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pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins

February 2015 , Volume 28 , Number  2
Pages  107 - 121

Xiaoyan Gong,1 Oscar Hurtado,1 Baohua Wang,1 Congqing Wu,1 Mihwa Yi,2 Martha Giraldo,2 Barbara Valent,2 Michael Goodin,1 and Mark Farman1

1Department of Plant Pathology, Plant Sciences Building, 1405 Veteran's Dr., University of Kentucky, Lexington, KY 40546, U.S.A.; 2Department of Plant Pathology, 4024 Throckmorton Plant Sciences Center, Kansas State University, Manhattan, 66506, U.S.A.

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Accepted 5 October 2014.

As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their “directly fused” counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

© 2015 The American Phytopathological Society