Functional significance of Argonaute 2 proteoforms in the plant antiviral immune response

RNA沉默或RNA干扰（RNAi）是植物抵抗病毒感染的免疫应答的核心组分。首先，Dicer样酶加工病毒RNA的双链元件，即，病毒基因组、-复制中间体和/或-mRNA，转化为小干扰RNA，siRNA。作为RNA诱导的沉默复合物（RISC）的一部分，Argonaute（AGO）核酸内切酶然后掺入单个siRNA链，并且这些通过互补碱基配对将RISC引导至靶RNA。靶RNA主要对应于早期产生siRNA的同源RNA。含有病毒衍生的“vsiRNA”的AGO/RISC然后通过内切核酸裂解降解病毒靶RNA或抑制它们的翻译。重要的是，在病毒感染的细胞中，RNAi不仅影响病毒，而且影响细胞基因表达。最近，发现后者主要通过产生一类新的siRNA而发生，所述siRNA产生自细胞mRNA并且被称为“病毒激活的”“vasiRNA”。有趣的是，其他人和我们的观察表明，在菊科中感染不同类型的病毒会诱导vasiRNA的形成，这些vasiRNA下调同一组基因的表达，包括在胁迫反应和光合作用中发挥重要作用的几个基因。作为免疫应答的重要部分，vasiRNA因此似乎通过调节应激应答或通过消耗能量资源来抑制病毒复制。抗病毒RNAi基本上涉及21 nt以及22 nt长的vsiRNA和vasiRNA。然而，特别是对于22 nt siRNA，其活性模式仍不清楚。AGO 2蛋白也是如此，在模式植物拟南芥（At）中鉴定的至少10种AGO蛋白中，AGO 2蛋白最近成为vsiRNA和vasiRNA介导的抗病毒沉默的关键组分。在这种情况下，出乎意料地发现，AGO 2，与同样具有抗病毒作用的AGO 1相反，受到强大的选择压力以多样化。最有趣的是，我们最近观察到，AGO 2在At中的蛋白质形式，即由单核苷酸多态性（SNP）引起的蛋白质变体，当与22 nt siRNA相关联时，其沉默活性显示出显著差异。由此产生的中心假设是AGO 2蛋白形式通过22 nt siRNA的差异活性在植物适应病毒感染中发挥重要作用。这种适应可以通过22 nt vsiRNA的差异活性在病毒基因表达和复制水平上发生和/或通过22 nt vsiRNA的差异活性在宿主基因表达水平上发生。我们提出的项目旨在通过以下方式证明这一假设：（i）鉴定介导AGO 2蛋白形式与22 nt siRNA的差异活性的分子决定因素，以及（ii）表征AGO 2 SNP蛋白形式在vsiRNA和vasiRNA介导的RNA沉默中的抗病毒活性植物。

RNA silencing or RNA interference (RNAi) is a central component of the immune response of plants against viral infections. First, Dicer-like enzymes process double-stranded elements of viral RNAs, i.e., viral genomes, - replicative intermediates and/or – mRNAs, into small interfering RNAs, siRNAs. As part of RNA-induced silencing complexes (RISC), Argonaute (AGO) endonucleases then incorporate single siRNA strands, and these direct RISC to target RNAs via complementary base pairing. The target RNAs mostly correspond to the cognate RNAs from which the siRNAs were earlier generated. AGO/RISC that contain virus-derived ‘vsiRNAs’ then either degrade viral target RNAs by endonucleolytic cleavage or inhibit their translation. Importantly, in virus infected cells, RNAi affects not only viral but also cellular gene expression. Recently, the latter was found to occur primarily through the generation of a new class of siRNAs that arise from cellular mRNAs and are termed ‘virus-activated’ ‘vasiRNAs’. Interestingly, observations by others and us showed that infections with different types of viruses in Brassicaceae induce the formation of vasiRNAs that down-regulate the expression of the same group of genes, including several that play important roles in stress response and photosynthesis. As an important part of the immune response, vasiRNAs thus appear to inhibit viral replication via modulation of the stress response or by depletion of energy resources. Antiviral RNAi essentially involves 21 nt as well as 22 nt long vsiRNAs and vasiRNAs. However, especially for 22 nt siRNAs, the modes of activity are still unclear. The same is true for the AGO2 protein, which, of the at least 10 AGO proteins identified in the model plant Arabidopsis thaliana (At) has recently come into focus as a crucial component of both vsiRNA- and vasiRNA-mediated antiviral silencing. In this context, it was unexpectedly found that AGO2, in contrast to the also antivirally acting AGO1, is subject to strong selection pressure to diversify. Most interestingly, we recently observed that proteoforms of AGO2 in At, i.e. variants of the protein caused by single nucleotide polymorphisms (SNPs), show marked differences in their silencing activity when associated with 22 nt siRNAs. The central hypothesis arising from this is that AGO2 proteoforms play an essential role in plant adaptation to viral infections through differential activity of 22 nt siRNAs. This adaptation could occur at the level of viral gene expression and replication via differential activity of 22 nt vsiRNAs and/or at the level of host gene expression via differential activity of 22 nt vasiRNAs. Our proposed project aims at proving this hypothesis by (i) identifying the molecular determinants, which mediate the differential activities of AGO2 proteoforms with 22 nt siRNAs and by (ii) characterizing the antiviral activity of AGO2 SNP proteoforms in vsiRNA- and vasiRNA-mediated RNA silencing in plant.