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The Arabidopsis (Arabidopsis thaliana) transcription factor WRKY33 is essential for defense toward the necrotrophic fungus Botrytis cinerea. Here, we aimed at identifying early transcriptional responses mediated by WRKY33. Global expression profiling on susceptible wrky33 and resistant wild-type plants uncovered massive differential transcriptional reprogramming upon B. cinerea infection. Subsequent detailed kinetic analyses revealed that loss of WRKY33 function results in inappropriate activation of the salicylic acid (SA)-related host response and elevated SA levels post infection and in the down-regulation of jasmonic acid (JA)-associated responses at later stages. This down-regulation appears to involve direct activation of several jasmonate ZIM-domain genes, encoding repressors of the JA-response pathway, by loss of WRKY33 function and by additional SA-dependent WRKY factors. Moreover, genes involved in redox homeostasis, SA signaling, ethylene-JA-mediated cross-communication, and camalexin biosynthesis were identified as direct targets of WRKY33. Genetic studies indicate that although SA-mediated repression of the JA pathway may contribute to the susceptibility of wrky33 plants to B. cinerea, it is insufficient for WRKY33-mediated resistance. Thus, WRKY33 apparently directly targets other still unidentified components that are also critical for establishing full resistance toward this necrotroph.
Plants are constantly challenged by pathogens, but only a few are capable of successfully colonizing a specific host, suggesting the existence of potent recognition and defense mechanisms. In general, there are two types of microbial pathogens that grossly differ in their lifestyles (Glazebrook, 2005). Biotrophic pathogens require living plant cells for growth and reproduction. They tend to keep the colonized plant tissue widely intact for long-term nutritional benefits. By contrast, necrotrophic pathogens need to kill plant cells to make nutrients accessible for growth and for completion of their life cycles. Whereas one general defense strategy of plants against biotrophic pathogens is to sacrifice infected cells by triggering cell death, the maintenance of plant cell viability is generally the defense strategy against necrotrophic pathogens (Spoel et al., 2007).
To discriminate between these distinctive pathogens and to activate appropriate responses, plants use phytohormones for signaling (Pieterse et al., 2009). Studies using transgenic and mutant lines impaired in hormone biosynthesis, perception, or signaling revealed that responses against biotrophic pathogens are in general regulated by salicylic acid (SA; Vlot et al., 2009), while responses to necrotrophs are mediated by jasmonic acid (JA) and ethylene (ET; Farmer et al., 2003). In case of multiple infections or when plants were treated simultaneously with methyl jasmonate and SA, substantial cross talk between the SA and JA-response pathways was observed (Spoel et al., 2007; Koornneef et al., 2008). Often, the SA pathway was shown to be dominant and to suppress the JA pathway. However, synergistic effects between both pathways have been reported when low concentrations of SA and JA were applied, indicating that the concentration and the balance of these hormones as well as their temporal activation kinetics may be decisive (Koornneef and Pieterse, 2008; Leon-Reyes et al., 2010). A recent comprehensive study revealed that synergistic and compensatory relationships exist among the response pathways and that they are important for optimal resistance to different pathogens (Tsuda et al., 2009).
Nectrotrophic fungi including Alternaria brassicicola, Botrytis cinerea, Fusarium oxysporum, and Sclerotinia sclerotiorum are the largest class of fungal plant pathogens and cause serious crop losses worldwide (Łaźniewska et al., 2010). In contrast to numerous biotrophic pathogens, specific recognition of necrotrophic pathogens by products of major plant resistance (R) genes is currently unknown. Indeed, the resistance of Arabidopsis (Arabidopsis thaliana) to B. cinerea appears to be under complex genetic control (Rowe and Kliebenstein, 2008). Numerous genetic and pharmacological studies have uncovered plant genes and products that influence the outcome of host-B. cinerea interactions. These include enzymes of cutin biosynthesis and secondary cell wall formation (Hernández-Blanco et al., 2007; Tang et al., 2007; Voisin et al., 2009; Raiola et al., 2011), the receptor-like kinase BIK1 (Veronese et al., 2006), the TIR domain-encoding gene RLM3 (Staal et al., 2008), the mitogen-activated protein kinase MPK3 (Ren et al., 2008), the RING E3 ligases HUB1 and BOI (Dhawan et al., 2009; Luo et al., 2010), and several autophagy genes (Lai et al., 2011; Lenz et al., 2011). Certainly, one critical component appears to be the phytoalexin camalexin, because plants deficient in CYP71B15 (PAD3), which catalyzes the last step in camalexin biosynthesis, are highly susceptible to B. cinerea (Ferrari et al., 2007).
Global transcriptional profiling studies also revealed that B. cinerea infection of Arabidopsis leaves results in massive transcriptional reprogramming in the host, indicating the involvement of key regulators in this process (AbuQamar et al., 2006; Ferrari et al., 2007; Rowe et al., 2010). As expected, several transcription factors (TFs) have been identified that positively or negatively affect the outcome of such an interaction. Loss of function of the helix-loop-helix Leu zipper TF MYC2, which antagonistically regulates two distinct branches of the JA pathway, rendered plants more resistant toward B. cinerea (Lorenzo et al., 2004). A similar resistant phenotype was observed in mutant plants defective in MYB46 function and in the double mutant anac019 anac055 encoding two NAC family proteins (Bu et al., 2008; Ramírez et al., 2011). In contrast, loss-of-function mutants of MYB108/BOS1 and of EIN3, encoding an AP2-type TF involved in ET signaling, showed enhanced susceptibility to B. cinerea infection (Mengiste et al., 2003; Van Baarlen et al., 2007). Moreover, overexpression of the AP2-type TF genes ERF1 and ORA59 rendered plants more resistant toward several necrotrophs, including B. cinerea (Berrocal-Lobo et al., 2002; Pré et al., 2008). ERF1 and ORA59 both appear to be key integrators of the JA and ET signaling pathways (Pieterse et al., 2009; Leon-Reyes et al., 2010; Zarei et al., 2011). In some cases, however, contradictory phenotypic results have been reported. This can be partly explained by the different experimental setups used, but recent reports also draw attention to the fact that they may also be due to isolate-specific differences of this pathogen (Rowe and Kliebenstein, 2010; Rowe et al., 2010).
The WRKY family of TFs also modulates host defenses toward various phytopathogens (Pandey and Somssich, 2009). In particular, WRKY33 was shown to be required for resistance toward the necrotrophs A. brassicicola and B. cinerea (Zheng et al., 2006). In that previous study, negative regulation of the SA pathway and positive regulation of the JA pathway by WRKY33 were concluded based on the higher expression of PR genes and the lower expression of JA-response genes in wrky33 compared with wild-type plants upon B. cinerea challenge. Clues as to how WRKY33 may transduce pathogen-triggered host signals to the nucleus were provided by work showing that WRKY33 interacts with MPK4 and the protein MKS1 within the nucleus and that, upon challenge with the hemibiotrophic pathogen Pseudomonas syringae or upon elicitation by the microbe-associated molecular pattern flg22, WRKY33 is released from the trimeric complex and subsequently binds to the PAD3 promoter (Andreasson et al., 2005; Qiu et al., 2008). Whether the same signal transduction cascade is utilized during infections with necrotrophic pathogens remains uncertain, since recent studies imply that WRKY33 is phosphorylated by the mitogen-activated protein kinases MPK3 and MPK6 in vivo upon B. cinerea infection (Mao et al., 2011). Finally, ATG18a, a critical autophagy protein, was also shown to interact with WRKY33 within the cell nucleus (Lai et al., 2011).
In this study, we performed a detailed transcriptomic analysis in order to obtain a deeper view of the transcriptional responses mediated by WRKY33 upon challenge with B. cinerea. We identified numerous genes encoding TFs, components of the SA, JA, and ET signaling pathways, and redox regulators that were misexpressed in the wrky33 mutant. By chromatin immunoprecipitation (ChIP), several direct in vivo target genes of WRKY33 were identified. In infected wrky33 plants, the SA response pathway was inappropriately activated, which later during infection resulted in SA-mediated repression of JA responses. This repression involved deregulated expression of the WRKY33 direct target genes TRX-H5, jasmonate ZIM-domain1 (JAZ1), JAZ5, ORA59, and GLIP1. Additional genetic studies, however, showed that down-regulation of JA responses by the premature activation of SA-mediated defenses on its own was insufficient to lead to susceptibility in wrky33 plants. Rather, WRKY33 appears to regulate the expression of various distinct components of defense pathways that are crucial in activating appropriate host responses toward B. cinerea.
Transition from Nonaggressive to Aggressive Growth of B. cinerea on the wrky33 Mutant
WRKY33 is involved in the regulation of defense against the necrotrophic fungi A. brassicicola and B. cinerea (Zheng et al., 2006). To analyze differences in the development of B. cinerea isolate 2100 on wrky33 compared with wild-type Columbia-0 plants, we collected spray-inoculated leaves at time points 16, 24, 40, 48, and 64 h post inoculation (hpi) and stained them either with 3,3-diaminobenzidine (DAB) to visualize hydrogen peroxide (H2O2) accumulation, a major component of reactive oxygen species (ROS), or with trypan blue to detect the fungus and dying host cells. Mock-treated leaves revealed no cell death or H2O2 production for both genotypes (Supplemental Fig. S1). On infected leaves, spotty trypan blue staining, indicative of dying cells, was clearly visible at 40 hpi predominantly in mesophyll cells of the wild type and more pronounced in wrky33. Microscopically, we could not detect significant differences in B. cinerea development between the two genotypes up to 40 hpi (Fig. 1, A–D). The spores germinated with the same high efficiency (nearly 100%), and hyphal growth was indistinguishable. Later than 40 hpi, the fungus switched from nonaggressive to aggressive growth only in infected wrky33 leaves, manifested by a boost of hyphal growth that gave rise to rapidly expanding lesions on the leaves. In wild-type plants during the same period, necroses became visible, which were associated with fungal growth arrest (Fig. 1, E and F). Conidiophores appeared 3 dpi only on the susceptible wrky33 plants. Accumulation of H2O2 in leaves was observed in both the wild type and wrky33 already at 16 hpi, mainly in the mesophyll cells. Moreover, consistent with findings by Temme and Tudzynski (2009), the fungus itself exhibited strong DAB staining in both genotypes up to 24 hpi, indicating that it produced ROS (Fig. 1, G and H).
Temporal Expression of WRKY33 during B. cinerea Infection
In order to monitor the WRKY33 protein in subsequent experiments, a complementation line was created expressing a WRKY33 genomic construct with a C-terminal hemagglutinin (HA) tag under the control of its native promoter in the wrky33 mutant (designated WRKY33-HA). Upon droplet inoculation with B. cinerea spores, this line exhibited the same resistance as wild-type plants, validating its functionality (Supplemental Fig. S2).
To identify changes in the transcriptome related to WRKY33 action during infection with B. cinerea, we first investigated the expression profile of WRKY33 itself. Samples from wild-type and wrky33 spray-inoculated or mock-treated plants taken at various time points were analyzed by quantitative real-time (qRT)-PCR. In the wild type, compared with its basal levels in mock-treated plants, WRKY33 RNA levels were induced about 6-fold at 8 hpi and increased further during the course of infection (Fig. 2A). In the wrky33 mutant, no WRKY33 transcript was detected.
The appearance and stability of the WRKY33 protein during infection were determined by western blotting using total leaf extracts of the spray-inoculated WRKY33-HA complementation line (Fig. 2B). WRKY33-HA protein was clearly detectable at 8 hpi, consistent with the onset of induction at the RNA level. Maximal protein accumulation was observed at 14 hpi and remained constant until 24 hpi, after which it decreased. Induction of WRKY33 mRNA upon B. cinerea inoculation after 8 h, and highest accumulation of the corresponding protein at 14 hpi, led us to set the time point for subsequent global expression profiling to 14 hpi.
B. cinerea Infection Massively Alters Gene Expression in Wild-Type and wrky33 Plants
To identify genes involved in the WRKY33-dependent response to B. cinerea on a genome-wide level, we used Arabidopsis whole genome (ATH1) microarrays. For this, total RNAs derived from wild-type or wrky33 plants 14 h after spray inoculation or mock treatment were compared. At this infection stage, B. cinerea conidia had already germinated but the hyphae were still only in the process of elongation and no infection hyphae were yet detected. In wild-type plants, the expression of 1,577 genes was altered 2-fold or more (P ≤ 0.05) compared with noninfected plants, with 1,154 genes being up-regulated and 423 genes being down-regulated. In wrky33, upon infection, the expression of 2,387 genes, 1,369 up-regulated and 1,018 down-regulated, was altered more than 2-fold, with a common set of 1,379 genes showing similar changes upon infection in wild-type and wrky33 plants (Supplemental Table S1). While upon infection the expression of 198 genes was changed only in the wild type, differential expression of 1,008 genes was triggered by the loss of WRKY33 function. Comparing the expression profiles from noninfected plants identified 309 genes that were constitutively differentially expressed between the wild type and wrky33, 212 of them up-regulated and 97 down-regulated (Fig. 3). Based on Gene Ontology terms, genes differentially expressed in the wild type and wrky33 upon infection were clearly overrepresented in the categories responsive to stress and responsive to abiotic or biotic stimuli (Supplemental Table S2).
The broad reprogramming of the host transcriptome at this early infection stage (14 hpi) suggested the involvement of many TFs. Indeed, B. cinerea infection altered the expression levels of 110 and 160 TF genes more than 2-fold in the wild type and wrky33, respectively, with an overlap of 97 TF genes affected in both (Supplemental Table S3). TF families described to be involved in pathogen defense (Libault et al., 2007) were similarly represented in both genotypes. Most prominent were genes encoding AP2-ethylene-responsive element-binding proteins, NAC TFs, and WRKY TFs (Supplemental Table S3). Among the 34 TF genes differentially expressed in infected wrky33 mutants relative to wild-type plants were, in particular, the AP2-ERF TF genes ERF2, CEJ1, and ORA59, which are involved in the regulation of JA and ET defense genes (McGrath et al., 2005), and CBF1 and CBF2, involved in the cold stress response (Novillo et al., 2007). Also included were the NAC genes NAC016, NAC036, and NAC090, WRKY18, WRKY38, and WRKY54, which are positive regulators of SA-mediated responses downstream of NPR1 (Wang et al., 2006), WRKY46 and WRKY60, and MYB2, which is responsive to dehydration and abscisic acid (Abe et al., 2003). WRKY50 and WRKY51, not represented on the ATH1 microarray but recently shown to negatively influence the outcome of Arabidopsis-B. cinerea interactions (Gao et al., 2011), were analyzed by qRT-PCR and found to be also differentially expressed (Supplemental Fig. S3). Except for ERF2 and ORA59, which appeared to be positively controlled by WRKY33, all other mentioned TF genes were up-regulated upon infection in the mutant, suggesting that they are under direct or indirect negative regulation by WRKY33 (Supplemental Table S3).
In wrky33 the JA/ET Signaling Pathways Are Functional Early during B. cinerea Infection but the JA Response Is Repressed at Later Stages
Our microarray studies revealed that genes involved in the biosynthesis of JA as well as the JA-response genes were up-regulated at 14 hpi in the wild type and wrky33 (Table I). There was no significant difference between wild-type and wrky33 plants, with the exception of the defensin PDF1.1 and the LIPOXYGENASE2 genes, whose mRNA levels were higher in wild-type plants.
To verify and extend the expression data for selected genes, total RNA samples collected at different time points after inoculation were subjected to qRT-PCR analysis. The induction profiles for two genes encoding key enzymes in JA biosynthesis, AOC3 and OPR3, showed no differences between the wild type and wrky33 up to 14 hpi (Fig. 4A). However, at 24 hpi, when fungal growth was still indistinguishable between the wild type and the mutant, there was a significant increase in the expression of both genes only in the mutant. While expression of JAR1, encoding the enzyme that converts JA to the bioactive JA-Ile conjugate, and the regulatory gene COI1 was similar in both genotypes and not affected by inoculation, the JA-response genes PDF1.1 and PDF1.2 were strongly up-regulated at 24 hpi only in the wild type. PDF1.1 and PDF1.2 expression levels were clearly also induced in wrky33 at 14 hpi but returned to their basal levels thereafter (Fig. 4A). Together, these data show that up to 14 hpi, the JA pathway was similarly induced in both genotypes, while later during B. cinerea inoculation, the JA response was repressed in the wrky33 mutant.
To investigate if enhanced expression of the JA biosynthetic enzymes led to higher JA levels in the infected mutant plants, we determined the amounts of free JA and the bioactive JA-Ile conjugate in leaves from infected wild-type and wrky33 plants (Fig. 4B). Prior to inoculation, the levels of both substances were low and nearly identical in the wild type and wrky33. At 24 h after inoculation, there was a 3-fold increase in JA in the wild type but a more than 6-fold increase in wrky33. For JA-Ile, a slight increase was observed upon infection, with similar levels reached in both genotypes. These data show that the repression of the JA response in wrky33 occurred downstream of JA biosynthesis. The fact that in wrky33 the JA-Ile levels were not higher than in the wild type, despite higher JA levels, may be due to the altered turnover of JA-Ile in the mutant, as has been reported for coi1 in wild tobacco (Nicotiana attenuata; Paschold et al., 2008).
Along with the down-regulation of the JA response in wrky33, a significant increase of transcripts encoding the jasmonate ZIM-domain proteins JAZ1, JAZ5, JAZ7, JAZ8, and JAZ10 was observed (Fig. 5). These proteins serve as negative regulators of the JA-signaling pathway (Chini et al., 2007). We performed ChIP using the WRKY33-HA complementation line in combination with qPCR to investigate direct WRKY33 binding to the promoter regions of these JAZ genes. By employing primer sets, representing different regions within their promoters and in the coding regions, we could detect significant B. cinerea-dependent binding to distinct promoter regions of JAZ1 and JAZ5 that contain varying numbers of WRKY factor-binding sites (W-boxes; Fig. 6, A and B), suggesting direct negative regulation of these JAZ genes by WRKY33 upon infection. For JAZ8 and JAZ10, no WRKY33-specific binding was observed (Fig. 6, C and D), while JAZ7 does not contain W-boxes in its promoter region. However, ChIP qPCR performed using an antibody raised against the conserved WRKY domain (anti-all WRKY) that recognizes many different WRKY proteins (Turck et al., 2004) revealed additional occupancy of all four JAZ promoters by other WRKY factors. In the case of JAZ8 and JAZ10, the binding of these WRKY proteins was clearly enhanced upon B. cinerea infection (Fig. 6, C and D). For JAZ1 and JAZ5, the WRKY33-specific promoter-binding sites appear to be occupied to some extent by other WRKY factors prior to infection in both genotypes (Fig. 6, A and B), an observation also found in parsley (Petroselinum crispum) for the PcWRKY1-binding site within the PcPR1 promoter and indicative of a complex and dynamic WRKY factor occupancy network (Turck et al., 2004).
Interaction between the JA and ET pathways can be synergistic (Lorenzo et al., 2003). In our infection experiments, at 14 hpi, ET responses, as indicated by up-regulation of many ET response factor (ERF) genes and the gene encoding the ET biosynthesis enzyme 1-aminocyclopropane-1-carboxylic acid synthase, were clearly induced, with only small differences observed for some transcript levels between the wild type and wrky33 (Table I). Upon B. cinerea infection, expression of the ERF gene ORA59 was induced in the wild type and wrky33, with a peak at 14 hpi. Its expression remained elevated in the wild type thereafter, while in wrky33, it returned to noninduced levels (Fig. 7A). This is consistent with the role of ORA59 as an activator of JA-response gene expression and could be one reason for the high JA response observed later than 14 hpi only in the wild type. The ORA59 promoter contains numerous W-boxes, and WRKY33 may prolong expression of the gene after initial induction. To test whether WRKY33 directly targets ORA59, we performed ChIP qPCR and observed strong B. cinerea-induced binding to two distinct W-box-containing promoter regions (Fig. 7C).
Another ET pathway-associated gene, GDSL LIPASE1 (GLIP1), shown to be involved in defense against the necrotroph A. brassicicola (Oh et al., 2005), was significantly higher induced upon B. cinerea infection in the wild type (Fig. 7B). ChIP PCR experiments revealed strong inducible WRKY33 binding to one W-box-containing site 3.5 kb upstream from the translation start site of GLIP1 (Fig. 7D).
In summary, the expression of several genes encoding components associated with JA and ET signaling is modulated by WRKY33 during B. cinerea infection. Of these, JAZ1, JAZ5, ORA59, and GLIP1 were found to be direct in vivo targets of WRKY33, with the first two likely being negatively regulated and the latter two positively regulated by WRKY33.
WRKY33 Is a Negative Regulator of the SA Pathway upon B. cinerea Infection
Most of the genes associated with the SA signaling pathway were higher expressed in leaves of the wrky33 mutant compared with wild-type plants at 14 hpi. This holds true for the genes encoding the key SA biosynthesis enzyme ICS1/SID2, the positive regulatory proteins EDS1, PAD4, EDS5/SID1, FMO1, and PBS3, the negative regulatory protein NIMIN1, and the SA response genes PR1, PR2, and PR3 (Table I).
For selected genes, we performed qRT-PCR to get a better understanding of their temporal expression behavior during the infection process. The data revealed that beyond 14 hpi, the expression of ICS1, PAD4, and PBS3 as well as PR1 and PR2 became strongly enhanced in wrky33 compared with wild-type plants, indicating that the loss of WRKY33 function abolished negative control over genes involved in the SA signaling pathway (Fig. 8A). Comparing the basal expression levels of SA-associated genes in uninfected plants revealed no significant differences between the wild type and wrky33.
We also determined the SA levels from infected plants of both genotypes. While prior to inoculation the levels of the bioactive free SA were nearly the same in wild-type and wrky33 plants, a stronger accumulation of SA was observed in wrky33 during infection, reaching three times higher levels in the mutant at 24 hpi. Similarly, four times higher levels of total SA were observed in the wrky33 mutant at 18 hpi (Fig. 8B). These results are consistent with the high induction of ICS1 observed in the mutant. Together, these data show that upon B. cinerea infection in wild-type plants, WRKY33 is involved in preventing the inappropriate induction of the SA pathway.
Production of Camalexin upon B. cinerea Infection Is Delayed in wrky33
Previous studies showed that wrky33 plants inoculated with B. cinerea accumulated significantly lower levels of the phytoalexin camalexin than wild-type plants (Qiu et al., 2008; Mao et al., 2011). Since WRKY33 is a positive regulator of the camalexin biosynthetic genes PAD3 (CYP71B15) and CYP71A13, and the pad3 mutant is similarly susceptible to this isolate of B. cinerea as wrky33 (Supplemental Fig. S4), the susceptibility of wrky33 may be solely due to a lack of camalexin. Thus, we determined the camalexin levels of wild-type, wrky33, and pad3 plants during the infection process (Fig. 9A). While in uninfected plants of all three genotypes and in infected pad3 almost no camalexin was detectable at 24 hpi, in the wild type, about three times higher camalexin accumulation was found than in wrky33. At 48 hpi, a strong increase in camalexin in both genotypes was observed, but at this stage, significantly higher levels accumulated in wrky33 plants.
The delay in camalexin biosynthesis in wrky33 early during infection and the strong increase at later time points are reflected at the mRNA levels for CYP71A13 and PAD3 (Fig. 9B). At 14 hpi, transcript levels of both genes in wrky33 were significantly lower than in the wild type, while at 24 hpi, when fungal growth was still indistinguishable in both genotypes, there was a sharp increase of both transcripts in wrky33. This increase very likely resulted in elevated camalexin levels, since PAD3 transcript and camalexin accumulation are highly correlated (Zhou et al., 1999).
Direct WRKY33 binding to the PAD3 promoter at around −500 bp (relative to the translation start site) has been shown previously (Qiu et al., 2008; Mao et al., 2011). Our ChIP analyses revealed an additional stronger WRKY33-binding site within the PAD promoter at about −1,800 bp at 24 hpi (Fig. 9C). Binding to both sites was clearly induced by fungal infection. For CYP71A13, we observed strong inducible binding at −2,800 bp, again indicating direct positive regulation by WRKY33. To test if the later increase in camalexin levels in wrky33 was related to the binding of other WRKY factors in the absence of WRKY33, we performed ChIP using the anti-all WRKY antibody. With this antibody, additional WRKY factors binding to the WRKY33-specific sites were detected not only in the wild type but also in wrky33 plants (Fig. 9C, bottom panels). This suggests that other WRKY factors play a partly redundant function in regulating camalexin biosynthesis, particularly in the absence of WRKY33.
Expression of Genes Required for Redox Homeostasis upon B. cinerea Infection
Another group of host genes affected by B. cinerea infection code for proteins involved in the regulation of cellular redox homeostasis. Among them were several up-regulated glutathione S-transferases, FAD-binding domain proteins, redoxins, reductases, and peroxidases (Supplemental Table S1). Moreover, the expression of RBOHD, the main cytosolic stress-induced ROS producing NADPH oxidase, recently shown to be an essential component in rapid systemic ROS signaling (Miller et al., 2009), was 3- to 5-fold elevated in the wild type and wrky33 upon B. cinerea infection. Altogether, 71 of the 81 genes previously reported to be induced in an RBOHD-dependent manner (Miller et al., 2009) were also up-regulated 14 h after B. cinerea inoculation in our experiments (Supplemental Table S1). With few exceptions, the induction of these genes was independent of WRKY33. This suggests that systemic ROS signaling, which is independent of SA, JA, and ET (Miller et al., 2009), is triggered upon B. cinerea infection, possibly also due to the observed ROS production by the fungus (Fig. 1).
Relevant to WRKY33 function were the redox-related genes differentially expressed between wrky33 and the wild type. Among them were the glutaredoxins GRX480 and GRXS13, thioredoxin TRX-H5, thioredoxin-dependent peroxidase TPX2, monodehydroascorbate reductase MDAR3, and cytokinin oxidase CKX4, all related to ROS signaling and scavenging (Van Breusegem et al., 2008). Upon B. cinerea inoculation, significantly higher expression levels of GRX480, TRX-H5, CKX4, and MDAR3 were observed in wrky33 compared with the wild type, indicating negative regulation by WRKY33 (Fig. 10A). Contrarily, TPX2 was much less induced in wrky33, suggesting positive regulation by WRKY33. In B. cinerea-infected WRKY33-overexpressing plants, the expression of GRX480, TRX-H5, CKX4, and MDAR3 was close to wild-type levels while the expression of TPX2 was massively enhanced, consistent with negative and positive regulation by WRKY33, respectively (data not shown).
We performed ChIP using the WRKY33-HA complementation line to investigate direct WRKY33 binding to the promoter regions of these genes. While for GRX480, CKX4, MDAR3, and TPX2 the data were inconclusive, significant B. cinerea-induced WRKY33 binding to a defined W-box-bearing TRX-H5 promoter region was observed (Fig. 10B). This suggests direct B. cinerea-dependent negative regulation of TRX-H5 by WRKY33.
Genetic Analysis of WRKY33-Regulated Genes
We tested mutants in hormone signaling, redox homeostasis, and antimicrobial metabolite biosynthesis with B. cinerea isolate 2100 and monitored susceptibility toward this fungus (Supplemental Fig. S4). Mutants in SA biosynthesis and signaling, npr1-1, sid2-1, eds1-1, and pad4-1, were nearly as resistant as the wild type, consistent with previous reports (Ferrari et al., 2003). Interestingly, the mutants opr3, jar1-1, jrg21, and ein2, corresponding to genes associated with the JA and ET pathways, were also resistant. These findings partly contrast with previous studies demonstrating the enhanced susceptibility of jar1-1 and ein2 toward this fungal pathogen (Thomma et al., 1999; Ferrari et al., 2003; Nickstadt et al., 2004; Van Baarlen et al., 2007) and highlight the fact that defenses against B. cinerea can be dependent on the fungal isolate (Rowe et al., 2010). Additionally, mutants in genes shown to act in fungal penetration resistance, pen1-1, pen3-3, and pen4-1 (Collins et al., 2003), were resistant, as was cyp81F2 (Pfalz et al., 2009), a key component in glucosinolate toxification. We also assayed the sensitivity of ora59, glip1, trx-h5, and grx480 plants. Somewhat unexpectedly, all mutants exhibited similar resistance as wild-type plants to this fungus (Supplemental Fig. S5). Thus, apart from wrky33, only the pad3-1 mutant exhibited enhanced susceptibility toward this fungal isolate. However, plants ectopically overexpressing GRX480 (GRX480ox) were strongly susceptible to this fungus, resulting in large lesion formation and conidiophore development (Supplemental Fig. S5). In contrast, overexpression of TRX-H5 (TRX-H5ox) did not alter the resistant phenotype. Thus, the WRKY33-dependent negative regulation of GRX480 upon infection is likely one required mechanism for resistance toward B. cinerea.
To test if the inappropriately induced SA responses are causal for the enhanced susceptibility of wrky33 toward B. cinerea, we generated double mutants of wrky33 with the SA biosynthesis mutant sid2-1, with npr1-1, a key response regulator of the SA pathway, with wrky70, an NPR1-dependent TF known to play a role in SA-JA cross talk (Li et al., 2004), and with rbohD and then inoculated them with B. cinerea spores. Both lesion morphology (data not shown) and fungal biomass determination at 3 dpi revealed that the sid2-1 wrky33 and npr1-1 wrky33 double mutants were nearly as susceptible as wrky33 (Fig. 11). This indicates that inappropriate induction of the SA pathway and early SA-mediated repression of the JA response alone are insufficient to cause the strong infection phenotype of wrky33 plants toward this fungus. The double mutant wrky70 wrky33 showed reduced fungal growth, indicating a possible contribution of this factor to the phenotype.
Earlier studies have shown that WRKY33 is required for resistance to B. cinerea and have implicated this factor to positively regulate JA- and ET-mediated defenses while negatively affecting SA-mediated signaling (Zheng et al., 2006). Our results, however, suggest that early JA signaling upon B. cinerea infection is independent of WRKY33 function, while WRKY33 acts as a negative regulator of SA signaling and thereby prevents subsequent SA-mediated suppression of the JA pathway. Thus, in wrky33 mutants, inappropriate activation of SA-mediated responses leads to a suppression of JA responses downstream of JA biosynthesis at later stages of B. cinerea infection, thereby contributing to the susceptibility of the plant toward this necrotroph.
Our conclusions were mainly derived from genome-wide transcript profiling obtained at 14 hpi (maximal WRKY33 protein levels) and at 24 hpi, when gene expression differences between wild-type and wrky33 plants were the largest. At these infection stages, no obvious differences in fungal growth and development were observed between plants of both genotypes. Thus, alterations in transcript and hormone levels are likely direct consequences of the loss of WRKY33 function. The partial discrepancy between our findings and those of Zheng et al. (2006) is very likely due to differences in sampling times, since 24 hpi was the earliest time point analyzed previously.
Different from a previous report (Mao et al., 2011), we found that WRKY33 is not absolutely essential to promote Botrytis-induced camalexin production. Rather, we observed a delay in camalexin biosynthesis in wrky33 that was fully restored at later stages of infection, reaching even higher camalexin levels than in the wild type. Consistent with this observation was the delayed temporal expression of the camalexin biosynthetic genes CYP71A13 and PAD3 in wrky33, which also reached higher transcript levels at later infection stages than in the wild type. Although both genes are direct targets of WRKY33, ChIP assays revealed that other WRKY factors can also bind to the same promoter regions and thus may functionally compensate in the wrky33 mutant (Fig. 9). Camalexin, however, is not absolutely required for the resistance of Arabidopsis toward B. cinerea, and its contribution to WRKY33-mediated resistance remains unclear (Denby et al., 2005; Veronese et al., 2006; Walley et al., 2008; Berr et al., 2010).
High SA levels in plants can favor the growth of necrotrophic fungi by triggering programmed cell death (Govrin and Levine, 2000) and by suppressing appropriate JA defense pathways. For instance, exogenously applied SA suppressed the expression of JA-response genes induced by infection with the necrotrophs A. brassicicola and B. cinerea and by insect herbivores (Koornneef et al., 2008). Similarly, infection with a virulent strain of the biotrophic pathogen P. syringae that induces SA-dependent defenses resulted in increased susceptibility to A. brassicicola (Spoel et al., 2007).
SA-mediated suppression of JA responses is also modulated by pathogen-triggered changes in cellular redox potential and by the cytosolic function of NPR1 (Spoel et al., 2003; Ndamukong et al., 2007; Koornneef et al., 2008). We identified TRX-H5 to be a direct target of WRKY33 and its expression to be exaggerated in wrky33 plants. TRX-H5 reduces disulfide bonds between cytosolic NPR1 molecules for monomerization, a prerequisite for nuclear NPR1 function (Tada et al., 2008). We hypothesize that TRX-H5 is negatively controlled by WRKY33 and that loss of WRKY33 function results in strong pathogen-dependent TRX-H5 expression, resulting in enhanced enzymatic activity. This may partly explain the preferential expression of NPR1-dependent genes in wrky33 compared with the wild type (Supplemental Table S4). Moreover, the glutaredoxin gene GRX480 was also massively induced by B. cinerea infection only in wrky33 plants. Expression of GRX480 is induced by SA, is dependent on NPR1, and interacts with TGA factors regulating SA-responsive genes. Plants overexpressing this gene suppressed the transcription of JA-responsive genes (Ndamukong et al., 2007) and resulted in the susceptibility of plants toward B. cinerea (Supplemental Fig. S5). Consistent with this, SA-mediated suppression of JA signaling has been shown to coincide with a cellular increase in glutathione levels, and inhibition of glutathione biosynthesis antagonized this effect (Koornneef et al., 2008). It is also in agreement with findings demonstrating that Columbia-0 plants overexpressing ROXY1, another GRX of the CC type, were highly susceptible to B. cinerea (Wang et al., 2009), whereas plants impaired in the glutaredoxin gene GRXS13 were resistant toward this nectrotroph (La Camera et al., 2011).
In wrky33, simultaneous with the up-regulation of SA-associated genes, only JA-response genes were repressed, while the expression of the JA-signaling genes JAR1 and COI1 remained unchanged, and the JA-biosynthesis genes OPR3 and AOC3 were even up-regulated, resulting in higher JA accumulation. JA-response genes are mainly under the negative control of JAZ repressors (Chung and Howe, 2009; Pauwels and Goossens, 2011). The expression of several JAZ genes was strongly and positively influenced in wrky33 upon pathogen challenge. For two, JAZ1 and JAZ5, in vivo enrichment of WRKY33 at specific W-box-containing promoter regions could be shown, strongly indicating direct negative regulation by this TF. We hypothesize that all identified JAZ genes, except JAZ7, are under additional positive regulation by NPR1-dependent WRKY factors. We deduce this from the fact that the expression peaks of these JAZ genes coincided with those of the differentially induced WRKY genes in wrky33 following B. cinerea infection (Supplemental Fig. S3) and from ChIP assays using an antibody raised against the conserved WRKY domain (Turck et al., 2004) that revealed additional occupancy of all four JAZ promoters by such factors (Fig. 6). Prime candidate interactors may be WRKY50, WRKY51, WRKY53, WRKY54, and WRKY70. WRKY50 and WRKY51 were recently shown to repress JA signaling in a SA- and low-oleic-acid-dependent manner in ssi2 plants (Gao et al., 2011). Mutations in the SSI2 gene, encoding a plastid-localized stearoyl-acyl carrier protein desaturase, resulted in dwarf plants with high levels of SA and defects in JA-mediated defense responses. Such plants showed high susceptibility to B. cinerea but enhanced resistance to bacterial and oomycete pathogens (Nandi et al., 2005). Overexpression of WRKY70 repressed several JA-responsive genes and compromised resistance toward the necrotrophic fungus A. brassicicola while enhancing SA-dependent resistance toward the biotrophic fungus Erysiphe cichoracearum (Li et al., 2006). Moreover, WRKY53 was demonstrated to be partly redundant to WRKY70, whereas WRKY54 and WRKY70 have dual roles in SA biosynthesis and SA signal transduction (Wang et al., 2006).
Based on the up-regulation of many ET response factor (ERF) genes in our study, the ET response was clearly induced in both genotypes early during B. cinerea infection. This was also true for ERF1 and ORA59, two principal integrators of JA and ET signaling (Lorenzo et al., 2003; Pré et al., 2008). ORA59 plays an important role in counteracting the SA-mediated repression of JA-response genes (Leon-Reyes et al., 2010). In the cases where the SA pathway is activated prior to or simultaneous with the JA pathway, SA suppresses ORA59 expression, resulting in the repression of JA-response genes such as PDF1.2 (Leon-Reyes et al., 2010). This is consistent with the decline in the expression of ORA59 and the subsequent reduced expression of the JA-response genes PDF1.1 and PDF1.2 observed in B. cinerea-infected wrky33 plants as a consequence of inappropriate early activation of the SA pathway. We identified ORA59 to be a direct target of WRKY33. Moreover, the expression of ORA59 and consequently PDF1.2 was further substantially reduced in B. cinerea-infected wrky33 sid2-1 and wrky33 npr1-1 double mutants, compared with sid2-1 and npr1-1 plants, providing additional evidence that WRKY33 directly regulates ORA59 in a positive manner (Supplemental Fig. S6). Since pathogen-triggered early activation of ORA59 was similar in wild-type and wrky33 plants, the binding of WRKY33 may be essential in maintaining the prolonged expression of this gene.
Another identified direct in vivo target of WRKY33 was GLIP1, encoding a secreted lipase associated with ET signaling. GLIP1 targets fungal cell walls and possesses antimicrobial activity, and glip1 mutants are susceptible to A. brassicicola (Oh et al., 2005; Kwon et al., 2009). B. cinerea infection in wrky33 plants significantly decreased GLIP1 transcript levels compared with wild-type plants, indicating a positive regulatory role of WRKY33 in its expression.
Whereas up-regulation of the SA pathway by Botrytis isolate B191 was identified as causal for susceptibility in tomato (Solanum lycopersicum; El Oirdi et al., 2011), our genetic studies clearly demonstrate that this was not the case in Arabidopsis wrky33 plants infected with the Botrytis isolate 2100. The wrky33 double mutants with sid2-1 and npr1-1 were similarly susceptible like wrky33. Accordingly, the JA and ET single mutants opr3, coi1, jar1, ein2, and ora59 appeared as resistant as wild-type plants, again indicating that loss of the JA/ET responses alone is not the major cause for wrky33 susceptibility. Rather, it seems that WRKY33 regulates various distinct components of the phytohormone and possibly other signaling pathways that, in their concerted action, are essential to maintain proper temporal/spatial coordination of defense responses toward this necrotrophic fungus. This is consistent with recent studies by Tsuda et al. (2009) demonstrating that the establishment of optimal resistance to the necrotroph A. brassicicola involves complex interactions and requires substantial cross talk between various signaling pathways.
In summary, our studies indicate that by regulating components of both the SA and JA pathways, WRKY33 provides a mechanistic link to how SA-mediated repression of the JA response is achieved during the course of B. cinerea infection. This is partly accomplished by directly regulating the expression of genes such as TRX-H5 and ORA59 or by WRKY-mediated transcriptional activation of JAZ genes leading to the de novo synthesis of JAZ repressors. Supplemental Figure S7 outlines the currently known WRKY33 interactions in Arabidopsis. It is obvious that the network is complex and will involve additional WRKY33-dependent regulatory components. In particular, one should note that loss of WRKY33 function without infection did not result in altered expression of the majority of defense genes identified. Rather, many of the differential responses that we observed were dependent on the pathogen stimulus. Such inducible plant responses may be the result of altered protein specificities/activities as a general consequence of changes in cellular redox homeostasis or of pathogen-triggered alterations of specific signaling pathways involving WRKY33 functions.
Plant Materials
All experiments were done with Arabidopsis (Arabidopsis thaliana) plants of cv Columbia-0. The wrky33 mutant line used was the GABI_324B11 insertion line. To generate the WRKY33 HA-tagged complementation line (WRKY33-HA), the genomic region from −1,218 to 2,370 bp relative to the start codon was PCR amplified and cloned into the XhoI and NotI sites of vector KWS-5 (B. Ülker and I.E. Somssich, unpublished data). In a second step, the PstI-NotI fragment containing the stop codon was removed and replaced by the PstI-NotI fragment containing an in-frame HA tag sequence. This construct was stably transformed into wrky33 as described (Logemann et al., 2006). The single-insertion homozygous line was indistinguishable from wild-type plants with respect to growth and response to B. cinerea.
The overexpression line WRKY33ox was generated by cloning HA-tagged full-length cDNA into the pDONR201 Gateway vector (Invitrogen). This entry clone was recombined into the Gateway-compatible binary vector pAM-Kan-2x35S-intron:GW-HA, a pPAM (GenBank accession no. AY027531) derivative. The construct was transformed into Arabidopsis wrky33 by Agrobacterium tumefaciens-mediated transformation as described (Logemann et al., 2006). The expression of WRKY33 was confirmed by western-blot analyses (data not shown).
Other mutants or plant lines used in this study were as follows: grx480 (SALK_031817), GRX480ox (Ndamukong et al., 2007), ora59 (GK_061A12), glip1 (SALK_130146), wrky70 (GK_752F08), pad3-1 (Zhou et al., 1999), rbohD (Torres et al., 2002), sid2-1 (Wildermuth et al., 2001), npr1-1 (Cao et al., 1997), eds1-1 (Falk et al., 1999), pad4-1 (Glazebrook et al., 1997), opr3 (Stintzi et al., 2001), jar1-1 (Staswick et al., 2002), ein2-1 (Guzmán and Ecker, 1990), jrg21 (Delker et al., 2007), pen1-1 (Collins et al., 2003), pen3-3 (Stein et al., 2006), and cyp81F2 (SALK_123882). The double mutants wrky33 npr1-1, wrky33 sid2-1, wrky33 rbohD, and wrky33 wrky70 were generated by crossing single mutants followed by PCR-based verification.
Plant Growth Conditions and Pathogen Inoculation Procedure
Plants were grown for 5 weeks under short-day conditions in closed cabinets (Schneijder chambers: 20°C, 16 h of light/8 h of darkness, 80% relative humidity) on 42-mm Jiffy-7 pots (Jiffy) to prevent pathogen infection from garden soil. Before sewing, the Jiffy pot peat pellets were soaked in water containing 0.1% liquid fertilizer Wuxal (Manna).
For inoculations, the Botrytis cinerea isolate 2100 (CECT2100; Spanish Type Culture Collection, Universidad de Valencia) was used, which was cultivated on potato dextrose plates at 22°C for 10 d. Spores were collected, washed, and frozen at −80°C in 0.8% NaCl at a concentration of 107 spores mL−1. For inoculation of Arabidopsis plants, the spores were diluted in Vogelbuffer (in 1 L: 15 g of Suc, 3 g of Na-citrate, 5 g of K2HPO4, 0.2 g of MgSO4·7H2O, 0.1 g of CaCl2·2H2O, and 2 g of NH4NO3) to 5 × 105 spores mL−1. For droplet inoculations, 5 μL was applied to single leaves of 5-week-old plants. The same spore suspension was used for spray inoculations. For mock treatment, Vogelbuffer alone was used. One day before inoculation and during the entire infection process, plants were incubated at high humidity under a hood.
Quantification of Fungal Growth by qPCR
Quantification of fungal biomass relative to plant biomass by qPCR was basically performed as described by Gachon and Saindrenan (2004). Leaves of the indicated Arabidopsis lines were inoculated with two 4-μL droplets of B. cinerea spores, and after 3 d, DNA from whole leaves of similar weight was extracted following the modified protocol of Edwards et al. (1991). The relative amounts of B. cinerea and Arabidopsis DNA were determined by qPCR employing specific primers for cutinase A and SKII, respectively.
Microarray Analysis
For microarray analysis, 100 mg of leaf tissue was collected and total RNA was isolated using TRI reagent following the supplier's instructions (Ambion). The samples were collected from spray-infected and mock-treated wild-type and wrky33 plants after 14 h. Leaves from three plants were pooled to make one biological replicate, and three independent biological replicates were isolated for each genotype and treatment for hybridization against a total of 12 Arabidopsis ATH1 GeneChip arrays (Affymetrix). Quality control, labeling, hybridization, and scanning were done at Integrated Functional Genomics, Interdisziplinäre Zentrum für Klinische Forschung, at the University of Muenster. Data were analyzed using the software package GeneSpring GX (Agilent Technologies). Differential expression of genes was calculated using a combination of ANOVA (Benjamini-Hochberg multiple testing) at P ≤ 0.05 and a cutoff of 2-fold or greater.
Total RNA was isolated from leaves at 0, 5, 8, 14, 24, and 48 hpi as described above and reverse transcribed with oligo(dT) primer to produce cDNA using the SuperScript First-Strand Synthesis System for Reverse-Transcription PCR following the manufacturer's protocol (Invitrogen). cDNA corresponding to 2.5 ng of total RNA was subjected to qPCR with gene-specific primers (Supplemental Table S5) using the Brilliant SYBR Green qPCR Core Reagent Kit accordingly (Stratagene). The qPCRs were performed on the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad) with two technical replicates in the same run and three biological replicates in different runs. The endogenous reference gene for normalization was At4g26410, which was described as being highly constant under varying stress conditions (Czechowski et al., 2005) and which also displayed very constant expression levels in our microarrays. The results were analyzed using the ΔΔCt method (Livak and Schmittgen, 2001). To simplify data interpretation, transcript abundance was expressed as a ratio relative to mock-treated wild-type plants, which was set to 1. Data shown are means ± sd from three biological replicates.
Five-week-old plants were spray inoculated or mock treated for 14 or 24 h before they were processed following the protocol by Gendrel et al. (2005) with some modifications. Each 2-g leaf sample was cross-linked by vacuum infiltration of 1% formaldehyde solution for three subsequent 7-min treatments. Sonication of the isolated nuclei was performed on a UP50H sonicator (Hielscher) equipped with an MS1 tip four times for 30 s each with 30 s of cooling on ice in between. The sheared chromatin (25 μg) was diluted 10-fold in ChIP dilution buffer (Gendrel et al., 2005) containing protease inhibitors (Roche) and phosphatase inhibitor (Sigma) to lower the SDS concentration to 0.1%. After preclearing with protein A-agarose beads, the chromatin was incubated overnight with rabbit polyclonal antibodies to HA (ChIP grade; ABCAM) at 4°C on a nutator. Immuncomplexes were collected by incubation with protein A-agarose. After washing, the beads were resuspended in 250 μL of Tris EDTA buffer, pH 8.0, containing 0.5% SDS with 2 μg of RNase (Roche) and 2 μg of proteinase K (Invitrogen) and incubated overnight at 65°C to reverse cross-links. The phenol-chloroform-extracted DNA was precipitated with ethanol and resolved in 50 μL of Tris EDTA buffer, pH 8.0. One microliter was used for qPCR.
Quantification of Phytohormones
Analysis of phytohormone levels was conducted as described (Diezel et al., 2009) with the only difference being that as one internal standard, (±)-jasmonic acid-9,10-D2 was added. To determine total SA levels, 0.1 mL of 0.1 m NaOAc, pH 5.0, containing β-glucosidase (final concentration, 100 units mL−1) was added to dried extract residues after solvent evaporation, and the samples were incubated for 3 h at 37°C. Fifty microliters of 10% trifluoroacetic acid was added, and the solution was extracted three times with 600 μL of ethyl acetate. The three ethyl acetate fractions were pooled, the solvent was evaporated in a Speed-Vac, and after adding 70% methanol, the levels of free SA were determined by HPLC.
Quantification of Camalexin
In three biological replicates, 100 mg of B. cinerea-inoculated or mock-treated leaves was harvested at various time points and homogenized in dimethyl sulfoxide (2.5 μL mg−1 fresh weight). After centrifugation (20 min, 15,000 rpm, 4°C), supernatants were collected in fresh tubes and subjected to HPLC analysis.
DAB and Trypan Blue Staining of Plant Material
To detect H2O2, B. cinerea spray-inoculated and mock-treated leaves were stained with DAB as described (Torres et al., 2002). To visualize fungal tissue and dying plant cells, the leaves were stained with trypan blue as described (Roetschi et al., 2001).
Microarray data have been deposited in the NASCArrays database under accession number NASCARRAYS-577.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. B. cinerea 2100-induced ROS production and cell death.
Supplemental Figure S2. WRKY33-HA complements wrky33 susceptibility to B. cinerea 2100 infection.
Supplemental Figure S3. Differential up-regulation of WRKY genes after B. cinerea 2100 infection.
Supplemental Figure S4. Sensitivity of selected mutants from hormone and metabolite pathways toward B. cinerea 2100.
Supplemental Figure S5. Susceptibility of mutant and overexpression lines to B. cinerea 2100 infection.
Supplemental Figure S6. Expression levels upon B. cinerea 2100 infection of hormone-response genes in double mutants containing the wrky33 mutation.
Supplemental Figure S7. Schematic representation of WRKY33 interactions.
Supplemental Table S1. Fold induction of all genes at 14 hpi with B. cinerea 2100 (P ≤ 0.05).
Supplemental Table S2. Gene Ontology counts of differentially expressed genes upon B. cinerea 2100 infection.
Supplemental Table S3. Differential expression of TFs upon B. cinerea 2100 infection at 14 hpi.
Supplemental Table S4. Differential expression of NPR1 target genes in wrky33 upon B. cinerea 2100 infection.
Supplemental Table S5. Primers used in qPCR.
We are grateful to Dr. C. Gatz (University of Göttingen) for providing the GRX480ox line, to Dr. O. Park (Korea University) for the glip1 mutant, and to Dr. J. Lorang (Oregon State University) for the trx-h5 mutant and the TRX-H5 overexpressor. We also thank Dr. A. Töller (Max Planck Institute for Plant Breeding Research Köln) for help in establishing the wrky33 double mutants and for critical comments on the manuscript.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Imre E. Somssich (somssich{at}mpipz.mpg.de).
↵1 This work was supported by the Deutsch Forschungsgemeinschaft (grant no. SO235/7–1) within the framework of the Arabidopsis Functional Genomics Network.
↵[W] The online version of this article contains Web-only data.
- salicylic acid
- jasmonic acid
- ethylene
- TFs
- transcription factors
- ChIP
- chromatin immunoprecipitation
- hpi
- h post inoculation
- 3,3-diaminobenzidine
- reactive oxygen species
- hydrogen peroxide
- hemagglutinin
- qRT
- quantitative real-time
- Received December 21, 2011.
- Accepted March 5, 2012.
- Published March 5, 2012.


text:
The Senate stepped in to save Cash for Clunkers Aug. 6, giving it a $2 billion extension. But on Aug. 7, CNN.com found a big difference between independent analysis and government claims of which cars were most popular buys.
Peter Valdes-Dapena explored the difference between government data on the clunkers program (claiming small cars were most popular) and Edmunds.com analysis which showed that "two full-size trucks and a small crossover SUV were actually among the top-ten buys."
"The discrepancy is a result of the methods used. Edmunds.com uses traditional sales measurements, tallying sales by make and model. The government uses a more arcane measurement method that subdivides models according to engine and transmission types, counting them as separate models," Valdes-Dapena wrote.
Based on those methods Edmunds ranked the Ford Escape crossover SUV as the best seller, while the government ranked it seventh.
According to Valdes-Dapena, "sales of truck models would tend to be heavily diluted using the government's method because practically each version counts as a different vehicle."
Ironically, just one day earlier CNN "Newsroom" reported that there had been a "big shift" to smaller cars. CNN's Felicia Taylor said, "critics feared some would actually use that money to buy another truck, but that's not what's happening. The top sellers so far: Toyota Corolla, Ford Focus, Honda Civic." She did mention that the Ford Escape made the top ten.
The national news media was outspokenly supportive of Cash for Clunkers, particularly on ABC, CBS and NBC. Reporters called the program "wildly popular," "too successful," and all three networks said it's a "victim of its own success" when it burned through the entire $1 billion budget in a mere week.
CNBC's Jim Cramer even praised the potential $2 billion extension as "money well-spent" on NBC's "Today."
However, Jeremy Anwyl, CEO of Edmunds.com said "it's not clear that cash for clunkers actually increased sales."

subset: commoncrawl


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We are teaming up with Pinup Style Worldwide to bring you an awesome new contest! How would you like to win a $100 gift certificate to www.poisoncandyfashion.com? The rules are simple:
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This was a 5 out of 5 stars read for me! "This is Claude. He's five years old, the youngest of five brothers, and
Tag: #reading
Castle Of Water
This was a 5 out of 5 stars novel for me; as near a perfect emotional ride as you can take. How do you review
Lying To Children
From the nine months it takes to create that perfect little life until the eighteen years and beyond it takes to raise them, children are
Postcards From A War
In our current everyday life, war has become the "normal." We have been at war for years with no end in sight. Every day thousands
New Boy
This was a 3.5 to 4 stars read for me, so I rounded up! The tragedy of bullying. One day. From the start of
The Stars Are Fire
I must say that one of the best experiences when reading a book is the sensory aspects. The quiet promise in the bookstore. The smell


text: The Novotel Glasgow is located in a central location within easy reach of Glasgow's shopping and nightlife. The hotel has 139 guest rooms all of which have internet access and satellite TV and are en suite. The Elements restaurant has a varied menu with unique Scottish hospitality in mind, the hotel also has a bar, a fitness centre and a sauna.


text:
Like Newton's laws of motion, the laws of karma are about actions and reactions. People and nations come together to repay past rna. If I steal from you, I only complete an action that you once started. My act does not create a fresh karmic debt for me.
It is only by my ego-identification with the act that I create rnanubandhana or debt bondage.
Abhay Ghiara is a teacher, writer, and Gandhian economist. He studied economics and yoga at Bombay University, graduating exactly 100 years after Mahatma Gandhi. He has practiced yoga all his life.
Abhay lectures on yoga and life in San Francisco, London, Bristol, Chicago, and Mumbai, India. photo: Maritstar
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Boba Fett
If badasses were to look up to someone then it would be Boba Fett. He is cruel, calculating and ruthless in his pursuit for a chosen target.
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text: Prices and promotions can change or expire at any time without notice. Always verify pricing before purchase. The information contained on this site is for general information purposes only. While we make every attempt to keep the information on this site correct, we make no representations or guarantees of any kind about the accuracy, pricing or availability of any deal, context, graphics or outside links on this site. Dixiedoesdeals.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.


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This trio includes all 3 of our favorite scents. The luscious lather will leave your skin feeling clean and pampered.
Banana Berry Almond (Vegan) - 4oz
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text:
Travelling to Iceland...March 19, 2016
The main activities started on Saturday (all of Friday was spent travelling) and we of course had to check out the main tourist attractions in the Golden Circle. Above is the amazing Gullfoss waterfall, which was followed up by visits to Geysirs and the National Park (below). This is were the North American plate meets the Eurasian plate, and that's basically as close to America as I've ever got so it wasn't just the geology students that got excited! At this point we were pretty cold so used any excuse to start a snowball fight or slide down hills, so that was all fun too. Lastly we went to a geothermal power plant (probably the least exciting part of the day, as you might have guessed!)
On Sunday we explored the south coast. Firstly we had a short walk to see the beautiful Solheimajokull glacier, followed by a visit to the black beach- although it was covered in snow so not very black at all!
4 comments
Iceland looks so beautiful! I would love to visit there one day. Did you catch the Northern Lights?ReplyDelete
Thank you Rue! We did actually - most of the time it was cloudy or snowing but one of the evenings it cleared so that we could see them, they were beautiful! I would definitely recommend visiting if you have the opportunity.Delete
Emma xo
Oh how wonderful! I'm so envious of you visiting Iceland. I very nearly decided to go this year for my birthday over a long weekend but decided on Venice instead, will be going in just a few weeks. It looks like a trip that couldn't be done in just a few days,, I would want to do and see so much! It looks truly incredible, very lucky xReplyDelete
Amber - ambambe.blogspot.co.ukReplies
Thank you Amber- Venice is beautiful too for a weekend too, I hope you have a wonderful time! Yes you'd definitely need more than a couple of days in Iceland, there's so much to see haha!Delete
Emma xo
Please keep the comments section a positive place, thank you! Have a lovely day!
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text:
For immediate release
Jessica Kohen
Marketing and Communications
Diane Adams-Graf
The KSTP-TV Archive holds over three million feet of news film (1948-1976) and 2,500 videotapes (1976-1993) now housed at the Minnesota Historical Society. This television news film collection covers the social, political, economic and natural landscape of Minnesota for the second half of the 20th-century. A pioneer television station, KSTP-TV was one of the first full-color stations, and the first station in the Midwest to air a daily newscast. A selection of news segments is available for viewing now at www.mnhs.org/collections/kstp including footage of a 1949 U.S. Navy blimp, the 1964 Como Lake ice skating races and the 1979 gas lines, among many others. Later this year, a total of 150 news segments and an index to the full collection will be posted online.
Hubbard Broadcasting, Inc., gave the KSTP-TV Archive to the Minnesota Historical Society in 1994. Over the next 10 years, with support from the Hubbard Broadcasting Foundation, the 16mm news film was cleaned, spliced, cataloged and re-housed in a secure and climate-controlled storage area. The Society continues to promote long-term care, create further information, and provide access to this important collection.
The KSTP-TV Archive is supported by funds from the Hubbard Broadcasting Foundation and by fees for research, copying and use. Reproductions or permissions for use of any material in the collection are provided under license agreement for purposes indicated and agreed upon by the user and the Minnesota Historical Society. Direct requests for research, copy or use, by submitting the KSTP Request Form to the Minnesota Historical Society. Commercial reproductions of the KSTP-TV Archive are also available through CONUS ARCHIVE at http://www.conus.com.
In addition to film, the Society has more than 195,000 photographs, art, posters and fine art photographs available through the Visual Resources Database. These images may be ordered online or through the Library's Copy Service. Items are continually added to this database as the Society's collections grow.
The Minnesota Historical Society is a non-profit educational and cultural institution established in 1849 to preserve and share Minnesota history. The Society collects, preserves and tells the story of Minnesota's past through museum exhibits, libraries and collections, historic sites, educational programs and book publishing.


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See the chart for estimated battery life for each item.
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The Nook might be discontinued, and the Spring Design Alex is long dead, but that's not going to stop the copycats.
The ebook reader at right is an unnamed Alex clone running Android. it has a 6" E-ink screen and a 3.6" (480x272) capacitive touchscreen. I'm not sure who made it (though the menu suggests it's a Rockchip design). It has Wifi, a SD card slot, and an indeterminate amount of Flash storage.
It's available from Paradigm Shift, and they want to sell it in quantities of 1k or more.


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Its Saturday. So theres no reason for me to wake up early Driving lesson? Nope! Not until 23rd Nov. But still, terbangun awal pagi bebenor tadi. Heh!! Go to the kitchen n make some nescafe n kuar kat kaki lima. The day still dark to see and its cold out there. Then go back to my room. Sit beside the bed for a couples of hours. Listening to my fav jiwang2 music sebelum matahari naik.
After dat, continue doin' my weekend chores. Basuh pakaian, then sidai. Cuci kasut! Jog? MALAS!!! Got couples of sms. Yes, of coz imma happy. Thanks for makin' me smiles early dis morning. Aftenun? Dun really doing anything pun other then enjoy playing computer game. Resident Evil 5. Abg bising2 kata jgn layan komputer saja. Yelah boring2, nak jugak pam darah kasi high the adrenalin level. Suspen ada, sakit hati pun ada sbab balik2 mati. But I'm enjoy most of the time. Yelah dari asyik pikir pasal hal2 yg tak best. Baik takyah.
Sometimes its better for u 2 run then to hide. U ran n they maybe cannot catch u. Dun care lah apa sebab. But if u hide, they wont see you, but how long u gonna stay like dat? If they dont move? Then u just gonna stay there lah forever??? So I made my decision. Whatever best for them, I will let they enjoy thier life. Its not mine tho so its up to you then. I never stop wishing for other peoples' happiness. I never wish something bad for them.
Same like me, I would rather enjoy being me than skit2 nak sakit hati bagai.. Tau lah panas baran kan. So I gotta be cool for some reason. Its not like I dun care pun, but still I dun want to do anything dat make other people think dat I'M DISTURBING. coz dats ANNOYING. Sure lah tak pasal aku ngamok bila dengar camtu. Yes. I cannot blame anyone pun if dat happen. I shud think much, I mean I must to think much before sayin' something.
arghhh.... 4get about dat..
Any last words??
Yeah, I think I shud leaving soon and change something in me. Change for better-ness. Kearah kemantapan camtu. Away from Sabah maybe. Dunno lah. I know dat someday I gotta do this. But now I think I'm..........
JRP aka. JR


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Toddler Angel Costume
DiscontinuedProduct # HRC885431
Product Includes
- Halo Headpiece
- Dress w/attached Wings
Purchase Product
Sorry, this item is Out of Stock
Product Measurements
|Toddler (2T-4T)
|Dress Chest
|Toddler (2T-4T)
|Dress Waist
|18" - 24"
|46cm - 61cm
|Toddler (2T-4T)
|Dress Length
Product Description
Your toddler can be a guardian angel in this adorable costume. The angel costume features a dress, wings and halo headband. The dress is made of blue velour with a center panel made of polyester and lined with ruffles that have silver star detailing. The elastic waist has gold trimmed ribbon with a golden star hanging from it. The sleeves also have elastic half way down and gold star accents. The dress secures up the back with Hook and Loop fastener and also has Hook and Loop fastener on the outside of the dress to attach the white angel wings. A blue halo headband with more gold stars and stripes completes the toddler angel costume. This is a unique detailed angel costume for Halloween or a Renaissance faire!
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