Induction of Hydrolytic Enzymes in Brassica campestris in Response to Pathovars of Xanthomonas campestris

Abstract

Inoculation of mature leaves of turnip (Brassica campestris) with the incompatible Xanthomonas campestris pv vitians resulted in the induction of 83-1,3-glucanase and chitinase/lysozyme (CHL) activity. No increase in the basal activity of 8-1,3-glucanase was observed after inoculation of leaves with heat-or nfampicin-killed X. c. vitians, Escherichia coii, or sterile water. Inoculation with the compatible X. campestris pv campestris resulted in a slower induction of glucanase than that seen with X. c. vitians. In contrast, all bacteria caused an induction of CHL activity. One major ,-1,3-glucanase (molecular mass 36.5 kilodaltons, isoelectric point [pi] -8.5) was purified from both inoculated and untreated leaves by ion-exchange chromatography. The enzyme degraded laminarin by an endo-glycolytic mechanism. Two major CHL isozymes (CHL 1 and CHL 2, molecular mass 30 kilodaltons and pi 9.4 and 10.2, respectively) were purified from X. c. vitians inoculated leaves by affinity chromatography on a chitin column followed by ion-exchange chromatography. Both enzymes degraded chitin by an endo-glycolytic mechanism although the ratio of lysozyme to chitinase specific activities for CHL 1 and CHL2 were different. The induction of CHL 1 was associated with the hypersensitive reaction caused by X. c. vitians whereas all other treatments induced largely CHL 2.gens. All plant lysozymes so far described have been shown to act as endochitinases (3). (3-1,3-Glucanase and chitinase are coordinately induced in a number of plant tissues by pathogen attack and elicitors (25).We are interested in the mechanisms of resistance of Brassica spp., in particular turnip (Brassica campestris) to incompatible pathovars of the Gram-negative phytopathogen Xanthomonas campestris. X. campestris pv campestris (hereafter referred to as X. c. campestris) is an important worldwide pathogen of almost all cultivated brassicas and noncrop crucifers (27) whereas X. c. vitians, a pathogen of lettuce (Lactuca sativa) is incompatible with brassica and triggers HR characterized by tissue collapse (6). As part of our study on defence mechanisms in Brassica, we have investigated the induction of CHL and (3-1,3-glucanase in response to X. c. campestris, X. c. vitians, and Escherichia coli (a nonpathogen). Lysozyme activity in turnip was originally described by Fleming (10) and the enzymes from turnip root (1) and cauliflower (9) have been partially characterized. In this paper we report on the characterization of the (-1,3-glucanase and of isoforms of CHL that show differential regulation in response to the different bacteria.The induction of the hydrolytic enzymes (3-1,3-glucanase and chitinase has been studied in a number of plants in response to infection, heat-killed pathogens, pathogen cell walls, or wounding (2,3,11). Some of the PR2 proteins induced in several different plants in response to infection have now been identified as (3-1,3-glucanases and chitinases (12,13,18). These hydrolases are of interest as plant defence products because of their...

Purification, characterization and differential hormonal regulation of a ?-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

“…Laminarin was used as the substrate for the fl-l,3-glucanase reaction. After various times of incubation, samples of the reaction products were applied to TLC silica-gel plates according to Conrads-Strauch et al (1990). N-Acetyl glucosamine, chitobiose and chitotriose (Sigma, Deisenhofen, FRG) were used as standards for the chitinase reaction, whereas glucose and laminaribiose (Sigma) were applied on TLC plates as standards for the fl-l,3-glucanase reaction.…”

“…N-Acetyl glucosamine, chitobiose and chitotriose (Sigma, Deisenhofen, FRG) were used as standards for the chitinase reaction, whereas glucose and laminaribiose (Sigma) were applied on TLC plates as standards for the fl-l,3-glucanase reaction. The TLC plates were developed as described by Conrads-Strauch et al (1990). Reaction products from both enzyme activities were detected using diphenylamine/ anilinie/phosphoric acid spray reagent (Chaplin 1986).…”

Plant chitinases

“…Induction of chitinase is often co-ordinated with the induction of specific p-1,3-glucanases and other pathogenesisrelated (PR) proteins (Bowles eta/., 1991 ;Brederode eta/., 1991 ;Conrads-Strauch et a/., 1990;Hedrick et a/., 1988;Joosten and De Wit, 1989;Kombrink et a/., 1988;Pegg and Young, 1982;Rasmussen eta/., 1992b;Shinshi eta/., 1987;Vad eta/., 1991: Vogeli etal., 1988Vogeli-Lange et a/., 1988). Differential regulation of individual chitinase isozymes has been reported from pea, barley, tobacco, turnip and cell cultures of peanut.…”

Pathogenicity Determinants and Global Regulation of Pathogenicity of Xanthomonas campestris pv. campestris

“…However, Xc. campestris is capable of substantial growth in the mesophyll tissue of crucifers and this is an experimentally easier system in which to study bacterial growth and concomitant plant defence responses (COLLINGE et al 1987;CONRADS-STRAUCH et al 1990). Assays in which seeds are soaked in bacterial suspensions before sowing may mimic another aspect of the disease cycle as contaminated seed is believed to be a major source of infection (SCHAAD and ALVAREZ 1993).…”