Identification and characterization of plant factors that counteract bacterial-mediated suppression of RNA silencing – PRIM

We have performed a mutagenesis by EMS to identify plants, that restore and/or increase RNA silencing with the presence of a bacterial effector. For that purpose, we have generated transgenic lines, that express both a reporter sensor of RNA silencing and a virulent bacterial effector, in a cell-type required for bacteria entry, namely the guard cells. This screen, realised with the naked eye, was based on the selection of mutants, that can exhibit chlorosis, indicating that the RNA silencing efficiency is increased in these mutants.

We have shown that the key components of the RNA silencing play a critical role at the level of stomata, in controlling stomata aperture upon bacterial infection, hence restricting bacteria invasion in host. In addition, we have performed a forward genetic screen to identify new repressors of the RNA silencing, whose inactivation should counteract bacterial suppressors effects in Arabidopsis. The phenotypical characterization of around twenty candidates is promising.

This project should allow a better understanding of the basic mechanisms underlying PTGS and host counter-counter defense in plants. It should also provide novel insights into strategies that can be used to control pathogens in plants, therefore opening-up new horizons in agricultural applications

A first paper, based on the impact of RNA silencing on the control of bacteria entry in host, is in preparation and would be likely submitted at the start of 2018.

The innate immune response is the first line of defense against pathogens, which plays a critical role in antimicrobial defense. This response is initiated by surface receptors that recognize Microbe- or Pathogen-Associated Molecular Patterns (MAMPs or PAMPs), and activate PAMP-triggered immunity (PTI). Plants and animals can also recognize adapted pathogens by detecting the presence of virulence determinants known as pathogen effectors. This process relies on the presence of divergent receptors that induce Effector-Triggered Immunity (ETI) upon recognition of pathogen effectors. Both MTI and ETI trigger massive transcriptional reprogramming, which results in the differential expression of hundreds of genes, including short interfering RNAs (siRNAs) and microRNAs (miRNAs). Recently, several siRNAs and miRNAs were found to orchestrate PTI and ETI, implying a key role of RNA silencing in the regulation of the plant immune system.

RNA silencing is a conserved eukaryotic mechanism that can repress gene expression in a sequence-specific manner via small RNA guides. RNA silencing controls gene expression at the transcriptional (TGS, Transcriptional Gene Silencing) and post-transcriptional (PTGS, Post-transcriptional Gene Silencing). In plants, PTGS plays a central role in antiviral resistance and has recently been shown to contribute to resistance against phytopathogenic bacteria, fungi and oomycetes. In turn, pathogens have evolved strategies to suppress PTGS as part of their pathogenicity. For example, many RNA viruses encode Viral Suppressors of RNA silencing (VSR) proteins that interfere with the biosynthesis and/or activity of small RNAs. The bacterium Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000) has also evolved type-three secreted proteins to suppress RNA silencing. However, the modes of action of such Bacterial Suppressors of RNA silencing (BSRs) remain elusive. Recent studies have filled in some gap by showing that two bacterial effectors from Pto DC3000 can directly interfere with the Arabidopsis ARGONAUTE 1 (AGO1) protein, a key component of the RNA-Induced Silencing Complex (RISC) in plants. For example, the Pto DC3000 effector HopT1-1 was found to interact with, and suppress the function of, Arabidopsis AGO1 through its glycine-tryptophan (GW) motifs. Such GW/WG motifs are present in some endogenous RNA silencing factors and are known to form AGO-binding platforms. Importantly, several key virulence determinants from RNA viruses and from the devastating Irish potato oomycete pathogen Phytophthora infestans also contain canonical GW/WG motifs suggesting that a wide range of phytopathogens have probably evolved an analogous virulence strategy to enable disease.

In the present proposal, we propose to conduct unbiased forward genetic screens in Arabidopsis thaliana to identify mutations that restore PTGS activity in the presence of the canonical GW/WG pathogen effector HopT1-1. Importantly, the proposed screens will be conducted at the levels of hydathodes and stomata, which represent two major entry sites for phytopathogens. Mutants that exhibit an altered pathogen entry phenotype will be further selected and the corresponding mutated genes identified using a mapping-by-sequencing approach. We are anticipating that some of these mutants will encode key negative regulators of PTGS, whose inactivation should counteract pathogen-mediated suppression of RNA silencing, thereby likely resulting in an enhanced disease resistance against pathogens. An in-depth characterization of these candidate genes will be further conducted in both RNA silencing and plant immunity. Overall, the proposed project should allow a better understanding of the basic mechanisms underlying PTGS and host counter-counter defense in plants. In addition, it should provide novel insights into strategies that can be used to control pathogens in plants, therefore opening-up new horizons in agricultural applications.