The OsGSK2 Kinase Integrates Brassinosteroid and Jasmonic Acid Signaling by Interacting with OsJAZ4

Abstract

The crosstalk between brassinosteroid (BR) and jasmonic acid (JA) signaling is crucial for plant growth and defense responses. However, the detailed interplay between BRs and JA remains obscure. Here, we found that the rice (Oryza sativa) Glycogen synthase kinase3 (GSK3)-like kinase OsGSK2, a conserved kinase serving as a key suppressor of BR signaling, enhanced antiviral defense and the JA response. We identified a member of the JASMONATE ZIM-domain (JAZ) family, OsJAZ4, as a OsGSK2 substrate and confirmed that OsGSK2 interacted with and phosphorylated OsJAZ4. We demonstrated that OsGSK2 disrupted the OsJAZ4-OsNINJA complex and OsJAZ4-OsJAZ11 dimerization by competitively binding to the ZIM domain, perhaps helping to facilitate the degradation of OsJAZ4 via the 26S proteasome pathway. We also showed that OsJAZ4 negatively modulated JA signaling and antiviral defense and that the BR pathway was involved in modulating the stability of OsJAZ4 protein in an OsCORONATINE INSENSITIVE1-dependent manner. Collectively, these results suggest that OsGSK2 enhances plant antiviral defenses by activating JA signaling as it directly interacts with, phosphorylates, and destabilizes OsJAZ4. Thus, our findings provide a clear link between BR and JA signaling.

Figure 1.

OsGSK2 Positively Regulates Antiviral Defense and JA Response. (A) RBSDV symptoms on Zh11, Go, and Gi plants. Bar = 10 cm. (B) Disease incidence in Zh11, Go, and Gi plants following RBSDV inoculation. The numbers of healthy and diseased plants in each treatment were determined by RT-PCR 30 d after inoculation, and the number of diseased plants was used to calculate the viral incidence (percentage of plants infected). Each treatment used at least 40 seedlings, and at least three biological replicates were performed. Different letters at the top of columns indicate significant difference between transgenic and control plants at P ≤ 0.05 by Fisher’s LSD test. (C) Expression levels of the RBSDV CP gene as measured by RT-qPCR at 30 dpi. Data are relative expression levels of CP in Go and Gi plants compared with that in the wild-type Zh11 plants. OsUBQ5 was used as the internal reference gene. Error bars indicate the sd of three biological replicates. Asterisk (*) indicates significant difference between transgenic and control plants at P ≤ 0.05 by Fisher’s LSD test. (D) Expression analysis of JA-responsive genes by RT-qPCR. Seven-day-old seedlings were collected for total RNA extraction. OsUBQ5 was used as the internal reference gene. Values are means ± se of three biological replicates. Asterisk (*) indicates significant difference between transgenic and the control plants at P ≤ 0.05 by Fisher’s LSD test. (E) Levels of endogenous JA in 7-d-old Zh11, Go, and Gi plants. The limit of quantification to JA was 1 ng/mL. Values are means ± sd of three biological replicates. Different letters at the top of columns indicate significant difference between transgenic and control plants at P ≤ 0.05 by Fisher’s LSD test. FW, fresh weight. (F) and (G) Images (F) and quantification of root length (G) of Zh11, Go, and Gi after MeJA treatment. The root lengths of 3-d-old seedlings grown in normal rice culture solutions supplemented with indicated concentrations of MeJA were measured. Data shown are the means from at least 10 seedlings for each indicated plant. Error bars represent sd. Different letters at the top of columns indicate significant difference between transgenic and control plants at P ≤ 0.05 by Fisher’s LSD test. Bar in (F) = 2 cm.

Figure 2.

OsGSK2 Interacts with OsJAZ4 in Vitro and in Vivo. (A) Y2H assay showing the interaction between OsGSK2 and OsJAZ1-15 proteins. Interactions were examined with SD base without Ade, His, Leu, and Trp. (B) Y2H assay showing the interaction between OsJAZ4 and OsGSK1-8 proteins. Transformed yeast cells were grown on SD-Ade-His-Leu-Trp medium. (C) Pull-down assay confirming that OsGSK2 interacts with OsJAZ4 in vitro. Immobilized GST and GST-OsGSK2 were used to pull down His-OsJAZ4, and immunoprecipitated fractions were detected using anti-His antibody. The bait proteins were probed with anti-GST antibody. (D) BiFC assay showing the interaction between OsGSK2 and OsJAZ4 in N. benthamiana leaves. OsJAZ11 was used as negative control. Bar = 20 µm. (E) Co-IP assays showing the interaction between OsGSK2 and OsJAZ4 in vivo. The proteins were extracted from N. benthamiana leaves and immunoprecipitated by anti-myc and anti-HA magnetic beads, respectively. The coimmunoprecipitated proteins were probed by either anti-Myc or anti-HA antibody. OsJAZ11-myc was used as negative control.

Figure 3.

OsGSK2 Phosphorylates OsJAZ4. (A) Immunoprecipitated OsJAZ4-myc protein from OsJAZ4-MYC plants was treated with CIP or water. OsJAZ4-myc protein was separated in a phos-tag SDS-PAGE gel and detected by anti-myc anti-body (top and middle panels). The slowly migrating band (red arrow) of OsJAZ4-myc in the phos-tag gel with short exposure time (Short exp.) or long exposure time (Long exp.) represents the phosphorylated form of OsJAZ4 (OsJAZ4-myc-P). As loading control (bottom), equal amounts of the immunoprecipitated OsJAZ4-myc protein were separated in a normal SDS-PAGE gel followed by immunoblot analysis. (B) Potential phosphorylation sites in OsJAZ4. (C) Immunoprecipitated OsJAZ4-myc and OsJAZ4Δ8-myc protein in N. benthamiana leaves transiently coexpressed with HA-empty vector, HA-OsGSK2, or HA-OsGSK2^K92R. The protein immunoprecipitated by anti-myc beads or CIP-treated OsJAZ4-myc protein from each combination were separated in a phos-tag SDS-PAGE gel and detected by anti-myc antibody (top). The slowly migrating band (red arrow) of OsJAZ4-myc in the phos-tag gel with short exposure time (Short exp.) or long exposure time (Long exp.) is the phosphorylated form of OsJAZ4 (OsJAZ4-myc-P). As loading controls, OsGSK2 (bottom) and different amounts of the immunoprecipitated OsJAZ4-myc protein (middle) were separated in a normal SDS-PAGE gel followed by immunoblot analysis.

Figure 4.

OsGSK2 Promotes OsJAZ4 Degradation. (A) Time course of OsJAZ4 degradation in the wild-type NIP protein extracts treated with GST, GST-OsGSK2, or GST-OsGSK2^K92R. Equal amounts of plant crude extracts were added to equal amounts of the recombinant proteins in the in vitro cell-free degradation assays. The Coomassie blue–stained ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) was used as a loading control. (B) Quantification analysis of (A). The relative levels of OsJAZ4 in the wild-type NIP plant protein extracts at 0 h were defined as 1. Data are means ± se (n = 3). (C) Time course of degradation of His-OsJAZ4 and His-OsJAZ4Δ8 in the wild-type NIP protein extracts with or without MG132. Equal amounts of the recombinant proteins were incubated with equal amounts of plant crude extracts in the in vitro cell-free degradation assays. (D) Quantification analysis of (C). The relative levels of His-OsJAZ4 or His-OsJAZ4Δ8 incubated with wild-type NIP plant protein extracts at 0 h were defined as 1. Data are means ± se (n = 3). (E) Protein levels of OsJAZ4 in Go, Gi, and Zh11 leaves. The OsJAZ4 protein was detected with anti-OsJAZ4 antibody and Rbc L was used as a loading control. Two independent pools of leaves are shown. (F) Quantification analysis of (E). The relative level of OsJAZ4 in the wild-type ZH11 was set as 1. Data are means ± se (n = 3).

Figure 5.

OsGSK2 Affects OsJAZ4-OsJAZ11 Interaction. (A) Y2H assay shows that the ZIM domain of OsJAZ4 is responsible for binding to OsGSK2. Schematic diagrams show the truncated versions of OsJAZ4. Interactions were examined with SD base without Ade, His, Leu, and Trp. aa, amino acids; NT, N-terminal domain. (B) Y2H assay shows the interaction between OsJAZ4 and OsJAZ1-15 proteins. Transformed yeast cells were grown on SD-Ade-His-Trp-Leu medium. (C) In vitro interaction between OsJAZ4-myc and His-JAZ11 is weakened by GST-OsGSK2. His-OsJAZ11 protein combined with GST-OsGSK2 was incubated with immobilized OsJAZ4-myc. The immunoprecipitated fractions were detected by anti-His antibody. The gradient indicates increasing amount of GST-OsGSK2. OsJAZ4-myc input was probed with anti-myc antibody, and the loading of His-OsJAZ11 and GST-OsGSK2 is shown in the lower panel by Coomassie blue (CBB) staining. (D) HA-OsGSK2 affects accumulation of OsJAZ4-myc and OsJAZ11-myc. The respective vectors were cotransiently expressed in the N. benthamiana leaves. The infiltrated leaves were collected at 48 h after infiltration, and at least four plants were pooled. The Coomassie blue–stained ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) was used as a loading control. (E) The protein levels of OsJAZ11 in Go, Gi, and Zh11 plants. The OsJAZ11 protein was detected with anti-OsJAZ11 antibody and Rbc L was used as a loading control. Two independent pools of leaves are shown.

Figure 6.

Effect of OsGSK2 on the OsJAZ4-OsNINJA Interaction. (A) OsNINJA and OsCOI1b interact with OsJAZ4. OsNINJA and OsCOI1b were fused to the GAL4 DNA BD, while OsJAZ4 and its mutants were fused to the GAL4 AD, respectively. Interactions were examined using SD base without Ade, His, Leu, and Trp. For the interactions between OsJAZ4 and OsCOI1b, 25 μM coronatine (COR) was added. (B) OsGSK2 competes with OsNINJA for binding to OsJAZ4. In vitro interaction between OsJAZ4-myc and His-OsNINJA is weakened by GST-OsGSK2. His-OsNINJA protein combined with GST-OsGSK2 was incubated with immobilized OsJAZ4-myc. The immunoprecipitated fractions were detected by anti-His antibody. The gradient indicates increasing amounts of GST-OsGSK2. OsJAZ4-myc input was probed with anti-myc antibody, and the loading of His-OsNINJA and GST-OsGSK2 is shown bottom by Coomassie blue (CBB) staining.

Figure 7.

OsJAZ4 Negatively Modulates JA Signaling and Rice Immunity. (A) JA-responsive gene expression in indicated transgenic plants. RT-qPCR analysis of the mRNA levels of JA-responsive genes in the wild-type NIP, OsJAZ4-OE lines (nos. 1 and 3), and OsJAZ4-RNAi lines (nos. 14 and 18). OsUBQ5 was used as the internal reference gene. Values are means ± se of three biological replicates. Asterisk (*) indicates significant difference at P ≤ 0.05 (n = 3) by Fisher’s LSD test. (B) and (C) Images (B) and quantification of root length (C) in indicated plants following MeJA treatment. The root lengths of 3-d-old seedlings grown in normal rice culture solutions supplemented with different concentrations of MeJA were measured. Data shown are the means from at least 15 seedlings for each plant type. Error bars represent sd. Different letters at the top of columns indicate significant difference at P ≤ 0.05 by Fisher’s LSD test. Bar = 2 cm. (D) Disease incidence. The numbers of healthy and diseased plants in each treatment were determined by RT-PCR 30 d after inoculation, and the number of the diseased plants was used to calculate the viral incidence (percentage of plants infected). Each treatment used at least 40 seedlings, and at least three biological replicates were performed. Different letters at the top of columns indicate significant difference between transgenic and control plants at P ≤ 0.05 by Fisher’s LSD test. (E) Relative expression levels of RBSDV CP gene measured by RT-qPCR at 30 dpi. Data represent relative expression levels of the CP gene in the mutant compared with that in the wild-type NIP plants. OsUBQ5 was used as the internal reference gene. Error bars represent sd. Asterisk (*) indicates significant difference at P ≤ 0.05 (n ≥ 3) by Fisher’s LSD test. (F) Viral symptoms in OsJAZ4-OE lines (nos. 1 and 3) and OsJAZ4-RNAi lines (nos. 14 and 18). Bar = 10 cm.

Figure 8.

Effect of OsGSK2-OsJAZ4 Interaction on JA and BR Pathway Crosstalk. (A) and (B) OsJAZ4 accumulation increases in response to BL (A) or Bikinin (B) treatment. The leaves of the 7-d-old wild-type NIP seedlings were treated with 1 μM BL or 20 μM Bikinin, and protein was extracted from the treated leaves 0, 3, 6, or 12 h later. The Coomassie blue (CBB)–stained ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) was used as a loading control. Two independent pools of leaves are shown. (C) and (D) Accumulation of OsJAZ4 induced by BL and Bikinin was inhibited in coi1-13 mutants. Leaves of the 7-d-old wild-type NIP and coi1-13 seedlings were treated with 1 μM BL (C) or 20 μM Bikinin (D), and the treated leaves were used for protein extraction at different times after treatment, and detected by anti-OsJAZ4 antibody. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) was used as a loading control. Two independent pools of leaves are shown. (E) Effect of Bikinin on MeJA hypersensitivity in OsJAZ4-RNAi plants. Germinated seeds were grown in normal rice culture solutions containing 0 or 1 μM MeJA, with or without 200 μM Bikinin for 5 d, and the root length was then measured. Relative root elongation is expressed as a percentage of root elongation in solutions with (right section) or without (left section) 200 μM Bikinin. Error bars represent se (n ≥ 20). Different letters at the top of columns indicate significant difference at P ≤ 0.05 by Fisher’s LSD test. (F) BL sensitivity test of the wild-type NIP, OsJAZ4-OE lines (nos. 1 and 3), and OsJAZ4-RNAi lines (nos. 14 and 18) by lamina joint assay. The plus and minus symbols indicate with and without BL (100 ng), respectively. (G) Quantification of the data shown in (E). Data shown are the means from at least 15 seedlings for each indicated plant. Error bars represent se. Different letters at the top of columns indicates significant difference at P ≤ 0.05 by Fisher’s LSD test.

Figure 9.

Model of OsGSK2-Mediated Plant Defense Signaling in Rice. OsGSK2 binds to OsJAZ4 to disrupt OsJAZ4-OsNINJA corepressor and OsJAZ4-OsJAZ11 dimerization, which promotes the degradation of phosphorylated OsJAZ4 and free OsJAZ11 by the 26S proteasome in an OsCOI1-dependent manner. The increased amount of OsGSK2 elevates the JA response but suppresses the BR response, thereby enhancing rice antiviral defense. Lines ending with arrows show activation; a solid line ending with a perpendicular line indicates suppression or an antagonistic interaction.