Ubiquitin E3 ligase activity of Ralstonia solanacearum effector RipAW is not essential for induction of plant defense in Nicotiana benthamiana

Abstract

As one of the most destructive bacterial phytopathogens, Ralstonia solanacearum causes substantial annual yield losses of many important crops. Deciphering the functional mechanisms of type III effectors, the crucial factors mediating R. solanacearum-plant interactions, will provide a valuable basis for protecting crop plants from R. solanacearum. Recently, the NEL (novel E3 ligase) effector RipAW was found to induce cell death on Nicotiana benthamiana in a E3 ligase activity-dependent manner. Here, we further deciphered the role of the E3 ligase activity in RipAW-triggered plant immunity. We found that RipAWC177A, the E3 ligase mutant of RipAW, could not induce cell death but retained the ability of triggering plant immunity in N. benthamiana, indicating that the E3 ligase activity is not essential for RipAW-triggered immunity. By generating truncated mutants of RipAW, we further showed that the N-terminus, NEL domain and C-terminus are all required but not sufficient for RipAW-induced cell death. Furthermore, all truncated mutants of RipAW triggered ETI immune responses in N. benthamiana, confirming that the E3 ligase activity is not essential for RipAW-triggered plant immunity. Finally, we demonstrated that RipAW- and RipAWC177A-triggered immunity in N. benthamiana requires SGT1 (suppressor of G2 allele of skp1), but not EDS1 (enhanced disease susceptibility), NRG1 (N requirement gene 1), NRC (NLR required for cell death) proteins or SA (salicylic acid) pathway. Our findings provide a typical case in which the effector-induced cell death can be uncoupled with immune responses, shedding new light on effector-triggered plant immunity. Our data also provide clues for further in-depth study of mechanism underlying RipAW-induced plant immunity.

The Ralstonia solanacearum Type III Effector RipAW Targets the Immune Receptor Complex to Suppress PAMP-Triggered Immunity

“…RipAW, a NEL effector in R. solanacearum , exhibits E3 ligase activity dependent on the cysteine 177 residue [ 17 ]. Recognition of RipAW and the relationship between RipAW-triggered ETI and its E3 ligase activity has been clarified [ 24 , 25 ]. In this study, we characterized the virulence function of RipAW in the host plant and explored its virulence mechanism, focusing on interference with the immune receptor complex.…”

“…Agrobacterium -mediated transient expression was performed in five-week-old N. benthamiana , according to Ouyang et al [ 25 ]. Flg22-induced ROS burst was monitored as previously described [ 8 ].…”

“…Total RNA was isolated using Trizol reagent (Sangon, Shanghai, China), according to the manufacturer’s recommendations. The expression of PTI-related genes WRKY29 and FRK1 [ 42 ] was analyzed by quantitative real-time PCR as previously described [ 25 ]. The ACTIN gene was used as an internal control.…”

“…Niu et al found that RipAW induced a strong hypersensitive response (HR) and programmed cell death (PCD) in Nicotiana benthamiana and Nicotiana tabacum , relying on its E3 ligase activity [ 24 ]. Ouyang et al further demonstrated that the E3 ligase activity of RipAW is not essential for the induction of plant defense [ 25 ]. Interestingly, RipAW has also been shown to impair PTI, although it can activate ETI in N. benthamiana [ 23 ].…”

A robust high‐throughput functional screening assay for plant pathogen effectors using the TMV‐GFP vector

“…However, the percentage of effectors affecting cell death is relatively low in most pathogen effector reservoirs. More importantly, effector-induced cell death can be uncoupled with immune responses, and therefore, HR-based screens may provide only a partial and perhaps biased view of ETI responses (Ouyang et al, 2023;Prautsch et al, 2023;Yang et al, 2022). Mitogen-activated protein kinase (MAPK) cascades play a vital role in plant defense against pathogen attack (Bi & Zhou, 2017;Meng & Zhang, 2013).…”