Submitted to: Molecular Plant Microbe International Symposium
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2002
Publication Date: 8/1/2002
Citation: Bailey, B.A., Apel-Birkhold, P.C., Luster, D.G. 2002. Expression of nep1 by fusarium oxysporum f. sp. erythoxyli after gene replacement and overexpression using peg-mediated transformation. Molecular Plant Microbe International Symposium. 92:283-841. Interpretive Summary: Invasive weeds are a major problem for land managers including farmers throughout the United States and the world. Innovative control measures such as the development of bioherbicides that take advantage of the weed's natural pests and pathogens offer environmentally friendly alternatives to traditional chemical control measures. The natural products produced by pathogens of weeds are also being considered as natural solutions to weed control problems. We have isolated a protein, Nep1, from the plant pathogen Fusarium oxysporum that causes necrosis in many dicot weed species including invasive weeds and has potential as a natural herbicide when applied to weeds as a foliar spray. We were able to increase production of this extracellular protein 64 to 128 times by overexpressing the gene for Nep1 in F. oxysporum using PEG mediated transformation techniques. Overexpression of the Nep1 gene did not influence pathogenicity of Fusarium oxysporum to Erythroxylem coca. The increased production of Nep1 by overexpressing isolate of F. oxysporum should facilitate scaled up production of Nep1 allowing more extensive study of its potential as a natural herbicide. The development of alternative weed control strategies will lessen the impact of invasive weeds and their control strategies on the environment improving the lives of people around the world on the farm and in the home.
Technical Abstract: The necrosis inducing extracellular protein Nep1 is produced by Fusarium oxysporum f. sp. erythroxyli in liquid culture. NEP1, the Nep1 protein structural gene, was disrupted in F. oxysporum f. sp. erythroxyli isolate EN-4 by gene replacement. NEP1-disruption was verified by PCR, Southern blot and Northern blot analysis. NEP1-disrupted transformants failed to produce Nep1 in liquid culture. NEP1-disruption did not affect the pathogenicity of isolate EN-4 towards Erythroxylum coca. Transformation of isolate EN-4 with construct pPB-FO11-45 carrying NEP1 between the trpC promoter and terminator resulted in increased production of Nep1 shale culture. Transformation of EN-4 with construct pPB-FO11-45 was verified by PCR and Southern blot analysis. Overexpression of NEP1 was confirmed by Northern blot and Tricine-SDS -PAGE analysis. NEP1-overexpressing transformant 15 produced 64 to 128 times as much Nep1 as EN-4 wildtype when grown in shake cultures. Transformants overexpressing Nep1 in liquid culture were no more or less pathogenic towards E. coca than wildtype isolates. In large-scale fermentations of NEP1-overexpressing transformant 15, the amount of secreted protein including Nep1 was 15.1 times that of the wild-type EN-4.