- Top of page
- Experimental procedures
- Supporting Information
Arabidopsis MPK4 has been implicated in plant defense regulation because mpk4 knockout plants exhibit constitutive activation of salicylic acid (SA)-dependent defenses, but fail to induce jasmonic acid (JA) defense marker genes in response to JA. We show here that mpk4 mutants are also defective in defense gene induction in response to ethylene (ET), and that they are more susceptible than wild-type (WT) to Alternaria brassicicola that induces the ET/JA defense pathway(s). Both SA-repressing and ET/JA-(co)activating functions depend on MPK4 kinase activity and involve the defense regulators EDS1 and PAD4, as mutations in these genes suppress de-repression of the SA pathway and suppress the block of the ET/JA pathway in mpk4. EDS1/PAD4 thus affect SA–ET/JA signal antagonism as activators of SA but as repressors of ET/JA defenses, and MPK4 negatively regulates both of these functions. We also show that the MPK4–EDS1/PAD4 branch of ET defense signaling is independent of the ERF1 transcription factor, and use comparative microarray analysis of ctr1, ctr1/mpk4, mpk4 and WT to show that MPK4 is required for induction of a small subset of ET-regulated genes. The regulation of some, but not all, of these genes involves EDS1 and PAD4.
- Top of page
- Experimental procedures
- Supporting Information
Plants are able to activate immune responses upon recognition of invading pathogens. Recognition may occur via gene-for-gene interactions in which a plant resistance (R) gene product interacts with or detects the action of a cognate pathogen avirulence (Avr) factor (Nimchuk et al., 2003). R–Avr interactions induce rapid resistance responses at the infection site that are often mediated by salicylic acid (SA). Many virulent pathogens also induce basal defense responses that involve SA, and loss of basal defense causes hyper-susceptibility to virulent pathogens.
In addition to initiation of local defenses, R protein activation can lead to an immune state in systemic tissues termed systemic acquired resistance (SAR). SAR development in Arabidopsis correlates with expression of the pathogenesis-related (PR) genes PR1, PR2 and PR5, and involves micro-oxidative bursts and SA accumulation in systemic tissues (Alvarez et al., 1998; Malamy et al., 1990; Uknes et al., 1992). The role of SA in plant immunity is supported by the fact that exogenous SA, or high-level endogenous SA accumulation by expression of bacterial SA synthases, induce SAR-like resistance and PR gene expression (Verberne et al., 2000). Conversely, SAR is impaired in the SA-deficient mutants eds5 and sid2 (Nawrath and Métraux, 1999). SA depletion by transgenic expression of bacterial nahG salicylate hydroxylase also impairs SAR induction, although nahG expression has pleiotropic effects beyond SA catabolism (Heck et al., 2003; van Wees and Glazebrook, 2003). Other defense-related hormones such as ethylene (ET) and jasmonic acid (JA) appear to be dispensable for SAR activation (Lawton et al., 1995; Pieterse et al., 1998).
Some signal transducers and transcriptional activators of SA-mediated responses have been identified. Many of these proteins are involved in local R-controlled responses, SAR, and maintenance of basal defenses, whereas others only have demonstrated roles in certain SA-mediated defense responses. Long-distance SAR signaling involves the activities of at least two apoplastic proteins. The non-specific lipid transfer-like protein DIR1 is required for an as yet undefined branch of SAR that is independent of systemic SA accumulation (Maldonado et al., 2002), while the CDR1 protease is involved in triggering SA accumulation (Xia et al., 2004). SA accumulation is negatively regulated by the MAP kinase MPK4 (Petersen et al., 2000), and in many cases requires the aminotransferase ALD1 and the action of the interacting EDS1, PAD4 and SAG101 proteins that are essential components of basal resistance (Falk et al., 1999; Feys et al., 2001, 2005; Jirage et al., 1999; Song et al., 2004). EDS1 and PAD4 participate in a defense amplification loop that responds to SA and reactive oxygen intermediate-derived signals (Rusterucci et al., 2001). Mechanisms of SA perception remain unclear, although a catalase, carbonic anhydrase and methylsalicylate esterase have been purified as SA-binding proteins (Forouhar et al., 2005; Slaymaker et al., 2002). The BTB/ankyrin repeat protein NPR1 is central to SA signal transduction, as npr1 mutants are non-responsive to exogenous SA (Cao et al., 1997). NPR1 translocates to the nucleus in the presence of SA and its actions include stimulation of the DNA-binding activity of the TGA family of leucine zipper transcription factors that bind to the PR1 promoter to activate transcription (Fan and Dong, 2002; Johnson et al., 2003). SA-dependent, NPR1- independent defense responses also exist, and may involve the transcription factor Why1 whose DNA-binding activity is induced by SA independently of NPR1 (Desveaux et al., 2004).
SA-mediated defense responses provide protection from biotrophic fungi, oomycetes and bacteria such as Erysiphe orontii, Peronospora parasitica and Pseudomonas syringae. In contrast, defense against many necrotrophic fungi does not involve SA, but relies on ET and JA accumulation and signaling. Although it is unclear how necrotrophic fungi are recognized by plants, infection by these pathogens initiates a systemic defense system mediated by ET and JA, and associated with expression of the defensin PDF1.2 (Penninckx et al., 1996). ET signaling involves a family of membrane-anchored receptors (ETR1, ETR2, EIN4, ERS1, and ERS2), the ETR1-associated protein kinase CTR1 that negatively regulates ET signaling, the family of labile EIN3-like transcription factors whose turnover is controlled by SCFEBP1/EBP2 ubiquitin ligases, and other factors whose biochemical functions are unclear (Guo and Ecker, 2004). JA signaling is less well understood, but involves the ubiquitin ligase SCFCOI1 and the JA-conjugating enzyme JAR1 (Devoto and Turner, 2003). ET and JA defense signaling converge on induction of the histone deacetylase HDA19 and the transcription factor ERF1. HDA19 is required for Alternaria brassicicola resistance, and its over-expression causes ERF1 induction (Zhou et al., 2005). ERF1 over-expression in wild-type (WT), ET- and JA-insensitive genetic backgrounds is sufficient to induce PDF1.2 expression and resistance to several necrotrophic fungi (Berrocal-Lobo et al., 2002; Lorenzo et al., 2003; Solano et al., 1998). The secreted lipase GLIP1 with anti-fungal activity is a physiologically relevant target of the ET/JA defense pathway, as GLIP1 is induced by both hormones, and glip1 mutants exhibit enhanced susceptibility to A. brassicicola infection (Oh et al., 2005).
PDF1.2 serves as a useful marker for ET/JA pathway activation, but defense responses mediated by ET and JA also involve aspects distinct from PDF1.2 induction. For example, the R2R3 Myb transcription factor BOS1 is induced in a JA-dependent manner by Botrytis cinerea infection, and is required for resistance to at least two necrotrophic fungi. Nonetheless, PDF1.2 induction occurs normally in bos1 mutants upon B. cinerea infection (Mengiste et al., 2003).
While distinct, the SA-, ET- and JA-mediated defense systems interact in complex ways. Overlap in gene induction between SA, JA and ET treatments is significant (Schenk et al., 2000), and the induction of some genes exhibits SA–JA and/or SA–ET synergism (Lawton et al., 1994; Xu et al., 1994), while some wound-related, JA-induced genes exhibit ET–JA antagonism (Norman-Setterblad et al., 2000). A third systemic defense system, induced systemic resistance (ISR), is an example of the compatibility and independence of SA and ET/JA signaling, as ISR requires JA and ET signaling as well as NPR1, and can be induced with SAR to produce additive resistance effects (Pieterse et al., 1998; van Wees et al., 2000). Nonetheless, antagonistic interactions between signaling via SA and ET/JA are well documented. For example, the necrotroph-induced genes ERF1, PDF1.2, b-CHI and PR4 are synergistically induced by ET and JA, but JA induction of PDF1.2 can be inhibited by SA (Lorenzo et al., 2003; Norman-Setterblad et al., 2000). Mutual antagonism between SA and ET/JA was also evident from a microarray study of defense-related mutants infected with P. syringae pv. maculicola (Glazebrook et al., 2003). This showed that expression of a cluster of SA-related genes, including PR1, was increased in ET- and JA-insensitive mutants, while ET/JA-related genes showed increased expression in SA pathway mutants. Inhibition of SA signaling by JA also occurs, as activation of JA signaling in tomato enhances susceptibility to virulent P. syringae pv. tomato DC3000 (Pst DC3000; Zhao et al., 2003), while JA-insensitive mutants exhibit increased pathogen-induced SA levels and resistance in both Arabidopsis and tomato (Kloek et al., 2001; Zhao et al., 2003). Pst DC3000 uses the JA agonist coronatine as a virulence factor, and may thereby hijack antagonistic functions in the host to suppress the SA defense mechanism that combats its infection.
Despite evidence for SA–ET/JA antagonism, the underlying molecular mechanisms remain ill-defined. In Arabidopsis, genetic evidence suggests involvement of NPR1, the transcription factors ERF1 and WRKY70, and the MAP kinase MPK4 in the control of antagonism (Berrocal-Lobo et al., 2002; Li et al., 2004; Petersen et al., 2000; Spoel et al., 2003). Unsaturated fatty acid-derived signals may also play a role, as ssi2 mutants, defective in a plastidic fatty acid desaturase, exhibit partially SA-dependent PR1 expression and Pst DC3000 resistance, and strongly reduced, but oleic acid-rescuable, PDF1.2 expression in response to JA (Kachroo et al., 2001; Shah et al., 2001). Formal genetic interpretations place NPR1 and WRKY70 as positive regulators of SA signaling, and as negative regulators of ET/JA signaling, while the opposite is true for ERF1 and MPK4. However, these observations do not clarify how antagonism is controlled, and, apart from a genetic interaction between WRKY70 and NPR1 in the suppression of PDF1.2, it is unclear how the actions of these factors are connected.
We showed previously that mpk4 mutants constitutively express SA-mediated resistance responses but are blocked in defensin expression by JA (Petersen et al., 2000). MAP kinases (MAPKs) are conserved in eukaryotic signal transduction where they orchestrate responses to extracellular stresses and developmental cues via phosphorylation of substrate proteins including transcription factors. In most cases, MAPK activity is controlled by sequential activation of three protein kinases, by which an MAPK kinase kinase (MAPKKK) activates an MAPK kinase (MAPKK) that in turn activates an MAPK by phosphorylation of conserved Thr and Tyr residues in the so-called MAPK T-loop (Madhani et al., 1997). We have recently described the MPK4 substrate MKS1, a nuclear protein that interacts with two WRKY transcription factors (Andreasson et al., 2005). The molecular phenotypes of plants over- or under-expressing MKS1 indicate that it mediates some effects of MPK4 on SA-mediated resistance responses but has little if any effect on responses mediated by JA.
Here we dissect the function of MPK4 in the SA–ET/JA defense network in further detail. We show that MPK4 kinase activity is central to both SAR repression and ET/JA defense induction, and that both processes involve EDS1 and PAD4 downstream of MPK4. Our data therefore place EDS1 and PAD4 as regulators of the antagonism between the SA- and ET/JA-mediated defense systems.