Mitogen-activated protein kinase (MAPK) signaling networks represent important means of signal transduction in plants and other eukaryotes, controlling intracellular signaling by linking perception of environmental or developmental cues to downstream targets. In the Arabidopsis MEKK subfamily, the MKKK19, 20, and 21 form a highly supported clade with the Solanaceous Fertilization-Related Kinases. In Arabidopsis, little is known about this group, except for MKKK20, which is involved in osmotic stress. Using a directed MKKK-MKK yeast two-hybrid (Y2H) screen, MKKK20 was found to interact only with MKK3, while a MKKK20 large-scale Y2H screen retrieved MPK18 as a direct interactant. In vitro phosphorylation assays showed that MKKK20 phosphorylates both MKK3 and MPK18. However, when all three kinases are combined, no synergistic effect is observed on MPK18 phosphorylation, suggesting a direct access to MPK18, consistent with the absence of interaction between MKK3 and MPK18 in protein–protein interaction assays. Since mpk18 mutant plants were previously shown to be defective in microtubule-related functions, phenotypes of mkkk20 single and mkkk20/mpk18 double mutants were investigated to determine if MKKK20 acts upstream of MPK18. This was the case, as mkkk20 root length was shorter than WT in media containing microtubule-disrupting drugs as previously observed for mpk18 plants. Surprisingly, mkk3 plants were also similarly affected, suggesting the presence of two non-complementary pathways involved in Arabidopsis cortical microtubule function, the first including MKKK20, MKK3 and an unknown MPK; the second, a non-canonical MAPK cascade made of MKKK20 and MPK18 that bypasses the need for an MKK intermediate.
Mitogen-activated protein kinase (MAPKs) constitute a major component in plant cellular signaling considering the sheer number of MAPKKKKs, MAPKKKs, MAPKKs and MAPKs when compared to yeast and animal systems. Nevertheless, only few complete MAPK cascades have been deciphered and the same hold true for their substrates. Furthermore, cascades often share kinase components, but little is known about their specific interactions and domains. The Arabidopsis thaliana MAP kinase kinase kinase 20 (MKKK20) was recently showed to interact with MKK3 and MPK18 in two non-complementary signaling cascades involved in root cortical microtubule functions. Here, MKKK20 and MKK3 proteins where dissected and tested in yeast two-hybrid assays followed by an in planta validation through bimolecular fluorescence complementation (BiFC) assays and showed that the MKKK20 C-terminal region interacted with MKK3 that comprised a typical DEF domain akin to MAPKs docking domains.
Mitogen-activated protein kinase (MAPK) cascades are critical signal transduction modules in stress responses, but how their composition and mode of activation induces a stress response is poorly understood. We showed in Arabidopsis that CRK21, a cysteine-rich receptor-like protein kinase (CRK), phosphorylates MAPK kinase kinase 20 (MKKK20) and thus directly activates a novel MAPK cascade, consisting of MKKK20, the MAPK kinase MKK3, and the MAPK MPK6. Furthermore, the protein phosphatase PP2C76 and the calmodulin CaM7 were identified as negative and positive modulators of the cascade, respectively. Loss-of-function in components of the MAPK cascade or in CaM7 led to susceptibility to the bacterial pathogen Pseudomonas syringae and the fungal pathogen Botrytis cinerea. In contrast, loss-of-function of PP2C76 as well as transient overexpression of the genes in the MAPK cascade and CaM7 conferred resistance to the pathogens. Moreover, seven additional CRKs interacted with MKKK20 in vivo, and four of these were highly expressed after inoculation with P. syringae. In summary, our findings demonstrate that the novel CRK21-MKKK20-MKK3-MPK6 signaling pathway functions in immunity to fungal and bacterial pathogens and that CRKs may function in directly activating MKKKs.
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