Contribution Of Hfq Protein And Srnas On Virulence In Pectobacterium Carotovorum Subsp.carotovorum

Bacteria from genera Pactobacterium usually have a broad host range,cause blight,decay and blackleg symptoms on the host plants.This kind of bacteria are recognised one of the top 10 most important bacterial pathogens.Plant cell wall degradation enzymes（PCWDEs）including pectate lyase,polygalacturonase,cellulase,and protease,produced by Pactobacterium are the key virulence determinents in these plant pathogens.All of Pectobacterium spp.infect potato,and P.carotovorum subsp.carotovorum（Pcc）has the widest host range worldwide.With the strategy of potato as a staple food is now being carried out nationally in China,the pathogenicity study on Pcc which causes severe disease on potato and might result in huge economcial losses is very important.Hfq is one of the most abundant RNA-binding proteins in bacteria.Almost half of all sequenced Gram-negative and Gram-positive species encode an Hfq homologue.There were a number of studies of Hfq in animal pathogens,it involved in regulating multiple cellular processes,metabolic pathways as well as virulence,however,few studies on Hfq protein were reported in plant pathogens.In order to explore the function of Hfq in P.carotovorum subsp.carotovorum PccS1,we constructed an hfq deletion mutant from PccS1.In the present study,the hfq deletion mutant significantly reduced virulence as well as PCWDEs activities.In addition to decreased biofilm formation and motility,the mutant exhibited greatly attenuated production of l-carbapen-2-em-3-acid carboxylic acid,carbapenem（Car）,and secretion of hemolysin co-regulated protein（Hep）compared to wild-type PccS1.Moreover,a higher level of callose deposition in the leaves of Nicotiana benthamiana was induced when the hfq mutant infiltrated into the host plant as compared to the wild-type.These mutant phenotypes were validated by the expression of related genes at the transcriptional level and the roles of Hfq in the production of PCWDEs and secretion of Hcp at the translational level.The result of RNA-seq showed that hfq regulated about 10%genes in strain PccS1 which can be classified into 16 categories,including lipid transport and metabolism,carbohydrate transport and metabolism,energy production and conversion,inorganic ion transport and metabolism,and general function prediction.Our results provide the first evidence that hfq plays a key role in Pectobacterium virulence by positively impacting the secretion of PCWDEs and Type VI secretion system（T6SS）,bacterial competition,and repression of host plant responses.Small non-coding RNA（sRNA）as a kind of gene transcription regulatory factors involved in many physiological process.To explore the function of sRNAs in P.carotovorum subsp.carotovorum PccS1,deletion mutants of 26 sRNAs deleted individually were constructed by homologous recombination.Then the pathogenic ability of the mutants was detected,the results showed that the virulence of ArcZ,CsrB,SraG deletion mutants declined significantly.Among these three sRNAs,ArcZ stabilization is dependent on Hfq protein.So some works on ArcZ were performed,and results showed that the activities of extracellular enzymes（pectase,cellulase,protease）in the arcZ mutant were reduced,especially the activity of protease was significantly decreased as compared to the wild-type.Meanwhile,the arcZ mutant was almost abolished in carbapenem secretion as well as the biofilm formation,in contrary,swimming ability of the mutant increased.In addition,compared with PccS1,the arcZ mutant completely suppressed Hcp secretion.ArcZ is a Hfq-binding sRNA,act by base-pairing to specific target mRNAs and changing their translation and/or stability.In strain PccSl,it has been shown that the virulence of arcZ deletion mutant significantly decreased.In order to find the target mRNAs of ArcZ in PccS1,we use Target RNA2 to predict 57 targets.To further screen for the potential targets,we contructed two strains,one is AarcZ（T1-PBAD-arcZ）which the expression of arcZ under the control of arabinose（ara）,the other one is ΔarCZ（T1-PBAD）which used as control.Samples of these two strains culture with or without arabinose were setted to RNA-seq analyses.The data showed that 23 genes were up-regulated while 14 genes were down-regulated by ArcZ.When the genes knocked out or over-expressed,our data showed that four genes PccS1₄093,PccS1₀318,PccS1₂987 and PccS1₀230（rpoS）supressed virulence.The result of qRT-PCR consistanted the regulation of ArcZ on these genes.In addition,we introducted a C-to-G pointmutation of ArcZ at the base-pairing site that disrupts base-pairing between arcZ and rpoS mRNA,the result of qRT-PCR analysis indicated that ArcZ activated the expression of rpoS.In conclusion,Hfq protein and ArcZ RNA in P.carotovorum subsp.carotovorum PccS1 regulated a lot of virulence factors and they were essential for accessory processes associated with virulence in PccS1.These findings will provide new evidence for our understanding of pleiotropic roles of Hfq and ArcZ RNA in P.carotovorum virulence.