Plant Molecular Biology 42: 387–396, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 387 Isolation and characterization of two pathogen- and salicylic acid-induced genes encoding WRKY DNA-binding proteins from tobacco Chunhong Chen and Zhixiang Chen ∗ Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID 83844-3052, USA ( ∗ author for correspondence) Received 28 July 1999; accepted in revised form 4 November 1999 Key words: gene expression, hypersensitive responses, plant defense responses, salicylic acid, tobacco mosaic virus, WRKY DNA-binding proteins Abstract A pathogen- and salicylic acid (SA)-induced DNA-binding activity has been recently identified in tobacco that is related to a previously identified class of WRKY DNA-binding proteins. To identify members of the WRKY gene family associated with this DNA-binding activity, we have attempted to isolate those WRKY genes that are induced by pathogen infection. Using a domain-specific differential display procedure, we have isolated two tobacco WRKY genes, tWRKY3 and tWRKY4, that are rapidly induced in resistant tobacco plants after infection by tobacco mosaic virus (TMV). Both tWRK3 and tWRKY4 encode proteins with a single WRKY domain that contain the conserved WRKYGQK sequence. Unlike other isolated WRKY proteins that contain the Cys 2 His 2 zinc motif, tWRKY3 and tWRKY4 appear to contain the Cys 2 HisCys zinc motif. Nonetheless, both tWRKY3 and tWRKY4 are capable of binding DNA molecules with the W-box (TTGAC) element recognized by other WRKY proteins. Expression of the tWRKY3 and tWRKY4 genes could be rapidly induced not only by TMV infection but also by SA or its biologically active analogues that are capable of inducing pathogenesis-related genes and enhanced resistance. Interestingly, induction of both genes by TMV infection was still observed in resistant tobacco plants expressing the bacterial salicylate hydroxylase gene (nahG), although the levels of induction appeared to be reduced. Identification of pathogen- and SA-induced genes encoding WRKY DNA-binding proteins should facilitate future studies on the regulation and functions of this novel group of DNA-binding proteins. Abbreviations: EMSA, electrophoresis mobility shifting assays; HR, hypersensitive responses; PR, pathogenesis- related; SA, salicylic acid; TMV, tobacco mosaic virus; TDBA, tobacco DNA-binding activity Introduction A key to plant disease resistance is the timely recogni- tion of the invading pathogen by a resistant plant and the rapid activation of plant host defense responses through a number of signal transduction pathways (Yang et al., 1997; van der Biezen and Jones, 1998). Like many other biological processes, pathogen- induced signal transduction pathways in plants often converge in the cell nucleus where a large number The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Dababases under the ac- cession numbers AF193770 (tWRKY3) and AF193771 (tWRKY4). of defense-related genes are transcriptionally acti- vated (Rushton and Somssich, 1998). Some of these defense-related genes encoding enzymes in a variety of primary and secondary metabolic pathways and their enhanced synthesis may result in reprogramming of cellular metabolisms (Somssich and Hahlbrock, 1998). For example, genes encoding enzymes of the phenylpropanoid pathways are often induced during plant defense responses that are responsible for en- hanced synthesis of a variety of antimicrobial com- pounds (phytoalexins). Some of the defense-related genes encode pathogenesis-related (PR) proteins in- cluding antimicrobial chitinases and glucanases that