Detection Of Geminiviruses By Rolling Circle Amplification And Influence Of AGO Proteins To Geminivirus Infection

A new method based on rolling circle amplification and restriction fragment length polymorphism (RCA-RFLP) was established to detect viral DNA components associated with geminiviruses. Our results demonstrate that RCA-RFLP has high efficiency for detection of different geminiviruses, including monopartite or bipartite geminiviruses and satellite DNA associated with geminiviruses. Analysis for field samples from Hangzhou, Zhejiang province, suggests that RCA-RFLP can be successfully used to identify geminiviruses in natural infected plants. In addition, RCA is more convenient and sensitive for detection geminiviruses than PCR.NbAGO-like genes named as NbAGO1 and NbAGO4 were amplified by RT-PCR from Nicotiana benthamiana using primers designed based on AGO1 and AGO4 homologue sequences of N. benthamiana. Sequence alignments show the amplified fragments are closely related to the AGO1 and AGO4 homologues reported in N. benthamiana. NbAGO1 and NbAGO4 were introduced into Tobacco rattle virus (TRV) RNA2 silencing vector to obtain recombinant vectors pTRV2-AGO1 and pTRV2-AGO4, respectively. Semi-quantitative RT-PCR assay demonstrated that NbAGO1 or NbAGO4 mRNA accumulation was inhibited in N. benthamiana plants co-agroinoculated with pTRV2-AGO1 or pTRV2-AGO4 and TRV RNA1 (pTRV1). These results suggest that inhibition of NbAGO1 and NbAGO4 via RNA silencing can enhance accumulation of geminiviruses in N. benthamiana plants.