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

• Published in: Plant pathology Vol. 58; no. 3; pp. 487 - 496
• Main Authors: de Silva, A.P, Bolton, M.D, Nelson, B.D
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.06.2009; Blackwell Publishing Ltd; Blackwell
• Subjects: bean; Biological and medical sciences; biomarkers; Brassica napus var. napus; canola; disease detection; dry beans; Fundamental and applied biological sciences. Psychology; Fungal plant pathogens; fungal wilt; genetic transformation; Glycine max; green fluorescent protein; Helianthus; Helianthus annuus; host-pathogen relationships; hyphae; microbial colonization; pathogenicity; Phytopathology. Animal pests. Plant and forest protection; plant pathogenic fungi; Sclerotinia sclerotiorum; sclerotinia wilt; soybean; soybeans; sunflower; transgenes; white mould; Plant pathology; Transformation; Plant pathogen; Fluorescence; Soybean; Compositae; Fungi; Inoculation; Gene; Cruciferae; Dicotyledones; Angiospermae; C3-Type; bean; sclerotinia wilt; Ascomycota; Vegetals; Biochemical marker; Sclerotinia sclerotiorum; Host; Glycine max; canola; sunflower; Grain legume; Brassica; Helianthus annuus; Leguminosae; Hypha; Green fluorescent protein; white mould; Spermatophyta; Oil plant (vegetal)
• DOI: 10.1111/j.1365-3059.2009.02022.x
• ISSN: 0032-0862

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 = 0·05) 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.