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...

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Complementary DNA clones encoding two isoforms of the acidic endochitinase (chitinase, EC 3.2.1.14) from tobacco were isolated. Comparison of amino acid sequences deduced from the cDNA clones and the sequence of peptides derived from purified proteins show that these clones encode the pathogenesis-related proteins PR-P and PR-Q. The cDNA inserts were not homologous to either the bacterial form of chitinase or the form from cucumber but shared significant homology to the basic form of chitinase from tobacco and bean. The acidic isoforms of tobacco chitinase did not contain the amino-terminal, cysteine-rich "hevein" domain found in the basic isoforms, indicating that this domain, which binds chitin, is not essential for chitinolytic activity. The accumulation of mRNA for the pathogenesis-related proteins PR-1, PR-R, PR-P, and PR-Q in Xanthi.nc tobacco leaves following infection with tobacco mosaic virus was measured by primer extension. The results indicate that the induction of these proteins during the local necrotic lesion response to the virus is coordinated at the mRNA level.

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The acidic, extracellular, glucan endo-1,3-t8-glucosidases (EC 3.2.1.39; j0-1,3-glucanases), pathogenesis-related proteins-2, -N, and -O (i.e. PR-2, PR-N, and PR-0) were purified from Nicotiana tabacum (tobacco) and their partial amino acid sequences determined. Based on these data, complementary DNA (cDNA) clones encoding the proteins were isolated. Additional cDNAs were isolated that encoded proteins approximately 90% identical with PR-2, PR-N, and PR-0. Although the proteins encoded by these cDNAs have not been identified, their deduced amino acid sequences have slightly basic or neutral calculated isoelectric points, as well as carboxy-terminal extensions. These physical characteristics are shared by the vacuolar form of ,B-1,3-glucanase and other vacuolar localized analogs of PR proteins, suggesting that the unidentified proteins may be similarly localized.A preliminary evolutionary model that separates the fl-1,3-glucanase gene family from tobacco into at least five distinct subfamilies is proposed. The expression of ,-1,3-glucanase messenger RNAs (mRNAs) in response to infection by tobacco mosaic virus was examined. Messages for the acidic glucanases were induced similarly to the mRNAs for other PR proteins. However, the basic glucanase showed a different response, suggesting that different isoforms are differentially regulated by tobacco mosaic virus infection at the mRNA level.