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Transformation of Sclerotinia sclerotiorum with the green fluorescent protein gene and fluorescence of hyphae in four inoculated hosts

Permanent URL:
http://handle.nal.usda.gov/10113/35872
Abstract:
To obtain a genetic marker to observe and study the interaction of Sclerotinia sclerotiorum with its hosts, isolates ND30 and ND21 were transformed using pCT74 and gGFP constructs, both containing genes for the green fluorescent protein (GFP) and hygromycin B phosphotransferase. Putative transformants were obtained using polyethylene glycol-mediated transformation of protoplasts. Seven stable gfp transformants were identified and evaluated for fluorescence in vitro and in planta, pathogenicity and colonization of host tissues. Real-time quantitative polymerase chain reaction detected a single copy of the gfp gene in transformants. Fluorescence was quantified directly from mycelium and protein extracted from hyphae. The seven transformants (four from ND30 and three from ND21) were pathogenic on dry bean, canola, soybean and sunflower. However, depending on the host, three transformants differed significantly (P = 0ยท05) in the length of lesions formed compared to the wild-type. Hyphae fluoresced in plant tissue and could clearly be distinguished from plant cells. Infection and colonization of tissues were clearly visible with a fluorescent microscope. Transformants differed in the intensity of GFP expression both in vitro and in planta.
Author(s):
de Silva, A.P. , Bolton, M.D. , Nelson, B.D.
Subject(s):
fungal wilt , Sclerotinia sclerotiorum , plant pathogenic fungi , hyphae , pathogenicity , microbial colonization , green fluorescent protein , transgenes , genetic transformation , biomarkers , host-pathogen relationships , dry beans , canola , Brassica napus var. napus , soybeans , Glycine max , Helianthus annuus , disease detection
Format:
p. 487-496.
Note:
Includes references
Source:
Plant pathology 2009 June, v. 58, no. 3
Language:
English
Publisher:
Oxford, UK : Blackwell Publishing Ltd
Year:
2009
Collection:
Journal Articles, USDA Authors, Peer-Reviewed
File:
Download [PDF File]
Rights:
Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.