3 EXTRACELLULAR PROTEASES FROM COCHLIOBOLUS-CARBONUM - CLONING AND TARGETED DISRUPTION OF ALP1

Three extracellular serine proteases (Alp1a, Alp1b, Alp2) from Cochlio bolus carbonum were purified and characterized, Of eight carbon/protei n substrates tested, total protease activity was highest when the fung us was grown on medium containing collagen, Alp1a and Alp1b are member s of the trypsin family (EC 3.4.21.4), and Alp2 is a member of the sub tilisin family (EC 3.4.21.62). Alp1a, Alp1b, and Alp2 have monomer mol ecular masses of 25, 30, and 38 kDa respectively. Alp1b is glycosylate d, whereas Alp1a is not, The gene encoding Alp1a, ALP1, was isolated u sing PCR primers based on two amino acid sequences: One obtained direc tly from the N-terminus of Alp1a and another that is highly conserved in other trypsins. The transcriptional start site was determined using RACE and the intron structure and polyadenylation site were determine d from a cDNA clone. An internal fragment of ALP1 was used to create A lp1a null mutants by transformation-mediated gene disruption. Total pr otease activity in the mutants was reduced by 35% to 45%. By chromatog raphic analysis, the mutants had lost two peaks of UV absorption and t he two protease activities corresponding to Alp1a and Alp1b, which, to gether with the biochemical data, indicates that Alp1a and Alp1b are p roducts of the same gene. The in vitro growth and disease phenotypes o f the ALP1 mutants were indistinguishable from the wild-type strain; t herefore, ALP1 is not by itself required for pathogenicity.