Plant Molecular Biology 33: 979–987, 1997. 979 c 1997 Kluwer Academic Publishers. Printed in Belgium. Molecular cloning, structure and expression of an elicitor-inducible chitinase gene from pine trees Haiguo Wu 1 , Craig S. Echt 3 , Michael P. Popp 2 and John M. Davis 12 1 Plant Molecular and Cellular Biology Program and 2 School of Forest Resources and Conservation, PO Box 110410, University of Florida, Gainesville, FL 32611, USA ( author for correspondence); 3 Forestry Sciences Laboratory, USDA-FS, 5985 Highway K, Rhinelander, WI 54501, USA Received 14 May 1996; accepted in revised form 3 January 1997 Key words: chitosan, class II chitinase, defense response, gymnosperm, Pinus, transgenic plants Abstract We have cloned, sequenced, and examined the expression of genes from pine trees that appear to encode extracellular class II chitinase. Nucleotide sequence analysis indicates a coding sequence composed of three exons interrupted by two introns at locations identical to those found in other chitinase genes that possess introns. One of the genes, Pschi4, potentially encodes a protein that shares 62% amino acid sequence identity through the catalytic domain with class II chitinase from tobacco. In contrast, Pschi1 contains a stop codon in the first exon and may be a pseudogene. Pschi4 genes are conserved in several species of pine, and appear to comprise a small multigene family. Treatment of pine cell suspension cultures with the general elicitor chitosan induced Pschi4 expression. The regulatory sequences associated with the Pschi4 gene were sufficient to direct chitosan-inducible expression of Pschi4 in transgenic tobacco plants, which indicates that Pschi4 is an actively expressed member of the multigene family. The observation that the Pschi4 gene from pine (a gymnosperm) was appropriately regulated by chitosan in tobacco (an angiosperm) suggests that the signaling pathways that mediate chitosan-induced transcription are highly conserved in the plant kingdom. Introduction Plants are constantly confronted by microbes and other pests that can cause tissue damage. The cell walls of many of these microbes share common structural fea- tures. Compounds like chitin and chitosan are found in the cell walls of many fungi, as well as the exoskeleton of arthropods, but these compounds are not found in plants. In most plants, a defense response is elicited when they are treated with chitin or chitosan [11, 14, 29]. The response to these general elicitors is thought to reflect the plant’s ability to recognize the general features of most potential pathogens (i.e., chitinous cell walls) and mount a defense response [11]. One component of the defense response to elicitors is the transcriptional activation of genes that encode The nucleotide sequences reported will appear in the GenBank Nucleotide Sequence Database under the accession numbers U57409 and U57410. pathogenesis-related (PR) proteins. One class of PR proteins is chitinase. Chitinases hydrolyze chitin, a lin- ear homopolymer of N-acetylglucosamine. Most of the characterized plant chitinases are endochitinases (EC 3.2.1.14), which randomly cleave internal -1,4 link- ages in chitin and consequently release oligomers of N-acetylglucosamine. Chitinases are therefore capable of inhibiting fungal growth by causing lysis of hyphal tips, particularly in combination with glucanase [18, 28]. Chitinase could also function to ‘amplify’ defense responses in cells surrounding a site of infection by lib- erating chitin oligomers from fungal cell walls [17]. In addition to potential roles in defense, chitinase can play important roles during early embryo development and potentially other developmental processes [6]. This is presumably because plant cells contain substrates for chitinase that are not chitin per se, but may resemble chitin structurally [6].