Functional Aanlysis Of LuxR/luxI In Acidovorax Citrulli

Bacterial fruit blotch of melons, caused by Acidovorax citrulli, is a seed borne disease that causesserious damages to the plants of cucurbits in worldwide. As most gram-negative bacteria, the pathogenhas luxR/luxI quorum sensing system to regulate its biological functions.Application the method of homologous recombination to construct three mutants related to the QSsignal systemic functional genes, containing luxR and luxI single gene deletion mutants and luxR/luxIdouble gene deletion mutant. luxR/luxI QS system function was studied by comparing the differnecebetween the wild type and mutant type strains. The results are as follows:(1) Results of AHLs detection：Compared with wide-type strain signal substances cannot bedetected in△luxI and△luxR/luxI because of the deletion of luxI, while△luxR can still display ablue chromogenic reaction due to the existence of luxI. This result is consistent with the conclusion thatluxI is the gene producing signal substances and luxR is the gene generating the carrier of the signalsubstances.(2) Results of pathogenicity determination：the mutants displayed the reduced in virulence, theresult showed that QS system played an important role in pathogenicity of Acidovorax citrulli.(3) Results of physiology and biochemistry bioasaay: the mutant strains are not attenuated ingrowth rate until stationary phase.There is no obvious difference in motility ability extracellularpolysaccharide secretion.(4) Results of Real-Time PCR：The expression of five genes related to virulence including luxI andluxR genes were detected quantitatively by Real-Time PCR, and the result revealed that the expressionof the seven genes we selected decreased in mutant strains, displayed negative regulation, similar withthe result of pathogenicity of the mutation-type strains. Results of Real-Time qPCR：The expression offive genes related to virulence and two gene including luxI and luxR related to quorum sensing werequantitatively detected by Real-Time qPCR. Results showed that the expression of the seven genesdecreased in mutant strains, displayed positive regulation. And the quorum sensing system related genesluxI was expressed at a lower level in△luxR and luxR was expressed at a lower level in△luxIrespectively.(5) The AHLs of bacterial fruit blotch of melons were extracted, and identified quantitatively byUPLC-MS/MS as OOHL. The pathogenicity of△luxI to melon seedlings was recovered after addingthe OOHL.