TY - JOUR
T1 - PFPL vectors for high-throughput protein localization in fungi
T2 - Detecting cytoplasmic accumulation of putative effector proteins
AU - Gong, Xiaoyan
AU - Hurtado, Oscar
AU - Wang, Baohua
AU - Wu, Congqing
AU - Yi, Mihwa
AU - Giraldo, Martha
AU - Valent, Barbara
AU - Goodin, Michael
AU - Farman, Mark
N1 - Publisher Copyright:
© 2015 The American Phytopathological Society.
PY - 2015/2/1
Y1 - 2015/2/1
N2 - 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.
AB - 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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U2 - 10.1094/MPMI-05-14-0144-TA
DO - 10.1094/MPMI-05-14-0144-TA
M3 - Article
C2 - 25390188
AN - SCOPUS:84937217468
SN - 0894-0282
VL - 28
SP - 107
EP - 121
JO - Molecular Plant-Microbe Interactions
JF - Molecular Plant-Microbe Interactions
IS - 2
ER -