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International audience; Burkholderiaceae are frequent and abundant colonizers of the rice rhizosphere and interesting candidates to investigate for growth promotion. Species of Paraburkholderia have repeatedly been described to stimulate plant growth.Burkholderia vietnamiensis LMG10929 and Paraburkholderia kururiensis M130 are bacterial rice growth-promoting models. Besides this common ecological niche, species of the Burkholderia genus are also found as opportunistic human pathogens, while Paraburkholderia species are mostly environmental and plant associated. In this study, we compared the genetic strategies used by B. vietnamiensis and P. kururiensis to colonize two subspecies of their common host, Oryza sativa subsp. japonica (cv. Nipponbare) and O. sativa subsp. indica (cv. IR64). We used high-throughput screening of transposon insertional mutant libraries (Tn-seq) to infer which genetic elements have the highest fitness contribution during root surface colonization at 7 days postinoculation. Overall, we detected twice more genes in B. vietnamiensis involved in rice root colonization than in P. kururiensis, including genes contributing to the tolerance of plant defenses, which suggests a stronger adverse reaction of rice toward B. vietnamiensis than toward P. kururiensis. For both strains, the bacterial fitness depends on a higher number of genes when colonizing indica rice compared to japonica. These divergences in host pressure on bacterial adaptation could be partly linked to the cultivars' differences in nitrogen assimilation. We detected several functions commonly enhancing root colonization in both bacterial strains, e.g., Entner-Doudoroff (ED) glycolysis. Less frequently and more strain specifically, we detected functions limiting root colonization such as biofilm production in B. vietnamiensis and quorum sensing in P. kururiensis. The involvement of genes identified through the Tn-seq procedure as contributing to root colonization, i.e., ED pathway, c-di-GMP cycling, and cobalamin synthesis, was validated by directed mutagenesis and competition with wild-type (WT) strains in rice root colonization assays. IMPORTANCE Burkholderiaceae are frequent and abundant colonizers of the rice rhizosphere and interesting candidates to investigate for growth promotion. Species of Paraburkholderia have repeatedly been described to stimulate plant growth. However, the closely related Burkholderia genus includes both beneficial and phytopathogenic species, as well as species able to colonize animal hosts and cause disease in humans. We need to understand to what extent the bacterial strategies used for the different biotic interactions differ depending on the host and if strains with agricultural potential could also pose a threat toward other plant hosts or humans. To start answering these questions, we used in this study transposon sequencing to identify genetic traits in Burkholderia vietnamiensis and Paraburkholderia kururiensis that contribute to the colonization of two different rice varieties. Our results revealed large differences in the fitness gene sets between the two strains and between the host plants, suggesting a strong specificity in each bacterium-plant interaction.

The burkholderia group is composed of multiple bacterial genera (including Burkholderia and Paraburkholderia). Some species indulge in diverse interactions with eukaryotic hosts that range from pathogenicity to symbiosis. Many burkholderia species dwell in association with rice, making this host and ideal model for burkholderia/plant interactions. The taxonomy of burkholderia delineates a frontier between the Burkholderia genus, grouping the majority of pathogenic species, and the Paraburkholderia genus, mainly phytobeneficial and environmental species. This thesis project strived to characterize the genetic frontiers between species of both genera that share the capacity to interact with rice. First, we explored the taxonomic diversity for 124 rice-isolates using dedicated markers (16S and recA), and went into a more detailed analysis for a subset of 12 supposedly different species suing whole genome sequencing. We developed the intraspecific diversity of B. cenocepacia strains which we had found to be rice associated This was unexpected for this human pathogen and led us to describe a new species, with improved plant interaction abilities (which we propose to name B. servocepacia). We then explored the genetic bases regulating plant-adaptation of Burkholderia and Paraburkholderia species using two complementary approaches. We first undertook a transcriptomic profiling of the reaction to root exudates for six rice-isolates belonging to either genus. We thus demonstrated a strain specific response of the early plant-sensing, independent of the strain’s taxonomic attribution. We also listed the principal functions involved in exudate sensing. Our second approach focused on the rice endophyte models B. vietnamiensis LMG10929 (Bv) and P. kururiensis M130 (Pk) for which we undertook a high-throughput genetic screening of insertion mutant libraries (Tn-seq), during the root surface colonization step of two rice cultivars belonging to Oryza sativa ssp. japonica and Oryza sativa ssp. indica. We noticed a strong disparity in the adaptation for rice colonization between Bv and Pk as the display genetic requisites that differ in nature and number. Furthermore, we demonstrated a host-dependent adaptation for root colonization from both strains. Finally, we investigated on the distribution of type III secretion systems (T3SS) within burkholderia. We describe two novel T3SS types and in one instance, demonstrate its role in plant colonization for Bv. The thesis is concluded by a general discussion, summarizing the results of this project and situating them in the context of adaptative frontiers for plant interactions between Burkholderia and Paraburkholderia.; Les burkholderias appartiennent à plusieurs genres bactériens (dont Burkholderia et Paraburkholderia). Certaines espèces interagissent avec de multiple hôtes eucaryotes avec lesquels elles développent une gamme d’interaction allant de la symbiose à la pathogénie. Une grande diversité d’espèces de burkholderias interagisse avec le riz, faisant de cette plante un modèle idéal pour l’étude des interactions burkholderias/plantes. Une frontière taxonomique semble se dessiner entre le genre Burkholderia, qui regroupe la majorité des espèces pathogènes, et le genre Paraburkholderia qui concentre des espèces phytobénéfiques et environnementales. Ce travail de thèse a porté sur l’étude des frontières génétiques chez des espèces de ces deux genres interagissant avec le riz. Nous avons d’abord exploré la diversité taxonomique de 124 souches isolées du riz par l’étude de marqueurs taxonomiques (16S et recA) et, pour un sous ensemble de 12 espèces potentiellement différentes, d’analyse de leurs génomes (ANI). Nous avons progressé dans la description de la diversité intraspécifique chez l’espèce Burkholderia cenocepacia dont nous possédions des isolats de racines de riz, fait étonnant pour ce pathogène humain, et qui nous a conduit à décrire une nouvelle espèce mieux adaptée à l’environnement végétale (que nous proposons de nommer B. servocepacia). Nous avons ensuite exploré les bases génétiques de l’adaptation à l’environnement plantes chez Burkholderia et Paraburkholderia par deux approches. La première portait sur les étapes précoces d’interaction avec la plante, en étudiant la réponse transcriptomique de six espèces, tirées des deux genres, stimulées aux exsudats racinaires de riz, qui nous a permis de montrer une réponse souche-spécifique indépendante de leur appartenance taxonomique, et de lister des catalogues de fonctions impliqués dans la perception des exsudats. La seconde approche a porté sur deux souches modèles endophytes du riz, Paraburkholderia kururiensis M130 (Pk) et Burkholderia vietnamiensis LMG10929 (Bv), pour lesquelles nous avons réalisé un crible génétique haut-débit de banques de mutants par Tn-Seq, visant à trouver les déterminants génétiques leur permettant de coloniser les racines des deux sous-espèces de riz, Oryza sativa ssp. japonica et Oryza sativa ssp. indica. Nous observons une forte disparité dans l’adaptation des deux souches à la colonisation racinaire puisque Bv et Pk requiert des gènes différents par leur nombre et leur nature. Par ailleurs, l’effet génotype de l’hôte induit une adaptation de la part des deux colonisateurs. En dernière partie, nous avons étudié la distribution des systèmes de sécrétion de type 3 (T3SS) chez les burkholderias. Nous décrivons deux nouveaux types de T3SS et démontrons que l’un d’eux est impliqué chez Bv dans la colonisation racinaire. Nous discutons finalement de l’ensemble des résultats de cette thèse dans le contexte de l’étude des frontières adaptatives à l’environnement plante chez les Burkholderia et Paraburkholderia.