The Mechanisms Of Transcription Factors HrpR/HrpS Involved In The Pathogenicity In Pseudomonas Syringae Pv. Actinidiae

Kiwifruit canker is a devastating bacterial disease that can lead to the death of the entire plant in severe cases.The disease is spreading worldwide,seriously constraint the healthy development of the kiwifruit industry.The pathogen is Pseudomonas sypingae pv.actinidiae(Psa).The type III secretion system(T3SS)is a key virulence factor widely present in Gram-negative bacteria and is involved in regulating the pathogenicity of pathogenic bacteria.hrpR and hrpS genes are important components of the hrpgene cluster in T3 SS,and T3 SS requires transcription factors Hrp R and Hrp S to mediate the secretion of proteins.In order to explore the function of hrpR and hrpS genes in T3 SS in Psa,hrpR and hrpS genes were cloned from the whole genome of kiwifruit canker pathogen M228;The mutant strains of hrpR and hrpS genes were constructed by homologous recombination,and the biological function experiments were carried out between the mutant strains and the wild-type strains to explore the biological function of hrpR and hrpS genes in M228;To further understand the regulatory mechanism of hrpR and hrpS on pathogenicity,ΔhrpR and ΔhrpS strains were subjected to high-throughput transcriptome sequencing;the expression of candidate genes was analyzed by real-time fluorescence quantitative PCR.The results are as follows:1.The wild-type strain was used as the test strain,and the hrpR and hrpS genes were cloned by designing specific primers hrpR-F/R and hrpS-F/R based on the conserved sequences of hrpR and hrpS genes,the fragment sizes were 1131 bp and 1108 bp.Bioinformatics analysis showed that Hrp R protein contained an AAA domain.Hrp S protein contains a low complexity region and an AAA domain.Phylogenetic tree analysis based on the amino acid sequence of Hrp S showed that the strain could be closely clustered with different biotypes of Psa strains(ICMP18884,ICMP19072 and MAFF212056),which were clearly distinguished from other pathogenic variants of Psa.2.and reduced biofilm formation ability.There was little change in swimming motility and swarming motility,and increased growth capacity and exopolysaccharide production outside the host.The deletion mutant strains ΔhrpR and ΔhrpS of the target gene were successfully constructed by homologous recombination double exchange method,and the biological function was detected with the wild-type strain.The results showed that compared with the wild-type strain,the pathogenicity of the gene deletion strains ΔhrpR and ΔhrpS to the host plant kiwifruit was weakened;And lost the ability to induce hypersensitive response(HR)of non-host plant tobacco;the biofilm formation ability and swimming ability were weakened;the production of extracellular polysaccharides and in vitro growth ability were enhanced;the swarming ability of ΔhrpS strain was enhanced,and the swarming ability of ΔhrpR strain did not change.3.To further understand the regulatory mechanism of hrpR and hrpS on the pathogenicity of kiwifruit canker,high-throughput transcriptome sequencing was performed on ΔhrpR,ΔhrpS strains and wild-type strain.The results showed that the bacterial secretion system,ABC transporters,Quorum sensing and protein export signaling pathway were significantly affected after hrpR and hrpS deletion.Common differentially expressed genes were mainly enriched in the cell components related to biofilm formation,and the CN228＿07645 related to Omp A family protein synthesis were significantly down-regulated in ΔhrpR and ΔhrpS strains.4.According to the data of transcriptome sequencing and the results of biological function experiments,the candidate pathogenicity-related genes were verified by q RT-PCR.The expression levels of T3SS-related genes(hrpL,hrpS and hrpK),harpin gene(hrpZ),type III effector gene(CN228＿04320),pilus-related gene(pil W),biofilm-related gene(CN228＿26180)and growth-related gene(rim M)in ΔhrpS strain were determined.The expression levels of rim M and pil W genes in ΔhrpS were significantly up-regulated,the expression levels of hrpL,hrpS,hrpK and CN228＿26180 genes were significantly down-regulated,and the expression level of CN228＿04320 gene was significantly down-regulated,while the expression level of hrpZ gene was not significantly different.