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UBC Theses and Dissertations

Characterization and functional analysis of two redundant MAPKKKs in plant immunity Zhang, Qian


Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules that transduce and amplify signals from upstream receptors in eukaryotes. In Arabidopsis, two MAP kinase cascades are activated upon treatment of flg22, a conserved peptide of 22 amino acids within the N terminus of bacteria flagellin. One cascade is composed of MEKK1-MKK1/2-MPK4. The other one is composed of MKK4/5-MPK3/6 and previously unknown MAPKKK(s). How signals are transduced to MAP kinase cascades was also not very clear. My Ph.D. research focuses on identification of the previously unknown MAPKKK(s) upstream of MKK4/MKK5-MPK3/MPK6 module and characterization of this MAP kinase cascade in plant immunity. It is known that YDA-MKK4/5-MPK3/6 cascade regulates stomatal development. I hypothesized that the close homologues of YDA, MAPKKK3 and MAPKKK5, function upstream of MKK4/MKK5-MPK3/MPK6 to regulate plant immunity. The mapkkk3 mapkkk5 double mutant was found to have significantly reduced MPK3 and MPK6 activation upon multiple elicitor treatment including flg22, suggesting that MAPKKK3 and MAPKKK5 are required for multiple elicitor-induced MPK3/MPK6 activation. The double mutant also shows enhanced susceptibility towards virulent pathogens, reduced cell death and enhanced susceptibility towards avirulent pathogens, suggesting that these two MAPKKKs are required for pathogen resistance. Using E.coli expressed proteins, MAPKKK3-MKK5-MPK6 cascade was reconstituted in vitro, biochemically confirming that MAPKKK3 is upstream of MKK5 and MPK6. Kinase assays using different mutant versions of MAPKKK3 protein show that the kinase domain and C terminal domain but not the N terminal regulatory domain of MAPKKK3 is required for signaling. Previously, PCRK1 (Pattern-triggered immunity Compromised Receptor-like cytoplasmic Kinase 1) and PCRK2 were shown to interact with flg22 receptor and the pcrk1 pcrk2 double mutant shows modestly reduced flg22-induced MPK3/MPK6 activation. Co-Immunoprecipitation and biotinylation assays using transient expressed proteins in Nicotiana benthaminana showed that MAPKKK3 and MAPKKK5 interact with PCRK2, suggesting PCRK2 may transduce signal from flg22 receptor to MAPKKK3/MPKKK5. Altogether, studies in this dissertation identified two MAPKKKs functioning upstream of MKK4/5-MPK3/6, characterized the roles of this MAP kinase cascade in immunity and provided insight on signal transduction from the flg22 receptor to this MAP kinase cascade.

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