article nature genetics • volume 26 • december 2000 403 The transcriptome of Arabidopsis thaliana during systemic acquired resistance Klaus Maleck 1 , Aaron Levine 2 , Thomas Eulgem 2 , Allen Morgan 1 , Jürg Schmid 1 , Kay A. Lawton 1 , Jeffery L. Dangl 2 & Robert A. Dietrich 1 Infected plants undergo transcriptional reprogramming during initiation of both local defence and systemic acquired resistance (SAR). We monitored gene-expression changes in Arabidopsis thaliana under 14 different SAR-inducing or SAR-repressing conditions using a DNA microarray representing approximately 25–30% of all A. thaliana genes. We derived groups of genes with common regulation patterns, or regulons. The regulon contain- ing PR- 1, a reliable marker gene for SAR in A. thaliana, contains known PR genes and novel genes likely to func- tion during SAR and disease resistance. We identified a common promoter element in genes of this regulon that binds members of a plant-specific transcription factor family. Our results extend expression profiling to defini- tion of regulatory networks and gene discovery in plants. 1 Syngenta, Research Triangle Park, North Carolina, USA. 2 Department of Biology and Curriculum in Genetics, University of North Carolina, Chapel Hill, North Carolina, USA. Correspondence should be addressed to J.L.D. (e-mail: dangl@email.unc.edu). Introduction Physiological flexibility is a prerequisite for life in a changing envi- ronment and requires elaborate regulatory mechanisms to simulta- neously alter the expression states of groups of genes. In prokaryotes, such regulons are often physically grouped into oper- ons. In eukaryotes, genes coregulated under specific conditions can be dispersed throughout the genome. Commonly, a limited set of key regulators, used combinatorially, is used to trigger defined tran- scriptional responses throughout an entire regulon. On recognition of avirulent pathogens, plants activate a set of transient local defence measures restricting pathogen growth (incompatible interactions) 1 . Infection with virulent pathogens that are not specifically recognized results in plant disease (compatible interactions). In addition to local defence responses, recognition of avirulent pathogens can trigger a long-lasting, broad-spectrum dis- ease resistance throughout the entire plant, referred to as systemic acquired resistance 2 (SAR). SAR is accompanied in uninfected ‘sec- ondary’ tissues by activation of a set of ‘pathogenesis-related’ (PR) marker genes. Some of these are also activated locally around infec- tion sites. Thus, the ‘pre-induction’ of a suite of proteins in systemic tissue is likely to confer broad-spectrum SAR (refs 2,3). A. thaliana and tobacco require salicylic acid accumulation to establish SAR (refs 3,4). Repression of salicylic acid accumulation by expression of a bacterial salicylic acid hydroxylase gene (NahG) abolishes SAR (ref. 5). Salicylic acid accumulation can be mimicked by chemical analogues including benzothiadiazol 6 (BTH) or 2,6- dichloroisonicotinic acid 7 (INA) and is stimulated in A. thaliana constitutive immunity (cim) or constitutive expression of PR genes (cpr) mutants 8,9 (K.M., unpublished data). In addition, genetic analyses identified regulators required to mediate SAR, such as NIM1/NPR-1 (refs 10,11), which appears to interact directly with transcription factors 3,12–15 (R.A.D., unpublished data). Salicylic acid accumulation functions upstream of NIM1/ NPR-1 (refs 3,11). The transcriptional consequences of pathogen infection and SAR-inducing conditions have been tested on a limited set of marker genes in a limited number of mutant backgrounds 2,16 . We analysed concerted transcriptional reprogramming in A. thaliana during the SAR response using cDNA microarrays. Our results extend the fundamental roles of salicylic acid accumula- tion and NIM1/NPR-1 in the control of the A. thaliana SAR tran- scriptome, but suggest that this key regulator cooperates with several types of transcription factors in the execution of the SAR transcriptional program. Results Related SAR conditions result in similar gene-activity patterns To examine gene-activity changes associated with the induction or maintenance of SAR in A. thaliana, we performed large-scale expres- sion profiling using cDNA arrays 17,18 . We used poly(A) + mRNA iso- lated from age- and environment-matched plants using 14 different treatments that either induce or repress SAR (Table 1) in mixed hybridizations with mRNA from genetically matched, untreated control plants on a DNA microarray with 10,000 expressed sequence tags (ESTs). This represents approximately 7,000 or 25–30% of all A. thaliana genes due to redundancy in the EST set. We included two control treatments that alter plant metabolism, but are unrelated to SAR (Table 1, treatments 1 and 2). One sample was taken during SAR induction (for example, 4 h after BTH treatment). Most sam- ples were derived from the SAR-maintenance phase (48 h after BTH treatment, cim mutants, or uninfected secondary tissue 44 h after infection of lower, primary leaves with avirulent Pseudomonas syringae bacteria). Thus, all but one of the time points for RNA col- lection correspond to fully induced SAR (refs 6,10,11). We required that ESTs included for analysis show differential expression equal to or greater than 2.5-fold in at least two SAR-relevant samples. © 2000 Nature America Inc. • http://genetics.nature.com © 2000 Nature America Inc. • http://genetics.nature.com