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

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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.