Functional Study Of The Type III Effector HopAZ1 Of P. Kiwifruit Canker

Kiwifruit bacterial canker,caused by Pseudomonas syringae pv.actinidiae(Psa),is a devastating disease in the main kiwifruit cultivating areas around the world.Unveiling the pathogenic mechanism of Psa would be useful for developing novel disease management strategies.Type?secretion system(T3SS),which deliver a variety of effector proteins(type?effectors,T3Es)into host plant cells,is required for the pathogenicity of Psa.However,the mechanisms of the interaction at T3Es with the host in causing disease remains unclearly.There are five genetically distinct Psa biogenic population,named as biovar 1,2,3,5,and 6,responsible for outbreaks of kiwifruit bacterial canker disease over the past four decades.Each biovar has ca.thirty T3Es genes,of which fourteen are conserved in all the five biovars.From the perspective of plant-pathogen co-evolution,these 14 T3Es may be the key factors for the compatible interaction between Psa and the host kiwifruit.In this study,we systematically investigated the pathogenic roles of HopAZ1 during Psa infection of kiwifruit.Meanwhile,a yeast two-hybrid library was constructed and the host target that interacts with HopAZ1 was screened.The results is as follows:(1)A phylogenetic analysis revealed that of hop AZ1 is accompanied by the divergence of P.syringae species.HopAZ1 was widely distributed in diverse pathovars of P.syringae,and horizontal gene transfer events occurred between different pathovars.However,the gene sequences of HopAZ1 in diverse Psa pathovars were identical,and showed only two SNPs to those in the closely-related pathovars P.syringae pv.actinidifoliorum and P.syringae pv.theae.This indicates that HopAZ1_Psa was acquired before biovar differentiation of Psa,and may suffered purify selection due to its potential important roles in pathogenesis.In addition,re-analysis of published transcriptome data revealed that expression of HopAZ1 was induced in the early stage of Psa infection in kiwifruit.q RT-PCR was used to detect the transcription of hop AZ1 in wild-type Psa and T3SS-deficient mutants,showing that the expression of hop AZ1 was regulated by the typical‘Hrp R/S-Hrp L-T3SS/T3E'cascade in P.syringae.(2)The marker-free in-frame knockout mutant Psa?hop AZ1 based on the suicide plasmid p K18mob Sac B was successfully constructed,and its pathogenicity on kiwifruit woody tissue was evaluated.The results showed that the virulence of the knockout mutant was increased significantly to the canes from different kiwifruit cultivars,and the hop AZ1expressed by plasmid could restore the mutant phenotype.This indicated that HopAZ1_Psa is involved in the pathogenesis of Psa and may stimulate a certain degree of host disease resistance.(3)A high-quality kiwifruit yeast two-hybrid library was constructed by using the m RNA extracted from Psa-infected leaves of Actinidia chinensis var.chinensis cv.‘Hongyang',and the bait vector p GBKT7-hop AZ1_Psa of HopAZ1_Psa was successfully constructed.The bait strain showed no self-activation effect and obvious toxicity.After screening the yeast library with the bait vector p GBKT7-hop AZ1_Psa,25 positive interactions were identified,including eight effective proteins and eight frameshift expressed host genes.After one-to-one identification,two prey clones have positive interactions with HopAZ1_Psa,Cp1(Acc23383)and PR5(Acc28852).CP1 and PR5 belong to the PLCP(papain-like cysteine peptidase)family,and TLP(Osmotin/thaumatin like protein)family,respectively.It may be related to disease resistance of host kiwi fruit.(4)Re-analyses of several published transcriptome data revealed that the expression of both Cp1 and PR5 genes were induced during Psa infection to kiwifruit,and the expression levels in diverse kiwifruit tissues were different.A q RT-PCR assay was performed to detect the effect of HopAZ1 on the expression of PR5 and CP1genes.The results showed that after inoculation on kiwifruit canes by wild-type Psa and the hop AZ1 mutant,both PR5 and Cp1showed similar transcriptional patterns with a trend of first increasing and then decreasing.However,the mutant treatment caused the PR5 gene to maintain a high expression level for a longer time(12 hpi and 48 hpi),and caused the Cp1 gene to express in significantly lower leveals than that of wild type treatment at 12 hpi and 48 hpi.This indicates that HopAZ1 also affect the transcription of host PR5 and Cp1 genes.In summary,the distribution and evolution characteristics of the T3Es-HopAZ1 in P.syringae were described in this article.The the pathogenicity roles of HopAZ1 in Psa was evaluated by gene deletion method,and two candidate HopAZ1-interacting proteins Cp1 and PR5 were identified from kiwifruit.This provides an important basis for the mode of action of HopAZ1 in infection process of Psa and for the discovery of disease resistance genes in kiwifruit.