No abstract

RNA silencing constitutes an important antiviral mechanism in plants. Small RNA guided Argonaute proteins fulfill essential role in this process by acting as executors of viral restriction. Plants encode multiple Argonaute proteins of which several exhibit antiviral activities. A recent addition to this group is AGO2. Its involvement in antiviral responses is established predominantly by studies employing mutants of Arabidopsis thaliana. In the virological model plant, Nicotiana benthamiana, the contribution of AGO2 to antiviral immunity is much less certain due to the lack of appropriate genetic mutants. Previous studies employed various RNAi based tools to down-regulate AGO2 expression. However, these techniques have several disadvantages, especially in the context of antiviral RNA silencing. Here, we have utilized the CRISPR/Cas9 technology to inactivate the AGO2 gene of N. benthamiana. The ago2 plants exhibit differential sensitivities towards various viruses. AGO2 is a critical component of the plants’ immune responses against PVX, TuMV and TCV. In contrast, AGO2 deficiency does not significantly influence the progression of tombusvirus and CMV infections. In summary, our work provides unequivocal proof for the virus-specific antiviral role of AGO2 in a plant species other than A. thaliana for the first time.

RNA guided ribonuclease complexes play central role in RNA interference. Members of the evolutionarily conserved Argonaute protein family form the catalytic cores of these complexes. Unlike a number of other plant Argonautes, the role of AGO2 has been obscure until recently. Newer data, however, have indicated its involvement in various biotic and abiotic stress responses. Despite its suggested importance, there is no detailed characterization of this protein to date. Here we report cloning and molecular characterization of the AGO2 protein of the virological model plant Nicotiana benthamiana. We show that AGO2 can directly repress translation via various miRNA target site constellations (ORF, 3′ UTR). Interestingly, although AGO2 seems to be able to silence gene expression in a slicing independent fashion, its catalytic activity is still a prerequisite for efficient translational repression. Additionally, mismatches between the 3′ end of the miRNA guide strand and the 5′ end of the target site enhance gene silencing by AGO2. Several functionally important amino acid residues of AGO2 have been identified that affect its small RNA loading, cleavage activity, translational repression potential and antiviral activity. The data presented here help us to understand how AGO2 aids plants to deal with stress.