| Pseudomonas syringae pv.actinidiae(Psa)caused Kiwifruit bacterial canker is currently the most devastating disease of kiwifruit in the world.P.syringae strain regulates through the GacS/GacA two-component system(TCS)expression,virulence,population sensing,biofilm and other pathogenic traits of the type III secretion system(T3SS)mainly;although the Gacsystem is highly conserved among different bacteria,its regulated functions show significant differences in different bacterial genera and different growth environments.Although the Gacsystem is highly conserved in different bacterial species,its regulatory functions are significantly different in different bacterial genera and growth environments.In this study,the complete gene sequences of gacS and gacA were cloned and analyzed based on the strong pathogenic strain M228;homologous recombination was used to construct gacS and gacA deletion mutant strains and explore the biological functions;transcriptome data were verified by RNA-seq and q RT-PCR and the expression of candidate pathogenicityrelated genes were detected.The main findings are as follows:1.Sequence analysis of gacS and gacA genes in P.syringae pv.actinidiae.In this study,the complete gene sequences of M228 gacS and gacA were cloned,and the results showed that the gacS and gacA genes were 2745 bp and 829 bp,respectively.the homology between M228 and Psa strains was more than 99.78%;the gacS gene encodes a 917 aa protein with two transmembrane structural domains,and contains five structural domains: HAMP,HISKA,HATPsae＿c,REC,HPT;the gacA gene encodes a 222 aa protein with two structural domains,REC and HTH,as shown by SMART structural domain analysis;phylogenetic analysis showed that GacS and GacA of M228 are most closely related to different biotype Psa strains.2.Functional analysis of the gacS and gacA genes of P.syringae pv.actinidiae.A homologous recombinant double exchange was used to perform deletion mutations in the gacS and gacA genes of wild type strain,and two deletion mutants were obtained by screening separately.The results showed that ΔgacS and ΔgacA strains had reduced pathogenicity and significantly increased growth rate and iron carrier production compared with wild type strain;motility and extracellular polysaccharide production ability were reduced but not significantly different,while extracellular protease and HR production were not affected;biofilm formation ability was not affected in ΔgacS strain compared with wild type strain,while in ΔgacA strain the ability of biofilm formation was not affected by theΔgacS strain compared to the wild type strain,while the biofilm formation ability was significantly reduced in the ΔgacA strain.3.Transcriptome and qRT-PCR analysis In this study,transcriptome sequencing analysis were carried out between ΔgacA,ΔgacS and M228 strains,respectively.Results showed that there were 2910 significantly different genes in ΔgacA＿vs＿WT,2453 significantly different genes in ΔgacS＿vs＿WT,and 1271 common differentially different genes between the two comparison groups.These significantly differential genes were subjected to GO and KEGG enrichment analysis,and the results showed that GO enrichment analysis showed that the up-regulation of differential genes mainly involved pathways such as response stimulation and metabolic processes,and the down-regulated pathways mainly involved cellular processes and biological regulation.KEGG enrichment analysis showed that the upregulated differential genes were mainly involved in aminoacyl-t RNA biosynthesis and ABC transporter,and the down-regulated pathways were mainly enriched in flagellar assembly and quorum sensing.The candidate genes hrp L,hrp R,hrp S,uvr C,gacS,fli S,clp B and fli C were detected by q RT-PCR.The results showed that the expression levels of hrp L,hrp S,gacS,fli S,clp B and fli C genes were down-regulated in ΔgacA strain,and the expression levels of hrp R and uvr C genes were up-regulated.The expression levels of clp B and fli C genes were down-regulated in ΔgacS strain. |
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