Journal
FUNGAL GENETICS AND BIOLOGY
Volume 49, Issue 6, Pages 483-497Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.fgb.2012.03.005
Keywords
Botrytis cinerea; Necrotroph; Virulence; Fluorescent protein; Bimolecular fluorescence complementation
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Funding
- German Federal Ministry of Education and Research (BMBF) [FKZ 0315455]
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Targeted gene inactivation is extensively used in the plant pathogenic fungus Botrytis cinerea for gene function analysis while strategies involving the expression of reporter genes have been rarely used due to the lack of appropriate expression vectors. Hence, an approach was initiated to establish an expression system for B. cinerea possessing the following features: (i) the targeted integration of constructs at defined gene loci which are dispensable under standard growth conditions, (ii) the use of promoter and terminator sequences allowing optimal gene expression, (iii) the use of codon-optimized reporter genes (Leroch et al., 2011), (iv) the use of multiple selection markers, and (v) the incorporation of a highly efficient cloning system. A set of basic vectors was generated by yeast recombinational cloning permitting a variety of protein fusions. The successful application of the expression system for labeling F-actin, the cytosol, the nuclei, the membrane, the ER and the peroxisomes was demonstrated. In addition, cloning vectors for bimolecular fluorescence complementation (BiFC) analyses for studying protein-protein interactions in situ were generated by splitting the codon-optimized gfp. The functionality of the constructed BiFC vectors was validated by demonstrating the interaction of the two white collar-like transcription factors BcWCL1 and BcWCL2 in the nuclei of growing B. cinerea hyphae. (C) 2012 Elsevier Inc. All rights reserved.
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