Journal
MOLECULAR PLANT-MICROBE INTERACTIONS
Volume 28, Issue 2, Pages 107-121Publisher
AMER PHYTOPATHOLOGICAL SOC
DOI: 10.1094/MPMI-05-14-0144-TA
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Funding
- United States Department of Agriculture National Institute of Food and Agriculture [2008-35600-18809]
- Direct For Biological Sciences
- Division Of Integrative Organismal Systems [0749519] Funding Source: National Science Foundation
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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.
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