烟草和水稻中水稻条斑病菌过敏反应激发子Ssb_x互作因子的鉴定

【目的】水稻条斑病菌(Xanthomonas oryzae pv.oryzicola)中单链DNA结合蛋白(single-stranded DNA-binding protein,SsbX)通过病原菌III型分泌系统(type-III secretion system,T3SS)分泌,在非寄主植物烟草上产生过敏反应(hypersensitive response,HR),研究旨在明确SsbX激发烟草产生HR的机理。【方法】将水稻条斑病菌RS105菌株注射水稻和烟草叶片,应用CreatorTM SMARTTM cDNA文库构建试剂盒构建水稻和烟草cDNA文库,借助pGADT-Sfi AB载体上特殊酶切位点SfiⅠ,酶切检测水稻和烟草cDNA文库质量;以SsbX为诱饵,通过酵母双杂交技术(Y2H)从水稻和烟草cDNA文库中筛选在SD/-Ade/-Leu/-Trp/-His培养基上能够生长的阳性克隆;并利用β-gal试验验证阳性克隆;序列测定后,使用MEGA 4序列分析软件,并利用邻位相连法(neighbor-joining,NJ)对筛选获得的互作因子进行同源序列和系统进化树分析;利用荧光双分子互补试验(BiFC)于488 nm处黄色激发光和520—550 nm透射光、20×物镜下,在激光共聚焦显微镜(Leica TCS SP5-II)观察SsbX与烟草和水稻中互作因子的互作位点。【结果】水稻和烟草cDNA文库构建和质量检测结果显示,随机挑选20个克隆中水稻来源的cDNA插入在pGADT-Sfi AB载体上,平均片段大小1.0kb;随机挑选20个克隆中烟草来源的cDNA均插入在pGADT-Sfi AB载体上,平均片段大小1.2 kb,说明水稻和烟草的cDNA文库构建质量较好;Y2H和β-gal试验结果显示,SsbX可与烟草和水稻的ADF2(actin-depolymerization factor 2)蛋白互作,使酵母AH109能够在SD/-Ade/-Leu/-Trp/-His平板上生长,并且β-gal染色显示为蓝色。水稻Os ADF2编码139氨基酸,蛋白质分子量为15.9 k D,而烟草Nb ADF2编码137氨基酸,蛋白质分子量为15.kD,两者同源性达69.02%。同源性以及系统进化树分析显示,植物中ADF2广泛存在,同源性高达60%以上。双子叶植物烟草和拟南芥的ADF2同源性最高,相似性达69.05%;单子叶禾本科植物水稻和狗尾草的ADF2同源性最高,相似性为88.81%。BiFC试验结果显示,在488 nm波长的激光共聚焦显微镜下,仅SsbX和Os ADF2以及SsbX和Nb ADF2共同存在时,可在烟草细胞膜上显示黄色荧光,与阳性对照显示黄色荧光一致,说明SsbX可与Os ADF2或Nb ADF2互作,互作位点发生在植物细胞膜上。【结论】通过T3SS分泌的水稻条斑病菌中的SsbX,在植物细胞膜上与ADF2互作,从而激发植物的免疫性。这为进一步揭示SsbX如何激发植物产生HR的机制提供了依据。

Identification of a Protein both in Tobacco and Rice that Interacts with an HR-elicitor SsbX of Xanthomonas oryzae pv. oryzicola

【Objective】A highly-conserved single-stranded DNA-binding protein (SsbX) of Xanthomonas oryzae pv. oryzicola, secreted through the type-III secretion system (T3SS), elicits hypersensitive response (HR) in non-host tobacco. The objective of this study is to clarify how it triggers HR. 【Method】 The appropriate period of rice and tobacco leaves were infiltrated with X. oryzae pv. oryzicola RS105 strain to construct rice and tobacco cDNA libraries using Creator^TM SMART^TM cDNA Library Construction Kit, then the qualities of rice and tobacco cDNA libraries were determined by using the special enzyme cutting site SfiⅠof pGADT- SfiAB vector. These cDNA libraries were applied to screen SsbX-interacting proteins on SD/-Ade/-Leu/-Trp/-His medium by yeast two-hybrid system (Y2H). The homology and phylogenetic tree analysis of SsbX-interacting proteins were operated by the method of neighbor-joining using MEGA.4 software. The sublocalization of SsbX with SsbX-interacting proteins in tobacco leaves was observed and imaged under a laser confocal microscope at 488 nm, the excitation wavelength for YFP, and 520 to 550 nm, the emission filter wavelength.【Result】Construction and detection of rice and tobacco cDNA libraries showed the average length of cDNAs inserted into pGADT-SfiAB was 1.0 kb among 20 randomly-selected rice cDNA library clones and 1.2 kb among 20 tobacco cDNA library clones, indicating good qualities of rice and tobacco cDNA libraries could be applied for subsequent research. The Y2H and β-gal assay results demonstrated that SsbX interacted with an actin-depolymerization factor 2 (ADF2) either from rice or tobacco and this couple made AH109 positively grow on SD/-Ade/-Leu/-Trp/-His plate with blue color. Hereby this factor was designated OsADF2 for rice and NbADF2 for tobacco correspondingly. OsADF2 encoded a 139 amino acid (aa) protein, 15.9 kD in molecular weight, and NbADF2 encoded a 137 aa in 15.8 kD. These two proteins were highly conserved with 69.02% identities to each other in protein sequences. Homology and phylogenetic tree analysis displayed that ADF2 proteins in plants were highly conserved in similarity up to 60%. NbADF2 had 69.05% similarities to the homolog of Arabidopsis, higher than orthologs in other dicotyledonous plants. OsADF2 showed 88.81% similarities to the homolog of Setaria italic, higher than orthologs in other monocots plant. Bimolecular fluorescence complementation (BiFC) assay results demonstrated that the presence of OsADF2 or NbADF2 together with SsbX, like a positive control, displayed yellow fluorescence in plant plasma membrane under a laser confocal microscope when the pair proteins were transciently expressed in plant cells mediated by Agrobacterium. 【Conclusion】 The SsbX protein of Xanthomonas, secreted through the T3SS, targets ADF2 in plant plasma membrane to trigger plant immunity. This novel finding provides a scientific clue to further understand how SsbX elicits HR in tobacco plants.