猕猴桃细菌性溃疡病菌致病关键基因的鉴定与作用机制研究

Bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), has been a big problem urgently to be solved in the development of kiwifruit industry. To develop novel effective management strategies, many efforts have been made to unveil the pathogenicity mechanisms of Psa via different methods. However, the key effector of the full bacterial pathogenicity has not been elucidated yet. Based on the inference that the pathogenicity divergence within each genetically monomorphic clonal-complex of Psa might be caused by limited number of genetic variations, we found comparative genomics to be a good method to identify the pathogenic determinants in Psa. Therefore, we will systematically investigate the population composition and pathogenic diversity of Psa strains isolated from the Southwest China, and to collect more pathogenicity-defective strains including non-pathogenic and host-selective strains. Then we will perform comparative genomic analysis and allele swaps between the pathogenicity-defective strain and its reference strain belonging to the same clonal-complex, in order to identify the pathogenicity locus. We will further conduct comparative transcriptome and secretome analyses between the reference strain and the pathogenicity-locus mutant after growing in hrp-inducing environment (mimicking the in-planta conditions) to unveil the pathway linking the locus to the pathogenic gene. Finally, the roles of the pathogenic gene during Psa infection will be evaluated via gene knock-out, host target identification, and interaction transcriptome analysis. In conclution, these results will provide insights into the molecular basis underlying the pathogenicity variation of Psa, and will be useful in disease management.

丁香假单胞菌猕猴桃致病变种引起的猕猴桃细菌性溃疡病是发展猕猴桃产业亟需克服的重要难题。为了开发新的高效防治策略，许多研究从多个角度尝试解析病菌致病机制，但是关键致病因子仍然未知。基于遗传结构单一的病菌克隆系内存在明显致病力分化这一生物学现象，我们前期证实了比较基因组学可用于鉴定致病关键位点。本项目拟系统分析西南地区病菌群体结构和致病力分化特征，发现更多致病力缺陷（不致病或寄主品种选择性）菌株；对同一克隆系的致病力差异菌株开展比较基因组和基因替换研究，以鉴定致病关键位点；进而通过参考菌株与其位点突变体的转录组和外泌蛋白组比较分析，明确该位点影响致病基因的作用方式；最后通过基因敲除、互作靶标鉴定及互作转录组等试验研究致病基因的功能。这将为深入认识病菌致病力变异的遗传基础和致病机制提供基础，也为针对性地制定病害防控策略提供科学依据。

对植物病原菌致病机制的解析有助于开发靶向防控策略。本项目从自然丧失致病力的猕猴桃溃疡病菌（丁香假单胞菌猕猴桃致病变种Pseudomonas syringae pv. actinidiae biovar 3，简称为Psa3）分离株入手，明确导致其致病力缺陷的遗传机制和其生物学特性，以鉴定病菌关键致病因子，并解析田间病菌致病力演化的驱动因子。通过项目实施，取得以下重要进展。明确贵州省及周边地区Psa群体结构与致病力分化特征，并鉴定到大量不致病菌株，发现不致病菌株形成频繁且普遍。解析了 32株不致病分离株和4株弱致病菌株形成的分子机制，发现病菌III型分泌系统（T3SS）调控基因hrpR/S和hrpL发生变异是不致病变异株产生的主要原因。32株不致病Psa3菌株中，24株由转座子ISPsy36插入hrpR基因导致T3SS功能缺陷（G4-like），2株由转座子ISPsy36插入破坏hrpS基因导致T3SS功能缺陷（G230-like），4株由单碱基插入hrpL基因启动子导致T3SS功能缺陷（G166-like），1株因hrpL基因移码导致T3SS功能缺陷（G411），1株因hrpR基因内部SNP导致T3SS功能缺陷（G126）。对4株弱致病菌株分析，发现其致病力下降也与T3SS表达水平降低有关，其中，有3株由hrpR基因上游调控区域被转座子ISPsy32插入导致（G25-like），1株由T6SS基因簇缺失导致（G282）。 T3SS调控基因变异导致的不致病菌株在混合群体中具有竞争优势，且不致病菌株分离于病菌可长期存活的木质溃疡组织，解释了不致病菌株分利率高的原因。发现 III型效应蛋白HopAZ1可能通过与寄主蛋白GAPC-2互作而激发一定的抗病性。上述研究结果不仅强调了T3SS在致病中的关键作用，也证实T3SS关键调控基因在病菌致病力演化中的关键作用，为后续研究不致病菌株在病害传播与流行中的作用，以及开展病害生物防治和开发长期控制策略奠定了理论基础。在项目资助下于《Molecular Plant Pathology》、《Phytopathology》、《Frontier in Microbiology》、《植物病理学报》等期刊发表研究论文4篇。