CLONING AND TARGETED DISRUPTION OF ENPG-1, ENCODING THE MAJOR IN-VITRO EXTRACELLULAR ENDOPOLYGALACTURONASE OF THE CHESTNUT BLIGHT FUNGUS, CRYPHONECTRIA-PARASITICA

The gene enpg-1, encoding the major extracellular endopolygalacturonas e (endoPG) purified from culture filtrates of the chestnut blight fung us, Cryphonectria parasitica, was cloned and characterized, The deduce d mature enpg-1 protein product, ENPG-1, had a calculated molecular ma ss of 34.5 kDa and a pi of 7.2, consistent with empirically derived va lues 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 phosphotrans ferase gene (hph) inserted into exon 1. enpg-1 disruption resulted in no reduction in canker formation on dormant American chestnut stems, U nexpectedly, the level of polygalacturonase (PG) activity measured in cankered bark tissue infected with enpg-1 disruptants was indistinguis hable from that found in canker tissue infected with virulent strain E P155. Isoelectric focusing and activity gel analysis of PG activity ex tracted from canker bark tissue revealed ENPG-1 to be a minor (less th an 5%) activity component in tissue infected with the virulent strain and to be absent in tissue infected with the disruption mutants, The p redominant activity in both canker samples consisted of two previously undetected acidic PG forms that appear absent in C. parasitica cultur e filtrates, We conclude from these results that the major C. parasiti ca extracellular endoPG produced in culture, ENPG-1, does not play a s ignificant role in fungal virulence, However, the identification of tw o acidic PG activities expressed predominantly, if not exclusively, in planta provides new opportunities for examining the importance of PGs in C. parasitica pathogenesis.