The roles of the small pMEKK subfamily comprising MAPKKK19, 20 and 21 in Arabidopsis thaliana

Mitogen-activated protein kinase (MAPK) signaling cascades participate in a wide range of biological activities in eukaryotes. Among them, variety of combinations of the “three-tiered modules” and related regulators are the key factors to ensure the diversity and accuracy of the signal transduction. To date, a great variety of different MAPK cascades involved in biotic and abiotic stresses have been discovered in plants.   Here, a complete MAPK cascade (MKKK20-MKK3-MPK6) that functions downstream of CRK21, a cysteine-rich receptor-like protein kinase (CRK) has been found in Arabidopsis. Furthermore, a number of interesting MKKK20-interacting regulator candidates, including a protein phosphatase 2C (PP2C) called MFH8.8; a calmodulin-like protein (CML10), and five calmodulins (CaM1, 4, 6, 7, and 9) have been identified by Y2H. The MFH8.8 (PP2C), and CaM7 were also identified as a negative and positive regulator of the MKKK20-cascade, respectively. Mutations in this MAPK cascade, as well as CaM7, leads to susceptibility to both bacterial and fungal pathogens (Pseudomonas syringae pv. tomato DC3000 (EV) [Pst3000] and Botrytis cinerea strain B191), while mutation of MFH8.8 (PP2C) confers resistance to the pathogens, indicating that CRK21-MKKK20-MKK3-MPK6 signaling pathway has an important role in plant immune responses under the regulation of MFH8.8 (PP2C) and CaM7. Moreover, another seven CRKs (CRK12, 14, 16, 18, 24, 30, and 33) sharing a kinase domain very similar to CRK21 are all interacting with MKKK20 in Y2H assays and regulate the phosphorylation status of MKKK20. Importantly, RT-PCR results showed that CRK12, 14, 18, 21, and 24 are highly expressed after pathogen (Pst3000) inoculation, but weakly (CRK14, 18, 21, and 24) or not expressed under normal conditions, suggesting that CRKs are a large upstream family of MKKK20 playing important roles in plant disease resistance.  MKKK19, 20, and 21 form a single clade in the Arabidopsis MEKK family. Multiple sequence alignments and protein structure predictions showed that these three MKKKs are very similar in amino acid sequence, secondary, and even tertiary structure. To dissect the relationships among MKKK19, 20, and 21, all MKKK20-interacting CRKs, MKK3, as well as the candidate regulators have been tested with MKKK19 and 21 in a Y2H system separately. The results indicated that these three MKKKs share some interaction partners, including upstream (CRK12, CRK21, CRK33) and downstream (MKK3) elements. MKKK19 and 20 also share CRK18, 24, and 30, as well as several candidate regulators including the PP2C MFH8.8 phosphatase, CML10, CaM4, 6, and 9. The results of kinase assays indicated that CRK14, 24, and MKKK19 are autophosphorylated proteins, and that the autophosphorylation of MKKK19 is inhibited by CRK12, 16, 18, 24, and 30. These results revealed that CRKs are also important in MKKK19 mediated signaling pathways, and MKKK19 and 20 may function in some similar biological activities under the control of the same regulators. Interestingly, MKKK19 and 21 are both phosphorylated by CRK21-MKKK20 in vitro, suggesting that MKKK19 and 21 can work at the same hierarchical level with MKKK20 in the cascade as well as acting downstream of MKKK20 in the signaling pathways.   Finally, the MKKK20 binding domain that anchors MKK3 was characterised through an in-depth protein-protein Y2H screen and BiFC, a first for a MAP kinase kinase kinase in plants. This C-terminal domain comprises two short subdomains with high percentage identity between the MKKK19, 20, and 21, but not with the other 18 Arabidopsis MEKKs, suggesting that this C-terminal is essential for the binding between MKKK20 and MKK3.  Taken together, this project uncovered a complete MAPK cascade signaling pathway involved in immune response, and identified numbers of upstream elements as well as regulators of MKKK19, 20, and 21. during the analyzing of the relationships of MKKK19, 20, and 21, that belong to a single clade in the Arabidopsis MEKK. Lastly, MKKK20 and MKK3 were finely dissected and used in Y2H assays to uncover potential docking domains interacting between these two kinases. Surprisingly, a short MKKK20 C-terminal sequence not only interacted with MKK3 but also harbored docking domain sites similar to MAPK substrates. Our results shed new light on the remarkably diversified working modes of MAPK signaling cascades and in the role of receptor kinases and MEKKs in plant disease resistance.

Keyphrases: CRK, MAPK, Cascade kinase, CaM, Phosphatase, Régulation, Arabidopsis, Kinase cascade, Immune response, Regulation, Binding domain