Epitope-tagging MAP kinases in Arabidopsis

Mitogen-activated protein (MAP) kinases regulate a wide variety of processes in all eukaryotes, from abiotic and biotic stress responses to development. The complex nature of the processes regulated by these kinases is reflected in the complexity of the gene families that make up the members of the MAP kinase signaling cascades We are presently characterizing T-DNA knock-outs of various members of the Arabidopsis MAP kinase family as part of an FWF-funded project. With 20 members it is a large family, and therefore distinguishing between the functions of the different members poses a problem. In the present proposal, we aim to address this in the first instance using epitope tagging of the MAP kinases. In a novel approach, each T-DNA knock-out line will be transformed with its respective gene, including its own promoter, that has been tagged with an epitope. Such an approach offers a number of advantages. The expression from its own promoter ensures the correct temporal and spatial expression of the gene, maintaining stoichiometry. There is no competition between the endogenous protein - that is eliminated by the knock-out - and the tagged protein, so the tagged protein will be optimal for localization studies, biochemical assays, and for use in protein-protein interaction studies. Commercially available high-quality antibodies against the tagged proteins avoid the problem of generating new antibodies, which may not always be successful, and different tags can distinguish between even highly related proteins. At the same time, transformation of T-DNA knock-outs with the proposed constructs serves as a complementation assay of any aberrant phenotype associated with the knock-out, Further, a novel approach for addressing the problem of functional redundancy between the different MAP kinases in Arabidopsis is presented based on this methodology.

Mitogen-activated protein (MAP) kinases regulate a wide variety of processes in all eukaryotes, from abiotic and biotic stress responses to development. The complex nature of the processes regulated by these kinases is reflected in the complexity of the gene families that make up the members of the MAP kinase signaling cascades We are presently characterizing T-DNA knock-outs of various members of the Arabidopsis MAP kinase family as part of an FWF-funded project. With 20 members it is a large family, and therefore distinguishing between the functions of the different members poses a problem. In the present proposal, we aim to address this in the first instance using epitope tagging of the MAP kinases. In a novel approach, each T-DNA knock-out line will be transformed with its respective gene, including its own promoter, that has been tagged with an epitope. Such an approach offers a number of advantages. The expression from its own promoter ensures the correct temporal and spatial expression of the gene, maintaining stoichiometry. There is no competition between the endogenous protein - that is eliminated by the knock-out - and the tagged protein, so the tagged protein will be optimal for localization studies, biochemical assays, and for use in protein-protein interaction studies. Commercially available high-quality antibodies against the tagged proteins avoid the problem of generating new antibodies, which may not always be successful, and different tags can distinguish between even highly related proteins. At the same time, transformation of T-DNA knock-outs with the proposed constructs serves as a complementation assay of any aberrant phenotype associated with the knock-out, Further, a novel approach for addressing the problem of functional redundancy between the different MAP kinases in Arabidopsis is presented based on this methodology.