Study On The Effect Of Type Ⅲ Effector Protein HopAZ1 Of Kiwifruit Canker Pathogen On Host Disease Resistanc

Bacterial canker of kiwifruit caused by Pseudomonas syringae pv.actinidiae(Psa)is a devastating disease in kiwifruit industry.The type Ⅲ secretion system(T3SS),which secret a variety of type III effectors(T3Es)into host cells,has been demonstrated to be required for pathogenicity of Psa.The T3 E HopAZ1 has been found to be important during Psa infection,but its mechanism remains unclear.In this study,the pathogenetic role of HopAZ1 in Psa was determined by bacterial gene deletion and transient overexpression in kiwifruit leaves;the host targets of HopAZ1 were screened and identified by yeast two-hybrid(Y2H)assay,nano-luciferase complementary assay,and co-immunoprecipitation(Co-IP)assay;and the underlying mechanism of host immunity induction by HopAZ1 and host target was determined by TMT-based proteomics of HopAZ1 and GAPC2-transgenic kiwifruit seedlings challenged by Psa inoculation.The following results are obtained:1)Inoculation assay on kiwifruit leaves revealed that deletion of hop AZ1 led to increased pathogenicity of Psa against kiwifruit leaves.Compared with empty vector,transient overexpression of HopAZ1 significantly reduced the prolificatio of Psa,and induced callose deposition and reactive oxygen species(ROS),indicating that HopAZ1 may elicit plant immunity in the host plant.2)Forty-four candidate host proteins interacting with HopAZ1 were screened in Y2 H experiment using the yeast library constructed with ‘Hongyang’ kiwifruit leaves infected by Psa.Then a nano-luciferase complementary assay was established and used to verify 7 candidates,but with negative results.However,the Co-IP assay confirmed that host GAPC2 protein could interact with HopAZ1 in vivo.3)To test whether GAPC2 participates in host defence against Psa,we performed transient overexpression of GAPC2 in kiwifruit leaves followed by Psa inoculation,and it was found that the phenotypes in GAPC2 overexpression treatment were similar to those of HopAZ1 overexpression,both stimulated host defence and limited pathogen proliferation.4)The transgenic technique for ‘ Hongyang ’ kiwifruit was established and optimized to construct hop AZ1 transgenic plants and GAPC2 overexpressing plants,and two hop AZ1 transgenic line and two GAPC2 overexpressing lines were obtained.5)The TMT-based proteomics of kiwifruit leaves overexpressing HopAZ1 and GAPC2 showed that overexpression of GAPC2 could lead to up-regulated expression of 32 proteins and down-regulated expression of 48 proteins;and HopAZ1-overexpression resulted in up-regulated expression of 17 host proteins and down-regulated expression of 28 host proteins.Among them,both HopAZ1 and GAPC2 can lead to the down-regulated expression of Major allergen Pru ar like,Poly(Hydroxyalkanoate)granule-associated protein and Alginate biosynthesis protein Alg X,and the up-regulated expression of UDP-galactose transporter like,which may be related to the activation of host defence response.In conclusion,our study confirmed that HopAZ1 in Psa may stimulate defence response of host kiwifruit and preliminarily explored its mechanism of action,providing effective evidence for analysis of the mechanism of HopAZ1 in kiwifruit-Psa interactions.