IMMUNOLOCALIZATION AND CHARACTERIZATION OF A BETA-1,3-GLUCANASE FROM SUGAR-BEET, DEDUCTION OF ITS PRIMARY STRUCTURE AND NUCLEOTIDE-SEQUENCEBY 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 su bcellular 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 prox imal to the necrosis. High levels of beta-1,3-glucanase 2 were observe d in the necrosis and in the vicinity of the necrotic lesions. Low lev els of the enzyme were found at distant sites from the necrosis. Two i soforms, beta-1,3-glucanase 2 and 4, with molecular masses of 33 and 2 9 kDa and pi values of approximately 9.4 and 9.5, respectively, were p urified to homogeneity on a laminarin-agarose column, followed by Mono S chromatography. Partial amino acid sequence data for beta-1,3-gluca nase 2 were used to design a cDNA probe. This probe was used to isolat e cDNA clones encoding beta-1,3-glucanase 2 by polymerase chain reacti on. Five cDNA clones were isolated. An apparently full length clone, d esignated Glu2(1), had an open reading frame of 1248 base pairs encodi ng a polypeptide of 336 amino acids, together with a presumed NH2-term inal 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 cl one, designated GenN1. This GenN1 clone contained the entire coding se quence for beta-1,3-glucanase 2, as well as the promotor and terminato r. Six clones (one genomic and five cDNA) contained a total of 15 nucl eotide 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 b eet glucanase gene were examined and compared with beta-1,3-glucanases reported from other plants. (C) 1998 Elsevier Science Ireland Ltd. Al l rights reserved.