Arabidopsis Mitogen-Activated Protein Kinase Kinase Kinase Family Members and Their Possible Role in Innate Immunity

Abstract

From an early developmental stage, plants are exposed to both beneficial and pathogenic microbes. Upon pathogen detection, receptor complexes on the cell surface can detect conserved microbial molecules, such as bacterial flagellin, to elicit an immune response. Several early immune outputs are triggered by receptor complex activation, such as the activation of MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascades and the production of extracellular reactive oxygen species, which ultimately confer broad-spectrum immunity. While this response is necessary for survival, it is energetically costly and can result in developmental trade-offs. The CALCIUM-DEPENDENT PROTEIN KINASE (CPK28) negatively regulates immune output downstream of multiple receptor complexes by promoting turnover of a key positive regulator of immunity. To identify novel positive and regulators of the plant immune response, putative CPK28-associated proteins were previously identified through affinity purification of wild-type and kinase-dead CPK28 from stable Arabidopsis thaliana lines both before and after immune elicitation, followed by liquid chromatography and tandem mass spectrometry. A putative MAPK KINASE KINASE (MAPKKK), MLK/RAF-RELATED PROTEIN KINASE 1 (MRK1), and two protein phosphatase regulatory subunits, PROTEIN PHOSPHATASE 2A (PP2A)-Bα and PP2A-Bβ, were identified in this screen as potential CPK28-associated proteins. The primary objective of my thesis was to examine the role of MRK1, MRK1-related family members, and the PP2A-B subunits in the plant innate immune response. Through functional characterization of double null lines, I found that MRK1 and two closely related proteins, Raf26 and Raf39, may negatively regulate immune-induced oxidative burst in a partially redundant manner. I also observed that MRK1 localizes to the cytosol in Arabidopsis, and present preliminary findings which suggest that MRK1 may play a role in normal rosette development. Taken together, this work suggests that MRK1 and related protein kinases may be acting as novel regulators of immunity, and primes future analysis on this previously unstudied family.