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

1. 土传小麦花叶病毒（SBWMV）是小麦花叶病毒属的典型种，在感染小麦植株的RNA2中形成自发缺失突变。对RNA2中缺失位点的详细分析表明，缺失的3'端直接位于p19启动子序列的上游。进一步进入启动子区域的缺失抑制了p19亚基因组RNA的转录和病毒RNA的细胞间运动，但单细胞中的RNA复制不受影响。在SBWMV感染性cDNA克隆中，将RNA1上的p37基因和RNA2上的CP、RT和p19基因替换为绿色荧光蛋白（GFP）基因。将体外转录本接种到小麦叶片上，用荧光显微镜检测接种叶片和上部叶片的GFP荧光。结果表明，p37是病毒RNA运动蛋白，p19是病毒RNA高效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被证明能促进病毒RNA的系统运动。野生型SBWMV的最佳侵染温度在20℃以下。然而，当系统感染的小麦或大麦植株在25℃下保存时，在有限的植株中出现了系统感染25℃的病毒变体。当对变异基因组进行彻底分析时，在p37运动蛋白基因中只发现了一个不相同的核苷酸变化。p37突变被证实是SBWMV变异在25℃下持续感染的原因。在RNA2.5上的p19基因上定位并鉴定了SBWMV突变体引起的毒力增强。将双诱饵基因整合到酵母染色体中，对酵母双杂交系统进行了改造。cDNA文库筛选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.