Contribution Of SRNA(ArcZ) On Virulence In Pectobacterium Carotovorum Subsp.Brasilience

Soft rot is an important disease of food crops(potatoes)and cash crops(vegetables and flowers),and such plant disease responsible for many $US billions each year in losses worldwide.The pathogens causing soft rot mainly belong to the genera Pactobacterium and Dickeya,which have widely distributing diverse hosts.A strian of P.carotovorum subsp.brasilience(Pcb)was isolated and identified from soft rotted potato plants collected in Inner Mongolia in 2018.Pcb has been reported in many countries and regions,causes stem rot and blackleg in potato.Previous studies in our laboratory showed that the non-coding small RNA(sRNA)chaperone Hfq is a global regulator being essential for the virulence of P.carotovorum subsp.carotovorum PccS1,and hexA is one of the potential target genes for sRNA(ArcZ)in PccS1,and ArcZ negatively regulates hexA.It is well known that sRNAs ragulate their target genes at the posttranscriptional level basing on base-pairing with the target mRNAs,and play important roles in many cellular processes.Details in regulatory mechanism of sRNA are crucial for deeply understanding pathogenesis of Pactobacterium.To reveal the regulatory of ArcZ on Pcb virulence,the strains with mutation in either or both of the hfq and arcZ fragment(s)were constructed.The results show that the virulence of?hfq and ?arcZ/hfq was almost completely lost and that of ?arcZ was significantly reduced.and activities of the exo-enzymes of these mutants were significantly decresed than that in the wild type.Additionally,a higher level of callose deposition was observed in the Solanum tuberosum leaves infiltrated with these mutants compared to those with the wild-type.It indicates that ArcZ is essential for the virulence of Pcb.We have known that sRNAs regulate target genes by base-pairing with 5'-UTR nonconding or coding regions of target mRNAs.To verify that hexA is one of the ArcZ target genes and the site(s)of sRNA-mRNA base-pairing between ArcZ and hexA,the strains with a mutation or overexpression in hexA were constructed.The results showed that hexA significantly negatively regulated the virulence of PeeS1.The strains with a mutation in segmented fragment of hexA 5'-UTR but then fusion with hexA-FLAG were obtained:?arcz/hexA-500?-124-hexA-FLAG.while a arabinose-inducible expression strain:?arcZ/hexA-500?-124-hexA-flag(TI-PBAD-areZ).Western blotting results Indicate that the base-pairing site of hexA mRNA and ArcZ is located in the 5'-UTR region of hexA.Based on the software predicted base-pairing sites between ArcZ and hexA mRNA,the strains with a mutation in the site of ArcZ were constructed as follows:?arcZ-hexA-FLAG(T1-PBAD-arcZ*1),?arcZhexA-FLAG(T1-PBAD-arcZ*2).Western blotting results indicate that hexA 5'-UTR region possesses two sites interacting with ArcZ.The sites in both hexA and ArcZ contain GC regions.We initially obtained the pattern in which ArcZ and its target gene hexA jointly regulated the virulence of Pectobacterium:ArcZ negatively regulates its target gene hexA that inhibits the activities of the exo-enzymes and negatively regulates the virulenc of Pectobacteri um.Systemic immune responses of host plants are facilitated by infection of pathogenic agents.Analysis of the changes in host immunity in the course of infection will be helpful to deeply understanding the pathogenesis of ArcZ.To investigate the response mechanism of Solanum tuberosum in infection of Pectobacterium,aseptic cultured Solanum tuberosum(DM 1-3)plants were properly inoculated with the strains of wild-type Pcb,AarcZ,Ahfq and AarcZ/hfq respectively,then the RNA samples of DM 1-3 were extracted before the presence of obvious symptoms,and RNA-seq was performed using Illumina sequencing.The data of comparetive transcriptome indicated that signaling pathways in DM 1-3 were activated to respond to Pcb infection through many processes including ethylene production,metabolism of phenylpropane,carbohydrates,amino acids and synthesis of various secondary metabolites in the cell components,such as cell wall,apoplast,extracellular region and chloroplast.The results also revealed that some photosynthesis-related pathways of DM1-3 might be inhibited by ArcZ in response to infection,including light energy capture in photosynthesis system ?(PSII)and synthesis of some important enzymes in calvin cycle.And the genes for leucine repeat receptor protein kinase(LRR RLKs)in DM1-3 were significantly up-regulated under the synergistic effect of ArcZ and Hfq.It is well known that callose deposition is one of the phenotypes of PTI(PAMPs trigged immunity).A increasement of callose deposition had been observed in the leaves of DM1-3 inoculated with the strains with mutation in arcZ and/or hfq than those with the wild type.it was agreeed with the differentially expressed genes related to PTI in Solanan tuherosum plants during the infection.Our results indicate that ArcZ might influence the response of host plant to regulate the pathogenicity of Pectohucterium and the mechanisms underling are waiting further studies.In conclusion.sRNA(ArcZ)negatively regulates its target gene hexA by base-pairing with two sites of hexA 5'-UTR,which significantly affects the defense response of Solanum tuberosum and regulates the virulence of Pcb.