WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in Nicotiana benthamiana

Abstract

Pathogen attack sequentially confers pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) after sensing of pathogen patterns and effectors by plant immune receptors, respectively. Reactive oxygen species (ROS) play pivotal roles in PTI and ETI as signaling molecules. Nicotiana benthamiana RBOHB, an NADPH oxidase, is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Here, we show that RBOHB transactivation mediated by MAPK contributes to R3a/AVR3a-triggered ETI (AVR3a-ETI) ROS burst. RBOHB is markedly induced during the ETI and INF1-triggered PTI (INF1-PTI), but not flg22-tiggered PTI (flg22-PTI). We found that the RBOHB promoter contains a functional W-box in the R3a/AVR3a and INF1 signal-responsive cis-element. Ectopic expression of four phospho-mimicking mutants of WRKY transcription factors, which are MAPK substrates, induced RBOHB, and yeast one-hybrid analysis indicated that these mutants bind to the cis-element. Chromatin immunoprecipitation assays indicated direct binding of the WRKY to the cis-element in plants. Silencing of multiple WRKY genes compromised the upregulation of RBOHB, resulting in impairment of AVR3a-ETI and INF1-PTI ROS bursts, but not the flg22-PTI ROS burst. These results suggest that the MAPK-WRKY pathway is required for AVR3a-ETI and INF1-PTI ROS bursts by activation of RBOHB.

Figure 1.

Induction of RBOHB by INF1 and R3a/AVR3a. (A) Expression of RBOHB in response to flg22 and INF1 proteins. INF1 (100 nM) and 100 nM flg22 were infiltrated into leaves by a needleless syringe, and total RNAs were used for RT-qPCR. (B) Expression of RBOHB in response to MEK2^DD, R3a/AVR3a, and INF1. Total RNAs were extracted from leaves at the indicated hours after agroinfiltration and were used for RT-qPCR. (C) Effects of single or multiple MAPK gene silencing on RBOHB expression induced by INF1 and R3a/AVR3a. Total RNAs were extracted from SIPK (S)- or NTF6 (N6)-silenced leaves 0, 36, and 48 h after agroinfiltration and were used for RT-qPCR. Asterisks indicate statistically significant differences compared with water (A), GUS (B), or TRV (C) (t test, *P < 0.05 and **P < 0.01). Data are means ± sd from at least three experiments.

Figure 2.

Promoter Activity of RBOHB Induced by MEK2^DD via cis-Element. (A) Deletion analysis of RBOHB promoter activity. The number indicates the distance from the RBOHB translation start site. A mixture of Agrobacterium cultures containing RBOHB promoter-GUSint (reporter), pER8:MEK2^DD (effector), and CaMV 35S promoter-LUCint (reference) was coinfiltrated into leaves and 24 h later 20 μM estradiol was injected into the leaves. MEK2^KR, MEK1^DD, and MEK1^KR were also expressed as the effector. GUS activities and LUC activities were determined 24 h after estradiol treatments, and RBOHB promoter activities were the values of GUS activities divided by LUC activities. (B) Analysis of RBOHB promoter and mutated promoters containing two-base substitutions. Mutant bases are shown on a black background. (C) Analysis of RBOHB −1000 and mB4-1000 promoter in response to MEK2^DD. (D) Analysis of three-tandem repeats of the cis-element and mB4 with 35S minimal promoter in response to MEK2^DD and MEK1^DD. (E) Effects of SIPK and WIPK silencing on the three-tandem promoter activity. SIPK (S)- and WIPK (W)-silenced leaves were inoculated with Agrobacterium carrying promoter assay constructs. Asterisks indicate statistically significant differences compared with MEK2^KR and MEK1^KR ([A] and [D]), −460 (B), mB4-1000 (C), and TRV (E) (t test, *P < 0.05 and **P < 0.01). Data are means ± sd from at least three experiments.

Figure 3.

Promoter Activity of RBOHB Induced by INF1 and R3a/AVR3a via the cis-Element. (A) Deletion analysis of the RBOHB promoter in response to flg22, INF1, and R3a/AVR3a. Leaves were treated with 100 nM INF1 and 100 nM flg22 or were inoculated with Agrobacterium carrying INF1 and R3a/AVR3a. Promoter activities were analyzed as described in Figure 2A. (B) Analysis of the RBOHB −1000 and mB4-1000 promoter in response to INF1 and R3a/AVR3a. (C) Activation of three tandem repeats of the cis-element by INF1 and R3a/AVR3a. Promoter activity was represented as a relative value, using the comparative 3 × cis of INF1. (D) Analysis of INF1- or R3a/AVR3a-induced three-tandem promoter activities in single or multiple MAPK gene-silenced leaves. (E) Analysis of INF1- or R3a/AVR3a-induced RBOHB −2000 promoter activities in single or multiple MAPK gene-silenced leaves. Asterisks indicate statistically significant differences compared with water and empty (Emp) (A), mB4-1000 (B), 35S minimal (C), or TRV ([D] and [E]) (t test, *P < 0.05 and **P < 0.01). Data are means ± sd from at least three experiments.

Figure 4.

Promotion of Cell Death by Phospho-Mimicking Mutations on the SP Cluster of WRKY Transcription Factors. (A) Phospho-mimicking mutations in a SP cluster. Putative phosphorylated Ser residues in SP cluster were substituted with Asp (nD). (B) Induction of cell death by overexpression of phospho-mimicking mutants. Leaves were infiltrated with Agrobacterium carrying the indicated gene expression constructs. Photographs were taken 4 d after agroinfiltration (left). After taking the photographs, WRKY-induced cell death was detected by trypan blue staining (right). (C) Detection of WRKY-HA-StrepII proteins by anti-HA antibody. Total proteins were prepared 48 h after agroinfiltration. Protein loads were monitored by Coomassie blue (CBB) staining of the bands corresponding to the ribulose-1,5-bisphosphate carboxylase large subunit (RBCL). Red asterisks indicate WRKY-HA-StrepII proteins. C, control plant; W, wild-type WRKYs; D, phospho-mimicking mutants. (D) Detection of WRKY10-HA-StrepII protein. Total proteins were purified by Strep-Tactin and then immunoblot analysis was done.

Figure 5.

Regulation of RBOHB Expression by Multiple WRKY Transcription Factors. (A) Induction of the RBOHB gene by phospho-mimicking mutants of SP cluster-carrying WRKYs. Total RNAs were extracted from leaves 24 h after agroinfiltration and were used for RT-qPCR. nD indicates phospho-mimicking mutants of WRKYs. (B) Involvement of multiple WRKY genes in MEK2^DD-induced RBOHB expression. Total RNAs were extracted from the WRKY-silenced leaves 0 and 36 h after agroinfiltration and were used for RT-qPCR. (C) Suppression of INF1- and R3a/AVR3a-induced RBOHB expression by silencing of multiple WRKY genes. Total RNAs were extracted from leaves 0 or 36 h after agroinfiltration and were used for RT-qPCR. (D) Expression of RBOHB in response to variants of WRKY8 and WRKY11. Total RNAs were extracted from leaves 24 h after agroinfiltration and were used for RT-qPCR. Anti-HA antibody was used to detect accumulations of WRKY-HA variants. nD and nA indicate that WRKY mutants mimic the phosphorylated form and nonphosphorylated form, respectively. Letters represent each significance group, determined through Tukey's multiple range test. Asterisks indicate statistically significant differences compared with TRV ([B] and [C]) (t test, *P < 0.05 and **P < 0.01). Data are means ± sd from at least three independent experiments.

Figure 6.

Involvement of Multiple WRKY Transcription Factors in INF1-PTI and AVR3a-ETI ROS Bursts. (A) Enhancement of flg22-triggered ROS burst in MAPK- or WRKY-silenced plants. Flg22-triggered ROS bursts were measured for 60 min in leaves silencing SIPK/WIPK (S/W), WRKY7, 8, 9, and 11 (7/8/9/11), or RBOHB (NbB). Total photon counts of each treatment during 60 min were graphed in the right panel. (B) Effects of multiple WRKY gene silencing on effector-triggered ROS burst. Silenced leaves were coinoculated with Agrobacterium carrying pER8:AVR3a-HA and pBinPlus:R3a and then were injected with 20 μM estradiol 24 h later. To detect ROS generation, inoculation sites were infiltrated with 0.5 mM L-012 solution 24 h after estradiol injection and were monitored using a CCD camera. Chemiluminescence intensities were quantified by a program equipped with a photon image processor. (C) Effects of multiple WRKY gene silencing on INF1-triggered ROS burst. INF1-ROS burst was detected in silenced leaves 24 h after agroinfiltration. Asterisks indicate statistically significant differences compared with TRV (t test, *P < 0.05 and **P < 0.01). Data are means ± se from at least five experiments.

Figure 7.

Involvement of WRKY Transcription Factors in MEK2^DD-, INF1-, or R3a/AVR3a-Dependent Activation of the RBOHB Promoter. (A) Yeast one-hybrid analysis using a three-tandem promoter as bait and WRKY7, 8, 9, 11, or 13 as prey. The representative growth status of yeast cells is shown on SD/-HTL agar media with or without 3-amino-1,2,4-triazole from triplicate independent trials. Numbers on the top of each photograph indicate relative densities of the cells. dpi, days postinoculation. (B) Analysis of RBOHB −1000 and mB4-1000 promoter in response to WRKY transcription factors. N. benthamiana leaves were inoculated with Agrobacterium carrying promoter assay constructs. WRKY genes were expressed as the effector. Promoter activities were analyzed as described in Figure 2A. (C) Involvement of multiple WRKY genes in MEK2^DD-induced RBOHB promoter activity. Silenced leaves were inoculated with Agrobacterium carrying promoter assay constructs. (D) Effects of multiple WRKY gene silencing on RBOHB promoter activity induced by INF1 and R3a/AVR3a. (E) ChIP-qPCR analysis in GFP-WRKY8^5D-expressed plants. Leaves were inoculated with Agrobacterium carrying pER8:GFP-WRKY8^5D and then were injected with 20 μM estradiol 24 h later. Input chromatin was isolated from the leaves 12 h after estradiol treatment. GFP-tagged WRKY8-chromatin complex was immunoprecipitated with an anti-GFP antibody. A control reaction was processed at the same time using rabbit IgG. ChIP- and input-DNA samples were quantified by qPCR using primers specific to the promoters of RBOHA and RBOHB genes. ChIP results are shown as percentages of input DNA. Asterisks indicate statistically significant differences compared with mB4-1000 promoter (B), TRV ([C] and [D]), and rabbit (E) (t test, *P < 0.05 and **P < 0.01). Data are means ± sd from three experiments.

Figure 8.

Involvement of Multiple WRKY Transcription Factors in RBOHB-Dependent ROS Burst. (A) ROS generation by overexpression of WRKY transcription factors. Leaves were inoculated with Agrobacterium carrying the indicated gene expression constructs. ROS generation was detected 24 h after agroinfiltration as described in Figure 6B. (B) WRKY-induced ROS generation via RBOHB. TRV control, RBOHB (NbB)-, and BAK1-silenced leaves were inoculated with Agrobacterium, and ROS were measured 24 h after agroinfiltration. (C) ROS generation by overexpression of phospho-mimicking mutants. (D) Detection of phospho-mimicking mutants by anti-HA antibody. C, control plant; W, wild-type WRKY11; D, phospho-mimicking mutant; A, nonphosphorylated mutant. Letters represent each significance group, determined through Tukey's multiple range test. Asterisks indicate statistically significant differences compared with TRV ([B] and [C]) (t test, **P < 0.01). Data are means ± se from at least three experiments.

Figure 9.

Increased Disease Susceptibility to a Virulent Strain of P. infestans by Silencing of Multiple WRKY Genes. (A) Susceptibility to P. infestans in the silenced plants. Inoculated leaves were photographed 6 d after the inoculation (left). N. benthamiana leaves were stained with lactophenol-trypan blue 6 d after inoculation (right). Arrowheads indicate hyphae of P. infestans. Asterisks indicate HR cell death of mesophyll cells. Bars = 20 μm. (B) Effects of single or multiple WRKY gene silencing on P. infestans infection. Biomasses were determined by qPCR 6 d after inoculation. Asterisks indicate statistically significant differences compared with TRV (t test, **P < 0.01). Data are means ± sd from three independent experiments.

Figure 10.

Model of the Regulatory Mechanism of INF1-PTI and AVR3a-ETI ROS Bursts. Sensing INF1 by the PRR triggers the early phase ROS burst, which is not required for de novo RNA synthesis, and the late phase ROS burst. Perception of INF1 and AVR3a by receptors leads to activation of the MAPK cascade. Activated MAPK phosphorylates and activates WRKY7, 8, 9, and 11. These WRKYs bind to the W-box in the RBOHB promoter, resulting in upregulation of the RBOHB gene. Supply of newly synthesized RBOHB to the plasma membrane may contribute to the late phase INF1-PTI and AVR3a-ETI ROS bursts.