Introduction générale

N° d’ordre Année 2006
THÈSE
Présentée
Devant l’UNIVERSITE CLAUDE BERNARD - LYON 1
Pour l’obtention du
DIPLÔME DE DOCTORAT
(arrêté du 25 avril 2002)
présentée et soutenue publiquement
le 29 septembre 2006
par
Alessandra PONTIROLI
DEVENIR DE L'ADN TRANSGÉNIQUE
DANS LES ENVIRONNEMENTS LIÉS À LA PLANTE ET AU SOL:
IMPLICATIONS POTENTIELLES DANS LES TRANSFERTS HORIZONTAUX
DE GÈNES ENTRE PLANTES TRANSGÉNIQUES ET BACTÉRIES
Directeur de thèse :
Mr Pascal Simonet
JURY
Mr R. Rohr Professeur à l’Université de Lyon1 Président
Me F. Binet Chargé de Recherches CNRS à Rennes Rapporteur
Mr D. Daffonchio Professeur à l’Université de Milan Examinateur
Mr F. Martin-Laurent Chargé de Recherches INRA à Dijon Examinateur
Me C. Morris Directeur de Recherches INRA à Avignon Rapporteur
Mr P. Simonet Directeur de Recherches CNRS à Lyon Directeur de thèse
Mr W. Wildi Professeur à l’Institut Forel de Geneve Examinateur

Liste des abréviations
ADN/DNA
Acide désoxyribonucléique
UFC/CFU
Unité Formant Colonie
DO/OD
Densité optique
g
Gramme
mg
Milligramme
µg
Microgramme
ng
Nanogramme
kb
Kilobase
pb
Paire de base
mM
Millimolaire
µM
Micromolaire
PCR
Polymerase Chaine Reaction
rpm
Rotation par minute
Km
Kanamycine
Spe
Spectynomicine
Str
Streptomycine
Rif
Rifampicine
Gm
Gentamycine
A. nal.
Acide nalidixique
LB
Milieu Luria Bertani

Table des matières
INTRODUCTION GÉNÉRALE ................................................................................................. 1
Références .............................................................................................................................................. 12
Chapitre 1. Ecology of transgenic DNA in the environment ...................................................... 16
I-INTRODUCTION .............................................................................................................................. 16
I.1 Genetically engineered plants: history, features and applications ........................................ 16
I.2 Potential ecological fallouts linked to field release of GEPs ................................................... 17
I.3 Current transgenic plants and selectable marker genes ......................................................... 19
II- HORIZONTAL GENE TRANSFER BETWEEN TRANSGENIC PLANTS
AND BACTERIA ............................................................................................................................ 26
II.1 HGT mechanisms ..................................................................................................................... 27
II.2 Requisites for natural transformation ................................................................................... 28
II.3 Barriers to natural transformation ......................................................................................... 32
II.3.1. Uptake of DNA into the cell ............................................................................................................... 32
II.3.2. DNA homology: the antagonistic role of mismatch repair system and the SOS system ..................... 33
II.4 In situ transformation of bacteria by plant DNA .................................................................. 34
III- KEY PARAMETERS AFFECTING TRANSGENIC DNA AVAILABILITY ........................ 35
III.1 Persistence of nucleic acids in the environment ................................................................... 35
III.2 Biological potential of DNA .................................................................................................... 38
IV-THE PHYTOSPHERE AND THE “HOTSPOTS” FOR HORIZONTAL GENE
TRANSFER ..................................................................................................................................... 39
V-IMPACT OF TRANSGENIC PLANTS ON THE BIODIVERSITY OF THE
SOIL MICROBIAL COMMUNITY .............................................................................................. 42
V.1 Effects of genetically engineered plants on microbial communities .................................. 43
VI-CONCLUDING REMARKS .......................................................................................................... 45
References .............................................................................................................................................. 48
Avant-propos du chapitre 2 ........................................................................................................ 61
Chapitre 2. Natural transformation and induction of spontaneous mutants
in Ralstonia solanacearum ........................................................................... 63
I-INTRODUCTION .............................................................................................................................. 63

Table des matières
II-MATERIALS AND METHODS ..................................................................................................... 67
II.1 Strains and transformation in vitro ......................................................................................... 67
II.2 Transforming DNA .................................................................................................................. 67
II.3 In planta transformation .......................................................................................................... 68
II.4 PCR analysis of spontaneous mutants .................................................................................... 68
II.5 Assessment of spontaneous mutation rates in different media ............................................. 69
III-RESULTS AND DISCUSSION ..................................................................................................... 69
III.1 In vitro transformation ........................................................................................................... 69
III.2 In planta transformation ........................................................................................................ 71
IV- CONCLUSIONS ............................................................................................................................. 72
References .............................................................................................................................................. 75
Avant propos du chapitre 3 ........................................................................................................ 77
Référénces.......................................................................................................................................78
Chapitre 3. Première partie: A strategy for in situ localization of horizontal gene transfer by
natural transformation ................................................................................... 79
I-ABSTRACT…………………………………………………………………………………...79
II-INTRODUCTION .................................................................................................................. 80
III-MATERIAL AND METHODS ............................................................................................ 81
III.1 Bacterial strains and plasmids .................................................................................................... 81
III.2 Plant material and growthconditions ......................................................................................... 82
III.3 DNA manipulations techniques ................................................................................................... 82
III.4 Transforming DNA ...................................................................................................................... 82
III.5 Construction of aadA::gfp-based constructs for cloning in A. baylyi BD413 .......................... 83
III.6 Construction of aadA::gfp-tagged A. baylyi BD413 strains ....................................................... 84
III.7 In vitro marker rescue experiments ............................................................................................ 84
IV-RESULTS………….. ............................................................................................................. 85
IV.1 Construction of a A. baylyi BD413-based marker rescue system ............................................. 85
IV.2 Transformation frequencies of marker rescue experiments in vitro ........................................ 86
V-DISCUSSION…. ..................................................................................................................... 86
ACKNOWLEDGEMENTS ........................................................................................................ 90
References……………………………………………………………………………………..... . 91

Table des matières
Deuxième partie:Hot spots for horizontal gene transfer from transplastomic plants to
Acinetobacter baylyi in the phytosphere ........................................................ 98
I-ABSTRACT...............................................................................................................................98
II-INTRODUCTION .................................................................................................................. 99
III-MATERIAL AND METHODS .......................................................................................... 101
III.1 Plant material ............................................................................................................................. 101
III.2 Bacterial strains, plasmids, and culture media ....................................................................... 101
III.4 DNA extraction ........................................................................................................................... 102
III.5 Plant inoculation and incubation .............................................................................................. 103
III.6 Growth and competence development of A. baylyi strain BD413 .......................................... 105
III.7 Natural transformation of A. baylyi strain BD413 in vitro and in the phytosphere .............. 105
III.8 Analysis of transformants .......................................................................................................... 106
III.9 Visualisation of bacterial cells on plant tissues ........................................................................ 107
IV-RESULTS..............................................................................................................................108
IV.1 Growth of A. baylyi BD413 in the phytosphere ........................................................................ 108
IV.2 Competence development of A. baylyi BD413 in vitro and in the phytosphere ..................... 108
IV.3 Natural transformation of A. baylyi BD413 by transgenic DNA in vitro
and in the phytosphere.............................................................................................................. 109
IV.4 Visualization of total and transformant bacteria in planta ..................................................... 110
V-DISCUSSION.. ...................................................................................................................... 113
ACKNOWLEDGEMENTS ...................................................................................................... 117
References.....................................................................................................................................118
Avant propos du chapitre 4 ...................................................................................................... 136
Chapitre 4. Long-term persistence and bacterial transformation potential of transplastomic plant
DNA in soil ................................................................................................... 138
I- ABSTRACT........ ................................................................................................................... 138
II-INTRODUCTION ................................................................................................................ 139
III-MATERIALS AND METHODS ........................................................................................ 140