| Mitogen-activated protein kinase (MAPK) signal transduction cascades are conserved in eukaryotes, including Arabidopsis thaliana (Arabidopsis). Loss-of-function mutations in genes encoding MAPK, MAPK kinase, and MAPK kinase kinase (MAP3K) proteins often do not reveal abnormal mutant phenotypes in Arabidopsis, potentially due to functional redundancy between members of these multi-gene families. I have used several reverse genetic strategies to attempt to overcome this redundancy. First, I isolated plants carrying genes encoding dominant negative versions of nine MAPKs. The dominant negative mutation alters the wild-type TEY or TDY activation loop to AEF or ADF, and prevents the MAPK from being phosphorylated and activated by upstream MAPK kinases. Plants carrying MPK3AEF or MPK6AEF have abnormal development including clustered stomata, a phenotype not present in mpk3 or mpk6 null mutant plants, indicating the utility of the dominant negative strategy to reveal instructive phenotypes. Second, I identified plants over-expressing the MAP3K genes MEKK1, MEKK2, and MEKK3, which display dwarfed phenotypes with increased salicylic acid signaling similar to the phenotype of loss-of-function mekk1 mutant plants. These results are consistent with the MEKK proteins acting as positive regulators of defense signaling, in contrast to the current model that MEKK1 acts to negatively regulate defense. Finally, I developed iTILLING as a novel high-throughput reverse genetic method to screen for mutations in specific genes of interest. iTILLING, a modification of traditional TILLING, where mutagenized seeds, collected in bulk, are screened in a 96-well format. Seedlings are screened for mutations using high-resolution melting of gene-specific PCR amplicons. I utilized iTILLING to identify point mutations in MEKK2 and MEKK3, closely linked to a T-DNA insertion in MEKK1. Observation of the phenotypes of mekk1; mekk2 and mekk1; mekk3 double mutant plants revealed that the mutations in MEKK2 and MEKK3 did not alter the dwarfed mekk1 mutant phenotype. These strategies of dominant negative proteins, over-expression of genes. and creation of plants carrying multiple mutations have provided a means to begin to overcome functional redundancy within the MAPK gene families in Arabidopsis. This thesis details my use of these reverse genetic approaches to interrogate the function of members of the MAPK and MAP3K gene families. |
