Functional Analysis Of Polyamine Transporter Signaling Pathway In Dickeya Zeae MS3

Dickeya zeae MS3,the causal agent of banana soft rot disease,is a gram negative bacterium with a wide host range and infects both monocotyledons and dicotyledons.Although this disease is not frequently reported in China,it can cause serious economical costs once outbreak and has been listed as No.302 quarantine objective.Studies around the complex signal communication involved in interactions between bacteria and host cellshave just begun.Here,we aim on the role of polyamine signaling pathwayin D.zeae MS3 during pathogen-host interaction,focus on the regulation of polyamine on important virulence factors,including cell wall degrading enzymes activity,cell motility,biofilm formation and pathogenicity,especially the relationship with T3 SS effectors,moreover,construct specific polyamine reporter system on the basis of expression of T3 SS effector genes to reveal the polyamine signal response cues and regulatory networks,elucidate pathogenic mechanism of D.zeae MS3,understand pathogen-host interactions at molecular level,and also establish theoretical foundation for development of new disease prevention and control strategy.The main results are as follows:1.By searching for the closely relative genome of D.zeae MS1,4 genes(spe A,spe C,spe D and spe E)associated with polyamine biosynthesis,16 genes encoding ABC transporter proteins associated with polyamine transport,and 2 other genes pla P and puu Ppredicted to encode membrane proteins specifically transport putrescine were selected for further study.In the light of the high homology between MS1 and MS3,we finally got 29 mutants except △spe C and △puu P of MS3 using primers designed based on MS1 sequence.2.Results of characteristic and phenotypic analyses showed that in comparison with the wild type strain MS3,all mutants showed no obvious difference in growth rate,extracellular enzyme production or biofilm formation,whereas reduced in swimming and swarming motility,and also virulence on Musa ABB seedlings and potato tubers.Amongst,mutants △ pot C2,△ spe A,△ pot F △ spe A,△ spe A △ pla P and △ pot F △ pla P △ spe A showed weakened in both motility and pathogenicity,e.g.swimming motility decreased to 76.7%,46.4%,31.9%,39.1% and24.9%andswarming motility decreased to81.7%,59.7%,45.3%,56.7% and 34.2% of MS3,respectively.Their virulence on potato tubers red uced by 13.5%,19.2%,22.3%,14% and 23.6%,respectively,and on banana seedlings reduced by 11.1% in mutants△pot C2 and △spe A△pla P,and 22.2% in the rest.These results suggest that the membrane protein Pot C2 and arginine decarboxylase Spe A are important for the motility and pathogenicity of MS3,and in the polyamine biosynthesis pathway,only putrescine regulates bacterial motility and virulence.3.Results from swimming and swarming motility showed that addition of low concentration of exogenous putrescinerestored the motility of △spe A to wild type level,whereas,△pot F△pla P△spe A did not loss the ability to transport exogenous putrescine,suggesting that there must be another transporter protein playing a key role in putrescine uptake.Virulence on banana seedlings and potato tubers were increased after induction by host extracts,revealing that putrescine could act as a signaling molecule and further induce and enhance the pathogenicity of D.zeae MS3.The consistent results of motility and pathogenicity illustrated the impact of putrescine during pathogenic processes through modulating bacterial motility.4.Data from transcriptome analyses revealed that after host extract induction,207 genes were up expressed,and 494 were down expressed,and the expression of spe A encoding arginine decarboxylase increased nearly 3 folds,suggesting that banana tissue could promote putrescine synthase.Furhtermore,expression of transporter genes pot D1,pot D2,pot A1,pot A2,pot D1,pot D2 and pot D3 increased to 2.05~8.84 folds,suggesting that polyamine transporters are induced by signals in banana tissue.Additionally,after host tissue induction,expression of all genes in T6 SS was significantly reduced by ranging from 2.6~8.7 folds.T6 SS was reported to be involved in the corporation and competition between bacteria and eukaryotes,the role of which in interactions between D.zeae MS3 and banana host waits for our further investigation.Expression of extracellular enzyme encoding genes increased after host extract induction,including 7 Pel genes,2 Cel genes and 1 Prt gene,in consistent,the virulence of all isolates in our study was enhanced after banana tissue induction,implicating cell wall degrading enzymes as major virulence factors in D.zeae MS3.5.The results of real-time quantative PCR showed that the expression of T3 SS effector genes increased when MS3 cultured with host extracts,whereas that of △spe A was very low;after adding banana tissue when cultured,expression of hrp A,hrp J,hrp N,hrp P,hrc S,hrp X and q2066 could increase up to the wild type level in MM medium,suggesting that MS3 could trigger the expression of T3 SS effector genes after induced by putrescine signal from medium or the bacterium.