Soutenance de thèse - Investigation of plant responses to foreign double-stranded RNA - Varusha Pillay Veerapen

Description :

Double-stranded RNA (dsRNA) is a common feature generated by both RNA and DNA viruses. While the recognition of dsRNA by Dicer-like (DCL) endoribonucleases in plants has been well-studied in the context of antiviral RNA silencing, the existence of other mechanisms by which plants recognize dsRNA is poorly understood. Antiviral RNA silencing consists of the recognition of viral dsRNA by DCLs, which process dsRNA into small interfering RNAs (siRNAs), which can in turn target homologous viral sequences. However, viruses have evolved with Viral Suppressors of RNA silencing (VSRs), which hinder the effectiveness of RNA silencing, allowing successful infection. In contrast to plants, mammals possess Protein Kinase R (PKR), capable of recognizing viral dsRNA and inducing translational shutdown. Our objective is to uncover a comparable response and explore other potential mechanisms akin to Pattern-Triggered Immunity (PTI) responses. In the first study, we investigated a potential PKR-like response in plants. Using hairpin constructs regulated by a dexamethasone (dex)-inducible promoter, we generated transgenic Arabidopsis thaliana lines in wild-type and Dicer-like (DCL) -deficient backgrounds. Dex treatment induced necrosis in hairpin-expressing dcl2/4 mutant plants. Analysis revealed a 40-fold increase in hairpin RNA levels in dcl2/4 mutants compared to wild-type, indicating a significant rise in dsRNA. This absence of DCL proteins allowed us to study dsRNA impact without interference from RNA silencing or viral proteins. To assess if dsRNA accumulation triggers an antiviral response, we inoculated transgenic plants with various viruses. Dex-treated ∆dcl2/4 and ∆dcl2/3/4 transgenics exhibited significantly reduced systemic virus accumulation. Similar results were observed in Nicotiana benthamiana when co-expressing a dex-inducible Luciferase hairpin construct with other viruses. Phosphoproteomic analysis in dex-treated dcl2/4 mutants unveiled distinct phosphorylation changes in Ribosomal Protein S27 (RPS27). Ribosome profiling demonstrated translational repression in dex-treated dcl2/4 transgenics, which is abolished in the same transgenics without gcn2. This suggests a PKR-like response in plants is dependent on GCN2 and RPS27. The well-explored mechanism of Pattern-Triggered Immunity (PTI) primarily focuses on Pattern-Associated Molecular Patterns (PAMPs) originating from bacteria and fungi. Recent studies have shown whether viral dsRNA could be recognized as a PAMP. Our second study indicates that certain MAPK signaling, a sign of PTI responses, might be involved, and there is no significant callose deposition after dex treatment in dcl2/4 mutants with the hairpin. This suggests that the dsRNA itself might not necessarily be identified as a PAMP. In summary, we emphasized the discovery of a novel antiviral system in plants, analogous to the PKR response observed in mammals. This intricate response holds considerable promise and offers various avenues for further exploration.

Voici les membres du jury qui participeront à la soutenance :

• Professeur Peter Moffett, directeur de recherche, Département de biologie, Université de Sherbrooke
• Professeur David Bisaro, évaluateur externe, Department of Molecular Genetics, Ohio State University
• Professeur Daniel Lafontaine, évaluateur interne, Département de biologie, Université de Sherbrooke
• Professeur Kamal Bouarab, président-rapporteur, Département de biologie, Université de Sherbrooke