Volume 23, Issue 2, Pages 241-253.e6 (February 2018) A Tyrosine Phosphorylation Cycle Regulates Fungal Activation of a Plant Receptor Ser/Thr Kinase  Jun Liu, Bing Liu, Sufen Chen, Ben-Qiang Gong, Lijuan Chen, Qi Zhou, Feng Xiong, Menglong Wang, Dongru Feng, Jian-Feng Li, Hong- Bin Wang, Jinfa Wang  Cell Host & Microbe  Volume 23, Issue 2, Pages 241-253.e6 (February 2018) DOI: 10.1016/j.chom.2017.12.005 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2018 23, 241-253. e6DOI: (10. 1016/j. chom. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Site-Directed Mutagenesis Screen Identifies Two Potential Tyr Phosphorylation Sites in CERK1 Important for Chitin Signaling (A) CERK1 undergoes Tyr autophosphorylation in the absence of chitin. Wild-type (WT) or catalytically inactive (ΔRD) CERK1 was enriched by immunoprecipitation (IP) from transgenic complementation plants before immunoblotting. (B) Pretreatment of Tyr kinase inhibitor tyrphostin A23 impairs the chitin-induced band shift of CERK1 and MAPK activation. Protoplasts isolated from cerk1/35S::CERK1-FLAG transgenic complementation plants were pretreated with A23 for 1 hr before chitin treatment. CBB (Coomassie brilliant blue) staining indicates equal protein loading. (C) Diagram of the CERK1 cytoplasmic domain with 16 Tyr phospho-deficient mutation sites indicated. JM, juxtamembrane region; CT, C-terminal tail. (D and E) Mutagenesis screen of potentially important Tyr phosphorylation sites in CERK1 kinase domain based on the chitin-induced MAPK activation (D) and ROS burst (E). WT and kinase-dead (K350N) CERK1 were used as positive and negative controls, respectively. Error bars represent SEM from means of six biological repeats. ∗∗∗p < 0.001, Student's t test. The experiments in (A) and (B) were repeated three times with similar results. See also Figure S1. Cell Host & Microbe 2018 23, 241-253.e6DOI: (10.1016/j.chom.2017.12.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Tyr428 and Tyr557 of CERK1 Are Potentially Important Phosphorylation Sites Regulating CERK1-Mediated Chitin Signaling (A) The Y428F, but not Y557F, mutation of CERK1 blocks the chitin-triggered MAPK activation. Two representative mutant complementation lines were treated with chitin. (B) Both Y428F and Y557F mutations of CERK1 impair the chitin-triggered ROS burst. Error bars represent SEM from means of six biological repeats. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001, Student's t test. (C) The Y428F and Y557F mutations of CERK1 compromise plant immunity against the fungal pathogen B. cinerea to different extents. Error bars represent SEM from means of eight different leaves. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001, Student's t test. (D) The Y428F, but not Y557F, mutation of CERK1 disrupts the chitin-induced resistance against the bacterial pathogen Pst DC3000. Error bars represent SEM from means of six different leaves. ∗∗p < 0.01; ∗∗∗p < 0.001, Student's t test. See also Figure S2. Cell Host & Microbe 2018 23, 241-253.e6DOI: (10.1016/j.chom.2017.12.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Tyr428 Phosphorylation of CERK1 Is Indispensable for Chitin-Triggered CERK1 Activation and Dynamic Interaction with Downstream RLCKs (A) CERK1 is autophosphorylated at Tyr428 in vivo in the absence of chitin. Wild-type (WT), Y428F mutant, or catalytically inactive (ΔRD) CERK1 was enriched by IP from transgenic complementation plants before immunoblotting. (B) A23 treatment dramatically reduces the levels of Tyr428 phosphorylation in CERK1. Protoplasts isolated from cerk1/35S::CERK1-FLAG transgenic complementation plants were treated with 50 μM A23 for 1 hr before CERK1 was enriched by IP for immunoblotting. (C) Recombinant CERK1 cytoplasmic domain (CD), but not its variant with Y428F or kinase-dead (K350N) mutation, shows Tyr428 phosphorylation in vitro. (D) Tyr428 phosphorylation of CERK1 occurs in a dynamic manner in vivo after chitin elicitation. The cerk1/35S::CERK1-FLAG transgenic seedlings were treated with chitin for indicated time before CERK1 was enriched by IP from 90% of crude protein extracts for immunodetection of Tyr428 phosphorylation. The rest (10%) of crude protein extracts were used to examine the dynamics of chitin-induced MAPK activation. (E) Quantification of Tyr428 phosphorylation of CERK1 at different time points after chitin elicitation by densitometric analysis of immunoblot signals. Error bars represent SEM from means of three biological repeats and the Tyr428 phosphorylation level at time point 0 was set as 1 in each repeat. (F) The Y428F mutation of CERK1 disrupts the chitin-triggered band shift of CERK1. Protoplasts from indicated transgenic complementation plants were treated with chitin and total proteins were resolved by an 8% SDS-PAGE gel. (G and H) The Y428F mutation of CERK1 blocks the chitin-induced BIK1 dissociation (G) and PBL27 recruitment (H). BIK1-GFP or PBL27-GFP was expressed for 12 hr in protoplasts isolated from cerk1/35S::CERK1-FLAG transgenic complementation plants before cells were treated with chitin and then subjected to coIP. Numbers indicate relative amounts of BIK1-GFP co-immunoprecipitated with CERK1- or CERK1Y428F-FLAG. The experiments were repeated three times with similar results. The other two replicates of (D) and (G) are shown in Figures S7A and S7B, respectively. See also Figure S3. Cell Host & Microbe 2018 23, 241-253.e6DOI: (10.1016/j.chom.2017.12.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Tyr557 Phosphorylation of CERK1 Is Required for the Chitin-Triggered CERK1-BIK1 Dissociation (A) Tyr557 phosphorylation of CERK1 is induced by chitin. WT or Y557F CERK1 was enriched by IP from transgenic complementation plants with or without chitin treatment before immunoblotting. (B) A23 treatment dramatically suppresses the chitin-induced Tyr557 phosphorylation of CERK1. Protoplasts isolated from cerk1/35S::CERK1-FLAG transgenic complementation plants were pretreated with A23 at different doses for 1 hr and then treated with chitin before CERK1 was enriched by IP for immunoblotting. (C) Chitin-induced Tyr557 phosphorylation is dependent on the Tyr428 phosphorylation in CERK1. WT or Y428F CERK1 was enriched by IP from corresponding transgenic complementation plants with or without chitin treatment before immunoblotting. (D) The Y557F mutation of CERK1 has no effect on the chitin-triggered band shift of CERK1. Protoplasts from indicated transgenic complementation plants were treated with chitin and total proteins were resolved by an 8% SDS-PAGE gel. (E and F) The Y557F mutation of CERK1 has no effect on the chitin-induced PBL27 recruitment (E) but blocks the chitin-induced BIK1 dissociation from CERK1 (F). Numbers indicate relative amounts of BIK1-GFP co-immunoprecipitated with CERK1- or CERK1Y557F-FLAG. PBL27-GFP or BIK1-GFP was expressed for 12 hr in protoplasts isolated from cerk1/35S::CERK1-FLAG transgenic complementation plants before cells were treated with chitin and then subjected to coIP. The experiments were repeated three times with similar results. The other two replicates of (F) are shown in Figure S7C. See also Figure S4. Cell Host & Microbe 2018 23, 241-253.e6DOI: (10.1016/j.chom.2017.12.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 CIPP1 Is Recruited to CERK1 under Chitin Elicitation for Dephosphorylation (A) Chitin induces strong interaction between CERK1 and CIPP1. Protoplasts co-expressing CERK1-GFP and CIPP1-FLAG for 12 hr were treated with chitin before coIP. (B) CIPP1 quickly associates with CERK1 after chitin treatment and then slowly dissociates. Transgenic seedlings co-expressing CERK1-FLAG and CIPP1-GFP were treated with chitin for different time before coIP. (C) CIPP1 interacts directly with CERK1 cytoplasmic domain (CD) in an in vitro pull-down assay. (D) Chitin fails to induce the interaction between CIPP1 and CERK1Y428F. Transgenic seedlings co-expressing CERK1Y428F- or CERK1-FLAG and CIPP1-GFP were treated with chitin for different time before coIP. (E) Chitin cannot induce the interaction between CIPP1 and CERK1 after pretreatment with A23. Protoplasts isolated from the transgenic plants co-expressing CERK1-FLAG and CIPP1-GFP were pretreated with 50 μM A23 for 1 hr and then treated with chitin before coIP. (F) CIPP1 is able to dephosphorylate Tyr428 of CERK1 in vitro. Purified recombinant GST-CIPP1, GST-PAPP2C (control phosphatase), or mock was incubated with GST-CERK1 CD for different times before Tyr428 phosphorylation of CERK1 was detected by immunoblotting. CBB, Coomassie brilliant blue. (G) Co-expression of CIPP1 inhibits the recovery of Tyr428 phosphorylation of CERK1 after chitin treatment. The cerk1/35S::CERK1-FLAG transgenic complementation plants with or without CIPP1 co-expression were treated with chitin for different time before CERK1 was enriched by IP for immunoblotting. (H) Quantification of immunoblot signals from three biological repeats in (G) by densitometric analysis. Error bars represent SEM from means of three biological repeats and, for each genotype, the Tyr428 phosphorylation level at the time point of 0 was set as 1 in each repeat. ∗p < 0.05; ∗∗∗p < 0.001, Student's t test. The experiments were repeated three times with similar results. The other two replicates of (G) are shown in Figure S7D. See also Figure S5. Cell Host & Microbe 2018 23, 241-253.e6DOI: (10.1016/j.chom.2017.12.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 CIPP1 Plays a Negative Regulatory Role in the Chitin-Triggered Plant Immunity (A and C) Two independent transgenic lines overexpressing CIPP1 show reduced MAPK activation (A) and ROS burst (C) by chitin. ∗p < 0.05; ∗∗∗p < 0.001, Student's t test. CBB, Coomassie brilliant blue. (B and D) Two independent CIPP1 RNAi silencing lines show enhanced MAPK activation (B) and ROS burst (D) by chitin. ∗p < 0.05; ∗∗∗p < 0.001, Student's t test. (E) Generation of the cipp1-1 mutant by CRISPR/Cas9. CRISPR/Cas9-mediated single-nucleotide insertion in the CIPP1 target sequence leads to a truncated CIPP1 protein at the catalytic domain. (F and G) The cipp1-1 mutant plants show enhanced MAPK activation (F) and ROS burst (G) by chitin. ∗∗∗p < 0.001, Student's t test. (H) The cipp1-1 mutant plants are more resistant to the fungal pathogen B. cinerea than wild-type plants. Error bars represent SEM from means of eight different leaves. ∗p < 0.05, Student's t test. (I) The cipp1-1 mutant plants show enhanced chitin-induced resistance against the bacterial pathogen Pst DC3000. Error bars represent SEM from means of six different leaves. ∗p < 0.05, Student's t test. (J) Model of CERK1- and CIPP1-regulated chitin signaling. See also Figure S6. Cell Host & Microbe 2018 23, 241-253.e6DOI: (10.1016/j.chom.2017.12.005) Copyright © 2017 Elsevier Inc. Terms and Conditions