Molecular characterization of a subtilase from the vascular wilt fungus Fusarium oxysporum

The gene prt1 was isolated from the tomato vascular wilt fungus Fusarium ox ysporum f. sp. lycopersici, whose predicted amino acid sequence shows signi ficant homology with subtilisin-like fungal proteinases, Prt1 is a single-c opy gene, and its structure is highly conserved among different formae spec iales of F. oxysporum. Prt1 is expressed constitutively at low levels durin g growth on different carbon and nitrogen sources and strongly induced in m edium containing collagen and glucose. As shown by reverse transcription-po lymerase chain reaction and fluorescence microscopy of E oxysporum strains carrying a propromoter-green fluorescent protein fusion, prt1 is expressed at low levels during the entire cycle of infection on tomato plants. F, oxy sporum strains transformed with an expression vector containing the prt1 co ding region fused to the inducible endopolygalacturonase pg1 gene promoter and grown under promoter-inducing conditions secreted high levels of extrac ellular subtilase activity that resolved into a single peak of pi 4.0 upon isoelectric focusing, The active fraction produced two clearing bands of 29 and 32 kDa in sodium dodecyl sulfate gels containing gelatin. Targeted ina ctivation of prt1 in E oxysporum f,sp. lycopersici had no detectable effect on mycelial growth, sporulation, and pathogenicity on tomato plants.