A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici
| Autor: | Nicholas J. Talbot, Sreedhar Kilaru, Martin Schuster, Darren M. Soanes, Congping Lin, David J. Studholme, Gero Steinberg |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
0106 biological sciences
Cys cysteine ROI region of interest Gene Expression 01 natural sciences Green fluorescent protein Gene expression n sample size dpi days post infection GFP green fluorescent protein 0303 health sciences RB and LB right and left border Virulence biology Protein subcellular localization prediction 3. Good health eGFP enhanced green fluorescent protein Mycosphaerella graminicola Val valine Aequorea victoria Intracellular AcGFP Aequorea coerulescens green fluorescent protein Green Fluorescent Proteins ZtGFP Z. tritici codon-optimized green fluorescent protein Ser serine Microbiology Article Leu leucine 03 medical and health sciences Ascomycota Live cell imaging Genetics Wheat pathogenic fungi Codon sdi1 succinate dehydrogenase 1 Ile isoleucine His histidine 030304 developmental biology Arg arginine Staining and Labeling Protein localization Tyr tyrosine tub2 α tubulin biology.organism_classification Molecular biology Microscopy Fluorescence FPs fluorescent proteins Septoria tritici blotch Heterologous expression 010606 plant biology & botany |
| Zdroj: | Fungal Genetics and Biology |
| ISSN: | 1087-1845 |
| DOI: | 10.1016/j.fgb.2015.03.022 |
| Popis: | Highlights • We generated a Z. tritici codon-optimized gene for green fluorescent protein (ZtGFP). • In epi-fluorescence and confocal microscopy, ZtGFP is brighter and more stable than eGFP. • We provide 3 vectors that carry AcGFP, eGFP and ZtGFP for yeast recombination-based cloning. • The vectors carry carboxin resistance for targeted integration. • The carboxin resistance conveying vectors integrate as single copies into the sdi1 locus. 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. |
| Databáze: | OpenAIRE |
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