Caffeine production in tobacco plants by simultaneous expression of three coffee N-methyltrasferases and its potential as a pest repellant Hirotaka Uefuji 1,3 , Yuko Tatsumi 1 , Masayuki Morimoto 1 , Pulla Kaothien-Nakayama 1 , Shinjiro Ogita 1,2 and Hiroshi Sano 1, * 1 Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Nara, 630-0192, Japan (*author for correspondence; e-mail sano@gtc.naist.jp); 2 Biotechnology Research Center, Toyama Prefectural University, Toyama, 939-0398, Japan; 3 Biotechnology Institute University of Minnesota, St. Paul, MN, 55108, USA Received 11 March 2005; accepted in revised form 6 June 2005 Key words: Caffeine, insect repellant, Nicotiana tabacum, Spodoptera litura, theobromine Abstract Caffeine (1,3,7-trimethylxanthine) is derived from xanthosine through three successive transfers of methyl groups and a single ribose removal in coffee plants. The methyl group transfer is catalyzed by N-zmethyltransferases, xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT) and 3,7-dimethylxanthine methyltransferase (DXMT). We previously cloned three genes encoding each of these N-methyltransferases from coffee plants, and reconstituted the final sequence of the caffeine synthetic pathway in vitro. In the present study, we simultaneously expressed these coffee genes in tobacco plants (Nicotiana tabacum), using a multiple-gene transfer method, and confirmed successful caffeine production up to 5 lgg )1 fresh weight in leaves of the resulting transgenic plants. Their effects on feeding behavior of tobacco cutworms (Spodoptera litura), which damage a wide range of crops, were then examined. Leaf disc choice test showed that caterpillars selectively fed on the wild-type control materials, or positively avoided the transgenic materials. The results suggest a novel approach to confer self-defense by producing caffeine in planta. A second generation of transgenic crops containing caffeine may save labor and agricultural costs and also mitigate the environmental load of pesticides in future. Introduction Caffeine is naturally produced in certain tropical and subtropical plants, including coffee, tea, mate´, guarana´ and cola (Ashihara and Crozier, 2001), and has long been used as an ingredient of pharmaceuticals (Schmeller and Wink, 1998). In addition, it was shown to be effective as a repellant and pesticide for slugs and snails (Hollingsworth et al., 2002) and also for insects (Nathanson, 1984; Mathavan et al., 1985). Caffeine is synthesized through multiple meth- ylation of xanthine derivatives (Figure 1A) (Ashihara and Crozier, 2001). The first step is methylation of xanthosine by xanthosine methyl- transferase (XMT), yielding 7-methylxanthosine (Figure 1A, step 1), of which ribose moiety is removed by 7-methylxanthosine nucleosidase (step 2). The resulting 7-methylxanthine is methy- lated by 7-methylxanthine methyltransferase (MXMT or theobromine synthase) to produce 3,7-dimethylxanthine (theobromine) (step 3), which is further methylated by 3,7-dimethyl- xanthine methyltransferase (DXMT or caffeine synthase) to give caffeine (1,3,7-trimethylxan- thine) (step 4). Plant Molecular Biology (2005) 59:221–227 Ó Springer 2005 DOI 10.1007/s11103-005-8520-x