Authors, Siodmak et al., conducted a study on Arabidopsis MPK4 to investigate the function of the common docking motif (CD) domain in MAPKs, which are signaling factors found in all eukaryotic organisms. They examined the interactions of MPK4 and analyzed its crystal structure when bound to ligands. To generate complementation and mutant lines in Arabidopsis ecotype Col-0, the mpk4-2 mutant (SALK_056245) was grown in soil under long-day conditions. Heterozygous plants were transformed and selected for three generations to obtain stable homozygous lines. Multiple lines were prepared for each mutant and complementation, except for C181D, which had two independent lines. Fresh weight, dry weight, and root length measurements were performed on plants grown on ½-strength Murashige & Skoog medium (½MS) at specific time points. Seedlings were treated with mock or flg22 after being grown vertically on ½MS solid medium. Wild-type Arabidopsis thaliana ecotype Columbia-0 (Col-0) plants served as controls.
Studying MPK4-2 Mutantation
Gene synthesis and cloning were performed using primers designed based on specific criteria. The mpk4-2 mutant in the Columbia-0 background, which had a T-DNA insert in the MPK4 gene, was genotyped using gene-specific primers. Genomic DNA was extracted from plant leaves, and PCR was carried out ; and successful amplicons were sequenced. The purified gene amplicons were then digested with specific restriction enzymes and ligated into the pGreen229-PC2 vector for protein expression. Moreover, transgenic lines were created by introducing the pGreen229-PC2 vector, controlled by the MPK4 native promoter, into the mpk4-2 mutant background. Complementation of the mpk4-2 mutants was accomplished by inserting either the wild-type MPK4 gene or MPK4 genes with mutations at C181S or C181D. Agrobacterium tumefaciens C58C1 was used for stable transformation via the floral dip method on flowering Arabidopsis plants. Positive transformation events were identified by applying a BASTA solution. The transformation rate was determined by comparing the number of non-resistant plants to the total number of plants. Differential-contrast observation of Arabidopsis ovules involved emasculating the oldest closed flower bud, followed by clearing the entire flower in a solution. The samples were then rehydrated, and the pistil was separated and mounted on an object slide. Visikol® for Plant Biology™ reagent was used for this purpose.
In pathogen assays, Pseudomonas syringae pv tomato-DC3000 (Pst DC3000) was grown on LB agar plates and sprayed onto Arabidopsis plants. Bacterial titers were estimated by counting colonies after plating leaf extracts. Methyl viologen-induced oxidative stress was introduced to plants, and root length, density, and fresh weight were measured. Protein extraction and Western blots were performed to analyze protein expression. Recombinant proteins were cloned, expressed, and purified. In vitro kinase assays and phosphosite identification were conducted. Isothermal titration calorimetry (ITC) was used to study protein interactions. Protein crystallization and structure determination were performed, and protein modeling was conducted.
Overall, Siodmak et al. highlight and shed light on the function of plant MAPKs, which relies on their phosphorylation status, particularly the TEY phosphorylation in their activation loop. The interaction between MAPKs and their upstream MAPKKs is crucial for MAPK activation, and crystallographic data of the MPK4-MKK1 complex highlights the importance of the MAPK CD motif in this interaction. Post-translational modifications, such as acetylation and phosphorylation, can affect MAPK activity. MAPKs also play a role in oxidative signaling and stress responses in plants. The MPK4-C181S protein does not show reduced kinase activity compared to the wild-type, suggesting that sulfenylation of C181 may not be essential for its regulation. Persulfidation of MAPKs can occur and have similar effects to sulfenylation. MPK4-C181D, which hinders interaction with its activating MAPKK, is constitutively inactive. An inactive MPK4 protein cannot fully substitute for a knockout mutant, emphasizing the importance of an activatable MPK4 for development and stress signaling.
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