Targeted mutants of Cochliobolus carbonum lacking the two major extracellular polygalacturonases.

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 Pgn1p 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.

Histone Hyperacetylation in Maize in Response to Treatment with HC-Toxin or Infection by the Filamentous Fungus Cochliobolus carbonum.

HC-toxin, the host-selective toxin produced by the filamentous fungus Cochliobolus carbonum, inhibits maize (Zea mays L.) histone deacetylases (HDs) in vitro. Here we show that HDs are also inhibited by HC-toxin in vivo, as demonstrated by the accumulation of hyperacetylated forms of the core (nucleosomal) histones H3.1, H3.2, H3.3, and H4 in both maize embryos and tissue cultures. Hyperacetylation of H4 and all isoforms of H3 in tissue cultures of inbred Pr (genotype hm/hm) occurred at 10 ng/mL (23 nM). The effect was host-selective; acetylation of histones in the near isogenic inbred Pr1 (genotype Hm/Hm) did not occur in tissue cultures or embryos treated with 0.2 [mu]g/mL or 10 [mu]g/mL HC-toxin, respectively. Hyperacetylation of histone H4 in embryos of Pr1 began to occur at 50 [mu]g/mL. HC-toxin, and 200 [mu]g/mL HC-toxin caused equal hyperacetylation in Pr and Pr1 embryos. Hyperacetylated core histones, especially of the isoforms of histone H3, accumulated in leaves of inbred Pr, but not Pr1, after infection by toxin-producing strains of C. carbonum. Accumulation of hyperacetylated histones began at 24 h after inoculation, before the development of visible disease symptoms. Hyperacetylation of H2A or H2B histones were not detected in any of the studies. The results are consistent with HD being a primary site of action of HC-toxin.