Regulation of a Chitinase Gene Promoter by Ethylene and Elicitors in Bean Protoplasts

Abstract

Chitinase gene expression has been shown to be transcriptionally regulated by a number of inducers, including ethylene, elicitors, and pathogen attack. To investigate the mechanism(s) responsible for induction of chitinase gene expression in response to various stimuli, we have developed a transient gene expression system in bean (Phaseolus vulgaris) protoplasts that is responsive to ethylene and elicitor treatment. This system was used to study the expression of a chimeric gene composed of the 5' flanking sequences of a bean endochitinase gene fused to the reporter gene ,-glucuronidase linked to a 3' fragment from nopaline synthase. Addition of 1-aminocyclopropane-1-carboxylic acid, the direct precursor of ethylene, or elicitors such as chitin oligosaccharides or cell wall fragments derived from Colletotrichum lagenarium, to transformed protoplasts resulted in a rapid and marked increase in the expression of the chimeric gene. The kinetics and dose response for these treatments were similar to those observed for the native gene in vivo. Analyses of 5' deletion mutants in the protoplast system indicated that DNA sequences located between -305 and -236 are important for both ethylene and elicitor induction of the reporter gene.Chitinase, a lytic enzyme found in most higher plants (1, 3), catalyzes the hydrolysis of chitin, a A-( 1,4)-linked polymer of 2-acetamido-2-deoxy-13-D-glucose. Although no endogenous substrate for this enzyme has been found in plants, chitin is a major component of the cell walls of many fungi (31). For this reason, Abeles et al. (1) proposed that chitinase functions as a defense against chitin-containing pathogens. This hypothesis is supported by studies which indicate that chitinase levels are increased in response to pathogen attack (19,20,24,25). In vitro studies have demonstrated further that plant chitinases are capable of hydrolyzing the cell walls ofplant pathogenic fungi (30) and releasing elicitors ofdefense reactions (14). In addition, plants treated with the phytohormone ethylene (1, 3) or elicitors such as isolated fungal cell walls (7,21,22), endogenous plant cell walls (21), or chitin oligosaccharides (14, 23), have also been shown to contain

Induction of the tobacco PR‐1a gene by virus infection and salicylate treatment involves an interaction between multiple regulatory elements

“…We have shown that for induction by TMV infection of tobacco genes encoding a glycinerich protein and an acidic PR-5 protein upstream sequences of 650 bp and over 1 kb are required, respectively (Albrecht et a/., 1992; Van de Rhee et a/., 1990). A 5' deletion analysis of a bean chitinase promoter showed that the region between -305 and -236 is necessary for induction by ethylene and fungal elicitor treatment in bean protoplasts (Roby et a/., 1991). For the Arabidopsis acidic chitinase promoter it was found that 192 bp upstream of the transcription start site are sufficient for inducible expression following fungal infection and salicylate treatment in transgenic Arabidopsis plants (Samac and Shah, 1991).…”

Tansley Review No. 86 Accumulation of phytoalexins: defence mechanism and stimulus response system

“…ln fact, a chitinase gene in rice cellsuspension cultures is induced by a fungal cell wall elicitor (Zhu & Lamb, 1991), whereas an endochitinase gene from bean is regulated by chitin oligomers und cell-wall fragments from Colletotrichum lage/iariurn (Roby et al, 1991), or infection with C. lindemuthianum (Hedrick et al, 1988).…”

Regulation of Sesquiterpene Cyclase Gene Expression (Characterization of an Elicitor- and Pathogen-Inducible Promoter)

“…As observed with the stable transgenic lines, deletions into the EAS4 promoter brought about a quantitative decrease in GUS expression. Moreover, in spite of the high background of GUS expression that was presumably due to the elicitation occurring during the preparation of the protoplasts (Roby et al, 1991;Mohan et al, 1993), the protoplasts were able to respond to a subsequent elicitor treatment. The levels of GUS activity for 5' deletions to -567, -212, and -160 were significantly elevated by the elicitor treatment and were approximately 2-fold greater than the GUS activity measured in protoplasts not receiving additional elicitor.…”