甘蔗白条病菌特有效应蛋白及其寄主靶标的筛选和鉴定

Leaf scald caused by Xanthomonas albilineans (Ashby) Downson is a destructive disease of sugarcane. Compared with most Xanthomonas, X. albilineans showed obvious different pathogenic mechanism, ecological characteristics and classification status. Studys on the molecular mechanism of X. albilineans -sugarcane interaction can provide the basis for safe and effective control strategies. Effectors are the key secretory proteins that determine whether the pathogen can infect host successfully, but the functions of X. albilineans effectors are largely unkown. In our previous studies, we obtained the full-length genome sequence of strain XaFJ1 in China via the Pacbio sequencing and identified 16 candidate secretory proteins specific to the subgroup of X. albilineans. In this project, we will characterize the pathogenicity and physiological indices of their mutants. Adenylate cyclase reporting system will be used to detect the secretion, translocation of the secretory proteins which contribute to pathogenicity. Thus, effectors affecting the pathogenicity will be screened out and their location in the plant cells will be identified by Agrobacterium mediated transient expression and PEG-mediated protoplast transformation. The target genes in sugarcane will be screened with sugarcane cDNA library by yeast two-hybrid assays, and the interaction will be confirmed by co-immunoprecipitation assay and bimolecular fluorescence complementation technology. The results are expected to reveal the role of effectors in the leaf scald pathogen-sugarcane interaction and provide a theoretical foundation for molecular breeding of sugarcane disease resistance.

由白条黄单胞菌引起的甘蔗白条病是甘蔗上毁灭性病害之一。与大多数黄单胞菌相比，白条病菌显示出明显不同的致病机制、生态特征和分类地位，明确白条病菌与甘蔗互作的分子机理可为制定安全有效的防控策略提供依据。效应蛋白是决定病菌能否成功侵染植物的关键分泌蛋白，目前对白条病菌效应蛋白的研究非常有限。前期研究中，申请人利用PacBio技术获得中国株系XaFJ1全长基因组序列，鉴定出16个白条病菌亚群特有的候选分泌蛋白。在此基础上，本项目拟通过检测其突变体致病性和生理指标，结合腺苷酸环化酶报告系统明确其分泌、转运情况，筛选出有致病性变化的效应蛋白；并进一步通过农杆菌介导瞬时表达和PEG介导原生质体转化实验明确效应蛋白在植物细胞的定位，通过酵母双杂交筛库筛选其甘蔗靶标蛋白，利用免疫共沉淀和双分子荧光互补技术确认其互作情况。研究结果有助于揭示效应蛋白在白条病菌与寄主互作中的作用，为甘蔗抗病分子育种提供理论基础。

效应蛋白是病原菌侵染过程中决定其能否成功定殖的关键分泌蛋白。虽然黄单胞菌效应蛋白对致病性的贡献得到了大量的研究，缺乏Hrp-T3SS的甘蔗白条病菌（Xanthomonas albilineans, Xa）效应蛋白研究尚属空白。申请人前期对白条病菌中国菌株Xa-FJ1进行了PacBio基因组测序，通过生物信息学分析筛选得到16个Xa亚群特有的候选分泌蛋白，命名为Xsu1-Xsu16。本项目对这些候选分泌蛋白进行研究，鉴定出白条病菌第一个效应蛋白Xsu11及其甘蔗靶蛋白。主要结果如下：.利用基于负选择标记基因sacB的两次同源交换敲除技术，成功获得13个基因的突变体。致病性鉴定结果表明与野生型Xa-FJ1相比，2个突变体Δxsu3、Δxsu11致病性减低，且接种叶片的细菌数量明显减少。此外，与野生型相比，突变菌株Δxsu11在人工培养条件下细菌生长能力和胞外酶分泌能力没有显著变化；Δxsu3纤维素酶分泌能力下降；蛋白酶分泌能力与野生型无差异。利用蔗糖酶缺陷型酵母分泌系统证明Xsu3、Xsu11均可分泌至胞外。.通过农杆菌介导，分别在烟草叶片细胞中瞬时表达Xsu3、Xsu11蛋白，表明Xsu3主要定位于细胞膜上，而Xsu11在细胞核和细胞膜上均有表达。结合Δxsu3致病性减低、纤维素酶分泌能力明显下降，推测白条病菌分泌Xsu3蛋白至胞外，主要作用于植物细胞壁，不进入植物细胞内。将xsu11基因全长序列构建在诱饵载体pGBKT7中，验证其不存在自激活现象后，酵母双杂交筛选受白条病菌侵染的cDNA文库。两次大规模筛库工作得到55个候选靶标蛋白。经酵母双杂实验点对点验证后，确认了双去甲氧基姜黄素合酶（bisdemethoxycurcumin synthase，SsBDMC）与Xsu11存在互作。后续开展特有效应蛋白Xsu11通过靶向SsBDMC影响寄主抗病性的机理研究，有助于解析白条病菌特有的致病机制。