BYDV GAV编码蛋白质的定位、互作及致病相关功能研究

Localization,Interaction and Pathogenicity of BYDV GAV Encoded Proteins

大麦黄矮病毒引起的小麦黄矮病是我国小麦生产上重要的病毒病。BYDV GAV株系是我国小麦黄矮病的主要病原,目前有关BYDV GAV编码蛋白质P1、P2和CP的功能研究鲜有报道,因此,研究P1、P2和CP的功能,可为深入研究BYDV GAV的致病机制奠定基础。通过Mega 7.0构建系统进化树,对BYDV GAV株系编码蛋白质P1、P2和CP的核苷酸序列进行同源进化分析;通过构建P1、P2和CP的YFP表达载体,转化GV3101农杆菌后浸润本氏烟,激光共聚焦显微镜观察确定3个蛋白质的亚细胞定位;构建BYDV GAV 5个编码蛋白质的双分子荧光互补载体,转化GV3101农杆菌后分别与P1、P2和CP共浸润本氏烟叶片,通过激光共聚焦显微镜观察确定这3个蛋白质与其他病毒蛋白质的体内互作情况;通过构建P1、P2和CP的马铃薯X病毒(PVX)异源表达载体,转化GV3101农杆菌后接种本氏烟,接种后5 d观察症状形成,并采集系统叶进行病毒积累量检测,研究3个蛋白质对该病毒致病性的影响。结果表明,BYDVs的4种分离物中,PAV与GAV在核苷酸水平上的亲缘关系最近。BYDV GAV编码的P1、P2和CP蛋白亚细胞定位均为核质;在植物体内P1存在自身互作;PVXCP接种后5 d即可观察到系统叶褪绿并能够检测到病毒的积累,而PVX、PVXP1、PVXP2处理未能观察到症状且不能检测到病毒的积累;接种后10 dPVX、PVXP1和PVXP2处理能够检测到病毒的积累,表明CP促进了PVX症状的形成和致病进程。综上,P1可能通过自身互作参与病毒的侵染过程,CP可以促进病毒的侵染。

Wheat yellow dwarf disease,caused by Barley yellow dwarf viruses(BYDVs),is an important viral disease in wheat production.BYDV GAV has become the main pathogen causing wheat yellow dwarf disease.Till now the studies on the function of BYDV GAV encoded proteins P1,P2 and CP are lacking.We focued on the function of P1,P2 and CP,which could lay the foundation for the pathogenic mechanism of BYDV GAV.Phylogenetic analysis of BYDV GAV encoded P1,P2 and CP was conducted using Mega 7.0.We constructed the YFP expression vector of P1,P2 and CP,and then transformed them into GV3101 and infiltrated the leaves of Nicotiana benthamiana.The subcellular localization of P1,P2 and CP was observed using confocal laser scanning microscopy(CLSM).We constructed the biomolecular fluorescence complementation assay(BiFC)vectors of five coding proteins,and then transformed them into GV3101 and infiltrated the leaves of Nicotiana benthamiana.CLSM was used to observe the interaction of P1,P2 and CP and other viral proteins in vivo.Furthermore,we constructed the Potato virus X(PVX)expression vectors of P1,P2 and CP,transformed them into GV3101 and infiltrated the leaves of Nicotiana benthamiana.At 5 days post inoculation(dpi)the symptom formation of PVX infection was observed.The systemic leaves were collected for detection of viral accumulation to determine the effects of the pathogenicity of P1,P2 and CP.Results showed that BYDV GAV was most closely related to BYDV PAV at the nucleotide level.Subcellular localization of P1,P2 and CP was cytoplasm and nuclear.P1 interacted with itself in vivo using BiFC.In the pathogenicity assay,the systemic leaves of PVXCP infection showed chlorosis at 5 dpi,and PVX accumulation was detected,while PVX,PVXP1 and PVXP2 infection showed no symptoms in systemic leaves and PVX accumulation was undetectable,which was detected at 10 dpi,indicating that CP promoted the formation of PVX symptoms.In brief,P1 possibly involves in viral infection via self-interaction in vivo,and CP can promote viral infection.