dans" une" voie"
spécifique"des"associations"bénéfiques."L’éventualité"d’un"tel"positionnement"de"DMI2"en"amont"de"
DMI1"paraît"plausible"puisque"le"premier"code"un"récepteur"membranaire,"tandis"que"le"second"est"
un" canal" ionique" positionné" dans" les" membranes" d’organites" intracellulaires." En" l’absence" de"
connaissances" à" propos" du" ou" des" messagers" secondaires" reliant" DMI2" à" DMI1,0 il" reste" difficile" de"
commenter" davantage" ces" données." En" dépit" d’un" phénotype" qui" nécessite" d’être" clarifié" par" des"
répétitions" supplémentaires," le" mutant" du" gène" DMI3" apparaît" plus" résistant" à" la" maladie." Cette"
observation"n’est"pas"nécessairement"en"contradiction"avec"la"séparation"des"voies"de"l’immunité"et"
du"mutualisme"en"aval"de"DMI2."En"effet,"plus"de"la"moitié"des"poils"absorbants"du"mutant"dmi3"sont"
capables"d’induire"le"calcium"spinking"en"présence"de"doses"de"facteurs"NOD"aussi"faibles"que"10P13"
molaire,"là"où"chez"la"plante"sauvage,"moins"de"la"moitié"de"ces"mêmes"cellules"répondent"dès"10P12"
molaire"(G."E."Oldroyd,"Mitra,"Wais,"&"Long,"2001).""Cette"observation"conduit"les"auteurs"à"proposer"
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l’existence"d’une"boucle"de"régulation"négative"de"la"perception"des"facteurs"NOD"qui"serait"induite"
par"DMI3"et"donc"supprimée"dans"le"mutant"et"permettrait"une"activité"accrue"des"acteurs"en"amont"
de"ce"gène."Transposée"à"notre"cas"de"figure,"la""mutation"de"dmi3""pourrait"lever"les"inhibitions"qui"
concernent"NFP"et"DMI2"dans"la"symbiose"et"permettre"une"meilleure"signalisation"de"l’immunité"par"
des"voies"alternatives"à"DMI3."Indépendamment"de"toute"signalisation"symbiotique,"les"gènes"NSP1"
et"NSP2"sont"nécessaires"à"la"synthèse"de"strigolactones"(W."Liu"et"al.,"2011)."Ce"constat"suggère"que"
la"réduction"sensible"des"symptômes"causés"par"Aphanomyces0euteiches"dans"nsp2"pourrait"être"liée"
au" défaut" de" ce" métabolisme" et" donc" que" ces" molécules" pourraient" favoriser" la" croissance" de"
l’oomycète"comme"elle"favorise"celle"des"champignons"symbiotiques"ou"des"plantes"parasites."Dans"
cette"perspective,"nous"avons"observé"une"induction"forte"et"précoce"par"A.0euteiches"d’un"marqueur"
requis"pour"l’établissement"de"la"mycorhization"nommé"MtDxs20(Mtr.43585.1.S1_at)"qui"code"une"1P
deoxyPDPxylulose"5Pphosphate"synthase"impliquée"dans"la"voie"des"apocaroténoïdes"dont"découlent"
les"strigolactones"(Floss"et"al.,"2008";"Floss""et"al.,"2008";"Walter"et"al.,"2010)."
"
B. Niveau de résistance des mutants de l’organogénèse
symbiotique et des mutants hormonaux
"
Une" autre" série" d’expérimentations" a" été" lancée" pour" cette" deuxième" collection" de" mutants." Cette"
fois,"neuf"mutants"ont"été"criblés,"dont""deux"mutants"allèliques"du"récepteur"aux"cytokinines"CRE1"
et"le"mutant"issu"de"R108"nommé"vapyrin."Cette"fois,"A17"a"été"noté"à"49%"de"brunissement"et"8%"de"
morts"sur"342"plantes."Des"phénotypes"très"voisins"ont"été"obtenus"sur"la"plupart"des"mutants" rpg"
(52%"de"brunissement"17%"de"morts"sur"n=115)"ou"encore0sunn"(51%"de"brunissement"4%"de"morts"
sur"n=51)"et"lin"(50%"de"brunissement"2%"de"morts"sur"n=43)."D’autres"semblent"plus"résistants"mais"
n’ont"malheureusement"pas"pu"être"testés"en"effectif"suffisant"en"raison"notamment"de"leur"faible"
qualité" de" germination" tels" que" latd" (46%" de" brunissement" et" 0%" morts" sur" n=19)," efd" (47%" de"
brunissement"et"0%"de"morts"sur"n=12)"ou"cre1%20(46%"de"brunissement""et"0%"de"morts"sur"n=15)."
Enfin" les" mutants" nf%ya1%1" (38%" de" brunissement" et" 2%" de" morts" sur" n=149)" et" cre1%10 (43%" de"
brunissement"et"0%"de"morts"sur"n=83)"ont"pu"être"identifiés"comme"clairement"plus"résistants"au"
travers" des" ANOVAs" réalisées" (p<0,001" pour" nf%ya1%1" sur" les" deux" critères" et" p<0,05" pour" cre1%
1)(Figure" 4a" et" 4b)." Enfin," aucune" différence" notable" n’a" été" détectée" entre" vapyrin" (31%" de"
brunissement" de" 1%" morts" sur" n=77)" et" le" sauvage" R108" (34%" de" brunissement" de" 4%" morts" sur"
n=294)."
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Figure"4":"Notation"de"symptômes"sur"les"mutants"de"l’oranogénèse"symbiotique"en"présence"d’A.euteiches.""
Pourcentage"de"tissus"présentant"du"brunissement"14"jours"après"inoculation"(a)."Pourcentage"de"plantes"mortes"à"21"jours"
après"inoculation"(b)."
Il"est"à"noter"que"contrairement"au"cas"précédent,"ces"gènes"ne"sont"pas"impliqués"dans"une"cascade"
d’événements"connus":"ainsi,"il"n’est"pas"possible"de"discuter"d’éventuelles"interconnexions"entre"les"
phénotypes."Les"résultats"sur"CRE1"apparaîtront"dans"un"article"sur"un"ensemble"de"phénotypes"non"
symbiotiques" contrôlés" par" CRE1," dans" le" cadre" d’une" collaboration" avec" Florian" Frugier" (Gif" sur"
Yvette)." Phénotypes" et" analyses" de" gènes" en" relation" avec" les" cytokinines" ont" été" rassemblés" dans"
une" planche" en" annexe." Les" liens" potentiels" entre" CRE1" et" d’autres" gènes" symbiotiques" sont"
cependant" encore" à" traiter." La" régulation" des" gènes" NSP1" et" NSP2" au" cours" de" l’organogénèse"
nodulaire" étant" sous" le" contrôle" de" CRE1,0 il" est" envisageable" qu’une" relation" épistatique" entre" ces"
fonctions"soit"à"l’origine"des"phénotypes"de"résistances"similaires"observés"entre"mutant"de"CRE1"et"
mutants" NSPs.0 Dans" le" même" ordre" d’idée," EFD" (transcriptionnellement" induit" par" Aphanomyces0
euteiches" dans" nos" données" transcriptromiques," cf." chapitre" suivant)" en" contrôlant" RR4" qui" est" un"
régulateur"de"la"réponse"aux"cytokinines"pourrait"aussi"intégrer"ce"réseau"de"gènes""(GonzalezPRizzo"
et"al.,"2006";"Plet"et"al.,"2011)."Bien"que"ces"données"nécessitent"des"validations"expérimentales,"il"
apparaît," au" sein" de" nos" analyses" de" transcriptomes," que" deux" autres" récepteurs" de" la" famille" de"
CRE1"et"quatre"des"septs"régulateurs"de"la"famille"de"RR4"situés"en"aval"dans"la"signalisation"soient"
transcriptionnellement"réprimés"en"présence"d’Aphanomyces0euteiches0(cf."Annexe"I.C)."Ceci"est"en"
concordance" avec" la" répression" de" NSP2" observée" sur" nos" puces" qui" est" une" cible" directe" des" RRs"
(Ariel"et"al.,"2012)."Ainsi,"il"paraît"probable"que"ces"acteurs"de"signalisation"liés"aux"cytokinines"soient"
défavorables" à" la" résistance" à" la" maladie," les" mutants" affectés" dans" cette" voie" exhibant" par"
conséquent" une" meilleure" tolérance" au" parasite." L’analyse" plus" détaillée" du" mutant" nf%ya%1%10 sera"
présentée"dans"le"troisième"chapitre"de"cette"partie."
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C. Développement des racines des mutants au cours de
l’invasion par Aphanomyces euteiches
"
Si" l’infection" par" Aphanomyces0 euteiches" implique" l’apparition" de" tissus" symptomatiques" et" de"
plantes"mortes"qui"peuvent"être"analysés"pour"déterminer"différents"niveaux"de"résistance"partielle,"
le" parasite" influence" aussi" des" phénotypes" développementaux" de" l’hôte." A" titre" d’exemple," la"
formation"de"racines"latérales"est"discriminante"entre"A17,"où"elles"sont"induites"après"infection,"et"
F83005.5"où"elles"sont"réprimées"(Djébali"et"al.,"2009)."
"
1. Mutants de la signalisation symbiotique
Ainsi,"en"parallèle"des"notations"symptomatiques,"le"nombre"de"racines"latérales"par"plante"d’A17"et"
des" mutants" symbiotiques" en" condition" contrôle" (n=30" pour" tous" les" génotypes)" et" inoculée" a" été"
mesuré"au"bout"de"21"jours"de"culture"in0vitro"(Figure"5)."En"condition"contrôle,"4,4"racines"latérales"
par"plante"ont"été"observées"chez"A17,"le"maximum"ayant"été"observé"chez"lyk300avec04,60racines"par"
plante"et"le0minimum0chez0nfp%10avec"2,6."En"condition"d’inoculation,"sept"racines"par"plantes"ont"été"
observées"chez"A17,"transcrivant"l’induction"de"racines"latérales"déjà"décrites"antérieurement,"tandis"
que" chez" F83," ce" nombre" d’organes" latéraux" passe" de" 3,3" chez" les" plantes" saines" à" 1,3" chez" les"
plantes"infectées."De"manière"frappante,"aucun"des"mutants"de"la"signalisation"symbiotique"n’arbore"
plus"de"4"racines"en"moyenne"en"présence"d’Aphanomyces0euteiches."Ces"jeux"de"données"ont"été"
étudiés" au" travers" d’un" test" non" paramétrique" de" rang" de" type" Wilkoxon" utilisé" pour" des"
comparaisons"deux"à"deux"entre"A17"et"les"autres"génotypes,"en"condition"contrôle"puis"en"condition"
d’inoculation." Des" différences" significatives" ont" été" observées" dans" les" conditions" contrôles" entre"
A17" et" F83" (p<0,001)" et" A17" et" nfp%1" (p<0,01)." En" condition" d’infection," l’induction" de" racines"
latérales"rend"A17"significativement"différente"de"toutes"les"autres"plantes"(p<0,001)."
83"
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Figure" 5" :" Comptage" des" racines" latérales" 21" jours" après" mise" en" culture" in0 vitro,0 en" condition" " contrôle" (Mock," noir)" ou"
après"inoculation"avec"A.0euteiches"(Ae,"gris)."30"plantes"de"chaque"génotype"ont"été"étudiées"en"conditions"contrôles,"en"
plus" des" plantes" inoculées" déjà" utilisées" pour" les" notations" de" symptômes." Un" test" de" rang" de" type" wilkoxon" a" été"
appliqué""entre"chaque"génotype"et"A17"pour"les"conditions"contrôles"et"inoculées."
En"plus"de"conduire"aux"symbioses"mutualistes,"la"voie"de"signalisation"symbiotique"a"été"associée"à"
une" modification" du" nombre" de" branchements" de" l’appareil" racinaire," en" réponse" au"
lipochitooligosaccharides" fongiques" et" bactériens" (Maillet" et" al.," 2011)." L’altération" globale" de"
l’induction" de" racines" secondaires" en" réponse" à" Aphanomyces0 euteiches" pour" ces" mutants" de"
signalisation"suggère"que"cette"voie"joue"également"un"rôle"dans"la"réponse"d’A17"à" Aphanomyces0
euteiches."Comme"ces"mutants"symbiotiques"ne"présentent"pas"de"réductions"flagrantes"du"nombre"
de"racines"latérales"en"condition"contrôle,"il"est"probable"que"le"rôle"de"cette"voie"dans"l’émergence"
de"nouvelles"racines"soit"activé"par"la"présence"du"parasite.""
2. Mutants hormonaux et d’organogénèse nodulaire
Les" mutants" d’organogénèse" symbiotique" et" hormonaux" présentant" une" résistance" supérieure" au"
sauvage" ont" aussi" vu" leur" architecture" racinaire" étudiée." Dans" nos" conditions" de" culture," latd"
présente" une" absence" de" racines" latérales" et" une" coloration" plus" sombre" des" tissus" infectés." Cette"
dernière"observation,"typique"de"la"résistance,"est"corrélée"aux"valeurs"de"symptômes"notés"pour"ce"
génotype" qui" sont" inférieures" à" celles" décrites" pour" A17." En" dépit" d’un" faible" nombre" de" plantes"
analysées," il" est" aussi" intéressant" de" noter" que" les" mutants" efd" déploient" une" forte" croissance" des"
84"
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racines" principales" et" secondaires," en" dépit" du" développement" concomittant" des" tissus"
symptomatiques." Enfin," les" mutants" du" gène" CRE10 semblent" présenter" une" accumulation"
sensiblement" plus" grande" du" nombre" de" racines" secondaires" au" cours" de" l’infection" par" rapport" à""
A17," bien" que" l’induction" de" racines" latérales" semble" avoir" été" mauvaise" dans" cette" série"
expérimentale"(4,3"racines"pour"les"mutants"contre"3,2"chez"A17";""p<0,05";"Cf."Annexe"I.C)(Figure"6)."
Le" mutant" nf%ya1%1" présente" aussi" des" originalités" de" développement" racinaire" en" réponse" à"
Aphanomyces0euteiches"qui"seront"développées"dans"le"troisième"chapitre"des"résultats."
"
Figure"6":"Phénotypes"21"jai"in0vitro0chez"les"mutants"d’organogénèse"montrant"une"résistance"accrue.""
De" manière" intéressante," les" mutants" d’organogénèse" symbiotiques" présentant" une" résistance"
accrue" à" la" maladie" déploient" aussi" des" modifications" de" leurs" appareils" racinaires." latd" est" déjà"
connu"pour"être"affecté"dans"l’initiation"d’organes"latéraux"et"l’élongation"racinaire"(Veereshlingam"
et" al.," 2004;" " Bright" et" al.," 2005" ;" Liang" et" al.," 2007";" Ding" et" al.," 2008)." Dans" le" même" temps,"
l’expression" de" LATD" est" induite" par" " Aphanomyces" euteiches" selon" nos" puces" et" la" voie" de"
signalisation" de" l’acide" abscissique" est" connue" pour" jouer" un" rôle" négatif" sur" la" résistance" à"
Aphanomyces0 euteiches" (Colditz" et" al.," 2007)." Ainsi," le" phénotype" observé" chez" latd" est"
potentiellement" une" balance" entre" deux" phénomènes" antagonistes," d’une" part" le" défaut" de"
développement"racinaire"et,"d’autre"part"l’immunité"accrue"provoquée"par"le"dysfonctionnement"de"
cette" signalisation" hormonale." Le" blocage" de" la" voie" cytokinine" conduit" à" une" induction" de" la"
formation" de" racines" secondaires" chez" Medicago0 truncatula" (GonzalezPRizzo" et" al.," 2006)":" ainsi" le"
phénotype" observé" pour" les" mutants" de" CRE10 pourrait" découler" du" comportement" de" la" plante"
indépendamment"de"l’invasion"par"le"parasite."Malheureusement,"cela"n’a"pu"être"vérifié,"faute"de"
plantes"en"nombre"suffisant"pour"faire"des"tests"sans"Aphanomyces0euteiches."
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D. Cytologie de l’invasion par Aphanomyces euteiches
des mutants symbiotiques
"
Bien" qu’il" soit" difficile" d’étendre" le" large" criblage" phénotypique" entrepris" du" niveau" visuel" à" la"
microscopie,"des"observations"de"coupes"transversales"chez"des"plantes"envahies"par"Aphanomyces0
euteiches"depuis"21"jours"ont"été"entreprises."Si,"pour"les"mutants"d’intérêts"nfp%1,"nfp%2"et"nf%ya%1%1,"
des" échantillons" plus" nombreux" et" des" cinétiques" détaillées" ont" été" réalisés" (cf," chapitre" suivant),"
nous"avons"aussi"réalisé"quelques"observations"dans"les"autres"mutants,"à"la"recherche"d’éventuelles"
caractéristiques"particulièrement"frappantes."En"premier"lieu,"les"observations"des"lignées"parentales"
ont" été" reproduites." A17" présente" une" colonisation" intermédiaire" du" cortex" qui" est" sensiblement"
supérieure"à"celle"de"R108,"tandis"que"F83"est"très"fortement"colonisée"tant"au"niveau"de"l’épiderme,"
que"du"cortex"et"de"la"vasculature."Les"renforcements"du"cylindre"central"qui"se"traduisent"par"une"
autofluorescence"rougePorangé"et"des"couches"surnuméraires"du"péricycle"ont"été"observés"dans"les"
mêmes"proportions"chez"A17"et"R108,"mais"sont"absents"de"F83005.5"(Figure"7a).""
En"ce"qui"concerne"les"mutants"de"la"signalisation"symbiotique,"il"apparaît"que,"comme"observé"au"
niveau"visuel,"dmi1"est"très"semblable"à"A17,"tout"comme"nsp1"et"nsp2,"avec"une"densité"mycélienne"
moyenne" dans" le" cortex" et" une" protection" de" la" stèle." Les" mutants" lyk3" et" dmi3" paraissent" moins"
colonisés,"certaines"cellules"n’étant"en"contact"avec"aucun"hyphe"et"les"divisions"péricycliques"étant"
particulièrement" visibles." Enfin," le" mutant" dmi2," s’il" n’est" pas" colonisé" au" niveau" des" tissus"
vasculaires," présente" en" revanche" des" tissus" corticaux" totalement" macérés" et" peu" de" divisions"
péricycliques," le" cylindre" restant" sans" doute" protégé" par" des" renforcements" pariétaux"
particulièrement"fluorescents"(Figure"7b)."
Pour" ce" qui" est" des" mutants" d’organogénèse" et" des" voies" hormonales," tous" exhibent" " une"
colonisation"du"cortex"semblable"à"A17,"à"l’exception"de"pub%1"qui"paraît"fortement"colonisé,"ce"qui"
est""en"contradiction"avec"les"symptômes"visuels"et"appelle"à"des"observations"supplémentaires."La"
protection"du"cylindre"central"est"aussi"semblable"à"A17"(Figure"7c)."
En"conclusion,"ces"observations"n’ont"pas"permis"de"mettre"en"évidence"des"altérations"originales"du"
mode" de" colonisation" des" tissus" des" hôtes" mutants." On" notera" au" passage" que" la" faculté"
d’Aphanomyces0euteiches0à"pénétrer"dans"les"tissus"des"mutants"nfp"nous"a"confortés"dans"le"choix"
de" focaliser" nos" efforts" sur" ces" plantes." Certaines" discordances" apparaissant" entre" les" observations"
visuelles" et" cytologiques," il" paraît" opportun" de" compléter" les" observations" microscopiques" dans"
86"
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l’avenir" pour" explorer" plus" avant" les" gènes" «"douteux"»," notamment" les" NSPs." Dans" le" but" de"
poursuivre"l’étude"des"connexions"symbiosePimmunitéPdéveloppement,"un"autre"point"intéressant"à"
travailler" dans" le" futur" serait" l’existence" d’un" lien" éventuel" entre" les" divisions" péricycliques" en"
réponse"à"A.0euteiches"et"au"cours"de"la"formation"des"primordia"nodulaires"ou"racinaires."L’influence"
de" traitement" avec" des" lipochitooligosaccharides" sur" ces" procédés" pourrait" aussi" être" étudiée,"
d’autant" que" NFP" est" crucial" pour" l’initiation" du" primordium" nodulaire" (Rival" et" al.," 2012)." Il" est"
probable"que"la"génétique"inverse"ne"soit"pas"la"plus"appropriée"pour"répondre"à"ces"questions,"aussi"
des" analyses" génétiques" de" type" GWAS" (Genome" Wide" Association)" sontPelles" en" cours" au" sein" de"
l’équipe."
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Figure"7:"Observation"cytologique"de"coupes"transversales"21"jai"des"différents"mutants"inoculés"par"A.0euteiches0qui"0est"
marqué"en"vert"à"la"WGAPFITC."Lignées"sauvages"(a),"mutants"de"signlalisation"(b)"et"d’organogénèse"symbiotique"(c).(Barre"
="100µm)"
88"
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E. Conclusion du chapitre
L’objectif"de"la"première"partie"de"ma"thèse"était"de"rechercher"l’implication"éventuelle"de"gènes"clés"
pour" l’établissement" des" symbioses" mutualistes," dans" la" résistance" ou" la" sensibilité" de" Medicago0
truncatula" à" Aphanomyces0 euteiches." Dans" cette" optique," une" quinzaine" de" mutants" symbiotiques"
ont"été"phénotypés"avec"Ae."L’analyse"de"l’interaction"de"ces"mutants"avec"l’oomycète"a"été"guidée"
par"les"connaissances"déjà"acquises"sur"les"lignées"A17"et"F83005.5"qui"développent"respectivement"
des" réponses" de" résistance" partielle" et" de" sensibilité." Ainsi," en" comparant" les" phénotypes" des"
mutants" à" ceux" des" deux" accessions," nous" avons" pu" détecter" une" éventuelle" contribution" du" gène"
analysé"dans"le"niveau"de"résistance"de"la"plante."Des"essais"de"phénotypages"reposant"sur"la"mesure"
des"symptômes"développés"et"la"mortalité"des"mutants"inoculés,"ont"révélé"le"rôle"positif"joué"par"
NFP"et"DMI2"dans"la"résistance"tandis"que"CRE1"et"NF%YA1"ont"un"rôle"négatif."Les"résultats"obtenus"
avec" les" mutants" dmi3," nsp2," efd" et" latd" semblent" aussi" indiquer" une" implication" possible" de" ces"
gènes" dans" la" réponse" de" la" plante" à" Ae." Des" validations" supplémentaires" utilisant" plus" de" plantes"
pour"mieux"évaluer"la"significativité"des"différences"observées"sont"encore"nécessaires"de"même"que""
des"analyses"cytologiques"permettant"également"de"mieux"préciser"le"rôle"de"ces"gènes."Les"résultats"
obtenus" dans" ce" chapitre" montrent" donc" sans" conteste" que" des" gènes" qui" avaient" été" identifiés" et"
associés" de" manière" très" spécifique" à" différentes" phases" d’établissement" des" interactions"
symbiotiques"interviennent"également"dans"la"modulation"de"l’immunité"végétale."Dans"les"chapitres"
qui"suivent,"nous"avons"approfondi"le"rôle"d’un"gène"qui"est"un"régulateur"positif"(NFP)0et"celui"d’un"
autre"(NF%YA1)"qui"est"impliqué"dans"la"sensibilité"de"la"plante."
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Chapitre II. Caractérisation du rôle de
NFP dans l’immunité
"
Les" deux" mutants" nfp%1" et" nfp%2" ont" montré" des" données" de" phénotypage" des" symptômes"
(brunissement" et" mort)" qui" sont" significativement" supérieures" à" celles" d’A17" (fonds" génétique"
sauvage)"après"inoculation"avec"Aphanomyces0euteiches0(cf"chapitre"1)."Compte"tenu"de"l’association"
d’éléments"originaux":""la"fonction"initiale"de"NFP"(récepteur"potentiel"des"Lipochitooligosaccharides"
bactériens"aussi"appelés"facteurs"NOD),"la"structure"de"la"paroi"d’Aphanomyces0euteiches0 (riche"en"
chitosaccharides" atypiques)" et" la" sensibilité" des" mutants" nfp" à" Aphanomyces0 euteiches," un" projet"
financé" par" l’ANR" et" nommé" Sympasignal" a" été" élaboré." Ce" chapitre" reprend" les" résultats" que" j’ai"
obtenus" dans" ce" cadre" et" qui" avaient" pour" but" de" préciser" le" rôle" de" NFP" dans" l’immunité" de"
Medicago0truncatula."
La"première"partie"de"ce"chapitre"est"constituée"par"l’article"dans"lequel"la"caractérisation"de"la"plus"
grande"sensibilité"des"mutants"a"été"complétée"par"des"méthodes"moléculaires"et"cytologiques."Les"
mutants" nfp" ont" été" également" confrontés" à" un" champignon" pathogène" pour" voir" si" l’on" pouvait"
généraliser" le" rôle" de" NFP" à" d’autres" parasites." Enfin," l’article" relate" également" des" analyses"
transcriptomiques" visant" à" mieux" comprendre" quels" sont" les" mécanismes" moléculaires" sous" la"
dépendance"de"NFP."
La" deuxième" partie" de" ce" chapitre" a" pour" but" d’élargir" la" compréhension" du" rôle" de" NFP," en"
complétant"les"analyses"transcriptomiques"qui"apparaissent"dans"l’article,"en"les"recoupant"avec"des"
comparaisons"d’autres"transcriptomes"disponibles"dans"la"littérature"scientifique"et"reposant"sur"les"
mêmes"lignées"de"Medicago0truncatula"et"les"mêmes"outils"d’analyses"transcriptomiques"utilisés"au"
cours" d’autres" traitements." Pour" savoir" si" NFP" a" aussi" un" rôle" dans" la" perception" d’Aphanomyces0
euteiches,"des"expériences"évaluant"la"production"de"ROS"des"mutants"en"présence"de"PAMPs"issus"
d’Aphanomyces0euteiches"ou"mélangés"avec"des"facteurs"NOD"seront"présentées."
"
A. Article
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94"
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B. Analyses transcriptomiques complémentaires
"
La"publication"récente"de"l’étude"de"transcriptome"de"Medicago0truncatula"en"réponse"aux"différents""
lipochitooligosaccharides" identifiés" dans" la" mise" en" place" des" associations" symbiotiques"
mycorhiziennes" par" Czaja" et" ses" collègues" repose" sur" les" mêmes" puces" Affymetrix" " que" celles" que"
nous" avons" utilisées," permettant" ainsi" une" comparaison" des" réseaux" de" gènes" régulés" entre" ces"
expériences" et" les" nôtres." Les" conditions" de" culture" des" plantes" et" les" modalités" d’échantillonnage"
différentes" de" celles" que" nous" avons" utilisées" impliquent" une" certaine" prudence" dans" l’analyse."
Toutefois,"un"traitement"eau"ayant"été"utilisé"comme"contrôle"dans"nos"propres"expériences"et"celles"
de" Czaja" et0 al.," une" comparaison" est" possible" entre" ces" jeux" de" données" représentant" les" gènes"
régulés" suite" à" l’inoculation" par" Aphanomyces0 euteiches" et" le" traitement" avec" les"
lipochitoologisaccharides" fongiques." Notre" travail" a" été" facilité" par" le" défaut" total" de" réponses"
transcriptionnelles" des" mutants" nfp" aux" lipochitooligosaccharides." Ainsi," les" auteurs" décrivent"
l’ensemble" des" réponses" transcriptionnelles" provoquées" par" les" lipochitooligosaccharides" comme"
strictement"dépendantes"de"NFP."En"utilisant"un"clustering"hiérarchique,"nous"avons"pu"comparer"la"
régulation" du" transcriptome" de" A17" et" de" nfp%2" en" réponse" à" Aphanomyces0 euteiches" avec" la"
régulation"du"transcriptome"de"A17"en"réponse"aux"facteurs"NOD"et"aux"facteurs"MYC"sulfatés,"non"
sulfatés"et"mélangés."Seuls"les"gènes"régulés"par"Aphanomyces0euteiches0d’une"part,"et"au"moins"au"
cours" d’un" des" traitements" LCOs" d’autre" part," ont" été" considérés" (valeur" seuil" d’induction" ou"
répression" de" 1" en" logarithme" de" base" 2" pour" Aphanomyces0 euteiches" et" 0,6" pour" les"
lipochitooligosaccharides" conformémént" au" choix" de" Czaja" et0 al.)." Ainsi," parmi" les" 750" gènes"
répondant" aux" critères" précédents" (Figure" 8)," un" cluster" de" gènes" induits" dans" A17," mais" pas" dans"
nfp%2" en" présence" d’Aphanomyces0 euteiches0 mais" aussi" au" cours" de" traitements" LCOs" a" pu" être"
identifié."Ils"sont"induits"chez"les"plantes"traitées"avec"les"MycLCO"sulfatés"(uniquement),"six"heures"
après" traitement," ce" qui" correspond" au" stade" d’activité" biologique" maximale" de" ces" composés." De"
façon"surprenante,"il"renferme"24"gènes"dont"12"sont"associés"au"métabolisme"lipidique"(Table"1)."Ce"
groupe" de" marqueurs" transcriptionnels," génétiquement" " dépendants" de" la" mutation" de" NFP,"
constitue"un"point"de"départ"vers"le"décryptage"de"voies"de"signalisations"contrôlées"par"NFP"qui"sont"
impliquées"dans"la"signalisation"mutualiste"et"celle"de"l’immunité."Il"pourrait"aussi"constituer"un"outil"
vers"la"recherche"des"signaux"produits"par"Aphanomyces0euteiches"dont"la"perception"passerait"par"
NFP." Le" recouvrement" du" transcriptome" entre" la" réponse" à" l’oomycète" et" celle" liée" au" traitement"
avec" les" MycLCOs" sulfatés" permet" d’envisager" une" recherche" de" tels" composés" ou" de" composés"
voisins"chez"Aphanomyces0euteiches.""
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"
"
Figure"8":"Clustering"hiérarchique"des"gènes"régulés"24"heures"après"inoculation"avec"Aphanomyces0euteiches"et"au"moins"
un" traitement" lipochitooligosaccharide" (s=" MycLCO" sulfaté," ns=" MycLCO" non" sulfaté" et" ns/s=" mélange" des" deux" types" de"
MycLCOs)." Les" gènes" induits" de" manière" dépendante" à" NFP" par" Aphanomyces" euteiches" et" les" traitements" de" 6" heures"
renfermant"des"MycPLCO"sulfatés"sont"encadrés"en"jaune."
Table"1":"Groupe"de"gènes"associés"au"métabolisme"lipidique"parmi"les"gènes"d’intérêt"sélectionnés"sur"la"figure"8."
Le"fait"que"les"marqueurs"communs"à"ces"deux"expériences"de"transcriptomes"soient"associés"à"des"
métabolismes" lipidiques" posent" des" questions" sur" les" rôles" joués" par" de" tels" acteurs" dans" ces"
interactions."Pour"l’heure,"le"peu"de"données"disponibles"pour"la"symbiose"suggère"que"ces"fonctions"
pourraient" être" associées" à" la" signalisation" de" la" nodulation" et" au" processus" de" formation" des"
cordons"infectieux"dans"les"poils"absorbants"(den"Hartog"et"al.,"2001)(den"Hartog"et"al.,"2003)(Pingret"
et"al.,"2004)."Du"côté"de"la"défense,"le"métabolisme"des"acides"gras,"notamment"les"VLCFA"(Very"Long"
103"
"
Chain"Fatty"Acid),"sont"connus"pour"être"des"acteurs"de"la"réponse"hypersensible,"ouvrant"la"porte"à"
un"rôle"de"ces"gènes"dans"l’immunité"(Berkey,"Bendigeri,"&"Xiao,"2012)(Canonne,"FroidurePNicolas,"&"
Rivas," 2011)." Une" nouvelle" hypothèse" particulièrement" attrayante" est" apparue" ces" dernières"
semaines" avec" l’identification" du" gène" RAM2" de" Medicago0 truncatula" qui" est" requis" pour" la"
colonisation"de"la"plante"par"les"champignons"mycorhiziens"mais"aussi"un"oomycète,"Phythophthora0
palmivora."CeluiPci"code"une"glycerolP3Pphosphate"acyl"transferase"(GPAT)"en"charge"de"la"synthèse"
de" monomères" de" cutine." L’apport" exogène" de" ces" comosés" étant" suffisant" pour" rétablir" la"
colonisation"de"la"plante"par"les"deux"microbes,"il"est"proposé"que"la"cutine"joue"un"rôle"de"signal"sur"
le"microbe"pour"activer"la"formation"des"structures"de"pénétration"appelées"appressoria"(E."Wang"et"
al.,"2012)."Si"RAM2"n’est"pas"régulé"en"présence"d’A.0euteiches"selon"nos"données"transcriptomiques,"
que" ce" soit" à" 1" ou" 6" jours" après" inoculation," un" gène" sélectionné" dans" les" analyses" précédentes"
appartient" à" sa" famille" (Mtr.11816.1.S1_at)." Le" métabolisme" des" cutines" pourrait" donc" être" mis" en"
jeu" au" cours" de" l’infection" par" A.0 euteiches." Cette" perspective" est" renforcée" par" la" forte" induction"
transcriptionnelle" du" gène" requis" pour" la" mycorhization" STR2" (Zhang" et" al.," 2010";" Gutjahr" et" al.,"
2011)" à" 6" jours" après" inoculation" avec" A.0 euteiches" puisqu’il" code," selon" Wang" et" ses" collègues," un"
transporteur" présentant" des" similarités" avec" des" transporteurs" de" lipides." A" notre" connaissance,0 A.0
euteiches"ne"développe"pas"de"structure"de"pénétration"à"la"surface"de"la"racine,"ce"qui"questionne"
sur"le"rôle"que"pourraient"jouer"les"signaux"cutiniques"dans"cette"interaction."
"
C. Vers l’identification de signaux d’Aphanomyces
euteiches perçus par NFP
"
NFP"étant"un"récepteur"potentiel"de"signaux,"une"démarche"a"été"initiée"pour"essayer"d’identifier"le"
ou" les" composés" d’Aphanomyces0 euteiches0 qui" seraient" reconnus" de" manière" NFP" dépendante." Le"
travail" a" été" réalisé" dans" l’équipe" en" collaboration" avec" Amaury" Nars" (étudiant" en" thèse" sous" la"
direction" d’Arnaud" Bottin)." Ainsi" l’ensemble" des" travaux" de" biochimie" et" la" culture" in0 vitro"
d’Aphanomyces0euteiches"ont"été"réalisés"par"Amaury"Nars."A"titre"personnel,"je"me"suis"investi"dans"
l’étude" de" l’activité" biologique" des" préparations" générées.0 Dans" un" premier" temps," des" extraits"
d’Aphanomyces0euteiches"issus"de"sa"paroi"et/ou"du"filtrat"de"culture"ont"été"récupérés"et"en"partie"
purifiés." Dans" un" deuxième" temps," ces" extraits" ont" été" mis" en" contact" avec" des" racines" de" plantes"
entières" dans" un" milieu" liquide" en" présence" des" extraits." Puis," les" " espèces" réactives" de" l’oxygène"
(Reactive"Oxygen"Scpecies,"ROS)"ont"été"mesurées"chez"le"sauvage"et"les"mutants"nfp."Comme"tous"
les" microorganismes," Aphanomyces0 euteiches" contient" des" MAMPs." La" présence" de" NP
104"
"
Acétylglucosamine" non" cristalline" dans" sa" paroi" est" dans" des" proportions" supérieures" à" ce" qui" est"
observé"chez"les"champignons"et"la"plupart"des"autres"oomycètes,"soulignant"ainsi"un"aspect"original"
de" cet" oomycète" avec" la" présence" de" ces" motifs" originaux" possiblement" perçus" par" l’hôte"
(Badreddine"et"al.,"2008)."Un"effort"important"a"été"consacré"à"l’étude"des"composés"issus"de"la"paroi"
du"microbe"au"sein"de"l’équipe."Des"travaux"d’enrichissement"de"ces"composés"pariétaux"à"NAG"ont"
permis"d’obtenir"des"glucanes"issus"des"parois"du"mycélium,"pour"ensuite"les"tester"dans"le"bioessai"
ROS." La" perception" des" composés" issus" d’Aphanomyces0 euteiches0 par0 NFP0 étant" centrale" pour"
l’hypothèse"de"ce"travail,"nous"avons"travaillé"conjointement"avec"Amaury"sur"l’étude"des"composés"
libérés" naturellement" par" Aphanomyces0 euteiches" au" cours" de" sa" croissance." Il" a" été" notamment"
entrepris" de" caractériser" l’activité" biologique" de" filtrats" d’Aphanomyces0 euteiches0 obtenus" dans" un"
milieu" de" culture" liquide" et" synthétique," afin" de" récupérer" les" signaux" produits" et" d’éviter" l’apport"
d’éliciteurs" exogènes" tels" que" ceux" qui" peuvent" être" contenus" dans" les" milieux" complexes." Les"
travaux" réalisés" pour" la" mise" au" point" du" bioessai" permettant" d’évaluer" l’activité" de"
chitooligosaccharides"ont"été"relatés"dans"l’article"en"annexe"où"j’apparais"en"coPpremier"auteur"avec"
Amaury."
1. Préparation de MAMPs issus d’Aphanomyces euteiches
Afin"de"calibrer"la"composition"de"la"préparation"au"travers"de"différentes"cultures"d’Ae,"la"teneur"en"
glucose"libre"et"NAG"a"été"évaluée."Les"filtrats"ont"été"prélevés"à"21"jours"au"moment"où"le"mycélium"
arrive" en" fin" de" croissance" dans" les" fioles" de" Roux" où" il" pousse." " Une" dilution" adaptée" a" alors" été"
appliquée" pour" obtenir" une" concentration" en" NAG" de" 35µg/ml." Par" la" suite," cette" préparation" est"
dialysée"avec"un"taille"d’exclusion"de"1"kDa"pour"dessaler"le"milieu"et"évacuer"les"petits"déchets"du"
métabolisme" sans" éliminer" peptides" et" polymères" saccharidiques." L’utilisation" de" colonnes"
échangeuses" d’ions" permettant" de" séparer" les" peptides" et" protéines" des" polymères" de" sucres" a"
révélé"que"la"majorité"de"la"NAG"reste"séquestrée"avec"les"protéines"(Figure"9)."La"caractérisation"de"
l’activité"élicitrice"de"ces"préparations"de"filtrats"de"culture"a"été"évaluée."
L’existence"de"NPglycosylation"des"protéines"renfermant"de"la"NAG"ou"bien"la"présence"de"protéines"
en" charge" de" séquestrer" les" MAMPs" à" NAG" pour" prévenir" leur" perception" sont" deux" hypothèses" à"
même" d’expliquer" l’association" de" la" NAG" aux" protéines" et" mériterait" d’être" étudiée" pour" mieux"
comprendre"ce"phénomène"et"les"répercussions"éventuelles"sur"la"perception"par"la"plante."
105"
"
"
Figure"9":"Représentation"schématique"de"l’étude"des"MAMPs"à"NPacetylglucosamine"contenus"dans"les"filtrats"de""culture"
d’"Aphanomyces0euteiches"
"
2. Analyse des réponses de Medicago truncatula aux filtrats de culture
d’Aphanomyces euteiches
La" réponse" des" plantes" aux" éliciteurs" se" matérialise" par" une" cascade" d’événements" décrite" en"
introduction" et" dont" les" événements" initiaux" se" manifestent" de" manière" rapide" et" souvent"
transitoire."Une"des"réponses"les"plus"précoces"est"la"production"de"ROS"extracellulaires"qui"survient"
dans" les" minutes" suivant" la" perception" des" PAMPs" microbiens." Ce" marqueur" d’élicitation" présente"
l’avantage" d’être" étudiable" expérimentalement" de" manière" relativement" simple" à" partir" de" la"
réduction"de"substrats"par"une"enzyme"catalysée"par"l’H2O2."Ainsi,"il"ouvre"la"porte"au"criblage"d’un"
grand" nombre" d’échantillons," des" analyses" d’expression" de" gènes" marqueurs" de" défense" pouvant"
être"réalisées"par"la"suite"pour"confirmer"les"résultats"encourageants"obtenus"à"ce"niveau."Ces"deux"
types"de"marqueurs"ont"été"employés"pour"tester"la"perception"des"filtrats"de"culture"d’A.0euteiches"
à"travers"NFP"et"la"signalisation"symbiotique."
a. Les espèces réactives de l’oxygène
Les" traitements" d’élicitation" ont" été" réalisés" sur" les" plantes" cultivées" en" milieu" liquide" et"
préalablement"rincées"à"l’eau"avant"traitement"avec"les"filtrats"d’Aphanomyces0euteiches.""Un"milieu"
de"culture"du"microbe"dialysé"n’ayant"pas"reçu"l’oomycète"a"été"utilisé"comme"contrôle."Des"analyses"
de" cinétique" de" production" de" ROS" après" traitement" avec" la" chitine" ou" les" filtrats" d’Aphanomyces0
106"
"
euteiches0 ont" révélé" une" accumulation" maximale" de" ROS" dans" les" vingt" minutes" après" traitement,"
après"quoi,"leur"quantité"décroît"puisque"métabolisée"par"notre"système"rapporteur"(Figure"10a)."
"
Figure" 10" :Cinétique" de" production" de" ROS" quantifiées" en" temps" réel" par" chémiluminescence" pendant" 40" minutes." La"
réponse" à" la" chitine" représentée" en" vert" est" utilisée" comme" témoin" positif." Celle" au" filtrat" de" culture" est" représentée" en"
rouge"et"le"Mock"en"bleu."Les"essais"ont"été"conduits"sur"A17"et"le"mutant"nfp%20(a)"et","Arabidopsis0thaliana,"plante"non"
hôte"d’A.0euteiches0(b)."
Pour"accélérer"l’expérimentation,"il"a"été"décidé"de"faire"des"mesures"en"point"final""trente"minutes"
après"le"traitement"dans"des"plaques"multipuits"où"l’on"dispose"les"surnageants"de"la"plante."Ainsi"le"
test" utilisé" est" davantage" reproductible" en" permettant" de" " réaliser" tous" les" traitements"
simultanément." De" plus," étant" non" destructif," il" permet" ultérieurement" de" tester" la" régulation"
transcriptionnellele" de" marqueurs" de" l’immunité." Dans" un" premier" temps," les" filtrats" de" culture"
dialysés"et"dialysés/déprotéinés"ont"été"testés"chez"A17,"révélant"que"seuls"les"premiers"possèdent"
une"activité"élicitrice"des"ROS"significative"(Figure"11a)."Ainsi,"A17"et"les"mutants"nfp,0lyk30et0dmi20ont"
été" caractérisés" pour" leur" réponse" ROS" aux" filtrats" de" cultures" dialysés." Il" est" apparu" que" seuls" les"
mutants"du"gènes"NFP"sont"affectés"dans"la"réponse"ROS"aux"deux"préparations"de"filtrats,"les"autres"
exhibant" une" induction" similaire" à" A17" (Figure" 11b)." Une" préincubation" d’A17" dans" des"
lipochitooligosaccharides" bactériens" (10P8M)" pendant" deux" heures" avant" traitement" avec" les" filtrats"
conduit"à"l’inhibition"de"la"production"de"ROS"par"les"filtrats"de"culture"(Figure"11c)."Néanmoins,"cet"
effet"inhibiteur"n’est"pas"observé"lors"de"la"coPinoculation"des"filtrats"et"des"lipochitooligosaccharides"
et"ne"répond"pas"à"un"effet"dosePréponse."Enfin"ces"filtrats"de"culture"ont"également"été"appliqués"
sur" Arabidopsis0 thaliana" (écotype" ColP0)" et" il" a" été" observé" que" cette" plante" n’induisait" pas" la"
production"de"ROS"en"leur"présence"(Figure"10b).""
La" comparaison" des" filtrats" " dialysés" et" dialysés/déprotéinés" a" révélé" une" plus" forte" activité" des"
premiers," suggérant" que" cellePci" pourrait" être" corrélée" à" la" quantité" de" NAG" ou" de" protéines." Le"
résultat" obtenu" sur" la" plante" non" légumineuse" Arabidopsis0 thaliana" suggère" que" le" ou" les" MAMPs"
induisant" les" ROS" sont" spécifiques" de" la" plante" hôte," et" suggère" que" si" les" composés" à" NAG" sont"
responsables"de"cette"élicitation,"ceuxPci"ne"sont"pas"renfermés"dans"de"la"chitine"classique"qui"aurait"
107"
"
été"perçue"par"Arabidopsis0thaliana.""En"ce"qui"concerne"le"décryptage"de"la"voie"de"signalisation"en"
aval"de"NFP"en"réponse"à"Aphanomyces0euteiches,"il"est"intriguant"d’obtenir"un"résultat"distinct"entre"
les" mutants" nfp" et" dmi2" puisque" ceuxPci" ont" un" phénotype" similaire" quant" à" la" résistance" à"
Aphanomyces0euteiches."En"revanche,"LYK3"se"voit"confirmer"dans"son"rôle"de"récepteur"spécifique"
de" la" reconnaissance" de" lipochitooligosaccharides" appropriés" puisqu’il" perçoit" normalement" les"
filtrats" dialysés." " Ayant" observé" l’implication" potentielle" de" NFP" dans" la" perception" des" filtrats" de"
cultures" " et" la" mise" en" place" de" la" réponse" ROS," il" a" été" entrepris" d’interférer" " avec" ce" procédé" en"
préincubant" les" plantes" dans" des" lipochitooligosaccharides" bactériens." L’inhibition" de" réponse" au"
filtrat" ainsi" obtenue" ne" plaide" pas" nécessairement" pour" une" compétition" des" signaux" symbiotiques"
avec" ceux" d’A.0 euteiches0 puisqu’il" a" " antérieurement" été" démontré" que" des" facteurs" NOD" peuvent"
inihiber" les" ROS" induites" par" des" éliciteurs" reconnus" via" des" récepteurs" complétement" différents,"
notamment"les"oligogalacturonanes"(Shaw"&"Long,"2003)."De"plus,"une"longue"préincubation"semble"
requise" et" cet" effet" n’est" pas" dosePdépendant," suggérant" qu’il" est" plutôt" indirect." A" l’issue" de"
l’ensemble"de"ces"tests,"les"plantes"A17"et"nfp%20traitées"avec"les"filtrats"dialysés"et"les"plantes"A17"
préincubées"dans"les"lipochitooligossacharides"avant"élicitation0ont"été"récoltées"au"bout"de"quatre"
heures"pour"des"analyses"de"marqueurs"de"défense.""
Les"résultats"de"mesure"de"ROS"présentés"dans"ce"travail"sont"ceux"que"j’ai"obtenus"avec"deux"filtrats"
de"culture"et"apparaissent"particulièrement"intéressants"puisqu’il"y"a"une"réponse"NFP"dépendante"
qui" aurait" pu" expliquer" les" résultats" différentiels" observés" en" phénotypage" ou" encore" au" cours" des"
analyses" transcriptomiques" comparatives" avec" les" LCOs." Cependant," d’autres" expériences" similaires"
menées"dans"le"laboratoire"avec"d’autres"filtrats"et"par"d’autres"expérimentateurs"ne"permettent"pas"
de"trancher"quant"au"rôle"de"perception"de"PAMPs"d’Aphanomyces0euteiches0par"NFP."En"raison"de"
ce" manque" de" reproductibilité" des" résultats," ces" expériences" de" ROS" n’ont" pas" été" incluses" dans"
l’article." D’autres" tests," d’autres" fractions" sont" actuellement" en" cours" d’étude" pour" permettre" de"
préciser"définitivement"l’implication"de"NFP"dans"la"perception"de"PAMPs"d’A.0euteiches."
108"
"
"
Figure"11":Mesure"de"ROS"en"point"final"par"fluorescence.""Test"des"filtrats"dialysés"et"dialysés/déprotéinées"(n=3)"(a)."Test"
de"mutants"de"la"signalisation"symbiotique"avec"les"filtrats"dialysés"contrôles"(gris)"et"inoculés"avec"A.0euteiches"(noir)"(n=3)"
(b)." Production" de" ROS" suite" à" des" incubations" avec" des" lipochitooligosaccharides" bactériens" selon" différentes"
concentrations" et" différents" temps" (n=3)(c)." " Les" valeurs" sont" issues" d’une" moyenne" entre" 9" échantillons" obtenus" en" 3"
répétitions"distinctes."
b. Les marqueurs transcriptionnels
"Toujours" dans" le" but" d’identifier" une" réponse" NFP" dépendante," un" groupe" de" marqueurs"
représentatifs"de"différentes"voies"de"défense"telles"que"les"PR"protéines"(thaumatine,"inhibiteurs"de"
protéases," chitinase)," le" stress" oxydant" (peroxydase," famille" des" NADPH" oxidases)," le" métabolisme"
secondaire" (chalcone" et" stilbène" synthase," phenylammonialyase," vestitone" reductase)" et" de" la" voie"
jasmonate" (lipase" et" lipoxygenase)" a" été" choisi." Sélectionnés" à" partir" des" gènes" répondant" à"
l’infection" par" Aphanomyces0 euteiches," ils" se" sont" révélés" être" " des" marqueurs" généraux" de"
l’immunité"induits"par"des"éliciteurs"tels"que"la"flagelline"ou"la"chitine,"au"cours"des"travaux"réalisés"
par" Amaury" Nars." Ainsi," la" régulation" de" ce" groupe" de" gènes" a" été" étudiée" sur" les" échantillons"
sélectionnés"à"l’issue"des"tests"ROS.""
Il" s’est" avéré" que" tous" sont" fortements" induits" chez" A17" en" réponse" au" filtrat" de" culture" de"
l’oomycète" de" 1,7" (RboH" G)" fois" à" 277" fois" (Thaumatine" et" Peroxydase" anionique)." De" manière"
intriguante,"cette"activation"transcriptionnelle"apparaît"même"exacerbée"par"la"préPincubation"avec"
109"
"
les"lipochitooligosaccharides"passant""pour"certains"d’entre"eux"de"277"à"284"fois"pour"la"thaumatine"
27"à"53"fois"pour"un"inhibiteur"de"protéase,"277"à"363"fois"pour"la"peroxydase"anionique"et"5,7"à"7,4"
fois" pour" une" lipoxygénase." De" manière" encore" plus" frappante," ces" gènes" sont" tous" drastiquement"
plus" induits" dans" nfp%20 que" dans" A17":" de" 2,5" fois" pour" la" RboHG" à" plus" de" 520" fois" pour" la"
thaumatine""et"la"peroxydase"anionique00(Figure"12)."
"
Figure"12":Sélection"de"marqueurs"de"l’immunité"et"de"la"production"de"ROS"dont"la"réponse"au"traitement"avec"les"filtrats"
de"culture"est"suivie"par"RTPqPCR"(n=3)."
Ainsi" ces" marqueurs" transcriptionnels" de" l’immunité" viennent" contrarier" les" observations" faites"
précédemment" avec" les" ROS." Dans" la" mesure" où" ces" derniers" paraissent" plus" robustes" car"
spécifiques,"il"est"tentant"de"considérer"ces"résultats"en"priorité."Il"est"vrai"qu’un"grand"nombre"de"
facteurs,"notamment"développementaux,"sont"susceptibles"d’affecter"l’homéostasie"des"ROS,"ce"qui"
peut"engendrer"des"artefacts"en"dépit"des"contrôles"positifs"introduits"systématiquement."Il"est"tout"
de" même" curieux" de" noter" que" les" plantes" dans" lesquelles" la" réponse" ROS" n’a" pas" été" observée"
exhibent" les" plus" fortes" régulations" géniques." En" ce" sens," il" est" vrai" que" la" mutation" NFP" ou"
l’application" de" lipochitooligosaccharides" a" un" effet" sur" l’expression" des" marqueurs" bien" que" ce" ne"
soit" pas" celui" intuitivement" attendu." Le" suivi" de" l’expression" de" ces" marqueurs" au" long" d’une"
cinétique"détaillée"après"traitement"avec"les"filtrats"pourrait"permettre"d’observer"des"nuances"qui"
nous"ont"échappé"avec"le"seul"temps"de"quatre"heures"après"élicitation."Ainsi,"un"délai"dans"la"mise"
en" place" de" la" réponse" chez" les" mutants" nfp" et" au" cours" des" préPtraitements"
lipochitooligosaccharides"pourrait"être"observé."A"l’inverse,"une"réponse"immunitaire"plus"soutenue"
dans" le" temps" chez" A17" en" l’absence" lipochitooligosaccharidique" pourrait" être" mise" en" évidence."A"
titre" indicatif," des" plantes" incubées" pendant" quatre" heures" avec" des" lipochitooligosaccharides"
bactériens"avant"d’être"inoculées"par"A.0euteiches"n’ont"révélé"aucune"altération"de"leur"niveau"de"
résistance,"que"ce"soit"A17"vers"la"sensibilité"ou"F83005.5"vers"la"résistance"(données"non"montrées)."
Ainsi," l’effet" des" lipochitooligosaccharides" observés" chez" A17" visPàPvis" de" la" perception" des" filtrats"
d’Aphanomyces0euteiches0ne"peut"pas"être"associé"à"une"modification"du"comportement"de"la"plante"
face"au"parasite."
110"
"
D.Conclusion du chapitre
"
L’objectif"de"ce"chapitre"était"de"caractériser"le"rôle"de"NFP"dans"l’immunité"de"Medicago0truncatula."
De" manière" complémentaire" à" l’approche" de" phénotypage" dans" laquelle" des" mutants" apparaissent"
plus" sensibles" à" A.0 euteiches," il" a" pu" être" mis" en" évidence" que" la" surexpression" de" NFP" permettait"
d’améliorer" la" résistance" à" l’oomycète." De" façon" intriguante," il" a" été" montré" que" l’expression" était"
rapidement"réduite,"y"compris"lorsque"le"gène"était"sous"le"contrôle"d’un"promoteur"constitutif."Ces"
résultats"suggèrent"l’existence"d’une"régulation"postPtranscriptionnelle"qui"pourrait"être"induite"par"
le"parasite"luiPmême,"mais"cette"dernière"hypothèse"reste"à"démontrer."Toujours"estPil"que"le"niveau"
d’expression" basal" du" gène" semble" être" important" pour" le" niveau" de" résistance" observée" dans" la"
plantes." Tous" ces" résultats" suggèrent" un" rôle" précoce" du" gène" dans" les" étapes" précoces" de"
l’interaction." Ce" rôle" dans" les" étapes" précoces" de" l’interaction" est" aussi" supporté" par" l’expression"
altérée" de" centaines" de" gènes" dans" le" mutant" nfp%2" par" rapport" au" sauvage" A17," 24" heures" après"
inoculation" avec" A.0 euteiches." Ces" gènes" ne" semblent" pas" être" associés" directement" à" la" réponse"
immunitaire" mais" plutôt" à" des" processus" de" dynamique" cellulaire." Les" mutants" nfp0 ont" aussi" été"
caractérisés" au" cours" de" l’interaction" avec" Colletotrichum0 trifolii" et" se" sont" également" révélés" plus"
sensibles" à" ce" champignon." Ces" observations" plaident" en" faveur" d’un" rôle" général" de" NFP" dans" la"
résistance"de"la"plante."Son"intervention"en"tant"que"récepteur"transmembranaire"à"domaines"LysM"
dans"la"perception"de"composés"microbiens"contenant"des"résidus"à"NPacetylglucosamine"paraît"être"
l’hypothèse"la"plus"directe"pour"expliquer"son"rôle"dans"l’immunité."Cependant,"la"perception"de"COs"
a" été" étudiée" chez" les" mutants" nfp" sans" révéler" d’altération" de" la" perception" de" ces" MAMPs" au"
travers" de" la" mise" en" place" de" réponses" telles" que" les" ROS" ou" l’activation" de" marqueurs"
transcriptionnels"de"défense."Si"les"préparations"des"filtrats"de"culture"d’A.0euteiches"se"sont"révélées"
être" activatrices" de" l’immunité," un" rôle" de" NFP" n’a" pu" être" décelé" dans" leur" perception." Enfin," la"
comparaison"de"nos"données"transcriptomiques"avec"celles"issues"de"traitements"avec"des"LCOs"dont"
la" perception" dépend" de" NFP" a" révélé" que" des" gènes" associés" au" métabolisme" lipidique" pourraient"
être" activés" chez" A17" de" manière" dépendante" de" ce" récepteur." Ce" dernier" résultat" constitue" une"
piste"de"choix"pour"élucider"la"contribution"de"ce"gène"au"cours"des"interactions"avec"des"parasites."
L’obtention" récente," au" sein" de" l’équipe," de" données" transcriptomiques" de" perception" des"
chitosaccharides"pariétaux"d’A.0euteiches"chez"A17"et"les"mutants"nfp"devrait"permettre,"de"préciser"
les" fonctions" dépendantes" de" NFP" au" cours" de" l’interaction" avec" A.0 euteiches" avant," peutPêtre," de"
généraliser"son"rôle"visPàPvis"de"Colletotrichum0trifolii,"par"exemple."
"
111"
"
"
Chapitre III. Caractérisation du
rôle de NF-YA1 dans la résistance
à Ae
"
Introduction : Les facteurs de transcription NF-Y chez
les plantes
"
La" famille" des" facteurs" de" transcription" de" type" NF%Y" est" présente" chez" l’ensemble" des" eukaryotes"
(Mantovani," 1999";" Matuoka" and" Chen," 2002)." Elle" se" compose" de" trois" sousPunités" de" structures"
protéiques"distinctes"qui"s’associent"en"hétérotrimères"pour"réguler"la"transcription."Les"unités"B"et"C"
forment"un"dimère"possédant"des"homologies"avec"celui"formé"par"les"histones"H2A"et"H2B."Ainsi"B"
et"C"sont"supposées"décompacter"la"chromatine"en"entrant"en"compétition"avec"les"histones"pour"la"
liaison" à" l’ADN" de" manière" aspécifique." Cette" interaction" physique" entre" B" et" C" est" nécessaire" au"
recrutement" de" l’unité" A" (Hackenberg" et" al.," 2011)." CellePci" se" lie" aux" boîtes" CCAAT" et" active" la"
transcription" en" recrutant" la" machinerie" transcriptionnelle." Si" la" présence" de" cette" séquence" est"
importante"pour"la"liaison"à"l’ADN,"les"séquences"bordantes"sont"aussi"déterminantes,"conférant"à"A"
un"rôle"dans"la"spécificité"des"cibles"contrôlées"(Dolfini"et"al.,"2012";"Calvenzani"et"al.,"2012)."Si"une"
grande" partie" de" la" caractérisation" de" cet" hétérotrimère" protéique" dans" le" contrôle" de" la"
transcription" provient" d’exemples" issus" du" monde" animal," beaucoup" reste" à" découvrir" en" ce" qui"
concerne" les" plantes." Les" multiples" événements" de" duplication" génomique" qui" ont" eu" lieu" chez" ces"
organismes" sont" à" l’origine" d’une" grande" expansion" du" nombre" de" sousPunités" A," B" et" C," là" où" les"
animaux" n’ont" qu’une" copie" de" chacune" (Ji" Yang," Xie," &" Glover," 2005)." Ainsi" les" plantes" peuvent"
théoriquement" proposer" jusqu’à" plusieurs" milliers" de" combinaisons" trimèriques" en" vue" du" contrôle"
de" processus" biologiques" variés" et" de" leur" plasticité" phénotypique" face" à" leur" environnement"
(Laloum,"De"Mita,"Gamas,"Baudin,"&"Niebel,"2012)."Néanmoins,"l’ensemble"de"ces"complexes"ne"sont"
pas" tous" réunis" in0 vivo" car" ces" gènes" possèdent" des" profils" d’expression" tissulaires" plus" ou" moins"
spécifiques" (Siefers" et" al.," 2009)." Ainsi," il" est" par" exemple" proposé" que" certaines" sousPunités"
s’expriment" de" manière" ubiquiste" et" contrôlent" des" fonctions" stables" à" travers" l’organisme" ou" au"
112"
"
cours"de"sa"vie,"comme"la"photosynthèse"("Kusnetsov"et"al.,"1999";"Stephenson"et"al.,"2011),""tandis"
que" d’autres" seraient" induites" au" cours" de" réponses" plus" ponctuelles" telles" que" la" floraison," la"
formation"des"graines"(Wenkel"et"al.,"2006";"Casson"and"Lindsey,"2006";"Warpeha"et"al.,"2007;"Cai"et"
al.,"2007;"Yazawa"and"Kamada,"2007";"Kumimoto"et"al.,"2008;"";""Mu"et"al.,"2008";"Yamamoto"et"al.,"
2009" ;" Kumimoto" et" al.," 2010;" Tiwari" et" al.," 2010;" Klimaszewska" et" al.," 2010)" " ou" la" réponse" à" des"
stress"abiotiques"("Nelson"et"al.,"2007";"Li"et"al.,"2008)"(Figure"13)."De"manière"intriguante,"le"rôle"de"
tels"facteurs"de"transcription"dans"la"réponse"immunitaire"n’est"pas"décrit"dans"la"bibliographie."En"
revanche," plusieurs" membres" de" cette" famille" sont" importants" pour" les" interactions" des"
légumineuses" avec" les" microorganismes" mutualistes," qu’il" s’agisse" des" champignons" mycorhiziens"
(Hogekamp"et"al.,"2011";"Gaude"et"al.,"2011)"ou"des"bactéries"fixatrices"d’azote"(Combier"et"al.,"2006";"
Combier" et" al.," 2008" ;" Zanetti" et" al.," 2010)" ." Chez" Medicago0 truncatula0 le" rôle" prépondérant" de" la"
sousPunité" MtNF%YA1" dans" la" persistance" du" méristème" des" nodosités" à" croissance" indéterminée" a"
été"caractérisé."En"effet,"celuiPci"présente"une"expression"extrêmement"localisée"à"l’apex"du"nodule"
nécessaire" à" son" développement" et" qui" est" restreinte" par" deux" mécanismes" de" régulation" postP
transcriptionnelle,"l’un"impliquant"le"micro"ARN"miR169q"et"l’autre"un"petit"peptide"nommé"uORF1,"
issu"d’un"épissage"alternatif"du"transcrit"codant" MtNF%YA1."Ainsi"le"travail"décrit"par"la"suite"révèle"
des"aspects"totalement"originaux"sur"le"rôle"joué"par"le"marqueur"de"l’organogénèse"nodulaire"MtNF%
YA1"dans"la"sensibilité"à"l’invasion"par"des"agents"pathogènes"racinaires."
"
Figure"13":"Figure"2."Relations"phylogénétiques"entre"les"NFPYA"d'Arabidopsis0thaliana"(10"protéines),"Oryza0sativa"(riz)"(10"
OsHAP2s),"Medicago0truncatula"(8)"et"Glycine0max"(soja)"(20)."Chez"le"soja,"le"doublement"du"nombre"de"NFPY"pourrait"être"
113"
"
dû"à"une"récente"duplication"de"génome."Les"expressions"tissulaires"de"chaque"gène"proviennent"de"bases"de"données""de"
microaaray" (Medicago0 truncatula":" http://mtgea.noble.org/v2/;" Glycine0 max":" http://" digbio.missouri.edu," " Oryza0 sativa"
http://ricexpro.dna.affrc.go.jp/GGEP/index.html," et" Arabidopsis":" https://" www.genevestigator.com" /" GV" /" plant.jsp)." Les"
gènes" appartenant" au" sousPgroupe" des" légumineuses" ont" un" profil" d'expression" spécifiques" des" nodules" et" des" racines."
Laloum,&T.&et&al."(2012)."CCAATPbox"binding"transcription"factors"in"plants:"Y"so"many?"Trends0in0plant0science""
"
A. Article
The&transcription&factor&MtNF2YA1,&a&regulator&of&symbiotic&nodule&development,&promotes&
Medicago)truncatula&susceptibility&to&a&root&pathogen.&
Thomas" Rey" a,b;" Philippe" Laporte" c,d;" Marie" Françoise" Jardinaud" c,d;" Maxime" Bonhomme" a,b;"
Stéphanie" Huguete," Sandrine" Balzerguee;" Bernard" Dumas" a,b;" Andreas" Niebel" c,d;" and" Christophe"
Jacquet"a,b,1.""
a+&
Université" de" Toulouse," UPS," Laboratoire" de" Recherche" en" Sciences" Végétales," BP42617,"
Auzeville,"FP31326,"CastanetPTolosan,"France"
b
"CNRS,"Laboratoire"de"Recherche"en"Sciences"Végétales,"BP42617,"Auzeville,"FP31326,"CastanetP
Tolosan,"France""
c
"INRA,"Laboratoire"des"Interactions"PlantesPMicroorganismes"(LIPM),"UMR441,"FP31326"CastanetP
Tolosan,"France."
d
" CNRS," Laboratoire" des" Interactions" PlantesPMicroorganismes" (LIPM)," UMR2594," FP31326"
CastanetPTolosan,"France."
e
" Unité" de" Recherche" en" Génomique" Végétale" (URGV)," UMR" INRA" 1165" P" " Université" d’Evry" Val"
d’Essonne"P"ERL"CNRS"8196,"CP"5708,"FP91057"Evry"Cedex,France"
Author"contributions:"""
designed"research":"CJ;"AN,"BD"
"performed"research":"TR,"PL,"SH,"SB"
"analyzed"data":"TR,"CJ,"MFJ,"MB,"AN"
"wrote"the"paper":"TR,"CJ,"AN,"BD"
"
1
":"To"whom"correspondence"should"be"addressed."EPmail":"[email protected]"
114"
"
Abstract&
Plant"NFPY"transcription"factors"control"a"wide"array"of"biological"functions"enabling"appropriate"
reproductive" and" developmental" processes" as" well" as" adaptation" to" various" abiotic" and" biotic"
environments."In"Medicago0truncatula,"MtNF%YA10was"previously"identified"as"a"key"determinant"
for" nodule" development" and" establishment" of" rhizobial" symbiosis." Here" we" highlight" a" new" role"
for" this" protein" by" showing" that" it" promotes" susceptibility" to" Aphanomyces0 euteiches,0 a" root"
oomycete."nf%ya1%1"mutant"plants"inoculated"with"this"pathogen"showed"both"reduced"symptoms"
and" an" increased" development" of" their" root" apparatus." The" role" of" MtNF%YA1" in" plant"
susceptibility" was" further" confirmed" by" silencing" approaches" mediated" by" overexpressing"
miR169q,"a"microRNA"previously"shown"to"control"MtNF%YA1"expression"(Combier"et0al.,"2006),"or"
through" RNAi." Studies" using" a" pMtNF%YA%1::uidA" reporter" construct" showed" that" this" gene" is"
expressed" in" the" central" cylinder" of" roots" as" well" as" in" lateral" root" primordia," in" addition" MtNF%
YA1" was" strongly" upregulated" in" the" susceptible" accession" F83005.5" upon" A.0 euteiches"
inoculation.""Comparisons"of"transcriptome"analyses"between"wildPtype"(WT)"and"nf%ya1%1"plants"
before"and"after"inoculation"led"to"the"identification"of"about"2,000"genes"that"are"misregulated"
in" the" mutant." Among" these" genes" major" differences" were" notably" detected" for" a" large" set" of"
defencePrelated"genes"which"are"constitutively"expressed"in"mutant"plants"but"also"for"genes"that"
may"be"involved"in"the"regulation"of"root"growth."In"summary,"all"this"data"reveals"an"unexpected"
dual" role" for" this" transcription" factor" as" a" key" player" in" the" compatibility" mechanisms" to" both" a"
pathogen"and"a"symbiont.""
&
Introduction&
"
The" NFPY" transcriptional" regulator" complex" binds" CCAAT" boxes" that" are" presents" in" almost"
one"third"of"eukaryotic"promoters"(Mantovani,"1999;"Matuoka"&"Chen,"2002)."NF%Y"complexes"are"
heterotrimeric" complexes" composed" of" three" subunits" called" NF%YA," NF%YB" and" NF%YC" and" are"
found" in" all" eukaryotes" tested" so" far." Mechanistic" analysis" of" their" transcriptional" regulatory"
properties" has" been" extensively" investigated" in" animals" (Dolfini0 et0 al.," 2012)." Briefly," NF%YA"
proteins" located" in" the" nucleus" form" a" heterotrimeric" complex" with" NF%YB" and" NF%YC" subunits"
that"interacts"in"the"cytoplasm"before"mooving"into"the"nucleus"(Hackenberg0et0al.,"2012)."Among"
NF%Y0proteins0,"the"A"subunit"is"thought"to"mediate"the"specificity"of"targets"on"genomic"DNA"by"
binding"CCAAT"motifs"while"B"and"C"are"thought"to"be"involved"in"local"chromatin"decompaction"
(Calvenzani0et0al.,"2012).""
"
Unlike"animals,"plants"possess"multiple"subunits"of"each"category"(Laloum0et0al.,"2012)."As"an"
example"Medicago0truncatula"possesses"8"NF%YA,"19"NF%YB"and"11"NF%YC"subunits."This"wealth"of"
potential" NFPY" subunit" combinations" suggests" extended" capabilities" for" these" transcriptions"
factors" to" set" into" motion" several" different" plant" biological" functions" including" embryogenesis,"
germination,"drought"resistance,"flowering,"root"development"or"nitrogen"nutrition"(Laloum0et0al.,"
2012)." As" some" subunits" are" ubiquitously" and" strongly" expressed" while" others" are" specifically"
induced" in" particular" plant" compartments," it" is" proposed" that" the" former" may" mediate"
constitutive"and"stable"functions"while"the"latter"should"control"more"specific"responses"(Siefers0
et0al.,"2009).""
115"
"
"
The"role"of"NF%Y"genes"in"plantPmicrobe"interactions"is"starting"to"be"uncovered"especially"in"
the" frame" of" symbiotic" interactions." In" Phaseolus0 vulgaris" a" gene" encoding" a" NF%YC" subunit" was"
shown"to"be"upregulated"only"by"efficient"bacterial"nitrogen"fixing"symbionts"to"promote"nodule"
development" (Zanetti0 et0 al.," 2010)." Another" NF%YC" subunit" was" shown" to" be" upPregulated" in"
arbusculated" root" cells" of" the" model" legume" Medicago0 truncatula" interacting" with" mycorrhizal"
fungi"(Hogekamp0et0al.,"2011;"Gaude0et0al.,"2012)."However"the"best"characterized"NF%Y"gene"in"
plantPmicrobe"interactions"is"probably"MtNF%YA1,"formerly"named"MtHAP2%10(El"Yahyaoui0et0al.,"
2004," Combier0 et0 al.,0 2006),0 and0 which" was" shown" to" play" a" central" role" in" the" Medicago0
truncatula0 symbiosis" with0 Sinorhizobium0 meliloti." Its" first" identification" in" 2004" as" an" early" and"
strong" nodulin" by" a" transcritptomic" approach," suggested" it" is" a" highly" specific" regulator" of"
nodulation"(El"Yahyaoui0et0al.,"2004)."Previous"functional"studies"showed"that"MtNFPYA1"controls"
late" steps" of" nodule" organogenesis" by" participating" in" meristem" maintenance" under" sequential"
control"of"two"post"transcriptional"regulators"(Combier0et0al.,"2006;"Combier0et0al.,"2008)."Recent"
analyses" on" the" nf%ya1%1" mutant" also" revealed" the" presence" of" abnormal" infection" threads"
(Laporte"et"al.,"in"preparation)"suggesting"a"putative"role"of"MtNF%YA1"in"the"control"of"symbiont"
colonization," reminding" the" conclusion" drawn" for" PvNFPYC1" in" bean" nodules" by" Zanetti" et" al"
(2010)."The"specific"role"of"MtNF%YA1"was"recently"supported"by"the"observation"of"its"induction"
by" lipochitololigosaccharides" (LCOs)" produced" by" Rhizobium" but" not" by" mycLCOs" purified" from"
mycorhizae"(Czaja0et0al.,"2012)."
"
If"NF%Y"has"been"involved"in"the"regulation"of"plant"development"or"symbiotic"plantPmicrobe"
interactions," no" study" has" yet" reported" on" the" role" of" a" NFPYPencoding" gene" during" a" plant"
pathogen" interaction." In" this" work," we" used" the" M.0 truncatula" P" Aphanomyces0 euteiches"
pathosystem"to"assess"a"putative"involvement"of"MtNF%YA1"in"plant"responses"to"this"pathogen."
A.0euteiches"is"a"major"pathogen"of"crop"and"forage"legumes"and"is"the"causal"agent"of"pea"root"
rot"disease"(Gaulin0et0al.,"2007)."M.0truncatula"is"a"natural"host"for"this"biotrophic"oomycete"and"
accessions" of" this" model" legume" have" been" shown" to" display" a" high" level" of" variability" in" their"
responsiveness" upon" A.0 euteiches" inoculation" (Moussart0 et0 al.," 2007)." A" pathosystem" with" the"
F83005.5" (susceptible)" and" the" A17" (partially" resistant)" lines" was" developed" to" identify" the"
molecular" components" and" characterise" the" mechanisms" involved" in" the" observed" quantitative"
resistance"to"A.0euteiches"(Djébali0et0al.,"2009)."While"A.0euteiches"is"able"to"accomplish"a"full"life"
cycle"and"to"colonize"root"cortex"in"both"lines,"its"development"into"the"stele"is"prevented"in"the""
A17"line"that"keeps"on""growing,"unlike"the"F83005.5"plants"whose"root"tissues"are"fully"invaded"
and" which" finally" dies." Supplementary" pericycle" cell" divisions," lignin" deposit" on" endodermis" and"
pericycle" cell" wall" along" with" the" accumulation" of" soluble" phenolic" compounds" were" associated"
with"resistance"characteristics"(Djébali0et0al.,"2009;"Djébali0et0al.,"2011).""
"
As"A17"is"the"genetic"background"of"the"nf%ya%1"mutant"plants,"we"carefully"compare"in"this"
work" mutant" and" WT" phenotypes" upon" A.0 euteiches" inoculation." Following" the" identification" of"
changes"in"resistance"level"displayed"by"the"mutant"plants,"the"role"of"NFPYA1"in"plant"response"
was"confirmed"by"loss"of"function"analyses"in"the"susceptible"plants."Following"expression"pattern"
studies" of" NF%YA1," along" with" microarray" experiments" we" finally" proposed" an" unexpected" new"
role"for"this"transcription"factor"in"the"regulation"of"a"plant"pathogen"interaction."
116"
"
Results&
A) MtNF2YA1& null+mutation) increases& resistance& to& A.) euteiches& and& modifies& root& architecture&
upon&pathogen&inoculation&
"
To"assess"a"putative"role"of"MtNF%YA1"in"plant"immunity"mechanisms,"we"took"advantage"of"
the""nf%ya1%10null"mutant"line"that"was"obtained"in"the"A17"genetic"background"(Laporte"et"al."In"
preparation)." Young" seedlings" of" the" nf%ya1%1" mutant" line" were" inoculated" with" A.0 euteiches"
zoospores," along" with" A17" and" F83005.5" seedlings" that" were" respectively" used" as" partially"
resistant"and"susceptible"lines"to"this"oomycete."Phenotypes"of"each"lines"are"shown"in"Figure"1A,"
14"days"post"inoculation"(dpi)":"while"most"F83005.5"plants"are"severely"attacked,"A17"(wild"type"P"
WT)" plants" keep" growing" despite" the" presence" of" disease" symptoms" on" the" entire" roots."
Inoculated"nf%ya1%1"plants"display"also"symptoms"but"parts"of"their"primary"roots"as"well"as"some"
newly"formed"secondary"roots"are"symptomless"at"that"stage,"indicating"a"delay"in"the"pathogen"
development."The"extent"of"rot"symptoms"along"with"percentages"of"dead"plants"(Figure"2B)"were"
measured"on"a"total"of"150"inoculated"plants"by"line"and"revealed"a"significant"increase"(PPvalue"
<0.001)"in"the"resistance"level"of"nf%ya1%10compared"to"WT"for"both"parameters."Additional"data"
were"also"collected"to"assess"putative"changes"in"root"architecture"of"each"line"inoculated"or"not"
by" A.0 euteiches." While" a" similar" root" architecture" was" observed" between" each" line" in" control"
conditions" (n=30" plants" for" each" line)" number" and" length" of" secondary" roots" were" significantly"
different" upon" A.0 euteiches" inoculation" (Figure" 1C)." While" in" F83005.5" a" strong" decrease" in" size"
and" number" of" roots" after" inoculation" was" observed," the" nf%ya1%10 mutant" plants" showed"
significantly"higher"parameters"compared"to"WT,"for"both"the"number"of"lateral"roots"(+29%"P"PP
value"<0,05)"and"the"root"length"(+37%"P"PPvalue"<0,001)."Hence,"these"data"suggest"that"i)"root"
branching" and" root" development" are" positively" correlated" to" resistance" to" A.0 euteiches" and" ii)"
MtNF%YA%1"is"a"negative"regulator"of"these"responses"in"presence"of"the"pathogen."
"
Cytological&analyses&indicate&a&lower&development&of&A.)euteiches&within&nf2ya121&root&tissues:&
"
Staining"of"A.0euteiches"mycelium"using"WGAPFITC"staining"allows"the"detection"of"pathogen"
development" outside" and" inside" the" root" tissues." In" the" susceptible" F83005.5" plants," the"
rhizodermis"was"widely"colonized"at"3dpi"by"A.0euteiches"hyphae."In"contrast,"mycelium"is"clearly"
less" developed" on" the" root" surface" of" A17PWT." No" significant" difference" was" observed" in" the"
hyphae"development"at"this"timePpoint"between"inoculated"nf%ya1%10and"WT"plants"(Figure"2A).""
Examination"of"cross"sections"at"6dpi"showed"that"all"F83005.5"root"tissues"were"invaded"by"the"
pathogen"while"the"central"cylinder"was"protected"in"both"mutant"and"WT"plants."Moreover,"in"
nf%ya1%1" roots," A.0 euteiches" hyphae" are" more" concentrated" in" the" outer" cortex" and" their"
development"was"weaker"than"in"WT"roots"(Figure"2B)."At"the"end"of"the"test,"21"dpi,"F83005.5"
root"tissues"were"heavily"colonized,"plant"cell"walls"were"degraded"by"A.0euteiches"and"numerous"
oospores" are" easily" observed," indicating" that" the" pathogen" has" accomplished" full" life" cycles." In"
A17"roots"numerous"intercellular"hyphae"invaded"the"entire"cortex"but"did"not"enter"the"stele,"as"
previously"described"(Djebali"et"al."2009"and"2011)."Several"oospores"were"also"observed."In"nf%
ya1%1" roots,"no"or"only"a"very"few"oospores"located"in"the"very"outer"cortex"of"the"root"can"be"
observed"along"with"a"weaker"density"of"cortical"hyphae"compared"to"inoculated"WT"roots"(Figure"
2C)."Taken"together"results"presented"in"Figures"1"and"2"showed"a"lower"pathogen"development"
and"a"decrease"of"symptom"extent"in"nf%ya1%1"plants"compared"to"WT,"indicating"therefore"that"
117"
"
this"mutant"line"is"more"resistant"than"WT,"which"leads"to"the"involvement"of"MtNFPYA1"in"plant"
susceptibility"to"A.0euteiches.""
"
Spatio2temporal)expression)of)MtNF2YA1)in&Medicago&roots"
"
To" gain" insights" into" the" role" of" Mt%NF%YA1," a" kinetics" of" its" expression" was" first" analyzed"
through" RTPqPCR" experiments;" its" promoter" was" thereafter" fused" with" the" GUS" gene" and" the"
resulting"pMtNF%YA1::uidA"reporter"construct"was"then"used"to"transform"A17"and"F83005.5"roots"
and"visualize"expression"of"Mt%NF%YA1"inside"the"transformed"root"tissues."
"
Mt%NF%YA10expression"was"first"monitored"in"the"two"control"lines"and"in"the"mutant"plants"
before"and"after"A.0euteiches"inoculation"(1,"3"and"6"dpi)."qRTPPCR"results"confirmed"that"Mt%NF%
YA10expression"is"absent"or"barely"detectable"(6dpi)"in"the"mutant"plant.""
Before"inoculation"MtNF%YA1"transcripts"were"twice"more"abundant"in"F83005.5"than"in"A17."This"
difference"in"the"level"of"MtNF%YA1"expression"between"the"two"lines"increases"upon"A.0euteiches"
inoculation,"3"dpi,"and"culminates"6dpi"with"a"ten"times"higher"expression"in"the"susceptible"line"
compared"to"A17"(Figure"3A)."
"
A" similar" level" of" GUS" expression" was" observed" in" A17" and" F83005.5" lines" in" control"
conditions"(Figure"3B)."In"both"lines,"a"staining"was"detected"in"the"root"central"cylinder"and"the"
blue" color" was" more" pronounced" in" lateral" root" primordia." Upon" inoculation," a" stronger"
expression"was"detected"6dpi"in"F83005.5"roots"confirming"qRTPPCR"results"and"showing"that"A.0
euteiches"infection"induced"MtNF%YA1"expression"in"susceptible"plants"but"not"in"A17"plants."
Taken" together," these" expression" results" showed" a" localized" expression" of" MtNF%YA1" notably" in"
the" lateral" root" primordia," revealing" therefore" a" putative" link" with" the" lateral" root" number"
variations"observed"in"each"inoculated"line,"and"suggesting"a"negative"correlation"between"MtNF%
YA1"expression"and"the"observed"level"of"resistance"to"A.0euteiches.""
"
"
Suppression&of&MtNF2YA21&expression&increases&resistance&of&the&susceptible&F83005.5&line."
"
To" validate" the" role" of" MtNF%YA10 in0 M.0 truncatula0 susceptibility," two" complementary"
approaches" were" used" to" suppress" its" expression" in" the" F83005.5" line." In" the" first" one," the"
miR169q" that" negatively" regulates" the" MtNF%YA10 transcript" levels" (Combier" et0 al.," 2006)" was"
overexpressed"in"hairy"roots"of"the"susceptible"line"and"in"the"second"one"a"construct"triggering"
RNA"silencing"was"introduced"in"composite"F83005.5"plants.""
"
To" assess" the" resistance" level" of" these" transformed" roots," we" quantified" pathogen"
development" by" a" RTPqPCR" method" (Rey" et" al," submitted)" quantifying" A.0 euteiches" tubulin"
transcripts"6"days"after"root"inoculation."Hairy"root"lines"were"generated"following"Agrobacterium0
rhizogenes" transformation" with" empty" vector" (controls)" or" the" 35S::miR169q" construct." In" both"
cases," three" independent" transformants" with" homogeneous" growth" speed" and" a" similar" level" of"
marker" gene" expression" were" selected." OverPexpression" of" miR169q" was" first" validated" in" the"
35S::miR169q" transformants" by" qRTPPCR" (Figure" 4A)." Results" indicated" that" miR169q" transcripts"
were" about" 305" times" (on" average)" higher" in" these" selected" lines" than" in" control" lines."
Consequently,"the"mir169q"overPexpressingPlines"showed"a"very"significant"decrease"(about"83%"
less)"in"MtNF%YA10transcript"accumulation"and"also"a"striking"reduction"(88%)"of"the"A.0euteiches"
transcripts"6"dpi,"compared"to"controls"(Figure"4A).""
"
Composite"plants"transformed"with"an"empty"vector"or"with"an"RNAi"construct"that"targets"a"
specific"region"located"in"the"3’UTR"of"MtNF%YA10(35S::aSIII’UTR)"were"also"generated."About"120"
118"
"
transformants" were" obtained" for" each" construct." They" were" subsequently" inoculated" with" A.0
euteiches"and"pooled"6"dpi"in"12"samples"for"qPCR"assays."We"first"confirmed"that"RNAi"strategy"
led" to" a" significant" decrease" of" the" MtNF%YA10 transcript" accumulation" (65%" lower" than" in"
controls)." Monitoring" the" pathogen" development" by" checking" A.0 euteiches" tubuline" transcript"
revealed" a" lower" amount" of" pathogen" (58%)" in" 35S::aSIII’UTR" transformants" vs." control" plants"
(Figure"3B).""
In"conclusion,"both"miRP"and"RNAiPbased"strategies"led"to"a"significant"downPregulation"of"MtNF%
YA10expression"which,"in"turn"increased"resistance"to"A.euteiches"and"validated"therefore"the"key"
role" of" MtNF%YA%1," previously" suggested" by" the" nf%ya1%1" mutant" phenotype," in" promoting" M.0
truncatula"susceptibility"to"A.0euteiches."
"
Constitutive&expression&of&immune&reponses&in&nf2ya2121)
Transcriptomic" analyses" were" performed" using" whole" genome" Affymetrix" chips" on" A17" and" nf%
ya1%1.0 RNAs0 extracted" from0 whole" roots" systems" were" harvested" in" control" conditions" and" one"
and"six"days"post"inoculation."An"ANOVA"analysis"was"performed"to"detect"significant"differential"
gene" expression" among" the" two" genotypes" (“Geneotype" effect“)" and" at" the" three" time" points"
(“infection"effect“)."For"each"regulated"gene,"the"parameters"(genotype"effect,"inoculation"effect,"
and"interaction"between"inoculation"and"genotype"effects)"affecting"significantly"expression"were"
determined." Following" this" statistical" test," we" only" selected" genes" displaying" a" two" times"
induction"or"repression"compared"to"control"(fold">1"or"<P1"in"log2)."According"to"this"treatment"
of" the" data," 5318" genes" were" found" to" be" regulated" by" at" least" one" of" the" effects" mentioned"
above," representing" about" 10%" of" the" overall" Affymetrix" chip" (50900" probes" on" the" chip)." " In"
healthy" plants," 1965" genes" were" differentially" regulated" (529" upPregulated," 1446" downP
regulated)."Importantly,"a"large"part"of"these"genes"(468"upPregulated;"1285"downPregulated)"was"
found" to" be" also" differentially" regulated" during" interaction" with" A.0 euteiches0 suggesting" an"
important" role" of" NF%YA10 in" the" transcriptomic" modifications" observed" upon" A." euteiches"
inoculation." (Figure" 5A)(Table" S2)." To" better" understand" the" function" of" this" group" of" genes"
differentially" expressed" between" A17" and" nf%ya1%1," " a" hierarchical" clustering" was" performed"
(Figure"5B)."The"most"striking"feature"observed"on"the"heatmap"representation"of"the"hierarchical"
clustering"was"the"strong"correlation"between"A17"6dpi"responses"and"the"control"ratios"in"the"nf%
ya1%1" mutant." Based" on" the" comparison" of" these" expression" ratios" we" defined" three" class" of"
regulation."Class"1"represents"193"genes"constitutively"upPregulated"in"nf%ya1%1"and"induced"upon"
infection"in"both"A17"and"nf%ya%1%1"at"one"and"six"dpi."Class"2"comprises"1436"genes"repressed"in"
control" condition" in" the" mutant" and" 6" dpi" in" A17." Class" 3" contains" 336" genes" induced" in" the"
mutant"and"also"induced"only"at"6"dpi"in"the"A17"plants.""
Class"1"and"class"3"contain"250"(117"+"133)"genes"with"a"predicted"function"and"among"these"119,"
more" than" 47%" of" these" genes" are" predicted" to" have" a" role" in" defensePassociated" signaling" or"
response" (Figure" 5C" and" Table" 1)." DefensePrelated" responses" included" genes" involved" in"
secondary"metabolite"pathway,"including"some"encoding"enzymes"belonging"to"phenylpropanoid"
and"flavonoid"pathways"or"cytochromes"P450;"genes"coding"Pathogenesis"Related"proteins"such"
as"protease"inhibitor,"thaumatin,"osmotin,"chitinase"or"glucanase;"genes"coding"Reactive"Oxygen"
Species"(ROS)Prelated"enzymes"including"nine"peroxidases,"four"gluthatione"sulfotransferases"and"
four"germin"like"protein."Other"genes"involved"in"signaling"classes"were"notably"represented"by"14"
kinases"or"signaling"GPproteins"and"a"total"of"23"transcription"factor"and"9"genes"associated"with"
119"
"
ethylene" synthesis" and" signaling" or" the" JA" pathway" display" also" a" modified" level" of" expression"
which"may"therefore"affect"immune"responses"of"the"mutant.""
"
Thus," all" this" analysis" showed" that" constitutive" expression" of" A.0 euteichesPinduced" defense"
genes" occurred" in" nf%ya%1%1" roots" before" inoculation" and" this" expression" is" further" amplified"
during" infection." It" is" also" worth" noticing" that" among" the" class" 2" genes," 36" are" associated" with"
various"hormone"signaling"pathways"including"abscissic"acid"(8"genes),"Auxins"(8"genes),"ethylene"
(14" genes)," jasmonates" (3" genes)," gibberellin," cytokinin" and" brassinosteroid" (1" gene" each)." All"
these" alterations" in" hormonePrelated" gene" expression" may" be" involved" both" in" defence"
modulations"but"also"in"the"modification"of"root"architecture.""
"
Discussion&
While" coping" with" their" biotic" environement," legumes" establish" either" symbiosis" with"
rhizobacteria" or" mycorrhizes" or" trigger" immunity" mechanisms" to" reject" encountered" pathogens."
Early" steps" in" both" types" of" interactions" involved" signals" and" receptor" that" share" structural"
homologies" (AntolínPLlovera" et0 al.," 2012)." Recently" the" putative" Nod" factor" receptor" was" also"
shown"to"play"a"key"role"in"M.truncatula0partial"resistance"to"the"oomycete"A.euteiches"(Rey"et0al."
submitted)" or" the" fungus" Colletotrichum0 trifolii,0 indicating" that" a" same" molecular" actor" can" be"
involved" in" both" symbiotic" and" pathogenic" interaction." Other" studies" described" antagonism" of"
symbiotic" related" genes" toward" immunity." Screening" of" additional" nodulation" mutants" with" the"
wilting" agent" Verticillium0 albo%atrum" revealed" that" SKL," DMI1," LIN" and" NSP1" contribute" to"
susceptibility"as"shown"by"reduced"disease"progression"(Ben"et0al.","2012)."DMI3,"a"gene"belonging"
to" the" common" symbiotic" pathway," was" shown" to" be" associated" with" M.0 truncatula" cytological"
responses"to"C.0trifolii"(Genre"et"al.,"2009)"and"more"recently"RAM2,0a"gene"required"for"initiation"
of"root"colonization"by"mycorrhizal"fungus"was"identified"as"a"component"of0"plant"compatibility"
with"the"oomycete"Phythophthora0palmivora0(Gobbato"et0al.,"2012).""
In" this" work," we" demonstrate" that" MtNF%YA1," a" gene" which" is" notably" involved" in" nodule"
organogenesis" is" clearly" involved" in" susceptibility" to" a" root" pathogen." Phenotyping" results" along"
with"cytological"observations"showed"that"nf%ya1%1"mutant"was"significantly"more"resistant"to"A.0
euteiches.0 Symptoms" on" mutant" plants" were" indeed" less" intense" than" in" inoculated" wild" type"
plants"and"the"pathogen"was"dampened"in"the"accomplishment"of"its"life"cycle"and"in"its"mycelium"
colonization." Since" this" gene" is" induced" in" F83005.5" following" inoculation," complementary"
approaches" performed" to" silence" MtNF%YA1" in" this" susceptible" line" were" able" to" trigger" a"
significant""increase"in"the"resistance"level"of"the"silenced"roots"and"therefore"confirmed"the"key"
role" played" by" this" transcription" factor" in" promoting" the" compatibility" of" the" interaction." New"
insights" in" the" role" of" MtNFPYA1" in" infectious" thread" progression" obtained" by" Lapporte" and"
colleagues" (submitted)" suggest" this" gene" also" facilitates" infection" by" Sinorhizobium0 melilotii." To"
give" a" hint" about" the" role" of" MtNFPYA1" in" plant" susceptibility" to" A.0 euteiches," microarrays" were"
performed" with" mutant" and" wild" type" plants" inoculated" or" not" with" the" oomycete." The" most"
striking"feature"was"the"strong"similarities"observed"in"the"expression"pattern"of"the"wild"type"6"
days" after" inoculation" transcriptome" and" the" one" recorded" for" the" mutant" in" non" inoculated"
conditions."An"interpretation"of"this"result"can"be"that"mutant"plants"display"a"constitutive"state"
of" defence" that" would" make" them" more" resistant" to" the" infection" by" A.0 euteiches" compared" to"
wild" type" that" needs" 6" days" to" mount" its" defences" at" a" similar" level" than" mutants." Analyses" of"
mutant"transcriptome"in"control"conditions"indicate"that"MtNFPYA1"is"indeed"a"negative"regulator"
of" many" defence" associated" genes" including" PR" proteins" genes" or" phenylpropanoid" and"
120"
"
isoflavonoid"pathways."Such"molecules"are"known"to"be"notably"secreted"in"the"apoplasm"where"
they"could"actively"hamper"the"intracellular"mycelium"development"in"the"mutant"plants."
"
While" NF%Y" genes" have" already" been" described" to" be" involved" in" regulation" of" genes" associated"
with" embryo" development" (Lotan" et0 al.," 1998)," flowering" (Wenkel" et0 al.," 2006)" or" nodule"
development"(J.PP."Combier"et"al.,"2006),"we"present"here"a"not"yet"described"development"role"
for"a"NF%Y"gene"at"the"root"level."In"this"work,"we"showed"that"there"are"significant"changes"in"the"
root" architecture" of" inoculated" mutant" plants," but" not" in" control" plants," highlighting" a" potential"
negative" role" of" MtNFPYA1" in" regulation" of" lateral" root" growth" in" presence" of" A.0 euteiches." This"
crosstalk" between" developmental" and" biotic" responses" suggests" an" involvement" of" plant"
hormones."With"about"fifty"genes"whose"expression"are"significantly"altered,"hormonal"pathway"
are" well" represented" in" the" transcriptome" results." This" includes" notably" ethylene" synthesis" or"
auxin" signalling" that" have" been" already" involved" both" in" root" development" and" in" responses" to"
pathogens"(Lucas"&"Brady,"2012)."It"is"also"worth"noticing"that"Absissic"acid"pathway"is"also"well"
detected" in" nf%ya1%1" transcriptome" in" control" plants" in" which" eight" ABA" related" genes" are"
constitutively" downregulated" compared" to" wild" type" plants" suggesting" a" lower" concentration" of"
this"hormone"in"the"mutant."Interestingly,"ABA"was"shown"to"be"involved"in"susceptibility"of"M.0
truncatula" to" A.0 euteiches" (Colditz" et" al.," 2005)." Moreover" the" hypothesis" of" links" between" this"
hormone" and" root" development" regulation" was" particularly" tackled" in" M.0 truncatula" where"
exogenous"ABA"treatment"rescued"the"lateral"root"development"in"an"latd/nip"mutant"defective"
for"a"nitrate"nitrate"transporter"also"required"for"nodule"development"(Bright"et0al.,"2005;"Liang"et0
al.," 2007)." Strikingly" this" gene" is" upPregulated" in" mutant" compared" to" wild" type" in" control"
conditions"and"induced"6"days"after"inoculation"in"A17."
"
In"conclusion,"the"genetic"analysis"previously"performed"on"a"RIL"population"derived"from"a"cross"
between"A17"and"F83"revealed"one"major"QTL"named"prAe1"that"explains"only"one"third"of"the"
observed"partial"resistance"in"the"A17"line."These"results"suggest"that"other"genes"not"detected"by"
this" analysis" are" involved" in" the" modulation" of" plant" resistance." Two" recent" reverse" genetic"
approaches"identified"a"gene"encoding"signalling"GPprotein"(Kiirika"et0al.,"2012)"and"the"NFP"gene"
as"positive"regulator"of"A17"line"immunity."By"a"similar"approach"we"have"now"identified"MtNF%
YA10 which" acts" as" a" negative" actor" of" M.0 truncatula" resistance" to" A.0 euteiches" by" promoting"
compatibility" of" the" interaction." Beyond" the" basic" interest" of" the" indentification" of" this"
multifaceted" gene" that" intervenes" both" in" developmental" responses" and" in" the" compatibility" of"
the"interaction"between"plant"and"microorganisms,"the"increase"of"resistance"to"A.0euteiches,"the"
major"pathogen"of"pea"in"France"and"in"Europe,"by"a"gene"mutation"open"the"way"for"a"new"non"
genetic"modification"based"method"to"improve"rapidly"pea"as"alfafa"resistance"to"A.0euteiches."
"
Materials&and&Methods&
Plant&material&and&growth&conditions&
A17"(WT)"and"F83005.5"Medicago0truncatula"lines"were"used"in"each"Ae0inoculation"experiment"
as"resistant"and"susceptible"to"Aphanomyces0euteiches,0respectively"(Djébali0et0al.,"2009)."nf%ya%1%
10"was"obtained"from"EMS"mutagenized"seeds"A"glutamine"substitution"in"position"133"out"of"322"
amino" acids" provides" a" truncated" non" functional" protein" depleted" of" DNA" and" proteinPprotein"
interactions" domain." Plants" were" grown" in0 vitro" with" a" 16Ph" light" at" 22°C" and" 8Ph" dark," 20°C," as"
121"
"
previously" described" (Djébali0 et0 al.," 2009)." For" root" transformation," we" used" Arqua1"
Agrobacterium"rhizogenes"as"described"by"BoissonPDernier"et"al."(2001)"("a"BoissonPDernier"et"al.,"
2001)."
"
""
Inoculations&procedures&and&symptoms&analysis&
Zoospores" of" the" Aphanomyces0 euteiches0 Drechs." strain" ATCC" 201684," a" pea" isolate," were"
produced" as" described" by" " Badreddine" et" al.," 2008" and" adjusted" to" 105" spores/ml" for" root"
inoculation." Plantlets" were" then" inoculated" one" day" after" transfer" on" M" medium" with" a" 5" μl"
droplet" of" spore" suspension" deposited" in" the" middle" of" the" root." " To" assess" mutant" resistance,"
four"independent"in0vitro0infection"assays"were"performed"(as"indicated"in"the"legends"of"figures)"
with"15"to"30"inoculated"plants/line"or"mutant"in"each"repeat."Relative"length"of"tissues"displaying"
symptoms" (15" dpi)," along" with" percentages" of" dead" plants" (21" dpi)" were" recorded" from" each"
inoculated"plant,"as"described"by"Djebali"et"al."(2009)."The"calculated"means"for"each"parameter"
were"compared"through"statistical"ANOVA"analyses."
"
"
Sample&preparation&for&microscopy&
For" optical" microscopy," 100" μm" root" sections" were" prepared" and" labelled" with" WGAPFITC," as"
described" by" Djébali" et" al." (2009)," to" localize" A.0 euteiches0 hyphae" using" epifluorescence"
illumination"(excitation"filter,"BP"450P490"nm)."Images"were"acquired"using"a"CCD"camera"(colour"
Coolview," Photonic" Science," Robertsbridge," UK)." Histochemical" GUS" staining" (using" 5PbromoP4P
chloroP3PindolylPbPglucuronic" acid;" MP" Biomedicals)" were" performed" as" described" by" BoissonP
Dernier" et" al.," 2005." Observations" were" done" at" least" on" 10" independent" roots" transformed" by"
Agrobacterium0rhizogenes"and"at"least"two"biological"repetitions."
Constructions&and&vectors& &
The" promoter" of" MtNF%YA0 10 (a" 2.2" kb" fragment" upstream" of" the" first" alternative" ATG)" was"
amplified"from"M.0truncatula"Jemalong"A17"DNA"by"PCR"using"Phusion"DNA"polymerase"(Thermo"
Fisher" Scientific," USA)" and" recombined" in" the" Gateway®" vector" pDONRP4PP1R" according" to" the"
manufacturer’s"instructions"(Invitrogen)."Entry"clones"for"the"GUS0open"reading"frame"(ORF)"and"
the" 3’UTR" of" MtNF%YA0 1" were" obtained" in" the" Gateway" vectors" pDONR207" and" pDONRP2RPP3,"
respectively."For"pMtNF%YA10promoter"analysis"in"M.0truncatula,"entry"clones"were"recombined"in"
the" binary" vector" pK7m34GW." The" resulting" chimeric" construct" was" transformed" into" M.0
truncatula"Jemalong"A17"roots"as"described"(BoissonPDernier"et"al.,"2001)."A"histochemical"assay"
of" GUS" activity" (2" hours)" was" carried" out" on" roots" of" M.0 truncatula" Jemalong" A17" composite"
plants." Briefly," GUS" expression" was" visualised" after" soaking" the" tissues" in" staining" buffer" and"
washing"several"times"in"70%"ethanol."To"improve"the"contrast"between"stained"and"nonreactive"
tissues," the" samples" were" briefly" cleared" with" sodium" hypochlorite." Using" this" method,"
endogenous" GUS" activity" was" not" observed" in" root" tissues" from" untransformed" M.0 truncatula"
122"
"
Jemalong" A17" plants." Observations" were" performed" using" a" Zeiss" Axioplan" 2" microscope" (Carl"
Zeiss"SAS)"equipped"with"a"digital"camera."
"
Primer"sequences"used"were:"
attB4"pMtNF%YA1,""GGGGACAACTTTGTATAGAAAAGTTGGTGCCAAATTCAGAGATACTACTTCC"
attB1r"pMtNF%YA1,"GGGGACTGCTTTTTTGTACAAACTTGATTCAAGTACTATGTTCTTCTCTATTC"
attB1"GUS,""GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTTACGTCCTGTAGAAACCCCAAC"
attB2"GUS,""GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATTGTTTGCCTCCCTGCTGCGGT"
attB2r"
NF%YA1_3’UTR,""
GGGGACAGCTTTCTTGTACAAAGTGGGGGTTTCGATTCAGAAAGGAAACAAGTG"
attB3"
NF%YA1_3’UTR,"
GGGGACAACTTTGTATAATAAAGTTGGTTACAGAATCCCAAGCCAACATGGTGTTG"
"
Affymetrix&Array&hybridization&and&data&analyses&&
Total"RNA"was"extracted"using"the"RNeasy"Kit"(Qiagen)."One"µg"of"total"RNA"was"used"to"produce"
labelled"cRNA"and"hybridise"Affymetrix"GeneChip®"Medicago"genome"arrays"at"INRAPURGV"(Evry,"
France)."The"raw"CEL"files"were"imported"in"R"software"for"data"analysis."Raw"and"normalized"data"
are"available"through"the"CATdb"database"(AFFY_aphanomyces_Medicago)" (Gagnot"et0al.,"2008)"
and"from"the"Gene"Expression"Omnibus"(GEO)"repository"at"the"National"Centre"for"Biotechnology"
Information" (NCBI)" (Turnpenny," 2008)" accession" number" GSE" 20587" and" GSE" 26046." Following"
normalisation," a" gene" is" declared" differentially" expressed" if" its" Bonferroni" pPvalue" is" lower" than"
0.001."Three"independent"biological"replicates"were"performed"for"WT"and"nfya1"mutants,"nonP
inoculated" or" harvested" one" and" six" dpi." For" each" biological" repetition" RNA" samples" were"
extracted" from" roots" of" ten" 15" dayPold" plants." Following" extraction,"RNA" samples" were" checked"
for" their" integrity" on" The" Agilent" 2100" bioanalyzer" according" to" the" Agilent" Technologies"
(Waldbroon,"Germany)."One"μg"of"total"RNA"was"used"to"synthesize"biotinPlabeled"cRNAs"with"the"
OnePcycle"cDNA"synthesis"kit"(Affymetrix,"Santa"Clara,"CA)."Superscript"II"reverse"transcriptase"and"
T7Poligo" (dT)" primers" were" used" to" synthesize" the" single" strand" of" cDNA" at" 42°C" during" 1" hour"
followed" by" the" synthesis" of" the" double" stranded" cDNA" by" using" DNA" ligase," DNA" polymerase" I"
and"RNaseH"during"2"hours"at"16°C."Clean"up"of"the"doublePstranded"cDNA"was"performed"with"
Sample"Cleanup"Module"(Affymetrix)"followed"by"in"vitro"transcription"(IVT)"in"presence"of"biotinP
labeled" UTP" using" GeneChip®" IVT" labelling" Kit" (Affymetrix," Santa" Clara," CA)." Quantity" of" the"
labelledPcRNA" with" RiboGreen®" RNA" Quantification" Reagent" (Turner" Biosystems," Sunnyvale," CA)"
was" determined" after" cleanup" by" the" Sample" Cleanup" Module" (Affymetrix)." Fragmentation" of"
15μg"of"labelledPcRNA"was"carried"out"for"35minutes"at"94°C,"followed"by"hybridization"during"16"
hours" at" 45°C" to" Affymetrix" GeneChip®" Medicago" Genome" Array" representing" approximately""
61,200" probe" sets:" 32,167" M.0 truncatula" EST/mRNAPbased" probe" sets;" 18,733" M.0 truncatula"
IMGAG"and"phase"2/3"BAC"predictionPbased"probe"sets;"1,896"M.0sativa"EST/mRNAbased"probe"
sets;"and"8,305"S.0meliloti"gene"predictionPbased"probe"sets.."After"hybridization,"the"arrays"were"
washed"with"2"different"buffers"(stringent:"6X"SSPE,"0.01%"TweenP20"and"nonPstringent:"100mM"
MES,"0.1M"[Na+],"0.01%"TweenP20)"and"stained"with"a"complex"solution"including"Streptavidin"RP
Phycoerythrin" conjugate" (Invitrogen/molecular" probes," Carlsbad," CA)" and" antiPStreptavidin"
biotinylated"antibody"(Vectors"laboratories,"Burlingame,"CA)."The"washing"and"staining"steps"were"
performed"in"a"GeneChip®"Fluidics"Station"450"(Affymetrix)."The"Affymetrix"GeneChip®"Medicago"
123"
"
Genome" Arrays" were" finally" scanned" with" the" GeneChip®" Scanner" 3000" 7G" piloted" by" the"
Command" Console" Launcher" Tool." The" raw" CEL" files" were" imported" in" R" software" for" data"
analysis.The" data" were" normalized" with" the" gcrma" algorithm," available" in" the" Bioconductor"
package"(Gentleman"&"Hornik,"2002)."To"determine"differentially"expressed"genes,"we"performed"
a"usual"two"group"tPtest"that"assumes"equal"variance"between"groups."The"variance"of"the"gene"
expression"per"group"is"a"homoscedastic"variance,"where"genes"displaying"extreme"variance"(too"
small" or" too" large)" were" excluded." The" raw" pPvalues" were" adjusted" by" the" Bonferroni" method,"
which"controls"the"Family"Wise"Error"Rate"(FWER)."
Transcriptomic&data&analysis&
&
Genes"significantly"displaying"genotype"and"interaction"between"genotype"and"innoculation"
effects" were" clustered" using" gene" expression" ratios" between" nf%ya%1%10 and" A17" both" in" control"
conditions" and" then" ratios" between" one" or" six" dpi" and" the" control" of" each" genotype" respectively."
Cluster"
"
3.0"
"
was"
used"
for"
the"
hierarchical"
clustering"
(http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm)" with" euclidean" distance" and"
average" linkage" parameters" (Eisen," Spellman," Brown," &" Botstein," 1998)" and" Java" Treeview"
(http://sourceforge.net/projects/jtreeview)"was"used"for"visualization"of"the"heatmap"and"to"retrieve"
class" contents." Annotations" used" in" this" study" are" based" on" the" work" of" published" by" Czaja" and"
colleagues"(Czaja"et"al.,"2012).""
"
RNA&extraction&and&quantitative&RT+PCR&
Total" RNAs" were" isolated" from" inoculated" and" nonPinoculated" roots" with" the" “RNeasy" for" plant"
and"fungi”"kit"(Qiagen)."Following"total"RNA"extraction"and"DNAse"treatment,"RNA"integrity"was"
checked" using" a" Bioanalyzer" (Agilent" technologies)" and" reverse" transcriptions" were" performed"
with" 1" µg" of" total" RNA" for" each" sample" using" the" HighPCapacity" cDNA" Reverse" Transcription" Kit"
(Applied"Biosystems)"with"random"primers"in"a"total"volume"of"20"µL."Gene"specific"primers"were"
designed" using" the" Quantprime" software" (S." Arvidsson," 2009)." Then," the" cDNAs" were" used" for"
highPthroughput"qPCR"using"the"BioMarkTM"HD"System"(Fluidigm):"first,"1.3"µL"of"a"1:40"dilution"
of" the" synthesized" cDNA" were" submitted" to" specific" target" amplification" (STA)" by" PCR"
amplification" in" a" 5" µL" reaction" containing" 96" Medicago0 truncatula" specific" primer" pairs" (50" nM"
each)"and"a"twoPfold"dilution"of"the"TaqMan®"PreAmp"Master"Mix"(Applied"Biosystems)."The"PCR"
program" consisted" of" 14" cycles" of" 15" sec" at" 95°C" followed" by" 4" min" at" 60°C." Then," 340" nL" of"
preamplified" cDNA" were" used" for" qPCR" array" analysis" in" a" 6.7" µL" reaction" using" EvaGreen"
chemistry" (Applied" Biosystems)." Data" were" analyzed" with" the" BioMark" RealPTime" PCR" Analysis"
Software" Version" 2.0" (Fluidigm)." Calculations" for" comparing" expression" data" were" performed"
using" the" ΔΔ" cycle" threshold" method" using" a" histone" (Medtr4g097170)" reference" genes," which"
was" selected" using" the" NormFinder" software" (Andersen," Jensen," &" Ørntoft," 2004)." or" relative"
pathogen" transcript" levels" in" each" analysed" sample." Gene" encoding" an" A.0 euteiches" tubuline"
transcript" were" used" to" detect" the" amount" of" each" inoculated" pathogen" at" 6dpi." The" 2PΔΔCT"
method" (Livak" &" Schmittgen," 2001)" was" applied" to" visualize" induction" or" repression" of" gene"
expression" or" to" measure" the" relative" amount" of" pathogen" transcript" accumulation." Primers,"
124"
"
amplicons" length" and" melting" temperature" are" listed" in" (Table" S3)." There" was" design" on"
quantprime"(http://www.quantprime.de/).&
Figures&and&legends&
&
"
Figure& 1:0 Visual" observations" on" nf%ya1%10 mutant," A17" partially" tolerant" wild" type" and" F83"
susceptible"genotype"along"invasion"by"Aphanomyces0euteiches.""
A"–Visuals"symptoms"on"F83,"A17"and"nf%ya%10mutants"14dpi,"bar"="1cm.""
B–"Symptoms"extent"(%)"14dpi"and"dead"plants"21dpi"(%)"(n=150"plants"for"each"genotype)."In"all"
assays,"error"bars"represent"standard"errors."Means"of"each"line"were"compared"by"the"tPtest"for"
symptoms"extent;"and"by"the"χ2" test"for"proportion"of"dead"plants."In"all"graphs"asterisks"indicate"
significant"differences"compared"with"WT"results"(***":"P<0.001;"*":"P<0.05)."
C–"Roots"number"and"length"(in"millimeters)"21"days"after"germination"in"control"conditions"(grey"
bars)" and" 21dpi" (black" bars)." " A" pairwise" wilkoxon" test" was" applied" for" number" and" length" of"
lateral"roots"between"A17"and"the"other"lines"results"(***":"P<0.001;"*":"P<0.05)."
125"
"
"
Figure& 2:" Cytological" observations" on0 nf%ya1%10 mutant," A17" partially" tolerant" wild" type" and" F83"
susceptible"genotype"along"invasion"by"Aphanomyces0euteiches."Ae0is"labelled"in"green"with"WGAP
FITC" (Bar" =" 100µm)." Agarose" embedded" root" cross" sections" are" 100µm" thick." Ae" oospores" are"
indicated"by"an"arrow."
A"–"Root"surface"observation"3"dpi""
B–"Root"cross"sections"6"dpi"(middle)"and"21"dpi"(bottom)"
126"
"
"
Figure& 3:" Complementary" silencing" approaches" on" MtNF%YA1" improve" resistance" level" of" the"
F83005.5" susceptible" line" resist." miR169q,0 MtNF%YA10 0 and" Ae" housekeeping" gene" tubuline"
transcripts"are"quantified"by"RTPqPCR"and"normalized"with"plant"housekeeping"gene"histone"with"
2PΔCT"method"for"miR169q"and"2PΔCT"×103"for"MtNF%YA1"and"Ae0tubuline."
A"–"Overexpression"of"miR169q"reduces"MtNF%YA100"transcripts"accumulation"6dpi"and"decrease"
Ae"development."Experiments"performed"on"three"independent"hairyroots"lines"for"both"control"
vector"and"mir169q"overexpressor."
B–" Silencing" of" MtNF%YA10 0 " by" the" overexpressing" construct" of" antisens" MtNF%YA10 0 " 3’UTR0
decreases" Ae" development." Experiments" performed" with" 12" independent" samples" containing"
each" 10" composites" plants" for" both" control" and" silencing" vector." Wilkoxon" rank" sum" test" was"
applied" for" hairyroots" qRTPPCR" results" (***" :" P<0.001)" and" ANOVA" was" applied" for" " composite"
plants"qRTPPCR"results"(***":"P<0.001)."
127"
"
"
Figure&4:"SpatioPtemporal"expression"pattern"of"MtNF%YA10in"A17"and"F83005.5"in"control"and"Ae"
inoculated"conditions."
A–MtNF%YA%10expression"level"by"RTPqPCR"with"2PΔCT"×103" method"relative"to"(Medtr4g097170)"2P
ΔCT
"
B"–""Histochemical&glucuronidase"staining"under"control"of"MtNF%YA%100promotor"
128"
"
"
Figure&5:"Transcriptomic"analysis"of"A17"and"nf%ya%1%10in"healthy"plants"and"upon"Ae0inoculation.""
Analyses"were"performed"on"gene"which"were"significantly"regulated"(Tuckey"test,"p<0,001)."
A"–"Venn"Diagram"showing"genes"significantly"regulated"upon"inoculation"(I)"and/or"differentially"
expressed"in"healthy"plants"(G"for"genotype"dependent"expression).""
B"–"Hierarchical"clustering"of"genotypePdependent"genes"regulated"upon"Ae"inoculation."Induced"
genes"(log2"ratio>1)"are"in"red,"repressed"genes"(log2"ratio<P1)"are"in"green.""
129"
"
"
Table&1:"List"of"genes"belonging"to"Class"1"and"Class"3"with"their"biological"functions,"Affymetrix"
identifier,"Genomic"identifier,"gene"expression"ratios"considered"in"the"study,"average"expression"
level"and"detailed"annotation."
"
130"
"
"
&
Table& 2:" List" of" genes" belonging" to" Class" 2" with" their" biological" functions," Affymetrix" identifier,"
Genomic" identifier," gene" expression" ratios" considered" in" the" study," average" expression" level" and"
detailed"annotation."
"
"
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
&
131"
"
&
&
Supplementary&Informations&:&
&
Table&S1:&Selection"of"genes"belonging"to"genotype"and"not"genotype"effect"from"transcriptomic"
data"(array)"for"validation"by"RTPqPCR"(Biomark)."Expression"ratios"versus"controls"are"expressed"
in"Log2."Induced"genes"(log2"ratio>1)"are"in"red,"repressed"genes"(log2"ratio"<P1)"are"in"green."
Table& S2:& Whole" dataset" of" Ae" regulated" genes" distributed" along" not" genotype" and" genotype"
effect"in"theirs"respectives"regulatory"groups"and"Mapman"BINs&
Table&S3:&Primers"used"for"pathogen quantification and transcriptomic data validation.&
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Lucas,"M."De"and"Brady,"S.M."(2012)"Gene"regulatory"networks"in"the"Arabidopsis"root."Current"
Opinion"in"Plant"Biology,"1–6."Available"at:"http://dx.doi.org/10.1016/j.pbi.2012.10.007."
Mantovani"R."1999."The"molecular"biology"of"the"CCAATPbinding"factor"NFPY."Gene"239(1):"15P27."
Matuoka"K,"Chen"KY."2002."Transcriptional"regulation"of"cellular"ageing"by"the"CCAAT"boxPbinding"
factor"CBF/NFPY."Ageing"Res"Rev"1(4):"639P651."
Moussart"A,"Onfroy"C,"Lesne"A,"Esquibet"M,"Grenier"E,"Tivoli"B."2007."Host"status"and"reaction"of"
Medicago"truncatula"accessions"to"infection"by"three"major"pathogens"of"pea"(Pisum"sativum)"and"
alfalfa"(Medicago"sativa)."European"Journal"of"Plant"Pathology"117:"57P69."
Siefers"N,"Dang"KK,"Kumimoto"RW,"Bynum"WEt,"Tayrose"G,"Holt"BF,"3rd."2009."TissuePspecific"
expression"patterns"of"Arabidopsis"NFPY"transcription"factors"suggest"potential"for"extensive"
combinatorial"complexity."Plant"Physiology"149(2):"625P641."
Turnpenny,"L."(2008)"What"ID"is","beyond"what"it"is"not"Reannotation"of"array"probes"at"NCBI"’"s"
GEO"database.","5,"2008."
Wenkel,"S.,"Turck,"F.,"Singer,"K.,"Gissot,"L.,"Gourrierec,"J."Le,"Samach,"A."and"Coupland,"G."(2006)"
CONSTANS"and"the"CCAAT"box"binding"complex"share"a"functionally"important"domain"and"
interact"to"regulate"flowering"of"Arabidopsis."The"Plant"cell,"18,"2971–84."Available"at:"
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1693937&tool=pmcentrez&renderty
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135"
"
Zanetti"ME,"Blanco"FA,"Beker"MP,"Battaglia"M,"Aguilar"OM."2010."A"C"subunit"of"the"plant"nuclear"
factor" NFPY" required" for" rhizobial" infection" and" nodule" development" affects" partner" selection" in"
the"common"beanPRhizobium"etli"symbiosis."The"Plant"Cell"22(12):"4142P4157"
Acknowledgements"
"
"
B. Conclusion du chapitre
"
"Au"début"de"ma"thèse,"NF%YA%1"était"considéré"comme"un"acteur"spécifique"de"la"symbiose"fixatrice"
d’azote,"compte"tenu"de"son"faible"niveau"d’expression"dans"des"conditions"de"culture"contrôle"et"de"
sa" forte" induction" transcriptionnelle" au" cours" de" la" symbiose" fixatrice" d’azote." Le" travail" que" j’ai"
réalisé" au" cours" des" trois" dernières" années" a" permis" de" révéler" des" rôles" inattendus" " pour" ce" gène"
dans" le" contrôle" de" l’immunité" végétale" et" le" développement" de" la" plante." Les" données" de"
phénotypage"et"de"transcriptomique"obtenues"visPàPvis"d’A.0euteiches"montrent"que"ce"gène"est"un"
régulateur" négatif" de" l’immunité" ." Son" absence" entraîne" " en" effet" l’activation" constitutive" de"
nombreux" gènes" de" défense." De" plus," les" résultats" indiquent" que" NF%YA1" apparaît" comme" un"
régulateur" négatif" du" développement" racinaire" en" présence" d’A.0 euteiches," une" réponse"
précédemment"associée"à"la"résistance"des"plantes."Le"rôle"de"NF%YA1"sur"les""racines"est"supporté"
par" l’observation" de" son" expression" dans" les" primordia" racinaires." L’altération" de" plusieurs" voies"
hormonales" dont" celle" de" l’acide" abscissique" et" de" l’éthylène" chez" le" mutant" peut" permettre" de"
réconcilier" les" phénotypes" de" l’immunité" et" du" développement" racinaire" observés" chez" le" mutant"
inoculé."La"mise"en"évidence"d’une"augmentation"du"niveau"de"résistance"d’une"plante"à"A.0euteiches0
par"une"simple"mutation"d’un"gène"laisse"entrevoir"des"possibilités"prometteuses"pour"l’amélioration"
génétique"de"la"résistance"du"pois"à"A.0euteiches."
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136"
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Matériels et
Méthodes
137"
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Matériels et Méthodes
0
I. Matériel Biologique
"
A. Microorganismes pathogènes : souche, culture et
inoculation
1. Aphanomyces euteiches
a. Souche et culture
Un" isolat" issu" du" pois" ATCC201684" ou" AePall" a" été" utilisé" au" cours" de" l’ensemble" de" ces" travaux,"
permettant"ainsi"de"s’appuyer"sur"les"résultats"des"travaux"de"Djébali"et0al.en"2009"et"la"description"
de" la" résistance" partielle" d’A17" à" cette" souche." La" culture" de" cet" oomycète" est" maintenue" par" des"
repiquages" successifs" (chaque" 15" à" 21" jours)" sur" milieu" CMA" (Corn" Meal" Agar," Sigma)" à" 24°C" et" à"
l’obscurité." Pour" la" production" de" zoospores," des" explants" pris" directement" sur" milieu" CMA" sont"
repiqués" dans" des" Erlenmeyers" 250" ml" contenant" 30" ml" de" milieu" peptone" glucose" (20g/l" de"
peptones"et"5g/l"de"glucose)"liquide"puis"sont"mis"dans"une"chambre"climatique"à"24°C,"à"l’obscurité"
et"sans"agitation."Après"3"jours"de"croissance,"le"mycélium"obtenu"est"carencé"par"3"lavages"espacés"
de" 2" heures" avec" de" l’eau" commerciale" de" VOLVIC" stérile." Entre" les" lavages," les" erlenmeyers" sont"
remis" dans" la" chambre" climatique" dans" les" mêmes" conditions" de" culture." Au" dernier" lavage," le"
mycélium" est" laissé" dans" 30ml" d’eau" de" VOLVIC" toute" la" nuit" (24°C," obscurité)." Les" zoospores" sont"
récoltées" le" lendemain" dans" l’eau" et" leur" concentration" en" zoospores," déterminée" à" l’aide" d’une"
cellule" FuchsPRosenthal," est" ajustée" à" 105" zoospores/ml" pour" inoculation" des" plantes" cultivées" in0
vitro."Les"filtrats"de"culture"ont"été"préparés"dans"un"milieu"synthétique"composé"(Table"1)"élaboré"
au" cours" de" la" thèse" d’Amaury" Nars." CeluiPci" est" aliquoté" par" 100" ml" dans" des" fioles" de" Roux" puis"
autoclavé"avant"ensemencement"avec"50"000"spores."Les"filtrats"ont"été"prélevés""au"plus"tard"à"21"
jours"ou"dès"lors"que"la"teneur"en"glucose"monomérique"tendait"vers"0."Le"mycélium"a"été"séparé"du"
surnageant"de"culture"par"une"filtration"sur"laine"de"verre"sousPvide."Une"dialyse"de"huit"heures"en"
changeant"régulièrement"l’eau"a"été"réalisée"dans"des"tubes"d’une"taille"d’exclusion"de"un"kilodalton"
(Spectrum)." La" mesure" des" polymères" contenant" du" glucose" et" de" l’hexoseamine" a" été" réalisée" sur"
138"
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hydrolysat"neutralisé"passé"en"HPAECPPAD."Ces"aspects"ainsi"que"les"essais"de"déprotéination"ont"été"
réalisés"par"Amaury"Nars."Il"en"est"de"même"pour"le"dosage"des"teneurs"en"glucose"libre."
"
Table"1":"Composition"du"milieu"synthétique"de"culture"V2"d’Aphanomyces0euteiches"
"
b. Inoculations et notations de symptômes
L’inoculation" in0 vitro" se" fait" sur" des" graines" germées" mises" sur" milieu" M" " (Table" 2)" à" raison" de" 5"
plantules" par" boîte" de" Pétri" carrée" (12cm" par" 12cm)." Au" lendemain" de" leur" transfert" en" boîte," 5μl"
d’une" suspension" de" zoospores" (105" zoospores/ml)," déposés" sur" les" racines" (au" milieu" de" l’assise"
pilifère" des" plantules)." Les" symptômes" sont" mesurés" à" 14" et" 21" jours" après" inoculation." Ces" deux"
temps"ont"été"choisis"par"rapport"aux"notations"des"symptômes"mises"en"place"sur"les"parents"A17"et"
F83005.5" par" Djébali" et0 al.," 2009." Ces" symptômes" correspondent" au" pourcentage" de" brunissement"
présent"sur"la"racine"et"la"tige,"ramené"à"la"longueur"totale"de"la"plante"à"14"jours"et"au"pourcentage"
de" plantes" mortes" relevé" à" 21" jours." A" l’issue" de" l’infection," un" tronçon" d’un" centimètre"
correspondant"à"la"zone"d’inoculation"est"prélevé"pour"mener"les"observations"cytologiques."
"
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139"
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Table"2":"Composition"du"milieu"M"de"culture"de"Medicago0truncatula"
"
"
2. Colletotrichum trifolii
a. Souche et culture
Colletotrichum0 trifolii" est" un" champignon" phytopathogène" parasite" de" la" luzerne" cutivée" Medicago0
sativa" et" de" Medicago" truncatula." Plusieurs" races" reposant" sur" des" spectres" de" compatbilité" de"
lignées" parentales" chez" la" luzerne" ont" été" définies." Nous" nous" sommes" focalisés" sur" la" race" 2" qui"
présente" une" agressivité" reproductible." Le" mycélium" est" maintenu" sur" un" milieu" synthétique" riche,"
appelé"milieu"Mathur"(Table"3),"à"une"température"constante"de"23°C"à"l’obscurité"pour"favoriser"sa"
croissance" ou" à" 4°C" pour" sa" conservation." Dans" le" but" d’obtenir" une" concentration" suffisante" en"
spores," 150ml" de" milieu" sont" déposés" en" fiole" de" Roux" puis" ensemencés" par" une" suspension" de"
conidies" (5ml" à" 106" conidiospores" par" ml)." La" fiole" est" ensuite" placée" à" 23°C" à" l’obscurité." Les"
conidies," produites" après" 7" jours," sont" collectées" dans" de" l’eau" stérile" et" servent" à" la" fois" au"
repiquage"de"chaque"souche"ainsi"qu’à"la"préparation"de"l’inoculum."Les"souches"peuvent"également"
être"conservées"sous"la"forme"de"suspension"conidienne"mélangée"à"du"glycérol"50%"et"stockées"à"P
80°C."Pour"la"production""d’exsudats"de"spores"germées,"les"conidiospores"ont"été"disposées"à"105/ml"
dans" un" erlenmeyer" contenant" 100ml" d’eau" stérile" à" 24°C" à" l’obscurité" pendant" 24" heures." Les"
exsudats" ont" été" récupérés" en" centrifugeant" les" spores" à" 5000g" pendant" 30" minutes" puis" en"
récupérant"le"surnageant"sans"décrocher"le"culot"(cf."article"en"annexe)."
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140"
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Table"3":"Composition"du"milieu"Mathur"de"culture"de"Colletotrichum0trifolii"
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b. Inoculations et notations de symptômes
L’inoculation"in0vitro"avec"C.0trifolii"suit"les"mêmes"modalités"que"celle"avec"A.0euteiches."Cette"fois,"
5µl"d’une"suspension"de"106"conidiospores/ml"est"déposée"au"milieu"de"l’assise"pilifère."Le"diagnostic"
moléculaire" de" C.0 trifolii" évaluant" son" niveau" de" développement" est" réalisé" à" 6" jours" après"
inoculation."Les"notations"de"symptômes"visuels"se"font"entre"15"et"30"jours."
"
"
B. Medicago truncatula
"
1. Origine des lignées sauvages et mutantes
Comme" décrit" en" introduction," les" mutants" utilisés" dans" cette" étude" sont" essentiellement" issus" du"
fonds"génétique"A17."La"mutagénèse"chimique"employée"a"permis"l’obtention"de"mutations"nulles,"le"
plus"souvent"en"introduisant"des"codons"d’arrêts"précoces"qui"aboutissent"à"l’absence"de"traduction"
ou" à" des" protéines" tronquées" non" fonctionelles." Seul" le" mutant" du" gène" VAPYRIN" a" été" obtenu" à"
partir"d’une"banque"issue"de"R108,"obtenue"par"insertion"de"transposons."Ces"différentes"lignées"ont"
été"rétrocroisées"deux"fois"avec"le"sauvage"de"manière"à"éliminer"75%"des"mutations"non"impliquées"
dans" les" phénotypes" symbiotiques." Ainsi," elles" ne" présentent" pas" de" phénotypes" évidents"
(germination," croissance," développement)" qui" puissent" laisser" supposer" une" altération" de" la"
résistance"aux"parasites."
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141"
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2. Germination
Les"gousses"du"genre"Medicago,"renferment"en"moyenne"7"à"8"graines"dont"la"dormance"doit"être"
levée"avant"la"mise"en"germination."Par"ailleurs,"les"plantes"étant"essentiellement"utilisées" in0vitro,"
une" étape" de" stérilisation" est" nécessaire." Dans" un" premier" temps," les" graines" sont" scarifiées" par"
traitement" chimique" à" l’acide" sulfurique" concentré" pendant" 5" minutes" puis" rincées" 3" fois" à" l’eau"
distillée" stérile." Elles" sont" ensuite" stérilisées" par" un" bain" de" 3" min" dans" l’eau" de" Javel" à" 2,5%" puis"
rincées"4"fois"à"l’eau"stérile."Le"dernier"bain"de"rinçage"est"maintenu"pendant"une"heure."A"la"fin"du"
temps" d’imbibition," les" graines" sont" transférées" sur" milieu" gélosé" (agar" 1%)" et" mises" à" sécher"
pendant"30"min."Les"boîtes"sont"ensuite"placées"dans"un"incubateur"à"22°C"et"à"l’obscurité"pendant"2"
jours" au" minimum." Dans" le" cas" de" culture" in0 vitro," des" boîtes" de" Pétri" carrées" (12cm" x" 12cm)"
contenant"du"milieu"de"culture"M,"dépourvu"en"sucre"afin"d’éviter"un"développement"saprophytique,"
sont"préparées"à"accueillir"les"germinations."Les"plantes"sont"alors"cultivées"verticalement"dans"une"
enceinte"climatique"(photopériode"jour"16h"/nuit"8"h,"température"22°/20°C)."
"
3. Transformation par Agrobacterium rhizogenes
Le" protocole" utilisé" est" basé" sur" celui" décrit" par" BoissonPDernier" et0 al.0 (2001)." Des" jeunes"
germinations" de" Medicago0 truncatula0 sont" sectionnées" à" quelques" mm" de" l'apex" racinaire" avec" un"
scapel" stérile," puis" inoculées" avec" des" souches" ARquaI,0 transformées" préalablement" avec" les"
constructions" d'intérêts." L’inoculation" est" réalisée" en" trempant" la" section" faite" sur" la" radicule" dans"
une" culture" liquide" de" ARquaI," préalablement" mise" en" culture" à" 28°C" pendant" la" nuit," dans" un"
erlenmeyer"contenant"100ml"de"milieu"LB"(+"6"mM"de"CaCl2"+"50"mg/l"de"Kanamycine)."Entre"500"et"
1000"jeunes"germinations"par"construction"ont"été"inoculées"et"placées"sur"du"milieu"Farhaeus"(Table"
4)"gélosé""dans"des"boîtes"de"Pétri"carrées"de"12"cm"x"12"cm."Les"boîtes"sont"placées"verticalement"
dans"des"chambres"de"culture"in0vitro0à"20°C"(16"heures"photopériode,"70"μE"sP1.mP2)"pendant"une"à"
deux"semaines"avant"d’être"transférées"à"25°C"jusqu'à"obtention"de"racines"transgéniques"de"2P3"cm"
de"long"(environ"1"à"2"semaines"après"transfert)."
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142"
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Table"4":"Composition"du"milieu"Farhaeus"de"transformation"de"Medicago0truncatula"
"
"
4. Traitements MAMPs
Les"plantules"âgées"de"sept"à"huit"jours"sont"cultivées"en"milieu"M"liquide"(sans"phytagel),"dans"des"
boîtes"de"Pétri"rondes"de"35mm"sous"16"heures"de"jour"à"22°C"et"8"heures"de"nuit"à"20°C. Elles"sont"
préalablement" lavées" dans" l’eau" pendant" deux" heures" avant" traitement." Les" essais" de" cinétique" en"
temps"réel"de"production"de"ROS"ont"été"réalisés""par"chemiluminescence,"grâce"à"un"essai"reposant"
sur" l’oxydation" du" luminol" par" une" péroxydase." Les" plantules" sont" préincubées" dans" 900µl"
peroxydase" de" raifort" à" 1U/ml" (HRP," FlukaPSigma)" et" 22µM" de" luminol" (3PAminophthalhydrazide,"
Sigma)" dans" un" luminomètre" Sirius" (Berthold" Detection" Systems)" avant" addition" de" l’éliciteur" à"
hauteur"de"100µL."Suite"au"traitement,"l’évolution"de"la"luminescence"dans"chaque"échantillon"a"été"
suivie" pendant" une" heure." Les" mesures" en" " point" final" reposent" sur" une" chimie" fluorescente" plus"
sensible"avec"de"l‘Amplex"Red"oxydé"par"une"péroxydase."Cette"fois,"les"plantules"sont"traitées"après"
lavage"avec"1"ml"d’éliciteur"pendant"20"minutes"à"température"ambiante"avant"que"20"µl"du"milieu"
ne" soient" transférés" dans" une" plaque" 96" puits" noire" (Greiner)." Vingt" microlitres" du" mélange"
réactionnel" " de" péroxydase" (4" U/ml" HRP)" et" d’Amplex" Red" (Acetylresofurine," 0.2" mM," Invitrogen)"
préparés" dans" un" tampon" SodiumPPhosphate" (pH=7,5)" sont" injectés" dans" chaque" puits" par"
l’instrument," juste" avant" analyse." La" réaction" étant" cette" fois" instantanée," la" fluorescence" est" lue" à"
600"nm"après"excitation,"à"530"nm"directement"après"agitation"de"la"plaque dans"le"Berthold"TriStar"
LB"941.""
143"
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"
II. Méthodes
A. Cytologie
"
1. Préparation de coupes fines
Les" tronçons" de" racines" prélevées" ont" été" inclus" dans" de" l’agarose" bas" point" de" fusion" à" 5%" de"
concentration" (Agarose" LMP," SIGMA)." La" température" de" solidification" très" basse" de" l’agarose" LMP"
(25°C)" permet" de" préserver" l’intégrité" des" tissus" épidermiques" et" ainsi" d’éviter" de" brûler" les"
premières"couches"de"tissus"lors"de"l’inclusion."Les"échantillons"de"racines"ainsi"préparés"sont"ensuite"
coupés"à"l’aide"d’un"vibratome"(Leica"VTA1000S)"selon"une"épaisseur"de"150"μm."
"
2. Microscopie à épifluorescence
Afin"de"bien"visualiser"l’envahissement"des"racines"par"A.0euteiches,"ce"dernier"a"été"marqué"par"une"
solution"de"WGA"(Wheat"Germ"Agglutinin)"couplée"au"FITC"(Fluorescéine"IsoThioCyanate)"qui"se"fixe"
sur" les" composés" contenant" des" groupements" NPacétylglycosamine" (Molano" et" al." 1980)" présents"
dans" les" chitosaccharides" de" la" paroi" du" mycélium." Après" inclusion" dans" l’agarose" et" coupe," des"
sections" de" racines" de" 150" μm" sont" incubées" dans" une" solution" tampon" pH" 8,2" (TrisPBase," 20mM;"
NaCl," 160mM)," pendant" 15" minutes." Les" sections" racinaires" sont" ensuite" mises" en" présence" de" la"
WGAPFITC" (50μg/ml)" à" l’obscurité." Trois" rinçages" de" 5" min" avec" le" même" tampon" permettent"
d’éliminer"l’excès"de"WGAPFITC."La"visualisation"des"structures"du"parasite"se"fait"par"excitation"des"
coupes,"sous"une"lumière"bleue"(filtre"d’excitation,"BP"450P490"nm,"filtre"de"suppression"LP"515"nm)."
Le" mycélium" d’Aphanomyces" euteiches0 apparaît" en" vert," couleur" due" au" FITC." Les" observations"
microscopiques" en" lumière" blanche" et" en" UV" ont" été" faites" à" l’aide" d’un" microscope" photonique" à"
épifluorescence"(DMIRBE,"Leica,"RueilPMalmaison,"France)."Les"images"sont"saisies"avec"une"caméra"
numérique"CCD"(colour"Coolview,"Photonic"Science,"Robertsbridge,"UK)."
"
3. Coloration glucuronidase
Les"racines"traitées"ou"inoculées"ont"été"prélevées"et"incubées"à"37°C,"dans"un"tampon"à"fort"pouvoir"
oxydant" (ferricyanure" de" potassium" 0,5" mM," ferrocyanure" de" potassium" 0,5" mM," Na2EDTA" 5" mM,"
144"
"
tampon"phosphate"de"potassium"KPi"0,1"M"pH"7.0)"contenant"1"mM"du"substrat"de"l’enzyme,"le"XP
Gluc" (5PbromoP4PchloroP3PindoxylPβPDPglucuronide," Biosynth" AG," Staad," Suisse)." Le" produit" de" la"
réaction"conduit"à"la"formation"de"précipité"bleu"qui"peut"être"détecté"in0situ."
"
B. Biologie moléculaire
"
1. RT-qPCR
La" reverse" transcription" d’ARNs" totaux," suivie" d’une" quantification" de" produits" PCR" au" travers" d’un"
intercalant"fluorescent,"est"utilisée"pour"étudier"les"régulations"géniques."Les""messagers"sont"ciblés""
par"des"couples"de"primers"spécifiques"permettant"de"déterminer"la"quantité"de"transcrits"issus"d’un"
gène"de"manière"absolue"ou"relative,"c'estPàPdire"par"rapport"à"un"transcrit"exprimé"stablement."Les"
trois" grandes" étapes" (extraction" ARN," reverse" transcription" et" PCR" quantitative)" de" cette" approche"
sont"décrites"ciPdessous."
a. Extraction des ARNs
L’extraction" de" l’ARN" a" été" faite" à" partir" de" racines" de" plantes" âgées" d’au" moins" 15" jours" afin"
d’obtenir" un" poids" frais" racinaire" de" 120" mg" nécessaire" à" l’obtention" d’une" quantité" suffisante"
d’ARNs." La" récolte" a" été" faite" à" 1," 3" et" 6" jours" après" inoculation." Le" diagnostic" moléculaire," par"
quantification"de"transcrits"des"parasites"est"réalisé"sur"des"racines"entières"prélevées"6"jours"après"
inoculation"et"immédiatement"congelées"dans"l’azote"liquide."Les"expressions"géniques"ont"pu"être"
réalisées"sur"les"plantes"utilisées"en"mesure"point"final""de"ROS"puiqu’elles"ne"sont"jamais"au"contact"
des" réactifs." Le" prélèvement" a" été" fait" quatre" heures" après" traitement" puis" les" échantillons" sont"
stockés"à"P80°C."L’extraction"des"ARNs"a"été"faite"avec"le"Kit"RNeasy"de"QUIAGEN"(minikit"for"plant"
and" fungi)" selon" les" instructions" du" fabriquant." Pour" optimiser" les" concentrations," les" acides"
nucléiques" sont" repris" dans" le" volume" minimal" de" 30µl" d’eau" UHQ," nuclease" free" et" doivent" être" à"
une" concentration" supérieure" à" 100ng/µl." Ce" rendement" total" obtenu" est" déterminé" par"
spectrophotomètre"à"une"longueur"d’onde"de"260nm,"ainsi"1"unité"d’absorbance"est"équivalente"à"
40μg"d’ARN"simple"brin/ml."L’absence"de"contamination"des"échantillons"d’ARN"par"des"protéines"est"
appréciée" en" calculant" le" rapport" A260/A280." Pour" une" bonne" qualité" des" échantillons," ce" rapport"
doit" être" compris" entre" 1,7" et" 2,1." L’absence" de" contamination" des" échantillons" d’ARN" par" des"
phenols"est"appréciée"en"calculant"le"rapport"A260/A230."Pour"une"bonne"qualité"des"échantillons,"
ce"rapport"doit"être"compris"entre"1,8"et"2,2."L’absence"de"contamination"des"échantillons"d’ARN"par"
l’ADN" génomique" est" assurée" à" l’aide" d’une" étape" DNAse" comprise" dans" le" kit." Enfin," l’intégrité" de"
145"
"
l’ARN" est" vérifiée" par" Bioanalyzer" (Agilent" technologies) avec" le" concours" de" la" plateforme"
génomique."
b. Reverse transcription
La" rétrotranscription" (ou" reversetransciption)" permet" d’obtenir" de" l’ADN" complémentaire" (ADNc)" à"
partir"de"l’ARN"grâce"à"la"Reverse"transcriptase."Cette"étape"a"été"réalisée"avec"le"kit""High"capacity"
reverse" transcription& (Applied" Biosystem)" en" utilisant" 1µg" d’ARN" selon" les" recommandations" du"
fournisseur." Les" 20µl" d’ADNc" obtenus" ont" été" dilués" 50" fois" pour" pouvoir" tester" l’expression" d’une"
centaine"de"gènes"différents"et"limiter"la"teneur"en"EDTA,"un"chélateur"d’ions"divalents"nécessaire"à"
la" reverse" transcription" mais" aussi" inhibiteur" de" la" PCR." A" noter" que" ce" kit" repose" sur" l’emploi"
d’hexanucléotides" aléatoires" permettant" l’amplification" de" tous" les" ARNs" de" manière" indifférenciée"
et"des"parties"3’"comme"5’."
c. qPCR
Une"étape"cruciale"de"la"PCR"quantitative"est"la"construction"des"couples"de"primers"ciblant"les"gènes"
d’intérêt." Ces" couples" ont" été" construits" en" utilisant" l’outil" Primer" Blast" du" NCBI"
(http://www.ncbi.nlm.nih.gov/tools/primerPblast/index.cgi?LINK_LOC=BlastHome)." Les" conditions"
standards"ont"été"adoptées"en"utilisant"des"amplicons"compris"entre"50""et"200"nucléotides"et"la"base"
de" données" nr," en" spécifiant" les" taxides" 3880" (Medicago0 truncatula)" et" 100881" (Aphanomyces0
euteiches)"et"5466"(Colletotrichum0trifolii)"pour"obtenir"des"primers"spécifiques"entre"ces"organismes."
Les" gènes" d’intérêt" ont" été" isolés" à" partir" des" identifiants" Affymetrix" issus" des" puces"
transcriptomiques." Les" séquences" utilisées" pour" la" recherche" d’amorces" sont" issues" des" séquences"
MTGI" correspondantes" isolées" à" l’aide" de" l’outil" «"nicknames"»" de" l’onglet" «"Gateway"»" de" Legoo"
(http://www.legoo.org)." La" PCR" quantitative" a" été" réalisée"sur" un" ABI7900HT" (Applied" Biosystem)"
selon" les" conditions" standard" d’utilisation" (95°C" 5’," 60°C" 30’’" puis" 95°C" 30’’" 40X)." La" spécificité" des"
amplicons" obtenus" par" qPCR" et" l’absence" de" contaminants" d’ADNg" ont" été" suivie" par" l’obtention"
d’une"courbe"de"fusion"des"amplicons"de"60"à"95°C."En"plus"de"la"détermination"de"la"quantité"d’ADN"
accumulée"après"un"nombre"déterminé"de"cycles"PCR,"la"PCR"en"temps"réel"détermine"aussi"le"point"
à" partir" duquel" les" amplicons" sont" détectés." Ceci" est" déterminé" par" le" nombre" de" cycles" après"
lesquels" l’émission" du" rapporteur" dépasse" le" bruit" de" fond." Ce" nombre" de" cycles" est" indiqué" par" le""
«cycle"threshold"»" (Ct)." Le" Ct" est" déterminé" au" cours" de" la" phase" exponentielle" de" la" PCR" et" il" est"
inversement"proportionnel"au"nombre"de"copies"de"l’ADN"cible."Ainsi,"plus"le"nombre"initial"de"copies"
est" élevé," plus" la" fluorescence" sera" détectée" rapidement" au" cours" des" cycles" successifs"
d’amplification"et"plus"le"Ct"sera"faible."Le"niveau"d’expression"des"différents"gènes"a"été"étudié"en"
utilisant" le" Kit" Power" SYBR" Green" " (Applied" Biosystem)" selon" les" instructions" du" fabricant."
L’accumulation"des"amplicons"d’un"gène"donné"est"déterminée"par"la"quantité"de"lumière"émise"par"
146"
"
le" SYBR" Green," couplé" aux" nucléotides," au" moment" de" son" incorporation" dans" les" brins" d’ADN"
néoformés."Plusieurs"gènes"de"calibration"ont"été"utilisés"pour"calibrer"l’expression"des"gènes"cibles"
entre" échantillons" et" conditions." L’analyse" des" données" de" l’amplification" des" cDNA" est" faite" par" le"
logiciel"SDS2.2"qui"donne"à"la"fin"un"tableau"qui"contient"le"Ct"pour"chaque"échantillon"amplifié."Le"
niveau" d’expression" des" gènes" est" obtenu" en" utilisant" la" technique" développée" par" Livak" et"
Schmittgen" (2001)" qui" permet" de" calculer" la" valeur" de" ΔΔCt" pour" chaque" gène" étudié." CellePci"
représente" un" ratio" entre" le" niveau" d’expression" d’un" gène" d’intérêt" standardisé" par" rapport" à" un"
gène"calibrateur"d’une"condition"expérimentale"par"rapport"à"la"condition"contrôle."La"qPCR"reposant"
sur"un"doublement"exponentiel"du"nombre"d’amplicons"cycle"par"cycle,"le"niveau"d’induction"ou"de"
répression" est" égal" à" 2(PΔΔCt)," cette" valeur" peut" ainsi" être" convertie" en" logarithme" de" base" 2" pour"
permettre" des" représentations" graphiques" conjointes" de" gènes" présentant" des" régulations"
héthérogènes." " Dans" ce" cas," un" gène" non" régulé" a" une" valeur" de" 0," un" gène" dont" l’expression" est"
doublée"a"une"valeur"de"1"et"un"gène"dont"l’expression"est"divisée"par"2,"une"valeur"de"P1."
d. Biomark (qPCR haut debit)
Le"set"de"primer"établi"pour"l’analyse"BioMarkTM"HD"System"(Fluidgm)"repose"sur"les"paramétrages"
par" défaut" du" logiciel" QuantPrime" (http://www.quantprime.de/)" en" utilisant" comme" base" de"
données"l’annotation"du"génome"de"Medicago0truncatula"Mt3.5v4."Les"cDNA"sont,"cette"fois,"dilués"
40"fois"et"1,3"µl"de"cette"dilution"est"soumis"à"une"préamplification"spécifique"(STA,"Specific"Target""
Amplification)"des"cibles"dans"un"volume"réactionnel"de"5µl"contenant"96"couples"d’amorces"à"50nM"
chacune" et" une" dilution" au" demi" du" TaqMan®" PreAmp" Master" Mix" (Applied" Biosystems)." Le"
programme"de"cette"préamplification"comprend"14"cycles"de"15’’"à"95°C"suivis"de"4’"à"60°C."Ensuite,"
340"nl"d’ADNc"sont"dilués"dans"6,7"µl"de"réactif"qPCR"EvaGreen"chemistry"(Applied"Biosystems)."Puis"
la"puce"est"chargée"par"les"soins"du"personnel"de"la"plateforme"génomique"de"Toulouse."Les"résultats"
sont"validés"et"visualisés"par"le"BioMark"RealPTime"PCR"Analysis"Software"Version"2.0"(Fluidigm)."La"
calibration"a"été"réalisée,""comme"précédemment,"en"utilisant"le"gène"de"ménage"Medtr4g097170,"
sélectionné"par"l’utilisation"de"Normfinder"(http://www.mdl.dk/publicationsnormfinder.htm)."
"
2. Analyse de données transcriptomiques
Concernant" le" projet" NFP," les" gènes" régulés" ont" été" identifiés" en" réalisant" des" ratios" d’expression"
entre" plantes" contrôles" et" inoculées" des" deux" génotypes," suivis" d’un" test" statistique" de" Bonferroni"
dont"la"valeur"de"pPvalue"a"été"corrigée"par"le"nombre"de"gènes"présents"sur"la"puce":"dans"notre"cas,"
le" seuil" de" significativité" retenu" était" donc" de" 0,05/50900" soit" 8,3×10P7." Les" fonctions" des" gènes"
différentiellement" régulés" entre" les" deux" plantes" ont" été" assignées" à" l’aide" du" logiciel" MAPMAN"
147"
"
(http://mapman.gabipd.org)." La" comparaison" des" transcriptomes" A17" et" nf%ya1%10 " a" nécessité" un"
autre"procédé"d’analyse,"en"raison"de"l’existence"de"3"points"de"cinétique"pour"chaque"génotype""(0,"
1" et" 6" jai)" ce" qui" augmente" le" nombre" de" conditions" à" traiter." On" recherche," pour" chaque" gène," le"
type"d’effet"qui"module"son"expression"au"travers"d’une"analyse"de"variance""sur"les"ratios"entre"tous"
les" temps" et" tous" les" génotypes," suivi" d’un" test" de" Tuckey" (p<0,001)(Collaboration" avec" Marie"
Françoise" Jardineau," LIPM," Toulouse)." Les" effets" recherchés" sont" l’inoculation," le" génotype" et"
l’interaction"entre"le"génotype"et"l’inoculation"."Une"fois"les"gènes"régulés"triés"en"fonction"des"effets"
qui"les"influencent,"on"peut"tirer"profit"des"trois"points"de"cinétique"obtenus"pour"le"sauvage"A17"et"
son"mutant"nf%ya1%1,"en"réalisant"des"ratios"entre"plantes"contrôles"et"aux"deux"points"d’inoculation"
pour" chaque" génotype" ainsi" qu’un" ratio" contrôle" à" t=0" entre" les" deux" plantes." Des"
clusterings"" hiérarchiques" reposant" sur" un" calcul" de" distances" euclidiennes" et" une" liaison" par" la"
moyenne"
de"
ces"
distances"
ont"
été"
réalisés"
à"
l’aide"
du"
logiciel"
Cluster3.0"""
(http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm)."Les"distances"entre"gènes"et"entre"
conditions" ont" été" utilisées" pour" construire" des" "heatmaps"" sous" Java" Treeview"
(http://sourceforge.net/projects/jtreeview)."Une"table"de"contingence"a"été"testée""au"travers"d’une"
loi"hypergéométrique"pour"déceler"les"classes"fonctionnelles"significativement"enrichies"au"sein"des"
clusters"de"régulations"identifiés"(Maxime"Bonhomme)."
148"
"
"
Conclusions
Générales et
Perspectives
149"
"
Conclusions générales et perspectives
"
Les" déterminismes" sousPtendant" le" dialogue" moléculaire," la" signalisation" et" la" plasticité" de" réponse"
des"plantes,"que"ce"soit"dans"la"mise"en"oeuvre"d’une"réponse"immunitaire"ou"dans"l’établissement"
d’une"symbiose"mutualiste,"présentent"de"nombreuses"similitudes."Ce"constat,"construit"au"cours"des"
vingt"dernières"années,"a"permis"de"soulever"l’hypothèse"de"l’existence"de"gènes"clés"qui"pourraient"
intervenir" dans" la" réponse" aux" microorganismes" parasites" et" mutualistes." Pour" répondre" à" cette"
hypothèse,"l’objectif"initial"de"mon"projet"de"thèse"était"de"confronter"des"plantes"déficientes"dans"
les" symbioses" mutualistes" à" des" parasites" pour" évaluer" l’impact" des" mutations" sur" la" réponse"
immunitaire."Les"données"générées"par"cette"approche"de"génétique"inverse"chez"des"mutants"de"M.0
truncatula"criblés"par"A.0euteiches"ont"permis"d’obtenir"des"indications"sur"la"contribution"éventuelle"
des"gènes"symbiotiques"mutés"sur"le"niveau"de"résistance"quantitative"qui"s’exprime"chez"A17."Les"
expériences"de"phénotypage"ont"pu"permettre"de"répondre"aux"deux"questions"suivantes":"""
•
La"voie"de"signalisation"nécessaire"à"l’établissement"des"symbioses"interagitPelle"avec"la"mise"
en"œuvre"de"l’immunité"?""
"
•
EstPce" que" les" acteurs" moléculaires" végétaux" qui" interviennent" dans" le" contrôle" de" la"
colonisation" " des" tissus" racinaires" par" les" microbes" bénéfiques" participent" au" processus"
d’invasion"par"les"agents"pathogènes"?""
Pour" ce" qui" est" de" la" première" question," il" est" apparu" que" les" mutations" des" gènes" NFP0 " et" DMI2"
entraînent"un"accroissement"de"la"sensibilité"à"Aphanomyces0euteiches."Ces"deux"gènes"codent"des"
récepteurs" membranaires," ce" qui" suggère" qu’ils" sont" capables" de" reconnaître" des" signaux" issus" du"
parasite" afin" de" promouvoir" une" réponse" immunitaire," en" parallèle" à" leur" activité" de" récepteurs""
nécessaires" à" la" réponse" aux" lipochitooligosaccharides" bactériens" et" mycorhiziens." La" découverte"
récente"du"rôle"des"petits"chitooligosaccarides"dans"l’activation"du"calcium"spiking"de"manière"NFP"
indépendante"mais"DMI2"dépendante"souligne"l’aptitude""similaires"des"petits"COs"et"LCOs"à"activer"
la" signalisation" symbiotique," pour" le" moins" jusqu’à" l’étape" du" calcium" spiking" situé" en" amont" de"
DMI3." Si" NFP" n’est" pas" le" récepteur" de" ces" petits" COs," il" est" probable" qu’un" autre" ou" d’autres"
récepteurs" de" sa" famille" jouent" ce" rôle." Dans" tous" les" cas" de" figure," DMI20 pourrait" tenir" une" place"
cruciale"en"aval"de"ces"récepteurs"dans"la"transmission"du"signal."Ainsi,"le"phénotype"de"sensibilité"de"
dmi2"pourrait"être"lié"à"l’incapacité"de"cette"plante"à"transmettre"le"signal"issu"de"la"perception"d’un"
composé" par" NFP" dont" la" mutation" accroît" aussi" la" sensibilité" ou" bien" à" transmettre" un" signal"
150"
"
déclenché" par" un" autre" récepteur," sans" doute" aussi" de" type" LysMPRLK." En" parallèle," des" travaux"
initiés" dans" l’équipe" étudient" l’existence" d’un" calcium" spiking" en" réponse" à" des" préparations" de"
chitosaccharides" d’Aphanomyces0 euteiches." Les" résultats" encourageants" obtenus" dans" le" cadre" de"
cette" collaboration" entre" Arnaud" Bottin" et" le" groupe" de" David" Barker" suggèrent" que" la" perception"
d’Apahanomyces0 euteiches0 est" effectivement" susceptible" d’emprunter" ce" type" de" signalisation"
calcique." Si" ces" essais" venaient" à" être" finalisés," il" serait" très" intéressant" de" caractériser" le" calcium"
spiking"en"réponse"à"A.0euteiches"dans"les"mutants"nfp"et"dmi2"pour"valider"le"rôle"de"ces"récepteurs"
dans"la"perception"et"la"signalisation"de"composés"produits"par"l’oomycète."
"L’étude"des"mutants"de"DMI3,"NSP1"et"NSP2"n’a"pas"permis"de"déceler"de"phénotypes"significatifs."
Néanmoins," les" moyennes" obtenues" pour" les" mutants" de" ces" trois" gènes" semblent" pourtant"
inférieures"à"celle"d’A17"et"mériteraient"probablement"d’être"étudiées"sur"un"plus"grand"nombre"de"
plantes"pour"voir"si"l’augmentation"de"résistance"suggérée"dans"ces"plantes"deviendrait"significative."
Dans"ce"cas,"les"gènes"en"amont"de"la"signalisation"symbiotique"apparaîtraient"comme"des"facteurs"
impliqués" dans" l’immunité" alors" que" ceux" en" aval" sembleraient" " plutôt" jouer" un" rôle" dans" la"
sensibilité" ou" dans" la" compatibilité" de" l’interaction." Plusieurs" études" " récentes" ont" pointé" pour" ces"
derniers" des" rôles" annexes" à" la" signalisation" symbiotique," fournissant" " par" la" même" occasion" des"
éléments" de" réponse" à" ce" découplage" de" l’effet" de" la" voie" de" signalisation" symbiotique" dans"
l’immunité." Par" exemple," les" NSPs" contrôlent" la" synthèse" de" strigolactones." DMI3," en" plus" de"
percevoir" les" lipochitooligosaccharides" à" des" seuils" de" concentration" inférieurs" à" la" plante" sauvage"
chez"Medicago0truncatula,"a"un"orthologue"impliqué"dans"la"signalisation"de"l’acide"absicique"et"dans"
l’homéostasie" des" ROS" (Shi" et" al.," 2012)." Ces" fonctions" variées" qui" ne" dépendent" pas" de" stimuli"
symbiotiques" pourraient" contrôler" des" mécanismes" relatifs" à" la" résistance" à" A.0 euteiches." Ainsi" les"
phénotypes"obtenus"pour"les"mutants"de"ces"gènes""et"l’existence"d’un"calcium"spiking"en"réponse"à"
des" signaux" de" l’oomycète" suggèrent" que" la" voie" de" signalisation" commune" à" la" symbiose" et" à"
l’immunité"bifurquerait"en"aval"du"calcium"spiking"mais"en"amont"de"DMI3."Les"gènes"en"aval"de"la"
voie"pourraient"tout"de"même"influencer"l’issue"de"l’interaction"avec"le"parasite"au"travers"des"rôles"
non"symbiotiques"qu’il"joue"sur"la"physiologie"de"l’hôte."
Les" acteurs" du" contrôle" de" la" colonisation" " des" tissus" racinaires" par" les" microbes" bénéfiques"
participentPils"au"processus"d’invasion"par"les"agents"pathogènes"?"Cette"interrogation""est"suscitée"
depuis" de" nombreuses" années" par" les" similitudes" observables" au" niveau" cytologique," par" exemple"
entre"le"développement"de"la"mycorhization"et"celui"de"parasites"filamenteux"biotrophes"ou"bien"des"
galles"provoquées"par"les"nématodes"et"les"nodosités"fixatrices"d’azote."Ici,"la"collection"de"mutants"
qui"a"pu"être"criblée""s’inscrit"dans"des"fonctions"variées,"allant"des"signalisations"hormonales"à"des"
régulateurs" de" réponses" cellulaires" de" l’hôte" qui" sont" nécessaires" au" développement" du" microbe."
151"
"
Outre" les" réarrangements" observés" au" sein" des" tissus" végétaux," il" apparaît" que" l’architecture"
racinaire" ellePmême" est" modifiée" afin" de" favoriser" les" interactions" bénéfiques" ou" de" survivre" aux"
parasites." En" ce" sens," les" phénotypes" distincts" mais" associés" à" une" meilleure" résistance" à"
Aphanomyces0 euteiches0 observés" chez" latd," efd0 et" cre1" appellent" une" caractérisation" fonctionnelle"
plus" poussée" des" réseaux" de" gènes" sousPjacents" à" cette" composante" développementale" de" la"
résistance" à" la" maladie." Parmi" ces" gènes" d’organogénèse" symbiotique," une" attention" particulière" a"
été" " accordée" à" NF%YA1." Initialement" identifié" comme" une" noduline" précoce," fortement" induite" en"
présence"de"Sinorhizobium0meliloti0ou"de"ses"lipochitochitooligosaccharides,"il"est"décrit"comme"un"
régulateur"du"développement"nodulaire."Les"études"entreprises"dernièrement"ont"révélé"le"rôle"de"
ce"gène"dans"la"progression"de"l’infection"par"la"bactérie"mais"aussi"sa"contribution"dans"l’activation"
de" la" signalisation" symbiotique." Ainsi" ce" gène" apparaît" comme" un" nœud" important" de" régulation"
entre" signalisation" et" organogénèse" de" la" symbiose" fixatrice" d’azote." La" résistance" accrue" à" A.0
euteiches," observée" en" l’absence" de" l’expression" de" ce" gène" couplée" au" meilleur" développement""
racinaire"comparé"au"sauvage,"suggère"que"ce"gène"puisse"aussi"contrôler"des"processus"biologiques"
allant"de"l’immunité"au"développement"du"système"racinaire."L’étude"détaillée"du"transcriptome"du"
mutant"de""NF%YA%1"en"condition"standard"et"d’interaction"avec"A.0euteiches"a"permis"de"pointer"la"
dérégulation" de" gènes" qui" pourraient" participer" à" l’expression" des" phénotypes" observés." La"
caractérisation" du" complexe" de" régulation" transcriptionnelle" NF%Y" est" actuellement" approfondie" au"
travers" d’approches" transcriptomiques," d’études" des" interactions" protéinesPprotéines" et" de"
séquençages" de" sites" de" liaison" à" l’ADN" des" facteurs" de" transcription." L’ensemble" de" ces" données"
pourrait"permettre"à"moyen"terme"d’identifier"les"cibles"de"NF%YA1"afin"de"comprendre"précisement"
son"rôle"dans"l’interaction"avec"A.0euteiches."
Outre" ces" résultats," les" perspectives" à" venir" dans" l’étude" des" relations" croisées" entre" symbioses"
mutualistes" et" parasitaires" pourraient" s’appuyer" sur" l’émergence" de" nouveaux" outils." Par" exemple,"
DMI2" fait" actuellement" l’objet" d’études" biochimiques" poussées" vers" l’identification" de" ses"
partenaires"de"signalisation"(Riely"et"al.,"2012)."Des"progrès"conséquents"dans"l’étude"des"liaisons"des"
ligands"à"NAG""sur"les"domaines"LysM"ont"été"accomplis"(Broghammer"et"al.,"2012),"l’étude"du"rôle"
dans" l’immunité" de" certains" récepteurs" LysMPRLKs" est" également" en" cours":" LYR3" et" LYK9"
(communications" personnelles," Delphine" Pitorre," Clare" Gough," Judith" Fliegmann)." De" même," les"
analyses" transcriptomiques" de" lyr3" " en" présence" " d’A." euteiches" et" des" mutants" nfp" visPàPvis" de"
chitosaccharides" classiques" de" différentes" longueurs" ou" issus" d’Aphanomyces0 euteiches0 devraient"
permettre"très"prochainement"de"mieux"comprendre"la"manière"dont"la"plante"discrimine"les"signaux"
contenant" des" COs" d’origines" différentes." En" vue" de" rechercher" des" MAMPs" produits" " de" manière"
naturelle"par"les"parasites"et"perçus"via"NFP,0nous"avons"étendu"l’étude"à"des"filtrats"de"culture"de"
152"
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l’oomycète"et"des"exsudats"de"spores"germées"de"Colletotrichum0trifolii."Une"des"conclusions"de"ces"
essais"est"qu’il"est"difficile"de"standardiser"la"composition"des"filtrats"d’A.0euteiches0sur"des"cinétiques"
de"croissance"longue,"et"que"la"grande"complexité"biochimique"des"échantillons"obtenus"rend"difficile"
l’identification"de"molécules"«"signal"»."A""l’inverse,"des"petits"chitosaccharides"ont"été"identifiés"dans"
les" préparations" issues" du" champignon" " sans" qu’ils" induisent" pour" autant" un" calcium" spiking." Pour"
avoir"des"exsudats"moins"complexes"à"analyser,"il"serait"sans"doute"opportun"d’étudier"les"MAMPs"
relargués" par" l’oomycète" dans" son" milieu," également" à" partir" de" spores" germées." La" mise" au" point"
récente" au" sein" du" groupe," de" méthodes" d’élicitation" de" protoplastes" de" Medicago0 truncatula" qui"
peuvent" être" transfectés" avec" des" constructions" de" surexpression" ou" de" silencing" devraient"
également" permettre" de" mieux" analyser" la" réponse" aux" MAMPs," quelle" que" soit" leur" origine." En"
résumé," une" combinaison" d’analyses" biochimiques" des" molécules" libérées" par" les" microbes" et" des"
apports" génétiques" et" transcriptomiques" " issus" de" la" plante" devrait" aider" à" clarifier" le" rôle" de" ces"
signaux"et"de"leurs"récepteurs"potentiels"dans"la"symbiose"et"l’immunité.""
Pour" conclure" de" manière" plus" générale," la" génétique" inverse" s’est" révélée" " être" un" point" d’entrée"
efficace"pour"l’étude"de"ma"thématique"de"recherche."Cependant,"nous"devons"garder"à"l’esprit"que"
seuls" des" acteurs" préPidentifiés" dans" le" mutualisme" ont" été" travaillés" quant" à" leur" rôle" dans"
l’immunité." Ainsi," il" est" fort" probable" que" des" fonctions" clés" à" la" fois" pour" la" résistance" ou"
l’établissement" des" maladies" et" pour" les" symbioses" mutualistes" restent" " à" découvrir." L’émergence""
des" études" de" type" Genome" Wide" Association" Study" (GWAS)" qui" reposent" sur" la" cartographie"
génétique" de" grandes" collections" de" lignées" sauvages" récoltées" au" sein" de" conditions"
environnementales"variées"(Trontin,"Tisné,"Bach,"&"Loudet,"2011)"avec"des"marqueurs"de"types"SNPs"
(Single" Nucleotide" Polymorphism)" doit" permettre" de" rechercher" de" tels" gènes" dans" une" démarche"
sans" a0 priori." Ainsi" la" variabilité" génétique" de" M.0 truncatula" et" une" puissance" d’analyse" sans"
précédent" vont" être" mises" au" service" de" la" recherche" de" régulateurs" globaux" des" interactions" des"
plantes" avec" leur" environnement" biotique." Plusieurs" types" d’analyses" peuvent" être" envisagés," à"
commencer" par" une" comparaison" des" loci" détectés" suite" à" des" criblages" séparés" visPàPvis" de" la"
symbiose"fixatrice"d’azote,"de"la"mycorhization"et"de"parasites"racinaires."Par"la"suite,"l’effet"de"coP
inoculations" de" symbiotes" parasites" et" mutualistes," commensaux" ou" indirectement" bénéfiques""
pourrait" être" étudié" pour" voir" si" la" présence" de" différents" couples" ou" groupes" de" partenaires"
influencent" l’identité" des" gènes" clés" contrôlant" les" phénotypes" détectés." Eventuellement," l’effet" de"
molécules" «"signals"»" tels" que" les" lipochitooligosaccarides," flavonoïdes" ou" strigolactones" pourrait"
aussi"être"évalué"sur"les"collections"d’accessions"en"choisissant""par"exemple"un"criblage"basé"sur"le"
développement" racinaire." Un" tel" criblage" sera" bientôt" mis" en" place" dans" l’équipe" et" les" résultats"
obtenus"seront"ensuite"comparés"à"ceux"obtenus"avec"A.0euteiches"afin"d’identifier"éventuellement"
153"
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des" acteurs" moléculaires" communs" dans" la" réponse" à" ces" deux" conditions." Enfin," pour" mieux"
comprendre" la" modulation" des" réponses" de" la" plante" au" mélange" de" stimulations" par" plusieurs"
facteurs"biotiques,"tels"qu’ils"peuvent"exister"dans"le"sol,"des"analyses"transcriptomiques"étudiant"la"
réponse"de"la"plante"à"plusieurs"microorganismes"simultanément"pourront"être"entreprises"(Schenk,"
Carvalhais,"&"Kazan,"2012).""En"conclusion,"les"travaux"présentés"dans"cette"thèse"ont"jeté"les"bases"
fondamentales" de" l’identification" des" ponts" moléculaires" qui" existent" chez" la" plante" pour" lui"
permettre" de" faire" face" aux" microorganismes" qu’elle" rencontre." Ces" données" devront" également" ,"
dans" le" futur," être" prises" en" compte" sur" un" plan" plus" appliqué," pour" éviter" par" exemple" que" la"
recherche"d’un"meilleur"niveau"de"résistance"à"un"parasite"chez"les"légumineuses"n’entraînent"une"
perte" d’efficacité" dans" la" faculté" qu’ont" ces" dernières" à" établir" efficacement" des" interactions"
symbiotiques."
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Annexes
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Annexes
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I. Participation à divers articles
"
Au"cours"de"mes"travaux"de"recherche,"j’ai"été"amené"à"utiliser"des"méthodes"en"développement""tel"
que"le"bioessai"mis"au"point"par"Amaury"Nars,"tout"comme"j’ai"pu"apporter"du"matériel"biologique"
utile"à"des"projets"proches"du"mien"comme"l’étude"de"la"perception"des"petits"chitosaccharides"par"
Medicago0 truncatula" ou" l’analyse" de" la" régulation" de" gènes" LysM" tel" que" LYR3" (Collaboration" avec"
Delphine"Pitorre,"groupe"de"Clare"Gough)"et"à"des"posters"lors"de"congrès."Ces"échanges"ont"donné"
lieu"à"des"manuscrits"en"cours"de"relecture"ou"de"préparation."D’autres"projets"tels"que"l’étude"du"
mutant" cre1" ne" sont" pas" encore" complètement" achevés" mais" un" supplément" d’information" est"
proposé"ici"en"attendant"que"ces"projets"se"finalisent."
"
A. Etude de la perception de chitosaccharides de petits
degrés de polymérisation chez Medicago truncatula
"
Ma"participation"à"ce"projet"a"consisté"à""obtenir,"pour"les"besoins"de"l’étude,"les"exsudats"de"spores"
germées" de" Colletotrichum0 trifolii," tirant" profit" de" mes" connaissances" sur" la" manipulation" de" ce"
champignon." Les" collaborateurs" de" Genre" et0 al." ont" ainsi" pu" mettre" en" évidence" que" le" parasite"
libère," lors" de" sa" germination," des" chitooligosaccharides" de" degré" de" polymerisation" inférieur" à" 6,"
comme" les" champignons" mycorhiziens." Si" les" exsudats" de" ce" dernier" sont" capables" d’induire" un"
calcium" spiking" chez" les" cellules" de" l’hôte," ceux" de" Colletotrichum0 trifolii" ne" le" sont" pas," y" compris"
lorsque"ces"exsudats"sont"supplémentés"en"petits"chitooligosaccharidess"exogènes."Il"a"aussi"été"mis"
en" évidence" que" la" libération" de" petits" chitooligosaccharides" par" le" champignon" mycorhizien" est"
stimulée" par" la" présence" de" strigolactones," ce" qui" ne" semble" pas" être" le" cas" chez" Colletotrichum0
trifolii." " Ces" résultats" suggèrent" aussi" l’existence" de" signaux" additionnels" libérés" par" l’agent"
pathogène" et" inhibant" la" signalisation" symbiotique." Il" est" en" outre" apparu" que" la" perception" des"
petits"chitooligosaccharides"emprunte"une"voie"dépendante"de"DMI20mais"pas"de"NFP."
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B. Mise au point du bioessai d’élicitation de Medicago
truncatula
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Mon" implication" dans" le" travail" d’Amaury" Nars" se" situe" au" niveau" du" choix" des" marqueurs"
transcriptionnels"de"l’immunité"qui"ont"déjà"été"évoqués"dans"les"résultats."J’ai"vérifié"l’induction"des"
gènes" qu’il" a" sélectionnés" par" des" études" bibliographiques" et" l’interrogation" de" bases" de" données."
Pour"ce"faire,"des"RTPqPCR"sur"mes"échantillons"de"plantes"infectés"par"Aphanomyces0euteiches0ont"
confirmé" que" ces" marqueurs" sont" induits" au" cours" de" la" réponse" immunitaire" de" Medicago0
truncatula."J’ai"également,"à"partir"de"mes"données"de"transcriptomes,"fourni"à"Amaury"de"nouveaux"
marqueurs."
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"""
C. Analyse des phénotypes non symbiotiques contrôlés
par CRE1
"
Ce" travail" s’inscrit" dans" un" article" de" screening" des" phénotypes" non" symbiotiques" des" mutants" " du"
gène" CRE1." Différents" stress" biotiques," dont" Aphanomyces0 euteiches" mais" aussi" abiotiques,"
notamment"le"stress"salin"seront"testés."Pour"notre"part,"nous"avons"observé"des"symptômes"limités"
dans" les" mutants" de" ce" gène," sans" doute" associés" à" une" ramification" plus" importante" du" système"
racinaire."La"recherche"de"la"régulation"transcriptionnelle"des"récepteurs"aux"cytokinines"et"des"RRs"
(Responses"Regulators)"a"révélé"au"niveau"transcriptomique"une"répression"globale"des"RRs"de"type"
A"et"B"et"de"CRE1"luiPmême"en"présence"d’Aphanomyces0euteiches,0suggérant"que"la"répression"de"
cette" voie" est" nécessaire" à" la" défense" et" peut" expliquer" la" meilleure" résistance" du" mutant" cre1." CiP
contre,"une"figure"transcrivant"les"données"qui"seront"incorporées"dans"la"future"publication."
183"
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184"
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II. Participations en congrès et
colloques
"
J’ai"eu"l’opportunité"de"participer"à"plusieurs"congrès"internationaux"au"cours"de"ma"thèse"où"j’ai"pu"
à"chaque"fois"donner"une"communication"orale."
"
A. Oomycetes Molecular and Genetic Network
"
La" onzième" édition" de" ce" colloque" s’est" tenue" à" Toulouse" en" 2010." Il" rassemblait" l’ensemble" des"
spécialistes"travaillant"la"plupart"du"temps"sur"des"parasites"végétaux"mais"aussi"animaux."
Screening& of& Medicago) truncatula& symbiotic& mutants& with& Aphanomyces) euteiches& revealed&
molecular&crosstalks&between&symbiotic&interactions&and&resistance&to&pathogen&
Thomas&Rey1,&Clare&Gough2,&Francis&Carbonne1,&Bernard&Dumas1&and&Christophe&Jacquet1&
10 UMR5546,0 Pôle0 de0 biotechnologie0 Végétale,0 240 chemin0 de0 Borde0 Rouge0 313260 Castanet%Tolosan,0
FRANCE0
2" LIPM," UMR" 441P2594" PINRAPCNRS" " BP" 52627" chemin" de" Borde" Rouge" P" Auzeville" 31326" Castanet"
Tolosan"FRANCE"
Interactions"between"plants"and"soilPborn"microorganisms"have"a"crucial"impact"on"ecosystems"and"
crop" productivity," as" they" may" lead" to" parasitism" or" symbiosis" depending" on" the" nature" of" the"
interacting" microorganism." During" the" last" few" years," several" results" obtained" in" different" plant"
species"underlined"structural"and"biochemical"similarities"between"microbial"signals"and"their"plant"
receptors,"required"either"for"symbiosis"establishment"or"for"defence"activation."As"an"example,"the"
Nod" factors" (NFs)" produced" by" soilPbacteria," called" Rhizobia," are" key" signal" molecules" to" promote"
symbiotic" interactions" with" Legumes." NFs" contain" a" chitosaccharidic" moiety" and" their" perception"
involved" LysM" receptors," such" as" the" Medicago0 truncatula" (M.t.)" protein" NFP." In" plant" pathogen"
interactions," LysM" receptors" play" also" a" key" role" in" the" perception" of" chitin" fragments" which" are"
considered"as"Pathogen"Associated"Molecular"Patterns"(PAMPs)."This"comparison"raises"the"question"
of"how"plants"are"able"to"distinguish"friends"and"foes"and"whether"molecular"crosstalk"is"involved"in"
185"
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mechanisms" leading" to" rejection" or" acceptance" of" the" microbe." To" answer" these" issues," the" model"
legume"Medicago0truncatula0(M.t.)"was"particularly"well"suited"as"i)"a"collection"of"mutants"impaired"
in"symbiotic"interactions"was"available"in"the"genetic"background"of"the"A17"M0t."reference"line"and"
ii)" A17" was" shown" to" be" resistant" to" the" root" oomycete" Aphanomyces0 euteiches." The" screening" of"
eleven" M.0 t0 symbiosis" mutants" upon" A.0 euteiches" infection" showed" that" some" of" them" displayed" a"
significantly"altered"level"of"resistance"to"this"oomycete"compared"to"the"A17"WT."We"will"focus"the"
presentation"on"the"cytological,"transcriptomic"and"genetic"analyses"that"were"performed"on"the"nfp"
and" hap2a" mutants" that" are" respectively" more" susceptible" and" more" resistant" than" WT" line." The"
novel"functions"for"these"two"genes"as"components"of"plant"resistance"will"be"discussed."
"
B. International Congress of Legume Genetic and
Genomic
"
La" cinquième" édition" de" ce" colloque" s’est" tenue" à" Asilomar." Elle" rassemblait" l’ensemble" des"
chercheurs"s’intéressant"à"la"génomique"et"à"la"génétique"des"légumineuses,"aussi"bien"modèles"que"
d’intérêt"agronomique."Ainsi,"des"sessions"de"recherches"fondamentales"sont"représentées"même"si"
les" aspects" agronomiques" sont" prévalents." Ayant" été" financée" par" l’ASSEDISO" et" la" SFP," ma"
participation"à"ce"congrès"a"aussi"donné"lieu"à"un"rapport"publié"par"la"SFP."
"
Screening& of& Medicago) truncatula& symbiotic& mutants& with& Aphanomyces) euteiches& revealed&
molecular&crosstalks&between&symbiotic&interactions&and&resistance&to&pathogen&
Thomas&Rey1,&Clare&Gough2,&Francis&Carbonne1,&Bernard&Dumas1&and&Christophe&Jacquet1&
10 UMR5546,0 Pôle0 de0 biotechnologie0 Végétale,0 240 chemin0 de0 Borde0 Rouge0 313260 Castanet%Tolosan,0
FRANCE0
2" LIPM," UMR" 441P2594" PINRAPCNRS" " BP" 52627" chemin" de" Borde" Rouge" P" Auzeville" 31326" Castanet"
Tolosan"FRANCE"
Interactions"between"plants"and"soilPborn"microorganisms"have"a"crucial"impact"on"ecosystems"and"
crop" productivity," as" they" may" lead" to" parasitism" or" symbiosis" depending" on" the" nature" of" the"
interacting" microorganism." During" the" last" few" years," several" results" obtained" in" different" plant"
species"underlined"structural"and"biochemical"similarities"between"microbial"signals"and"their"plant"
receptors,"required"either"for"symbiosis"establishment"or"for"defence"activation."As"an"example,"the"
186"
"
Nod" factors" (NFs)" produced" by" soilPbacteria," called" Rhizobia," are" key" signal" molecules" to" promote"
symbiotic" interactions" with" Legumes." NFs" contain" a" chitosaccharidic" moiety" and" their" perception"
involved" LysM" receptors," such" as" the" Medicago0 truncatula" (M.t.)" protein" NFP." In" plant" pathogen"
interactions," LysM" receptors" play" also" a" key" role" in" the" perception" of" chitin" fragments" which" are"
considered"as"Pathogen"Associated"Molecular"Patterns"(PAMPs)."This"comparison"raises"the"question"
of"how"plants"are"able"to"distinguish"friends"and"foes"and"whether"molecular"crosstalk"is"involved"in"
mechanisms" leading" to" rejection" or" acceptance" of" the" microbe." To" answer" these" issues," the" model"
legume"Medicago0truncatula0(M.t.)"was"particularly"well"suited"as"i)"a"collection"of"mutants"impaired"
in"symbiotic"interactions"was"available"in"the"genetic"background"of"the"A17"M0t."reference"line"and"
ii)" A17" was" shown" to" be" resistant" to" the" root" oomycete" Aphanomyces0 euteiches." The" screening" of"
eleven" M.0 t0 symbiosis" mutants" upon" A.0 euteiches" infection" showed" that" some" of" them" displayed" a"
significantly"altered"level"of"resistance"to"this"oomycete"compared"to"the"A17"WT."We"will"focus"the"
presentation"on"the"cytological,"transcriptomic"and"genetic"analyses"that"were"performed"on"the"nfp"
and" hap2a" mutants" that" are" respectively" more" susceptible" and" more" resistant" than" WT" line." The"
novel"functions"for"these"two"genes"as"components"of"plant"resistance"will"be"discussed."
C. Model Legume Congress
"
Il" s’agissait" de" la" première" édition" autonome" de" ce" congrès" qui" était" antérieurement" satellite" de"
congrès"plus"importants."
Medicago) truncatula& Immune& Response& To& A& Root& Pathogen& & Involve& MtNFP,& A& LysM& Protein&
Acting&In&&Symbiotic&Nod&Factor&Perception&
Thomas& Rey1,& Clare& Gough2,& Julie& Cullimore2,& & Jean& Jacques& Bono1& ,Maxime& Bonhomme1,& Amaury&
Nars1,&Bernard&Dumas1&and&Christophe&Jacquet1&
10 UMR5546,0 Pôle0 de0 biotechnologie0 Végétale,0 240 chemin0 de0 Borde0 Rouge0 313260 Castanet%Tolosan,0
FRANCE0
2" LIPM," UMR" 441P2594" PINRAPCNRS" " BP" 52627" chemin" de" Borde" Rouge" P" Auzeville" 31326" Castanet"
Tolosan"FRANCE"
Interactions"between"plants"and"soilPborn"microorganisms"have"a"crucial"impact"on"ecosystems"and"
crop" productivity," as" they" may" lead" to" parasitism" or" symbiosis" depending" on" the" nature" of" the"
interacting" microorganism." During" the" last" few" years," several" results" obtained" in" different" plant"
species"underlined"structural"and"biochemical"similarities"between"microbial"signals"and"their"plant"
receptors,"required"either"for"symbiosis"establishment"or"for"defence"activation."As"an"example,"the"
187"
"
Nod" factors" (NFs)" produced" by" soilPbacteria," called" Rhizobia," are" key" signal" molecules" to" promote"
symbiotic" interactions" with" Legumes." NFs" contain" a" chitosaccharidic" moiety" and" their" perception"
involved" LysM" receptors," such" as" the" Medicago0 truncatula" (M.t.)" proteins" NFP" and" LYK3." In" plant"
pathogen" interactions," LysM" receptors" play" also" a" key" role" in" the" perception" of" chitin" fragments"
which" are" considered" as" Pathogen" Associated" Molecular" Patterns" (PAMPs)." This" comparison" raises"
the"question"of"how"plants"are"able"to"distinguish"friends"and"foes"and"whether"molecular"crosstalk"is"
involved"in"mechanisms"leading"to"rejection"or"acceptance"of"the"microbe."To"answer"these"issues,"
the"model"legume"Medicago0truncatula0(M.t.)"was"particularly"well"suited"as"mutants"impaired"in"NF"
perception" was" available" in" the" genetic" background" of" the" A17" M0 t." reference" line" and" ii)" A17" was"
shown"to"be"resistant"to"the"root"oomycete"Aphanomyces0euteiches."Screening"of""several"NFP"and"
LYK3" " allelic" mutants" as" well" as" a" double" mutant" upon" A.0 euteiches" infection" showed" that" all" nfp"
plants"displayed"a"significantly"reduced"resistance"level"to"this"oomycete"compared"to"the"A17"WT."
Further"cytological,"transcriptomic,"and"elicitation"assays"highlight"the"role"of"this"LysM"receptor"in"
perception" of" compounds" released" by" Ae" and" improved" our" understanding" of" the" mecanisms"
controlled"by"NFP"for"resistance"to"this"parasite."These"last"recent"advances"will"be"discussed"there."
"
This" work" was" funded" in" part" by" the" French" Agence" Nationale" de" la" Recherche" (contractANRP08P
BLANP0208P01“Sympasignal”)."
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AUTEUR : Thomas Rey
TITRE : Mise en évidence et caractérisation d'interconnexions moléculaires entre symbiose et résistance
chez la légumineuse modèle Medicago truncatula
DIRECTEURS DE THESE : Professeur Christophe Jacquet
DATE ET LIEU DE SOUTENANCE : le Mercredi 20 février 2013 à Castanet-Tolasan
RESUME
La# rhizosphère,# définie# comme# l’espace# du# sol# qui# entoure# les# racines,# héberge# de# nombreux#
microorganismes# à# l’origine# d’une# grande# variété# d’interactions# avec# la# plante.# Au# cours# des# dix##
dernières#années#,#un#grand#nombre#de#gènes#végétaux#impliqués#dans#le#dialogue#moléculaire#qui#
s’établit#dans#le#cadre#des#différentes#symbioses#ou#lors#de#la#réponse#de#la#plante#aux#parasites#ont#
été# identifiés.# Les# résultats# de# ces# travaux# ont# notamment# révélé# l’existence# de# fortes# similitudes#
entre# les# signaux# et# les# récepteurs# impliqués# dans# les# deux# types# d’interactions.# Toutefois,#
l’identification#de#gènes#essentiels#à#la#fois#aux#réponses#immunitaires#et#mutualistes#n’a#pas#encore#
été#décrite,#en#raison#notamment#de#l’incapacité#de#la#plante#modèle#Arabidopsis*thaliana#à#établir#
des# interactions# symbiotiques.# Pour# étudier# l’existence# d’interconnexions# moléculaires# entre#
symbioses#et#résistances#aux#parasites,#la#légumineuse#modèle#Medicago*truncatula#a#été#utlisée.#En#
exploitant# des# mutants# symbiotiques# et# deux# pathosystèmes# développés# avec# cette# plante,# les#
résultats# décrits# dans# ce# travail# relatent# la# découverte# de# l’implication# de# plusieurs# gènes# avec# un#
rôle#clé#dans#la#mise#en#place#de#la#symbiose#et#l‘immunité#de#la#plante.#L’implication#du#récepteur#
putatif#aux#facteurs#NOD#NFP#et#celle#du#facteur#de#transcription#NF7YA1,*marqueur#de#la#symbiose#
fixatrice#d’azote#ont#été#particulièrement#étudiées#après#inoculation#d’Aphanomyces*euteiches*(Ae),#
un#oomycète#racinaire.#Des#approches#de#phénotypage#complémentaires#(notations#des#symptômes,#
cytologie,#détection#moléculaire#du#parasite)#ont#montré#que#NFP#est#impliqué#dans#la#résistance#de#
la#plante#tandis#que#NF7YA1#est#associé#à#la#sensibilité#de#celleKci.#Le#comportement#des#mutants#nfp#
visKàKvis#de#Colletotrichum*trifolii,*un#champignon#parasite,#s’est#révélé#similaire#à#celui#observé#visKàK
vis#d’Ae,*permettant#de#généraliser#son#rôle#face#aux#organismes#filamenteux#pathogènes.#En#ce#qui#
concerne# NF7YA1,# il# est# apparu# que# sa# mutation# implique# aussi# un# meilleur# développement# des#
racines# latérales.# Son# expression# a# d’ailleurs# pu# être# détectée# dans# les# primordia# de# racines,#
suggérant# qu’il# possède# un# rôle# régulateur# de# ces# organes.# Des# analyses# transcriptomiques#
comparant# les# mutants# de# ces# deux# gènes# et# la# plante# sauvage# inoculée# par# Ae* ont# révélé# des#
modifications# non# seulement# sur# l’expression# de# gènes# dans# la# signalisation# et# la# mise# en# place# de#
réponses# immunitaires# mais# aussi# dans# des# fonctions# associées# à# des# processus# de# dynamique#
cellulaire.#Ainsi#l’analyse#détaillée#du#rôle#de#NFP#et#NF7YA1,#ainsi#que#l’initiation#de#plusieurs#autres#
travaux# sur# d’autres# mutants# symbiotiques# suggèrent# un# nombre# d’interconnexions# moléculaires#
beaucoup#plus#important#qu’imaginé#initialement#entre#symbiose#et#immunité.#
#
MOTS-CLES
Medicago truncatula, Aphanomyces euteiches, Oomycètes, légumineuse, symbiose, immunité, MAMPs,
LCOs, COs, LysM-RLK, NFP, NF-Y, MtNF-YA1, CSP, CRE1, ROS, Transcriptome, Hormones
#