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Ralstonia solanacearum causes bacterial wilt disease in a broad range of plants, primarily through type Ⅲ secreted effectors. However, the R. solanacearum effectors promoting susceptibility in host plants remain limited. In this study, we determined that the R. solanacearum effector RipV2 functions as a novel E3 ubiquitin ligase (NEL). RipV2 was observed to be locali in the plasma membrane after translocatio into plant cells. Transient expression of RipV2 in Nicotiana benthamiana could induce cell death and suppress the flg22-induced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, mediating such effects as attenuation of the expression of several PTI-related genes and ROS bursts. Furthermore, we demonstrated that the conserved catalytic residue is highly important for RipV2. Transient expression of the E3 ubiquitin ligase catalytic mutant RipV2 C403A alleviated the PTI suppression ability and cell death induction, indicating that RipV2 requires its E3 ubiquitin ligase activity for its role in plant-microbe interactions. More importantly, mutation of RipV2 in R. solanacearum reduces the virulence of R. solanacearum on potato. In conclusion, we identified a NEL effector that is required for full virulence of R. solanacearum by suppressing plant PTI., Competing Interests: Declaration of competing interest The authors have declared no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bacterial wilt caused by Ralstonia solanacearum is a destructive plant disease, particularly in potato (Solanum tuberosum). R. solanacearum deploys a diverse and potent arsenal of type III effectors to inhibit the plant immune system. However, the understanding of individual effectors promoting susceptibility in host plants and interfering with plant immunity responses is still limited. Here, we demonstrated that the type III effector RipV1 functioned as a novel E3 ubiquitin ligase (NEL) effector and exhibited E3 ubiquitin ligase activity in vitro. Transient expression of RipV1 suppressed plant pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) responses in Nicotiana benthamiana, such as the expression of PTI‐related genes and the reactive oxygen species (ROS) burst. Prolonged expression of RipV1 induced cell death in N. benthamiana leaves. Notably, mutating the conserved cysteine residue of RipV1 abolished its E3 ligase activity and its ability to suppress plant PTI responses. This study also revealed the indispensability of RipV1 for R. solanacearum's full virulence in potato. Transgenic potato plants overexpressing ripV1 but not the catalytic mutant ripV1‐C444A displayed enhanced susceptibility to R. solanacearum. RipV1 was observed to localize specifically to the plant plasma membrane, with its N‐terminus being pivotal in determining this localization. These findings showcase that RipV1 acts as a NEL effector and contributes to R. solanacearum virulence by suppressing plant PTI responses through its E3 activity. [ABSTRACT FROM AUTHOR]

Ralstonia solanacearum is one of the most destructive plant‐pathogenic bacteria, infecting more than 200 plant species, including potato (Solanum tuberosum) and many other solanaceous crops. R. solanacearum has numerous pathogenicity factors, and type III effectors secreted through type III secretion system (T3SS) are key factors to counteract host immunity. Here, we show that RipBT is a novel T3SS‐secreted effector by using a cyaA reporter system. Transient expression of RipBT in Nicotiania benthamiana induced strong cell death in a plasma membrane‐localization dependent manner. Notably, mutation of RipBT in R. solanacearum showed attenuated virulence on potato, while RipBT transgenic potato plants exhibited enhanced susceptibility to R. solanacearum. Interestingly, transcriptomic analyses suggest that RipBT may interfere with plant reactive oxygen species (ROS) metabolism during the R. solanacearum infection of potato roots. In addition, the expression of RipBT remarkably suppressed the flg22‐induced pathogen‐associated molecular pattern‐triggered immunity responses, such as the ROS burst. Taken together, RipBT acts as a T3SS effector, promoting R. solanacearum infection on potato and presumably disturbing ROS homeostasis. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]