Cloning And Identification Of Pathogenic Genes Of D.zeae MS2

As one of the top ten important bacterial phytopathogens,Dickeya spp.cause bacterial soft rot disease on many crops and ornamental plants all over the world.Among them,banana bacterial soft rot disease caused by Dickeya spp.is one of the three major bacterial diseases of banana.At present,there are few studies related to the virulence factors of Dickeya banana strains and the pathogenic mechanism of the pathogens is still unclear.In this study,D.zeae strain MS2 was used to construct a library of Tn5 transposon insertion mutants.By artificial inoculation on different host plants,mutants with reduced virulence were isolated,whose flanking sequences of inserted genes were obtained using the technology of hi TAIL-PCR.After gene knockout and complementation,as well as phynotypic analyses,functions of the target genes were identified.The main results of the study are as follows:(1)By Tn5 transposon mutagenesis,a library of 29403 muants was constructed,in which,2673 mutants were inoculated on cabbage blades.Mutants with defective virulence were screened out and then applied for at least three rounds of pathogenicity tests on different host plants.Finally,a total of 178 mutants with obviously reduced virulence was obtained.(2)Integration of Tn5 insertion fragemts with MS2 genomic DNA was verified by PCR amplification using primers located in both regions.Flanking sequences of 17 mutants were obtained using hi TAIL-PCR amplification,2 of which were used for further functional analysis,including mutant M392 with Tn5 inserted at the 53 bp of the open reading frame(ORF)of a gene encoding for a capsular biosynthesis protein(C1O30＿02590),and mutant M506 with Tn5 inserted at the 231 bp of the ORF of a gene encoding for an Eam A family transporter(C1O30＿20515).(3)Gene knockout mutants and the complementary strains of mutants M392 and M506 were obtained.Phynotypic analyses showed that the growth rate of both the Tn5 inserted mutants(M392 and M506)and the knock-out mutants(Δ392 and Δ506)was basically similar to the wild-type strain MS2,whereas,the production of mutant cell wall degrading enbzymes was slightly decreased,and the swimming and swarming motility as well as the virulence of the mutants were significantly reduced,and both the complementary strians C392 and C506 recovered the changed phynotypes.In addition,mutation of C1O30＿02590 gene(encoding a capsular biosynthesis protein)dramacticlly decreased the production of exopolysaccharides(EPS)and slightly decreased the biofilm formation.Findings in this study identify the pathogenic genes in D.zeae MS2,helping clarify the pathogenic regulatory networks of D.zeae MS2 and provide the targets for disease control.