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

Abstract

A basic β-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 β-1,3-glucanase 2 in Cercospora infected sugar beets was studied by immunohistological analysis. β-1,3-Glucanase 2 was primarily deposited in extracellular globuli proximal to the necrosis. High levels of β-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, β-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 β-1,3-glucanase 2 were used to design a cDNA probe. This probe was used to isolate cDNA clones encoding β-1,3-glucanase 2 by polymerase chain reaction. Five cDNA clones were isolated. An apparently full length clone, designated Glu2 1 The EMBL accession no. of Glu2 is X75946. 1 , had an open reading frame of 1248 base pairs encoding a polypeptide of 336 amino acids, together with a presumed NH 2-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 β-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 β-1,3-glucanases reported from other plants.