Potato mop-top virus: Variability, Movement and Suppression of Host Defence
Abstract: Potato mop-top virus (PMTV) causes an economically damaging disease called potato spraing. Despite being reported across the potato growing regions in the world, very little genetic variability has been reported for the virus. Also, the knowledge on how PMTV suppresses host defence mechanism, and how it interacts with the host during the cellto-cell and long-distance movement is still insufficient to develop successful preventive measures against the PMTV infection. This thesis work identified high diversity of the PMTV in the Andean region of Peru compared to the rest of the world. Among the PMTV genome, CP-RT and 8K genomic regions accumulated the largest number of mutations. Through phylogenetic analysis of the RNA-CP segment we identified two prevailing genotypes around the world. Based on the pathobiological differences, we named these lineages as S (severe), and M (mild) types. The phylogenetic relationship determined in this study helped us to propose a novel classification of PMTV isolates. Our analysis to address the selection pressure on the PMTV genome revealed that the ORF encoding the 8K protein, a viral suppressor of RNA silencing (VSR) is under strong positive selection. Characterization of the RNA silencing suppression activity of the 8K protein from seven highly diverse isolates revealed that the 8K encoded by a Peruvian isolate, P1 exhibits stronger RNA silencing suppression activity compared to that of other isolates. Through mutational analysis, we identified that Ser-50 is necessary for these differences. Through deep sequencing for sRNAs, we identified that VSRs reduce the sRNA accumulation. We observed lower amount of siRNAs with U residue at the 5’-terminus suggesting that P1 8K might affect AGO1-mediated RNA silencing. The present work also identified key host factors necessary for the cell-to-cell and long distance movement of the virus. We showed that the actin network and certain class VIII myosins motors are important for the cell-to-cell movement of PMTV. The dependency on the acto-myosin network for PMTV movement was further demonstrated by the fluorescence recovery after photo bleaching experiments that resulted in compromised delivery of the TGB1 at the plasmodesmata upon disrupting actin and inhibiting two class VIII myosins. In contrast, class XI myosins did not have a significant effect on the cell-to-cell movement of the PMTV, although they appear to be important viral long-distance movement. Analysis of PMTV TGB1 interactions the with host proteins revealed that TGB1 interacts with Nicotiana benthamiana HIPP26 protein, a vascular expressed, metallochaperone that acts as a plasma membrane to nucleus stress signalling relay. PMTV infection upregulated the expression of HIPP26 and altered its subcellular localization from plasmodesmata to the nucleus. Knockdown of NbHIPP26 expression resulted in inhibition of virus long-distance movement, but not the cell-to-cell movement. Together, this data suggests that PMTV hijacks NbHIPP26 to facilitate the long-distance movement of the virus.
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