Molecular mechanisms underlying host-induced gene silencing.

Host-induced gene silencing (HIGS) refers to the silencing of genes in pathogens and pests by expressing homologous double-stranded RNAs (dsRNA) or artificial microRNAs (amiRNAs) in the host plant. The discovery of such trans-kingdom RNA silencing has enabled the development of RNA interference-based approaches for controlling diverse crop pathogens and pests. Although HIGS is a promising strategy, the mechanisms by which these regulatory RNAs translocate from plants to pathogens, and how they induce gene silencing in pathogens, are poorly understood. This lack of understanding has led to large variability in the efficacy of various HIGS treatments. This variability is likely due to multiple factors, such as the ability of the target pathogen or pest to take up and/or process RNA from the host, the specific genes and target sequences selected in the pathogen or pest for silencing, and where, when, and how the dsRNAs or amiRNAs are produced and translocated. In this review, we summarize what is currently known about the molecular mechanisms underlying HIGS, identify key unanswered questions, and explore strategies for improving the efficacy and reproducibility of HIGS treatments in the control of crop diseases.

Arabidopsis apoplastic fluid contains sRNA- and circular RNA-protein complexes that are located outside extracellular vesicles.

Previously, we have shown that apoplastic wash fluid (AWF) purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these sRNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside EVs but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they include both sRNAs and long noncoding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the posttranscriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in AWF, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the sRNA-binding protein ARGONAUTE2 (AGO2). These two proteins coimmunoprecipitated with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of AWF, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that exRNAs located outside of EVs mediate host-induced gene silencing, rather than RNA located inside EVs.

AvrRpm1 Functions as an ADP-Ribosyl Transferase to Modify NOI-domain Containing Proteins, Including Arabidopsis and Soybean RPM1-interacting Protein 4.

The Pseudomonas syringae effector protein AvrRpm1 activates the Arabidopsis intracellular innate immune receptor protein RPM1 via modification of a second Arabidopsis protein, RIN4. Prior work has shown that AvrRpm1 induces phosphorylation of AtRIN4, but homology modeling indicated that AvrRpm1 may be an ADP-ribosyl transferase. Here we show that AvrRpm1 induces ADP-ribosylation of RIN4 proteins from both Arabidopsis and soybean within two highly conserved nitrate-induced (NOI) domains. It also ADP-ribosylates at least ten additional Arabidopsis NOI domain-containing proteins. The ADP-ribosylation activity of AvrRpm1 is required for subsequent phosphorylation on threonine 166 of Arabidopsis RIN4, an event that is necessary and sufficient for RPM1 activation. We also show that the C-terminal NOI domain of AtRIN4 interacts with the exocyst subunits EXO70B1, EXO70E1, EXO70E2 and EXO70F1. Mutation of either EXO70B1 or EXO70E2 inhibited secretion of callose induced by the bacterial flagellin-derived peptide flg22. Substitution of RIN4 threonine 166 with aspartate enhanced the association of AtRIN4 with EXO70E2, which we posit inhibits its callose deposition function. Collectively, these data indicate that AvrRpm1 ADP-ribosyl transferase activity contributes to virulence by promoting phosphorylation of RIN4 threonine 166, which inhibits the secretion of defense compounds by promoting the inhibitory association of RIN4 with EXO70 proteins.