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 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-beta1,3-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 (beta1,3-beta1,4) glucanase with low activity against beta1,4-glucan and no activity against beta1,3-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 beta1,3-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 beta1,3-glucan or mixed-linked glucan. None of the mutants showed any decrease in virulence.
Kim
H
H
Department of Energy Plant Research Laboratory, Michigan State University, East Lansing 48824, USA.
Ahn
J H
JH
Görlach
J M
JM
Caprari
C
C
Scott-Craig
J S
JS
Walton
J D
JD
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D015966:Gene Expression Regulation, Fungal; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology; D018515:Plant Leaves / Q000382:microbiology; D010944:Plants / Q000382:microbiology*; D015183:Restriction Mapping; D014158:Transcription, Genetic; D003313:Zea mays / Q000382:microbiology*
D006026:Glycoside Hydrolases; C028020:licheninase