Interactional Analysis Of Plant Host Factor RBCS1 With RaMoV NSP

Ramie mosaic virus(RaMoV)is a bipartite begomovirus, the occurrence of plant disease has been reported in Hunan, Jiangsu, Zhejiang and Fujian provinces of China. This virus can infect Nicotiana tabacum and induce symptoms including leaf curling, mosaic , enation and plant stunting.The nuclear shuttle protein (NSP) from the DNA-B of bipartite geminiviruses facilitates the intracellular transport of viral DNA from the nucleus to the cytoplasm and acts in concert with the movement protein (MP) to promote the cell-to-cell spread of the viral DNA. In this study, the full-length coding sequence of BV1 was cloned into the prokaryotic expression vector pET-28a(+) to construct the desired plasmid pET-NSP, which was then transformed into Escherichia coli BL21(DE3) to get the BL21/pET-NSP. After induction with IPTG(Isopropylβ-D-1-Thiogalactopyranoside), the expressed protein was identified by SDS-PAGE and Western blotting. The recombinant NSP was employed for immunizing rabbits and the antiserum was obtained. The antibody titer was about 1:32000. Western blotting showed that the antibody was specific.The role in pathogenicity of RaMoV was investigated by heterologous expression with the PVX vector. The results showed that AV2 and BV1 were pathogenicity factors of RaMoV. Necrosis was observed in the newly developed leaves of Nicotiana benthamiana infected by PVX-AC2 and PVX-BC1 at 10 and 13 day post-inoculation respectively. Later, most of these plants died because of the disease. The N. benthamiana infected by PVX-AC4 displayed mosaic symptoms at 8 day post-inoculation. The symptoms induced by PVX-AC2 or PVX-BC1 were similar to that induced by PVX, which include mild mosaic and leaf curling.Total RNA was extracted from systemic leaves of N. benthamiana infected by different PVX vectors. Northern blot analysis using PVX CP as a probe indicated that AV2 significantly enhanced the accumulation of PVX. However, NSP showed no or undetectable effects on PVX replication.The cellular localization patterns of the proteins encoded by DNA-B of RaMoV were observed under a confocal microscope. As expected, it was found that the NSP encoded by BV1 mainly accumulated in the nucleus, whereas the MP encoded by BC1 was mostely localized to the cytoplasm.The interaction of the NSP of RaMoV and Clerodendrum yellow mosaic China virus(ClYMCNV)with Ribulose-1,5-bisphosphate carboxylase/oxylase small subunit(RBCS1)from N. benthamiana was studied by using yeast two- hybrid system. Total RNA was extracted from N. benthamiana. RT-PCR was used to clone the full-length ORF of RBCS1. pGBK-T7 and pGAD-T7 vectors harbouring RBCS1 or BV1 were transformed into the yeast AH109. Then, the cotransformants were plated on different synthetic dropout nutrient medium and their growth performance was observed. The results showed that the NSPs of both RaMoV and ClYMCNV could interact with RBCS1. The results of bimolecular fluorescence complementation(BiFC)showed that the interaction between the NSP of RaMoV and RBCS1 mainly occurred in the nucleus. Real-time PCR was conducted to detect the expression of RBCS1 in RaMoV infected N. benthamiana. The results showed the expression of RBCS1 was reduced by 55% in RaMoV infected plants.To investigated the biological implications of the interaction between RaMoV NSP and RBCS1. Virus induced gene silencing(VIGS)was used to silence RBCS1 in N. benthamiana. Then, an infectious clone of RaMoV was used to inoculate RBCS1 silenced N. benthamiana. Southern blot analysis revealed that the replication of RaMoV was greatly enhanced in these plants than in wild type N. benthamiana. This indicated that RBCS1 might play a negative role in RaMoV infection. PVX-GFP was also used to infect RBCS1 silenced N. benthamiana. 12 days after infection, the GFP fluorescence was observed under UV light. It was found that the fluorescence intensity was higher in RBCS1 silenced N. benthamiana than in wild type plants both in the inoculated and in the systemic leaves. Accordingly, Southern blot analysis showed that replication of PVX was elevated in RBCS1 silenced N. benthamiana. These results indicated that RBCS1 might play a general role in plant defense responses against viral infection.