Immunolocalization and characterization of a β-1,3-glucanase from sugar beet and deduction of its primary structure and nucleotide sequence by cDNA and genomic cloning

A basic beta-1,3-glucanase was found to accumulate in sugar beet (Beta vulgaris) leaves infected with the fungus Cercospora beticola. The subcellular distribution of the basic beta-1,3-glucanase 2 in Cercospora infected sugar beets was studied by immunohistological analysis. beta-1,3-Glucanase 2 was primarily deposited in extracellular globuli proximal to the necrosis. High levels of beta-1,3-glucanase 2 were observed in the necrosis and in the vicinity of the necrotic lesions. Low levels of the enzyme were found at distant sites from the necrosis. Two isoforms, beta-1,3-glucanase 2 and 4, with molecular masses of 33 and 29 kDa and pi values of approximately 9.4 and 9.5, respectively, were purified to homogeneity on a laminarin-agarose column, followed by Mono S chromatography. Partial amino acid sequence data for beta-1,3-glucanase 2 were used to design a cDNA probe. This probe was used to isolate cDNA clones encoding beta-1,3-glucanase 2 by polymerase chain reaction. Five cDNA clones were isolated. An apparently full length clone, designated Glu2(1), had an open reading frame of 1248 base pairs encoding a polypeptide of 336 amino acids, together with a presumed NH2-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 33 252 Da and a predicted pi of 9.74. Glu2 was used to isolate an approximately 5 kb genomic clone, designated GenN1. This GenN1 clone contained the entire coding sequence for beta-1,3-glucanase 2, as well as the promotor and terminator. Six clones (one genomic and five cDNA) contained a total of 15 nucleotide substitutions in the coding region, only four of which resulted in changes in amino acids, and those that did were of a conservative nature. The nucleotide and derived amino acid sequences of the sugar beet glucanase gene were examined and compared with beta-1,3-glucanases reported from other plants. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.