低温增强甜菜黑色焦枯病毒致病性的分子机制研究

In the field, Beet black scorch virus(BBSV)usually elicit serious viral disease in the sugar beet plant, especially in late spring season with cold weather. Similarly, BBSV could induce severer viral symptoms under low temperature in the laboratory. For understanding the phenomena, the preliminary works have been carried out in the three respects with the transcriptome sequencing, small RNA cloning, as well as viral mutant screening and verification. Accordingly, it is proposed that low efficiency of antiviral silencing,improvement of viral pathogenesis, reduction of suppressing activitys on the viral pathogenic factors by host genes, are the three conceivable mechanisms underlying the regulation of low temperature upon the pathogenesis of BBSV. For clarifying the regulation of temperature on the interaction between viral pathogenic factors and host antiviral genes, the proposal here is consisted of three aims that (i) mining of the high throughput data from deep sequence of transcriptome and small RNA, to analysis the transcriptional level of the genes especially with regard to RNA silencing, calmodulin-binding, and targeted mRNA by some small RNA,(ii) carrying out the transient and stable expression, virus inoculation, localization, and biochemistry assays, to reveal the dominant and recessive antiviral activity of host genes, (iii) screening the new viral mutants, evaluation of the viral pathogenesis and factor in either cell or plants level, to explore the viral factors regulated by low temperature.

在田间晚春季节低温气候的年份，甜菜黑色焦枯病毒（BBSV）造成的甜菜病害明显加重；在实验室中也发现，BBSV在低温时能高效侵染本生烟，并造成严重病毒症状，与田间危害情况一致。针对此现象，前期工作中对病毒侵染转录组深度测序、小RNA克隆分析及病毒自发突变体的筛选鉴定开展了深入分析，提出了低温导致"寄主RNA沉默能力降低"、"病毒编码蛋白致病能力增强"和"抑制病毒致病因子的寄主基因活性下降"三个可能的分子机制。本申请课题拟从BBSV致病因子和本生烟抗病基因受温度调控角度出发，通过生物信息学挖掘本生烟转录组和小RNA组分的测序结果，重点分析RNA沉默和钙调蛋白等基因的转录水平，以及小RNA的靶标基因变化；通过瞬时或稳定表达、病毒接种、生化分析等方法，探索受温度调控的寄主基因显性或隐性抗病机制；通过病毒突变体筛选、细胞和植株整体的侵染力鉴定及致病因子功能分析，明确低温致病相关的病毒因子。

RNA沉默是寄主植物的主要抗病毒策略，一些病毒携带的卫星RNA对其致病性和寄主RNA沉默也有显著影响。本项目首先利用sRNA的深度测序和分子检测分析了甜菜黑色焦枯病毒(BBSV)及其卫星RNA(sat-RNA)引发寄主RNA沉默的机理进行研究。结果表明低温和卫星RNA共侵染都能不同程度的降低BBSV产生siRNA的效率，从而这两种因素共同促进BBSV的侵染能力。病毒在受到寄主植物抗病反应后，也会通过自发突变产生毒力更强的病毒株系。本研究通过连续在高温抗病条件下接种BBSV,所获得77个BBSV突变体侵染性cDNA克隆中，26个突变体比野生型病毒的致病力更强，并且通过重组实验证明BBSV 3'UTR区域的第3477位核苷酸由U到G的改变是导致突变体病毒致病力增强的主要原因。此外，我们还通过透射电子显微镜 (TEM) 观察BBSV侵染本生烟后形成的内质网小泡结构是BBSV的复制场所。最后，我们发现内质网上发生的未折叠蛋白反应 (UPR) 在BBSV侵染过程中发挥重要作用，并且UPR反应是受到温度调节的。本研究得到的结果说明病毒的复制增殖、寄主RNA沉默反应，以及病毒进一步自发突变产生强毒株都是病毒-寄主-环境温度综合作用的结果。