Cloning And Functional Analysis Of Hfq Gene From Xanthomonas Citri Subsp.citri

Citrus bacterial canker caused by Xanthomonas citri subsp.citri(Xcc)is an important disease,which seriously impacts on fruit quality and poses a great threat to the citrus industry worldwide.At present,the control of citrus canker mainly depends on chemical.However,the excessive use of pesticides for a long period of time not only produces the drug resistance of pathogenic bacteria,but also affects the safety of fruit.Dissecting gene function and pathogenesis of Xcc will lay the foundation for the development of novel control strategies.The genome sequence of Xcc 306 was published in 2002 and has greatly facilitated the molecular research on the pathogen.Hfq gene is largely conserved gene in Gram-negative and Gram-positive bacteria,which plays important roles in regulating virulence,motility,growth and extracellular enzyme formation.Hfq protein(Host Factor RNA Phage Q beta)is a chaperone which interact with small non-coding RNA to regulate expression of mRNA.The hfq gene of Xcc contain 279 bp,encoding 93 amino acids.In order to explore the function of hfq in two strains(Xcc29-1 and Xcc49)with different virulence,two hfq deletion mutants named 29-1?hfq and 49?hfq,and two complementary strains named 29-1?hfq-C and 49?hfq-C were constructed and biological characteristics including growth metabolism,biofilm formation,extracellular enzyme activity and extracellular polysaccharide formation,bacterial motility and pathogenicity were evaluated.Furthermore,the specific roles of hfq gene in Xcc were confirmed through strand-specific RNA sequencing.The main results are as follows:1.Flanking sequences of hfq gene were amplified and linked to the suicide vector pK18 mobSacB.The recombinant plasmid pK18 mob SacB-hfq was electroporated into the competent cells of Xcc wide type by homologous recombination and hfq gene deletion mutants were obtained.The complementary strains were also constructed accordingly.2.The wild type strains,mutant strains,and complementary strains were used for biological identification.Compared with wild-type strains and complementary strains,the hfq gene deletion mutants showed slower growth and motility,significantly reduced extracellular protease,extracellular amylase and biofilm.The mutant strains were much sensitive to the H2O2 and pH stress.No significant difference on pathogenicity was observed among the strains tested.The biological characteristics revealed that hfq gene in Xcc plays important roles in growth,extracellular enzyme production,bacterial motility,stress response.However,it might be no significant effect on pathogenicity.3.To further clarify the role of hfq gene in Xcc,strand-specific RNA sequencing analysis of wild-type strain Xcc29-1/Xcc49 and the hfq deletion mutant 29-1?hfq /49?hfq was performed.There are 746 difference expressed genes between the mutant 29-1?hfq and the wide type strain Xcc29-1,in which 662 genes were down regulated and 84 genes were up-regulated in 29-1?hfq.There are 257 difference expressed genes between mutant 49?hfq and wide type strain Xcc49,in which 172 genes were down regulated and 85 genes were up-regulated in 49?hfq.Down regulated genes of hfq mutant mainly enriched in chemotactic behavior,quorum sensing,flagellar component,two-component system and secretion system,and up-regulated genes mainly distributed in the ribosome synthesis and secretion system.The down-regulation of the flagellar components and chemotaxis related genes through RNA-seq and the low mobility of hfq mutant strains on the semi-solid medium suggested that the hfq gene was involved in the regulation of movement-related genes.The down-regulation of two component system genes combined with the biological phenotype on H2O2 and pH responses revealed that hfq gene might play an important role in stress response in Xcc.The up-regulated expression of ribosome synthesis-related genes suggested that hfq gene was involved in the regulation of post-transcription in Xcc.4.The transcriptome analysis showed that there are a large number of difference expressed genes distributed in secretion systems,especially in type II secretion system and type IV secretion system between wild type strain Xcc29-1 and Xcc49.Type II secretion system could secrete some enzymes which can degrade cell wall of plants and type IV secretion system can transfer toxicity DNA.The differences in pathogenicity of Xcc29-1 and Xcc49 may associate with the difference expression of secretion system-related genes.