Molecular Plant Advance Access originally published online on May 8, 2008
Molecular Plant 2008 1(3):459-470; doi:10.1093/mp/ssn020
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Stress- and Pathogen-Induced Arabidopsis WRKY48 is a Transcriptional Activator that Represses Plant Basal Defense
a Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907–2054, USA
b Present address: Department of Botany, Miami University, Oxford, Ohio 45056, USA
1 To whom correspondence should be addressed. E-mail zhixiang{at}purdue.edu, fax 765-494-5896.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Plant WRKY transcription factors can function as either positive or negative regulators of plant basal disease resistance. Arabidopsis WRKY48 is induced by mechanical and/or osmotic stress due to infiltration and pathogen infection and, therefore, may play a role in plant defense responses. WRKY48 is localized to the nucleus, recognizes the TTGACC W-box sequence with a high affinity in vitro and functions in plant cells as a strong transcriptional activator. To determine the biological functions directly, we have isolated loss-of-function T-DNA insertion mutants and generated gain-of-function transgenic overexpression plants for WRKY48 in Arabidopsis. Growth of a virulent strain of the bacterial pathogen Pseudomonas syringae was decreased in the wrky48 T-DNA insertion mutants. The enhanced resistance of the loss-of-function mutants was associated with increased induction of salicylic acid-regulated PR1 by the bacterial pathogen. By contrast, transgenic WRKY48-overexpressing plants support enhanced growth of P. syringae and the enhanced susceptibility was associated with reduced expression of defense-related PR genes. These results suggest that WRKY48 is a negative regulator of PR gene expression and basal resistance to the bacterial pathogen P. syringae.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Plants have evolved sophisticated defense mechanisms that are activated in response to pathogen attack. Upon infection by a virulent pathogen, basal defense mechanisms can be activated by microbe-derived molecules such as bacterial flagellin and lipopolysaccharides, collectively called pathogen or microbe-associated molecular patterns (PAMPs) (Jones and Dangl, 2006). Gram-negative bacterial pathogens such as Pseudomonas syringae can deliver effector proteins to plant cells to interfere with PAMP-triggered resistance to promote pathogen virulence. Some of the effectors are specifically recognized by plant resistance proteins and activate strong effector-triggered resistance (Jones and Dangl, 2006). Both PAMP- and effector-triggered resistance are associated with extensive transcriptional reprogramming of plant host genes.
A large body of evidence has shown that WRKY transcription factors play important roles in regulation of genes associated with plant defense responses (Eulgem and Somssich, 2007). First, pathogen infection or treatment with pathogen elicitors or salicylic acid (SA) induces rapid expression of WRKY genes from a number of plants (Rushton et al., 1996; Eulgem et al., 1999; Chen and Chen, 2000; Dellagi et al., 2000; Hara et al., 2000; Dong et al., 2003; Kalde et al., 2003; Eckey et al., 2004; Kim and Zhang, 2004; Turck et al., 2004). In Arabidopsis, expression of 49 out of 72 tested WRKY genes was differentially regulated after pathogen infection or SA treatment (Dong et al., 2003). Secondly, a number of defense or defense-related genes, including several well studied Pathogenesis-Related (PR) genes and the regulatory NPR1 gene, contain TTGACC/T W-box elements in their promoters (Rushton et al., 1996; Willmott et al., 1998; Yang et al., 1999; Yu et al., 2001; Turck et al., 2004; Yamamoto et al., 2004; Rocher et al., 2005). A number of studies have shown that these W-box sequences are specifically recognized by WRKY proteins and are necessary for the inducible expression of these genes (Rushton et al., 1996; Willmott et al., 1998; Yang et al., 1999; Yu et al., 2001; Turck et al., 2004; Yamamoto et al., 2004; Rocher et al., 2005). Microarray analysis of gene-expression changes in Arabidopsis under different systemic acquired resistance (SAR)-inducing or -repressing conditions with 10 000 ESTs has provided additional evidence that WRKY proteins play important roles in regulating expression of SAR-associated genes (Maleck et al., 2000). A group of 26 genes including PR1 was identified to be coordinately induced by various pathogens and defense-inducing conditions. Within the 1.1-kb regions upstream of the predicated translation start sites, only the binding site for WRKY proteins (W-boxes; TTGAC) were found in all 26 promoters, with an average of 4.3 copies per promoter that are often organized in clusters (Maleck et al., 2000). By contrast, a randomly selected set of genes contained, on average, fewer than two W-boxes per promoter (Maleck et al., 2000).
More recent studies have provided direct evidence for the involvement of specific WRKY proteins in plant defense responses. For example, Arabidopsis WRKY22 and WRKY29 are induced by a MAPK pathway that confers resistance to both bacterial and fungal pathogens and expression of WRKY29 in transiently transformed leaves led to reduced disease symptoms (Asai et al., 2002). In tobacco, virus-induced silencing of three WRKY genes compromises N-gene-mediated resistance to tobacco mosaic virus (Liu et al., 2004). It has been shown that mutations of Arabidopsis WRKY70 enhances plant susceptibility to both biotrophic and necrotrophic pathogens, including the bacterial pathogen Erwinia carotovora as well as fungal pathogens Erysiphe cichoracearum and Botrytis cinerea (Li et al., 2004; AbuQamar et al., 2006; Li et al., 2006). In addition, wrky70 mutants are compromised in both basal and resistance gene (RPP4)-mediated disease resistance to the oomycete Hyaloperonospora parasitica (Knoth et al., 2007). Thus, some of the pathogen-induced WRKY proteins function as important positive regulators of plant disease resistance.
Other WRKY proteins can function as negative regulators of plant disease resistance. For example, mutations of Arabidopsis WRKY7, WRKY11, and WRKY17 enhance basal plant resistance to virulent P. syringae strains (Park et al., 2005; Journot-Catalino et al., 2006; Kim et al., 2006). Likewise, mutations of Arabidopsis WRKY25 enhance tolerance to P. syringae and overexpression of either WRKY25 or closely related WRKY33 enhances susceptibility to the bacterial pathogen and suppresses SA-regulated PR1 gene expression (Zheng et al., 2006, 2007). The structurally related WRKY18, WRKY40, and WRKY60 also function partially redundantly as negative regulators in plant resistance against the biotrophic bacterial pathogen P. syringae and fungal pathogen E. cichoracearum (Xu et al., 2006; Shen et al., 2007). In addition, barley HvWRKY1 and HvWRKY2 function as PAMP-inducible suppressors of basal defense (Shen et al., 2007). The WRKY domain in WRKY52/RRS1 R protein that confers resistance toward the bacterial pathogen Ralstonia solanacearum may also play a negative role in defense signaling (Noutoshi et al., 2005). The diverse roles of WRKY proteins may reflect the complex signaling and transcriptional networks of plant defense that require tight regulation and fine-tuning.
In Arabidopsis, there are more than 70 genes encoding WRKY proteins (Eulgem et al., 2000; Dong et al., 2003). In a previous study, we have analyzed expression of 72 identified WRKY genes and found that 49 of them were differentially regulated in response to pathogen infection and SA treatment (Dong et al., 2003). WRKY48 is one those genes that are highly responsive to an avirulent strain of P. syringae but not to SA treatment. In the present study, we show that WRKY48 is a stress- and pathogen-induced gene encoding a nuclear localized WRKY protein with strong DNA-binding and transcriptional activation activities. Growth of a virulent strain of the bacterial pathogen P. syringae was reduced in the wrky48 T-DNA insertion mutants, while transgenic WRKY48-overexpressing plants support enhanced growth of P. syringae. The altered resistance of the loss-of-function mutants and overexpression lines was associated with altered induction of SA-regulated PR genes by the bacterial pathogen. These results strongly suggest that pathogen-induced WRKY48 functions as a negative regulator of defense-related PR genes and resistance to the bacterial pathogen P. syringae.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Protein Structure, DNA Binding and Subcellular Localization
Arabidopsis WRKY48 (At5g49520) encodes a protein of 399 amino acid residues with a predicted molecular weight of 44.726 kD and a predicted isoelectric point of 6.5143 (Figure 1A). Based on the number and structure of WRKY domains, WRKY48 is classified as a group II WRKY protein. The N-terminus of WRKY48 contains a glutamine-rich domain (Figure 1A) that is found in some transcriptional activators in multi-cellular organisms such as Sp1 (Escher et al., 2000). The glutamine-rich activation domain of Sp1 selectively binds and targets core components of the transcriptional machinery such as TFIID, a multi-protein complex composed of the TATA-box binding protein (TBP) and TBP-associated factors (Escher et al., 2000). WRKY48 also contains a number of serine tracts and, therefore, may be regulated by protein phosphorylation by protein kinases.
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A number of isolated WRKY proteins have been shown to bind the TTGACC/T sequence (W-box) (Rushton et al., 1996; Chen and Chen, 2000; Yu et al., 2001). To examine the DNA-binding activity of WRKY48, we expressed the gene in E. coli, purified the recombinant protein, and assayed its binding to an oligo DNA molecule with W-box elements (Figure 1B). As shown in Figure 1C, the purified recombinant WRKY48 protein bound the oligo DNA molecule with two direct TTGACC repeats (Pchn0). When directly compared with other recombinant Arabidopsis WRKY proteins at the same protein concentrations (WRKY18, 40, 60, 25, 26, 33, and 51), WRKY48 had the highest in-vitro binding activity for the oligo DNA molecule (data not shown). Thus, WRKY48 binds W-box sequences with a high affinity in vitro. To determine whether the TTGACC sequence is essential for the sequence-specific binding activity, we tested a mutant probe (mPchn0) in which the two TTGACC sequences were changed to TTGAAC (Figure 1B). As shown in Figure 1C, binding of WRKY48 to the mutant probe was drastically reduced. Thus, binding of WRKY48 to the TTGACC W-box sequence is highly specific.
WRKY48, as a putative transcription factor, is likely to be localized in the nucleus. To test this, we constructed a GFP protein fusion of WRKY48 and demonstrated that this transiently expressed WRKY48-GFP fusion protein was localized exclusively to the nuclei of onion (Allium cepa) epidermal cells (Figure 2). By contrast, the GFP protein was found in both the nucleus and cytoplasm because of its small size (Figure 2). Nuclear localization of the WRKY protein supports its role as a transcriptional regulator.
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Analysis of WRKY48 Expression
We have previously shown that WRKY48 is rapidly induced in Arabidopsis plants upon infection by an avirulent strain of P. syringae (Dong et al., 2003). To analyze the role of WRKY48 in plant basal defense, we investigated the expression of WRKY48 after inoculation with the virulent P. syringae pv. tomato strain DC3000 (PstDC3000). As shown in Figure 3A, WRKY48 transcript levels increased rapidly in wild-type plants after infiltration with either the control MgCl2 solution (mock inoculation) or the bacterial suspension. However, WRKY48 induction was stronger in pathogen-infected plants than in MgCl2-treated control plants (Figure 3A). In the four independent experiments performed, WRKY48 transcripts in PstDC3000-inoculated plants were higher than those in mock-inoculated plants at 2 h post infiltration (hpi) or at both 2 and 4 hpi (Figure 3). After rapid induction during the first 4 h after mock or pathogen inoculation, the levels of WRKY48 transcripts declined and, in three of the four experiments, reached near basal levels by 24 hpi (Figure 3). WRKY48 induction after infection by the avirulent PstDC3000avrRpt2 strain was similar to or slightly stronger than that after infection by the virulent PstDC3000 strain (Figure 3A). On the other hand, the PstDC3000hrcC mutant, which is defective in the type III secretion system (Deng et al., 1998; Deng and Huang, 1999), did not enhance WRKY48 transcript levels above those in MgCl2-treated control plants (Figure 3A). Thus, the bacterial type III secretion system plays an important role in pathogen-enhanced expression of WRKY48. We also analyzed the induction of WRKY48 by defense-inducing molecules SA, 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene (ET) biosynthesis, and methyl jasmonic acid (JA). Unlike PstDC3000, these defense-inducing molecules induced little expression of WRKY48 (Figure 3B).
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To determine whether stress- and pathogen-induced WRKY48 expression is mediated by the SA, ET, and/or JA signaling pathways, stress- and pathogen-induced WRKY48 expression was monitored in various signaling mutants. MgCl2- and pathogen-induced WRKY48 expression was not significantly affected in the sid2 and npr1-3 mutants, which are defective in SA biosynthesis and signaling, respectively (Cao et al., 1997; Wildermuth et al., 2001) (Figure 3C). The levels of WRKY48 transcripts were higher in the JA-insensitive coi1-1 mutant plants (Xie et al., 1998) than those in the wild-type plants (Figure 3C). In addition, while WRKY48 transcripts were detected at 24 hpi in wild-type plants only in one of the four experiments, they were present at substantial levels in the coi1 mutant plants at this hpi in all four experiments (Figure 3C). The enhanced and prolonged induction of WRKY48 by MgCl2 and PstDC3000 was even more pronounced in the ET-insensitive ein2-1 mutant (Alonso et al., 1999) (Figure 3C). These results suggest that JA and ET signaling pathways have a negative role in stress- and pathogen-induced WRKY48 expression.
WRKY48 is a Transcriptional Activator
To determine the transcriptional regulatory activity of WRKY48 in planta, we employed a previously established transgenic system in which the transcriptional regulatory activity of a protein is determined through assays of a reporter gene in stably transformed plants (Kim et al., 2006). The reporter gene in the system is a GUS gene driven by a synthetic promoter consisting of the –100 minimal CaMV 35S promoter and eight copies of the LexA operator sequence (Figure 4A). Transgenic Arabidopsis plants harboring the reporter gene constitutively expressed similarly low levels of the GUS reporter gene due to the minimal 35S promoter used, thereby making them useful for assays of transcription activation or repression by determining increase or decrease in GUS activities following co-expression of an effector protein.
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To generate the WRKY48 effector, we fused its coding sequence with that of the DNA-binding domain (DBD) of LexA (Figure 4A). The fusion construct was subcloned behind the steroid-inducible Gal4 promoter in pTA7002 (Aoyama and Chua, 1997) and transformed into transgenic plants that already contain the GUS reporter construct. Unfused WRKY48 and LexA DBD genes were also subcloned into pTA7002 and transformed into transgenic GUS reporter plants as controls (Figure 4A). Transgenic plants containing both the reporter and an effector construct were identified through antibiotic resistance screens. To determine how the effectors influence GUS reporter gene expression, we determined the changes of GUS activities in these transgenic plants following induction of the effector gene expression by spraying 20 µM dexamethasone (DEX), a steroid. In the transgenic plants that expressed unfused WRKY48 or LexA DBD effector, the ratios of GUS activities measured before DEX treatment to those measured after DEX treatment were close to 1 (Figure 4B). These results indicated that induced expression of WRKY48 or LexA DBD alone had no significant effect on expression of the GUS reporter gene. In the transgenic plants harboring the LexA DBD-WRKY48 effector gene, induction of the fusion effector after DEX treatment resulted in an
25-fold increase in GUS activity (Figure 4B). These results strongly suggest that WRKY48 is a transcriptional activator in plant cells.
Disrupting or Altering WRKY48 Expression Affect Basal Disease Resistance
To analyze its biological roles directly, we identified and characterized two T-DNA knockout mutants for WRKY48. Both wrky48-1 (Salk_066438) and wrky48-2 (Sail_1267_D04) contain a T-DNA insertion in the first exon of the WRKY gene (Figure 5A). Homozygous mutant plants were identified by PCR with WRKY48-specific primers flanking the insertion sites. Northern blotting analysis confirmed the absence of the normal, full-length WRKY48 transcript in pathogen-infected mutant plants (Figure 5B). The wrky48 mutants grew and flowered at the same rates as the wild-type plants and exhibited no detectable alteration in morphology.
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To determine possible altered phenotypes in disease resistance, we examined the response of wild-type and wrky48 mutant plants to PstDC3000, which is virulent on Arabidopsis ecotype Columbia (Whalen et al., 1991). Plants were inoculated with the bacteria and the growth of the pathogen was monitored. At 1 and 2 dpi, there was a marked decrease (approximately nine-fold) in the bacterial growth in the wrky48 mutants when compared with the wild-type plants (Figure 6A). The reduced bacterial growth during the first 2 d after inoculation was recovered substantially during the third day, as there was only a three- to five-fold difference in bacterial titers between wild-type and wrky48 mutants at 3 dpi (Figure 6A). Nonetheless, the inoculated leaves of the wrky48 mutant consistently displayed less chlorosis than wild-type plants at 3 dpi (Figure 6C). Thus, disruption of WRKY48 resulted in enhanced resistance, particularly during the early stages after infection, to the bacterial pathogen.
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We also attempted to express WRKY48 constitutively in transgenic Arabidopsis plants. A full-length cDNA for WRKY48 was placed behind the CaMV 35S promoter (35S:WRKY48) and transformed into Arabidopsis. Northern blotting showed that several transgenic plants contained elevated levels of the WRKY48 transcript even in the absence of pathogen infection (Figure 5C). Those plants that constitutively express WRKY48 exhibited significantly smaller plant sizes and more serrated leaves than the wild-type (Figure 5D). A similar alteration in growth and morphology has been previously observed in transgenic Arabidopsis plants that constitutively express pathogen-induced WRKY18 (Chen and Chen, 2002). Two transgenic lines with constitutive WRKY48 expression and a single T-DNA locus in their genomes were identified from the ratio of antibiotic resistance phenotypes and chosen for further analysis.
There was a marked increase (8–15-fold) in bacterial growth in the transgenic 35S:WRKY48 overexpression lines when compared with the wild-type plants (Figure 6A). The transgenic plants also developed more severe disease symptoms than the wild-type plants after infection by the bacterial pathogen (Figure 6B). Thus, constitutive overexpression of WRKY48 led to increased growth of the bacterial pathogen and enhanced development of disease symptoms in the transgenic plants.
Expression of Defense-Related Genes
To further analyze the defense responses in the loss-of-function mutants and overexpression lines for WRKY48, we compared their defense gene expression with that of wild-type plants after pathogen infection. First, we examined pathogen-induced expression of PR1. The wild-type, transgenic 35S:WRKY48 plants and the wrky48-1 mutant plants were inoculated with PstDC3000. Total RNA was isolated from the inoculated leaves harvested at 0 and 1 dpi and probed with a PR1 gene probe. At 0 dpi, no PR1 transcripts were detected in wild-type, wrky48 mutants or WRKY48-overexpressing plants (Figure 7A). At 1 dpi, PR1 transcripts were detected in both wild-type and wrky48 mutant plants but the level was significantly enhanced in the mutants when compared to those in the wild-type plants (Figure 7A). By contrast, the level of the PR1 transcripts was decreased in the transgenic 35S:WRKY48 plants when compared with that in the wild-type plants (Figure 7A).
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We also compared the wild-type, transgenic 35S:WRKY48 plants and the wrky48 mutants for SAR-associated defense gene expression. Three lower leaves of plants were first inoculated with an avirulent strain of P. syringae (PstDC3000avrRpt2) and total RNA was isolated from upper leaves at 1, 2, and 3 dpi. As shown in Figure 7B, in SAR-induced wild-type plants, transcripts for PR genes started to increase at 1 dpi, peaked at 2 dpi and declined at 3 dpi. In the wrky48 mutants, a similar trend was observed but the transcript levels of the PR1, PR2, and PR5 genes were significantly higher than those in the wild-type plants (Figure 7B). By contrast, induction of these PR genes was greatly reduced in the upper leaves of the transgenic 35S:WRKY48 plants after inoculation of their lower leaves with the avirulent bacterial strain. These results indicated that overexpression of WRKY48 blocked PR gene expression while knockout of the WRKY gene enhanced induction of these PR genes during the development of SAR.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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WRKY48 as a Negative Regulator of Plant Basal Defense
Arabidopsis WRKY48 is a stress- and pathogen-induced WRKY gene that encodes a nuclear localized WRKY transcriptional activator with a high binding affinity for DNA molecules with W-box sequences (Figures 1–3). Through analysis of both transgenic overexpressing lines and T-DNA insertion mutants, we have provided strong evidence that this pathogen-induced WRKY gene functions as a negative regulator of plant basal disease resistance. In the wrky48 knockout mutants, the growth of the bacterial pathogen was reduced, particularly during the first 2 d after inoculation, when compared with that in the wild-type plants (Figure 6A). The stronger effect on the bacterial growth during the early stage of infection is likely a reflection of the rapid but transient expression of WRKY48 in pathogen-infected plants (Figure 1A). We have also shown that constitutive overexpression of WRKY48 led to enhanced susceptibility to the bacterial pathogen P. syringae as manifested by enhanced growth of the bacterial pathogen and development of disease symptoms (Figure 6). It should be noted that overexpression of WRKY48 resulted in significantly smaller size and more serrated leaves of transgenic plants (Figure 5D). In a previously reported study, we have shown that overexpression of another pathogen-induced WRKY gene, WRKY18, also resulted in significantly smaller plant size and more serrated leaves of transgenic plants (Chen and Chen, 2002). However, unlike transgenic 35S:WRKY48 plants, transgenic 35S:WRKY18 plants constitutively express PR genes at their mature stages and were more resistant to the bacterial pathogen P. syringae (Chen and Chen, 2002). Therefore, the more susceptible phenotype of the transgenic 35S:WRKY48 plants is unlikely to be caused by their altered growth and morphology.
It is tempting to speculate why plants activate expression of a negative regulatory gene in response to pathogen infection that would make them more susceptible to the invading pathogen. This could simply reflect the fact that the defense mechanisms induced in plants upon pathogen infection are a massive network of different pathways comprising both positive and negative regulators. Negative regulators may play roles in preventing over-activation of defense mechanisms that could be detrimental to other biological processes important for plant growth and development, thereby reducing the cost of plant defense to overall plant fitness. In addition, different defense mechanisms mediated through different signaling pathways may have differential effects on plant resistance to different types of microbial pathogens (Glazebrook, 2005). Different signaling pathways can exhibit not only positive but also negative interactions, resulting in both cooperative and antagonistic relationships. A negative regulator for one pathway may be important for maintaining proper balancing of a signaling network so that activation of one signaling pathway effective for combating one type of microbial pathogens does not greatly compromise other signaling pathways important for resistance to other types of microbial pathogens.
There is also a possibility that pathogen-induced expression of a negative regulator of plant defense, such as WRKY48, may result from the counter-defense mechanism of an invading pathogen. A number of studies have recently shown that gram-negative bacterial pathogens such as P. syringae have various mechanisms of suppressing innate immunity of plant hosts (Alfano and Collmer, 2004). For example, a number of type III effector proteins from P. syringae suppress hypersensitive cell death of plant host cells (Alfano and Collmer, 2004; Abramovitch and Martin, 2005). A P. syringae type III effector can compromise defense-related callose deposition in the host cell wall (Hauck et al., 2003; DebRoy et al., 2004). In addition, P. syringae type III effectors and phytotoxin coronatine can augment a COI1-dependent pathway in plants to promote parasitism (He et al., 2004). A number of studies have reported that PAMP-induced MAPK signaling pathways can be inactivated by specific effector proteins of P. syringae (He et al., 2006; Zhang et al., 2007). We have found that unlike wild-type virulent and avirulent PstDC3000 strains, the PstDC3000hrcC mutant strain defective in the type III secretion system did not enhance WRKY48 expression (Figure 1A). Thus, certain virulence factors from the bacterial pathogen may actively promote expression of negative regulatory genes such as WRKY48 as an active counter-defense mechanism to compromise the defense mechanism and promote parasitism.
WRKY48 is rapidly induced by infiltration with MgCl2 (Figure 3), likely due to osmotic and/or mechanical stresses generated from the infiltration. The stress-induced expression suggests that WRKY48 may play a role in plant responses to abiotic stresses. Notably, highest levels of WRKY48 transcripts were detected during the first few hours after infiltration but then declined steadily, reaching near basal levels by 24 hpi (Figure 3). WRKY48 induction was substantially enhanced and sustained in the JA and ET signaling mutants (Figure 3), suggesting that ET- and JA-mediated signaling plays a role in suppressing WRKY48 expression following its rapid induction after pathogen inoculation. By suppressing expression of negative regulators of basal defense such as WRKY48, JA and ET may play a positive role in some aspects of Arabidopsis responses to P. syringae. Several studies have indeed shown that systemic immunity of Arabidopsis responses to P. syringae is dependent on ET and JA (Verhagen et al., 2004; Ahn et al., 2007; Truman et al., 2007). Both positive and negative regulation of WRKY48 expression in pathogen-inoculated plants may, therefore, reflect the complex and dynamic nature of plant defense and pathogen counter-defense during plant–pathogen interactions. Upon pathogen inoculation, PAMP-induced defense responses are rapidly activated. At the same time, the osmotic and/or mechanical stresses generated from inoculation induce certain stress-induced regulatory genes such as WRKY48 with a negative role in basal disease resistance. As a counter-defense mechanism, invading pathogens may actively promote expression of such negative regulators through the action of certain secreted effector proteins. To counter the counter-defense mechanism, plants may rely on the ET- and JA-dependent pathways to repress the expression of the negative regulatory genes to promote defense responses.
Mechanisms for the Repression of Plant Basal Defense by WRKY48
SA plays an important role in Arabidopsis basal resistance to P. syringae, as mutants defective in SA biosynthesis or signaling are more susceptible to the bacterial pathogen than the wild-type plants (Glazebrook, 2005). However, SA accumulation in the wrky48 mutants is normal (data not shown), suggesting that the stress- and pathogen-induced WRKY transcription factor does not play an important role in SA biosynthesis. On the other hand, SA-regulated PR gene expression was altered in both the transgenic overexpression lines and the T-DNA insertion mutants for WRKY48 (Figure 7). Expression of the defense-related PR1 gene was reduced significantly in the leaves of transgenic overexpression plants after infection by a virulent strain of P. syringae (Figure 7). Furthermore, unlike in the wild-type plants, where infection of lower leaves by an avirulent strain of P. syringae led to induction of PR genes in upper uninoculated leaves, this SAR-associated PR gene expression was almost completely abolished in the transgenic 35S:WRKY48 plants (Figure 7B). By contrast, expression of PR genes during pathogen-induced SAR was enhanced in the wrky48 mutant plants when compared with that in wild-type plants (Figure 7B). These results strongly suggest that WRKY48 is a negative regulator of pathogen-induced PR gene expression.
Several previous studies have indeed suggested that WRKY proteins may play negative roles in the regulation of plant defense genes. For example, the Arabidopsis PR1 gene, a reliable marker for SA-mediated defense responses, contains a W-box sequence in its promoter (Lebel et al., 1998). Mutation of the W-box resulted in enhanced promoter activity when assayed with a reporter gene, suggesting that the W-box sequence acts as a negative cis-acting element in the expression of the defense-related gene (Lebel et al., 1998). Likewise, we have shown that a cluster of three W-box sequences in the promoter of the Arabidopsis WRKY18 gene reduced its promoter activity (Chen and Chen, 2002). Using chromatin immuno-precipitation, it has been shown in cultured parsley cells that the promoter sites of elicitor-induced genes such as PcWRKY1 are constitutively occupied by certain WRKY proteins but displaced by other WRKY proteins in a stimulus-dependent manner (Turck et al., 2004). It appears that different WRKY proteins act in a mutually competing manner with dynamic displacement upon pathogen infection or elicitor treatment. We have recently reported that WRKY7 from Arabidopsis, like WRKY48, is a negative regulator of plant defense gene expression and basal resistance to P. syringae based on the phenotypes of both loss-of-function mutants and overexpression lines (Kim et al., 2006). Arabidopsis WRKY7 is a transcriptional repressor in plant cells and, therefore, may function as a negative regulator of plant basal defense by directly repressing expression of plant defense genes (Kim et al., 2006).
Although it suppresses disease resistance and defense gene expression (Figures 6 and 7), WRKY48 acts as a transcriptional activator in plant cells (Figure 3). Thus, WRKY48 does not appear to repress defense genes directly. Instead, WRKY48 may first activate certain unknown negative regulators that, in turn, repress defense genes. Further characterization of in-vivo binding sites and identification of downstream target genes of WRKY48 will provide valuable insights into how the pathogen-induced transcription factor negatively regulates plant PR gene expression and compromise disease resistance to the bacterial pathogen.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Materials
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-32P]dATP (>3000 Ci mmol–1) was obtained from New England Nuclear; other common chemicals were purchased from Sigma. Arabidopsis plants were grown in a growth chamber at 22°C and 150 µEm–2s–1 light on a photoperiod of 12 h light and 12 h dark. SA was dissolved in water as 100 mM stock solutions and adjusted to pH 6.5 with KOH. SA, MeJA, and ACC treatments were performed by spraying the plants with solutions at indicated concentrations.
Recombinant Protein and DNA-Binding
Preparation of recombinant WRKY48 proteins and DNA-binding assays were performed as previously described (Yu et al., 2001).
Subcellular Localization
Onion epidermal cell layers were peeled and placed inside up on the MS plates. Plasmid DNAs of appropriate fusion genes (0.5 µg) were introduced to the onion cells using a pneumatic particle gun (PDS 1000, Du Pont). The condition of bombardment was a vacuum of 28 inch Hg, helium pressure of 1100 or 1300 psi, and 6 cm of target distance using 1.1 µm of tungsten microcarriers. After bombardment, tissues were incubated on the MS plates for 24 h at 22°C. Samples were observed directly or transferred to glass slides.
Pathogen Infection
Pathogen inoculations were performed by infiltration of leaves of at least six plants for each treatment with the P. syringae pv. tomato DC3000 strain (OD600 = 0.001 in 10 mM MgCl2). Inoculated leaves were harvested at indicated dpi and homogenized in 10 mM MgCl2. Diluted leaf extracts were plated on King's B medium supplemented with rifampicin (100 µg ml–1) and kanamycin (25 µg ml–1) and incubated at 25°C for 2 d before counting the colony-forming units.
Northern Blot Analysis
For Northern blot analysis of plant gene expression, total RNA was isolated from treated leaves using the TRIZOL reagent (BRL Life Technologies, Rockville, MD). The RNA was separated on agarose (1.2%)-formaldehyde gels and blotted onto nylon membranes. Hybridization was performed with random-primed 32P-labeled DNA probes in PerfectHyb plus hybridization buffer (Sigma) overnight at 68°C. The membranes were washed at 68°C once in 2 SSC and 0.1% SDS for 5 min, twice in 0.5 SSC and 0.1% SDS for 20 min and once in 0.1 SSC and 0.1% SDS for 20 min. DNA probes for WRKY48 were isolated from its full-length cDNA clone. DNA probes for PR genes were prepared from PCR-amplified DNA fragments using the following gene-specific primers: PR1: 5'-TTCTTCCCTCGAAAGCTCAA-3'/5'-CGTTCACATAATTCCCACGA-3’; PR2: 5'-TGGTGTCAGATTCCGGTACA-3'/5'-TCGGTGATCCATTCTTCACA-3’; PR5: 5'-GCGATGGAGGATTTGAATTG-3'/5'-GCGTAGCTATAGGCGTCAGG-3'.
Assays of Transcriptional Regulatory Activity of WRKY48
Transgenic Arabidopsis plants containing a GUS reporter gene driven by a synthetic promoter consisting of the –100 minimal CaMV 35S promoter and eight copies of the LexA operator sequence were previously described (Kim et al., 2006). To generate effector genes, the DNA fragment for the LexA DBD was digested from the plasmid pEG202 (Clontech) using HindIII and EcoRI and cloned into the same sites in pBluescript. The full-length WRKY48 cDNA fragment was subsequently subcloned behind the LexA DBD to generate translational fusion. The LexA DBD-WRKY48 fusion gene was cloned into the XhoI and SpeI site of pTA2002 behind the steroid-inducible promoter (Aoyama and Chua, 1997). As controls, the unfused LexADBD and WRKY48 genes were also cloned into the same sites of PTA7002. These effector constructs were directly transformed into the transgenic GUS reporter plants and double transformants were identified through screening for antibiotic (hygromycin) resistance. Determination of activation or repression of GUS reporter gene expression by the effector proteins was performed as previously described (Kim et al., 2006).
Identification of the wrky48 T-DNA Insertion Mutants
The wrky48-1 (Salk_066438) and wrky48-2 (Sail_1267_D04) each contain a T-DNA insertion in the first exon the WRKY48 gene. Confirmation of the T-DNA insertions was done by performing PCR using a combination of a gene-specific primer and a T-DNA border primer. The nature and location of the T-DNA insertions were confirmed by sequencing the PCR products. Homozygous wrky48 mutant plants were identified by PCR using a pair of primers corresponding to sequences flanking the T-DNA insertion sites (pW48F: 5'-CCCTTTTGCTCTTGTTGTTGA-3’ and pW48R: 5'-TCAGATCATCATCCGTTGGA-3’). To remove additional T-DNA loci or mutations from the mutants, backcrosses to wild-type plants were performed and plants homozygous for the T-DNA insertion were again identified.
Construction of Transgenic Overexpression Lines
To generate the 35S:WRKY48 construct, the cDNA fragment that contained the full coding sequence and 3’ untranslated region of WRKY48 was excised with KpnI and SalI from a cloning plasmid and subcloned into the same restriction sites of the Agrobacterium transformation vector pOCA30 (Chen and Chen, 2002) in the sense orientation behind the CaMV 35S promoter. Arabidopsis transformation was performed by the floral dip procedure (Clough and Bent, 1998). The seeds were collected from the infiltrated plants and selected in MS medium containing 50 µg ml–1 kanamycin. Kanamycin-resistant plants were transferred to soil 9 d later and grown in a growth chamber.
Accession Numbers
The Arabidopsis Genome Initiative identifiers for the genes described in this article are as follows: WRKY48 (At5g49520), PR1(At2g14610), PR2 (At3g57260) and PR5 (At1g75040).
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This work was supported in part by the US National Science Foundation grant MCB-0209819. This is journal paper 2007–18186 of the Purdue University Agricultural Research Program.
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We would like to thank RIKEN Bioresource Center for the full-length cDNA clone for WRKY48 and the Arabidopsis Resource Center at the Ohio State University and Syngenta Biotechnology, Inc. for the wrky48 T-DNA insertion mutants. We would also like to thank Drs Shengyang He (Michigan State University, East Lansing, Michigan) and Xiaoyan Tang (Kansas State University, Manhattan, MA) for the PstDC3000hrcC mutant and Dr Walter Gassmann (University of Missouri, Columbia, MO) for other P. syringae strains.
No conflict of interest declared.
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2 These two authors contributed equally to this work.
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