AtMYB44 regulates WRKY70 expression and modulates antagonistic interaction between salicylic acid and jasmonic acid signaling

Authors


For correspondence (e-mail choiyngd@snu.ac.kr).

Summary

The role of AtMYB44, an R2R3 MYB transcription factor, in signaling mediated by jasmonic acid (JA) and salicylic acid (SA) is examined. AtMYB44 is induced by JA through CORONATINE INSENSITIVE 1 (COI1). AtMYB44 over-expression down-regulated defense responses against the necrotrophic pathogen Alternaria brassicicola, but up-regulated WRKY70 and PR genes, leading to enhanced resistance to the biotrophic pathogen Pseudomonas syringae pv. tomato DC3000. The knockout mutant atmyb44 shows opposite effects. Induction of WRKY70 by SA is reduced in atmyb44 and npr1-1 mutants, and is totally abolished in atmyb44 npr1-1 double mutants, showing that WRKY70 is regulated independently through both NPR1 and AtMYB44. AtMYB44 over-expression does not change SA content, but AtMYB44 over-expression phenotypes, such as retarded growth, up-regulated PR1 and down-regulated PDF1.2 are reversed by SA depletion. The wrky70 mutation suppressed AtMYB44 over-expression phenotypes, including up-regulation of PR1 expression and down-regulation of PDF1.2 expression. β-estradiol-induced expression of AtMYB44 led to WRKY70 activation and thus PR1 activation. AtMYB44 binds to the WRKY70 promoter region, indicating that AtMYB44 acts as a transcriptional activator of WRKY70 by directly binding to a conserved sequence element in the WRKY70 promoter. These results demonstrate that AtMYB44 modulates antagonistic interaction by activating SA-mediated defenses and repressing JA-mediated defenses through direct control of WRKY70.

Introduction

To cope with pathogen challenge, plants rapidly activate defense responses, which are regulated by the major signaling molecules salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). Accumulation of SA, JA or ET in response to pathogen invasion or herbivore attack activates distinct but overlapping sets of defense genes; complex networking among these signaling pathways also modulates defense responses to maximize effective defenses while minimizing cost to the plant (Reymond and Farmer, 1998; Kunkel and Brooks, 2002; Spoel et al., 2003; Robert-Seilaniantz et al., 2011).

JA plays a role in defense signaling against necrotrophic pathogens and herbivore attack (Thomma et al., 1998; Turner et al., 2002; Browse and Howe, 2008). Arabidopsis JA-mediated defense responses require the F-box protein CORONATINE INSENSITIVE 1 (COI1), which is a jasmonyl-isoleucine receptor (Xie et al., 1998; Devoto et al., 2002; Yan et al., 2009; Sheard et al., 2010). COI1 acts as part of the SCFCOI1 complex to activate JA signaling by 26S proteasome-mediated degradation of jasmonate ZIM-domain (JAZ) proteins. JA induces degradation of JAZ proteins, and this degradation results in activation of JA-responsive gene expression (Chini et al., 2007; Thines et al., 2007; Chung and Howe, 2009). These JA-activated genes include that encoding the anti-microbial defensin PDF1.2, which acts against necrotrophic pathogens (Penninckx et al., 1996). Ethylene, often together with JA, activates plant defenses to necrotrophic pathogens such as Alternaria brassicicola (Shan et al., 2012).

SA plays a role in defense signaling distinct from that mediated by JA (Feys and Parker, 2000; Durrant and Dong, 2004). Accumulation of SA leads to up-regulation of defense-related genes including the pathogenesis-related (PR) genes PR1, PR2 and PR5, and results in enhanced disease resistance against biotrophic pathogens (Gaffney et al., 1993; Delaney et al., 1994). SA-induced defense responses are mediated by an ankyrin repeat protein, NONEXPRESSOR OF PR1 (NPR1) (Cao et al., 1997; Spoel et al., 2003). However, NPR1-independent pathways have also been reported (Bowling et al., 1997; Li et al., 2004). For example, constitutive expression of PR genes in cpr6 and ssi2 was not compromised by the npr1-1 mutation (Clarke et al., 1998; Shah et al., 2001).

In some cases, various defense signaling pathways act synergistically to enhance resistance against pathogen attack (van Wees et al., 2000). In other cases, antagonistic interactions between defense signaling pathways provide focused resistance against pathogens (Kunkel and Brooks, 2002). One well-documented antagonistic interaction involves cross-talk between JA and SA. Early studies of the role of SA in tomato wounding responses revealed that exogenous SA suppressed JA-induced wound responses (Doherty et al., 1988). In Arabidopsis, exogenous SA suppresses JA-dependent gene expression and defense responses against A. brassicicola infection (Spoel et al., 2007). Transgenic plants harboring the NahG transgene encoding SA hydroxylase, which converts SA to catechol, showed enhanced expression of JA biosynthesis genes and defense genes during infection with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) (Delaney et al., 1994; Spoel et al., 2003).

JA also suppresses SA signaling (Kunkel and Brooks, 2002). Treatment with exogenous JA inhibits the expression of SA-dependent genes (Niki et al., 1998). Arabidopsis mpk4 and ssi2 mutants, which are impaired in JA-responsive gene expression, constitutively express SA-dependent genes and show enhanced disease resistance against Pst DC3000 and Peronospora parasitica (Petersen et al., 2000; Kachroo et al., 2001). The JA-insensitive mutant coi1 also shows similar gene expression and disease resistance against Pst DC3000 (Kloek et al., 2001).

A large set of transcription factors is involved in the regulation of plant defense (Riechmann et al., 2000; Eulgem, 2005), and antagonistic interaction between SA and JA involves transcriptional reprogramming by a subset of these transcription factors. For example, WRKY transcription factors are defined by the highly conserved amino acid sequence WRKYGQK and are involved in plant defense responses. Several WRKY transcription factors, including WRKY11, WRKY17 and WRKY70, play roles in antagonistic interaction between SA and JA (Li et al., 2004; Journot-Catalino et al., 2006). WRKY70 plays a pivotal role in JA and SA responses (Li et al., 2004). Expression of WRKY70 is activated by SA but suppressed by JA. Over-expression of WRKY70 leads to up-regulation of PR genes and down-regulation of PDF1.2, leading to enhanced resistance against biotrophic pathogens and enhanced susceptibility to necrotrophic pathogens.

MYB transcription factors contain a MYB domain consisting of up to four imperfect repeats of a 52 amino acid motif. Most plant MYB transcription factors belong to the R2R3-MYB family (Dubos et al., 2010). Arabidopsis R2R3-MYB transcription factors have been implicated in abiotic stress responses and development (Dubos et al., 2010). They also act in biotic stress responses; for example, AtMYB30 acts as positive regulator of hypersensitive cell death (Vailleau et al., 2002) and AtMYB96 positively regulates accumulation of SA by activating SID2 expression (Seo and Park, 2010). However, the contribution of R2R3-MYB transcription factors to regulation of the antagonistic interaction between JA and SA remains unclear.

AtMYB44 belongs to sub-group 22 of the R2R3 MYB transcription factor family. AtMYB44 was rapidly induced by methyl jasmonate (MeJA) in Arabidopsis (Jung et al., 2008). In this study, we characterized the role of AtMYB44 in the defense responses mediated by SA and JA. It is shown that AtMYB44 directly regulates WRKY70, and thus regulates PR genes. AtMYB44 mediated modulation of the antagonistic interaction between SA and JA is also demonstrated by over-expression and mutation analysis.

Results

AtMYB44 down-regulates defense responses against A. brassicola

To understand the function of AtMYB44 in JA-mediated defense responses, we examined two previously characterized AtMYB44 over-expressing lines: OX18 and OX21 (Jung et al., 2008). To examine the JA-mediated defense responses of these plants, we challenged wild-type, atmyb44 mutants and OX18 and OX21 over-expressing lines with the necrotrophic pathogen A. brassicicola. Wild-type plants showed limited necrosis at inoculation sites (Figure 1a). However, OX18 and OX21 over-expressing lines showed more severe disease symptoms with extended necrosis. The mean diameter of lesions in OX18 and OX21 plants caused by A. brassicicola infection was approximately six times larger than that of wild-type plants. By contrast, atmyb44 mutant plants showed reduced lesion size.

Figure 1.

Suceptibility of AtMYB44 over-expressing and atmyb44 knockout mutant plants to the necrotrophic pathogen A. brassicicola. (a) Four-week-old Arabidopsis plants were inoculated with Abrassicicola. OX18 and OX21 are AtMYB44 over-expressing lines (Jung et al., 2008). The photograph was taken 10 days after inoculation. (b) Quantification of fungal growth by determination of the relative amount of fungal DNA compared with plant DNA using quantitative PCR. Values are means ± standard deviation. The experiments were repeated twice with similar results. The statistical significance of the measurements was determined using Student's t test (*< 0.01) for comparison with the wild-type value. (c) Four-week-old Arabidopsis plants were inoculated with A. brassicicola. Inoculated plants were collected for RNA extraction and analyzed by Northern blot.

To determine whether the altered lesion size and necrosis resulted from changes in the growth of fungi in plants, the amount of fungal DNA in infected leaves was measured by quantitative PCR using A. brassicicola and Arabidopsis gene-specific primers. The level of A. brassicicola-specific DNA in OX18 and OX21 plants was approximately 30 times higher than in wild-type plants (Figure 1b). Consistent with their reduced lesion formation phenotype, the amount of fungal DNA in atmyb44 knockout plants was lower than in wild-type plants by a small but significant amount (< 0.01).

The enhanced susceptibility to A. brassicicola in OX18 and OX21 plants and the increased resistance in atmyb44 knockout mutants suggest that AtMYB44 negatively regulates JA-mediated defenses responses to necrotrophic A. brassicicola. To test whether these altered disease responses depend on JA signaling, expression of the defense marker gene PDF1.2 was examined (Figure 1c). The AtMYB44 transcript was detected 12 h after infection in wild-type plants (Figure 1c), and expression of PDF1.2 was induced later, 48 h after infection (Figure 1c). PDF1.2 induction was clearly reduced in OX18 plants. By contrast, PDF1.2 expression was induced earlier in atmyb44 knockout plants than in wild-type plants. These results show that resistance against A. brassicicola and PDF1.2 induction are inversely correlated with AtMYB44 expression.

AtMYB44 is a negative regulator in JA signaling pathways

We next tested whether gene expression patterns induced by MeJA treatment are consistent with those produced by A. brassicicola infection in AtMYB44 over-expression lines and atmyb44 knockout mutants. Over-expression of AtMYB44 led to delayed and reduced expression of the JA-responsive genes ALLENE OXIDE SYNTHASE (AOS), VEGETATIVE STORAGE PROTEIN 1 (VSP1) and PDF1.2 after MeJA treatment (Figure 2). In contrast to the over-expression phenotype, knockout mutation of AtMYB44 enhanced MeJA-mediated expression of these genes. Basal expression levels of VSP1 and PDF1.2 were constitutively up-regulated in atmyb44 knockout plants.

Figure 2.

AtMYB44 negatively regulates expression of JA-mediated genes. Two-week-old plants were treated with MeJA and analyzed by Northern blot with the indicated probes. rRNA was visualized by ethidium bromide staining as a loading control.

AtMYB44 also affected JA-mediated growth responses. Compared to wild-type, OX18 plants are less sensitive and atmyb44 knockout plants are more sensitive to JA-mediated root growth inhibition and root hair formation at sub-micromolar concentrations (Figure S1). Taken together, these results demonstrate that AtMYB44 negatively regulates various JA-mediated responses.

AtMYB44 enhances disease resistance against Pst DC3000

JA and SA signaling pathways mutually antagonize each other (Kunkel and Brooks, 2002), and AtMYB44 acts as a negative regulator of JA responses. Therefore, we next tested whether AtMYB44 is involved in SA-mediated bacterial defense responses by testing AtMYB44 mutant and over-expressing lines for resistance to the biotrophic bacterial pathogen Pst DC3000. We found that over-expression of AtMYB44 led to enhanced resistance to Pst DC3000 (Figure 3a), and atmyb44 knockout mutation led to slightly increased susceptibility. To test whether resistance resulted from inhibited pathogen growth, bacterial growth in infected leaves was measured by a colony-counting assay. We found that the titer of Pst DC3000 in OX18 and OX21 plants was approximately ten times lower than that in wild-type plants (Figure 3b). In atmyb44 knockout plants, the bacterial titer was slightly increased compared to wild-type plants. This result indicates that AtMYB44 increases resistance to Pst DC3000.

Figure 3.

Resistance of AtMYB44 over-expressing and atmyb44 knockout mutant plants against the biotrophic pathogen Pst DC3000. (a) Four-week-old Arabidopsis Col-0, AtMYB44 over-expressing plants (OX18 and OX21) and atmyb44 knockout mutant plants were inoculated with Pst DC3000. The photograph was taken 3 days after inoculation. (b) Bacterial growth in leaves was determined 1 and 3 days after inoculation. The statistical significance of the measurements was determined using Student's t test (*< 0.01) for comparison with the wild-type value. Values are means ± standard deviation for eight independent plants. The experiments were repeated twice and showed similar results.

PR genes are up-regulated in AtMYB44 over-expressing plants

To investigate the enhanced resistance of OX18 and OX21 plants to Pst DC3000, we examined the expression levels of PR genes, which participate in SA-mediated defense responses. Northern blot analysis demonstrated that PR1, PR2 and PR5 were constitutively over-expressed in AtMYB44 over-expressing plants but were not expressed in untreated knockout and wild-type plants (Figure 4a).

Figure 4.

SA-mediated response in AtMYB44 over-expressing and atmyb44 knockout plants. (a) Northern blot showing basal level expression of PR genes in Arabidoposis Col-0, AtMYB44 over-expression (OX18 and OX21) and atmyb44 knockout mutant plants. (b) Effect of AtMYB44 on induction of PR1 by SA treatment. After SA treatment, total RNA was analyzed by Northern blot and rRNA was visualized by ethidium bromide staining as a loading control.

Gene expression patterns induced by SA treatment were consistent with the observed changes in resistance against Pst DC3000 infection in AtMYB44 over-expression and atmyb44 knockout mutant plants. In wild-type plants, AtMYB44 was induced rapidly, within 30 min after SA treatment, but PR1 was induced after approximately 3 h (Figure 4b). Even though the level of PR1 was already higher in OX18 plants than that in wild-type plants, PR1 was induced to a much higher level after SA treatment. In contrast, activation of PR1 by SA treatment was reduced in atmyb44 knockout plants. These results demonstrate that AtMYB44 positively regulates SA-mediated defense responses, including activation of PR genes.

AtMYB44 regulates SA-mediated defense responses

AtMYB44 activates SA-mediated PR genes; to determine whether the effect of AtMYB44 on PR gene expression is through SA signaling, we tested AtMYB44 expression in npr1-1 mutant and NahG plants. AtMYB44 was rapidly induced by SA treatment in wild-type plants. Its induction was not affected in npr1-1 plants, but was significantly reduced in NahG plants (Figure 5a). Therefore, AtMYB44 is induced by SA but does not depend on NPR1. WRKY70, a key regulator in the SA signaling pathway, was also highly induced in wild-type plants, but was reduced in npr1-1 and totally abolished in NahG plants.

Figure 5.

Role of AtMYB44 in SA signaling. (a) Expression of AtMYB44 in SA signaling mutants. Plants were treated with SA for the indicated times and analyzed by Northern blot. (b) WRKY70 and PR1 gene expression in SA signaling mutants in the background of AtMYB44 over-expression. The OX18 AtMYB44 over-expression line was crossed with SA signaling-deficient mutants or lines (npr1-1, NahG and sid2-2). (c) Disease resistance against biotrophic pathogen Pst DC3000 in SA signaling mutants in the background of AtMYB44 over-expression. Four-week-old Arabidopsis Col-0, AtMYB44 over-expression plants (OX18), OX18 npr1-1 and OX18 NahG plants were inoculated with Pst DC3000. The photograph was taken 3 days after inoculation. Bacterial growth in leaves was determined 1 and 3 days after infection. The statistical significance of the measurements was determined using Student's t test (*< 0.01) for comparison with the wild-type value. Values are means ± standard deviation for eight independent plants. The experiments were repeated twice and showed similar results.

To dissect the molecular components upstream and downstream of AtMYB44 in the SA signaling pathway leading to PR gene expression, we determined the epistatic relationships between various components in the SA signaling pathway. OX18 plants were crossed with npr1-1, NahG or sid2-2 mutant plants. SID2 encodes an isochorismate synthase acting in SA biosynthesis. Activation of PR1 expression by AtMYB44 over-expression was slightly reduced in OX18 npr1-1 plants but totally abolished in OX18 NahG and OX18 sid2-2 plants (Figure 5b). These results indicate that AtMYB44 depends on a basal level of SA to activate PR1 expression.

To understand the role of NPR1 and SA in AtMYB44-mediated disease resistance, we next challenged the OX18 npr1-1 and OX18 NahG plants with Pst DC3000. Consistent with the expression patterns of PR1 in Figure 5(b), bacterial resistance was enhanced in OX18 plants but completely compromised in the OX18 NahG plants. In OX18 npr1-1 plants, resistance to Pst DC3000 was less than in OX18 but not as low as in OX18 NahG and wild-type (Figure 5c). Resistance of npr1-1 and NahG to Pst DC3000 was decreased compared with Col-0 plants (Figure S2). The decreased resistance of OX18 npr1-1 is probably due to disruption of NPR1-dependent pathways. NPR1 is required for full-scale activation of PR1 transcription mediated by AtMYB44. We measured the degree of resistance by quantification of bacterial growth in infected leaves (Figure 5c), and found that resistance varies with the level of PR1 gene expression (Figure 5b).

Over-expression phenotypes of AtMYB44 are reversed by SA depletion

Because PR1 activation in OX18 plants was abolished in the NahG background, we studied the growth phenotypes of OX18 NahG plants. Growth of OX18 and OX18 npr1-1 plants was severely retarded, similar to plants over-expressing PR genes (Bowling et al., 1997; Li et al., 2004). However, the OX18 growth retardation was abolished in OX18 NahG plants (Figure 6a).

Figure 6.

Effect of npr1 mutation or NahG expression on AtMYB44-mediated responses. (a) Growth of plants over-expressing AtMYB44 in various mutant backgrounds. Photographs were taken 2 or 5 weeks after germination. (b) JA response of plants over-expressing AtMYB44 in various mutant backgrounds. Two-week-old plants were treated with MeJA for 6 h, and total RNA was analyzed by Northern blot. rRNA was visualized by ethidium bromide staining as a loading control. (c) Quantification of free SA and SA glucosides in AtMYB44 over-expressing plants and atmyb44 mutant plants. Values are means ± standard deviation for three replicates. One-way anova revealed no difference in SA content among control and mutant plants at a confidence level of < 0.05.

We also examined the JA response of OX18 NahG plants. In OX18 plants, expression of JA-responsive genes such as VSP1 and PDF1.2 was not substantially induced by treatment with MeJA; however, in OX18 NahG plants, VSP1 and PDF1.2 were strongly induced by MeJA treatment (Figure 6b). By contrast, OX18 npr1-1 plants showed similar growth retardation and repression of JA-responsive genes to OX18 plants, because the NPR1-independent AtMYB44 pathway leading to PR1 is still functioning, as shown in Figure 5(b).

Because the effects of AtMYB44 over-expression were reversed in the NahG background, we next measured the levels of SA in wild-type, OX18 and atmyb44 mutant plants (Figure 6c). One-way anova revealed that the endogenous levels of free SA and glucosylated SA were not significantly different among all genotypes tested at a confidence level of < 0.05. Surprisingly, enhanced expression of PR genes in OX18 plants is not a result of enhanced SA biosynthesis.

AtMYB44 drives WRKY70 and PR1 expression

Because activation of PR genes by AtMYB44 over-expression required SA but did not result from SA accumulation, we investigated the expression of various genes related to expression of PR genes in AtMYB44 over-expression plants. We used RT-PCR, with a limited number of cycles, to screen the expression of 18 regulatory factors and SA-biosynthesis-related genes. In this assay, WRKY70 was the only gene affected by AtMYB44 over-expression or knockout mutation (Figure S3). SA biosynthesis and signaling genes, other WRKY transcription factors, and TGACC MOTIF-BINDING PROTEIN (TGA) genes, which are induced by SA and biotrophic pathogens, were not affected by AtMYB44 over-expression or knockout mutation. We also used Northern blot analysis to confirm that WRKY70 was constitutively over-expressed in OX18 and OX21 plants but was not expressed in atmyb44 mutants (Figure 7a).

Figure 7.

Role of AtMYB44 in expression of WRKY70 and PR1. (a) Northern blot showing basal expression of WRKY70 in Arabidopsis Col-0, AtMYB44 over-expression (OX18 and OX21) and atmyb44 knockout plants. (b) Induced expression of WRKY70 and PR1 by AtMYB44. Twelve-day-old seedlings of transgenic Arabidopsis harboring AtMYB44 under the control of a β-estradiol-inducible promoter (XVE:AtMYB44) were induced by transferring to MS plates containing β-estradiol. Total RNA was analyzed by Northern blot. (c) Effect of WRKY70 mutation on PR1 and PDF1.2 induction in AtMYB44 over-expressing plants. Two-week-old plants were treated with MeJA for 6 h and total RNA was analyzed by Northern blot. rRNA was visualized by ethidium bromide staining as a loading control.

To demonstrate activation of WRKY70 by AtMYB44 over-expression, we produced transgenic Arabidopsis plants expressing AtMYB44 under the control of the β-estradiol-inducible promoter. AtMYB44 was induced within 6 h after β-estradiol treatment (Figure 7b). WRKY70 was induced approximately 6 h after induction of AtMYB44, and PR1 was induced after another 12 h, suggesting a hierarchical relationship among these genes. This observation supports the hypothesis that induction of AtMYB44 mediates increased expression of WRKY70, which in turn mediates PR1 gene expression.

To test whether the activation of PR1 and suppression of PDF1.2 (Figures 2 and 4) in AtMYB44 over-expression plants required WRKY70, we generated OX18 wrky70 double mutants. Constitutive expression of PR1 in OX18 plants was completely abolished in OX18 wrky70 double mutants. Also, in contrast to the OX18 plants, PDF1.2 was induced by MeJA in the OX18 wrky70 double mutant as in the wild-type. These results demonstrate that expression of PR1 and suppression of PDF1.2 in OX18 plants are mediated by WRKY70 (Figure 7c).

AtMYB44 activates WRKY70 independently of NPR1

WRKY70 is known to be regulated by NPR1 (Li et al., 2004). To define the contribution of AtMYB44 to WRKY70 expression, we generated an atmyb44 npr1-1 double mutant. In wild-type plants, WRKY70 was activated 15 min after SA treatment and increased continuously (Figure 8). By contrast, the increase in WRKY70 expression was slightly delayed in atmyb44 mutants and did not occur at all in atmyb44 npr1-1 double mutants. In npr1-1 mutants, WRKY70 still appeared at an early time point but did not accumulate. These data demonstrate that WRKY70 is regulated through both AtMYB44 and NPR1, but these two factors act independently of each other.

Figure 8.

Effect of atmyb44 or npr1 mutation on expression of WRKY70. Two-week-old Arabidopsis plants were treated with SA, and WRKY70 expression was analyzed by Northern blot. rRNA was visualized by ethidium bromide staining as a loading control.

AtMYB44 binds to the promoter region of WRKY70

AtMYB44 regulates transcriptional activation of WRKY70 (Figures 7 and 8). To test the possibility that AtMYB44 acts as a direct transcriptional activator for WRKY70, we used a GAL4/β-galactosidase assay to determine whether AtMYB44 contains a transcriptional activation domain. Various truncated forms of AtMYB44 were fused to a GAL4 DNA binding domain and tested to determine whether they could activate transcription from a GAL4/β-galactosidase reporter plasmid (Figure S4a). The AtMYB44 C-terminal domain without the DNA binding R2R3 domain showed the highest transcriptional activation activity (Figure S4b). Therefore, AtMYB44 acts as a transcriptional activator of target gene expression. However, transcriptional activation was not observed with full-length AtMYB44 fused to the GAL4 DNA binding domain. This suggests that the structure of the DNA binding domains from two proteins may be affected by juxtaposition, and thus may have lost their DNA binding activities (Gourrierec et al., 1999; : Yu et al., 2011).

To determine the consensus binding sequence of AtMYB44, we performed systematic evolution of ligands by exponential enrichment (SELEX). The core binding sequence of AtMYB44, 5'-CNGTTA-3', was deduced by alignment of the sequences identified by SELEX (Figure 9a). This consensus sequence is similar to the previously reported MYB binding consensus sequence (CNGTTA/G) (Romero et al., 1998).

Figure 9.

AtMYB44 binds to the WRKY70 promoter region. (a) Nucleotide frequency distribution of the AtMYB44 core binding consensus sequence as determined by SELEX. The sizes of the letters represent the relative frequency of occurrence. (b) Electrophoretic mobility shift assay shows binding of the R2R3 domain of AtMYB44 to the WRKY70 promoter region (−381 to −284) in vitro. Probe sequences of the wild-type WRKY70 promoter region (wt) and mutant versions of the promoter (m1, m2 and m3) are shown at the top. Radiolabeled probe was competed for using an excess of unlabeled probe (left panel). Mutant probes were compared with wild-type probe (right panel). (c) Fragmented chromatin DNA of Arabidopsis Col-0 and AtMYB44–GFP over-expressing plants were immunoprecipitated using anti-GFP antibody. For quantitative analysis, PCR products amplified by a primer set containing the AtMYB44 core binding sequence were analyzed by quantitative PCR. The statistical significance of the measurements was determined using Student's t test (*P < 0.01) for comparison with the wild-type value. Values are means ± standard error of the mean of three measurements for each sample. (d) The WRKY70 promoter sequence from −328 to −299 containing the core binding sequence (RE44) or its mutant version (mRE44) was repeated four times and fused to the GUS reporter gene. A cDNA encoding the whole AtMYB44 protein was fused to the CaMV 35S promoter as an effector. The reporter and effector constructs were infiltrated into Nicotiana benthamiana. Transactivation activity was detected by GUS staining assay.

To test binding of AtMYB44 to the WRKY70 promoter region in vitro, the DNA fragment from −381 to −284, which contains the core binding sequence, was selected and tested by electrophoretic mobility shift assay (EMSA). As the full-length protein was not as stable, the AtMYB44 R2R3 DNA binding domain (AtMYB44R2R3) was fused to glutathione S-transferase (GST) and expressed in Escherichia coli for the EMSA. The EMSA showed that AtMYB44R2R3 bound specifically to the probe from the WRKY70 promoter. The GST protein did not bind to the probe containing the AtMYB44 core binding sequence. Binding of AtMYB44 R2R3 to the labeled probe was competed off in the presence of excess unlabeled probe. Probes containing a mutated binding motif did not bind to AtMYB44R2R3 (Figure 9b).

Binding of full-length AtMYB44 to the promoter region of WRKY70 was confirmed using a yeast one-hybrid (Y1H) assay (Figure S5a). Thirty nucleotides from the WRKY70 promoter region (−328 to −299) containing the AtMYB44 core binding sequence were repeated four times and placed upstream of the HIS3 selectable marker gene. Transformation with the core binding site–HIS3 construct and full-length AtMYB44 fused to the GAL4 activation domain made auxotrophic yeast viable on histidine selective medium. However, a mutant version of the promoter fragment did not activate HIS3 in the Y1H assay.

Chromatin immunoprecipitation (ChIP) experiments were used to test whether AtMYB44 binds directly to WRKY70 in vivo. Extracts from plants over-expressing AtMYB44–GFP were subjected to ChIP analysis and compared with wild-type plants. ChIP from AtMYB44–GFP over-expressing plants with anti-GFP antibody showed enrichment of the WRKY70 promoter region containing the AtMYB44 core binding sequence (Figure 9c). A control ChIP product from wild-type plants did not show enrichment of the WRKY70 promoter region. AtMYB44 also bound to an upstream region of the WRKY70 promoter containing the AtMYB44 core binding sequences (sites 1 and 2, Figure S5b). However, three negative control regions that did not contain the core binding sequence also were not enriched by ChIP; these regions include another promoter region (site 3), a coding region (site 5) and part of the 3' UTR of WRKY70 (site 6).

We performed transient GUS assays in Nicotiana benthamiana to confirm transactivation of WRKY70 through the core binding sequence. The WRKY70 promoter sequences from −328 to −299 was repeated four times (4xRE44) and fused to the GUS reporter gene. The reporter plasmid and effector plasmid, 35S:AtMYB44, were co-infiltrated into N. benthamiana. The GUS reporter gene was expressed by co-infiltration of 35S:AtMYB44 with 4xRE44. However, the mutant reporter containing a mutated version of the WRKY70 promoter (4xmRE44) was not expressed (Figure 9d).

Discussion

WRKY70 is regarded as a pivotal regulator in the antagonistic interaction between SA and JA. Activation or suppression of WRKY70 is critical step in developing an effective defense response against pathogen attack. Here we report that AtMYB44 contributes to establishing appropriate plant defense responses by direct regulation of WRKY70 expression in cross-talk between SA and JA.

Role of AtMYB44 in SA- and JA-mediated defense responses

Here we show that AtMYB44 is induced by MeJA (Figure 2). JA signaling is required for disease resistance against necrotrophic pathogens such as A. brassicicola (Thomma et al., 1998; Seo et al., 2001); expression of AtMYB44 was also induced by A. brassicicola (Figure 1c). However, over-expression of AtMYB44 led to increased susceptibility to Abrassicicola by suppression of JA-mediated defense gene expression. By contrast, a knockout mutation of AtMYB44 increased resistance to Abrassicicola by activation of JA-mediated defense gene expression (Figures 1 and 2). In atmyb44 plants, expression of VSP1 and PDF1.2 were also up-regulated without JA treatment (Figure 2). These data indicate that AtMYB44 acts as a negative regulator of JA-mediated defense responses. The negative effects of AtMYB44 over-expression on JA signaling were not limited to the defense response, but also affected JA-mediated root growth inhibition, root hair development (Figure S1) and anthocyanin accumulation (Jung et al., 2010).

AtMYB44 over-expressing plants showed enhanced resistance against a biotrophic pathogen, but atmyb44 knockout plants showed decreased resistance compared to wild-type (Figure 3). Suppression of the JA-mediated defense response was balanced with activation of the SA-dependent defense response (Gupta et al., 2000; Kunkel and Brooks, 2002; Spoel et al., 2003). The enhanced disease resistance established in over-expressing plants was accompanied by activation of SA-dependent PR genes (Figure 4) (Li et al., 2004). Moreover, PR1 was rapidly and strongly activated in AtMYB44 over-expressing plants by exogenous SA treatment (Figure 4). This demonstrates that AtMYB44 acts as a positive regulator of SA-mediated defense responses. Moreover, the antagonistic effect of SA on the JA pathway was reduced in the atmyb44 mutant (Figure S6). Mutual antagonism between JA- and SA-mediated responses is thus observed in over-expression lines and knockout mutants of AtMYB44.

AtMYB44 directly regulates expression of WRKY70

NPR1 and TGA factors directly regulate PR1 expression in SA signaling (Zhang et al., 1999; Spoel et al., 2003). However, data presented here shows that the expression levels of these direct regulators (NPR1 and TGA factors) were not affected by AtMYB44 over-expression (Figures 4a and S3). Our data show that WRKY70 was up-regulated, thus up-regulating PR1 in AtMYB44 over-expressing plants (Figure 5b). PR genes are activated by WRKY70 in SA signaling, and WRKY70 was identified as an important regulatory component in the antagonistic interaction between SA and JA (Li et al., 2004, 2006). Activation of PR genes and suppression of JA-dependent defense genes were reported in WRKY70 over-expressing plants (Li et al., 2004, 2006). AtMYB44 over-expressing plants showed a similar pattern of disease resistance to WRKY70 over-expressing plants; both were resistant to a biotrophic pathogen (Pst DC3000) and susceptible to a necrotrophic pathogen (A. brassicicola). These data show that AtMYB44 modulates SA- and JA-mediated defense responses through WRKY70. This conclusion is also supported by the OX18 wrky70 double mutant phenotype comprising induction of PDF1.2 expression and elimination of PR1 expression (Figure 7c).

Transcription factors regulate target gene expression by binding to promoter regions and interacting with the transcription complex to effect transcriptional activation or repression. By trans-activation analysis, we showed that AtMYB44 acts as a transcriptional activator (Figure S4). AtMYB44 binds to the promoter of WRKY70, which contains the AtMYB44 core binding sequence CNGTTA (Figure 9). ChIP, Y1H and EMSA analyses demonstrate that AtMYB44 binds to the core binding sequence in the WRKY70 promoter. These results show that AtMYB44 directly regulates WRKY70 expression. These results were consistent with elevated WRKY70 expression in AtMYB44 over-expressing plants and β-estradiol-induced trans-activation by AtMYB44 (Figure 7). Transient expression of the GUS reporter driven by the core binding sequence of the WRKY70 promoter provides more evidence that AtMYB44 regulates expression of WRKY70 (Figure 9d).

The enhanced expression of PR genes in OX18 is not a result of increased SA content (Figure 6c). The phenotype is reminiscent of WRKY70 over-expression plants, in which PR1 was constitutively over-expressed without a change in SA content (Li et al., 2004). Even though AtMYB44 directly regulates expression of WRKY70 and PR genes, activation of WRKY70 by AtMYB44 was abolished in the NahG or sid2-2 background (Figure 5b). These results suggest that a basal level of SA may be essential to activate AtMYB44 and WRKY70. There have been reports that expression of WRKY70 was totally abolished in NahG transgenic plants even after SA treatment and biotrophic pathogen infection (Li et al., 2004; Knoth et al., 2007).

AtMYB44 is an NPR1-independent component of SA signaling

WRKY70 is associated with both NPR1-dependent and NPR1-independent pathways in SA signaling (Li et al., 2004). For example, WRKY70 over-expression resulted in activation of PR genes in the npr1-1 mutant background. It has also been reported that NPR1-independent expression of PR genes in snc2-1D npr1-1 was activated through WRKY70 (Zhang et al., 2010). Therefore, WRKY70 can trigger SA-mediated activation of PR genes through an NPR1-independent pathway. Here we show that SA-induced expression of AtMYB44 does not require NPR1 (Figure 5a). By double mutant analysis, we show that activation of WRKY70 and PR1 by AtMYB44 over-expression also did not require NPR1 (Figure 5b). Moreover, NPR1-independent expression of WRKY70 is abolished in the atmyb44 npr1 double mutant (Figure 8). Our data demonstrate that AtMYB44 is an NPR1-independent regulatory component that directly regulates expression of WRKY70. It has been reported that WHIRLY TRANSCRIPTION FACTOR 1 is also induced by SA through an NPR1-independent pathway, which also activates PR1 (Desveaux et al., 2004). Moreover, constitutive expression of PR1 in cpr6 and ssi2 mutants was not diminished in the npr1-1 mutant background (Clarke et al., 1998; Shah et al., 2001). Therefore, an NPR1-independent branch of the SA signaling pathway does exist.

Regulation of defense responses by AtMYB44

The network of SA- and JA-responsive gene expression mediated by AtMYB44 is summarized in Figure 10. Plant resistance is triggered by recognition of the invading pathogen. Plants have developed an effective defense response against pathogen attack by changing the levels of endogenous defense hormones such as SA and JA (De Vos et al., 2005; Koo et al., 2007; Tsuda et al., 2008). AtMYB44 transcripts are detected 12 h after A. brassicicola infection and 6 h after Pst DC3000 infection (Figures 1c and S7). AtMYB44 is induced by SA and directly activates WRKY70, which activates PR genes; SA also independently activates PR genes through NPR1. These SA-dependent signals confer resistance against biotrophic pathogens such as Pst DC3000 (Cao et al., 1997; Li et al., 2004). AtMYB44 is also induced by JA through COI1 (Figure S8). JA also induces expression of JA-responsive genes such as PDF1.2 through COI1, conferring resistance against necrotrophic pathogens (Thomma et al., 1998; Seo et al., 2001). COI1 represses WRKY70, a negative regulator of the JA response, to maintain the transcription of JA-responsive downstream genes (Li et al., 2004). At the same time, JA-induced expression of AtMYB44 activates WRKY70 (Figure S8). The expression of AtMYB44 in response to JA is reminiscent of the JAZ repressor genes, which are induced in response to JA (Chini et al., 2007; Thines et al., 2007).

Figure 10.

Role of AtMYB44 in antagonistic interaction between SA- and JA-mediated defense signaling. In the SA-mediated defense response, AtMYB44 is induced by NPR1-independent SA signaling. Expressed AtMYB44 regulates activation of PR1 and suppression of JA-mediated defense genes (VSP1 and PDF1.2) by direct transcriptional activation of WRKY70. JA-mediated expression of AtMYB44 occurs through a COI1-dependent pathway. AtMYB44 activated by JA acts as a negative regulator to fine-tune the JA signal. COI1 also negatively regulates WRKY70 (Li et al., 2004).

Plants pay significant costs to activate and maintain defense responses. For example, SA mutants in which defense genes are constitutively activated show growth retardation (Bowling et al., 1997; Clarke et al., 1998; Li et al., 2004). Therefore, the plant defense response must be under tight and finely tuned regulation (Spoel et al., 2003; Moore et al., 2011). Induction of a negative regulator in response to signal molecules contributes to fine-tuning of defense responses (Journot-Catalino et al., 2006; Chini et al., 2007). Thus, the biological role of AtMYB44 may be to fine-tune JA-mediated defense signals for balanced allocation of resources in plant defense responses (Spoel et al., 2003; Journot-Catalino et al., 2006). This possibility is supported by constitutive expression of JA-responsive genes in atmyb44 knockout mutants (Figure 2). AtMYB44 acts as a point of intersection for coordination of signals from JA and SA to allow cross-talk.

The function of AtMYB44 in promoting the SA signal may be counter-balanced by the function of the other WRKYs promoting the JA signal. For example, WRKY7, WRKY8, WRKY11 and WRKY17 are induced by the biotrophic pathogen Pst DC3000, but these WRKY transcription factors suppress the SA-mediated defense response (Kim et al., 2006; Chen et al., 2010). Furthermore, WRKY11 and WRKY17 up-regulate expression of JA biosynthesis genes such as LOXII and AOS (Journot-Catalino et al., 2006). These findings also support the occurrence of fine-tuned regulation of the defense response.

Another potential role of AtMYB44 in the plant defense response was also described previously. Upon activation by Agrobacterium or pathogen-associated molecular patterns, mitogen-activated protein kinase 3 (MPK3) phosphorylates VirE2 interacting protein 1 (VIP1). Activated VIP1 regulates the expression of PR1 (Djamei et al., 2007). AtMYB44 was shown to be a direct target of VIP1 (Pitzschke et al., 2009). It has also been reported that AtMYB44 is phosphorylated by MPK3 (Nguyen et al., 2012). These results suggest that AtMYB44 and WRKY70 may mediate activation of PR1 by VIP1.

In summary, we examine here the function of AtMYB44 in defense responses. The differential modulation of SA- and JA-mediated defense responses by AtMYB44 provides evidence that AtMYB44 is a regulatory component in the antagonistic interaction between the SA and JA signaling pathways.

Experimental Procedures

Plant materials and growth conditions

Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col-0) was used throughout this study. The 35S:AtMYB44, 35S:AtMYB44-GFP and T-DNA insertion atmyb44 mutants (SALK_039074) have been described previously (Jung et al., 2008). The Arabidopsis sid2-2 mutant, the transgenic line expressing NahG, and the coi1-1 mutant were kindly provided by Frederic M. Ausubel (Harvard Medical School, Boston, MA), J. Ryals (Ciba-Geigy Agricultural Biotechnology, Research Triangle Park, NC) and J. Turner (University of East Anglia, Norwich, UK), respectively. The Arabidopsis jar1-1, atmyc2, npr1-1 and wrky70 mutants were obtained from the Arabidopsis Biological Resource Center (stock numbers CS8072, SALK_061267, CS3726 and SALK_025198, respectively). Strains of Pst DC3000 and Abrassicicola were kindly provided by Ingyu Hwang and Yong Hwan Lee, respectively (Seoul National University, Seoul, Korea).

Plants were grown on soil or half-strength Murashige and Skoog (MS)/agar plates in a growth chamber maintained at 22°C and 60% relative humidity under long-day conditions (16 h light/8 h dark cycle).

To examine the effect of plant hormones on gene expression, a solution of 50 μm MeJA (Sigma, http://www.sigmaaldrich.com) or 1 mm SA (Sigma) was applied to the surface of MS/agar plates in which 2-week-old seedlings were growing. For inducible expression of AtMYB44, an AtMYB44 cDNA was inserted into the XhoI and SpeI sites in the pER8 vector (Zuo et al., 2000). A full-length AtMYB44 cDNA (EST 119B8) was obtained from the Arabidopsis Information Resource. Twelve-day-old transformants grown on MS/agar plates were transferred to medium containing 5 μm β-estradiol for induction of AtMYB44.

Plant transformation and analyses of transgenic plants

Agrobacterium tumefaciens strain C58C1 containing plasmid constructs was used to transform plants by the floral-dip method (Clough and Bent, 1998). Transgenic plants were identified by selection on half-strength MS agar medium containing 20 μg ml−1 hygromycin (Duchefa, http://www.duchefa-biochemie.nl/).

Analysis of transcript levels

For Northern blot analysis, total RNA was extracted from frozen sample using the phenol/SDS/LiCl method (Carpenter and Simon, 1998). Total RNA (5 μg) was separated on 1.3% formaldehyde agarose gels and transferred to Genescreen Plus hybridization transfer membranes (Perkin-Elmer, http://www.perkinelmer.com/). [α-32P]-labeled cDNA probes containing gene-specific sequences were hybridized to detect signal.

For quantitative real-time PCR, a SYBR kit (Philekorea, http://www.philekorea.co.kr/) was used. Forty cycles of amplification (15 sec at 95°C, annealing for 60 sec at 68°C) after an initial step (10 min at 95°C) were performed in a Rotor-gene 2000 (Corbett, http://www.corbettlifescience.com). Primer sequences are listed in Table S1. Actin1 was included in the assay for normalization. The quantitative real-time PCR reactions were performed using two or three biological and three technical repeats. The comparative ΔΔCT method was used for relative quantification of each amplified product.

Pathogen infection assay

A. brassicicola was cultivated on potato dextrose agar plates. Preparation and inoculation of fungi were performed as described previously (Li et al., 2006). Relative fungal DNA levels of A. brassicicola were determined by quantitative real-time PCR using primers specific for genomic 5.8S ribosomal RNA (GenBank accession number U05198) and primers for Arabidopsis genomic ACT2.

Pst DC3000 was grown in King's B medium (King et al., 1954) and adjusted to 5 × 105 cfu ml−1 in 10 mm MgCl2 solution. Inoculation of Pst DC3000 and determination of bacterial growth were performed as described previously (Li et al., 2004).

Quantification of SA and glucosylated SA

SA and glucosylated SA were extracted from the leaves and quantified as described previously (Koo et al., 2007). Extracts were separated using a Symmetry C18 HPLC column (4.6 mm internal diameter, 15 cm long, particle size 5 μm; Waters, http://www.waters.com) in a LC-6A HPLC (Shimadzu, http://www.shimadzu.com/), and quantified using an RF-10A XL fluorescence detector (excitation 301 nm, emission 412 nm; Shimadzu). The amount of glucosylated SA was quantified by the difference between SA quantities with and without glucosidase treatment (160 units for 60 min at 37°C, Sigma). To monitor sensitivity and recovery, a known amount of SA was added to the sample and analyzed in parallel. The measurements for three biological replicates were averaged.

Systematic evolution of ligands by exponential enrichment (SELEX)

SELEX was performed as previously described (Grotewold et al., 1994). After five rounds of selection, the amplified DNA was inserted into a pGEM-TEasy vector (Promega, http://www.promega.com/), and nucleotide sequences of 36 DNA fragments were determined using an ABI PRISM® 377 DNA sequencer (Applied Biosystems, http://www.appliedbiosystems.com). The obtained sequences were aligned and visualized using the Weblogo package (Crooks et al., 2004; http://weblogo.berkeley.edu/).

Electrophoretic mobility shift assay (EMSA)

The R2R3 domain of AtMYB44 (amino acid residues 1–111) was fused with the GST coding sequence through the BamHI and EcoRI sites of the pGEX-5x-1 expression vector (GE healthcare, http://www.gelifesciences.com). The GST–AtMYB44 fusion protein was purified according to the manufacturer's instructions. DNA fragments labeled with [α-32P]dCTP were incubated with 0.5 μg of purified GST–AtMYB44 protein for 20 min at 23°C in 25 μl binding buffer (20 mm HEPES pH 7.8, 50 mm KCl, 1 mm EDTA, 0.5 mm dithiothreitol, 5 μg BSA, 200 μg poly[dI-dC] and 10% glycerol). The reaction mixture was separated on 6% gels by native PAGE.

Chromatin immunoprecipitation

Two-week-old 35S:AtMYB44-GFP transgenic plants grown on MS/agar plates were used for chromatin immunoprecipitation (ChIP). ChIP assays were performed as described previously (Saleh et al., 2008). Fragmented chromatin was immunoprecipitated using anti-GFP antibody (Clontech, http://www.clontech.com/). DNA extracts separated from the DNA–protein complex were used for quantitative real-time PCR analysis. The primer sets used in this analysis amplify various regions of the WRKY70 locus (Figure S5b and Table S1). The ChIP experiments were performed three times.

Transient GUS assay by agroinfiltration of Nicotiana benthamiana

Agrobacteria were infiltrated into intact leaves of Nicotiana benthamiana as previously described (Kane et al., 2007). After infiltration, plants were kept at 24°C for 3 days. Histochemical GUS assays were performed as previously described (Jung et al., 2008).

Acknowledgements

This work was supported by a grant from the Next-Generation BioGreen 21 Program (project numbers PJ008053 to Y.D.C. and PJ007971 to J. -K.K.), Rural Development Administration, Republic of Korea through the National Center for GM Crops. A graduate research assistantship to J.S.S. and S.J.L. from the Brain Korea 21 project of the Ministry of Education, Science and Technology is also acknowledged.

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