1146 Nucleic Acids Research, 2007, Vol. 35, No. 4 In addition, WRKY genes are involved in abiotic stress, such as drought (14–17), cold (14,17,18), heat (15), salinity (17) and wounding (19); and in biosynthesis of anthocya- nin (20) and starch (21). Some WRKY genes can also regulate developmental process such as embryogenesis (22), senescence (4,23), tricome development (20) as well as seed size (24). There are more than 70 WRKY family members in Arabidopsis (1) and more than 100 in rice (25). The name of WRKY is derived from the conserved WRKY sequence motif which is present in all WRKY members. The WRKY domain is a stretch of about 60 amino acids with strictly conserved WRKYGQK sequence at its N-terminal followed by a putative novel zinc-binding motif with features of C–C–H–H or C–C–H–C (26–28). The existence of Zn2þ is crucial for the DNA-binding activity, which implies the importance of the putative zinc- binding motif (29,30). WRKY proteins can be classiﬁed into three groups according to the number of WRKY domains and zinc-binding pattern (26). Group I WRKY proteins are distinct from groups II and III by containing two WRKY domains. The zinc-binding motif of group III proteins is C–X7–C–X23–H–X–C, diﬀerent from the common pattern of C-X4–5-C-X22–23-H-X-C of group I and II proteins. It has been reported that substitutions of the invariable WRKYGQK residues in the WRKY domain decreased the DNA-binding aﬃnity, and any mutations of the conserved cysteine and histidine of the zinc-binding motif abolished the protein–DNA inter- action (29). The two WRKY domains in group I proteins play diﬀerent roles in DNA-binding activities (27). It has been shown that the speciﬁc binding to W-box is mediated mainly by the C-terminal WRKY domain (27,30), whereas the function of N-terminal WRKY domain remains unclear. Arabidopsis thaliana WRKY1 protein (also known as ZAP1), the ﬁrst identiﬁed WRKY transcription factor from Arabidopsis, is localized in Chromosome 2 as a single copy and belongs to group I WRKY family (30). The recognition of AtWRKY1 with W-box mainly depends on the C-terminal WRKY domain while the N-terminal WRKY domain just slightly aﬀect the protein–DNA interaction (30). The structural information of WRKY proteins and how they interact with W-box were lacking until recently when an NMR structure of C-terminal WRKY domain of AtWRKY4, AtWRKY4-C was determined (31). However, the domain division of the NMR sample seemed to be short and may not represent the structure of the whole domain. After the whole genome cloning of the transcription factors of A. thaliana (32), we have carried out functional and structural studies on AtWRKY1. In this report, we have showed that AtWRKY1 can be induced by SA treatment and the induction was partially dependent on NPR1, suggesting that this protein may be involved in the defense response. Most importantly, we present the crystal structure of the AtWRKY1-C at 1.6 A˚ and a model of protein–DNA complex from site-directed mutagenesis studies. MATERIAL AND METHODS Plant material and growth conditions Wild-type Arabidopsis plants (ecotype Col-0) were grown in the soil at a growth room (23/208C, 12-h-light/ 12-h-dark cycle). All the seeds were vernalized at 48C for at least 2 days before placement in a growth environment. The non-functional NPR1 mutant npr1–3 (12) and the transgenic line overexpressing NPR1 gene (33) were kindly provided by Dr Frederick M. Ausubel (Harvard Medical School, USA) and Dr Xin-Nian Dong (Duke University, USA), respectively. SA treatment and northern blotting Arabidopsis plants were sprayed with 2 mM SA diluted from stock solution (100 mM, adjusted to pH 6.5 with KOH). Leaves from diﬀerent 4-week-old plants were harvested at indicated time points for total RNA preparation with RNAzol (Vigrous, Beijing) according to the manufacturer’s instruction. Ten micrograms of total RNA was separated on 1.5% agrose–formaldehyde gels and transferred to a nylon membrane to hybridize with digoxigenin (DIG) labeled DNA probes. DIG DNA probes labeling, hybridization and detection were per- formed according to DIG Application Manual for Filter Hybridization (Roche, Penzberg, Germany). The follow- ing primer combinations were used to amplify DNA probes by PCR: PR1: forward 50-ATGAATTTTACTGG CTATTCTCG-30, reverse 50-TTAGTATGGCTTCTCGT TCAC-30; AtWRKY1-C: forward 50-ATGGCTGAGGT GGGAAAAGTTCTG-30, reverse 50-GCTTTGGGCAG GCTCTGTCTTGGG-30. Equal loading was conﬁrmed by staining the gel with ethidium bromide (EB). Protein preparation and crystallization The preparation and crystallization of the AtWRKY1-C protein was described previously (34). Brieﬂy, the cDNA fragment covering the C-terminal WRKY domain of AtWRKY1 was ampliﬁed by polymerase chain reaction (PCR) from a full-length cDNA clone of AtWRKY1 obtained from Arabidopsis (32) and cloned into the expression vector pET21aDEST. The protein was expressed in E. coli strain Rosetta and was puriﬁed to homogeneity in a two-step procedure of Ni2þ chelating and size exclusion chromatography. The mutant proteins were prepared similarly. The protein sample was kept in 20 mM Tris, 200 mM NaCl, pH 7.5 prior to crystallization. Crystals of the AtWRKY1-C were obtained at 277 K from condition containing 1.2 M succinic acid, 0.1 M Tris–HCl pH 7.0 and 1% w/v PEG MME 2000, by using the hanging-drop vapor diﬀusion methods. Data collection and structure determination Two crystal forms were obtained from the same growth condition. One belongs to the space group P21, with diﬀraction to 2.5 A˚ resolution at home X-ray sources as published before (34), whereas the other crystal form