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Applied and Environmental Microbiology, July 2002, p. 3408-3415, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3408-3415.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Improving the Pathogenicity of a Nematode-Trapping Fungus by Genetic Engineering of a Subtilisin with Nematotoxic Activity

Johan Åhman,^1, Tomas Johansson,¹ Maja Olsson,¹ Peter J. Punt,² Cees A. M. J. J. van den Hondel,² and Anders Tunlid^1*

Department of Microbial Ecology, Lund University, S-223 62 Lund, Sweden,¹ Department of Applied Microbiology and Gene Technology, TNO Voeding, 3700 AJ Zeist, The Netherlands²

Received 26 December 2001/ Accepted 10 April 2002

Nematophagous fungi are soil-living fungi that are used as biological control agents of plant and animal parasitic nematodes. Their potential could be improved by genetic engineering, but the lack of information about the molecular background of the infection has precluded this development. In this paper we report that a subtilisin-like extracellular serine protease designated PII is an important pathogenicity factor in the common nematode-trapping fungus Arthrobotrys oligospora. The transcript of PII was not detected during the early stages of infection (adhesion and penetration), but high levels were expressed concurrent with the killing and colonization of the nematode. Disruption of the PII gene by homologous recombination had a limited effect on the pathogenicity of the fungus. However, mutants containing additional copies of the PII gene developed a higher number of infection structures and had an increased speed of capturing and killing nematodes compared to the wild type. The paralyzing activity of PII was verified by demonstrating that a heterologous-produced PII (in Aspergillus niger) had a nematotoxic activity when added to free-living nematodes. The toxic activity of PII was significantly higher than that of other commercially available serine proteases. This is the first report showing that genetic engineering can be used to improve the pathogenicity of a nematode-trapping fungus. In the future it should be possible to express recombinant subtilisins with nematicidal activity in other organisms that are present in the habitat of parasitic nematodes (e.g., host plant).

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* Corresponding author. Mailing address: Department of Microbial Ecology, Lund University, Ecology Building, S-223 62 Lund, Sweden. Phone: 46 46 2223757. Fax: 46 46 2224158. E-mail: Anders.Tunlid{at}mbioekol.lu.se.

Present address: Department of Ecology and Molecular Biology, The Royal Veterinary and Agricultural University, 1871 Frederiksberg C, Denmark.

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Applied and Environmental Microbiology, July 2002, p. 3408-3415, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3408-3415.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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