Identification And Functional Study Of Pantoea Ananatis DZ-12 T6SS And Its Effector TseG

Pantoea ananatis is a Gram-negative facultative anaerobic bacterium belonging to the genus Pantoea and family Enterobacteriaceae.In 1928,it was first discovered as a pathogen of pineapples in the Philippines.P.ananatis is a pathogen of many plants,including mazie,rice,onion,potato,etc.,which brings great losses to agricultural production in several areas.Previously,our research group isolated and identified a Pantoea ananatis strain DZ-12 that causes brown rot in mazie.Genome sequencing and bioinformatics analysis was conducted and we found that the major pathogenicity-related gene cluster T3 SS was incomplete in DZ-12,while it possessed other two complete T6 SS gene clusters which was named as T6SS-1and T6SS-2,respectively.Therefore,the T6 SS gene clusters of DZ-12 was speculated to be involved in its’ pathogenicity to maize.Type Ⅵ Secretion System(T6SS)was first discovered and proposed in 2006 in the research about V.cholerae,and bioinformatics analysis indicated that more than 25% of Gram-negative bacteria have T6 SS.T6SS is a novel needle-like device which can infect eukaryote or prokaryote via injecting effector proteins into their cells.It colud act as pathogenic factors to directly attack host cells,mediate interspecific competition among bacteria,and may affect the environmental adaptability of bacteria.Meanwhile,it has been found recently that for plant pathogenic bacteria,T6 SS plays a vital role in its pathogenicity.Consequently,the study of bacterial T6 SS is of significantly scientific meaning and application value.The results of this study are as follows:1.Identification and genome sequencing analysis of Pantoea ananatis DZ-12 showed that the genome of DZ-12 contains a 4.85 Mb chromosome and a resident plasmid p DZ-12 with size of 304 kb.DZ-12 possesses three T6 SS gene clusters which were named as T6SS-1,T6SS-2 and T6SS-3,respectively.T6SS-1 and T6SS-3 is located on the chromosome,T6SS-2 is located on p DZ-12,T6SS-1 and T6SS-2 are complete while T6SS-3 is lack of some structural genes.Fluorescence microscopy was used to observe the pathogenic process of DZ-12 on maize leaves,it was found that DZ-12 was concentrated in the vascular bundle of maize leaves,which suggests DZ-12 might affect the growth of host plants by interfering with its’ transport of substances.Scanning electron microscopy(SEM)observation indicated that P.ananatis DZ-12 may invade maize leaves through stomata.2.T6SS-1 and T6SS-2 of P.ananatis DZ-12 were correlated with pathogenicity to plants.Hcp protein is the inner sheath structural protein of T6 SS which encoded by hcp gene,we hence mutated hcp to destroy T6 SS gene cluster.T6SS-1 mutant △hcp1,T6SS-2mutant △hcp2 and double mutant △hcp1△hcp2 were constructed successfully and the pathogenicity of DZ-12 mutants on maize,onion and potato was significantly reduced,which indicates T6 SS was an important pathogenic factor of DZ-12 on host plants.Experiments about biofilm formation and EPS production were conducted to found that the ability of biofilm formation and EPS production of T6 SS mutants was significantly suppressed than that of DZ-12.3.P.ananatis DZ-12 can utilize T6 SS to kill E.coli and make itself in an advantageous position in the competition with bacteria.In order to investigate whether DZ-12 takes advantage of T6 SS for interspecific competition,wild type DZ-12 and T6 SS mutants were used.Results showed that △hcp1 and △hcp1△hcp2 almost lose the antagonism ability against E.coli,while the competitiveness of △hcp2 is not much different from that of wild type.It indicaed that T6 SS,especially T6SS-1,function in the interspecific competitiveness for strain DZ-12 against E.coli.4.TseG of T6SS-1 from P.ananatis DZ-12 is an effector for plant pathogenicity.There are few reports so far on T6 SS of P.ananatis.Effectors of T6 SS generally have their homologous immunity proteins to neutralize its’ toxicity,some of them have chaperones proteins for better excretion.In this study,we found a protein with domain DUF2169 on T6SS-1 which might be a potential chaperone,named Tec G.Hence downstream of Tec G was infer as a T6 SS effector and homologous immunity protein,named TseG and Tsi G,respectively.Firstly,extracellular and total protein of DZ-12 was extracted and TseG was identified as an extracellular protein which is mainly secreted by T6SS-1.Mutants △tse G,△tsi G,△tse G△tsi G and △tec G were constructed and inoculated on mazie,onion and potato.The results showed that the virulence of the strain to host plants decreased significantly after TseG knockout.Additionally,pathogenicity of △tsi G and △tec G on host plants decreased as well,possibly because chaperone Tec G and immunity protein Tsi G assist the secretion of TseG,but the detailed mechanism need to be further studied.5.TseG of T6SS-1 from P.ananatis DZ-12 is also an effector with antagonism activity against E.coli.The toxicity experiment of TseG against E.coli was conducted to verify TseG has bactericidal effect on E.coli.After the introduction of immunity protein Tsi G into E.coli cells,the toxicity of TseG was almost lost,which demonstrated that Tsi G was the immunity protein of TseG.Furthermore,immunity protein Tsi G and chaperone Tec G were proved to interact with effector TseG via Double-Bacterial Assay,Co-IP and Pull-down,respectively.Research above is of reference significance to study the interaction between effector,immunity protein and chaperone in T6 SS from pathogenic bacteria.