Establishment of the monomeric yellow-green fluorescent protein mNeonGreen for life cell imaging in mycelial fungi

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.

Tracking Fungal Growth: Establishment of Arp1 as a Marker for Polarity Establishment and Active Hyphal Growth in Filamentous Ascomycetes

“…The application of a higher imaging contrast allowed for the further determination of dot-like structures apart from hyphal tips ( Figure 1 a). To confirm that the SmArp1 localization was independent of the used fluorophore, we replaced TagRFP-T by a C-terminal fused mNG, a monomeric green fluorescent protein derived from the lancelet Branchiostoma lanceolatum [ 59 ]. SmArp1-mNG localized dynamically to growing hyphal tips ( Figure S5 and Video S1 ).…”

“…The fragment containing the native promoter and coding sequence of Smarp1 was amplified from S. macrospora wt gDNA using the Arp1_promotor_f/Arp1-Neo_r primer pair for the generation of the pSmarp1-mNG plasmid. The amplification of mNG was done with the Neo_f/Neo_TtrpC_r primer combination using the pmn-xyl plasmid [ 59 ]. The terminator of the anthranilate synthase gene trpC of Aspergillus nidulans , TtrpC , was amplified from the p1783-1 plasmid [ 60 ] with the TtrpC_f/TtrpC_pRS_r primer pair.…”