中国甜菜坏死黄脉病毒进化过程中RNA3新tetrad 基序的致病机制分析

Beet necrotic yellow vein virus (BNYVV) is a serious threat to sugar beet industry by significantly inducing rhizomania disease worldwidely. Recent studies have demonstrated that the viral RNA3 encoded p25 protein played a pivotal role in the induction of classical rhizomania symptom on sugar beet plants, and the highly variation tetrad motif present at amino acid residues 67–70 could mediate the improving viral pathogenesis to overcome the resistance of some sugar beet varieties in fields. Our preliminary data revealed that a complicated and genetic diversity of BNYVV populations distributed across China. In addition, a more aggressive variant containing the VCHG tetrad motif was detected in some serious virus disease fields of Xijiang and Heilongjiang provinces, implying a potential outbreak and spread of some resistance breaking variants. Accordingly, the proposal here will mainly focus on the molecular variability of BNYVV in China, as well as the pathogenicity of RNA3 encoded p25. Firstly, the detection is performed continuously to survey the genetic evolution of BNYVV throughout China. Furthermore, the pathogenicity and RB ability of the p25 containing different tetrads will be explored in local lesion host and the partial resistant sugar beet cultivar. Finally, the p25 sequence variation, subcellular localization and the interaction between p25 and other proteins encoded by BNYVV will be conducted to elucidate molecular mechanism of the p25 pathogenicity in either leaf or root. This project will provide new scientific support for the breeding and planting of sugar beet varieties.

甜菜坏死黄脉病毒（Beet necrotic yellow vein virus，BNYVV）在世界范围内引起严重的甜菜丛根病。该病毒RNA3编码的p25是导致甜菜丛根症状的决定因子，其67-70位氨基酸（tetrad基序）的高度变异与甜菜抗病品种的抗性丧失有关。我国BNYVV分子变异复杂，在新疆和黑龙江的某些重病田中已出现了tetrad基序为VCHG的强病毒株系能够侵染抗病甜菜品种，因此存在重病田进一步扩大的潜在风险。本申请课题拟进一步跟踪分析我国甜菜主产区病毒序列的变异特点和系统进化，通过构建病毒侵染性克隆和接种等方法明确含有不同tetrad基序的突变株分别在枯斑寄主的致病性差异和在甜菜的抗性突破能力。在此基础上，从序列变异、亚细胞定位以及其和病毒其他关键蛋白互作等实验入手，解析p25参与病毒在叶部和根部致病的分子机制，为抗病品种的选育和合理种植布局提供科学依据。

BNYVV RNA3编码的p25是导致甜菜丛根症状的决定因子，其67-70位氨基酸（tetrad基序）的高度变异与甜菜抗病品种的抗性丧失有关。本项目首先采用RT-PCR 技术对中国各甜菜产区甜菜及土壤样品进行了检测，跟踪监测了BNYVV的田间自然变异情况，研究表明中国BNYVV群体遗传多样性复杂，基于4 个基因（CP、RNA3-p25、RNA4-p31 和RNA5-p26）的系统发生分析结果表明，全球BNYVV 分离物被分成2–4组，这与之前的报道结果相似。鉴定出BNYVV中国分离物p25和p26蛋白存在两个新亚组和一个新组。鉴定出12种 p25 tetrad基序类型，其中4种为首次报道，选择压分析表明p25处于正选择压，其他蛋白属于负选择压；其次，构建了BNYVV全长cDNA侵染性克隆，并在此基础上建立并稳定了BNYVV侵染本生烟和甜菜的研究体系，该系统可用于不同tetrad RNA3在甜菜上的致病性的鉴定、室内抗丛根病甜菜品种的快速筛选、多种异源蛋白的同时表达和寄主蛋白与病毒蛋白的作用研究；最后，初步鉴定了含新tetrad 基序RNA3的致病性，通过酵母双杂交和BiFC筛选获得与p25互作的寄主因子，对p25的致病机制进行了初步研究。我们的研究为抗病品种的选育和合理种植布局提供科学依据。