OsGSK2 Integrates Jasmonic Acid and Brassinosteroid Signaling in Rice

Plant defenses against herbivore or pathogen attack involve the coordination of multiple hormone-mediated signaling networks, including the jasmonate (JA) and brassinosteroid (BR) pathways. JA is an oxylipin phytohormone that triggers the transcription of defense-related proteins and secondary metabolites. In general, JA-responsive genes are controlled by MYC family transcription factors, which are themselves repressed when bound by JASMONATE ZIM-DOMAIN (JAZ) proteins (Major et al., 2017). BRs are steroid hormones that primarily regulate plant growth. They function mainly by interacting with BR-specific membrane-bound receptors in the BRASSINOSTEROID INSENSITIVE1 family. This initiates an intracellular phosphorylation cascade that activates transcription factors that regulate BR-responsive genes (Nolan et al., 2020). There is considerable crosstalk between JA and BR signaling, but despite recent improvements in the elucidation of these two pathways, which proteins are involved remains unclear.

In this issue of The Plant Cell, He et al. (2020) established a link between JA and BR signaling in rice by performing a functional characterization of OsGSK2, a Glycogen Synthase Kinase3 (GSK3)-like kinase found in rice (Oryza sativa). GSK-like kinases are versatile, constitutively active enzymes found in many eukaryotes. They participate in signal transduction by phosphorylating substrates that regulate the transcription or repression of downstream targets. In plants, GSK3s have long been known to participate in BR signaling, but recent functional studies have identified their involvement in other pathways (Youn and Kim, 2015). OsGSK2 is the rice homolog of Arabidopsis (Arabidopsis thaliana) BRASSINOSTEROID INSENSITIVE2, which plays a key role in BR signaling (Tong et al., 2012).

He et al. (2020) first examined OsGSK2 expression (see figure). To do so, they compared the OsGSK2 levels of Go rice plants (which overexpress OsGSK2) and Gi plants (where OsGSK2 is knocked down by RNAi) after plants were inoculated with Rice black-streaked dwarf virus (RBSDV). Thirty days after inoculation with RBSDV, the authors found that OsGSK2 levels had increased in wild-type plants and the symptoms of RBSDV infection were less severe in Go plants than in Gi plants. Crucially, the authors found that the expression of genes encoding proteins involved in JA biosynthesis and signaling was higher in Go than in Gi or control plants, and this was confirmed by directly measuring JA levels. Next, they used OsGSK2 as bait in yeast two-hybrid assays with a prey library sourced from a rice cDNA library and identified OsJAZ4, a member of the rice JAZ protein family, as a potential interactor of OsGSK2. Localization experiments in Nicotiana benthamiana epidermal cells revealed that GFP-labeled OsGSK2 and mCherry-labeled OsJAZ4 both colocalized in the cytoplasm and nucleus. Moreover, coimmunoprecipitation assays revealed that tagged OsGSK2 specifically coprecipitated with tagged OsJAZ4 but not OsJAZ11.

Overexpression of OsGSK2 Is Associated with Increased Antiviral Defense and Higher Endogenous JA Content.

(A) RBSDV symptoms in mock and infected Go (OsGSK2 overexpression), Zh11 (control), and Gi (OsGSK2 RNAi) rice plants.

(B) Endogenous JA content in 7-d-old Go, Zh11, and Gi plants. FW, fresh weight.

(Adapted from He et al. [2020], Figure 1.)

The authors then examined how OsGSK2 interacts with OsJAZ4. They found that OsJAZ4 proteins are phosphorylated in vivo and identified eight specific Ser sites where this may occur. They used a mutated protein, OsJAZ4Δ8, that contained alterations in each of these sites to test whether phosphorylation took place preferentially at these sites. Yeast two-hybrid assays showed that OsGSK2 was able to interact with OsJAZ4Δ8, but myc-bead immunoprecipitation revealed differences in phosphorylation between OsJAZ4 and OsJAZ4Δ8. The authors concluded that OsGSK2 directly phosphorylates OsJAZ4, primarily at the eight Ser sites. Next, the authors examined OsGSK2’s functional role by examining whether it affects the stability of OsJAZ4. They found that OsGSK2 appears to selectively phosphorylate OsJAZ4, prompting its degradation. The authors also established that OsGSK2 can prevent OsJAZ4-OsJAZ11 dimerization as well as the formation of a complex between OsJAZ4 and a protein called OsNINJA (NOVEL INTERACTOR OF JAZ) that negatively regulates JA signaling. The authors then used OsJAZ4 overexpression and RNAi transgenic plants to determine the specific function of OsJAZ4. They found that 30 d after RBSDV infection, OsJAZ4 expression was lower in infected than in uninfected control plants and that plants overexpressing OsJAZ4 were more susceptible to attack from RBSDV. They also found that OsJAZ4 expression was associated with a suppression of JA-responsive genes.

Taken together, these results show that OsGSK2 plays a role in both JA- and BR-mediated antiviral defense. By prompting the degradation of OsJAZ4, OsGSK2 positively regulates JA signaling, thus boosting plant resistance to viral attack. At the same time, OsGSK2 has also been found to downregulate BR signaling, thereby negatively regulating plant growth (Tong et al., 2012). Previous work has identified antagonistic effects of JA-mediated RBSDV resistance and BR-mediated RBSDV susceptibility (He et al., 2017). Thus, OsGSK2 is implicated in both the JA and BR signaling pathways and is a major factor coordinating antiviral immunity in rice.