Functional Analysis of a Virulence Gene of Ralstonia Solanacearum

Abstract: We previously found that gene ORF3 cloned from one race 1 peanut strain T2015 of Ralstonia solanacearum could enable one race 3 potato strain, which were nonpathogenic to peanut, to cause bacterial wilt on peanut. ORF3 was thus defined as the host specific virulence gene to peanut (hsvP). In this work, we found that the ORF3 had apparent effect on the ability of pathogen to cause disease on not only peanut but also other crops including potato态tomato and eggplant. This confirmed that the ORF3 is a general gene in Ralstonia solanacearum.The encoded product of hsvP acts as lipidA core-O-antigen ligase to form the bacterial whole LPS structure. In this work, we found that the ORF3 had no apparent effect on the the growth of Ralstonia solanacearum in GBM medium, Bacterial growth assay in medium containing peanut water extract showed that the ORF3 non-polar mutant of T2015 and T2014 grew less well than T2015 and T2014 carrying T2015 ORF3 did, respectively. We speculated that the role of the complete LPS structure in bacterial virulence may be protecting the bacterial cells from the damage of antibacterial substances of plants and be propitious to the growth of bacterial in plants and the ability to causing disease on plants. We had tested the expression of the strain containing the recombinant plasmids that the promoter of ORF3 was fused to gusA reporter gene. The test of GUS expression activity in different medium revealed that poor medium and some component in peanut water extract could induce the expression of ORF3.GUS activity assay within plant confirmed that ORF3 has been expressed actively in plant.The test of the virulence and the SDS-PAGE of LPS of Ralstonia solanacearum revealed that the whole LPS structure were not necessary to the pathogenicity. ORF3 was transferred into Ralstonia solanacearum P053 and M4 lacked O-antigen. The result revealed that ORF3 was unable to enhance the pathogenicity of P053 and M4, and not more O-antigen were detected in the resultant transconjugant. Sequence analysis of the ORF3 homologous sequence in P053 and M4 revealed that the ORF3 sequence in P053 and M4 were integrity. The lack of O-antigen of P053 and M4 may be the result of the mutation of other gene involved in the synthesization of LPS. These further confirmed that the ORF3 is a general gene in Ralstonia solanacearum…
Key words: Ralstonia solanacearum; virulence gene; lipopolysaccharide

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