title: A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici.
abstract: Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20-30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue.
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: Schuster
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: D
authors.initials: D
authors.lastname: Studholme
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: D
authors.initials: D
authors.lastname: Soanes
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.
authors.forename: N J
authors.initials: NJ
authors.lastname: Talbot
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 / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D003062:Codon; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*; D014774:Virulence
chemical_list: D003062:Codon; D049452:Green Fluorescent Proteins
keywords: Green fluorescent protein; Mycosphaerella graminicola; Protein localization; Septoria tritici blotch; Wheat pathogenic fungi