CLONING AND TARGETED DISRUPTION OF MLG1, A GENE ENCODING 2 OF 3 EXTRACELLULAR MIXED-LINKED GLUCANASES OF COCHLIOBOLUS-CARBONUM

Mixed-linked glucanases (MLGases), which are extracellular enzymes abl e to hydrolyze beta 1,3-1,4-glucans (also known as mixed-linked glucan s or cereal beta-glucans), were identified in culture filtrates of the plant-pathogenic fungus Cochliobolus carbonum. Three peaks of MLGase activity, designated Mlg1a, Mlg1b, and Mlg2, were resolved by cation-e xchange and hydrophobic-interaction high-performance liquid chromatogr aphy (HPLC). Mlg1a and Mlg1b also hydrolyze beta 1,4-glucan (laminarin ), whereas Mlg2 does not degrade beta 1,3-glucan but does degrade beta 1,il-glucan to a slight extent. Mlg1a, Mlg1b, and Mlg2 have monomer m olecular masses of 33.5, 31, and 29.5 kDa, respectively, The N-termina l amino acid sequences of Mlg1a and Mlg1b are identical (AAYNLI). Mlg1 a is glycosylated, whereas Mlg1b is not. The gene encoding Mlg1b, MLG1 , was isolated by using PCR primers based on amino acid sequences of M lg1b. The product of MLG1 has no close similarity to any known protein but does contain a motif (EIDI) that occurs at the active site of MLG ases from several prokaryotes. An internal fragment of MLG1 was used t o create mlg1 mutants by transformation-mediated gene disruption. The total MLGase and beta 1,3-glucanase activities in culture filtrates of the mutants were reduced by approximately 50 and 40%, respectively. W hen analyzed by cation-exchange HPLC, the mutants were missing the tao peaks of MLGase activity corresponding to Mlg1a and Mlg1b. Together, the data indicate that Mlg1a and Mlg1b are products of the same gene, MLG1. The growth of mlg1 mutants in culture medium supplemented with m acerated maize cell walls or maize bran and the disease symptoms on ma ize were identical to the growth;th and disease symptoms of the wild t ype.