A systematic screen of conserved Ralstonia solanacearum effectors reveals the role of RipAB, a nuclear-localized effector that suppresses immune responses in potato

Both Solanum tuberosum and Ralstonia solanacearum phylotype IIB originated in South America and share a long-term co-evolutionary history. However, our knowledge of potato bacterial wilt pathogenesis is scarce as a result of the technical difficulties of potato plant manipulation. Thus, we established a multiple screening system (virulence screen of effector mutants in potato, growth inhibition of yeast and transient expression in Nicotiana benthamiana) of core type III effectors (T3Es) of a major potato pathovar of phylotype IIB, to provide more research perspectives and biological tools. Using this system, we identified four effectors contributing to virulence during potato infection, with two exhibiting multiple phenotypes in two other systems, including RipAB. Further study showed that RipAB is an unknown protein with a nuclear localization signal (NLS). Furthermore, we generated a ripAB complementation strain and transgenic ripAB-expressing potato plants, and subsequent virulence assays confirmed that R. solanacearum requires RipAB for full virulence. Compared with wild-type potato, transcriptomic analysis of transgenic ripAB-expressing potato plants showed a significant down-regulation of Ca2+ signalling-related genes in the enriched Plant-Pathogen Interaction (PPI) gene ontology (GO) term. We further verified that, during infection, RipAB is required for the down-regulation of four Ca2+ sensors, Stcml5, Stcml23, Stcml-cast and Stcdpk2, and a Ca2+ transporter, Stcngc1. Further evidence showed that the immune-associated reactive oxygen species (ROS) burst is attenuated in ripAB transgenic potato plants. In conclusion, a systematic screen of conserved R. solanacearum effectors revealed an important role for RipAB, which interferes with Ca2+-dependent gene expression to promote disease development in potato