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Cloning and targeted disruption, via Agrobacterium tumefaciens-mediated transformation, of a trypsin protease gene from the vascular wilt fungus Verticillium dahliae

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Abstract

A gene encoding a trypsin protease was isolated from a tomato isolate of Verticillium dahliae. The gene, designated VTP1, contains two introns and is predicted to encode a protein of 256 amino acids. The gene is present in V. dahliae isolates from different host plants and in V. albo-atrum; weakly hybridizing sequences are present in V. tricorpus. VTP1 cDNA sequences were identified in a sequence tag analysis of genes expressed under growth conditions that promote microsclerotia development. Replacement of the gene, by Agrobacterium tumefaciens-mediated transformation (ATMT), with a mutant allele construct did not noticeably alter either pathogenicity or growth in culture. Searches of expressed sequence tag databases showed that, in addition to the VTP1 gene, V. dahliae contains two genes encoding subtilisin-like proteases similar to those produced by pathogenic Aspergillus spp. This is the first description of the application of ATMT to the molecular analysis of phytopathogenic Verticillium spp.

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Acknowledgements

We thank E. Antone for technical assistance, I. van Grinsven and S. Millar for sequence analysis, A. Molnar for preparation of Figs. 3A and 4, S. Evans for assistance with preparation of other figures, and M. Neumann for critical reading of the manuscript. This study was supported in part by funding from the Natural Sciences and Engineering Research Council of Canada.

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Authors and Affiliations

  1. Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 1391 Sandford St., London, Ontario, N5V 4T3, Canada

    Katherine F. Dobinson & Sandra J. Grant

  2. Department of Biology, The University of Western Ontario, London, Ontario, Canada

    Katherine F. Dobinson

  3. Department of Plant Pathology, The Pennsylvania State University, University Park, PA 16802, USA

    Seogchan Kang

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  1. Katherine F. Dobinson
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  2. Sandra J. Grant
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  3. Seogchan Kang
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Correspondence to Katherine F. Dobinson.

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Communicated by J. Heitman

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Dobinson, K.F., Grant, S.J. & Kang, S. Cloning and targeted disruption, via Agrobacterium tumefaciens-mediated transformation, of a trypsin protease gene from the vascular wilt fungus Verticillium dahliae . Curr Genet 45, 104–110 (2004). https://doi.org/10.1007/s00294-003-0464-6

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  • DOI: https://doi.org/10.1007/s00294-003-0464-6

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