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Molecular Characterization of a Subtilase from the Vascular Wilt Fungus Fusarium oxysporum

May 2001 , Volume 14 , Number  5
Pages  653 - 662

Antonio Di Pietro , 1 M. Dolores Huertas-González , 1 J. Felix Gutierrez-Corona , 2 Guadalupe Martínez-Cadena , 2 Emese Méglecz , 1 and M. Isabel G. Roncero 1

1Departamento de Genética, Facultad de Ciencias, Universidad de Córdoba, 14071 Córdoba, Spain; 2Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Mexico

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Accepted 23 January 2001.

The gene prt1 was isolated from the tomato vascular wilt fungus Fusarium oxysporum f. sp. lycopersici, whose predicted amino acid sequence shows significant homology with subtilisin-like fungal proteinases. Prt1 is a single-copy gene, and its structure is highly conserved among different formae speciales of F. oxysporum. Prt1 is expressed constitutively at low levels during growth on different carbon and nitrogen sources and strongly induced in medium containing collagen and glucose. As shown by reverse transcription-polymerase chain reaction and fluorescence microscopy of F. oxysporum strains carrying a prt1-promoter-green fluorescent protein fusion, prt1 is expressed at low levels during the entire cycle of infection on tomato plants. F. oxysporum strains transformed with an expression vector containing the prt1 coding region fused to the inducible endopolygalacturonase pg1 gene promoter and grown under promoter-inducing conditions secreted high levels of extracellular 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 inactivation of prt1 in F. oxysporum f. sp. lycopersici had no detectable effect on mycelial growth, sporulation, and pathogenicity on tomato plants.

© 2001 The American Phytopathological Society