• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of plntphysLink to Publisher's site
Plant Physiol. Sep 2008; 148(1): 358–368.
PMCID: PMC2528098

A Role for AtWRKY23 in Feeding Site Establishment of Plant-Parasitic Nematodes1,[W]

Abstract

During the interaction between sedentary plant-parasitic nematodes and their host, complex morphological and physiological changes occur in the infected plant tissue, finally resulting in the establishment of a nematode feeding site. This cellular transformation is the result of altered plant gene expression most likely induced by proteins injected in the plant cell by the nematode. Here, we report on the identification of a WRKY transcription factor expressed during nematode infection. Using both promoter-reporter gene fusions and in situ reverse transcription-polymerase chain reaction, we could show that AtWRKY23 is expressed during the early stages of feeding site establishment. Knocking down the expression of WRKY23 resulted in lower infection of the cyst nematode Heterodera schachtii. WRKY23 is an auxin-inducible gene and in uninfected plants WRKY23 acts downstream of the Aux/IAA protein SLR/IAA14. Although auxin is known to be involved in feeding site formation, our results suggest that, during early stages, auxin-independent signals might be at play to activate the initial expression of WRKY23.

Sedentary plant-parasitic nematodes (e.g. Heterodera and Meloidogyne ssp.) have developed a fascinating way to parasitize plants. After locating and migrating toward their host, they penetrate the root and find their way to the vascular tissue. There they manipulate plant root cells, resulting in the establishment of highly specialized nematode feeding sites (NFS). Depending on the nematode species, the initial feeding cell develops into either a syncytium (for cyst nematodes such as Heterodera spp.) or a system of giant cells (for the root-knot nematodes such as Meloidogyne spp.; Vanholme et al., 2004). The nematodes depend strictly on these feeding cells for food supply during their entire life cycle. Although it is not yet understood how nematodes establish syncytia or giant cells, glandular secretions produced by the nematode and injected into plant cells are suspected of interacting directly or indirectly with the plant nuclear genome, causing a whole cascade of altered gene expression that ultimately results in the complex NFS (Vanholme et al., 2004). To study how NFS become established, different molecular approaches were used to identify plant genes/promoters that are induced upon nematode infection. Previously, the promoter-tagging strategy was used, which led to the identification of six Arabidopsis (Arabidopsis thaliana) lines exhibiting GUS activity in nematode feeding structures (Barthels et al., 1997). A detailed molecular analysis of one of these lines (Att0001, ARM1) revealed that the tagged promoter belongs to a member of the WRKY transcription factor family (this study). In Arabidopsis, there are 72 expressed WRKY genes (Eulgem and Somssich, 2007) and the majority are transcriptionally inducible upon pathogen infection and other defense-related stimuli. One common feature of all WRKY proteins is the occurrence of a conserved WRKY domain of approximately 60 amino acids. The WRKY domain contains a highly conserved group of seven invariant amino acids WRKYGQK followed by a Cys-2His-2 or Cys-2His-Cys zinc-finger binding motif. Additional conserved domains and the type of the zinc-finger motif divide the family in different subgroups (Eulgem et al., 2000). During the past 10 years, a large body of evidence has accumulated implicating WRKY proteins in the transcriptional reprogramming during plant defense responses. For example, loss-of-AtWRKY33 function rendered plants susceptible to infection by Botrytis cinerea and Alternaria brassicicola (Zheng et al., 2006), whereas overexpressing AtWRKY18 resulted in plants with activated pathogenesis-related (PR) gene expression and enhanced resistance to the bacterial pathogen Pseudomonas syringae (Chen and Chen, 2002). However, the role of WRKY proteins seems to be far more complicated because AtWRKY7, AtWRKY11, and AtWRKY17, members of the IIb subgroup, can act as negative defense regulators and loss-of-function mutants showed increased resistance toward avirulent and virulent P. syringae strains. Moreover, members of subgroup IIa can play both positive and negative roles in plant defense and multiple studies have demonstrated interactions of WRKY proteins with either their own promoters or those of other family members. All these studies clearly show that WRKY proteins act in a complex transcriptional network, which is often referred to as the WRKY web (Eulgem, 2006). WRKY expression is, however, not exclusively associated with pathogen attack. It has been shown that WRKY genes were also up-regulated upon other stresses, such as wounding (Hara et al., 2000; Cheong et al., 2002), low temperatures (Huang and Duman, 2002), and during senescence (Hinderhofer and Zentgraf, 2001). Interestingly, WRKY proteins have been characterized with a defense-independent role in plant growth and development. TTG2 encodes AtWRKY44 and functions in the development of trichomes and root hair cells (Johnson et al., 2002; Ishida et al., 2007), whereas AtWRKY10, identified through the miniseed3 mutant, is reported as a regulator of seed size (Luo et al., 2005). In this study, we report on the involvement of WRKY23 during plant-parasitic nematode infection. The expression of WRKY23 is up-regulated almost immediately upon nematode infection and WRKY23 knock-down lines show a decreased sensitivity toward the cyst nematode Heterodera schachtii. Furthermore, we show that the expression of WRKY23 is inducible by the plant hormone auxin, a known player in the plant-nematode interaction. However, the early WRKY23 up-regulation upon infection seems to be independent from auxin signaling.

RESULTS

Up-Regulation of a WRKY Transcription Factor upon Infection with Plant-Parasitic Nematodes

A fundamental question in studying the plant-nematode interaction is how nematodes alter plant gene regulation in a cell-specific manner. To gain insight into this process, we previously used a promoter trap strategy in the model plant Arabidopsis (Barthels et al., 1997). One line, Att0001, showed very strong GUS activity in giant cells during the early stages of root-knot nematode (Meloidogyne incognita) infection (Barthels et al., 1997; Fig. 1, A and C). Similar results were obtained with the cyst nematode H. schachtii (Fig. 1B). Southern analysis of the Att0001 plants, using the GUS- or the NPTII-coding region as probe, showed that two T-DNAs were inserted as an inverted repeat over the right border (Supplemental Fig. S1A). Analysis of the T-DNA flanking sequences isolated from the tagged locus revealed that the tagged promoter belonged to AtWRKY23 (At2g47260), a member of the WRKY transcription factor family. The WRKY23 protein consists of 337 amino acids from three exons encoding 146, 48, and 143 amino acids, respectively, and is a typical group-II WRKY protein, containing only one WRKY domain and having a Cys-2His-2 zinc-finger motif (Fig. 1F). Due to the presence of the conserved A-motif and the KAKKxxQK sequence, which probably functions as a nuclear localization signal, WRKY23 is categorized in the IIc subgroup (Eulgem et al., 2000). A WRKY23-GFP fusion protein construct driven by the 35S promoter in tobacco (Nicotiana tabacum) BY-2 cells localized to the nucleus, consistent with the assumed function of WRKY23 as a transcription factor (Fig. 1G). The full-length WRKY23 promoter of 3.2 kb was isolated and WRKY23::GUS and WRKY23::GFP lines confirmed the strong activity of the promoter in NFS as initially observed in the tagged line (Fig. 1, D and E; Supplemental Fig. S1, B and C). This was further analyzed at the mRNA level using in situ reverse transcription (RT)-PCR. Transcripts of WRKY23 strongly accumulated in young syncytia (Fig. 1H), whereas only weak staining could be observed in 10-d-old syncytia (Fig. 1I). No WRKY23 product was detected with the control reactions without Taq polymerase (Fig. 1, K–M). These in situ RT-PCR results entirely mimicked the promoter activity studies.

Figure 1.
Expression of AtWRKY23 during nematode infection. A to E, Infection of the promoter trap line Att0001 with M. incognita (A and C) and H. schachtii (B) and of WRKY23::GUS seedlings with H. schachtii at 2 dpi (E) and 5 dpi (D). All images show high ...

Expression of WRKY23 Is Inducible by Auxin in an IAA14-Dependent Way

WRKY proteins are well known for their involvement in biotic and abiotic stress responses and one plausible reason for the up-regulation of WRKY23 in NFS could be an induction by a wound response mechanism. To investigate this, wounding experiments were performed on leaves and hypocotyls of WRKY23::GUS seedlings. When leaves were transversely dissected in an apical and a basal half, GUS staining could only be detected at the cutting edge of the apical part and not in the basal part (Fig. 2B). The same was observed when hypocotyls were cut (Fig. 2C). The plant hormone auxin is known to be transported throughout the plant in a basipetal way. As a result of cutting experiments, the auxin flow is disrupted, resulting in an accumulation of auxin at the basal side of the cut tissue. Auxin is reported as a major player during NFS establishment (Goverse et al., 2000); therefore, we questioned whether WRKY23 is an auxin-responsive transcription factor and thus whether the strong GUS activity at the basal end of the cut segment would be auxin related. To sort this out, we repeated the above-mentioned experiment using WRKY23::GUS seedlings treated with the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) and observed an obvious reduction in GUS accumulation at the basal side of the injured segment (Fig. 2, D and E), indicating that the activation of the WRKY23 promoter is due to auxin accumulation instead of being a response to wounding. The expression of AtWRKY23 in response to auxin was further tested at the mRNA level in a time course experiment using semiquantitative RT-PCR. An increase of WRKY23 transcript level was detected within 4 h of auxin treatment and from 8 h the WRKY23 transcript reached a very high level (Fig. 2A). A detailed promoter analysis also revealed the presence of four elements that have been shown to be involved in auxin-regulated expression (Supplemental Fig. S1A). Next, the auxin-induced activation of the WRKY23 promoter was compared to that of the auxin response marker DR5 (Ulmasov et al., 1997). Seeds were germinated on 1-N-naphthylphthalamic acid (NPA) and 72 h postgermination transferred to 10 μm naphthaleneacetic acid (NAA; according to Himanen et al., 2002). Germination on NPA ensures auxin depletion in the root and results in the absence of DR5 and auxin-responsive promoter activity at the onset of the auxin treatment. Two hours after transfer to auxin, DR5 activity was strongly induced, whereas the WRKY23 promoter was only slightly activated at 6 h after transfer (Fig. 2, F–M). This suggests that WRKY23 acts downstream of the primary auxin response.

Figure 2.
Auxin-inducible expression of WRKY23. A, Semiquantitative RT-PCR analysis of WRKY23 using 10-d-old seedlings. Seedlings were treated for 0, 2, 4, 8, 12, and 24 h with 10 μm NAA. B to E, Wounding experiment on leaf (B) and hypocotyls (C–E) ...

Auxin-responsive genes are controlled by the auxin response factors (ARFs) that activate or repress their expression and the Aux/IAA proteins that negatively regulate the ARFs at low auxin concentrations. To examine whether WRKY23 expression depends on the Aux/IAA-ARF control mechanism, we checked the auxin inducibility of WRKY23 in a solitary root1 (slr1) background. slr1 gain-of-function mutants produce a stabilized mutant IAA14 protein resulting in a complete lack of lateral roots (Fukaki et al., 2002). WRKY23::GUS was introduced in the slr1 background and the F1 seedlings were treated with auxin. In contrast to WRKY23::GUS plants, no GUS staining could be detected in the pericycle and vascular tissue of WRKY23::GUS × slr1 roots (Fig. 2, N and O), strongly suggesting that WRKY23 is responsive to auxin in an IAA14-dependent way.

Knocking Down WRKY23 Expression Decreases Susceptibility to H. schachtii

To examine further the role WRKY23 might play during nematode parasitism, we screened all public available T-DNA insertion databases for wrky23 knockout mutants. All five insertions found were situated in the promoter region and, unfortunately, the respective homozygous T-DNA insertion lines showed no or only a slight reduction in WRKY23 transcript levels. In an attempt to obtain knock-down plants, WRKY23 RNAi lines were generated based on a hairpin construct of a 108-bp WRKY23-specific fragment. Several lines with a clear reduction in WRKY23 expression were obtained, but no obvious phenotypical differences compared to wild type were retrieved. Two lines with a 50% reduction in WRKY23 transcript levels were infected with the cyst nematode H. schachtii. Interestingly, both lines reproducibly showed a 20% to 30% reduction in nematode infection (Fig. 3), indicating that an up-regulation of WRKY23 might be required for successful induction of the NFS.

Figure 3.
Infection of WRKY23 knock-down lines. Top graph shows the number of cysts per plate counted 6 weeks postinoculation with H. schachtii. Bars with different letters denote a significant difference at P < 0.05. Bottom image shows the expression of ...

NFS-Specific WRKY23 Up-Regulation Is Independent of Auxin

WRKY23 is highly expressed during early NFS establishment and it was demonstrated that auxin accumulates in nematode-infected cells that develop into NFS (Karczmarek et al., 2004). In uninfected plants, the expression of WRKY23 is auxin inducible in an IAA14-dependent way. Therefore, we questioned whether WRKY23 induction upon nematode infection might also be controlled by an Aux/IAA-ARF-dependent mechanism. IAA14 promoter activity was analyzed during the early stages of feeding site induction. At both 2 and 5 d postinfection (dpi), strong GUS staining could be observed in the syncytium (Fig. 4, A and B), indicating that IAA14 is expressed during feeding site establishment. To investigate the involvement of IAA14 in nematode infection in more detail, the slr1 mutant was infected with H. schachtii and 6 weeks postinoculation the number of cysts was recorded both in wild-type and mutant plants. Consistent with previous observations that highlighted the importance of efficient auxin signaling for feeding site establishment (Goverse et al., 2000), the slr1 mutant showed more than 60% reduction in infection (Fig. 4C). To further address the question of whether WRKY23 acts downstream of IAA14 in NFS, WRKY23 expression was analyzed in the slr/IAA14 mutant background upon nematode infection. Surprisingly, in all syncytia observed, the WRKY23 promoter could still be activated by the nematodes in the presence of a stabilized Aux/IAA14 protein (Fig. 4, D and E). To further evaluate whether the induction of WRKY23 upon infection could occur independently from Aux/IAA signaling, the exact timing of auxin response upon infection was compared to the temporal expression pattern of WRKY23 in a time course experiment using DR5::GUS and WRKY23::GUS plants (Fig. 5). The earliest DR5 promoter activity could be observed 21 h postinoculation (hpi; Fig. 5I), which is consistent with previous reports by Karczmarek et al. (2004). The GUS staining remained in the developing syncytium until 2 d upon inoculation. At later time points, the GUS staining became less specific to the syncytium and was more pronounced in the periphery of the NFS (Fig. 5O). Consistent with the above-mentioned results, the WRKY23 promoter did not follow the DR5 promoter activity and was already activated at 12 hpi (Fig. 5D). Moreover, at 5 dpi, WRKY23 expression could still be observed in the NFS (Fig. 5P). These results show that, during nematode infection, WRKY23 is activated prior to the enhanced auxin response reported by the DR5::GUS construct, whereas in uninfected plants the auxin-induced WRKY23 expression is significantly later than the enhanced DR5 activity (Fig. 2, F–M). In the above-mentioned wounding experiments, application of a polar auxin transport inhibitor resulted in strong reduction of the WRKY23 promoter activity at the cutting edges (Fig. 2, D and E). If the expression of WRKY23 during nematode infection would be due to auxin accumulation in the initial feeding site, a similar reduction should be expected when infecting NPA-treated WRKY23::GUS plants. However, examining NFS at 18 hpi and 2 dpi revealed that NPA treatment did not influence WRKY23 expression (Fig. 4, F–I). All these data together strongly indicate that accumulation of auxin in the initial NFS cannot be solely responsible for WRKY23 up-regulation and imply the existence of other nematode-dependent signals in regulating WRKY23 expression.

Figure 4.
WRKY23 promoter activation in syncytia is sustained in slr background. A and B, IAA14 promoter activity at 2 dpi (A) and 4 dpi (B) using H. schachtii. C, Susceptibility against H. schachtii of wild type compared to slr/IAA14. Numbers of cysts per plate ...
Figure 5.
WRKY23 promoter is activated in syncytia prior to the auxin response visualized by DR5 promoter activity. Time course experiment comparing WRKY23 (B, D, F, H, J, L, N, and P) and DR5 (A, C, E, G, I, K, M, and O) promoter activity during cyst nematode ...

DISCUSSION

A WRKY Protein of the IIc Subgroup Is Involved in Nematode Infection

Proteins belonging to the WRKY family of transcription factors are known to regulate a multiplicity of biotic and abiotic stress responses. They have been implicated in the regulation of genes involved in pathogen-induced stress, as well as drought and cold stresses (Dong et al., 2003; Pnueli et al., 2002; Rizhsky et al., 2002). Some WRKY proteins also play an important role during senescence (Miao et al., 2004) and plant development (Johnson et al., 2002). Their regulatory effect is primarily through their binding with conserved W-box elements present in the promoters of specific genes (Ulker and Somssich, 2004; Supplemental Fig. S1A). Here, we report the involvement of WRKY23 in nematode infection. Using promoter::GUS/GFP fusions and in situ RT-PCR, we could show that its expression is rapidly and highly induced upon infection with both root-knot and cyst nematodes. At later stages of the infection (10 dpi), the expression fades away, hinting at a role for WRKY23 during the early stages of nematode infection. The up-regulation of WRKY23 could be confirmed by several transcript-profiling studies. Ithal et al. (2007) analyzed the expression of soybean (Glycine max) genes in feeding sites of Heterodera glycines using laser capture microdissection. The soybean probe sets Gma.8336 and Gma.16547 both showed high sequence similarity with AtWRKY23 and were 7- to 17-fold up-regulated in syncytia (2 dpi), respectively (M. Mitchum, personal communication). WRKY23 transcripts were also found to be up-regulated in microaspirated cytoplasm of syncytia at 3 dpi (K. Wieczorek and F. Grundler, unpublished data).

WRKY23 is a member of the subgroup IIc. Little is known about this rather large subgroup of 17 WRKY proteins. The only report so far is the functional characterization of AtWRKY75, which suggests that it acts as a modulator of phosphate starvation responses (Devaiah et al., 2007). To find regulatory elements in the promoters of group-IIc WRKY genes that might be shared, we performed a phylogenetic footprinting. However, except for the W-box elements, which are characteristic for the promoters of WRKY genes, we did not find any elements specific for group-IIc WRKYs (data not shown).

Is WRKY23 a Negative Regulator of Basal Defense or Is Its Expression Hijacked by Nematodes?

Sedentary plant-parasitic nematodes are able to influence their host's gene expression to establish feeding sites. For example, the cyst nematode H. schachtii induces the expression of several expansin genes of its host during syncytium formation (Wieczorek et al., 2006). Expansins are cell wall-loosening proteins and enable the cyst nematode to install its feeding site by cell wall degradation and fusion of root cells. Besides this manipulation by the nematode, the infection process coincides with a plethora of plant defense responses. Genes encoding proteins related to stress and defense responses, such as PR proteins, are consistently up-regulated during all infection stages (Ithal et al., 2007). Also during the migration through the root and during expansion of the feeding site, nematodes cause wounding of the plant tissue. By performing wounding experiments on WRKY23::GUS seedlings, we were able to ensure that WRKY23 is not a part of the wound response accompanying the infection process. Also, cold treatments did not influence WRKY23::GUS activity (data not shown). Paradoxically to the role of WRKY proteins in plant defense, WRKY23 knock-down lines showed reduced infection of the cyst nematode H. schachtii. This paradox might be explained by two different trains of thought. First, WRKY23 could act as a negative regulator of basal defense. Plant defense responses need to be under tight and fine-tuned regulation. Defense genes should only be activated during pathogen attack because constitutively activated plant defenses are energy consuming and hypersensitive responses, often accompanying plant defense, reduce the fitness of the plant. Thus, one hypothesis could be that WRKY23 tones down the expression of plant defense responses upon pathogen infection. Reducing the expression of WRKY23 could therefore increase the resistance to cyst nematodes. Other WRKY genes have been reported with a similar function. AtWRKY11 and AtWRKY17 are both induced upon infection with P. syringae pv tomato, whereas wrky11wrky17 double mutants showed increased resistance toward virulent and avirulent strains (Journot-Catalino et al., 2006). Also, WRKY7 acts as a negative regulator of plant defense toward P. syringae as could be demonstrated by the enhanced susceptibility of WRKY7-overexpressing plants and enhanced resistance of loss-of-function mutants (Kim et al., 2006).

The molecular mechanisms controlling plant defense responses are exceedingly complex. Necrotrophic pathogens, which benefit from host cell death, rather activate jasmonic acid (JA) signaling, whereas the salicylic acid (SA)-signaling pathway is generally effective against biotrophic pathogens that feed on living host tissue (Glazebrook, 2005). Considerable genetic evidence suggests that SA- and JA-signaling pathways are antagonistic (Gupta et al., 2000; Kloek et al., 2001). Consistent with their role in plant defense, at least 36 AtWRKY genes were differentially regulated upon SA treatment (Dong et al., 2003) and, interestingly, at least some WRKY proteins play crucial roles at the convergence of SA and JA. Overexpression of WRKY70 results in constitutive SA signaling, whereas knocking down the expression results in activation of the JA pathway (Li et al., 2006). Plant pathogens can take advantage of this plant defense antagonism as could be demonstrated by the coronatine-producing bacterial plant pathogen P. syringae. Coronatine is a phytotoxin that activates JA signaling, thereby inhibiting SA-mediated defenses that normally limit growth of P. syringae within host tissue (Laurie-Berry et al., 2006). Because plant-parasitic nematodes depend on a nutrient-delivering host, it is tempting to speculate that their hosts' SA-signaling pathway would be activated. Recently, Wubben et al. (2008) could show enhanced susceptibility of SA-deficient mutants to H. schachtii, whereas SA-treated wild-type plants showed decreased susceptibility. It will be very interesting to investigate whether the JA defense pathway is also involved in nematode infection and whether WRKY23 or other members of the WRKY family would have a role in the interplay between both hormones. However, the findings that application of SA does not change WRKY23 mRNA levels (Dong et al., 2003) or WRKY23::GUS activity (data not shown) argue against a defense-related role of WRKY23. Moreover, in a transcript-profiling search for JA-responsive genes, WRKY23 transcript levels also did not change upon methyl jasmonate treatment (Pauwels et al., 2008; L. Pauwels and A. Goossens, personal communication).

A second hypothesis to explain the enhanced resistance of WRKY23 knock-down lines to H. schachtii might be the hijacking of WRKY23 by the nematode. It has been proposed that plant-parasitic nematodes use the developmental programs of their hosts to induce the NFS (Bird, 1996). In an attempt to do this, the nematode manipulates the plant gene expression for its own benefit. WRKY23 is an auxin-inducible gene and in uninfected plants the auxin-inducible expression of WRKY23 is controlled by the Aux/IAA protein SLR/IAA14. Our observations are consistent with a transcript-profiling study set up to identify auxin-inducible genes downstream of Aux/IAA14 (Vanneste et al., 2005; Supplemental Fig. S1D). Auxin is involved in almost every aspect of plant development and is the rooting hormone par excellence (for review, see Grunewald et al., 2007). The idea that auxin could be involved in the nematode infection process was launched as early as the 1960s (Balasubramanian and Rangaswami, 1962; Bird, 1962; Yu and Viglierchio, 1964; Cutler and Krusberg, 1968). Since then, an increasing amount of information points toward an important role for auxin in NFS establishment (Kochba and Samish, 1971; Glazer et al., 1986; Goverse et al., 2000). More recently, in Arabidopsis, the up-regulation of the auxin-responsive DR5 reporter could be demonstrated shortly after nematode infection (Karczmarek et al., 2004; this study). Whether this local auxin accumulation is due to auxin directly secreted by the nematode (De Meutter et al., 2005) and/or is a result of directional auxin transport toward the initial feeding cell is still an unsolved question. Notwithstanding the importance of auxin, our results suggest that WRKY23 can be activated by the nematode in an auxin-independent way. In the slr1 mutant carrying a dominant-negative mutation that inhibits auxin-inducible expression of WRKY23 in uninfected plants, the WRKY23 promoter could still be activated in syncytia. WRKY23 promoter activity could also be detected in NFS upon treatment with NPA, inhibiting polar auxin transport along the root and thus toward the initial feeding sites. This demonstrates that, during the infection process, WRKY23 is not induced solely by the auxin accumulating in NFS. Moreover, a time course experiment revealed that the WRKY23 promoter is activated prior to the auxin response visualized using the DR5 promoter. One plausible explanation could be that the nematode is capable of bypassing the auxin-induced WRKY23 pathway to initiate its feeding site more rapidly. A detailed study on the endogenous role of WRKY23 in the future should clarify this problem. Using all publicly available mutant databases, we were not able to find knockout lines and the RNAi lines only showed <50% reduction. The lack of a viable full knockout might suggest that WRKY23 is indeed an important developmental gene.

During H. schachtii infection, WRKY23 is activated within the first 12 hpi. Given that, during this period, the nematode must penetrate its host's root, migrate toward the vascular tissue, and select an initial feeding cell, the activation of the WRKY23 promoter can be considered as extremely fast. This observation propounds the idea that WRKY23 could be directly activated by the nematode secretions. Recently, a CLE peptide with a function similar to CLAVATA3 of Arabidopsis has been characterized from the soybean cyst nematode H. glycines (Wang et al., 2005). The HgCLE peptide is able to complement the Arabidopsis mutant clv3 and was shown to be involved in root development. However, when WRKY23::GFP seedlings were treated with HgCLE peptides, the GFP signal did not change (data not shown). It will be a challenge for the future to sort out whether nematode-secreted proteins can stimulate the WRKY23 promoter by bypassing the rigid Aux/IAA-mediated control on auxin response to initiate NFS formation.

MATERIALS AND METHODS

Plant Lines and Growth Conditions

The tagged line Att0001 of Arabidopsis (Arabidopsis thaliana L. Heyhn.) ecotype C24 was transformed by the promoter trap vector pΔgusBin19 (Topping et al., 1991). slr1 (Col-0 background) and IAA14::GUS (Col-0 background) lines were described by Fukaki et al. (2002), and DR5::GUS (Col-0 background) by Ulmasov et al. (1997). For in vitro growth studies, seeds were vernalized, surface sterilized (2 min in 70% ethanol and 10 min in 5% sodium hypochlorite), and plated on Murashige and Skoog medium (0.5× Murashige and Skoog; 1% Suc; 0.8% agar; pH 5.7). In general, plants were grown in vitro at 21°C under a 16-h-light/8-h-dark photoperiod. Wounding experiments were done on 10-d-old seedlings. For wounding experiments, hypocotyls and leaf blades of WRKY23::GUS (C24 background) plants were cut and plant segments were placed on solidified Murashige and Skoog medium. GUS assays were performed on samples 2 d after treatment. For auxin transport inhibition experiments, 10-d-old WRKY23::GUS seedlings were transversally cut at the hypocotyl and incubated upside down (leaf surface touching the agar) on solidified B5 medium with or without 50 μm TIBA. The GUS histochemical assay was performed after 2 d of incubation in the growth room. NPA treatments prior to Heterodera schachtii inoculations were done according to Goverse et al. (2000).

Nematode Culture and Infection Tests

Cultures of the root-knot nematode Meloidogyne incognita were maintained in vitro on roots of tomato (Lycopersicon esculentum ‘Marmande’) transformed with Agrobacterium rhizogenes and grown on Gamborg's B5 medium (Duchefa) at 28°C. Stage-2 juveniles (J2s) were obtained from egg masses hatched in sterile water 6 to 8 weeks after inoculation. Cyst nematodes (H. schachtii) were grown in vitro on roots of mustard (Sinapis alba) on Knop medium (Sijmons et al., 1991). J2s were obtained from cysts hatched in 3 mm ZnCl2 6 to 8 weeks after inoculation. For infection tests, 2-week-old seedlings were transferred to Knop medium on 12 × 12 cm2 petri dishes (Greiner), resulting in five plants/plate and six replica plates/line. The petri dishes were placed slightly tilted to promote unidirectional root growth. One week later, two isolated root tips of each plant were inoculated with 5 μL of H. schachtii or M. incognita juveniles, with a total of about 200 J2s per plate. Therefore, the hatched nematodes were collected in 50-mL falcon tubes and centrifuged 3 min at 1,000 rpm. The supernatant was removed and the nematodes were resuspended in sterile 0.3% low-melting-point agarose. The solution was diluted until the desired nematode concentration (20 J2s/5 μL) was reached. For the first 2 weeks, the plates were sealed with urgopore and afterward parafilm was used. Six weeks after infection, the number of cysts (in the case of H. schachtii) or galls and egg masses (in the case of M. incognita) was counted on each plate and compared with the control lines. The data were statistically analyzed in SPSS (version 12.1) with the Levene test for homogeneity of variance and the independent sample t test to compare means.

Identification of WRKY23 and Isolation of WRKY23 cDNA

To search for fragments containing T-DNA/plant DNA, we first carried out a Southern-blot analysis using the GUS-coding region as a probe. Therefore, 3 μg of purified DNA of the Att0001 line was digested with a range of restriction enzymes and separated in a 0.8% agarose gel. According to the Southern analysis, the SspI and EcoRI digests resulted in T-DNA/plant DNA fragments and were chosen for an inverse PCR (iPCR). The iPCR was performed using 2 μg of digested DNA and the fragments were ligated overnight at 14°C by 50 units of T4 ligase in a volume of 1 mL to generate monomeric circles. Ligated DNA was extracted with phenol-chloroform, then precipitated in ethanol, and resuspended in 100 μL of distilled water. PCR was performed using primer 1 (5′-CCAGCGTGGACCGCTTGCTGGACC-3′) and primer 2 (5′-GTATTGCCAACGAACCGGATACCCG-3′) for the SspI circle and primer 1 and primer 3 (5′-CCCAGTCACGACGTTGTAAAAC-3′) for the EcoRI circle. Both iPCR products were cloned into pGEMT (Promega) (ARM1-a and ARM1-b), sequenced, and used to screen a genomic library of Arabidopsis. Four positive plaques out of 105 were identified (Ch\ARM1-A, Ch\ARM1-B, Ch\ARM1-C, and Ch\ARM1-D) and, after purification, they were mapped based on restriction and hybridization analysis. The Ch\ARM1-D3500, a subclone from Ch\ARM1-D containing the insertion site of the T-DNAs, was sequenced and used in a BLAST program to screen the Arabidopsis database.

To obtain WRKY23 cDNA, a cDNA library from M. incognita-infected Arabidopsis roots was screened using PCR. Two primers from the cloning region of the phage were used (forward 5′-GGTGGCGACGACTCCTGGAGCCCG-3′, reverse 5′-TTGACACCAGACCAACTGGTAATG-3′) in combination with primer 4 (5′-ACAATGGAGTTTACAGATTTC-3′) and primer 5 (5′-GCGTGGCTATTAAGGTACT-3′). Amplified bands were cloned into pGEMT (Promega) according to the manufacturer's instruction, and clones with either the 5′ end or the 3′ end of the cDNA (reverse/primer 4 and forward/primer 5, respectively) were identified. To obtain the full-length cDNA of WRKY23, a clone containing the 3′ end and a clone containing the 5′ end were digested with ClaI (cuts the insert only once and has no recognition site in the vector) and SpeI from the polycloning site of pGEMT. Digests were separated on a 0.8% gel and vector fragments containing either the 3′ or the 5′ end of the cDNA were isolated and purified using a gel extraction kit (Qiagen). Purified fragments were ligated to obtain the full-length WRKY23.

Generation of Constructs and Transgenic Lines

For isolation of the WRKY23 promoter, DNA of the plaque genomic clone Ch\ARM1-D was digested by XbaI and the resulting 6.7-, 3.5-, 2.3-, and 0.7-kb fragments were cloned into pBluescriptII KS− (Ch\ARM1-D6663, Ch\ARM1-D3500, Ch\ARM1-D2299, and Ch\ARM1-D688). PCR was done on Ch/ARM1-D3500 DNA and the PCR product was cloned. The cloned fragment that contained 975 bp from the 3′ end of the WRKY23 promoter was sequenced (pW975). The pW395 construct was made from pW975 by removing all DNA 5′ from the BsaI site. The pW3195 was constructed by fusion of the pW395 fragment to the DNA 5′ of the BsaI site of Ch\ARM1-D3500. The promoter fragment from the pW3195 construct was fused to the coding sequence of GUS in the pBI101 binary vector to generate the WRKY23::GUS construct.

To construct the WRKY-GFP fusion, the full length of WRKY23 cDNA was amplified using forward and reverse primers from WRKY23 extended with the attB1 and attB2 recombination sequences of the Gateway system. The PCR product was recombined with the pDONOR201 to generate pW23ENTRY. The pW23ENTRY clone was recombined to the pK2GWF7 to create pK2W23F7, a C-terminal GFP fusion construct. The CATMA program was used to make a gene-specific tag (GST) for WRKY23. A 108-bp-long fragment of the WRKY23 open reading frame was amplified using primer W23GS5 (5′-TCCACCGTCAGAGCAATTAGT-3′) and primer W23GS3 (5′-AAGCAGACGAAATCGATGA-3′). The PCR product was recombined into pK2GWIWG7 vector to generate pK2WIW7 for RNAi-mediated gene silencing. Using the three-parental method, the vectors were transferred into Agrobacterium tumefaciens strain C58C1 (pMP90) and the pK2W23F7 into A. tumefaciens strain LBA4404. Arabidopsis plants were transformed by the floral-dip method (Clough and Bent, 1998) using ecotype Col-0 for the RNAi construct (pK2WIW7 vector) and ecotype C24 for the WRKY::GUS and WRKY::GFP constructs.

GUS Histochemical Assay and in Situ RT-PCR

For histochemical localization of GUS activity, plants were incubated in 90% acetone for 30 min at 4°C and then washed twice with NT buffer (100 mm Tris HCl/50 mm NaCl solution, pH 7.5). Subsequently, samples were incubated at 37°C in NT buffer supplemented with 2 mm ferricyanide. After 30 min, the GUS staining reaction was performed at 37°C in fresh NT buffer supplemented with ferricyanide and X-Glu (26.1 mg dissolved in dimethyl sulfoxide for 25 mL staining solution). After the GUS staining, the samples were washed in NT buffer, cleared using lactic acid, and analyzed using a digital interference contrast light microscope (Olympus) and photographed using a Nikon digital camera. The in situ RT-PCR procedure was performed according to Wieczorek et al. (2006). Syncytia at 5 and 10 dpi, as well as root fragments above syncytia, were dissected from roots and immediately put into cold fixation solution (63% ethanol [v/v]; 2% formalin [v/v]). After 48 h, syncytia were embedded in 4% low melting agarose and 25-μm-thick sections were prepared using a vibratome (VT 100; Leica). Further, a RT-PCR with specific primers and digoxigenin-labeled dUTP was carried out. After staining reaction with NBT/BCIP substrate, cross sections were photographed under an inverted microscope (Axiovert 200 M; Zeiss) with an integrated camera (AxioCam MRc5; Zeiss).

RNA Extraction, cDNA Synthesis, and RT-PCR

Ten-day-old seedlings were ground in liquid N2 and total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. First-strand cDNA was prepared from 2 μg total RNA sample in a volume of 40 μL using a Superscript II reverse transcriptase first-strand cDNA synthesis kit (Invitrogen). Primers used to detect the endogenous WRKY23 gene are primer WRKY_ssp5 and primer 3′-UTRB (5′-CTAGATTAAAACTATAAGACTATAT-3′). The ACTIN-2 gene was used as a control, with primers Act2F (5′-GTTGCACCACCTGAAAGGAAG-3′) and primer Act2R (5′-CAATGGACTAAAACGCAAAA-3′). Various PCR cycles were tested to verify that the PCR conditions were not saturated. For auxin treatments, 10-d-old seedlings were transferred to Murashige and Skoog medium supplemented with 10 μm NAA (Sigma-Aldrich) for 2, 4, 8, 12, and 24 h. For WRKY23 amplification, RT-PCR was performed using 24 cycles and for ACTIN-2 using 22 cycles.

Localization in Tobacco BY-2 Cells

WRKY23 cDNA was cloned in the pK7WGF2 and pK7FWG2 vectors (Karimi et al., 2002) using the Gateway cloning facilities (Invitrogen). The obtained vectors were stably transformed in BY-2 cells using the A. tumefaciens strain LBA4404 harboring the construct. Four milliliters of a 3-d-old BY-2 suspension culture was mixed with 100 μL from an overnight grown Agrobacterium suspension and incubated for 3 d at 28°C. After incubation, the mixture was plated on BY-2 medium (4.302 g Murashige and Skoog, 0.2 g KH2PO4, 30 g Suc, 6.6 g agar, 0.02 mg, 2,4-dichlorophenoxyacetic acid [auxin], 0.05 mg thiamine, 5 mg/L myoinositol, pH 5.8) containing carbenicillin (500 mg/L) and kanamycin (100 mg/L). Growing calli were subsequently screened for fluorescence and the selected calli were imaged using a Zeiss LSM 510 confocal microscope.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers NM_130234 (WRKY23) and NM_117535 (IAA14).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure S1. Schematic representation of the WRKY23 promoter, which is activated by plant-parasitic nematodes as well as by auxin.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Bartel Vanholme for helpful discussions and Bernard Cannoot for maintaining the in vitro nematode culture.

Notes

1This work was supported by Ghent University (grant nos. 01G00805 and 01J11602) and Research Foundation-Flanders (grant no. 3G003108).

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: Wim Grunewald (eb.tnegu@dlawenurg.miw).

[W]The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.119131

References

  • Balasubramanian M, Rangaswami G (1962) Presence of indole compounds in nematode galls. Nature 194 774–775
  • Barthels N, van der Lee FM, Klap J, Goddijn OJM, Karimi M, Puzio P, Grundler FMW, Ohl SA, Lindsey K, Robertson L, et al (1997) Regulatory sequences of Arabidopsis drive reporter gene expression in nematode feeding structures. Plant Cell 9 2119–2134 [PMC free article] [PubMed]
  • Bird AF (1962) The inducement of giant cells by Meloidogyne javanica. Nematologica 8 1–10
  • Bird DM (1996) Manipulation of host gene expression by root-knot nematodes. J Parasitol 82 881–888 [PubMed]
  • Chen C, Chen Z (2002) Potentiation of developmentally regulated plant defense response by AtWRKY18, a pathogen-induced Arabidopsis transcription factor. Plant Physiol 129 706–716 [PMC free article] [PubMed]
  • Cheong YH, Chang HS, Gupta R, Wang X, Zhu T, Luan S (2002) Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiol 129 661–677 [PMC free article] [PubMed]
  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16 735–743 [PubMed]
  • Cutler HG, Krusberg LR (1968) Plant growth regulators in Ditylenchus dipsaci, Ditylenchus triformis and host tissues. Plant Cell Physiol 9 479–497
  • De Meutter J, Tytgat T, Prinsen E, Gheysen G, Van Onckelen H, Gheysen G (2005) Production of auxin and related compounds by the plant parasitic nematodes Heterodera schachtii and Meloidogyne incognita. Commun Agric Appl Biol Sci 70 51–60 [PubMed]
  • Devaiah BN, Karthikeyan AS, Raghothama KG (2007) WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant Physiol 143 1789–1801 [PMC free article] [PubMed]
  • Dong J, Chen C, Chen Z (2003) Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol Biol 51 21–37 [PubMed]
  • Eulgem T (2006) Dissecting the WRKY web of plant defense regulators. PLoS Pathog 2 128–130 [PMC free article] [PubMed]
  • Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5 199–206 [PubMed]
  • Eulgem T, Somssich IE (2007) Networks of WRKY transcription factors in defense signaling. Curr Opin Plant Biol 10 366–371 [PubMed]
  • Fukaki H, Tameda S, Masuda H, Tasaka M (2002) Lateral root formation is blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis. Plant J 29 153–168 [PubMed]
  • Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43 205–227 [PubMed]
  • Glazer I, Epstein E, Orion D, Apelbaum A (1986) Interactions between auxin and ethylene in root-knot nematode (Meloidogyne javanica) infected tomato roots. Physiol Mol Plant Pathol 28 171–179
  • Goverse A, Overmars H, Engelbertink J, Schots A, Bakker J, Helder J (2000) Both induction and morphogenesis of cyst nematode feeding cells are mediated by auxin. Mol Plant Microbe Interact 13 1121–1129 [PubMed]
  • Grunewald W, Parizot B, Inzé D, Gheysen G, Beeckman T (2007) Developmental biology of roots: one common pathway for all angiosperms? International Journal of Plant Developmental Biology 1 212–225
  • Gupta V, Willits MG, Glazebrook J (2000) Arabidopsis thaliana EDS4 contributes to salicylic acid (SA)-dependent expression of defense responses: evidence for inhibition of jasmonic acid signaling by SA. Mol Plant Microbe Interact 13 503–511 [PubMed]
  • Hara K, Yagi M, Kusano T, Sano H (2000) Rapid systemic accumulation of transcripts encoding a tobacco WRKY transcription factor upon wounding. Mol Gen Genet 263 30–37 [PubMed]
  • Himanen K, Boucheron E, Vanneste S, de Almeida-Engler J, Inzé D, Beeckman T (2002) Auxin-mediated cell cycle activation during early lateral root initiation. Plant Cell 14 2339–2351 [PMC free article] [PubMed]
  • Hinderhofer K, Zentgraf U (2001) Identification of a transcription factor specifically expressed at the onset of leaf senescence. Planta 213 469–473 [PubMed]
  • Huang T, Duman JG (2002) Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA-binding activity from winter bittersweet nightshade, Solanum dulcamara. Plant Mol Biol 48 339–350 [PubMed]
  • Ishida T, Hattori S, Sano R, Inoue K, Shirano Y, Hayashi H, Shibata D, Sato S, Kato T, Tabata S, et al (2007) Arabidopsis TRANSPARENT TESTA GLABRA2 is directly regulated by R2R3 MYB transcription factors and is involved in regulation of GLABRA2 transcription in epidermal differentiation. Plant Cell 19 2531–2543 [PMC free article] [PubMed]
  • Ithal N, Recknor J, Nettleton D, Maier T, Baum TJ, Mitchum MG (2007) Developmental transcript profiling of cyst nematode feeding cells in soybean roots. Mol Plant Microbe Interact 20 510–525 [PubMed]
  • Johnson CS, Kolevski B, Smyth DR (2002) TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell 14 1359–1375 [PMC free article] [PubMed]
  • Journot-Catalino N, Somssich IE, Roby D, Kroja T (2006) The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana. Plant Cell 18 3289–3302 [PMC free article] [PubMed]
  • Karczmarek A, Overmars H, Helder J, Goverse A (2004) Feeding cell development by cyst and root-knot nematodes involves a similar early, local and transient activation of a specific auxin-inducible promoter element. Mol Plant Pathol 5 343–346 [PubMed]
  • Karimi M, Inzé D, Depicker A (2002) GATEWAYTM vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7 193–195 [PubMed]
  • Kim KC, Fan B, Chen Z (2006) Pathogen-induced Arabidopsis WRKY7 is a transcriptional repressor and enhances plant susceptibility to Pseudomonas syringae. Plant Physiol 142 1180–1192 [PMC free article] [PubMed]
  • Kloek AP, Verbsky ML, Sharma SB, Schoelz JE, Vogel J, Klessig DF, Kunkel BN (2001) Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms. Plant J 26 509–522 [PubMed]
  • Kochba J, Samish RM (1971) Effect of kinetin and 1-naphthylacetic acid on root-knot nematodes in resistant and susceptible peach rootstocks. J Am Soc Hortic Sci 96 458–461
  • Laurie-Berry N, Joardar V, Street IA, Kunkel BN (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae. Mol Plant Microbe Interact 19 789–800 [PubMed]
  • Li J, Brader G, Kariola T, Palva ET (2006) WRKY70 modulates the selection of signaling pathways in plant defense. Plant J 46 477–491 [PubMed]
  • Luo M, Dennis ES, Berger F, Peacock WJ, Chaudhury A (2005) MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a leucine-rich repeat (LRR) kinase gene, are regulators of seed size in Arabidopsis. Proc Natl Acad Sci USA 102 17531–17536 [PMC free article] [PubMed]
  • Miao Y, Laun T, Zimmermann P, Zentgraf U (2004) Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Mol Biol 55 853–867 [PubMed]
  • Pauwels L, Morreel K, De Witte E, Lammertyn F, Van Montagu M, Boerjan W, Inzé D, Goossens A (2008) Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells. Proc Natl Acad Sci USA 4 1380–1385 [PMC free article] [PubMed]
  • Pnueli L, Hallak-Herr E, Rozenberg M, Cohen M, Goloubinoff P, Kaplan A, Mittler R (2002) Molecular and biochemical mechanisms associated with dormancy and drought tolerance in the desert legume Retama raetam. Plant J 31 319–330 [PubMed]
  • Rizhsky L, Liang H, Mittler R (2002) The combined effect of drought stress and heat shock on gene expression in tobacco. Plant Physiol 130 1143–1151 [PMC free article] [PubMed]
  • Sijmons PC, Grundler FMW, von Mende N, Burrows PR, Wyss U (1991) Arabidopsis thaliana as a new model host for plant-parasitic nematodes. Plant J 1 245–254
  • Topping JF, Wei W, Lindsey K (1991) Functional tagging of regulatory elements in the plant genome. Development 112 1009–1019 [PubMed]
  • Ulker B, Somssich IE (2004) WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol 7 491–498 [PubMed]
  • Ulmasov T, Murfett J, Hagen G, Guilfoyle T (1997) Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9 1963–1971 [PMC free article] [PubMed]
  • Vanholme B, De Meutter J, Tytgat T, Van Montagu M, Coomans A, Gheysen G (2004) Secretions of plant-parasitic nematodes: a molecular update. Gene 332 13–27 [PubMed]
  • Vanneste S, De Rybel B, Beemster GTS, Ljung K, De Smet I, Van Isterdael G, Naudts M, Iida R, Gruissem W, Tasaka M, et al (2005) Cell cycle progression in the pericycle is not sufficient for SOLITARY ROOT/IAA14-mediated lateral root initiation in Arabidopsis thaliana. Plant Cell 17 3035–3050 [PMC free article] [PubMed]
  • Wang X, Mitchum MG, Gao B, Li C, Diab H, Baum TJ, Hussey RS, Davis ED (2005) A parasitism gene from a plant-parasitic nematode with function similar to CLAVATA3/ESR (CLE) of Arabidopsis thaliana. Mol Plant Pathol 6 187–191 [PubMed]
  • Wieczorek K, Golecki B, Gerdes L, Heinen P, Szakasits D, Durachko DM, Cosgrove DJ, Kreil DP, Puzio PS, Bohlmann H, et al (2006) Expansins are involved in the formation of nematode-induced syncytia in roots of Arabidopsis thaliana. Plant J 48 98–112 [PubMed]
  • Wubben MJE, Jin J, Baum TJ (2008) Cyst nematode parasitism of Arabidopsis thaliana is inhibited by salicylic acid (SA) and elicits uncoupled SA-independent pathogenesis-related gene expression in roots. Mol Plant Microbe Interact 21 424–432 [PubMed]
  • Yu PK, Viglierchio DR (1964) Plant growth substances and parasitic nematodes. I. Root knot nematodes and tomato. Exp Parasitol 15 242–248 [PubMed]
  • Zheng Z, Qamar SA, Chen Z, Mengiste T (2006) Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J 48 592–605 [PubMed]

Articles from Plant Physiology are provided here courtesy of American Society of Plant Biologists
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...