Abstract
The engineered monomeric version of the lancelet Branchiostoma lanceolatum fluorescent protein, mNeonGreen (mNG), has several positive characteristics, such as a very bright fluorescence, high photostability and fast maturation. These features make it a good candidate for the utilization as fluorescent tool for cell biology and biochemical applications in filamentous fungi. We report the generation of plasmids for the expression of the heterologous mNG gene under the control of an inducible and a constitutive promoter in the filamentous ascomycete Sordaria macrospora and display a stable expression of mNG in the cytoplasm. To demonstrate its usefulness for labeling of organelles, the peroxisomal targeting sequence serine-lysine-leucine (SKL) was fused to mNG. Expression of this tagged version led to protein import of mNG into peroxisomes and their bright fluorescence in life cell imaging.
Highlights
Fluorescence microscopy is a potent method for studying in vivo protein localization, cytoskeleton dynamics, infections and protein–protein-interactions in filamentous fungi (Berepiki et al 2010; Chapuis et al 2019; Hoff and Kück 2005; Lorang et al 2001).Fluorescent proteins can be expressed in fungal cells by inserting genes encoding fluorescent proteins into the genome under the control of constitutive or inducible promoters (Oda et al 2016; Pöggeler et al 2003)
Construction of fungal mNeonGreen reporter plasmids The visualization of fluorescent reporter proteins is an important technique in fungal cell biology
The yellow-green fluorescent protein mNG was predicted to be superior to enhanced green fluorescent protein (eGFP) with regard to its brightness and photostability (Shaner et al 2013), these effects were not that evident when the mng gene was expressed in S. macrospora
Summary
Fluorescence microscopy is a potent method for studying in vivo protein localization, cytoskeleton dynamics, infections and protein–protein-interactions in filamentous fungi (Berepiki et al 2010; Chapuis et al 2019; Hoff and Kück 2005; Lorang et al 2001).Fluorescent proteins can be expressed in fungal cells by inserting genes encoding fluorescent proteins into the genome under the control of constitutive or inducible promoters (Oda et al 2016; Pöggeler et al 2003). The tetrameric fluorescent protein DsRed, its monomeric variant mRFP from the marine anemone Discosoma striata as well as brighter versions of mRFP (mCherry and tdTomato) emitting in the red light spectrum have been successfully expressed in filamentous fungi (Campbell et al 2002; Mikkelsen et al 2003; Schuster et al 2015; Toews et al 2004; Xiao et al 2018). The new and very bright yellow fluorescent protein LanYFP with a high quantum yield (~ 0.95) and extinction coefficient (~ 150,000 M − 1 cm−1) was isolated from lancelet Branchiostoma lanceolatum. In comparison to LanYFP, mNG harbors 21 substitutions and contains modified N- and C-termini of additional 11 and 7 amino acids derived from eGFP. mNG has a blue-shifted excitation maximum of 506 nm and an emission maximum of 517 nm with a high quantum yield excitation coefficient
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
