Identification Of PSA Type ? Effectors And Priliminary Analysis Of Effector HopX3 In Pathogenicity

Bacterial disease of the kiwifruit caused by Pseudimunass syringae pv.actinidiae?PSA?is an important destructive disease to kiwifruit production,which was first reported in Japan and later broke out.Since 2008 it has become a world disease,happening in the cultivated areas of Italy,New Zealand,Chile and so on.Because of its high incidence and high prevalence rate,it may cause the whole tree to die and a large pieces of garden destroyed.However,at present,people know little about the disease,neither do they know how it happens,so there is no any effective way to solve the problem.In the early stage,we found that it is Type 3 secretion system,T3 SS that is in charge of the disease of PSA,but we didn't make clear its mechanism of action.Fortunately many studies have shown through a needle of device,T3 SS injects various effectors into plant cell to interfere host immune response or normal physiological process so as to achieve successful infection.30 type3 effectors exist in PSA,some of which work when infecting host cell.Besides,different roles make different contributions to the disease.In the research,we used the agrobacterium transient expression system to evaluate and screen PSA T3 SS effector and preliminarily explore the effector of interaction between the non-host tobaccos and other possible functions.In order that the pathogenesis of PSA is further clarified and the evidence for the discovery of resisting disease gene is provided,preliminary functional analysis of effector HopX3?BAX induced necrosis?is performed with the method of biological information analysis,gene knockout,RT-PCR and other techniques.The main results are as follows:1)We successfully constructed 19 recombinant vectors by enzyme cutting and connecting 32 genes obtained from the clones,which can interfere with the defense response of plant body;Having screened 32 T3 SS effectors,we found 10 effectors gene expression proteins causing interference with tobacco defense response;Five of them called 23315?HopZ5-1?,3610?HopBB1-1?,3555?HopBB1-12?,29455?HopAU1?,HopF2 promote cell necrosis in tobacco leaf cells.Five effectors which can inhabit BAX to induce cell necrosis,are 7905?selected effector HopW1?,3510?HopX3?,11535?AvrPto5?,3480?AvrB4|1?,3560?HopAF1?.2).By analyzing the biological information of HopX3,the results of the phylogenetic tree suggest HopX3?IYO₀3510?lies in EEC?exchangeable effector locus?of PSA and exists in Psa biovar 1,2,3 regularly.HopX3 belongs to Hop X family,but HopX3 has N-terminal?-70aa?,similar to the effectors such as HopF2,HopBB1 and part of AvrRpm.Under the condition of T3 SS culture,the expression of HopX3 was up-regulated.After the structure gene of T3 SS is knocked out,bacteria will not be able to secrete active proteins outside the cell while the expression of HopX3 was still up-regulated,but its pattern is different from that of wild type.HopX3 can be secreted by T3 SS.Its pathogenicity is equal to that of wild type if the knockout mutant M7?HopX3 is inoculated with the branches of the kiwifruit.