1. Red fluorescent proteins for imaging Zymoseptoria tritici during invasion of wheat
- Author
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Gero Steinberg, Martin Schuster, M. Sommerauer, Congping Lin, Sreedhar Kilaru, and M. Guo
- Subjects
0106 biological sciences ,ROI, region of interest ,Gene Expression ,01 natural sciences ,Green fluorescent protein ,RFP, red fluorescent protein ,Genes, Reporter ,Gene expression ,n, sample size ,Triticum ,dpi, days post infection ,2. Zero hunger ,Recombination, Genetic ,0303 health sciences ,Ascomycota ,biology ,Virulence ,food and beverages ,Colocalization ,Protein subcellular localization prediction ,Fluorescence ,3. Good health ,Cell biology ,eGFP, enhanced green fluorescent protein ,Mycosphaerella graminicola ,Wheat pathogenic fungus ,mCherry, monomeric cherry ,mRFP, monomeric red fluorescent protein ,Genetic Vectors ,Microbiology ,Article ,03 medical and health sciences ,Transformation, Genetic ,Red fluorescent protein ,Genetics ,sdi1, succinate dehydrogenase 1 ,030304 developmental biology ,Plant Diseases ,Staining and Labeling ,Protein localization ,tub2, α tubulin ,biology.organism_classification ,Molecular biology ,Luminescent Proteins ,Microscopy, Fluorescence ,FPs, fluorescent proteins ,Septoria tritici blotch ,mCherry ,TagRFP, monomeric red (orange) fluorescent protein ,tdTomato, tandem dimeric red fluorescent protein ,010606 plant biology & botany - Abstract
Highlights • We investigate brightness and photo-stability of RFPs in live Z. tritici cells. • mCherry is most useful in epi-fluorescence and confocal laser scanning microscopy. • The combination of mCherry and an orange-shifted filter set proves to provide brightest signals. • We provide 4 vectors with various mRFPs for yeast recombination based cloning. • The vectors carry carboxin resistance and integrate as single copies into the sdi1 locus., 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.
- Published
- 2015