title: Red fluorescent proteins for imaging Zymoseptoria tritici during invasion of wheat.
abstract: The use of fluorescent proteins (FPs) in plant pathogenic fungi provides valuable insight into their intracellular dynamics, cell organization and invasion mechanisms. Compared with green-fluorescent proteins, their red-fluorescent "cousins" show generally lower fluorescent signal intensity and increased photo-bleaching. However, the combined usage of red and green fluorescent proteins allows powerful insight in co-localization studies. Efficient signal detection requires a bright red-fluorescent protein (RFP), combined with a suitable corresponding filter set. We provide a set of four vectors, suitable for yeast recombination-based cloning that carries mRFP, TagRFP, mCherry and tdTomato. These vectors confer carboxin resistance after targeted single-copy integration into the sdi1 locus of Zymoseptoria tritici. Expression of the RFPs does not affect virulence of this wheat pathogen. We tested all four RFPs in combination with four epi-fluorescence filter sets and in confocal laser scanning microscopy, both in and ex planta. Our data reveal that mCherry is the RFP of choice for investigation in Z. tritici, showing highest signal intensity in epi-fluorescence, when used with a Cy3 filter set, and laser scanning confocal microscopy. However, mCherry bleached significantly faster than mRFP, which favors this red tag in long-term observation experiments. Finally, we used dual-color imaging of eGFP and mCherry expressing wild-type strains in planta and show that pycnidia are formed by single strains. This demonstrates the strength of this method in tracking the course of Z. tritici infection in wheat.
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: M
authors.initials: M
authors.lastname: Schuster
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: S
authors.initials: S
authors.lastname: Kilaru
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: M
authors.initials: M
authors.lastname: Guo
authors.affiliation: AHF Analysentechnik AG, Kohlplattenweg 18, DE-72074 Tübingen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Sommerauer
authors.affiliation: Mathematics, University of Exeter, Exeter EX4 3QF, UK.
authors.forename: C
authors.initials: C
authors.lastname: Lin
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK. Electronic address: G.Steinberg@exeter.ac.uk.
authors.forename: G
authors.initials: G
authors.lastname: Steinberg
mesh_terms: D001203:Ascomycota / Q000254:growth & development* / Q000472:pathogenicity; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics*; D008856:Microscopy, Fluorescence / Q000379:methods; D010935:Plant Diseases / Q000382:microbiology*; D011995:Recombination, Genetic; D013194:Staining and Labeling / Q000379:methods*; D014170:Transformation, Genetic; D014908:Triticum / Q000382:microbiology*; D014774:Virulence; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: Colocalization; Mycosphaerella graminicola; Protein localization; Red fluorescent protein; Septoria tritici blotch; Wheat pathogenic fungus