Type III effectors RipA5 and RipAM of Ralstonia pseudosolanacearum mainly activate the host plant defense system

Raslonia solanacearum species complex (RSSC) contains a relatively large number of T3 effectors (T3Es), typically ranging from 60 to 75. There are genetic and functional redundancies among the different T3Es. This redundancy complicates the functional characterization of individual effectors. Ralstonia pseudosolanacearum strain OE1-1, belonging to RSSC phylotype-I, contains 70 T3E candidates. Fifty T3Es were deleted from OE1-1, and the resultant poly-mutant strain, OE1-1D50E, was used as an effector-depleted mutant. Twenty-one conserved T3E genes with the promoter region of OE1-1 were integrated into the OE1-1 glmS locus to construct strains with a reintroduced single T3E. These strains infiltrated eggplant and tobacco leaves, and several phenotypes were observed. None of the strains caused diseases on Nicotiana benthamiana as OE1-1D50E. Several single-T3E-reintroduced strains multiplied less than the effector-depleted strain in the leaves of eggplant and N. tabacum. The ripA5-expressing strain caused electrolyte leakage from infiltrated eggplant and N. tabacum leaves. A large amount of electrolytes leaked from N. tabacum leaves with the ripAM-expressing strain inoculation. Reactive oxygen species (ROS) production was observed in N. benthamiana leaves inoculated with the ripA5-expressing strain. These results indicate that RipA5 and RipAM of R. pseudosolanacearum are primarily involved in the induction of the host plant defense system.