Pine chitinase gene structure, expression and regulation: Analysis in pine cells and in heterologous systems

Chitinases are plant enzymes that hydrolyze chitin, which is the major component of the cell walls of many pathogenic fungi but absent in higher plants. Chitinases belong to Group III PR-proteins which are believed to play important roles during plant-pathogen interactions. The present study describes the structures of several genomic clones from pine trees that appear to encode extracellular class II chitinase, and examines the expression of these genes in pine cells as well as in transgenic tobacco plants. 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. The corresponding Pschi4 cDNA was cloned by RT-PCR. Nucleotide sequence analysis indicated that the Pschi4 coding sequence is composed of three exons interrupted by two introns at locations identical to those found in other chitinase genes that possess introns. 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 at both mRNA and protein levels. The regulatory sequences associated with the Pschi4 gene were sufficient to direct chitosan-and wound-inducible expression of Pschi4 in transgenic tobacco plants, which indicated that Pschi4 is an actively expressed member of the multigene family. The region 5{dollar}spprime{dollar} to the putative transcription start site of Pschi4 was fused to the GUS reporter gene to further analyze these inducible regulatory elements. The 200 bp 5{dollar}spprime{dollar}-upstream sequence of Pschi4 was demonstrated to contain active promoter sequences capable of wound-induced expression in transient assays (particle bombardment) as well as in stably transformed tobacco plants. This region was also sufficient to induce transcription in pollen of transgenic tobacco. The putative pine promoter showed higher promoter activity than the widely used CaMV 35S in the transient expression in pine cells, which implies that the Pschi4 promoter could be a good candidate to regulate transcription of other genes in transgenic pine cells. 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.