NATURE BIOTECHNOLOGY VOL 18 JULY 2000 http://biotech.nature.com 779 For centuries it was known that chewing the bark of the willow tree (Salix) relieved headaches and fevers, and in the 19th century sali- cylic acid (SA) was identified as the active component in the bark extract 1 . Only recently it was found that in plants SA is involved in various physiological processes like stomatal closure, flower induc- tion, and heat production, and that it plays a central role in defense against pathogen attack 2–5 . After the formation of a necrotic lesion to restrict the spread of an invading pathogen, plants activate a path- way leading to systemic acquired resistance (SAR) 6 . Characteristics of SAR are resistance extending to plant tissues distant from the ini- tial infection site, persistence for weeks to months, and plant protec- tion against secondary infection by a broad spectrum of pathogens 7 . Exogenous SA can induce SAR 8 , and SA appears to be the endoge- nous signal that triggers SAR 9,10 . Systemic acquired resistance is accompanied by the induced expression of genes encoding pathogenesis-related (PR) proteins. Among these are antifungal chitinases, β-1,3-glucanases, and PR- 1 and PR-5 proteins with anti-oomycete activity. PR proteins with a specific antiviral activity have not yet been characterized; how- ever, SA has been implicated in development of virus resistance by modulation of the alternative oxidase pathway 11 . In tobacco, sub- groups of basic and acidic PR proteins accumulate in the vacuole and intercellular space, respectively. In particular, the genes encoding acidic PR proteins are systemically induced in leaves with acquired resistance 12 . Additional evidence for a role of SA in plant defense came from tobacco plants transformed with the nahG gene from Pseudomonas putida. The nahG gene product encodes salicylate hydroxylase, which converts SA to the biologically inactive catechol. In these plants SA can no longer accumulate, which results in the inability to induce SAR 7,13 . Currently, genetic and biochemical data are accumulating on components of the signaling pathway acting upstream or downstream of SA. In plants the biosynthesis of SA is thought to proceed by the phenylpropanoid pathway 14 . After biosynthesis, most SA is conju- gated to SA 2-O-β-D-glucoside (SAG) 15,16 . In addition, SAG may play a role in SAR, possibly serving as an inactive storage form that can be rapidly cleaved to release active SA at the site of infection 17 . Microorganisms, among them Pseudomonas fluorescens and Escherichia coli, produce SA and related compounds to serve as building blocks for iron-chelating siderophores 14 . For example, cer- tain Pseudomonas species produce SA by means of a short biosyn- thetic pathway from chorismate, the general precursor of aromatic compounds. This substrate is converted by isochorismate synthase (ICS) to isochorismate, which is subsequently cleaved by isochoris- mate pyruvate lyase (IPL) to yield SA. Here, we report the transformation of tobacco with bacterial ICS and IPL genes fused to a strong plant promoter. Overproduction of SA in the transgenic plants resulted in constitutive expression of defense genes and an enhanced resistance to various pathogens. Results and discussion Chorismic acid, the end product of the shikimate pathway, is mainly produced in the chloroplasts 18 . Therefore, we fused the plastid targeting sequence (ss) of the small subunit of tobacco ribulose bisphosphate carboxylase precursor to the ICS coding sequence of the E. coli entC gene. Using the pmsB coding sequence from P. fluorescens, we constructed two types of IPL genes, one containing and the other lacking the plastid targeting sequence ss (Fig. 1). The resulting genes, cloned behind the constitutive 35S promoter from cauliflower mosaic virus (CaMV), were used to transform tobacco. For each construct three randomly chosen pri- Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance Marianne C. Verberne 1 , Rob Verpoorte 1 , John F. Bol 2 , Jesus Mercado-Blanco 3,4 and Huub J.M. Linthorst 2 * 1 Leiden/Amsterdam Center for Drug Research and 2 Institute of Molecular Plant Sciences, Leiden University, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands. 3 Department of Plant Ecology and Evolutionary Biology, PO Box 800.84, 3508 TB Utrecht University, Utrecht, The Netherlands. 4 Present address: Dept. Proteccion de Cultivos Instituto Agricultura Sostenible (C.S.I.C.), Apdo 4084, 14080 Cordoba, Spain. *Corresponding author (linthors@chem.leidenuniv.nl). Received 25 January 2000; accepted 9 April 2000 After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens. There is compelling evidence that SA plays a crucial role in trig- gering SAR. We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process. When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumula- tion of SA and SA glucoside compared to control plants. Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants. This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants. Keywords: salicylic acid, systemic acquired resistance, tobacco, Tobacco mosaic virus, Oidium lycopersicon, PR gene expression RESEARCH ARTICLES © 2000 Nature America Inc. • http://biotech.nature.com © 2000 Nature America Inc. • http://biotech.nature.com