Construction Of Virulence Deficient Mutants Of Dickeya Zeae MS3 Using PLG107 Plasmid

Banana soft rot disease caused by Dickeya zeae,spreads fast and has great harm on banana yield.In recent years,it has resulted in seriously economic losses in banana planting areas in China.At present,few studies are reported about the pathogenicity mechanism of D.zeae banana strain.In this study,we constructed a library of virulence deficient mutants of D.zeae MS3 using transposon insertion mutagenisis of plasmid pLG107,used hiTAIL-PCR technique to amplify and identify the mutated genes in MS3 genome,and finally verify the functions of target genes by gene knockout and complementation.Results from this study may help to understand the pathogenicity of D.zeae MS3 and provide new targets for controlling the banana bacterial soft rot disease.The main results are as follows:(1)A library of 1767 mutants was constructed using pLG107 plasmid,after several rounds of pathogenicity tests on cabbage and potato slices,145 mutants were confirmed as virulence deficient mutants for further analysis.(2)hiTAIL-PCR was performed on 11 mutants with significantly decreased virulence on banana seedlings,sequencing results revealed that the nested primers(LG107-0a/1a/2a)designed on the basis of 3' terminal of T24 transposon sequence are not suitable for amplification of the flanking sequences of the mutants,since other sequences in pLG107 randomly combine with the T24 transposon 3' terminal.Thus,the nested primers(LG107-0b/1b/2b)at the 5' terminal were designed and used to amplify the flanking sequences of the mutants,results showed that T24 transposon was respectively inserted at the 562 nd bp of efeO gene(encoding an iron uptake system component EfeO)in mutant M688,at the 333 rd bp of a gene encoding Phenylproprionate permease(PPP)in mutant M753,at the 17 th bp of togM gene(encoding an oligogalacturonide transport system permease)in mutant M848,and at the 311 st bp of a kinetochore protein encoding gene in mutant M853.(3)Further analysis of togM revealed that there is a pelW(encoding pectate disaccharide-lyase)gene upstream,and another oligogalacturonide transport system permease encoding gene togN,an ATP-binding protein encoding gene togA,and substrate-binding protein encoding gene togB downstream.This TogMNAB operon constructs an oligogalacturonide transport system.qRT-PCR analysis showed that T24 tranposon insertion in togM resulted in significantly decreased expression of all the tog genes.After knocking-out genes togM and togN,we found that both the single-and the double-knocked out mutants reduced the pathogenicity on banana seedlings,and as well as the swarming motility,comparing with the wild type strain MS3.The pathogenicity was basically restored to the wild-type level after gene complementation.