Evidence for a third structural class of β-1,3-glucanase in tobacco

Abstract

Glucan endo-1,3-beta-glucosidases (beta-1,3-glucanases) have been implicated in several developmental processes and they may also play a direct role in the plant's defense against fungal pathogens. In an effort to characterize the glucanase gene family, complementary DNA clones encoding an acidic form of beta-1,3-glucanase have been isolated from tobacco. The cDNA was expressed in E. coli and shown to encode a beta-1,3-glucanase activity. The protein sequence encoded by the cDNA was found to match the partial protein sequence of PR-35, a previously characterized beta-1,3-glucanase. The protein encoded by the cDNA was purified from the extracellular fluid of TMV-infected tobacco leaves and found by immunological methods to correspond to glucanase PR-Q'. From a detailed analysis of the cDNA it is clear that this glucanase represents a third structural class of enzyme which differs substantially from both the basic, vacuolar glucanase and the acidic, extracellular forms (PR-2, PR-N and PR-O). It has previously been demonstrated that the basic form of beta-1,3-glucanase is synthesized as a pre-pro-enzyme and upon maturation the 21 amino acid signal peptide and a 22 amino acid carboxy-terminal peptide are removed. This processing event has been proposed to be involved with the vacuolar localization of the enzyme. By comparing the deduced protein structure of PR-Q' to that of the basic form it is evident that this extracellular enzyme is missing the carboxy-terminal 22 amino acids. The role of a conserved phenylalanine-glycine dipeptide in the processing of glucanases and other pathogenesis-related proteins from tobacco is discussed.

Differential Regulation of β-1,3-Glucanase Messenger RNAs in Response to Pathogen Infection

“…diverged more recently than did the others. In the suggested nomenclature for differentiating the different glucanases, class I refers to the basic, vacuolar forms, class II refers to the acidic, extracellular glucanases, and class III refers to the acidic, extracellular glucanases similar to PR-Q' (21). We suggest that the class II glucanases can be further divided into class Ila comprising the nearly identical proteins PR-2 and PR-N, class lIb comprising PR-O, which "links" PR-2/-N to GL153 and GLi61, and class lIc comprising the proteins encoded in GL153 and GL161.…”

“…At least three major classes of the enzyme have been identified in tobacco (21). The class I enzymes include three related isoforms, characterized by basic pI values, that accumulate predominantly in the vacuole (1 1, 27).…”

“…4). To date, one class III enzyme has been identified, PR-Q' (9,21), an acidic protein that also accumulates in the apoplast when infected with a pathogen. cDNA clones have been isolated for the class I and III enzymes (21,24), but the lack of cDNAs for the class II enzymes has limited detailed studies of their gene structure and regulation.…”

“…To date, one class III enzyme has been identified, PR-Q' (9,21), an acidic protein that also accumulates in the apoplast when infected with a pathogen. cDNA clones have been isolated for the class I and III enzymes (21,24), but the lack of cDNAs for the class II enzymes has limited detailed studies of their gene structure and regulation. Here, we describe the isolation and character-DIFFERENTIAL p3-1, 3-GLUCANASE REGULATION IN TOBACCO ization of cDNAs encoding the class II 0-1,3-glucanases PR-2, -N, and -0, as well as cDNAs encoding two novel glucanases closely related to these PR proteins.…”

Molecular Cloning and Characterization of a Novel β-1,3-Glucanase Gene from Rice

“…5,6) Based on amino acid similarities, tobacco b-1,3-glucanases have been classiˆed into three structural classes. 7) Monocotyledonous b-glucanases together with nine reported rice glucanases (gns) have been classiˆed into four subfamilies based on the structure and function of the genes, which are comprised of b-1,3-gns subfamily A, b-1,3;1,4-gns subfamily B, and b-gns subfamilies C and D with structures distinct from other subfamilies. 8) In this study, we ampliˆed a novel b-1,3-glucanase sequence from rice anther cDNA and characterized the structures and the expression patterns.…”