1Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan; 2Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-0867, Japan

Background and objectives
Plant defences against pathogen attack involve an array of inducible responses that contribute to resistance. In many plant-pathogen interactions, defence responses include a rapid burst of active oxygen species, the hypersensitive response (HR), increased expression of defence-related genes, and production of antimicrobial compounds. The defences are activated upon recognition of a pathogen and successive signal transduction including GTP-binding protein, phospholipases, protein kinases and cytoskeletal proteins [1]. In order to elucidate the molecular mechanisms by which plant defence systems are activated, it is necessary to clone the genes encoding components of signalling pathways that ultimately lead to defence responses. We performed differential screening for tobacco plants responsive to elicitor treatment.

Materials and methods
Tobacco plants (Nicotiana tabacum cv. Samsun NN) carrying the N gene, were cultivated at 25C for 5 weeks after sowing. Fungal elicitor, hyphal wall components (HWC), was prepared from Phytophthora infestans, the potato late blight fungus. Tobacco leaves were injected with 1 mg/ml HWC or water and incubated at 20C in the dark. For preparations of total protein, tobacco leaves were homogenized with extraction buffer and centrifuged to remove tissue residue after various periods. 10 g of total proteins were separated by SDS-PAGE on 12.5% polyacrylamide gel and transferred to a nitrocellulose membrane in a semi-dry electroblotting system. Immunoreactions were performed using antisera against PR proteins as the first antibody and peroxidase-conjugated antibodies against rabbit immunoglobulin as the second antibody. Total RNAs from tobacco leaves treated with HWC or water were prepared by the SDS-phenol method and subjected to RT-PCR with random primers. RT-PCR products specific for RNAs from HWC-treated leaves were labelled with 32P by random primer labelling. 20 g of total RNAs were electrophoresed in a 1% agarose gel containing formaldehyde, blotted to a nylon membrane and hybridized to a radiolabelled DNA probe. PolyA+ RNA was prepared from HWC-treated tobacco leaves and used for the construction of a cDNA library.

Results and conclusions
We estimated that tobacco plants injected with HWC showed typical defence responses. The induction of localized necrosis at the site of HWC injection was observed. We prepared total proteins from leaf tissues treated with HWC and subjected then to Western analysis using antisera against PR-proteins, PR-N, P and S. PR proteins were detected at 12 h and accumulated at 36 h after treatment with HWC. Thus we prepared total RNA from tobacco leaves at 0, 12 and 36 h after the elicitor treatment and performed differential screening. PCR products amplified specific to HWC-treated leaf RNA were obtained by RT-PCR with 132 random primers. Next, Northern hybridization using the PCR products as probes confirmed two transcripts were induced by the HWC treatment. One transcript encoded an amino-acid sequence homologous to that of cytochrome P450, which is involved in the general phenylpropanoid pathway, whereas we found no known sequence similar to that for the other gene. We obtained an additional transcript, which was detected by RT-PCR using newly designed specific primers. The third HWC-induced gene was similar to known disease resistance gene(s). The cloning of these genes is a first step toward the identification of factors leading to defence responses.

1. Takemoto D, Furuse K, Doke N, Kawakita K, 1997. Plant and Cell Physiology 38, 441-448.