Cloning and targeted disruption of enpg-1, encoding the major in vitro extracellular endopolygalacturonase of the chestnut blight fungus, Cryphonectria parasitica

Abstract

The gene enpg-1, encoding the major extracellular endopolygalacturonase (endoPG) purified from culture filtrates of the chestnut blight fungus, Cryphonectria parasitica, was cloned and characterized. The deduced mature enpg-1 protein product, ENPG-1, had a calculated molecular mass of 34.5 kDa and a pI of 7.2, consistent with empirically derived values for the purified enzyme, and had 66% identity with an endoPG from the maize pathogen Cochliobolus carbonum. Targeted disruption of enpg-1 was accomplished by homologous recombination with a cloned copy of the gene that contained the Escherichia coli hygromycin B phosphotransferase gene (hph) inserted into exon 1. enpg-1 disruption resulted in no reduction in canker formation on dormant American chestnut stems. Unexpectedly, the level of polygalacturonase (PG) activity measured in cankered bark tissue infected with enpg-1 disruptants was indistinguishable from that found in canker tissue infected with virulent strain EP155. Isoelectric focusing and activity gel analysis of PG activity extracted from canker bark tissue revealed ENPG-1 to be a minor (less than 5%) activity component in tissue infected with the virulent strain and to be absent in tissue infected with the disruption mutants. The predominant activity in both canker samples consisted of two previously undetected acidic PG forms that appear absent in C. parasitica culture filtrates. We conclude from these results that the major C. parasitica extracellular endoPG produced in culture, ENPG-1, does not play a significant role in fungal virulence. However, the identification of two acidic PG activities expressed predominantly, if not exclusively, in planta provides new opportunities for examining the importance of PGs in C. parasitica pathogenesis.