Three Extracellular Proteases from Cochliobolus carbonum: Cloning and Targeted Disruption of ALP1. Jenifer M. Murphy. DOE-Plant Research Laboratory, Michigan State University, East Lansing, Ml 48824-1312 U.S.A. Jonathan D.Walton DOE-Plant Research Laboratory, Michigan State University, East Lansing, Ml 48824-1312 U.S.A. MPMI 9:290-297. Accepted 2 February 1996. Copyright 1996 The American Phytopathological Society.

Three extracellular serine proteases (Alpla, Alplb, Alp2) from Cochliobolus carbonum were purified and characterized. Of eight carbon/protein substrates tested, total protease activity was highest when the fungus was grown on medium containing collagen. Alpla and Alplb are members of the trypsin family (EC 3.4.21.4), and AIp2 is a member of the subtilisin family (EC 3.4.21.62). Alpla, Alplb, and Alp2 have monomer molecular masses of 25, 30, and 38 kDa respectively. Alplb is glycosylated, whereas Alpla is not. The gene encoding Alpla, ALP1, was isolated using PCR primers based on two amino acid sequences: One obtained directly from the N-terminus of Alpla and another that is highly conserved in other tryp-sins. The transcriptional start site was determined using RACE and the intron structure and polyadenylation site were determined from a cDNA clone. An internal fragment of ALP1 was used to create Alpla null mutants by transformation-mediated gene disruption. Total protease activity in the mutants was reduced by 35% to 45%. By chromatographic analysis, the mutants had lost two peaks of UV absorption and the two protease activities corresponding to Alpla and Alplb, which, together with the biochemical data, indicates that Alpla and Alplb are products of the same gene. The in vitro growth and disease phenotypes of the ALPI mutants were indistinguishable from the wild-type strain; therefore, ALPI is not by itself required for pathogenicity.