The viral silencing suppressor P19 interacts with the receptor‐like kinases BAM1 and BAM2 and suppresses the cell‐to‐cell movement of RNA silencing independently of its ability to bind sRNA

Abstract

To guarantee a successful invasion of their host plant, viruses need to co-opt the cell molecular machinery in order, not only to allow for viral replication and spread, but also to effectively suppress defence. In plants, RNA silencing is considered the main layer of anti-viral defence. RNA silencing encompasses post-transcriptional gene silencing (PTGS), a conserved response to the presence of double-stranded RNA (dsRNA) molecules produced during the viral infection, targeting viral genomes and transcripts in a sequence-specific manner and leading to their degradation or repression of translation (reviewed in Li & Wang, 2019). The efficacy of PTGS to fend off viruses is illustrated by the fact that virtually all plant viruses described to date encode at least one protein acting as a suppressor of RNA silencing, which is essential to guarantee the establishment of a full infection. One important property of RNA silencing is its ability to move between cells, which depends on 21-nt small interfering (si) RNA duplexes (reviewed in Zhang et al., 2018). This mobile nature of RNA silencing allows for the information of a viral invasion to spread ahead of the front of the infection, hence ‘immunizing’ distant tissues; the functional relevance of mobile siRNA-mediated non cell-autonomous RNA silencing for plant recovery during a viral infection has been recently demonstrated (Kørner et al., 2018). Phylogenetically unrelated virus-encoded silencing suppressors interfere with the cell-to-cell movement of RNA silencing; however, most of these silencing suppressors also suppress silencing locally, and this ability is assumed to underlie their effect on the intercellular movement of the silencing signal. One exception is C4 from the geminivirus Tomato yellow leaf curl virus (TYLCV), which does not act as a strong local silencing suppressor, but can interfere with the intercellular spread of silencing mediated by siRNA (Rosas-Diaz et al., 2018). C4 interacts with the intracellular domain of two plasma membrane-localized receptor-like kinases (RLKs), BAM1 and BAM2, this interaction being particularly strong at plasmodesmata. BAM1 and BAM2 are redundantly required for the cell-to-cell spread of RNA silencing (Rosas-Diaz et al., 2018), suggesting that C4 inhibits this function of BAM1/2. In Arabidopsis, BAM1/2 are strongly expressed in the vasculature, through which plant viruses spread to establish a systemic infection. Interfering with RNA silencing and its movement is supposed to be conditio sine qua non for a successful viral infection. Although this interference can take place at different steps of the pathway, since BAM1/2 has been proposed to play a role in the cell-to-cell spread of RNA silencing and is targeted by a geminivirus-encoded protein, we reasoned that other plant viruses might also have evolved to target BAM1/2 or BAM1/2-containing protein complexes. To test this idea, we selected the well-known silencing suppressor P19 from Tomato bushy stunt virus (TBSV; Fam. Tombusviridae) (Qu & Morris, 2002). Strikingly, we found that P19 associates with BAM1 and BAM2 in co-immunoprecipitation (co-IP) assays, can be found at the cell periphery, and interacts with BAM1/2 in bimolecular fluorescence complementation (BiFC) experiments (Fig. 1a–d; Supporting Information Figs S1a, S2a–c,e); notably, P19 interacts with BAM1/2 at the plasma membrane, but preferentially at plasmodesmata (Figs 1d, S1a, S2c,e). These results strongly suggest that BAM1/2 are targeted by independently evolved viral silencing suppressors, therefore suggesting a potential central role of these RLKs in plant–virus interactions. P19 binds sRNA duplexes and inhibits RNA silencing in a cell-autonomous manner (Silhavy et al., 2002; Vargason et al., 2003; Ye et al., 2003); notably, P19 also suppresses the intercellular spread of silencing, and this property is assumed to be the direct result of sRNA sequestration by the viral protein, an idea supported by immunoprecipitation experiments (Papp et al., 2003; Chapman et al., 2004; Brosnan et al., 2019; Devers et al., 2020). Structural studies unveiled that one homodimer of P19 binds one 21-nt RNA duplex, and identified two tryptophan residues, W39 and W42, as involved in the contact (Vargason et al., 2003; Ye et al., 2003); mutations in W39/W42 compromise the ability of P19 to suppress RNA silencing locally (Vargason et al., 2003; Fig. 1e). Our results indicate these do not, however, affect the interaction between P19 and BAM1/2 (Figs 1f–h, S1b, S2a,b,d,f). We reasoned that, since P19 targets BAM1, the ability of this viral protein to suppress RNA silencing movement might be underpinned by two superimposed independent mechanisms, namely the sequestration of sRNA molecules and the interaction with BAM1/2. With the aim of determining if P19 can interfere with RNA silencing movement in a sRNA binding-independent manner, we generated transgenic Arabidopsis plants expressing wild-type P19 or the potentially sRNA binding-compromised version P19W39/42R (Vargason et al., 2003) in the SUC-SUL background (Himber et al., 2003). Transgenic SUC-SUL plants express a partial inverted repeat of the SUL gene (At4g18480) (SUC2-SUL-LUS), essential for chlorophyll biosynthesis, in phloem companion cells, leading to the production of 21- and 24-nt siRNA against SUL and the subsequent gene silencing; cell-to-cell movement of 21-nsiRNA results in a bleaching phenotype around the vasculature (Himber et al., 2003). We selected three independent transgenic lines for each version of P19, displaying different levels of expression of this transgene (Fig. 1i); none of the lines is affected in the accumulation of the SUC2-SUL-LUS transcript (Fig. 1i). Strikingly, P19W39/W42R could suppress the spread of RNA silencing from the vasculature to a similar extent as the wild-type version (Fig. 1j,k), indicating that the capacity of P19 to block the intercellular movement of RNA silencing does not require wild-type-like sRNA binding and is therefore most likely independent of sRNA sequestration. Thus, it is probable that the abilities exhibited by P19 to suppress RNA silencing cell-autonomously and non cell-autonomously can be uncoupled. This opens up the possibility that P19 halts the cell-to-cell spread of RNA silencing through its interaction with BAM1/2, following a similar strategy to that displayed by the C4 protein encoded by TYLCV (Rosas-Diaz et al., 2018) (Fig. 1l). Taken together, our results shed new light onto the molecular mechanisms underpinning the role of P19 in silencing suppression, and uncover BAM1/2 as proteins convergently targeted by independently evolved plant viruses, making them emerge as potentially interesting candidates for anti-viral breeding and editing strategies to be explored in the future. The authors thank all members in the Lozano-Duran laboratory (GeminiTeam) for useful comments and suggestions and insightful discussions. Work in the Lozano-Duran laboratory is supported by the Shanghai Centre for Plant Stress Biology from the Chinese Academy of Sciences, the National Science Foundation China (NSFC grant nos. 31671994 and 31870250), and the Chinese Academy of Sciences Strategic Pilot Science and Technology Special (B) funding (grant no. XDB27040206). BGG is funded by President’s International Fellowship Initiative (PIFI) postdoctoral fellowship (no. 2020PB0082) from the Chinese Academy of Sciences, and is the recipient of a Talent-Introduction Grant (Chinese Postdoctoral International Exchange Programme). JSR is funded by PIFI postdoctoral fellowships (nos. 2018PB0057 and 2020PB0088) from the Chinese Academy of Sciences. RL-D conceived and supervised the project; BGG, TR-D, CS, PF, DZ and JSR generated/analysed data; BGG made the figures; RL-D wrote the manuscript with input from all the authors. Fig. S1 P19 interacts with BAM1. Fig. S2 P19 and P19W39/42R interact with BAM2. Methods S1 Extended description of Materials and Methods used in this work. Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Topics & Concepts