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. 2015 Jun;79:132-40.
doi: 10.1016/j.fgb.2015.03.025.

Red Fluorescent Proteins for Imaging Zymoseptoria Tritici During Invasion of Wheat

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Free PMC article

Red Fluorescent Proteins for Imaging Zymoseptoria Tritici During Invasion of Wheat

M Schuster et al. Fungal Genet Biol. .
Free PMC article

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.

Keywords: Colocalization; Mycosphaerella graminicola; Protein localization; Red fluorescent protein; Septoria tritici blotch; Wheat pathogenic fungus.

Figures

Fig. 1
Fig. 1
Vectors for integration of various red-fluorescent proteins into the genome of Z. tritici. (A) Cloning vectors for controlled integration of various RFPs into the sdi1 locus of Z. tritici. TagRFP: generated from the wild-type RFP from sea anemone E. quadricolor (Merzlyak et al., 2007); mRFP: a derivative of the red fluorescent protein from Discosoma corals (Campbell et al., 2002); tdTomato and mCherry: mutated versions of the red fluorescent protein from Discosoma corals (Shaner et al., 2004). After integration into the sdi1 locus, the vector confers carboxin resistance due to a point mutation in the succinate dehydrogenase gene sdi1, which changes a histidine to a leucine (H267L). For more details of this integration into the “carboxin locus” (Kilaru et al., 2015a). Left and right border enable Agrobacterium tumefaciens-based transformation of Z. tritici. Note that fragments are not drawn to scale. For more accurate information on fragment sizes see main text. (B) Image illustrates the integration of any vector shown (A) into the native sdi1 locus of Z. tritici. This co-integrates a carboxin-resistant sdi1H267L allele and cytoplasmic RFPs, expressed under the control of α-tubulin promoter (tub2). (C) Southern blot, showing integration of vectors into the sdi1 locus. After digestion of the genomic DNA with BglII and subsequent hybridisation with a labelled DNA probe, a shift in the DNA fragments from 2.3 kb to 5.3 kb and 6.0 kb is detected. Size standards are given at the left. (D) Transmission spectra of the emission and excitation filters of various filter sets, tested in this study. For details see main text. (E) Cloning vector for ectopic integration of enhanced GFP for cytoplasmic expression in Z. tritici. The vector is compatible with yeast recombination-based cloning, expresses cytoplasmic eGFP and confers resistance to hygromycin. Note that the vector pHeGFP was derived from carboxin resistance conferring vector pCeGFP (Kilaru et al., 2015a, 2015c). As such they contain part of the succinate dehydrogenase gene, carrying the mutation H267L and succinate dehydrogenase terminator. However, these fragments are of no significance.
Fig. 2
Fig. 2
Signal intensity and bleaching behavior of red-fluorescent proteins in epi-fluorescence microscopy. (A) Images showing cytoplasmic expression of TagRFP, mRFP, tdTomato and mCherry. Note that Z. tritici shows virtually no auto-fluorescence (see B). All images were acquired and processed identically. Bar represents 10 μm. (B) Bar chart showing intensity of cytoplasmic fluorescence of various RFPs in 4 different filter sets (see Fig. 1E for fluorescent spectra). Autofluor.: background fluorescence without expressing a RFP; TagRFP: a mutant protein, generated from the wild-type RFP from sea anemone E. quadricolor; mRFP: a derivative of the red fluorescent protein from Discosoma corals; tdTomato and mCherry: mutated versions of the red fluorescent protein from Discosoma corals. Mean ± standard error of the mean is shown, sample size n (=number of cells) is indicated. Single asterisk indicates significant difference at P = 0.0282, triple asterisk at P < 0.0001, Student t-test. (C) Graph showing decay of fluorescent signals due to photo-bleaching in 4 fluorescent filter sets. TagRFP: a mutant protein, generated from the wild-type RFP from sea anemone E. quadricolor; mRFP: a derivative of the red fluorescent protein from Discosoma corals; tdTomato and mCherry: mutated versions of the red fluorescent protein from Discosoma corals. Each data point is given as mean ± standard error of the mean, sample size n (=number of cells) is indicated. Note that mRFP is most stable whereas mCherry shows the brightest signal.
Fig. 3
Fig. 3
Signal intensity and bleaching behavior of RFP proteins in confocal laser-scanning microscopy. (A) Bar charts showing intensity of cytoplasmic fluorescence of various RFPs, observed with a confocal laser scanning microscope in liquid culture and in infected wheat tissue (in planta). Autofluor. = background fluorescence without expressing a RFP; TagRFP = a mutant protein, generated from the wild-type RFP from sea anemone E. quadricolor; mRFP = a derivative of the red fluorescent protein from Discosoma corals; tdTomato and mCherry = mutated versions of the red fluorescent protein from Discosoma corals. Mean ± standard error of the mean is shown, sample size n (=number of cells) is indicated. Double asterisk indicates significant difference at P = 0.0064, triple asterisk indicates significant difference at P < 0.0001, Student t-test. (B) Graph showing decay of fluorescent signals due to photo-bleaching in confocal laser scanning microscopy, both in infected wheat tissue (in planta) and in liquid culture (Liquid culture). TagRFP: a mutant protein, generated from the wild-type RFP from sea anemone E. quadricolor; mRFP: a derivative of the red fluorescent protein from Discosoma corals; tdTomato and mCherry: mutated versions of the red fluorescent protein from Discosoma corals. Each data point is given as mean ± standard error of the mean, sample size n (=number of cells) is indicated. In confocal microscopy, tdTomato shows the brightest signal, but undergoes rapid decay due to photo-bleaching (see inset). (C) Images of infected wheat tissue at 14 dpi. Hyphal cells express cytoplasmic TagRFP and mCherry. Bar represents 10 μm.
Fig. 4
Fig. 4
Co-visualization of Z. tritici strain IPO323, expressing mCherry and IPO94269, expressing eGFP. (A) Image showing mCherry and eGFP fluorescence of both strains in liquid culture. Bar represents 20 μm. (B and C) Images showing mCherry and eGFP fluorescence of both strains in infected wheat tissue at 14 dpi. Note that hyphae of either strain colonize the stomatal cavity to form pre-pycnidia. Either IPO323 or IPO94269 was found (B), suggesting that they mutually exclude each other. Occasionally, single hyphae were crossing a colonized stomata space (C). Bar represents 30 μm.

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References

    1. Albermann S., Linnemannstons P., Tudzynski B. Strategies for strain improvement in Fusarium fujikuroi: overexpression and localization of key enzymes of the isoprenoid pathway and their impact on gibberellin biosynthesis. Appl. Microbiol. Biotechnol. 2013;97:2979–2995. - PubMed
    1. Baird G.S., Zacharias D.A., Tsien R.Y. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA. 2000;97:11984–11989. - PMC - PubMed
    1. Berepiki A., Lichius A., Shoji J.Y., Tilsner J., Read N.D. F-actin dynamics in Neurospora crassa. Eukaryot. Cell. 2010;9:547–557. - PMC - PubMed
    1. Bielska E., Higuchi Y., Schuster M., Steinberg N., Kilaru S., Talbot N.J., Steinberg G. Long-distance endosome trafficking drives fungal effector production during plant infection. Nat. Commun. 2014;5:5097. - PMC - PubMed
    1. Bloemberg G.V., Wijfjes A.H., Lamers G.E., Stuurman N., Lugtenberg B.J. Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent proteins in the rhizosphere: new perspectives for studying microbial communities. Mol. Plant. Microbe. Interact. 2000;13:1170–1176. - PubMed

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