Open AccessMutational Analysis of β-Glucanase Genes from the Plant-Pathogenic Fungus Cochliobolus carbonum
Author(s) -
Hoon Kim,
JoongHoon Ahn,
Jenifer M. Görlach,
Claudio Caprari,
John S. Scott-Craig,
Jonathan D. Walton
Publication year - 2001
Publication title -
molecular plant-microbe interactions
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.565
H-Index - 153
eISSN - 1943-7706
pISSN - 0894-0282
DOI - 10.1094/mpmi.2001.14.12.1436
Subject(s) - glucanase , mutant , gene , biology , fungus , glucan , genetics , microbiology and biotechnology , biochemistry , botany
Two new β-glucanase-encoding genes, EXG2 and MLG2, were isolated from the plant-pathogenic fungus Cochliobolus carbonum using polymerase chain reaction based on amino acid sequences from the purified proteins. EXG2 encodes a 46.6-kDa exo-β1,3-glucanase and is located on the same 3.5-Mb chromosome that contains the genes of HC-toxin biosynthesis. MLG2 encodes a 26.8-kDa mixed-linked (β1,3-β1,4) glucanase with low activity against β1,4-glucan and no activity against β1,3-glucan. Specific mutants of EXG2 and MLG2 were constructed by targeted gene replacement. Strains with multiple mutations (genotypes exg1/mlg1, exg2/mlg1, mlg1/mlg2, and exg1/exg2/mlg1/mlg2) were also constructed by sequential disruption and by crossing. Total mixed-linked glucanase activity in culture filtrates of mlg1/mlg2 and exg1/exg2/mlg1/mlg2 mutants was reduced by approximately 73%. Total β1,3-glucanase activity was reduced by 10, 54, and 96% in exg2, mlg1, and exg1/exg2/mlg1/mlg2 mutants, respectively. The quadruple mutant showed only a modest decrease in growth on β1,3-glucan or mixed-linked glucan. None of the mutants showed any decrease in virulence.