Mutational analysis of beta-glucanase genes from the plant-pathogenic fungus Cochliobolus carbonum

Two new beta -glucanase-encoding genes, EXG2 and MLG2, were isolated from t he plant-pathogenic fungus Cochliobolus carbonum using polymerase chain rea ction based on amino acid sequences from the purified proteins. EXG2 encode s a 46.6-kDa exo-beta1,3-glucanase and is located on the same 3.5-Mb chromo some that contains the genes of HC-toxin biosynthesis. MLG2 encodes a 26.8- kDa mixed linked (beta1,3-beta1,4) glucanase with low activity against beta 1,4-glucan and no activity against beta1,3-glucan. Specific mutants of EXG2 and MLG2 were constructed by targeted gene replacement. Strains with multi ple mutations (genotypes exg1/mlg1, exg2/mlg1, mlg1/mlg2, and exg1/exg2/mlg 1/mlg2) were also constructed by sequential disruption and by crossing. Tot al mixed-linked glucanase activity in culture filtrates of mlg1/mlg2 and ex g1/exg2/mlg1/mlg2 mutants was reduced by approximately 73%. Total beta1,3-g lucanase activity was reduced by 10, 54, and 96% in exg2, mlg1, and exg1/ex g2/mlg1/mlg2 mutants, respectively. The quadruple mutant showed only a mode st decrease in growth on beta1,3-glucan or mixed-linked glucan. None of the mutants showed any decrease in virulence.