Thèse Regulation de l'Adn-T Pendant l'Interaction Agrobacterium-Arabidopsis H/F

Doctorat.Gouv.Fr

L'ADN-T (T-DNA) constitue un élément génétique mobile transféré par Agrobacterium dans le génome de nombreuses plantes dicotylédones, notamment Arabidopsis thaliana. À la suite de ce transfert, l'ADN-T s'intègre au génome de l'hôte et induit une reprogrammation des cellules infectées, conduisant à la formation de tumeurs (galles) favorables à la prolifération bactérienne. En biologie végétale, des versions désarmées de l'ADN-T, dépourvues de gènes de virulence, sont largement exploitées pour des approches de mutagenèse et de transgénèse. En raison de son origine exogène, les transcrits issus de l'ADN-T ont été parmi les premières cibles identifiées des mécanismes de silencing de l'ARN, et la majorité des travaux s'est concentrée sur la régulation des transgènes. En revanche, les modalités de régulation de l'ADN-T natif au cours d'une infection naturelle, ainsi que la dynamique de son expression, demeurent insuffisamment caractérisées, malgré leur rôle central dans la pathogenèse. Une compréhension approfondie de ces mécanismes apparaît essentielle pour éclairer les interactions plante-pathogène ainsi que la biologie de l'ADN-T.
En contexte de la maladie dans les tissus somatiques (tumeur), nous proposons que des approches génomiques et unicellulaires permettront d'identifier des mécanismes de régulation de l'hôte (y compris des voies encore non décrites), impliquées dans le contrôle ou la réponse à l'introduction d'ADN exogène tel que l'ADN-T, ainsi que des stratégies bactériennes favorisant son expression efficace et le succès de l'infection. Le projet s'articule autour de deux objectifs. L'objectif 1 repose sur des approches de profilage génomique visant à déterminer si l'ADN-T natif est ciblé par les voies de silencing post-transcriptionnel des gènes de l'hôte (silencing de l'ARN médié par les petits ARN) et à évaluer si certains gènes de l'ADN-T, en particulier les oncogènes, échappent à ce contrôle. Parallèlement, le séquençage des ARN à l'échelle unicellulaire au niveau des noyaux(snRNA-seq) de tissus tumoraux infectés permettra en particulier de caractériser l'hétérogénéité cellulaire de l'expression de l'ADN-T et d'identifier des signatures transcriptionnelles de l'hôte associées aux cellules positives pour l'expression de l'ADN-T. L'objectif 2 visera à tester fonctionnellement l'impact des différentes couches de régulation identifiées dans l'objectif 1 sur l'expression de l'ADN-T ainsi que sur l'issue de l'infection, et réciproquement. À cette fin, des mutants d'Arabidopsis et d'Agrobacterium seront analysés afin d'établir des relations causales entre les voies de régulation de l'hôte, l'activité des gènes de l'ADN-T et les phénotypes associés à l'infection.

T-DNA is a mobile genetic element transferred from Agrobacterium tumefaciens into the genome of many dicotyledonous plants, including Arabidopsis thaliana. T-DNA transfer is mediated by the tumor-inducing (pTi) plasmid of A. tumefaciens, a facultative pathogen that alternates between a commensal lifestyle on roots and a pathogenic lifestyle upon contact with wounded plant tissues resulting in to a gall (tumour) on which the bacteria grow. The Agrobacterium-Arabidopsis interaction therefore provides a unique and tractable system to study natural horizontal DNA transfer and its regulation within host cells.
In plant research, T-DNA is primarily known in its disarmed form and has been extensively exploited as a tool for mutagenesis and transgenesis. Studies on transgene expression revealed that T-DNA-derived transcripts are efficiently recognized as foreign and targeted by host RNA silencing pathways, leading to the production of small RNAs that mediate post-transcriptional gene silencing and, in some cases, DNA methylation. In contrast, the regulation of wild T-DNA during natural infection-particularly in relation to its expression and dynamics-has received comparatively little attention, despite its central role in tumor formation and disease progression.
During infection, wild T-DNA is expected to be a major target of host defense mechanisms, including RNA silencing. However, the nature and extent of these defenses, and how Agrobacterium counteracts them, remain poorly defined. Preliminary data from our laboratory (as well as published data) indicate that DNA methylation of T-DNA genes is negligible in Arabidopsis root galls, whereas small RNAs accumulate massively on specific T-DNA genes but not others. This gene-specific silencing suggests a complex and selective regulatory landscape that is compatible with successful tumor development. Whether this differential regulation is shaped by the developmental reprogramming associated with the tumor state, by bacterial proteins encoded within the T-DNA, or by both remains unknown.
More broadly, plant genes co-expressed with T-DNA during infection have not been systematically identified. These host factors could either promote or restrict T-DNA expression, or be secondarily induced by T-DNA activity. Similarly, potential regulatory interactions among T-DNA genes themselves remain largely unexplored. Together, these observations suggest that multiple, layered regulatory mechanisms-particularly RNA silencing pathways-act on wild T-DNA, yet their roles in infection biology are still poorly understood.

The objective of this thesis is to elucidate how wild T-DNA is regulated during
natural Agrobacterium-Arabidopsis infection by examining its expression dynamics and identifying host regulatory pathways and bacterial counter-defense mechanisms that control T-DNA expression and shape infection outcomes.

To achieve this objective, we will combine genomics and functional approaches to investigate T-DNA regulation during infection. We will profile small RNAs and apply single-nuclei transcriptomics to identify cellular signatures associated with T-DNA transcription (Aim 1: Genomics). Functional analyses using both plant and bacterial mutants will then be used to test the roles of host and bacterial factors in T-DNA regulation and infection outcomes (Aim 2: Functional analyses).

AIM 1. Genomic dissection of T-DNA regulation in tumors

1.1 Small RNA profiling
Small RNA sequencing will be performed on pooled tumors (n = 30) with colonized, non-infected roots of equivalent weight as controls, using three biological replicates per condition. Based on small RNA size (21 nt vs 24 nt), we will distinguish post-transcriptional from transcriptional regulation. We expect predominantly 21-nt small RNAs, as DNA methylation on T-DNA is negligible (unpublished data from the team); in addition, northern blot performed at two genes indicate 21-nt smallRNA presence and only at one gene out of two . Small RNA data will be produced early at the beginning of the thesis. Associations between small RNA accumulation, T-DNA gene expression, and existing RNA-seq datasets will be analyzed.

1.2 Single-cell transcriptomics
Single-nucleus RNA sequencing will be used to characterize transcriptional heterogeneity in roots and galls and to identify cellular signatures associated with T-DNA expression or silencing. At least 10,000 plant nuclei per condition (roots and galls at 21 dpi) will be sequenced. Cells will be clustered based on gene expression using CellRanger, and compared with bulk RNA-seq data for T-DNA genes. Lists of co-expressed host genes with T-DNA genes will be generated. Experiments and analyses will be conducted with sequencing platform, with data available early in the thesis (first sequencing experiment is planned during the summer).

AIM 2. Functional analyses of T-DNA regulation

2.1 Role of plant RNA silencing pathway
Plant mutants: Arabidopsis mutants impaired in the small RNA pathway generating populations of smallRNAs (e.g. dcl2/3/4, rdr6) will be infected with Agrobacterium along WT plants and analyzed for :
-transcript levels of various T-DNA genes
-bacterial titers and gall weight,
in order to assess the contribution of RNA silencing to infection control.

Bacterial mutants: In parallel, in order to assess bacterial modulation of host silencing, we will infect WT plants with an Agrobacterium mutant lacking the 6b protein (a proposed silencing suppressor), a mutant for oncogenes required for tumor formation (the tumor state was also proposed to dampen RNA silencing and the Ipt-Tms1-Tms2 mutant has been already constructed), as well as a mutant control for another T-DNA gene. Small RNA profiles, T-DNA expression, and bacterial titers will be assessed upon infection of these various strains along wild type Agrobacterium.
To test, whether differential small RNA accumulation at T-DNA expression reflects promoter strength promoter swaps between two genes will tested, and smallRNA accumulation tested again.
Finally, if time allows it, fluorescent reporters will be introduced in translational fusion with two T-DNA genes to monitor one smallRNA-targeted and one non-targeted T-DNA gene; alternatively, smRNA FISH could be used in collaboration with F. Pontvianne, partner of the ANR project.

2.2 Role of T-DNA-coexpressed host genes
Plant mutants: Three to four host genes co-expressed with T-DNA will be selected, and corresponding mutants (either published or isolated by us) will be analyzed for effects on T-DNA genes expression (by RT-qPCR, on several T-DNA genes as above), bacterial growth, and gall development. We predict they could be genes expressed in specific cell states and belonging to specific developmental or signaling pathways.

Bacterial mutants: Conversely, bacterial mutants affecting tumor formation or specific T-DNA genes (same as in 2.1) will be used to test reciprocal regulation between T-DNA expression and host pathways (RT-qPCR on the choosen plant genes selected and corresponding pathway if relevant).
If time permits , spatial expression of one or two host gene products (co-expressed with T-DNA) will be examined using reporter constructs, ideally those available in the literature (transcriptional reporters/smRNA Fish by our collaborators).