ARS | Publication request: Transformation of Sclerotinia Sclerotiorum with the Green Fluorescent Protein Gene and Fluorescence of Hyphae in Four Inoculated Hosts

Submitted to: Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 4, 2008
Publication Date: March 8, 2009
Repository URL: http://hdl.handle.net/10113/35872
Citation: deSilva, A.P., Bolton, M.D., Nelson, B.D. 2009. Transformation of Sclerotinia Sclerotiorum with the Green Fluorescent Protein Gene and Fluorescence of Hyphae in Four Inoculated Hosts. Plant Pathology. 48:487-496.

Interpretive Summary: Sclerotinia sclerotiorum is an important pathogen of a wide variety of crops. To gain a better understanding of the interaction between S. sclerotiorum and its hosts, a gene encoding the green fluorescent protein (GFP) was transformed into isolates ND30 and ND21. Seven stable transformants were identified and evaluated for fluorescence in culture and during colonization, and were evaluated for colonization of host tissues. Each transformant was shown to have a single copy of the GFP gene. Fluorescence was quantified directly from mycelium and protein extracted from hyphae. The seven transformants were pathogenic on dry bean, canola, soybean, and sunflower. However, depending on the host, three transformants had variation in the length of lesions formed compared to the non-transformed isolate. 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 in culture and during colonization.

Technical Abstract: Sclerotinia sclerotiorum is an important pathogen of a wide variety of crops. To obtain a genetic marker to observe and study the interaction of the pathogen 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 (4 from ND30 and 3 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.