摘要
【目的】以水稻条纹病毒(Rice stripe virus,RSV)编码的NSvc4蛋白和CP蛋白的致病功能鉴定为切入点,研究RSV的致病机理。【方法】通过农杆菌介导在本氏烟中对RSV编码的6个蛋白进行细胞定位。采用双分子荧光互补(Bimolecular fluorescence complementation,BiFC)技术鉴定NSvc4蛋白与其余5个蛋白间的互作关系,并用酵母双杂交体系(Yeast two-hybrid,YTH)对NSvc4与CP蛋白间的互作再次验证。利用马铃薯X病毒(Potato virus X,PVX)系统在本氏烟叶片研究NSvc4蛋白和CP蛋白的致病性,采用实时荧光定量PCR(Real-time PCR)技术验证致病性鉴定结果。【结果】RSV NSvc4蛋白能与CP蛋白互作,且蛋白复合体在本氏烟细胞中能够定位到叶绿体。致病性鉴定显示,PVX-RSV-NSvc4侵染的本氏烟表现为花叶症状;PVX-RSV-CP能够在侵染叶上诱导花叶症状,但是系统叶却恢复健康;PVX-RSV-NSvc4与PVX-RSV-CP共侵染的本氏烟和PVX-RSV-NSvc4CP侵染的本氏烟的侵染叶、系统叶均表现出了严重病症。Real-time PCR结果显示,PVX载体在各侵染本氏烟中的累积量均较低,而RSV CP和NSvc4基因的表达量与本氏烟的发病症状紧密相关。【结论】NSvc4和CP蛋白均有致病性。CP蛋白的致病力不能持久,但是与NSvc4蛋白互作后具有持久致病力,表明二者协同在RSV致病过程中发挥作用。
[Objective] The objective of this study is to investigate the roles of NSvc4 and CP proteins of Rice stripe virus （RSV） in pathogenicity to reveal the mechanisms underlying RSV pathogenesis. [Method] The subceUular localization patterns of six RSV proteins were studied by expressing them in epidermal ceils ofNicotiana benthamiana through Agrobacterium inoculation. The interaction between NSvc4 and five other proteins of RSV was investigated using Bimolecular fluorescence complementation （BiFC）, and the interaction between NSvc4 and CP was further confirmed by yeast two-hybrid experiments. The possible involvement of NSvc4 and CP in pathogenicity was exploited by expressing them individually or together in N. benthamiana employing the vector Potato virus X （PVX）. [Result] NSvc4 interacted with CP and they formed protein complexes in chloroplasts of N. benthamiana when co-expressed, both NSvc4 and CP could induce leaf mosaic symptoms when they were expressed using PVX. However, leaf mosaic symptoms caused by CP was recovered in systemic leaves of PVX inoculated plants. When expressed together or as a fused protein using PVX, NSvc4 and CP induced severe symptoms in both inoculated and systemic leaves of N. benthamiana. Real-time PCR experiments showed that NSvc4 and CP had no effects on the accumulation of PVX, and the severity of the symptoms induced by the recombinant virus correlated with the expression levels of NSvc4 and CP. [ Conclusion] Both NSvc4 and CP have pathogenicity properties and they might have synergistical function in RSV pathogenesis.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第1期45-53,共9页
Scientia Agricultura Sinica
基金
国家自然科学基金重点项目(3113044)
