甘蔗线条花叶病毒RNA沉默抑制子的寄主互作蛋白鉴定

甘蔗线条花叶病毒（Sugarcane streak mosaic virus, SCSMV）是引起甘蔗花叶病的一种主要病原。SCSMV编码的P1蛋白是RNA沉默抑制子,在SCSMV抑制寄主的RNA沉默防御中发挥关键作用,但其作用机制尚未清楚。与寄主蛋白互作是RNA沉默抑制子发挥其抑制作用的主要途径之一,因此鉴定与病毒RNA沉默抑制子互作的寄主蛋白是研究抑制子作用机制的一个重要方法。为探究SCSMV P1抑制寄主RNA沉默的机制,本研究首先将SCSMV P1基因连接到质粒pGBKT7上构建诱饵质粒pGBKT7-P1,然后对pGBKT7-P1进行毒性和自激活检测,最后以pGBKT7-P1为诱饵,采用酵母双杂交技术从甘蔗cDNA文库中筛选与P1互作的寄主蛋白。结果显示,成功构建pGBKT7-P1诱饵质粒。将pGBKT7-P1诱饵质粒转入Y2H Gold酵母菌株后,酵母菌株在SD/-Trp平板及液体培养基中生长良好,表明pGBKT7-P1诱饵质粒对Y2H Gold酵母菌株无毒性。含pGBKT7-P1诱饵质粒的酵母菌株在SD/-Trp/X-α-Gal平板上长出白色菌落未变蓝,在SD/-Trp/X-α-Gal/AbA和SD/-Trp/-Leu/X-α-Gal/AbA平板上无菌落生长,表明pGBKT7-P1诱饵质粒无自激活活性。用pGBKT7-P1诱饵质粒对甘蔗cDNA文库进行筛选,经SD/-Trp/-Leu/X-α-Gal/AbA平板筛选1次及SD/-Trp/-Leu/-His/-Ade/X-α-Gal/AbA平板筛选3次后,获得42个阳性酵母克隆,提取阳性酵母质粒并导入大肠杆菌中扩大培养,经测序及blastx比对分析,获得13个可能与SCSMV P1互作的寄主甘蔗蛋白,分别是生长素应答蛋白IAA1、IAA15,转录因子NAC、GATA4、OFP4,真核翻译起始因子eIF5A,分子伴侣DnaJ,辅分子伴侣SBA1,重金属相关异戊二烯化植物蛋白HIPP35,mec-8和unc-52蛋白同系物抑制子SMU2,外膜孔蛋白OEP24,及2个未表征蛋白。基于这些蛋白的功能,推测P1可能通过与寄主蛋白互作来调控寄主防御反应相关基因的表达,和/或通过与寄主蛋白互作来影响寄主RNA沉默相关蛋白的合成、加工或转运,从而发挥其RNA沉默抑制子的功能。研究结果为后续深入解析P1抑制RNA沉默的作用机制奠定了重要基础。

Identification of Host Proteins Interacting with RNA Silencing Suppressor of Sugarcane Streak Mosaic Virus

Sugarcane streak mosaic virus (SCSMV) is a major pathogen of sugarcane mosaic disease. P1 protein encoded by SCSMV is an RNA silencing suppressor, which plays a key role in suppressing the host’s RNA silencing defense. However, its mechanism is not yet clear. Interaction with host proteins is one of the main pathways for RNA silencing suppressors to exert the suppression functions. Therefore, identifying host proteins that interact with viral RNA silencing suppressors is an important method to study the mechanism of suppressors. In order to explore the mechanism of SCSMV P1 suppressing the host RNA silencing, in this study, SCSMV P1 was ligated to the plasmid pGBKT7 to construct the bait plasmid pGBKT7-P1, then pGBKT7-P1 was tested for toxicity and self-activation, and finally pGBKT7- P1 was used as a bait to screen the host proteins that interact with P1 from the sugarcane cDNA library by yeast two-hybrid technology. The results showed that the bait plasmid pGBKT7-P1 was successfully constructed. After the pGBKT7-P1 bait plasmid was transferred into the Y2H Gold yeast strain, the yeast strain grew well in the SD/-Trp plate and liquid medium, indicating that the pGBKT7-P1 bait plasmid was non-toxic to the Y2H Gold yeast strain. The yeast strain containing the pGBKT7-P1 bait plasmid grew white rather than blue colonies on the SD/-Trp/X-α-Gal plate, and did not grow on the SD/-Trp/X-α-Gal/AbA and SD/-Trp/-Leu/X-α-Gal/AbA plates, indicating that the pGBKT7-P1 bait plasmid had no self-activation activity. The sugarcane cDNA library was screened with pGBKT7-P1 bait plasmid. After screening on SD/-Trp/-Leu/X-α-Gal/AbA plate once and SD/-Trp/-Leu/-His/-Ade/X-α-Gal/AbA plate three times, 42 positive yeast clones were obtained. The positive yeast plasmids were extracted and introduced into Escherichia coli for amplification. After sequencing and blastx comparison analysis, a total of 13 host sugarcane proteins that may interact with SCSMV P1 were obtained, namely auxin-responsive proteins IAA1, IAA15, transcription factors NAC, GATA4, OFP4, eukaryotic translation initiation factor eIF5A, chaperone DnaJ, co-chaperone SBA1, heavy metal-associated isoprenylated plant protein HIPP35, suppressor of mec-8 and unc-52 protein homolog SMU2, outer envelope pore protein OEP24, and two uncharacterized proteins. Based on the functions of the proteins, it was speculated that P1 may interact with host proteins to regulate the expression of host defense response-related genes, and/or interact with host proteins to affect the synthesis, processing, or transport of host RNA silencing-related proteins, thereby exerting the RNA silencing suppressor function of P1. The research results would lay an important foundation for the subsequent in-depth analysis of the RNA silencing suppression mechanism of P1.