Journal of Phytopathology (2013) 161, 800-806

Chang-Yan Ge, Ya-Bing Duan, Ming-Guo Zhou and Chang-Jun Chen (2013)
A protoplast transformation system for gene deletion and complementation in Sclerotinia sclerotiorum
Journal of Phytopathology 161 (11-12), 800-806
Abstract: Protoplast transformation is an important technique for establishing a mutation library and determining gene function for Sclerotinia sclerotiorum and other plant pathogenic fungi. In this study, we determined the effect of various conditions on preparation of protoplasts for transformation. These conditions included the age of the culture providing the hyphae to be digested; enzyme composition, buffer solution and concentration; and digestion time and temperature. The optimum conditions for preparing protoplasts were as follows: 10 ml of enzyme solution (1.5% lysing enzyme in 0.8 m mannitol and citric acid-sodium citrate buffer) reacting with 0.1 g of hyphae (cultured for 36 h) at 30°C for 2.5 h. The transformation efficiency was 60-85 transformants per microgram of DNA. In addition, an expression vector for gene complementation was constructed, and an additional dominant selectable marker (neomycin) was demonstrated. To verify the reliability of the expression vector, we constructed and transformed the complementation vector of Shk1 for gene complementation based on the Shk1 deletion mutant Delta-Shk1. The results showed that the expression level and biological phenotypes of Shk1 were restored in the complementary strain Delta-Shk1+Shk1. The techniques and procedures described will improve our ability to study gene function in S. sclerotiorum and are likely applicable to other plant pathogens.
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Link to article at publishers website
Database assignments for author(s): Chang-Jun Chen, Ming-Guo Zhou, Yabing Duan

Research topic(s) for pests/diseases/weeds:
molecular biology - genes

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Pest and/or beneficial records:

Beneficial	Pest/Disease/Weed	Crop/Product	Country	Quarant.
	Sclerotinia sclerotiorum