猕猴桃溃疡病菌不致病菌株G230的鉴定及形成原因分析

为探索田间猕猴桃溃疡病菌Pseudomonas syringae pv. actinidiae(Psa)致病力丧失的分子机制，针对从猕猴桃果园中分离获得的1株不致病菌株G230，通过特异性引物检测和多基因序列分析明确其分类地位，并设计引物检测其是否由已知遗传变异引起，通过比较基因组学、基因表达、超敏反应和荧光素酶报告菌株检测确定引起菌株G230致病力丧失的原因。结果表明，不致病菌株G230为Psa生物型3(Psa3)，其致病缺陷不是由已报道的遗传变异引起；基于基因组比较分析发现菌株G230中的hrpS基因被转座子ISPsy36插入破坏，导致Ⅲ型分泌系统（type Ⅲ secretion system,T3SS）不能正常表达；而在不致病菌株G230中表达hrpS基因后能恢复其T3SS功能，使其具备致病能力及激发非寄主超敏反应的能力。表明转座子ISPsy36插入hrpS基因内部可以破坏Psa的T3SS功能进而使其丧失致病力，这是自然条件下Psa3丧失致病力的一种新型机制。

Determining the molecular basis for the loss-of-virulence in bacterial pathogen Pseudomonas syringae pv. actinidiae isolate G230

To gain insight into the molecular basis underlying spontaneous loss-of-virulence in natural populations of Pseudomonas syringae pv. actinidiae (Psa), the causative agent of kiwifruit bacterial canker, a non-pathogenic isolate G230 from a kiwifruit orchard was identified using Psa-specific PCR detection and multi-locus sequence analysis, and the genetic cause of the non-pathogenic phenotype was determined with PCR detection of several known transposon-insertion events, comparative genomics and gene expression methods, followed by the detection of type III secretion system (T3SS) via a nanoluciferase reporter system and the hypersensitive response (HR) indicator on the non-host Nicotiana benthamiana leaves. The non-pathogenic isolate G230 was identified as Psa biovar 3 (Psa3), and results revealed that its loss of virulence was not caused by known transposon-insertion events in Psa3. Moreover, a novel transposon-insertion event inside hrpS gene by ISPsy36 was found in G230, and after expression of the hrpS gene in non-pathogenic isolate G230 resulted in a functional T3SS, which was necessary for pathogenicity in Psa3 and HR induction in non-host plants. Taken together, the insertion of ISPsy36 into the hrpS gene, which results in T3SS deficiency in the non-pathogenic isolate G230, represents a novel mode of action underlying spontaneous loss-of-virulence in natural populations of Psa3.