Studies on pathogenicity and soil-transmissibility of plant RNA viruses in the genus Furovirus

1.土传小麦花叶病毒（Soil-borne Wheat Mosaic Virus，SBWMV）是糠病毒属（Furovirus）的模式种，在受感染的小麦植株中会自发形成RNA 2的缺失突变。对RNA 2中缺失位点的详细分析表明，缺失的3'末端终止于p19启动子序列的上游。启动子区的进一步缺失抑制了p19亚基因组RNA的转录和病毒RNA的细胞间移动，但不影响RNA在单细胞中的复制.在SBWMV的感染性cDNA克隆中，RNA 1上的p37基因和RNA 2上的CP、RT和p19基因被绿色荧光蛋白（GFP）基因取代。将体外转录物接种到小麦叶片中，用荧光显微镜检查接种叶片和上部叶片中的GFP荧光。结果表明，p37是病毒RNA运动蛋白，p19是病毒RNA有效的细胞间运动和长距离运动所必需的，CP是病毒持续感染所必需的。CP-RT能促进病毒RNA的系统性移动.野生型SBWMV的最适感染温度低于20℃。然而，当将系统感染的小麦或大麦植株保持在25℃时，在有限的植株中出现了在25℃下系统感染植株的病毒变异体。当对变异基因组进行全面分析时，仅在p37运动蛋白基因中发现了一个不同名的核苷酸变化。证实p37突变是SBWMV变异株在25℃下持续感染的原因.在RNA2.5上的p19基因中定位并鉴定了SBWMV突变体引起的毒力增加。将BD：bait基因整合到酵母染色体上，对传统的酵母双杂交系统进行了改造。从cDNA文库筛选中获得真阳性克隆的效率显著提高。

1. Soil-borne wheat mosaic virus (SBWMV), the type species of the Genus Furovirus, forms spontaneous deletion mutations in RNA2 in infected wheat plants. Detailed analysis of deleted sites in RNA2 showed that the 3' termini of deletions end immediately upstream of the p19 promoter sequence. Deletion further into the promoter region inhibited transcription of p19 subgenomic RNA and cell-to-cell movement of viral RNA although RNA replication in single cells was not affected.2. The p37 gene on RNA1 and CP, RT and p19 genes on RNA2 were replaced with a green fluorescence protein (GFP) gene in infectious cDNA clones of SBWMV. In vitro transcripts were inoculated to wheat leaves and GFP fluorescence in inoculated leaves and upper leaves were examined with a fluorescence light microscopy. Results showed that p37 is the viral RNA movement protein, which p19 is required for efficient cell-to-cell movement and long-distance movement of viral RNA, and that CP is required for persistent infection of the virus. CP-RT was shown to facilitate movement of viral RNA systemically.3. The wild-type SBWMV has optimal temperatures for infection below 20℃. However, when systemically infected wheat or barley plants were kept at 25℃, virus variants that infects plants systemically at 25℃ appeared in limited plants. When the variant genome was thoroughly analyzed, only one asynonymous nucleotide change was found in p37 movement protein gene. The mutation in p37 was confirmed to be responsible for persistent infection of SBWMV variants at 25℃.4. Increased virulence caused by mutants of SBWMV was mapped and identified in p19 gene on RNA2.5. A conventional yeast two-hybrid system was modified by integration of BD:bait genes into yeast chromosome. Efficiency to get true-positive clones from cDNA library screening was significantly increased.