Volume 10, Issue 4, Pages 619-633 (April 2017) OsCERK1-Mediated Chitin Perception and Immune Signaling Requires Receptor-like Cytoplasmic Kinase 185 to Activate an MAPK Cascade in Rice  Chao Wang, Gang Wang, Chi Zhang, Pinkuan Zhu, Huiling Dai, Nan Yu, Zuhua He, Ling Xu, Ertao Wang  Molecular Plant  Volume 10, Issue 4, Pages 619-633 (April 2017) DOI: 10.1016/j.molp.2017.01.006 Copyright © 2017 The Author Terms and Conditions

Figure 1 Phylogenetic Analysis of Plant MEKK Family Genes and Interactions between OsRLCK185 and OsMAPKKKs. (A) Phylogenetic tree of MEKK subfamily genes. Bootstrap values (%) obtained from 1000 trials are indicated at nodes. O. sativa MAPKKK genes are named according to the Arabidopsis orthologs in the same clade. A1–A4 represent four subgroups of the MEKK subfamily. Eight OsMAPKKKs cloned for yeast two-hybrid (Y2H) assays are shown in magenta. (B) Eight OsMAPKKKs from the MEKK subfamily were cloned for testing interaction with OsRLCK185 or OsCERK1 using the Y2H system. Interactions were assessed by evaluating yeast growth on plates lacking leucine, tryptophan, histidine, and adenosine (-LWHA) or plates lacking leucine, tryptophan, and histidine (-LWH) plates, indicating strong and weak interaction, respectively. OsCERK1-IC refers to the intracellular domain of OsCERK1. (C) Schematic illustration of the Y2H assays. Yeast colonies grown on -LWHA and -LWH plates indicate strong and weak interaction, respectively. No interaction was found between OsCERK1 and OsMAPKKKs. The original data are presented in Supplemental Figure 1. Molecular Plant 2017 10, 619-633DOI: (10.1016/j.molp.2017.01.006) Copyright © 2017 The Author Terms and Conditions

Figure 2 OsRLCK185 Interacts with and Phosphorylates OsMAPKKKε. (A) Diagram of the domain features of OsMAPKKKε. The kinase domain and ARM (armadillo/β-catenin-like) repeats are indicated by a circle and rhombuses, respectively. Gray boxes indicate relatively conserved regions between MAPKKKε orthologs. (B) Dissection of the functional domain of OsMAPKKKε required for interaction with OsRLCK185. OsRLCK185 interacts with the partial C-terminal domain, OsMAPKKKε (aa 769–1357). A series of truncated OsMAPKKKε proteins were fused to GAL4 activation domain and tested for interaction with OsRLCK185. Interaction strength was assessed on -LWHA plates and -LW plates containing X-gal (80 μg/mL). The empty vector (Ev) showed no interaction with OsRLCK185, which served as a negative control. -LWHA indicates a plate lacking leucine (L), tryptophan (W), histidine (H), and adenosine (A). (C) In vitro pull-down assay reveals direct interaction between OsMAPKKKε and OsRLCK185. Proteins retained on the maltose binding protein (MBP) affinity resins were separated on SDS–PAGE and then immunoblotted with anti-His antibody. (D) Coimmunoprecipitation (CoIP) assays show that OsMAPKKKε forms a complex with OsRLCK185 in N. benthamiana leaves. Proteins were immunoprecipitated (IP) with FLAG-M2 beads, and analyzed by western blot using HRP-conjugated anti-FLAG or anti-HA antibody. Asterisks and arrows indicate full-length and degraded OsMAPKKKε, respectively. (E) CoIP assay using transgenic rice seedlings expressing FLAG-tagged OsRLCK185. Wild-type seedlings that do not express OsRLCK185-FLAG served as the control. Immunoprecipitated proteins were analyzed by western blot using HRP-conjugated anti-FLAG or anti-MAPKKKε antibody. (F and G) OsRLCK185 phosphorylates full-length OsMAPKKKε (F) and its C terminus (aa 769–1357) (G) in vitro. The C terminus (aa 769–1357) of OsMAPKKKε is the domain that interacts directly with OsRLCK185. The in vitro phosphorylation reaction was performed using purified proteins and [γ-32P]ATP, and detected by autoradiography. Kinase-inactive MAPKKKεK49M was used as the substrate to avoid autophosphorylation of MAPKKKε. Values at the side of SDS–PAGE gels in (C) to (G) indicate the molecular weight (kDa) of proteins. CBB, Coomassie brilliant blue staining. Molecular Plant 2017 10, 619-633DOI: (10.1016/j.molp.2017.01.006) Copyright © 2017 The Author Terms and Conditions

Figure 3 OsMAPKKKε Interacts with and Phosphorylates MKK4. (A) The kinase domain (KD) of OsMAPKKKε interacts with OsMKK4 and OsMKK5 in the Y2H system. Yeast colonies grown on -LWH plates or -LWH plates containing 12 mM 3AT indicated positive interactions. -LWH indicates a plate lacking leucine (L), tryptophan (W), and histidine (H). (B) CoIP assays show that OsMAPKKKε forms a complex with OsMKK4 or OsMKK5 in N. benthamiana leaves. FLAG-tagged OsMAPKKKε, and HA-tagged OsMKK4 and OsMKK5 were coexpressed in N. benthamiana leaves. Proteins were immunoprecipitated (IP) with FLAG-M2 beads, and analyzed by western blot using HRP-conjugated anti-FLAG or anti-HA antibody. Asterisks and arrowheads indicate the full-length and degraded OsMAPKKKε, respectively. (C) OsMAPKKKε phosphorylates OsMKK4 in vitro. OsMAPKKKε or OsMAPKKKε-KD was expressed to the C terminus of maltose binding protein (MBP), generating MBP-OsMAPKKKε and MBP-OsMAPKKKε-KD. The in vitro phosphorylation reaction was performed using purified proteins and [γ-32P]ATP, and detected by autoradiography. Kinase-inactive OsMAPKKKεK49M, which cannot phosphorylate OsMKK4, served as a negative control. (D) OsMAPKKKε induces OsMKK4 phosphorylation in N. benthamiana leaves. Kinase-inactive OsMKK4K122M was coexpressed with OsMAPKKKε-KD or OsMAPKKKε-KDK49M. OsMKK4 phosphorylation was detected by a mobility shift on a Phos-tag SDS–PAGE gel. Values at the side of SDS–PAGE gels in (B) to (D) indicate the molecular weight (kDa) of proteins. CBB, Coomassie brilliant blue staining. Molecular Plant 2017 10, 619-633DOI: (10.1016/j.molp.2017.01.006) Copyright © 2017 The Author Terms and Conditions

Figure 4 OsMAPKKKε Activates an MAPK Cascade. (A) OsMAPKKKε-KD induces MAPK activation in N. benthamiana. OsMAPKKKε-KD was expressed in N. benthamiana leaves. Total proteins were extracted and immunoblotted with anti-FLAG antibody, which demonstrated normal expression of OsMAPKKKε-KD. MAPK activation was detected using anti-phospho-p44/42 (pThr-X-pTyr) MAPK antibody. Kinase-inactive OsMAPKKKε-KDK49M cannot induce MAPK activation, indicating that kinase activity is required for OsMAPKKKε-KD to induce MAPK activation. Leaves infiltrated with infiltration buffer served as a mock control. hpi, hours post infiltration. (B and C) Overexpression of the kinase domain of OsMAPKKKε, OsMAPKKKε-KD, induces MAPK activation. Kinase-inactive mutant OsMAPKKKε-KDK49M, which cannot induce MAPK activation, was used as a control. Gene overexpression in transgenic plants was induced with 20 μM dexamethasone (DEX). Ethanol was used as solvent control. dpt, days post treatment. Relative expression levels (Rel.) are shown as the average of two or three independent biological replicates ± SD. (D and E) Overexpression of OsMAPKKKε-KD strongly induces defense gene expression. Expression level of PBZ1 (GenBank accession D38170) and Chitinase1 (D16221) was induced with 20 μM DEX for 4 days. The result could be observed in a replicated experiment using another transgenic line. (F) Overexpression of OsMAPKKKε-KD by DEX treatment enhances resistance to the rice blast fungal pathogen. All tested transgenic plants expressing OsMAPKKKε-KD showed resistance to blast infection, while transgenic plants expressing OsMAPKKKε-KDK49M were infected by blast fungi. The experiment was repeated twice, suggesting the same result. Scale bar, 5 mm. (G) OsMAPKKKε lacking the C-terminal regulatory domain induces MAPK activation and cell death. Cell death symptoms (%) were calculated 6 days post infiltration by the ratio of the necrotic area relative to the infiltrated area. Error bars represent SD of cell death symptoms (%) obtained from 30 infiltrated areas. Scale bar, 1 cm. Values at the side of SDS–PAGE gels in (A), (C), and (D) indicate the molecular weight (kDa) of proteins. CBB, Coomassie brilliant blue staining. Molecular Plant 2017 10, 619-633DOI: (10.1016/j.molp.2017.01.006) Copyright © 2017 The Author Terms and Conditions

Figure 5 A Linear Signaling Pathway, OsCERK1-OsRLCK185-OsMAPKKKε, Induces MAPK Activation. (A) OsCERK1 enhances phosphorylation of OsMAPKKKε by OsRLCK185 in vitro. The in vitro phosphorylation reaction was performed using purified proteins and [γ-32P]ATP, and detected by autoradiography. Kinase-inactive OsCERK1-KDK351E and OsRLCK185K108E served as controls. Note that the amount of OsRLCK185 protein is restricted to a low level (see Methods), so that OsRLCK185 inputs on the CBB gel and phosphorylation signals cannot be visible. However, this low amount of OsRLCK185 can phosphorylate OsMAPKKKεK49M. (B) Phosphomimetic OsRLCK185-3D exhibited enhanced interaction with OsMAPKKKε in the Y2H system. Interaction strength was assessed by the β-galactosidase activity either on -LW plates containing X-gal (80 μg/mL) or by quantification assay. Values represent means ± SD of interaction strength of five individual colonies of each interaction combination. Letters on the top of the bars indicate significant difference. -LWHA indicates plate lacking leucine (L), tryptophan (W), histidine (H), and adenosine (A). (C) CoIP assays showed that phosphomimetic OsRLCK185-3D enhanced interaction with OsMAPKKKε in N. benthamiana leaves. (D) Phosphomimetic OsRLCK185-3D enhances MAPK activation when coexpressed with OsMAPKKKε. Proteins were coexpressed in N. benthamiana leaves. After 3 days, total proteins were extracted and immunoblotted with anti-phospho-p44/42 MAPK antibody to indicate MAPK activation. Proteins immunoblotted with anti-FLAG and anti-MAPKKKε antibodies suggested normal expression of OsRLCK185 and OsMAPKKKε. Values at the side of SDS–PAGE gels in (A) and (C) indicate the molecular weight (kDa) of proteins. CBB, Coomassie brilliant blue staining. Molecular Plant 2017 10, 619-633DOI: (10.1016/j.molp.2017.01.006) Copyright © 2017 The Author Terms and Conditions

Figure 6 OsMAPKKKε Functions in the Chitin Signaling Pathway and Fungal Pathogen Resistance. Relative expression levels (Rel.) of OsMAPKKKε were detected by qRT–PCR (A, C, E, and I). MAPK activation was analyzed by immunoblotting with anti-phospho-p44/42 MAPK antibody (B, D, and F). Chitin treatments were performed with 1 × 10−6 M (GlcNAc)8 for 7.5 min (B, D, and F). (A and B) Overexpression of OsMAPKKKε enhances chitin-induced MAPK activation. The kinase-inactive mutant OsMAPKKKεK49M was used as a control. Relative expression levels are shown as the mean ± SD of two or three independent biological replicates. Ethanol was used as solvent control. (C and D) OsMAPKKKε-RNAi transgenic plants show reduced chitin-triggered MAPK activation. For each transgenic line (L), the stem was used for chitin treatment and total RNA isolation. NT, nontransgenic plants isolated from tissue culture. Error bars in (C) indicate SD of three technical replicates. (E and F) Silencing of MAPKKKε in N. benthamiana leaves reduces chitin-triggered MAPK activation. Values in (E) indicate mean ± SD of relative expression levels in three independent leaves. Representative data from the biological replicates are presented (F). (G–I) OsMAPKKKε-RNAi reduces resistance to the rice blast fungal pathogen. Lesion sizes in OsMAPKKKε-RNAi leaves were evidently larger than those of transgenic lines without obvious silencing of OsMAPKKKε isolated from tissue culture. Values in (H) and (I) indicate mean ± SE of lesion diameter. Transgenic lines without obvious silencing of OsMAPKKKε served as the control (Ctrl). n indicates numbers of the transgenic lines tested. Asterisks show significant difference assessed by Student's t-test (*P < 0.05; ***P < 0.001). Scale bar, 5 mm. Values at the side of SDS–PAGE gels in (B), (D), and (F) indicate the molecular weight (kDa) of proteins. CBB, Coomassie brilliant blue staining. Molecular Plant 2017 10, 619-633DOI: (10.1016/j.molp.2017.01.006) Copyright © 2017 The Author Terms and Conditions

Figure 7 Schematic Model for Chitin-Triggered MAPK Activation. The fungal chitin elicitor is perceived by the OsCEBiP/OsCERK1 complex, leading to chitin-induced OsCERK1 phosphorylation. Activated OsCERK1 phosphorylates OsRLCK185, which is a direct downstream target of OsCERK1 (Yamaguchi et al., 2013a). OsRLCK185 interacts with and phosphorylates OsMAPKKKε as described in this work. This interaction links chitin perception to activation of an MAPK cascade consisting of OsMAPKKKε, OsMKK4/5, and OsMAPK3/6. TF, transcription factor; PTI, PAMP-triggered immunity. Molecular Plant 2017 10, 619-633DOI: (10.1016/j.molp.2017.01.006) Copyright © 2017 The Author Terms and Conditions