RESUMO
In plants and some animal lineages, RNA silencing is an efficient and adaptable defense mechanism against viruses. To counter it, viruses encode suppressor proteins that interfere with RNA silencing. Phloem-restricted viruses are spreading at an alarming rate and cause substantial reduction of crop yield, but how they interact with their hosts at the molecular level is still insufficiently understood. Here, we investigate the antiviral response against phloem-restricted turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana. Using a combination of genetics, deep sequencing, and mechanical vasculature enrichment, we show that the main axis of silencing active against TuYV involves 22-nt vsiRNA production by DCL2, and their preferential loading into AGO1. Moreover, we identify vascular secondary siRNA produced from plant transcripts and initiated by DCL2-processed AGO1-loaded vsiRNA. Unexpectedly, and despite the viral encoded VSR P0 previously shown to mediate degradation of AGO proteins, vascular AGO1 undergoes specific post-translational stabilization during TuYV infection. Collectively, our work uncovers the complexity of antiviral RNA silencing against phloem-restricted TuYV and prompts a re-assessment of the role of its suppressor of silencing P0 during genuine infection.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Argonautas/genética , Proteínas de Ciclo Celular/genética , Interações Hospedeiro-Patógeno/genética , Luteoviridae/genética , Doenças das Plantas/genética , Ribonuclease III/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Arabidopsis/imunologia , Arabidopsis/virologia , Proteínas de Arabidopsis/imunologia , Proteínas Argonautas/imunologia , Proteínas de Ciclo Celular/imunologia , Resistência à Doença/genética , Regulação da Expressão Gênica , Genes Supressores , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/imunologia , Luteoviridae/crescimento & desenvolvimento , Luteoviridae/metabolismo , Floema/genética , Floema/imunologia , Floema/virologia , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Interferência de RNA , Ribonuclease III/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteínas Virais/metabolismoRESUMO
RNA silencing is a major antiviral defense mechanism in plants and invertebrates. Plant ARGONAUTE1 (AGO1) is pivotal in RNA silencing, and hence is a major target for counteracting viral suppressors of RNA-silencing proteins (VSRs). P0 from Turnip yellows virus (TuYV) is a VSR that was previously shown to trigger AGO1 degradation via an autophagy-like process. However, the identity of host proteins involved and the cellular site at which AGO1 and P0 interact were unknown. Here we report that P0 and AGO1 associate on the endoplasmic reticulum (ER), resulting in their loading into ER-associated vesicles that are mobilized to the vacuole in an ATG5- and ATG7-dependent manner. We further identified ATG8-Interacting proteins 1 and 2 (ATI1 and ATI2) as proteins that associate with P0 and interact with AGO1 on the ER up to the vacuole. Notably, ATI1 and ATI2 belong to an endogenous degradation pathway of ER-associated AGO1 that is significantly induced following P0 expression. Accordingly, ATI1 and ATI2 deficiency causes a significant increase in posttranscriptional gene silencing (PTGS) activity. Collectively, we identify ATI1 and ATI2 as components of an ER-associated AGO1 turnover and proper PTGS maintenance and further show how the VSR P0 manipulates this pathway.