Institut de Biologie de l'École Normale Supérieure ENS . CNRS UMR8197 . Inserm U1024 Directeur : Pierre Paoletti

Authors
Antinéa Ravet^#, Jérôme Zervudacki^#, Meenu Singla-Rastogi, Magali Charvin, Odon Thiebeauld, Alvaro L Perez-Quintero, Lucas Courgeon, Adrien Candat, Liam Lebeau, Antonio Emidio Fortunato, Venugopal Mendu, Lionel Navarro

Abstract

Extracellular plant small RNAs (sRNAs) and/or double-stranded RNA (dsRNA) precursors act as triggers of RNAi in interacting filamentous pathogens. However, whether any of these extracellular RNA species direct gene silencing in plant-interacting bacteria remains unknown. Here, we show that Arabidopsis transgenic plants expressing sRNAs directed against virulence factors of a Pseudomonas syringae strain, reduce its pathogenesis. This Antibacterial Gene Silencing (AGS) phenomenon is directed by Dicer-Like (DCL)-dependent antibacterial sRNAs, but not cognate dsRNA precursors. Three populations of active extracellular sRNAs were recovered in the apoplast of these transgenic plants. The first one is mainly non-vesicular and associated with proteins, whereas the second one is located inside Extracellular Vesicles (EVs). Intriguingly, the third population is unbound to proteins and in a dsRNA form, unraveling functional extracellular free sRNAs (efsRNAs). Both Arabidopsis transgene- and genome-derived efsRNAs were retrieved inside bacterial cells. Finally, we show that salicylic acid (SA) promotes AGS, and that a substantial set of endogenous efsRNAs exhibits predicted bacterial targets that are down-regulated by SA biogenesis and/or signaling during infection. This study thus unveils an unexpected AGS phenomenon, which may have wider implications in the understanding of how plants regulate microbial transcriptome, microbial community composition and genome evolution of associated bacteria.

More information

Fig.10 sub-title

Model depicting the extracellular sRNA populations that are active against Pto DC3000 and/or internalized by these bacterial cells
Model depicting the characterized Arabidopsis-encoded artificial sRNA species directing gene silencing in interacting Pto DC3000 cells. The Arabidopsis IR-CFA6/HRPL#4 transgenic plants produce an inverted repeat transcript that is processed by DCL proteins into anti-cfa6/hrpL sRNAs (in green). The cytoplasmic anti-cfa6/hrpL sRNA species are further incorporated into protein complexes containing RNA Binding Proteins (RBPs), or potentially present in a free and dsRNA form in the cytosol, or in EVs. Both sRNA species are then exported in the apoplast through unknown mechanisms (dashed arrows). Three populations of active anti-cfa6/hrpL sRNAs have been characterized in this study : a first pool that is bound to proteins, which are either non-associated to PEN1-positive EVs, or potentially located on their surface. A second pool of sRNAs that is presumably located inside TET8-positive EVs, and a third pool that is unbound to proteins and in a free and dsRNA form, named efsRNAs. Active plant extracellular-sRNA species (e.g. anti-hrpL sRNAs), are subsequently transferred towards Pto DC3000 cells (dashed arrows), to direct sequencespecific gene silencing of hrpL through as-yet unknown mechanisms. Finally, the model depicts the endogenous sRNAs (in red), produced from various Arabidopsis endogenous genomic origins, including specific tRFs, which are internalized by Pto DC3000 cells.