PFPL vectors for high-throughput protein localization in fungi: Detecting cytoplasmic accumulation of putative effector proteins

Xiaoyan Gong, Oscar Hurtado, Baohua Wang, Congqing Wu, Mihwa Yi, Martha Giraldo, Barbara Valent, Michael Goodin, Mark Farman

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)107-121
Number of pages15
JournalMolecular Plant-Microbe Interactions
Volume28
Issue number2
DOIs
StatePublished - Feb 1 2015

Bibliographical note

Publisher Copyright:
© 2015 The American Phytopathological Society.

ASJC Scopus subject areas

  • Physiology
  • Agronomy and Crop Science

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