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Reference : UMR8197-NATBOI-025
Workplace : PARIS 05
Date of publication : Wednesday, September 11, 2019
Scientific Responsible name : Lionel Navarro
Type of Contract : PhD Student contract / Thesis offer
Contract Period : 36 months
Start date of the thesis : 15 October 2019
Proportion of work : Full time
Remuneration : 2 135,00 € gross monthly
Description of the thesis topic
RNA interference (RNAi) is a conserved gene regulatory mechanism that has been characterized as an antiviral defense response by repressing translation, accumulation and/or replication of viral RNAs. In plants, RNAi has also been shown to control resistance against bacterial, fungal and oomycete pathogens partly by fine-tuning the expression of immune-responsive genes. The core mechanism of RNAi involves the processing of double-stranded RNAs (dsRNAs) or single-stranded RNAs (ssRNAs) carrying stem loop structures (e.g. primary microRNA –miRNA– transcripts) by Dicer-Like (DCL) proteins leading to the production of 20-25 nt long short interfering RNAs (siRNAs) or miRNAs. SiRNAs or miRNAs are then loaded into Argonaute (AGO) proteins to direct post-transcriptional silencing of sequence complementary mRNA targets through endonucleolytic cleavage and/or translational inhibition.
An important feature of plant small RNAs, and particularly of siRNAs, is their ability to trigger non-cell autonomous silencing in adjacent cells as well as in distal tissues. This phenomenon is essential to prime antiviral response ahead of the infection front but also to translocate silencing signals between plant cells and their non-viral eukaryotic interacting (micro)organisms. Although this trans-kingdom RNAi has never been reported in plant-bacterial interactions, a recent study from our team has demonstrated that Arabidopsis-encoded small RNA species directed against the coding region of virulence factors from Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000) can trigger post-transcriptional silencing of these bacterial genes. This Antibacterial Gene Silencing (AGS) phenomenon was associated with a reduced bacterial pathogenesis, which was also observed upon external application of corresponding plant-derived small RNAs onto wild-type Arabidopsis and tomato leaves prior to infection. Furthermore, we have demonstrated that small RNA species, were causal for both AGS and pathogenesis suppression. This implies that this Gram-negative bacterium is capable of taking-up –passively and/or actively– small RNAs despite the double membrane composition of its cell envelope. It also indicates that bacterial cells can trigger post-transcriptional gene silencing upon assimilation of exogenous small RNAs, although the mechanisms involved in this process remain elusive.
The present PhD research program aims to identify and characterize bacterial factors involved in the uptake of external small RNA species or in the silencing of bacterial genes upon assimilation of these antibacterial small RNAs. For this purpose, we will use an unbiased Tn-seq approach, which couples a barcoded library of transposon (Tn) mutants with high-throughput sequencing strategies to measure relative mutant abundance. This approach will be conducted in vitro using small RNAs directed against essential genes from Pto DC3000 or Xanthomonas campestris pv. campestris (Xcc), which we have already shown to be effective in reducing bacterial titer in those conditions. Briefly, the collections of barcoded transposon mutants will be inoculated in liquid bacterial culture medium containing either control or antibacterial siRNA species and we will select mutants exhibiting the most significant changes in their relative abundance. It is anticipated that mutants depicting normal bacterial titer in the presence of antibacterial small RNAs will be impaired in factors required for the uptake and/or the action of these small RNA species, while mutants showing an enhanced deficiency in bacterial growth will be altered in negative regulators of these processes. Functional validation of dozens of candidates will be subsequently achieved using a CRISPR interference (CRISPRi) approach and the most promising candidates genes will be further validated by classical deletion and complementation assays. We will further select two or three promising bacterial factors and further characterize them in the processing, degradation and/or translational inhibition mediated by small RNAs. RNA-seq and pRNA-seq genomic approaches will also be conducted from wild-type and bacterial mutant strains in the context of in planta infection. The latter analysis will allow us to identify the extent to which bacterial genes are targeted by AGS in a physiological context of infection.
Overall, this project should unveil novel mechanisms by which external small RNA species can reprogram the expression of genes in prokaryotic cells and thus makes significant advances in our understanding of host-bacterial interactions, bacterial gene expression and trans-kingdom RNAi. We are also anticipating that this basic research program will open-up completely new perspectives in biotechnological, agricultural and medical applications.
The PhD work will be conducted in the team of Lionel Navarro based at the “Institut de Biologie de l'Ecole Normale Supérieure” (IBENS) and under the supervision of Lionel Navarro. The Tn-seq approach will be carried out in collaboration with Jennifer Lewis (UC Berkeley) and Laurent Noël (LIPM, Toulouse). The student will have access to all the IBENS facilities and he / she will benefit from a very stimulating intellectual environment and will exchange on a daily basis with students, engineers, technicians and scientists of different nationalities.
A good knowledge of microbiology and molecular biology techniques is requested.
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