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 RipAW^C177A, 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 RipAW^C177A-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.

Keywords: Ralstonia solanacearum; RipAW; SGT1; cell death; plant immunity.

Figure 1

Plant immunity triggered by RipAW^C177A in Nicotiana benthamiana. (A) The RipAW^C177A mutant failed to induce cell death in N. benthamiana. RipAW and RipAW^C177A were expressed in the same leaf of N. benthamiana. RipP1 and LTI6b were the positive control and negative control, respectively. Photographs were taken 2 days post inoculation (dpi). Circles indicate the infiltrated areas. The fractions in brackets represent the number of leaves displaying HR over the total number of infiltrated leaves. (B) Detection of RipAW and RipAW^C177A protein expression by western blot using anti-FLAG antibody. Total protein was extracted from the leaves 2 days after agroinfiltration and detected by western blot. The blot was stained with Coomassie brilliant blue (CBB) to confirm equal loading. (C) Expression of defense-related genes NbHIN1 and NbPR1a in N. benthamiana leaves expressing LTI6b (control), RipAW, or RipAW^C177A by qRT–PCR. (D,E) Growth of Ralstonia solanacearum CQPS-1 (D) and Pseudomonas syringae DC3000 ΔhopQ1-1 (E) in N. benthamiana leaves expressing RipAW^C177A and LTI6b. RipAW^C177A and LTI6b were agroinfiltrated into N. benthamiana leaves. 48 h later, CQPS-1 or DC3000 ΔhopQ1-1 was infiltrated into the same areas. The bacterial population was determined at 2 dpi for CQPS-1 and 3 dpi for DC3000 ΔhopQ1-1. Values are means ± standard errors (SEs) from six biological replicates. *** and **** indicate significant differences at p ≤ 0.001 and p ≤ 0.0001, respectively.

Figure 2

Induction of cell death by RipAW and its mutants. (A) Schematic illustration of RipAW and its derivatives RipAW^C177A (cysteine at position 177 of RipAW is substituted by alanine), RipAW ΔN, RipAW ΔC, RipAW^N1-90, RipAW^NEL and RipAW^C322-448. The N-terminus, NEL domain and C-terminus of RipAW are indicated as blue, gray and yellow boxes, respectively. The positions of amino acid residues are indicated by numbers. (B) RipAW induces cell death in N. benthamiana, but all its derivatives do not. RipAW and its derivatives were expressed in the same leaf of N. benthamiana. LTI6b was the negative control. Photographs were taken at 3 dpi. Circles indicate the infiltrated areas. The fractions in brackets represent the number of leaves displaying HR over the total number of infiltrated leaves. (C) Cell death in (B) was evaluated by the degree of ion leakage. The degree of ion leakage from the leaf discs was measured 44 h after agroinfiltration using a conductivity meter. Values are means ± SEs from three biological replicates. Different letters indicate significant differences at p ≤ 0.01. (D) Detection of RipAW and its derivatives with western blot using anti-FLAG antibody. Total protein was extracted from the leaves 2 days after agroinfiltration. The blot was stained with Coomassie brilliant blue (CBB) to confirm equal loading.

Figure 3

Plant immunity triggered by RipAW mutants. (A,B) Bacterial population of R. solanacearum CQPS-1 (A) and P. syringae DC3000 ΔhopQ1-1 (B) in N. benthamiana leaves expressing corresponding RipAW derivatives. Agrobacterial cells harboring RipAW mutants or LTI6b were infiltrated into N. benthamiana leaves. 48 h later, CQPS-1 or DC3000 ΔhopQ1-1 was infiltrated into the same areas. The bacterial population was determined at 2 dpi for CQPS-1 and 3 dpi for DC3000 ΔhopQ1-1. (C) NbHIN1 expression in N. benthamiana leaves expressing LTI6b (control) or RipAW’s derivatives by qRT–PCR. Values are means ± SEs from three biological replicates. *, **, ***, and **** indicate significant differences at p ≤ 0.05, p ≤ 0.01, p ≤ 0.001 and p ≤ 0.0001, respectively.

Figure 4

Silencing NbSGT1 impairs defense triggered by RipAW and RipAW^C177A. (A) Both RipAW and RipAW^C177A cannot induce cell death in NbSGT1-silenced N. benthamiana leaves. Photographs were taken at 3 days after infiltration. Circles indicate the infiltrated areas. The fractions in brackets represent the number of leaves displaying HR over the total number of infiltrated leaves. (B) Cell death in (A) was evaluated by the degree of ion leakage. The degree of ion leakage from the leaf discs was measured 2 days after agroinfiltration using a conductivity meter. (C) NbSGT1 expression in leaves of TRV2::NbSGT1 and TRV2::GFP N. benthamiana. (D) Growth of R. solanacearum CQPS-1 in TRV2::NbSGT1 and TRV2::GFP N. benthamiana leaves expressing RipAW and RipAW^C177A. LTI6b, RipAW and RipAW^C177A were agroinfiltrated into TRV2::NbSGT1 and TRV2::GFP N. benthamiana leaves, respectively. 48 h later, CQPS-1 was infiltrated into the same areas. The bacterial population was determined at 2 dpi. (E) NbHIN1 expression in TRV2::NbSGT1 and TRV2::GFP N. benthamiana leaves expressing LTI6b (control), RipAW or RipAW^C177A by qRT–PCR. Values in (B–E) are means ± SEs from at least three biological replicates. Different small letters in (B,E) and ** in panels (C,D) indicate significant differences at p ≤ 0.001. ns indicates no significant difference. (F) Detection of RipAW, RipAW^C177A and INF1 in TRV2::NbSGT1 and TRV2::GFP N. benthamiana by western blot using anti-FLAG or anti-HA antibody. Total protein was extracted from the leaves 2 days after agroinfiltration. The blot was stained with Coomassie brilliant blue (CBB) to confirm equal loading.

Figure 5

RipAW^C177A-triggered immunity in eds1 and mutants of Helper NLRs. (A) RipAW-induced cell death in wild-type (WT) N. benthamiana and eds1, nrg1, nrc2/3/4 mutants. RipAW and LTI6b (control) were expressed in the same leaf of wild-type N. benthamiana, eds1, nrg1 and nrc2/3/4 mutants, respectively. Photographs were taken at 3 dpi. Circles indicate the infiltrated areas. The fractions in brackets represent the number of leaves displaying HR over the total number of infiltrated leaves. (B) Growth of R. solanacearum CQPS-1 in WT and mutant leaves expressing LTI6b or RipAW^C177A. LTI6b and RipAW^C177A were agroinfiltrated into WT and mutant leaves. 48 h later, CQPS-1 was infiltrated into the same areas. The bacterial population was determined at 2 dpi. Values are means ± SEs from six biological replicates. ** and *** indicate significant differences at p ≤ 0.01 and p ≤ 0.001, respectively. (C) Detection of RipAW and RipAW^C177A in WT and mutants by western blot using anti-FLAG antibody. Total protein was extracted from the leaves 2 days after agroinfiltration and subjected to western blot analysis. The blot was stained with Coomassie brilliant blue (CBB) to confirm equal loading.

Figure 6

RipAW^C177A-triggered immunity is independent of SA pathway. (A) RipAW induces cell death when co-expressed with NahG. RipAW and RipE1 were co-expressed with LTI6b (control) or NahG in the same leaf. Photographs were taken at 3 dpi. Circles indicate the infiltrated areas. The fractions in brackets represent the number of leaves displaying HR over the total number of infiltrated leaves. (B) Cell death in (A) was quantified by the degree of ion leakage. The degree of ion leakage from the leaf discs was measured 2 days after agroinfiltration using a conductivity meter. (C) Detection of proteins in N. benthamiana by western blot using anti-FLAG (for RipAW and RipE1) or anti-GFP (for LTI6 and NahG) antibody. Total protein was extracted from the leaves 2 days after agroinfiltration. The blot was stained with Coomassie brilliant blue (CBB) to confirm equal loading. (D) NbHIN1 expression in N. benthamiana leaves co-expressing LTI6b (control) or NahG with RipAW by qRT–PCR. (E) NbHIN1 expression in N. benthamiana leaves co-expressing LTI6b (control) or NahG with RipAW^C177A by qRT–PCR. (F) Growth of R. solanacearum CQPS-1 in N. benthamiana leaves co-expressing NahG with LTI6b (control) or RipAW^C177A. LTI6b and RipAW^C177A were agroinfiltrated into NahG-expressed N. benthamiana leaves, respectively. 24 h later, CQPS-1 was infiltrated into the same leaf areas. The bacterial population was determined at 2 dpi. Values are means ± SEs from six biological replicates. *** indicates significant differences at p ≤ 0.001 and ns indicates no significant difference.