| Summary: | 博士 === 國立中興大學 === 生物科技學研究所 === 99 === Abstract
Bamboo mosaic virus (BaMV), a positive-sense RNA virus with the length of 6.4-kb, contains five ORFs. ORF1 of the BaMV encodes a 155-kDa replicase consisting of mRNA capping enzyme domain, helicase-like domain, and RNA-dependent RNA polymerase (RdRp) domain. In the previous study, the interaction between the BaMV RdRp domain and a host putative methyltransferase (PMtsNb1) was identified from a yeast two-hybrid screening against a leaf cDNA library of Nicotiana benthamiana. Over expression of PMtsNb1 in N. benthamiana protoplasts by CaMV 35S promoter significantly decreased the viral coat protein accumulation by 40% and the viral in vitro RdRp activity also by 50%. To assure that these inhibitions were related to the interaction of PMtsNb1 and RdRp, the PNbMts1 fused with GFP and HA-tagged RdRp were co-expressed in protoplast. In this experiment, the HA tagged RdRp could be recognized in the immunoprecipitation of PNbMts1-GFP fusion protein. In addition, mutations at the putative AdoMet-binding motifs abolished the BaMV-inhibition effect. Furthermore, addition of AdoMet in the incubation medium of protoplast enhanced the inhibition effect of PNbMts1. Besides, the accumulation of viral coat protein was less in PNbMts1-overexpressing N. benthamiana than in the wild type plant. In contrast, knock-down of PNbMts1 by virus-induced gene silencing in N. benthamiana increased the accumulation of the viral coat protein about four to six times. In summary, we have identified a novel virus-resistant protein, PNbMts1, which can inhibit the accumulation of BaMV.
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