| The Mitogen-activated protein kinases are highly conserved in all eukaryotic organisms. There are 10 genes encoding MAP Kinase Kinase Kinase Kinases (MAP4K), ca. 80 genes encoding MAP Kinase Kinase Kinases (MAP3K), 10 genes encoding MAP Kinase Kinases (MAP2K), and 20 genes encoding MAP Kinases (MAPK) in Arabidospsis. However, the biological roles have only been identified for a subset of these kinases due to the lack of mutant phenotypes observed when individual genes from these families are inactivated via mutagenesis. In my project, I focused on studying genetic interactions between pairs of mutant loci affecting these MAPK pathway genes as a strategy for uncovering functional associations that are not apparent from studying single-mutants. First, I performed a pilot genetic interaction experiment using two well-known MAPK pathway single-mutants, mekk1 (a MAP3K) and mpk4 (a MAPK). These two genes have been previously suggested to function in a pathway that serves to negatively regulated defense responses. Detailed phenotypic analysis of the single-mutants revealed that these two mutants respond differently to NaCl treatment and their lateral root development. The mekk1;mpk4 double-mutant caused synthetic lethality. These results suggested that the functions of MEKK1 and MPK4 are not limited to a single, linear pathway, and indicated that genetic interaction analysis has the potential to highlight new functions for genes. Based on these initial findings, I expanded my study by performing a large-scale genetic interaction analysis of MAPK pathway related genes. To facilitate this project I developed a high-throughput double-mutant generating pipeline based on a system for growing Arabidopsis seedlings in 96-well plates for genotyping. A quantitative root growth-rate assay as well as qualitative phenotyping were used to screen for evidence of genetic interactions in the 289 double-mutants that I produced. In this screen, I identified seven novel genetic interactions. In particular, I found five mutant loci that caused synthetic enhancement of the root growth defects in mpk4 single-mutant. I also found two mutants that rescue the mpk4 dwarfism. My results have provided new insights into the functional roles of a number of MAPK pathway genes in Arabidopsis and also demonstrate the value of systematic genetic interaction analysis. |
