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NPR1 enhances the DNA binding activity of the Arabidopsis bZIP transcription factor TGA71 Heather L. Shearer, Lipu Wang, Catherine DeLong, Charles Despres, and Pierre R. Fobert
Abstract: Pathogen-induced transcriptional reprogramming of the plant genome is mediated predominantly by the cofactor NPR1 (NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1). NPR1 lacks any known DNA-binding domain and is proposed to regulate transcription through interactions with TGA transcription factors that bind to as-1-like promoter elements. Previous studies have focused on the interaction of NPR1 with subgroup I (TGA1, TGA4) or subgroup II (TGA2, TGA5, TGA6) factors. Using the yeast two-hybrid system, we showed that a member of subgroup III (TGA7) interacts with wild-type NPR1 but not with mutants in the ankyrin repeats that are important for disease resistance. Mutations in the NPR1 BTB/POZ domain also greatly reduced interaction with TGA7. NPR1 substantially increased the binding of TGA7 to cognate promoter elements in vitro, including a salicylic-acid-inducible element of the PR-1 promoter. While TGA7 interacted with all TGA factors tested, interactions were not observed between TGA2 and subgroup I factors, indicating that cross-clade interaction is not a general property of the family. Transcripts from subgroup III TGA factors were weakly inducible by salicylic acid and pathogens, but only TGA3 expression was dependent on NPR1. These results suggest that NPR1-mediated DNA binding of TGA7 could regulate the activation of defense genes. Key words: plant defense response, transcription, pathogenesis-related genes, salicylic acid, protein–protein interaction. Re´sume´ : La reprogrammation transcriptionnelle du ge´nome ve´ge´tal, induite par des agents pathoge`nes, s’effectue surtout sous l’effet du cofacteur NPR1 (NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1). Le NPR1 ne posse`de aucun domaine de liaison de l’ADN et on le propose comme re´gulateur de transcription par des interactions avec les facteurs de transcription TGA qui s’attachent a` des e´le´ments de promoteurs ressemblant au as-1. Des e´tudes ante´ce´dentes se sont concentre´es sur l’interaction du NPR1 avec les facteurs du sous-groupe I (TGA1, TGA4) ou du sous-groupe II (TGA2, TGA5, TGA6). En utilisant le syste`me hybride double de la levure, les auteurs montrent qu’un membre du sousgroupe III (TGA7) interagit avec le NPR1 du type sauvage, mais pas avec les mutants dans les ite´rations de l’ankyrine qui sont importantes pour la re´sistance aux maladies. Des mutations dans le domaine du NPR1 BTB/POZ diminuent aussi fortement les interactions avec le TGA7. Le NPR1 augmente substantiellement in vitro la liaison du TGA7 a` des e´le´ments de promoteur connus, y compris un e´le´ment du promoteur PR-1 inductible par l’acide salicylique. Alors que le TGA7 interagit avec tous les facteurs TGA teste´s, on n’observe pas d’interactions entre le TGA2 et les facteurs du sous-groupe I, ce qui indique que l’interaction croise´e entre clades ne constitue pas une proprie´te´ ge´ne´rale de la famille. Les transcriptions des facteurs TGA du sous-groupe III sont faiblement inductibles par l’acide salicylique et les agents pathoge`nes, mais seule l’expression du TGA3 de´pend du NPR1. Ces re´sultats sugge`rent que la liaison TGA7 a` l’ADN, sous l’effet du NPR1, pourrait re´guler l’activation des ge`nes de de´fense. Mots-cle´s : re´action de de´fense de la plante, transcription, ge`nes relie´s a` la pathogene`se, acide salicylique, interaction prote´ine–prote´ine. [Traduit par la Re´daction]
Introduction TGA factors are a class of basic leucine-zipper (bZIP) transcription factors originally isolated based on their ability to bind to the activating sequence-1 (as-1) element found in
the cauliflower mosaic virus 35S promoter (Katagiri et al. 1989). Both as-1 and related elements contain the core sequence TGACG and often confer responsiveness to stress or phytohormones, particularly salicylic acid (SA), jasmonic acid (JA), and auxin (Xiang et al. 1996). These cis elements
Received 30 June 2008. Published on the NRC Research Press Web site at botany.nrc.ca on 10 June 2009. H.L. Shearer,2 C. DeLong, and P.R. Fobert.3 National Research Council Canada, Plant Biotechnology Institute, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada. L. Wang.2 Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK S7N 5E2, Canada. C. Despres. Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St. Catharines, ON L2S 3A1, Canada. 1This
paper is one of a selection of papers published in a Special Issue from the National Research Council of Canada – Plant Biotechnology Institute. 2These authors contributed equally to this work. 3Corresponding author (e-mail:
[email protected]). Botany 87: 561–570 (2009)
doi:10.1139/B08-143
Published by NRC Research Press
562 Fig. 1. Model showing the position of TGA factors in the signal transduction pathway leading to salicylic-acid-mediated disease resistance. Perception of a virulent biotrophic pathogen triggers the accumulation of salicylic acid (SA) and generation of reactive oxygen species (ROS), changing the cellular oxido-reduction (DRedox) balance. In response to a net reduction in the redox status, certain cysteine residues in key regulators, including NPR1 and clade I TGA factors, become reduced. This permits nuclear localization of NPR1 and facilitates the interaction between clade I TGA factors and NPR1. NPR1 also interacts with clade II and clade III TGA factors and has been shown to enhance the DNA-binding activity of clade I and clade II TGA factors (black block arrows) to cognate TGACG-containing cis-acting regulatory elements. This is thought to be critical for the activation of defense-related genes, and ultimately resistance to disease. The NPR1-mediated enhancement of DNA-binding activity has not been previously reported for clade III TGA factors (open block arrow).
are present frequently in promoters of stress-regulated plant genes (see Rama et al. 2006). Of note, linker-scanning (LS) mutagenesis identified two important as-1-like sequences in the Arabidopsis pathogenesis-related1 (PR-1) promoter; LS7 is a positive element contributing to activation of PR-1 in response to SA and its functional analog 2,6-dichloroisonicotinic acid (INA), while LS5 is a negative regulatory element (Lebel et al. 1998). Chromatin immunoprecipitation (ChIP) has confirmed that TGA factors bind to the PR-1 promoter in planta, presumably on these same as-1-like elements (Johnson et al. 2003; Rochon et al. 2006). The Arabidopsis genome encodes 10 TGA factors (Jakoby et al. 2002), 6 of which were divided into subgroups based on sequence similarity (Xiang et al. 1997). Subgroup I consists of TGA1 and TGA4, subgroup II of TGA2, TGA5 and TGA6, and subgroup III contains TGA3. TGA7, the subject of the current study, represents a second member of subgroup III. Most studies to date have focused on subgroup II factors. Members of this clade have been shown to interact with NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), also known as NIM1, within the nucleus and in vitro (Despre´s et al. 2000; Subramaniam et al. 2001; Fan and Dong 2002; Rochon et al. 2006). Interaction with NPR1 stimulates the DNA-binding activity of TGA2 to cognate cis-acting elements in vitro (Despre´s et al. 2000, 2003) and in vivo (Fan and Dong 2002). Furthermore, the NPR1TGA2 complex constitutes an enhanceosome in planta, capable of transactivating a PR-1 promoter–reporter fusion gene in an SA-dependent manner (Rochon et al. 2006). Given NPR1’s role as a key positive regulator of SAmediated gene expression and defense responses (reviewed by Dong 2004), these findings implicate subgroup II TGA factors as potential mediators of defense gene expression (summarized in Fig. 1). Consistent with this hypothesis, an Arabidopsis mutant containing deletions in all subgroup II genes failed to accumulate PR-1 transcripts in the presence of INA and were compromised in systemic acquired resistance (SAR) (Zhang et al. 2003). NPR1 contains two known protein–protein interaction motifs: ankyrin repeats and a BTB/POZ (for Broad complex, Tramtrack, and Bric-a`-brac/Pox virus and Zinc finger) domain (Cao et al. 1997; Ryals et al. 1997; Rochon et al. 2006). The ankyrin repeats mediate interactions with subgroup II
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Pathogen
PERCEPTION
SA
ROS
ΔRedox
NPR1
Clade I Clade II (TGA1/4) (TGA2/5/6)
Clade III (TGA3/7)
TGACG
DEFENSE GENE EXPRESSION
Resistance TGA factors and TGA3, and their mutation abolishes the stimulatory effect of NPR1 on TGA2 DNA-binding, blocks PR gene expression, and compromises disease resistance (Cao et al. 1997; Ryals et al. 1997; Zhang et al. 1999; Despre´s et al. 2000). Together with C-term cysteines, the BTB/POZ domain is required for the SA-dependent transactivation of the PR-1 promoter by the NPR1-TGA2 enhanceosome (Rochon et al. 2006). Mutations of the NPR1 BTB/POZ domain Published by NRC Research Press
Shearer et al.
substantially reduce, but do not abolish, interaction with TGA2 (Rochon et al. 2006). Unlike subgroups II and III, subgroup I factors do not interact with NPR1 in yeast or in vitro (Zhang et al. 1999; Despre´s et al. 2000; Zhou et al. 2000; Despre´s et al. 2003), although they interact weakly in uninduced Arabidopsis leaves (Despre´s et al. 2003). This failure to interact is due to the inhibitory effects of two oxidized cysteine residues conserved only in TGA factors of this clade (Despre´s et al. 2003). Mutation of these cysteines to amino acids that mimic the reduced state of cysteine enables interaction between subgroup I factors and NPR1. Furthermore, in vivo reduction of subgroup I cysteines, triggered by SA-mediated redox changes, also facilitates interaction between this clade of TGA factors and NPR1 in Arabidopsis leaves (Despre´s et al. 2003). The DNA-binding activity of subgroup I factors is not directly affected by redox conditions; this property is conferred by interaction with NPR1. We previously reported the isolation of a then-novel member of the TGA family, encoded by At1g77920, through its ability to interact with NPR1 in the yeast twohybrid system (Despre´s et al. 2000). This protein was originally named TGA7 and corresponds to bZIP50 using the nomenclature of Jakoby et al. (2002). Unfortunately, a different member of the TGA family, PERIANTHIA (bZIP57), is also named TGA7 according to the TIGR database. However, bZIP50 is listed as corresponding to TGA7 in the TAIR and Swiss-prot databases, and has appeared under the name TGA7 in several publications (e.g., Hepworth et al. 2005; Liu et al. 2005; Kesarwani et al. 2007; Potlakayala et al. 2007). Accordingly, we have continued to make use of this name. TGA7 is most closely related to TGA3 (At1g22070) and the two proteins share substantial amino acid identity throughout their N-terminus before the basic domain, which is the least conserved region among TGA factors. Thus, TGA7 can be categorized as a group III factor with TGA3 according to the nomenclature of Xiang et al. (1997). Although TGA7 has been tested for its ability to interact with Arabidopsis NPR1-related proteins (Hepworth et al. 2005; Liu et al. 2005) and an NPR1 ortholog from Brassica napus (Potlakayala et al. 2007), it has remained largely uncharacterized since its first isolation. Furthermore, analysis of the interaction between subgroup III TGA factors and NPR1 has lagged behind that of other family members. Thus, the aim of this study was to characterize TGA7, particularly with respect to protein–protein interaction and gene expression pattern in response to pathogens.
Materials and methods Protein–protein interactions Yeast two-hybrid assays were performed as previously described (Kohalmi et al. 1997; Despre´s et al. 2000). All genes were inserted into pBI880 or pBI881 as Sal1-Not1 fragments containing entire coding regions without 5’ or 3’ untranslated sequences. Quantitative b-galactosidase assays as described in the Clontech Yeast Protocols Handbook (Clontech, Palo Alto, Calif.). The in vitro protein–protein interaction assays and the electrophoretic mobility shift assays (EMSA) were performed exactly as described by Despre´s et al. (2000).
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Plant materials All Arabidopsis thaliana plants used in this study, wildtype and the npr1–3 mutant, were in the ecotype Columbia (Col-0) genetic background. The npr1–3 mutant was obtained from the Arabidopsis Biological Resource Center (ABRC; Columbus, Ohio). The npr1–3 mutation introduces a premature stop at codon, resulting in truncation of the last 194 amino acids of NPR1 (Cao et al. 1997). Seeds were surface sterilized and germinated on 1 Murashige and Skoog medium (M-5519, Sigma, St. Louis, Mo.) as previously described (Liu et al. 2005), except that 4.5 gL–1 phytagel (Sigma) was used as gelling agent and plates were sealed with micropore tape (3M, St. Paul, Minn.). Seedlings were germinated and grown under the cabinet conditions described below. At 7 or 8 d post-germination, seedlings were transferred individually to sterilized Sunshine No. 4 soil (Sun Gro, Bellvue, Wash.) in 72-cell flats. Plants were grown at 70% relative humidity, under 150 mE cool white fluorescents, with a 10 h – 21 8C light period, and a 14h – 19 8C dark period. Salicylic acid experiment Twenty-five days post-germination, 8 and 1 h prior to harvest, flats were thoroughly misted with either water or a solution of 1 mmolL–1 SA (Sigma), covered, and returned to the growth cabinet. Whole leaves, excluding the petiole, were harvested into liquid nitrogen between 75 and 45 min before the end of the light cycle. Leaves from three individual plants grown at the same time were combined into each sample. The experiment was repeated three times. Bacterial infection time-course Twenty five days post-germination, all leaves large enough to handle were inoculated in a random order with either 10 mmolL–1 MgCl2, or with 1 106 cfumL–1 of either Pseudomonas syringae pv. tomato DC3000 (Pst) or Pst harboring AvrRpt2. Inoculations were performed using a 1 mL syringe during the light cycle, 24 and 6 h before harvest; the 0 h time point plants were not treated. Whole leaves were harvested into liquid nitrogen in chronological order of inoculation between 90 and 15 min before the end of the light cycle. Two 9-cell packs of plants, grown and treated on independent occasions, were combined into each sample. RNA extraction and kinetic polymerase chain reaction Total RNA was extracted from leaves using the RNeasy plant mini kit (QIAGEN, Mississauga, Ont.) according to the supplier’s instructions. After treatment with DNase I (Invitrogen, Carlsbad, Calif.), first strand cDNA synthesis was generated using SuperScript II reverse transcriptase (Invitrogen), and the (dT)17VN oligo in the presence of 0.4 U RNasin (Fisher Scientific, Pittsburg, Penn.). The newlysynthesized cDNA was diluted 1/200 to reflect a concentration of 10 ngmL–1 input total RNA. Kinetic polymerase chain reaction (PCR) was performed on an MX3000 spectrofluorometric thermal cycler (Stratagene, LaJolla, Calif.) using a two temperature cycling regime initiated with a 15 min activation at 95 8C, followed by 40 cycles of 2 min of annealing, and extension at 66 8C, and 10 s denaturation at 95 8C. Each assay contained Published by NRC Research Press
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0.5 pmol oligonucleotides (see supplementary data,4 Table S1), 5 ng cDNA, and 1 SYBR Green1 (Quantitech; QIAGEN), prepared as described in Rutledge and Stewart (2008). The fluorescence data collected at the end of each PCR cycle was analyzed by the absolute quantification via Ct method (Rutledge and Stewart 2008). Values for TGA3 and TGA7 were normalized against UBIQUITIN5.
Results Interaction between TGA7 and NPR1 To confirm the results from the yeast two-hybrid analysis and to study the physical interaction between TGA7 and NPR1, purified biotinylated wild-type NPR1 bound to streptavidin-agarose beads was incubated with in vitro-translated radiolabeled TGA7. As a control, the assay was also performed with TGA4, which does not interact with NPR1 either in the yeast two-hybrid system or in vitro (Despre´s et al. 2000). Analysis of the bound fraction using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS– PAGE) revealed the presence of a signal only when TGA7 was used, demonstrating a specific physical interaction between TGA7 and NPR1 (Fig. 2A, lane 2). To further probe the interaction between TGA7 and NPR1, NPR1 mutants and NPR1-related proteins were tested using the yeast-two hybrid system. Yeast cells cotransfected with TGA7 fused to the GAL4 transcriptional activation domain (GTA) together with full-length, wild-type NPR1 fused to the GAL4 DNA binding domain (GDB) were capable of growth on selective medium lacking tryptophan, leucine, and histidine, and supplemented with 3amino-1’,2’,3’-triazole (HIS–/3AT), and of activating the lacZ reporter gene (see supplementary data,4 Fig. S1A), indicative of protein–protein interaction. In contrast, cells cotransfected with TGA7-GTA together with GDB fusions to one of two NPR1 ankyrin repeat point mutants [npr1–1, which contains a H-to-T mutation at His 334 (Cao et al. 1997) and nim1–2, which contains a H-to-T mutation at His 265 (Ryals et al. 1997)], failed to grow on HIS–/3AT medium or activate the lacZ reporter gene (see supplementary data,4 Fig. S1A). Quantitative b-galactosidase assays confirmed the lack of interaction between TGA7 and the npr1 ankyrin repeat mutants (100-fold; data not shown). Levels of subgroup III transcripts were also found to increase slightly 8 h after SA treatment (*1.5-fold; Fig. 5B). When compared to each other, levels of TGA3 were consistently higher than those of TGA7. Our results are consistent with those found in the Genevestigator (Genevestigator.com) and eFP (bar.utoronto.ca) microarray databases. We also tested whether SA-induced subgroup III transcript accumulation required NPR1. In the npr1–3 mutant, the pattern of TGA7 transcript accumulation was similar to wild-type; however, no increases in TGA3 transcripts were observed (Fig. 5B). These data suggest that increased levels of TGA3, but not TGA7, in response to SA are dependent on NPR1.
Discussion We describe a previously uncharacterized member of the Arabidopsis TGA family of bZIP transcription factors (TGA7) with respect to its ability to interact with NPR1, a key regulator of systemic inducible disease responses in plants. We demonstrate that the DNA binding activity of TGA7 to cognate promoter elements, including the SAresponsive element (LS7) of the PR-1 promoter, is substantially enhanced by NPR1 but not by an SAR-impaired mutant form of NPR1 (nim1–2), and that TGA7 is capable of interacting with other TGA factors, thus providing a possible mechanism by which NPR1 could control gene activation. We also demonstrate that the abundance of TGA7 transcripts increases in response to both SA and pathogen challenge. Taken together, these results suggest that TGA7 (this study) Published by NRC Research Press
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Fig. 5. Expression of subgroup III TGA factors in response to SA and Pseudomonas syringae. (A) Pseudomonas syringae. Arabidopsis leaves were infiltrated with 10 mmolL–1 MgCl2, or 106 cfumL–1 of virulent Pst or avirulent Pst harboring AvrRpt2. Values represent averages (±1 SE) of three independent biological samples, each analyzed twice (technical replicates), and normalized against UBQ5. (B) Salicylic acid. Arabidopsis wild-type (Col-0) and npr1–3 mutants were sprayed with 1 mmolL–1 SA and tissue collected at the times indicated. Values represent averages (±1 SE) of three independent biological samples, each analyzed twice (technical replicates), and normalized against UBQ5. The experiment was repeated three times with similar results.
and TGA2 (Despre´s et al. 2000) could regulate expression of the Arabidopsis PR-1 gene, and possibly other genes involved in defense responses, through an NPR1-mediated DNA binding enhancement. A clustering analysis of seven members of the Arabidopsis TGA family showed that members of subgroup III are more similar to subgroup I than to those of subgroup II. The obvious questions that arise from these observations are whether the clustering of the TGA factors reflects functional differences and whether the in vivo functions of the different TGA factors are redundant. Two lines of evidence, which are summarized in Fig. 6, indicate a correlation between phylogenetic position and functional divergence. Firstly, members of subgroup II and III show many similarities in their interactions with NPR1;
members of both subgroups interact with NPR1 and their DNA-binding activity is enhanced by this regulator (Fig. 3 and Despre´s et al. 2000). Furthermore, members of subgroup II and III are unable to interact with ankyrin repeat mutants of NPR1 (Fig. 2A, 2C, see also supplementary data,4 Fig. S1A, and Despre´s et al. 2000), show reduced interaction with NPR1 BTB/POZ domain mutants (Fig. 2D, see also supplementary data,4 Fig. S1B, and Rochon et al. 2006), but are able to interact with NPR1-related proteins (see supplementary data,4 Fig. S1C; Hepworth et al. 2005; Liu et al. 2005). In contrast, TGA factors of subgroup I fail to interact with NPR1 or NPR1-like proteins, and their DNA-binding activity is not enhanced by NPR1 (this study and Despre´s et al. 2000; Despre´s et al. 2003). However, in Arabidopsis leaves, SA can trigger reduction of certain cysteine residues Published by NRC Research Press
Shearer et al. Fig. 6. Map of known interactions between TGA factors and NPR1. Block arrows indicate that an interaction occurs, but do not indicate specific interacting regions. Residues in NPR1 important to interaction between NPR1 and the TGA factors are shown. C260 and C266 of TGA1 are regulators of the interaction with NPR1 with the reduced forms of these residues permitting interaction (Despre´s et al. 2003). The oxidized form of C521 and C529, located in the transcriptional activation domain of NPR1, are required to form an enhanseosome with TGA2, but their importance in interactions with other TGAs has not yet been assessed (Rochon et al. 2006). C150, located in the POZ domain of NPR1, as well as H265 and H334, located in ankyrin repeat domain of NPR1, have been shown to be important in interactions with members of all subgroups of TGA factors. POZ, BTB/POZ domain of NPR1; ANK, ankyrin repeats of NPR1; TAD, transcriptional activation domain of NPR1 (Rochon et al. 2006); zip, leucine zipper domain; basic, basic domain, which binds DNA; N-term, amino-terminus; 30 a.a. domain, a region of TGA2 which is capable of promoting interaction with NPR1 (Despre´s et al. 2003).
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capacity for cross-clade interaction. It remains to be determined which of the possible TGA-TGA association suggested by our yeast two-hybrid analysis contributes to SAdependent gene expression in planta. It appears that there is some functional redundancy to the TGA factors, at least within each subgroup. Zhang et al. (2003) demonstrated that although single and double mutants of subgroup II displayed no altered phenotypes, a triple mutant in the TGA II subgroup was defective in INA-induced PR-1 expression and SAR. These phenotypes could be complemented by expression of either TGA2 or TGA5. Furthermore, a group I double mutant shows impaired basal resistance to P. syringae, while either of single mutants show a lesser degree of impairment (Kesarwani et al. 2007; our unpublished data). Mutation of TGA7 does not result in a noticeable change in PR gene or disease resistance (Kesarwani et al. 2007). Lack of a phenotype may be attributed to genetic redundancy between TGA7 and TGA3, and resolving this question will require the analysis of the corresponding double mutant.
Acknowledgements We are grateful to Catherine Chuback, Jamey Kalanack, and Kristen Hahn for technical assistance; to Dr. Robert Rutledge, Natural Resources Canada, for guidance on kPCR; and to Dr. Guosheng Liu, University of Saskatchewan, for critical reading of this manuscript. This research was supported by a grant from the Canada–Saskatchewan AgriFood Innovation Fund (P.R.F.), the NRC-PBI core program (P.R.F.), and the Natural Sciences and Engineering Council Discovery Grants Program (C.D. and P.R.F.). This publication is National Research Council of Canada publication number 50110.
References
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