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First published online September 8, 2006; 10.1104/pp.106.082487 Plant Physiology 142:1180-1192 (2006) © 2006 American Society of Plant Biologists
Pathogen-Induced Arabidopsis WRKY7 Is a Transcriptional Repressor and Enhances Plant Susceptibility to Pseudomonas syringae1,[W]Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907–2054
The Arabidopsis (Arabidopsis thaliana) WRKY7 gene is induced by pathogen infection and salicylic acid (SA) treatment and may therefore play a role in plant defense responses. Here, we show that WRKY7 is localized in the nucleus, recognizes DNA molecules with the W-box (TTGAC) elements, and functions as a transcriptional repressor in plant cells. To study its biological functions directly, we have characterized both loss-of-function T-DNA insertion and RNAi mutants and gain-of-function transgenic overexpression plants for WRKY7 in Arabidopsis. The T-DNA insertion and RNAi mutant plants displayed enhanced resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae as measured by significant decrease in both bacterial growth and symptom development as compared to those in wild-type plants. The enhanced resistance in the loss-of-function mutants was associated with increased induction of SA-regulated Pathogenesis-Related 1 (PR1) by the bacterial pathogen. Transgenic plants that constitutively overexpress WRKY7 have altered leaf growth and morphology strikingly similar to those observed in the previously isolated eds8 mutant plants. Like eds8 mutant plants, WRKY7-overexpressing plants supported more growth of P. syringae and developed more severe disease symptoms than wild-type plants. The enhanced susceptibility of both the WRKY7-overexpressing plants and the eds8 mutant correlated with reduced expression of defense-related genes, including PR1, but significantly increased accumulation of SA after pathogen infection, probably due to reduced negative feedback of SA synthesis. Thus, pathogen-induced WRKY7 transcription factor play a negative role in defense responses to P. syringae.
Pseudomonas syringae is a bacterial pathogen that infects a wide variety of plants (Hirano and Upper, 2000  ). Several strains of P. syringae infect Arabidopsis (Arabidopsis thaliana), and the establishment of the Arabidopsis-Pseudomonas model pathosystem has greatly contributed to our understanding of the molecular basis of plant-pathogen interactions (Katagiri et al., 2002). In the well-studied R gene-mediated disease resistance, recognition of specific avirulence factors from the bacterial pathogen by the products of corresponding R genes from Arabidopsis plants can trigger local plant defenses that are usually associated with programmed plant cell death, known as the hypersensitive responses (Nimchuk et al., 2003). Pathogen-induced hypersensitive responses are often associated with accumulation of salicylic acid (SA) and activation of defense mechanisms in the surrounding or even distal parts of the plants, leading to the development of systemic acquired resistance (SAR; Durrant and Dong, 2004).
A large number of Arabidopsis mutants with enhanced susceptibility to P. syringae have been isolated (Glazebrook et al., 1996
In Arabidopsis, resistance to P. syringae can be enhanced not only by SA-mediated SAR but also by induced systemic resistance activated by biocontrol rhizobacteria (Pieterse et al., 1996
A growing body of evidence suggests that WRKY proteins have regulatory functions in plant defense responses including activation of SAR (Ulker and Somssich, 2004
Other WRKY genes, on the other hand, may function to repress SA-mediated signaling. In a recently reported study using yeast two-hybrid screening, Arabidopsis MAP kinase 4 (MPK4), a repressor of SA-dependent resistance (Petersen et al., 2000
Arabidopsis WRKY7 is induced by SA and P. syringae (Dong et al., 2003
Protein Sequence, Subcellular Localization, and DNA Binding As shown in Figure 1A , Arabidopsis WRKY7 contains a single WRKY domain located at its C terminus. Inspection of the amino acid sequences of WRKY7 also revealed a typical nuclear localization sequence, with a cluster of basic residues (Fig. 1A). WRKY7 contains a number of Gly-, Glu-, or Gln-rich motifs that are often found in transcription factors with possible roles in transcriptional activation or repression (Fig. 1A). A Ser/Thr-rich region is also found at the middle of the WRKY protein (Fig. 1A), suggesting possible regulation of the transcription factor through posttranslational protein modification.
To examine the subcellular localization of WRKY7, its coding region was fused to the 3' end of a green fluorescent protein (GFP) reporter gene and expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The GFP gene alone under the control of the 35S promoter was used as a control. The GFP constructs were introduced separately by particle bombardment into onion (Allium cepa) epidermal cells. As shown in Figure 1B, this transiently expressed WRKY7-GFP fusion protein was localized exclusively to the nuclei of onion epidermal cells. The GFP protein was found in both the nuclei and cytoplasm, presumably due to its small size. This experiment suggests that WRKY7 is localized in the nucleus.
The novel type of the Cis2-His2 zinc finger of plant WRKY proteins serves as a DNA-binding domain (DBD) that specifically recognizes the TTGAC (W-box) elements (Ulker and Somssich, 2004
WRKY7 Is a Transcriptional Repressor
To determine the transcriptional regulatory activity of WRKY7 in planta, we developed a transgenic system in which the transcriptional regulatory activity of a protein can be determined through assays of a reporter gene in stably transformed plants. First, we generated a synthetic promoter consisting of the –100 minimal CaMV 35S promoter and eight copies of the LexA operator sequence. The promoter was fused with the
To generate the WRKY7 effector, we fused its coding sequence with that of the DBD of LexA. The fusion construct was subcloned behind the steroid-inducible Gal4 promoter in pTA7002 (Aoyama and Chua, 1997
T-DNA and RNA Silencing wrky7 Mutants To analyze the role of WRKY7 in plant defense, we identified a T-DNA insertion mutant, wrky7-1, that carries a T-DNA insertion in the second intron of the WRKY7 gene (Fig. 4A ). Northern-blot analysis revealed that SA induced WRKY7 transcripts of expected size in wild type but not in wrky7-1 (Fig. 4B). In addition, we generated RNAi mutant plants that harbor inverted repeats of a WRKY7 fragment under control of the CaMV 35S promoter. Using RNA-blot analysis, we identified several RNAi lines that accumulated little WRKY7 transcripts following SA treatment (Fig. 4C). A representative line containing a single T-DNA insertion locus (based on 3:1 antibiotic resistance segregation in T2 generation) was chosen for further investigation. The T-DNA insertion mutant and the RNAi lines grew and developed normally.
To determine the effect of disruption or suppressed expression of WRKY7 on plant disease resistance, we examined their response to P. syringae pv tomato DC3000 (PstDC3000), a strain virulent to Columbia-0 ecotype Arabidopsis plants (Whalen et al., 1991 ). Plants were inoculated through infiltration with the bacteria (OD600 = 0.001 in 10 mM MgCl2), and the growth of the pathogen was monitored 3 d later. As shown in Figure 5A
, the bacterial growth in the T-DNA insertion and RNAi mutant plants was 6- to 7-fold lower than that in wild-type plants. The mutant plants also developed less severe disease symptoms than wild-type plants after bacterial infection (Fig. 5B). These results indicate that WRKY7 is a negative regulator of plant defense against the bacterial pathogen.
Resistance of Arabidopsis plants to P. syringae is dependent of SA-mediated defense signaling that is often associated with enhanced expression of PR genes including PR1. To determine whether enhanced resistance of the T-DNA insertion and RNAi mutants for WRKY7 to P. syringae was associated with enhanced SA signaling, we analyzed their SA accumulation before and after bacterial infection but failed to find significant difference between the mutants and wild type. We then compared wild-type and wrky7 mutant plants for pathogen-induced PR1 expression. There was no PR1 transcript accumulation in wild type, the T-DNA insertion, or RNAi mutant plants prior to bacterial infection. At 2 and 3 d postinoculation (dpi), however, PR1 transcripts accumulated at higher levels in the mutant plants than in wild-type plants (Fig. 5C). Thus, enhanced resistance to the bacterial pathogen conferred by disruption or suppressed expression of WRKY7 correlated with enhanced induction of SA-regulated PR1 gene expression by the pathogen.
Arabidopsis WRKY70 has been shown to play a role in cross talk between SA- and JA-mediated signaling pathways (Li et al., 2004
To further analyze its biological function, we overexpressed WRKY7 constitutively in transgenic Arabidopsis plants. A cDNA with the full-length coding region of WRKY7 was placed behind the CaMV 35S promoter and transformed into Arabidopsis. Northern blotting showed elevated level of WRKY7 transcripts in the transgenic plants harboring the construct (Fig. 6A ). Interestingly, all those plants with substantially elevated WRKY7 transcripts have broader and more serrated leaves than wild-type plants, whereas transgenic plants with little or no elevation in WRKY7 transcripts do not exhibit such phenotypes (Fig. 6B). The altered morphology of WRKY7-overexpressing plants was strikingly similar to that of the previously isolated eds8 mutant (Fig. 6B). Because of the similarity in both leaf morphology and defense responses (see below), we also transformed the eds8 mutant plants with the 35S:WRKY7 construct, and WRKY7-overexpressing eds8 mutant plants were identified through RNA-blotting analysis (Fig. 6A). As shown in Figure 6B, when compared with the untransformed eds8 mutant plants and the WRKY7-overexpressing plants in the wild-type background, WRKY7-overexpressing eds8 mutant plants had an enhanced phenotype in leaf morphology; the sizes of these plants were also reduced, and seed setting substantially decreased. Thus, WRKY7 overexpression and mutation of EDS8 had additive effects on plant growth and development.
To determine the effect of WRKY7 overexpression on plant disease resistance, we examined the response of the overexpression plants to PstDC3000. For comparison, we included the npr1-3 and eds8 mutants that are known to exhibit enhanced susceptibility to the bacterial pathogen. Plants were inoculated through infiltration with the bacteria (OD600 = 0.001 in 10 mM MgCl2), and the growth of the pathogen was monitored 3 d later. As shown in Figure 7A , the bacterial growth in the npr1-3 mutant plants was approximately 7-fold higher than that in wild-type plants. The eds8 mutant also supported a significantly higher bacterial growth than wild-type plants, although the mutant was not as susceptible as the npr1 mutant based on the bacterial growth. The growth of the bacterial pathogen in the WRKY7-overexpressing plants was, at least, as high as in the eds8 mutant (Fig. 7A). When WRKY7 was overexpressed in the eds8 mutant background, the bacterial growth was further enhanced relative to those in the eds8 mutant and the WRKY7-overexpressing plants in the wild-type background. The npr1-3, eds8 mutants, and WRKY7-overexpression plants (in both the wild-type and eds8 backgrounds) also developed more severe disease symptoms than wild-type plants after infection (Fig. 7B). These results indicated that overexpression of WRKY7 had a negative effect on plant resistance to PstDC3000. It also appeared that overexpression of WRKY7 and mutation of EDS8 had an additive effect on plant susceptibility to the bacterial pathogen.
To determine whether WRKY7-induced susceptibility to PstDC3000 is associated with defects in SA signaling, we determined SA-regulated PR1 gene expression in Arabidopsis plants that overexpressed WRKY7. For comparison, we again included the npr1-3 and eds8 mutants in these analyses. As shown in Figure 8A , in wild-type plants the level of PR1 gene transcripts was elevated at 1 dpi, continued to increase at 2 dpi, and then declined at 3 dpi. By contrast, there was little induction of PR1 in the npr1-3 mutant after infection (Fig. 8A). In the eds8 mutant, PR1 transcripts were almost undetectable at 1 dpi and substantially reduced at 2 dpi as compared to those in the wild-type plants (Fig. 8A). Thus, pathogen-induced PR1 expression was both delayed and reduced in the eds8 mutant. Overexpression of WRKY7 also reduced pathogen-induced PR1 expression. In the wild-type background, WRKY7 overexpression did not appear to delay the induction based on the significant level of PR1 transcripts detected at 1 dpi in the overexpression plants (Fig. 8A). However, PR1 transcript levels were substantially lower at both 2 and 3 dpi in the transgenic overexpression plants than those in wild-type plants (Fig. 8A). In the eds8 mutant background, WRKY7 overexpression reduced PR1 transcript accumulation at both 1 and 2 dpi as compared to that in wild-type plants (Fig. 8A). It appeared that overexpression of WRKY7 in the eds8 mutant background both delayed and reduced expression of the SA-regulated defense gene.
We also examined the accumulation of SA in these plants. SA levels were very low in uninfected wild-type plants but increased approximately 20-fold at 24 h after infection. In the npr1-3 mutant plants, the basal SA levels were similarly low, but the induced SA levels after pathogen infection were approximately 3 to 4 times higher than those in pathogen-infected wild-type plants (Fig. 8B). The enhanced SA accumulation upon pathogen infection in the SA-signaling mutant has been attributed to negative feedback regulation of SA biosynthesis by NPR1-mediated SA signaling (Shah et al., 1997 ; Wildermuth et al., 2001). In the eds8 mutant, induction of SA levels after infection was also significantly higher than that in wild-type plants, although the magnitude of increase was lower than that in the npr1 mutant (Fig. 8B). There were significantly higher SA levels in WRKY7-overexpression plants than in wild-type plants (Fig. 8B). Overexpression of WRKY7 and mutation of EDS8 also had a significantly additive effect on pathogen-induced SA accumulation; the SA levels in the WRKY7-overexpressing eds8 mutant plants were significantly higher than those in the eds8 mutant and in WRKY7-overexpressing wild-type plants (Fig. 8B). Thus, pathogen-induced SA accumulation in the eds8 mutant and transgenic WRKY7 overexpression was not impaired.
Global expression phenotyping of wild-type and signaling-defective mutant plants has shown that EDS8 is involved in JA or ET signaling (Glazebrook et al., 2003
Interactions between Arabidopsis and the bacterial pathogen P. syringae have been extensively analyzed, and we therefore used this model system to investigate the role of WRKY7 in plant defense responses. In the T-DNA insertion and RNAi mutants for WRKY7, the growth of the bacterial pathogen and development of the disease symptom were significantly reduced when compared with those in wild-type plants (Fig. 5). Constitutive overexpression of WRKY7, on the other hand, led to enhanced susceptibility to P. syringae as manifested by enhanced bacterial growth and development of more severe disease symptom (Fig. 7). These results indicate that pathogen- and SA-induced WRKY7 plays a negative role in plant defense against the bacterial pathogen.
Arabidopsis mutants defective in SA biosynthesis or signaling, including eds1, pad4, eds5, sid2, and npr1, allow increased growth of P. syringae, indicating that SA-mediated signaling mechanisms are important for defense against P. syringae. In the T-DNA insertion and RNAi mutants for WRKY7 that exhibited enhanced resistance to P. syringae, SA-regulated PR1 gene expression was enhanced but SA accumulation was normal after infection. This observation suggests that WRKY7 might function as a negative regulator of SA signaling. In support of this hypothesis, overexpression of WRKY7 reduced SA-regulated PR1 gene expression without compromising SA accumulation. In fact, SA levels in the transgenic overexpression plants were significantly elevated after pathogen infection relative to those in wild-type plants. Enhanced SA accumulation is also observed in the SA signaling-defective npr1 mutant and is attributed to negative feedback regulation of SA biosynthesis by SA signaling (Durrant and Dong, 2004
We have previously shown that overexpression of Arabidopsis WRKY18 led to constitutive expression of PR genes and enhanced resistance to P. syringae. Likewise, overexpression of Arabidopsis WRKY70 resulted in enhanced resistance to P. syringae and stronger induction of SA-regulated PR genes (Li et al., 2004
How WRKY7 negatively regulates SA-regulated PR1 gene expression and compromised plant defense against P. syringae is unclear. A number of WRKY proteins have been shown to act as positive transcriptional regulators (de Pater et al., 1996 WRKY7-overexpression plants and the eds8 mutant exhibited not only similar changes in leaf morphology but also similar phenotypes in increased susceptibility to P. syringae and reduced expression of pathogen-induced PR1. In addition, there was a similar increase in the accumulation of SA in both the eds8 mutant and the WRKY7-overexpression plants. The strikingly similar phenotypes of the eds8 and transgenic WRKY7-overexpression plants raised the possibility that the two genes might function in the same signal transduction pathway. However, when WRKY7 expression was silenced in the eds8 mutant, neither the altered leaf morphology nor the enhanced disease susceptibility of eds8 was reversed (K.-C. Kim and Z. Chen, unpublished data). Thus, WRKY7 does not appear to be a downstream component of the EDS8 signaling pathway directly responsible for the mutant phenotypes of the eds8 mutant. Nevertheless, the strikingly similar phenotypes of the eds8 mutant and the WRKY7-overexpressing plants strongly suggest that they may share the same or similar molecular basis for enhanced disease susceptibility to P. syringae.
In this study, we have shown that in the eds8 mutant, induction of SA-regulated PR1 was also significantly reduced but SA accumulation was not reduced after infection by P. syringae. These observations suggested that the eds8 mutant might also be defective in SA signaling. However, global gene expression analysis has revealed that the eds8 mutant is defective in induction of genes regulated by JA, suggesting that EDS8 affects JA signaling (Glazebrook et al., 2003
Plant Growth Conditions The Arabidopsis (Arabidopsis thaliana) wild-type, mutant, and transgenic plants used in the study were all in the Columbia-0 genetic background and were grown in growth chambers at 22°C and 120 µE m–2 s–1 light on a 12-h-light and 12-h-dark photoperiod.
The WRKY7 cDNA was amplified with the following primers: 5'-ATGGAATTCATGACTGTTGAGCTGATGATGAG-3' and 5'-ATCGCCATGGAGAGTTTTGTCATGATTCATCGTCG-3'. The amplified fragment was digested with EcoR1 and NcoI and cloned into a GFP vector. The empty GFP plasmid was used as a control. The plasmid was isolated using Qiagen kits, concentrated to about 1 µg/µL, and used to coat the gold particles for bombardment experiments. Transient expression of the GFP fusion genes in onion (Allium cepa) epidermal cells through particle bombardment and subsequent localization of the proteins was performed essentially as described (Xu et al., 2006
To generate the WRKY7 recombinant protein, its full-length cDNA was cloned into pET32a (Novagen) and transformed into Escherichia coli strain BL21 (DE3). Induction of expression and purification of recombinant His-tagged WRKY7 protein were performed according to the protocol provided by Novagen. The purified proteins were dialyzed overnight against a nuclear extraction buffer (25 mM HEPES/KOH, pH 7.5, 40 mM KCl, 0.1 mM EDTA, 10% glycerol, 1 mM dithiothreitol, and 30 mg/L phenylmethylsulfonyl fluoride) at 4°C. Double-stranded synthetic oligonucleotides were labeled to specific activities of approximately 105 cpm/ng using the Klenow fragment of DNA polymerase I. DNA and protein complexes were allowed to form at room temperature for 30 min and resolved on a 10% polyacrylamide gel in 0.5x Tris-borate/EDTA at 4°C.
The transcriptional regulatory activity of WRKY7 was determined in stably transformed plants through assays of 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. The LexA operator sequence was PCR amplified from pER8 (Zuo et al., 2000
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 WRKY7 cDNA fragment was subsequently subcloned behind the LexA DBD to generate a translational fusion. The LexA DBD-WRKY7 fusion gene was cleaved from the plasmid with SalI/XbaI digestion and cloned into the XhoI/SpeI site of pTA2002 behind the steroid-inducible promoter (Aoyama and Chua, 1997
For determining activation or repression of GUS reporter gene expression by the effector proteins, one fully expanded leaf was harvested immediately prior to DEX treatment, and three additional leaves were harvested 18 h after treatment of DEX (20 µM) from each double-transformed plant (5 weeks old). Two of the three leaves harvested after DEX treatment were used for isolation of total RNA that was subsequently probed with a LexA DBD or WRKY7 DNA fragment for determining DEX-induced effector gene expression. For those double transformants with positive effector gene expression revealed from RNA blotting, the GUS activities from the third leaves harvested after DEX treatment were determined and compared with the GUS activities determined from the leaves harvested immediately before DEX treatment. GUS activity was measured through a 4-methylumbellifery-
The wrky7-1 mutant (GABI_356A10) contains a T-DNA insertion in the second intron of the WRKY7 gene. The T-DNA insertion in the wrky7-1 mutant was confirmed by PCR using a T-DNA-specific primer (5'-CCCATTTGGACGTGAATGTAGACAC-3') and a WRKY7-specific primer (pW7-1, 5'-AGAGTTTTGTCATGATTCATCGTCG-3'). Homozygous wrky7-1 mutant plants were identified by PCR using a pair of primers corresponding to sequences flanking the T-DNA insertion (pW7-1 and pW7-1R, 5'-ATGACTGTTGAGCTGATGATGAG-3'). The wrky70-1 mutant (Salk_025198) contains a T-DNA insertion in the first exon of WRKY70. Homozygous wrky70-1 mutant plants were identified by PCR using a pair of primers corresponding to sequences flanking the T-DNA insertion (pW70-1, 5'-CATGTGATAACGACGGCAAG-3' and pW70-1R, 5'-AAAGGACCTTGGGAATTTGG-3').
An EcoRI/HindIII fragment that contains the 35S promoter with double enhancers, multiple cloning sites, and 35S terminator was excised from pFF19 and cloned into the same sites of the transformation vector pOCA28 to generate pOCA30. To generate the 35S-WRKY7 construct, the cDNA fragment that contains full coding sequence and 3' untranslated region of WRKY7 was excised with KpnI and SalI from a cloning plasmid and subcloned into the same restriction sites of pOCA30 in the sense orientation behind the 35S promoter. To generate the WRKY7 RNAi construct, the first intron of the Arabidopsis Cat3 gene was first cloned between the CaMV 35S promoter and the 35S terminator of pOCA30 to generate an RNAi vector pAA1. An approximately 400-bp DNA fragment of WRKY7 was amplified with primers (5'-GACTCGAGTGGAGGAGAAGAAGCCAGAA-3' and 5'-AGTCTAGACCTCTCGAACCAGAGAGCTG-3') and digested with XhoI and XbaI. The XhoI/XbaI WRKY7 fragment was cloned into XbaI/SalI sites in sense orientation before the Cat3 intron in pAA1 and was cloned again into the XhoI/SpeI sites in antisense orientation behind the Cat3 intron in the resulting plasmid. The final construct contains two WRKY7 inverted repeats separated by the Cat3 intron.
Arabidopsis transformation was performed by the floral-dip procedure (Clough and Bent, 1998
For northern-blot analysis, total RNA (5 µg) was separated on agarose-formaldehyde gels and blotted to nylon membranes. Blots were hybridized with [
Pathogen inoculations were performed by infiltration of leaves of at least six plants for each treatment with the PstDC3000 strain (OD600 = 0.001 in 10 mM MgCl2). Inoculated leaves were harvested 3 d after infiltration and homogenized in 10 mM MgCl2. Diluted leaf extracts were plated on King's B medium supplemented with rifampicin (100 µg/mL) and kanamycin (25 µg/mL) and incubated at 25°C for 2 d before counting the colony forming units.
Total SA was extracted and quantified as described previously (Freeman et al., 2005 Arabidopsis Genome Initiative numbers for the genes discussed in this article are as follows: WRKY7, At4g24240; PR1, At2g14610.
The following materials are available in the online version of this article.
We thank the Arabidopsis Resource Center at The Ohio State University and Max Planck Institute for Plant Breeding Research for Arabidopsis mutants. We also thank Jixin Dong for efforts during the initial stage of the work and John Freeman for help with SA measurement. Received April 24, 2006; accepted August 30, 2006; published September 8, 2006.
1 This work was supported by the National Science Foundation (grant no. MCB–0209819 to Z.C.). This is journal paper 2006–18006 of the Purdue Agricultural Research Program.  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: Zhixiang Chen (zhixiang{at}purdue.edu).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.082487 * Corresponding author; e-mail zhixiang{at}purdue.edu; fax 765–494–5896.
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ABSTRACT
RESULTS
-glucuronidase (GUS) reporter gene, subcloned into a plant transformation vector, and transformed into Arabidopsis plants. Transgenic lines harboring a single insertion locus (based on kanamycin antibiotic resistance segregation) were identified, and homozygous T3 progeny lines were obtained. Due to the minimal 35S promoter used, these transgenic plants constitutively expressed similarly low levels of the GUS reporter gene, thereby making them useful for assays of transcription activation or repression by determining increase or decrease in GUS activities following coexpression of an effector protein. 
-32P]dATP-labeled gene-specific probes. Hybridization was performed in PerfectHyb plus hybridization buffer (Sigma) overnight at 68°C. The membrane was then washed for 10 min twice with 2x SSC and 1% SDS and 10 min with 0.1x SSC and 1% SDS at 68°C. 
