Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase.

Abstract

An extracellular, acidic chitinase was purified to homogeneity from tobacco necrosis virus-infected leaves of Cucumis sativis. The amino acid sequences of the intact protein and of peptides isolated following endoproteinase Lys-C digestion, cyanogen bromide cleavage, and trypsin digestion were determined. Oligonucleotide probes derived from this sequence were used to isolate a cDNA clone encoding this protein. No significant homology was found between this chitinase and either the basic chitinase isolated from bean or tobacco or the chitinase isolated from Serratia marcescens; however, strong homology was found between the cucumber chitinase and a lysozyme/chitinase from Parthenocissus quinquifolia. The induction of the protein by tobacco necrosis virus infection or salicylate was found to be at the level of RNA accumulation. Genomic Southern analysis indicates that a single gene in the cucumber genome encodes this protein.Many plants infected with necrotizing pathogens develop local or systemic resistance against subsequent infections (1, 2). This induced resistance is accompanied by a number of biochemical changes in the host plant, including the production of pathogenesis-related (PR) proteins (for review see refs. 3 and 4). The accumulation of these acid-extractable, low molecular weight, protease-resistant proteins correlates with induced resistance, but their function has largely remained elusive. Recently, it has been shown that of the 10 well-characterized PR proteins in tobacco, two (PR-P and -Q) have chitinase activity (5-7), three (PR-O, -N, and -2) have /3-1,3-glucanase activity (8), and two (PR-R and -S) are structurally similar to a maize protease/a-amylase inhibitor (9, 10).From cucumber, we have recently purified one PR protein and identified it as a chitinase (11). After infection with tobacco necrosis virus (TNV), this Mr 28,000 protein accumulates in the intercellular space of the infected, as well as the uninfected, parts of the plant (11, 12). Here, we report the purification of this protein to homogeneity and the determination of 55% of the amino acid sequence (148 of 267 residues) of the mature protein. Oligonucleotide probes were synthesized based on the protein sequence analysis and used to isolate cDNA clones encoding this protein from a library constructed with RNA isolated from TNV-infected cucumber leaves. We have sequenced the cDNA clones and find no significant homology to known chitinase genes. However, striking homology was found between the deduced amino acid sequence and the partial amino acid sequence of a bifunctional lysozyme/chitinase purified from Parthenociccus quinquifolia (13).In preliminary studies on the regulation of chitinase gene expression, we show that there is one gene encoding chitinase in the cucumber genome and the accumulation of this protein after TNV infection or salicylic acid induction correlates with the accumulation of mRNA. MATERIALS AND METHODSProtein Purification and Sequencing. Chitinase protein was isolated from TNV-infected cucumber (Cucu...

Systemic Acquired Resistance.

“…These include cell death and the oxidative burst (Low and Merida, 1996), deposition of callose and lignin (Vance et al, 1980;Kauss, 1987), and the synthesis of phytoalexins (Dixon, 1986) and novel proteins ( BOI et al, 1990;Bowles, 1990;Linthorst, 1991; see also Dangl et al, 1996;Hammond-Kosack and Jones, 1996, in this issue). Recently, however, marker genes termed SAR genes have been identified whose induction is tightly correlated with the onset of SAR in uninfected tissue (Métraux et al, 1989;Ward et al, 1991;Uknes et al, 1992), and these are described in more detail below.…”

Study of the Intercellular Fluid of Healthy Lupinus albus Organs (Presence of a Chitinase and a Thaumatin-Like Protein)

“…In addition, all of the Figure 3. Comparison of the amino acid sequence of IF3 with those of lysozyme/chitinases from Parthenocissus quinquefolia (Bernasconi et al, 1987), Cucumis sativus chitinase (Metraux et al, 1989), Arabidopsis thaliana chitinase (Samac et al, 1990), lysozyme/chitinase from Hevea brasiliensis (Jekel et al, 1991), and Phaseolus vulgaris chitinase (Margis-Pinheiro et al, 1993). X, No amino acid derivative was detected by HPLC.…”

Purification and Characterization of Abundant Secreted Protein in Suspension-Cultured Pumpkin Cells

“…The amino-terminal amino acid sequence of SP32 was similar to that of chitinase isolated from tobacco (27) or bean (13), although it was quite different from that of chitinase isolated from cucumber (21). As shown in Figure 5, there was an 80% identity (8 of 10 residues) between the amino-terminal amino acid sequence of SP32 and that of chitinase isolated from tobacco, and was a 71% identity (10 of 14 residues) between that of SP32 and that of chitinase from bean.…”