Functional Identification Of The Host Peanut Specific Virulence Gene Of Ralstonia Solanacearum

We previously found that one 4.5 kb DNA fragment (pGX1404) cloned from one race 1 peanut strain T2005 of Ralstonia solanacearum could enable one race 3 potato strain, which were nonpathogenic to peanut, to cause bacterial wilt on peanut. In this work, we subcloned ORF3 of pGX1404 in pLAFR6 and found that it still retained the ability of pGX1404 to extend the host range of strain T2014 to include peanut, confirming that ORF3 is the host specific virulence gene (hsvP) to peanut.We constructed a hsvP" mutant of T2015 by complementing its downstream gene ORF2, which formed one operon with ORF3. Virulence test on different crops including peanut and HR test on tobacco for T2014, T6418, T2015, T2135/R etc. indicated that hsvP was required for full virulence of T2015 on not only peanut but also other crops including potato, but had no apparent effect on the ability of T2015 to cause HR on nonhost plant tobacco. The colony size of T6418 is bigger than that of T2014, but the colony of 2135/R is smaller than that of T2015. This suggested hsvP may have relation with the size of colony. Sequence analysis of hsvP showed that its encoded product has 23% identity and 44% similarity with E.coli lipidA core-O-antigen ligase encoded by rfaL SDS-PAGE analysis of LPS revealed that the LPS of T2135/R lacked 0-antigen, confirming that the encoded product of hsvP acts as lipidA core-O-antigen ligase. Kinetics study of bacterial within plant revealed that the number of T2135/R in peanut plant was significantly lower than that of T2015, and the number of T6418 wassignificantly higher than that of T2014, indicating that the role of whole LPS in virulence may be protecting the bacterial cells from the damage of antibacterial substances of plants or suppressing the plant defense response.Sequence analysis of the hsvP homologous sequence in T2014 revealed that it lacked the four bases between nucleotide 1033 and 1036 of T2015 ORF3, generating an early in-frame stop codon in the gene. SDS-PAGE analysis of LPS revealed that more LPS of T6418 have O-antigen compared with that of T2014. These further confirmed that the protein encoded by hsvP acts as lipidA core-O-antigen ligase.