In plants, RNA silencing regulates gene expression through the action of Dicer-like (DCL) and Argonaute (AGO) proteins via micro RNAs and RNA-dependent DNA methylation (RdDM). In addition, RNA silencing functions as an antiviral defense mechanism by targeting virus-derived double-stranded RNA. Plants encode multiple AGO proteins with specialized functions, including AGO4-like proteins that affect RdDM and AGO2, AGO5, and AGO1, which have antiviral activities. Here, we show that AGO4 is also required for defense against the potexvirus Plantago asiatica mosaic virus (PlAMV), most likely independent of RdDM components such as DCL3, Pol IV, and Pol V. Transient assays showed that AGO4 has direct antiviral activity on PlAMV and, unlike RdDM, this activity does not require nuclear localization of AGO4. Furthermore, although PlAMV infection causes a decrease in AGO4 expression, PlAMV causes a change in AGO4 localization from a largely nuclear to a largely cytoplasmic distribution. These results indicate an important role for AGO4 in targeting plant RNA viruses as well as demonstrating novel mechanisms of regulation of and by AGO4, independent of its canonical role in regulating gene expression by RdDM.

24RNA silencing functions as an anti-viral defence in plants through the action of DICER-like 25 (DCL) and ARGONAUTE (AGO) proteins. However, there are few known examples of 26 functional variation in RNA silencing components. The AGO2 protein is important for antiviral 27 defense against multiple viruses and has been shown to be a major limiting factor to infection 28 by potato virus X (PVX) of Arabidopsis thaliana but not Nicotiana benthamiana. We show that 29 the AGO2 proteins from these two plants have differential activity against PVX, suggesting that 30 variation in AGO2 is important in plant-virus interactions. Consistent with this, we find that the 31 Arabidopsis thaliana AGO2 gene shows a high incidence of polymorphisms between 32 accessions, with evidence of selective pressure. AGO2 protein variants can be assigned to two 33 groups, in near equal frequency, based on an amino acid change and small deletions in the 34 protein N-terminus. Inoculation of a large number of Arabidopsis accessions shows strong 35 correlation between these alleles and resistance or susceptibility to PVX. These observations 36 were validated using genetic and transgenic complementation analysis, which showed that one 37 type of AGO2 variant is specifically affected in its antiviral activity, without interfering with other 38 AGO2-associated functions such as anti-bacterial resistance or DNA methylation. Our results 39 demonstrate a novel type of genetically-encoded virus resistance and suggest that plant-virus 40 interactions have influenced natural variation in RNA silencing components. 41 42 43 44Here, we show that AGO2 proteins from A. thaliana and N. benthamiana manifest 68 differing activities against PVX, suggesting that inter-specific differences in AGO2 may 69 contribute to differing outcomes of PVX infection in these species. Furthermore, we tested 70 whether intra-specific differences in AGO2 might affect plant-virus interactions by taking 71 advantage of the natural genetic variation of wild Arabidopsis accessions. We show the AGO2 72 gene presents a high level of polymorphism and shows evidence of having been subject to 73 selective pressure. Furthermore, unlike the commonly used Arabidopsis accession Col-0, 27 74 out of 63 accessions analysed were found to susceptible to PVX. Through genetic and 75 transgenic analysis, we show that this susceptibility is determined by two polymorphisms found 76 in the N-terminus of the AGO2 protein. Our results have uncovered a novel form of genetically-77 encoded virus resistance. Likewise, it suggests that natural variation in AGO2 may be important 78 for determining plant-virus interaction outcomes and that in turn these pressures may have 79 shaped the RNA silencing machinery in ways similar to other defense mechanisms.80

Road ecology studies are beneficial for informing sustainable development plans. Our ecological focus lies in developing crossing structures at five sites in the Kingston area to enable safe turtle crossing during the breeding season from May-September. Locations have been selected based on previous evidence of animal casualties and the potential for culvert construction. The Kingston Society of Conservation Biology hopes to implement a solution, over the course of 3-5 years that has commenced with the installation of turtle crossing signs at all locations and will be completed once culverts are constructed. We will investigate the success of culvert development by other conservation groups in order to determine if this is a viable solution for the four sites being investigated in this study. Our group will propose adjustments to culvert construction and alternate, feasible solutions where conventional culvert development is expected to be ineffective in reducing turtle mortality. This study aims to ensure that culverts will be effective in increasing the safe transfer of turtles between wetland areas. Ultimately, we hope to propose a long-term sustainable solution that will decrease the effects of anthropogenic activities and urbanization on the Kingston turtle population.