The Ralstonia solanacearum E3 ligase effector RipV1 targets subfamily IXb receptor-like cytoplasmic kinases that negatively regulate immunity in Nicotiana benthamiana

Plants detect microbe-associated molecular patterns from pathogens via plasma membrane- localized receptors which activate multiple signaling cascades that lead to pattern-triggered immunity (PTI). Receptor-like cytoplasmic kinases (RLCKs) are essential hubs of plant immune signaling, associating with receptors and intracellular proteins through phosphorylation events. As a consequence, RLCKs have emerged as common targets of pathogen effectors. To improve our knowledge on Solanaceae responses to the bacterial wilt pathogen Ralstonia solanacearum , we conducted a yeast two-hybrid screen between tomato RLCKs and effectors conserved in R. solanacearum Korean isolates. Several members of RLCK subfamily IXb, which contain a ubiquitin-ligase plant U-box domain in addition to the kinase domain, interacted with RipV1, an effector containing a novel E3 ligase domain (NEL). In vitro assays revealed that RLCK-IXb-1 displayed ubiquitin-ligase activity but no detectable kinase activity. RipV1 could trans- ubiquitinate RLCK-IXb-1 in vitro and promote its stability in planta . Using virus-induced gene silencing of RLCK-IXb homologs in Nicotiana benthamiana , we could further show that several RLCK-IXb proteins act as negative regulators of early PTI signaling. RipV1 was previously reported to contribute to R. solanacearum virulence in potato and to elicit cell death in an E3 ligase activity-dependent manner in N. benthamiana . Here we show that RipV1-induced cell death occurred in plants impaired for effector recognition but could be suppressed by over-expression of RLCK-IXb-1, suggesting that this response is related to the virulence function of RipV1. Altogether, our work identifies possible substrates of an NEL effector and underlines the complex roles of RLCKs in plant immune signaling.