pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins.
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.
Xiaoyan
X
Gong
Oscar
O
Hurtado
Baohua
B
Wang
Congqing
C
Wu
Mihwa
M
Yi
Martha
M
Giraldo
Barbara
B
Valent
Michael
M
Goodin
Mark
M
Farman
D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000502:physiology; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D020082:Magnaporthe / Q000166:cytology / Q000378:metabolism*; D012275:Oryza / Q000166:cytology / Q000378:metabolism / Q000382:microbiology*; D059828:Plant Cells; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport / Q000502:physiology*; D014170:Transformation, Genetic
D005656:Fungal Proteins