Targeted mutants of Cochliobolus carbonum lacking the two major extracellular polygalacturonases

• Published in: Applied and Environmental Microbiology 1998-04, Vol.64 (4), p.1497-1503
• Main Authors: Scott-Craig, J.S. (Michigan State University, East Lansing, MI.), Cheng, Y.Q, Cervone, F, De Lorenzo, G, Pitkin, J.W, Walton, J.D
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; AMINO ACID SEQUENCES; Ascomycota - enzymology; Ascomycota - genetics; Ascomycota - growth & development; Base Sequence; Biological and medical sciences; CHEMICAL COMPOSITION; Cloning, Molecular; COCHLIOBOLUS CARBONUM; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; DNA, Fungal - genetics; Fundamental and applied biological sciences. Psychology; Fungal plant pathogens; Fungi; GENBANK/L48982; Gene Expression; Gene Targeting; Genes; Genes, Fungal; Glycoside Hydrolases - genetics; Glycoside Hydrolases - isolation & purification; Microbiology; MOLECULAR SEQUENCE DATA; Mutation; Mycology; NUCLEOTIDE SEQUENCE; Pectins - metabolism; Phenotype; Phytopathology. Animal pests. Plant and forest protection; POLIGALACTURONASA; POLYGALACTURONASE; Polygalacturonase - genetics; Polygalacturonase - isolation & purification; Proteins; Restriction Mapping; SECUENCIA NUCLEOTIDICA; SEQUENCE NUCLEOTIDIQUE; Variation, races, biotypes, parasitic specialization, genetics; Virulence - genetics; Zea mays - microbiology
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.64.4.1497-1503.1998

The filamentous fungus Cochliobolus carbonum produces endo-alpha 1,4-polygalacturonase (endoPG), exo-alpha 1,4-polygalacturonase (exoPG), and pectin methylesterase when grown in culture on pectin. Residual activity in a pgn1 mutant (lacking endoPG) was due to exoPG activity, and the responsible protein has now been purified. After chemical deglycosylation, the molecular mass of the purified protein decreased from greater than 60 to 45 kDa. The gene that encodes exoPG, PGX1, was isolated with PCR primers based on peptide sequences from the protein. The product of PGX1, Pgx1p, has a predicted molecular mass of 48 kDa, 12 potential N-glycosylation sites, and 61% amino acid identity to an exoPG from the saprophytic fungus Aspergillus tubingensis. Strains of C. carbonum mutated in PGX1 were constructed by targeted gene disruption and by gene replacement. Growth of pgx1 mutant strains on pectin was reduced by ca. 20%, and they were still pathogenic on maize. A double pgn1/pgx1 mutant strain was constructed by crossing. The double mutant grew as well as the pgx1 single mutant on pectin and was still pathogenic despite having less than 1% of total wild-type PG activity. Double mutants retained a small amount of PG activity with the same cation-exchange retention time as Pg1p and also pectin methylesterase and a PG activity associated with the mycelium. Continued growth of the pgn1/pgx1 mutant on pectin could be due to one or more of these residual activities