|Schenk, Susan - HARC|
|Aoki, Ayumi - HARC|
Submitted to: International Society of Sugar Cane Technologists Pathology Workshop
Publication Type: Abstract Only
Publication Acceptance Date: December 20, 2005
Publication Date: January 20, 2006
Citation: Schenk, S., Albert, H.H., Aoki, A. 2006. Transformation of Ustilago scitaminea with a gfp gene. VIIIth International Society of Sugar Cane Technologists Pathology Workshop, Programme, and Abstracts, p. 47. Technical Abstract: Improved Detection of Ustilago scitaminea in Sugarcane. Breeding sugarcane for resistance to smut, Ustilago scitaminea, would be more cost effective if susceptible varieties could be pinpointed and eliminated sooner, before the appearance of whips. This can be done by sectioning meristematic tissues, staining, and observing sections microscopically, however that is too time consuming and costly for the Hawaii breeding program to carry out on a routine basis. One of the goals of our research has always been to develop a fast, easy method for detection of the fungus in sugarcane plants. A new technique is being developed that will enable us to screen many stalks at an early age. Other pathogenic fungi including Fusarium graminearum (Scadsen and Hohn, 2004) and Ustilago maydis (Spellig, et al. 1996) have been transformed with a GFP reporter gene to facilitate microscopic observation of plant infection experiments. We now report transformation of U. scitaminea with a constitutive GFP expression construct, resulting in brightly fluorescent sporidial colonies and dikaryon mycelia. Cultures of plus and minus haploid sporidia were obtained from dilution plates of suspensions of germinating teliospores. Plus and minus sporidial cultures were from both the Maui and the Oahu pathovars of Hawaiian smut. The cultures were stored on V8 juice agar and grown in YEPS liquid media for the transformation procedure. Spheroplasts were produced following the method of Wang, et al. (1988). Haploid sporidia were treated with Lytic Enzymes from Trichoderma sp. (Sigma L1412) yielding spheroplasts that were transformed by polyethylene glycol mediated transfection with linearized plasmid DNA. The expression vector was pOTEF-SG that contains the GFP gene under the control of the artificial OTEF promoter, obtained as a generous gift from Dr. Jan Schirawski, University of Marburg, Germany. The vector also contains an hptI cassette conferring resistance to Hygromycin. Selection was on solid YEPS plates with Hygromycin B at 200 'g/ml. The growth medium YEPS supported vigorous growth with minimal levels of solids that interfered with spectrophotometric determination of culture density. We have now obtained several U. scitaminea colonies producing a strong green fluorescence when viewed with UV light. Molecular characterization/confirmation is currently proceeding. Mixtures of transformed plus and minus sporidial colonies produced dikaryon mycelia that were also fluorescent. A paste of mixtures of transformed plus and minus sporidia were applied to the eyes of cut stalk pieces of the smut susceptible Hawaiian sugarcane variety, 50-7209. These were allowed to germinate and were planted in pots.