| Dickeya zeae(Erwinia chrysanthemi pv.zeae) is an important pathogenic bacterium and the wide range of host in the world. Rice bacterial foot rot caused by D.zeae is one of the important bacterial diseases in rice, contributing to serious occurrence in local rice areas of China and other Southeast Asian countries, resulting in yield reduction. The pathogenicity and regulation mechanism of D.zeae are unknown at present, hfq gene(code molecular chaperone of s RNA, Hfq) and crp gene(code c AMP receptor protein, CRP) and they are not clear about the role in D.zeae.In order to clarify the pathogenic of Hfq and CRP protein in D.zeae, this paper use D.zeae DZJS-4 as study material, by non-trace knockout method, we have obtained the gene deletion mutants of hfq and crp from DZJS-4, which named △hfq and △crp respectively. The results of phenotypic determination were those: the growth rate and bioflim of mutants were less than those of the wild strain; the activities of cellulase, pectate lyase in these mutants were decreased obviously. The symptoms of soft rot in Chinese cabbage, and loss pathogenicity on rice. △hfq loss of the activity of extracellular protease and toxin, not induce hypersensitive reaction(HR) on tobacco, the inhibitory effect on the rice seed germination was also reduced significantly. Compared with the wild strains, the survival ability of under different environmental pressures(low temperature, high temperature, 10% ethanol, 10% sucrose, 20% sucrose) is correspondingly reduced. The activities of extracellular protease, pectate lyase and toxin in △crp were decreased obviously, and less influence of rice seed germination and hypersensitive reaction(HR) on tobacco. Further functional complementation analysis showed that, the phenotypic characteristics of all mutants nearly recovered after complement.To further understand the Hfq regulation relationship, This paper adopts the Illumina Hi Seq200 sequencing platform on DZJS-4 and △hfq two strains have transcribed sequencing refs, and to compare the difference in the level of gene transcription between them. Transcriptome comparison revealed 3537 differentially expressed genes, 710 differentially expressed genes(|log2(Fold Change)|>1 and qvalue<0.005), of which 347 were up-regulated in △hfq whereas 363 were down-regulated. GO functional enrichment analysis and KEGG pathway analysis of these genes, illustrated that the differentially expressed genes were enrich in Flagellar assembly,Two-component system,Fatty acid metabolism,Bacterial chemotaxis,ABC transporters,Bacterial secretion system. △hfq has 22 T6 SS secretion system genes and 30 flagella system gene regulated by Hfq. 32 chemotaxis gene clusters also showed different transcription level. Moreover, there are 43 candidate s RNAs which have significant difference in DZJS-4 and △hfq strain. Targets prediction of s RNA show that each candidate s RNA corresponds to a number of target genes. We chose some differentially expressed genes and candidate s RNA genes to conduct Real-time PCR, the results show that the trend between Real-time PCR and transcriptome sequencing analysis are basically identical. Hfq positive regulation hcp、vgr G、vas J、vas D、imp B、imp C、zms A、pel E、cel Z、cyt B、prt C and related genes in bacteria metabolic pathways, negative regulation Flagella gene cluster yli B、ycg G、vfm E、rbsk genes.Based on the results above, we consider that hfq and crp with global regulatory function involves in the virulence and are required for the pathogenicity in D.zeae. |
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