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Title: EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity}
Article:
Abstract:
Mitogen-activated protein( MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase( MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that edr1 mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the mpk3, mkk4 and mkk5 mutations suppress edr1-mediated resistance, and over-expression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity.
Introduction:
Mitogen-activated protein kinase( MAPK) cascades are highly conserved signaling modules that control diverse signal transduction pathways in eukaryotes, including defenses against infection[1]. Activation of MAPK cascades is thought to be one of the earliest events in plant immunity[1]–[3]. For instance, treatment of Arabidopsis with a conserved 22-amino acid peptide fragment of bacterial flagellin, a pathogen-associated molecular pattern( PAMP), specifically recognized by the pattern recognition receptor FLS2, can trigger activation of MKK4/MKK5 and MPK3/MPK6[4],[5], which subsequently promotes expression of the downstream target gene FRK1 and activates plant defenses[5]. Other PAMPs, such as EF-Tu, chitin, harpin, oligogalacturonides and xylanase, also trigger the activation of MPK3 and/or MPK6[6]–[9]. In addition, MPK3 and MPK6 regulate phytoalexin biosynthesis by activating the transcription factor WRKY33, which is required for resistance to necrotrophic fungal pathogens[10]–[12]; also, the full priming of stress responses in Arabidopsis requires MPK3 and MPK6[13]. MKK4 and MKK5 function redundantly and act upstream of MPK3 and MPK6. Constitutive activation of MKK4 and MKK5 in Arabidopsis leads to HR-like cell death associated with the generation of reactive oxygen species[14]. Plants expressing active forms of MKK4 and MKK5 have enhanced resistance to Pseudomonas syringae and Botrytis cinerea[5]. Plant basal defenses require the MKK4/MKK5-MPK3/MPK6 kinase cascade. To effectively invade plants, bacterial pathogens block PAMP-induced defenses with effectors, such as AvrPto, AvrPtoB, HopAI1, and HopF2, that directly repress MKK4/MKK5 and MPK3/MPK6 activities or inhibit the components that act upstream of MAP kinase cascades[15]–[20]. In Arabidopsis, five phosphatases have been reported that regulate MPK3 and MPK6 kinase activity by dephosphorylation. For instance, the PP2C-type phosphatase AP2C1 inactivates stress-induced kinase activity of MPK4 and MPK6[21]; PP2C5 also regulates ABA-mediated activation of MPK3 and MPK6[22]. Also, MKP1 and PTP1 phosphatases repress salicylic acid biosynthesis and SNC1-mediated responses by inactivating MPK3 and MPK6[23]. Also, MKP2 interacts with and dephosphorylates MPK3 and MPK6 to regulate oxidative stress and plant defense responses[24],[25]. However, the mechanisms of the negative regulation of MKK4/MKK5 in Arabidopsis remain unclear. The Raf-like MAPK kinase kinase( MAPKKK) EDR1 functions as a negative regulator of plant defense. For example, edr1 mutants have enhanced resistance to pathogens including powdery mildew fungus, bacteria and oomycetes[26],[27]. The edr1 mutants also show enhanced ethylene-induced senescence[28]. EDR1 protein consists of an N-terminal functionally unknown domain and a C-terminal kinase domain. The kinase activity of EDR1 has been demonstrated in vitro[29]. KEEP ON GOING( KEG) encodes a protein containing RING E3 ligase domain, kinase domain, ankyrin repeats and HERC2-like repeats[30] and the recessive missense mutant keg-4 suppresses the phenotype of edr1 mutants. KEG could directly interact with EDR1 and recruit EDR1 to the trans-Golgi network/early endosome[31]. In Arabidopsis, the EDR1 homolog CTR1( Constitutive Triple Response 1), a Raf-like MAPKKK[29], plays an essential role in the negative regulation of ethylene signaling[32]. CTR1 and the MKK9-MPK3/MPK6 cascade antagonistically regulate ethylene responses[33]. However, whether EDR1 affects the regulation of one specific MAPK cascade pathway remains unknown. The edr1 mutants are constitutively primed for salicylic acid-inducible defenses and enhanced callose deposition, which may be mediated by the regulation of MPK3 and MPK6 in Arabidopsis[13],[34]. However, the molecular mechanisms leading to enhanced resistance and cell death in edr1 are still not well understood. Here, we report that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade pathway through direct interaction with MKK4 and MKK5 to modulate plant defense and cell death.
Discussion:
The essential MKK4/MKK5-MPK3/MPK6 kinase cascade transduces extracellular stimuli in many different response pathways. Activation of this cascade must be tightly controlled, as inappropriate activation of MAPKs can inhibit growth or even cause lethality. For instance, in Arabidopsis, constitutively activated MKK4 and MKK5 cause an accelerated cell death phenotype[14]. Over-expression of MPK3 may be lethal for the plant, as we( and others) have found it difficult to obtain MPK3 over-expressing plants([13],[40] and this study). Therefore, tight regulation of MKK4/MKK5 and MPK3/MPK6 kinase activity and protein accumulation seems essential for survival and adaptation to environmental challenges. The kinase activity of MPK3/MPK6 can be repressed through dephosphorylation mediated by several different phosphatases[21]–[25]. Modulation of MKK4 and MKK5 protein levels by EDR1 could be another mechanism for fine-tuning the activity of the MKK4/MKK5-MPK3/MPK6 kinase cascade in response to pathogen attack. How EDR1 affects accumulation of MKK4 and MKK5 protein remains unresolved. Our study showed that the N-terminal regulatory domain of EDR1 interacts with MKK4 and MKK5, and negatively affects MKK4 and MKK5 accumulation. One possible mechanism for this effect may be that EDR1 acts as a scaffold protein that keeps MKK4 and MKK5 in a catalytically inactive state. While MKK4 and MKK5 are not active, the plants would deploy a feedback regulation mechanism to keep MKK4 and MKK5 protein levels low in order to avoid detrimental spontaneous activation of defense responses. Low levels of MKK4 and MKK5 protein in turn would lead to low accumulation of MPK3 and MPK6 protein. An alternative explanation for our finding is that EDR1 affects the stability of MKK4 and MKK5 directly, perhaps through interactions with the 26S proteasome degradation machinery. Consistent with this possibility, EDR1 has been reported to associate with the E3 ubiquitin ligase KEG[30],[31], a component of the SCF ligase complex, and that mutations in KEG suppress edr1-mediated resistance. Furthermore, we recently showed that edr1-mediated defense responses require RPN1a, a subunit of the 26S proteasome[41], suggesting that 26S proteasome degradation machinery is involved in the EDR1 pathway. Although MKK4 and MKK5 are commonly considered, by implication, to be important components of plant immunity pathways, little genetic support for this notion has been reported. Here, we identified mkk4 and mkk5 loss-of-function mutants, and showed that they display enhanced susceptibility to powdery mildew. We also showed that edr1-mediated resistance requires MKK4 and MKK5 function, providing direct genetic evidence that MKK4 and MKK5 are key players in plant immunity responses. The mkk4 and mkk5 alleles described in this study will be valuable tools for dissecting the role of the MKK4/5 mediated MAPK cascade in biotic and abiotic stresses. The MKK4 and MKK5 proteins are considered to be functionally equivalent[1],[42]. Interestingly, our study revealed that single mutation of either MKK4 or MKK5 significantly affect edr1-mediated defense. Consistent with this observation, both mkk4 and mkk5 single mutant showed enhanced susceptibility to powdery mildew, indicating that loss-of-function of either one of these two genes causes a defect in immune responses. Dosage effects may be a possible explanation for these observations. In this scenario, the accumulation of MKK4 and MKK5 protein is limiting, such that the activity of both genes is required for normal immune responses. This view is supported by our finding that over-expression of either MKK4 or MKK5 enhanced resistance to powdery mildew and resulted in edr1-like cell death. However, the possibility that MKK4 and MKK5 have non-identical roles in plant immunity has not been rigorously tested and cannot be dismissed at this time. Arabidopsis has more than sixty MAPKKKs, which can be divided into two subfamilies, including 12 MEKK1-like kinases and approximately 50 Raf-like kinases[43]. Several members of the MEKK1 family have been shown to function as MAPKKKs upstream of MAPKKs in MAP kinase cascades[1]; in contrast, no evidence has implicated any of the Raf-like kinases as canonical MAPKKKs. EDR1 and CTR1 are the only two well-characterized Raf-like kinases in plants. CTR1 directly regulates EIN2 by phosphorylation; EIN2, in turn, inactivates EIN3/EIL1-dependent ethylene responses. This indicates that CTR1, at least in this context, does not function as a MAPKKK[44],[45]. Here, we show that the N-terminus of EDR1 associates with MKK4/MKK5 and negatively affects the accumulation of MKK4/5 protein, suggesting that EDR1 does not act as a MAPKKK either. Instead, EDR1 may fine-tune responses to biotic and abiotic stresses by controlling this MAP kinase cascade. Several proteins that were originally identified as negative regulators of plant immunity, such as LSD1 and ACD11, have on closer analysis turned out not be true negative regulators, as loss-of-function alleles lead to inappropriate activation of NBS-LRR proteins, suggesting that LSD1 and ACD11 may be guarded pathogen targets[46],[47]. However, EDR1 appears to be different from those proteins, as over-expression of EDR1 leads to enhanced susceptibility and lower activation of MAPK cascade, indicating that EDR1 could serve as a negative regulator of defense by repressing the MAPK pathway. In the absence of pathogen, EDR1 inactivates the MAPK pathways; however, upon pathogen infection, plants quickly activate defenses, possibly by de-repression of the inhibition of MAPK pathways by EDR1. In conclusion, we show that EDR1 directly associates with MKK4 and MKK5, and negatively affects protein levels of MKK4, MKK5, MPK3 and MPK6, which may represent an important mechanism that fine-tunes plant defense responses.
Plant immunity must be tightly regulated , as over- or constitutive activation of plant defenses can cause detrimental effects , such as dwarf stature and enhanced cell death . EDR1 , a Raf-like mitogen-activated protein kinase ( MAPK ) kinase kinase , negatively regulates defenses in Arabidopsis . The highly conserved MAPK cascades modulate diverse biological processes , including plant immunity . However , whether EDR1 affects the regulation of one of the MAPK pathways was not previously known . Here , we show that EDR1 physically associates with MKK4 and MKK5 , two MAP kinase kinases , and negatively regulates the protein levels of MKK4 , MKK5 , MPK3 and MPK6 . We further show that edr1-mediated disease resistance requires MKK4 , MKK5 and MPK3 function . Over-expression of MKK4 or MKK5 in wild-type increased resistance to powdery mildew and caused mildew-induced cell death . Our study suggests that EDR1 negatively regulates defenses and directly modulates the MKK4/MKK5-MPK3/MPK6 cascade to fine-tune plant immunity .