The T6SS Structural Genes TssC,tssM And TssJ Pathogenetic Determints In Pseudomonas Syringae Pv.Actinidiae

The bacterial secretion system is a bacterial-dependent secretory pathway for the transmembrane membrane transport system of proteins,the protein or protein-like virulence factors present in the secretory system are closely related to the survival,reproduction and spread of bacteria.At present,six kinds of secretion systems have been found in Gram-negative bacteria.The type VI secretion system?T6SS?is a newly discovered secretion system in gram-negative bacteria,which plays an important role in the interaction between bacteria and host.Kiwifruit bacterial canker,which caused by Pseudomonas syringae pv.Actinidiae?Psa?,had become the destructive disease of kiwi in worldwide.The Psa can cause branch canker,leaf spot and flower rot,and the Psa frequently lead to branch blight,tree death and no harvest,resulting in huge economic losses.Therefore,revealing the pathogenic mechanism of Psa as soon as possible which is great significance to the formulation of disease prevention and control strategies,and the development of disease prevention and control technology.In the early stage,our laboratory carried out studies on the epidemic law and control of diseases,the species and population structure of Psa,the pathogenic process and mechanism of infection of Psa,and screened a highly pathogenic strain M228 from the population of Psa.In this study,the strain M228 was used as material to reveal the role of T6SS in the pathogenic process of Psa.The pathogenetic determints of three T6SS structural genes tss C,tss M and tss J in M228 was analyzed based on the pathogenicity,histological and cytological observation with host,T6SS activity,biological characteristics,and the effect on the expression of genes related to type?secretion system?T3SS?and so on.The detailed results listed below.1. The three deletion mutants M228?tss C,M228?tss M and M228?tss J were constructed by homologous recombination.Besides,the corresponding complementation mutants M228?tss C-C,M228?tss M-C and M228?tss J-C were constructed by electroporating the expression vectors of the corresponding deletion genes into the three deletion mutants.It provides experimental materials for subsequent studying the influence of three genes on the pathogenetic determints in M228.2. In this study,the effects of three genes on the pathogenicity of M228 were studied by measuring the pathogenicity of all mutants and wild-type strains on host branches and leaves.The results of pathogenicity test of branches and leaves were consistent.Compared with the wild type strain M228,the pathogenicity of the three deletion mutants M228?tss C,M228?tss J and M228?tss M reduced significantly,and the degree of decline was 92.5%,94.4%and 92.5%respectively.The pathogenicity of the three complementation mutants returned to the same level as wild-type strain M228.The results of test indicated that the deletion all of the three genes would cause a significant reduce in the pathogenicity of M228.In order to further study the reasons leading to the decline of its pathogenicity,the tissue of the host leaves was observed in histocytology at the microscopic level.The observation results showed that:After 4 days of inoculation the host of the kiwifruit leaves using wild-type strain M228,a large number of pathogens could be observed in the host tissue,serious plasmolysis occured in the host cells of the pathogenic bacteria colonized area,and electron density of the host cell wall decreases and its chloroplast were degrades.While,after inoculation of the three deletion mutants strain,only a small amount of bacterial colonization was found in the host tissue at 4 days.The host cell was intactand the organelles were not obviously degraded in the pathogen colonization,indicating that its colonization and infectivity was obviously reduced which compared with the wild-type strain M228.However,the three complementation mutants can cause a similar phenomenon with wild-type strains to the host cell,indicating that its colonization and infectivity was restored to the wild-type level.3. The hemolysin co-regulatory protein?Hcp?is secreted by T6SS which is a reliable indicator of T6SS activity detection?Pukatzki et al.2009?.Therefore,the expression vector of Hcp-linked VSV-G tag was constructed and electrotransformed into M228 and the three deletion mutants.Then the exocrine protein and intracellular protein of each strain were extracted and the western blot hybridization technique was used to analyze whether the deletion of genes tss C,tss M and tss J affected the activity of T6SS.The results of experiment showed that only the deletion of genes tss M and tss J affected the activity of T6SS,indicating that the genes tss M and tss J were required for T6SS to exert its activity.In order to further study the function of the three genes and the reasons for the decline of pathogenicity of their deletion mutants,the biological characteristics such as growth curve,biofilm forming ability and tolerance to metal ions of all mutants and wild type strains were determined.The result of experiment showed that the three structural genes tss C,tss M and tss J of T6SS in M228also affect the biological characteristics of M228.Compared with the wild-type strain M228,the logarithmic phase and stationary phase of the growth curve in M228?tss C and M228?tss M appeared slightly later;M228?tss J and M228?tss M had a obviously higher tolerance to copper sulfate in the later period;M228?tss C,M228?tss J,and M228?tss M had significantly reduced of biofilm formation capacity,resistance to hydrogen peroxide?H₂O₂?,and the ability to hydrolyze extracellular proteins;However,M228?tss C,M228?tss J,and M228?tss M had no significant changes in exopolysaccharide-producing ability,motility,and tolerance to 3%sodium chloride?w/v?;while all of these properties of the three complementation mutants were restored to the same level as wild-type strain M228.The above results showed that the three genes had significant effects on the biofilm formation ability,the tolerance to H₂O₂,the tolerance to copper ion and the ability to hydrolyze extracellular proteins of strain M228.These factors may be closely related to the decrease of pathogenicity of their mutants.4. In order to study the effects of three genes on the expression of T3SS-related genes,qRT-PCR technique was used to analyze the changes of the expression of T3SS-related genes hrp R,hrp L,hrp Z,hrc C,hop M1,hop H1 and hop P1 in three deletion mutants.The results showed that compared with the wild-type strain M228,the expression of T3SS-related genes of M228?tss C and M228?tss M was significantly reduced,while the expression of T3SS-related genes of M228?tss J was significantly increased,indicating that there is a complex regulatory relationship between T6SS and T3SS in M228.In order to further explore the relationship between the decreased pathogenicity of the deletion mutants M228?tss C and M228?tss M and their change of T3SS-related genes expression.In this study,the T3SS transcriptional regulatory genes hrp L and hrp R were overexpressed in mutants M228?tss C and M228?tss M,and their pathogenicity were determined.The result of test found that compared with two deletion mutants M228?tss C and M228?tss M,it had no significant effect on the pathogenicity of the strains which overexpressed the transcriptional regulation genes hrp L and hrp R of T3SS in M228?tss C and M228?tss M.It indicated that the changes in the expression of T3SS-related genes in two mutants are not the key factors which caused their pathogenicity to reduce.