Genome Analysis And Functional Study Of LuxR Systems In The Soft Rot Banana Pathogen Dickeya Zeae MS2

Banana is a very important economic plant all over the world especially in tropical and subtropical regions.In recent years,there were cases of soft rot banana disease reported in Guangdong and Yunnan Provinces,China.It spreads very fast and causes huge losses in banana production.The disease becomes more and more threatening.Previously,we isolated a particularly virulent Dickeya zeae strain MS2 from the pseudo-stem of a soft rot banana plant collected from Nansha district in Guangzhou,China.MS2 strain showed different phenotypic characteristics from strains EC1(from rice)and MS2(also from banana),manifested as a kind of novel phytotoxin produced by MS2.Thus,to better understand the difference between strain MS2 and other D.zeae strains at genetic level,and further study on the pathogenicity mechanisms of MS2,we sequenced the genome of it,analyzed the genome by bioinformatics means and performed in-depth analysis on the Lux R systems.The results are as follows:1.The genome of D.zeae MS2 is 4.75 Mbp in size with 53.44% of GC content,encoding4275 genes and harboring 7 r RNA operons,2 prophages and 2 CRISPR gene clusters.A nonribosomal peptide synthesis(NRPS)coding gene cluster was found to be uniquely present in the genomes of MS2,D.paradisiaca NCPPB2511 and D.dadantii Ech703.We deleted the key gene C1O30＿05075 in this gene cluster and found that the mutant completely loses the antibiotic activity,therefore,we considered that this gene cluster is responsible for the production of the NRPS novel phytotoxin.2.Given that the Lux R-Lux I circuit plays an important role in the quorum sensing(QS)pathway of soft rot pathogens,we searched the Lux R homologs in the MS2 genome using homologous Blastp and found two genes encoding the Lux R proteins.One is adjacent to an AHL type QS signal encoding gene lux I,which was named as Exp IR system according to the high identity to the well-known Exp IR system in rice strain EC1.3.We identified the exp I encoding products and found that strain MS2 could produce two types of AHL signal.One is N-3-oxo-hexanoyl-homoserine lactone(3OC6-HSL)and the other is 3OC8-HSL,accounting for about 80% and 20% of the total AHL,respectively.No obvious difference of virulence on banana seedlings was observed in the exp I and exp R mutants compared with the wild type MS2 in greenhouse trial.Whereas,the exp I mutant significantly enhanced cell motility and reduced cell clumping and the production of pigment indigoidine.4.We also identified a gene encoding a proline iminopeptidase(PIP)downstream to the Lux R-solo coding gene.We named it as pip A and the lux R coding gene as pip R.Deletion of pip A significantly reduced the virulence on banana seedlings,while deletion of pip R showed similar virulence as MS2.In this system,expression of Pip A is under the control of Pip R,which was induced by the host extracts.In summary,this article focuses on the production of a strain of Dickeya zeae MS2 isolated from a soft-rot banana tree.We assembled its genome,analyzed and verified the gene function.Among them,the non-ribosomal polypeptide synthase encoded by NRPS can synthesize a bacterial toxin that inhibits the growth of E.coli around D.zeae MS2 and has an effect on the pathogenicity of pathogens.Exp R / I have no obvious effect on pathogenicity,yet knocking out Exp I can obviously improve the motility of pathogenic bacteria,reduce cell aggregation and the production of blue pigment Indigoidine.The pip A in the Lux R-solo gene cluster has a significant effect on pathogenicity and can be positively regulated by Pip R.The plant tissue extracts we tested also further showed that plants can induce the regulation by Pip R.This research provides a scientific experimental basis for further understanding the regulatory mechanism of the pathogenicity of MS2.