柑橘溃疡病菌效应蛋白PthA4诱导柑橘CsAPC7基因表达的分子机制及其在抗病育种中的应用基础研究

Citrus canker is an important bacterial disease of citrus plants. The effector protein PthA4 is the major virulence factor in Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. Transient expression of a single PthA4 can induce cell hypertrophya, generating a canker-like symptom on citrus plant. Our previous work aimed to clone citrus susceptible gene based on the PthA4 function. The results suggested that anaphase-promoting complex 7 (CsAPC7) is activated by PthA4. Most importantly, CsAPC7 induces cell hypertrophya on citrus, a similar phenotype caused by PthA4. This project aims to prove the contribution of CsAPC7 for cell division in overexpression and RNAi citrus plants, which will help to conclude that CsAPC7 is the major susceptible gene targeted by PthA4; Identify the specific binding sequence of PthA4 within CsAPC7 promoter region through series of truncated mutations; Take advantage of a CRISPR/Cas9-targeted editing strategy to modify the PthA4 binding sequence. Thereafter, assess the resistance of engineered citrus plant to canker disease. This project will facilitate to explore the promising usage of CsAPC7 gene in citrus resistance breeding.

柑橘溃疡病是柑橘植物上的一个重要细菌病害。柑橘溃疡病菌Xanthomonas citri subsp. citri (Xcc)的效应蛋白PthA4是起致病作用的TALE，并且单个PthA4即可诱导柑橘细胞膨大，形成类似溃疡的症状。我们以PthA4为切入点，克隆与溃疡症状形成相关的柑橘感病基因，发现PthA4靶定的细胞分裂后期促进复合物CsAPC7在表型上与PthA4一致。本项目拟在干扰和过表达柑橘中研究CsAPC7对溃疡症状形成的作用，进一步明确CsAPC7是PthA4靶定的关键感病基因；对CsAPC7基因启动子序列进行突变，分析PthA4诱导CsAPC7基因表达的分子机制；利用CRISPR/Cas9对CsAPC7启动子的PthA4结合位点进行编辑，通过精准调控感病基因CsAPC7的表达，评价对溃疡病的抗性效果，探索CsAPC7在柑橘抗病育种的应用价值。

柑橘溃疡病菌的PthA4是一个起致病作用的效应分子，靶定柑橘转录调节因子CsLOB1调控溃疡病的发生。实验室基于转录组学和qRT-PCR分析，从柑橘植物中筛选到受PthA4调控的柑橘细胞分裂后期促进复合物基因CsAPC7。本项目通过CTV载体介导的表达系统，构建了CsAPC7的干扰和过表达柑橘植株，接种柑橘溃疡病菌后过表达植株更有利于病害发展，而干扰植株症状减轻，明确了CsAPC7在溃疡症状形成中的作用，是病害形成的一个感病基因。通过CsAPC7启动子序列分析，根据PthA4和CsLOB1结合DNA的序列特征，找到PthA4和CsLOB1调节CsAPC7启动子的关键位点，证明了PthA4和CsLOB1都可以结合在CsAPC7启动子上；对结合位点序列进行突变研究，发现PthA4-CsLOB1模块调控CsAPC7表达的分子机制；在溃疡病菌侵染过程中，PthA4的过量表达反馈抑制CsAPC7的表达量。构建CRISPR/Cas9重组载体，通过农杆菌介导的遗传转化对CsLOB1的调控关键位点进行编辑，基因编辑植株产生溃疡病斑数量显著少于野生型，暗示该位点在抗病育种中的应用价值。