Constitutively Active Mitogen-Activated Protein Kinase Versions Reveal Functions of Arabidopsis MPK4 in Pathogen Defense Signaling

Abstract

Plant mitogen-activated protein kinases (MAPKs) are involved in important processes, including stress signaling and development. In a functional yeast screen, we identified mutations that render Arabidopsis thaliana MAPKs constitutively active (CA). Importantly, CA-MAPKs maintain their specificity toward known activators and substrates. As a proof-ofconcept, Arabidopsis MAPK4 (MPK4) function in plant immunity was investigated. In agreement with the phenotype of mpk4 mutants, CA-MPK4 plants were compromised in pathogen-induced salicylic acid accumulation and disease resistance. MPK4 activity was found to negatively regulate pathogen-associated molecular pattern-induced reactive oxygen species production but had no impact on callose deposition, indicating that CA-MPK4 allows discriminating between processes regulated by MPK4 activity from processes indirectly affected by mpk4 mutation. Finally, MPK4 activity was also found to compromise effector-triggered immunity conditioned by the Toll Interleukin-1 Receptor-nucleotide binding (NB)-Leu-rich repeat (LRR) receptors RPS4 and RPP4 but not by the coiled coil-NB-LRR receptors RPM1 and RPS2. Overall, these data reveal important insights on how MPK4 regulates plant defenses and establishes that CA-MAPKs offer a powerful tool to analyze the function of plant MAPK pathways.

MYB75 Phosphorylation by MPK4 Is Required for Light-Induced Anthocyanin Accumulation in Arabidopsis

“…CA-MPK4 was able to phosphorylate the standard MAP kinase substrate myelin basic protein, which confirms the previous report that CA-MPK4 is a constitutively active form of MPK4 (Berriri et al, 2012). MYB75 protein was also strongly phosphorylated by CA-MPK4 ( Figure 5A).…”

“…This makes it difficult to identify MAPK substrates by genome wide proteomic or yeast two-hybrid approaches (Feilner et al, 2005). To circumvent this difficulty, we exploited a recently described, constitutively active MPK4 form (CA-MPK4), which results from two mutations (D198G/E202A) and retains its substrate specificity and physiological functions (Berriri et al, 2012). In control Y2H assays, when a BD-CA-MPK4 bait construct in plasmid pGBKT7 was cotransformed with the empty pGADT7 prey vector, expression of selectable markers was not activated ( Figure 1A).…”

The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-Related Protein Kinases Are Required for Elicitor-Induced Oxidative Burst and Immunity

“…From our study, ANPs clearly emerge as key positive regulatory elements for response to both PAMPs and DAMPs and for PTI, rather than as mere repressors of defense responses, as initially hypothesized on the basis of the phenotypic similarities with the mpk4 mutant (Berriri et al, 2012). The action of MPK4 is placed downstream of MEKK1 in one branch of the MAPK cascade that is activated in response to flagellin, and accordingly, loss-of-function mutants in these kinases display similar PTI-related defects (Ichimura et al, 2006;Suarez-Rodriguez et al, 2007).…”

“…Three immunerelated MAPK modules have been identified. A module comprising the MAP3K MEKK1, the MAP2Ks MKK1 and MKK2, and the MAPK MPK4 (MEKK1/MKK1-MKK2/ MPK4) negatively controls defense responses (Kong et al, 2012;Rasmussen et al, 2012) by negatively regulating the expression of the MAP3K MEKK2, which triggers a salicylate (SA)-dependent autoimmunity response when the cascade is compromised (Berriri et al, 2012;Su et al, 2013). Two other modules, MEKK1-MAPKKKa/MKK4-MKK5/MPK3-MPK6 and MKK9/ MPK3-MPK6 (Xu et al, 2008), mediate the activation of defense responses, including the synthesis of ethylene and camalexin, i.e.…”

Identification of Constitutively Active AtMPK6 Mutants Using a Functional Screen in Saccharomyces cerevisiae

“…Until recently, no strategy to render MAPKs (Mitogen-Activated Protein Kinases) was available for plants. In a recent work, we took advantage of a functional expression screen in yeast to identify point mutations triggering MAP2K-independent constitutive activity to a model MAPK of Arabidopsis , MPK6 [ 2 ]. In this chapter, we provide a detailed protocol for the screening of CA MAPKs in yeast.…”