Establishment of in vivo proximity labeling with biotin using TurboID in the filamentous fungus Sordaria macrospora.

Proximity-dependent biotin identification (BioID) has emerged as a powerful methodology to identify proteins co-localizing with a given bait protein in vivo. The approach has been established in animal cells, plants and yeast but not yet in filamentous fungi. BioID relies on promiscuous biotin ligases fused to bait proteins to covalently label neighboring proteins with biotin. Biotinylated proteins are specifically enriched through biotin affinity capture from denatured cell lysates and subsequently identified and quantified with liquid chromatography-mass spectrometry (LC-MS). In contrast to many other affinity capture approaches for studying protein-protein interactions, BioID does not rely on physical protein-protein binding within native cell lysates. This feature allows the identification of protein proximities of weak or transient and dynamic nature. Here, we demonstrate the application of BioID for the fungal model organism Sordaria macrospora (Sm) using the example of the STRIPAK complex interactor 1 (SCI1) of the well-characterized striatin-interacting phosphatase and kinase (SmSTRIPAK) complex as proof of concept. For the establishment of BioID in S. macrospora, a codon-optimized TurboID biotin ligase was fused to SCI1. Biotin capture of the known SmSTRIPAK components PRO11, SmMOB3, PRO22 and SmPP2Ac1 demonstrates the successful BioID application in S. macrospora. BioID proximity labeling approaches will provide a powerful proteomics tool for fungal biologists.

A 20-kb lineage-specific genomic region tames virulence in pathogenic amphidiploid Verticillium longisporum.

Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassica napus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same-sex mating-type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage-specific (LS) region apparently originating from the Verticillium dahliae-related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence-reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent.

The Glyoxysomal Protease LON2 Is Involved in Fruiting-Body Development, Ascosporogenesis and Stress Resistance in Sordaria macrospora.

Microbodies, including peroxisomes, glyoxysomes and Woronin bodies, are ubiquitous dynamic organelles that play important roles in fungal development. The ATP-dependent chaperone and protease family Lon that maintain protein quality control within the organelle significantly regulate the functionality of microbodies. The filamentous ascomycete Sordaria macrospora is a model organism for studying fruiting-body development. The genome of S. macrospora encodes one Lon protease with the C-terminal peroxisomal targeting signal (PTS1) serine-arginine-leucine (SRL) for import into microbodies. Here, we investigated the function of the protease SmLON2 in sexual development and during growth under stress conditions. Localization studies revealed a predominant localization of SmLON2 in glyoxysomes. This localization depends on PTS1, since a variant without the C-terminal SRL motif was localized in the cytoplasm. A ΔSmlon2 mutant displayed a massive production of aerial hyphae, and produced a reduced number of fruiting bodies and ascospores. In addition, the growth of the ΔSmlon2 mutant was completely blocked under mild oxidative stress conditions. Most of the defects could be complemented with both variants of SmLON2, with and without PTS1, suggesting a dual function of SmLON2, not only in microbody, but also in cytosolic protein quality control.

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

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.

Impact of each individual component of the mutated PTS(Nag) on glucose uptake and phosphorylation in Ralstonia eutropha G⁺1.

A recent study of the UV-generated glucose-utilizing mutant Ralstonia eutropha G⁺1 comprising transcriptomic and proteomic analyses revealed clear evidence that glucose is transported by the N-acetylglucosamine-specific phosphotransferase system (PTS(Nag)), which is overexpressed in this mutant due to a derepression of the encoding nag operon by an identified insertion mutation in nagR (Raberg et al., Appl Environ Microbiol 77:2058-2070, 2011). The inability of the defined deletion mutant R. eutropha G⁺1∆nagFEC to utilize glucose confirms this finding. Furthermore, a missense mutation in nagE (membrane component comprising the cell membrane spanning EIIC(Nag) and the cytosolic domain EIIB(Nag)) was identified, which yields a substitution of an alanine by threonine at aa 153 of NagE and may affect glucose specificity of the mutated PTS(Nag) in R. eutropha G⁺1. The investigation of various generated deletion and substitution mutants of R. eutropha H16 and G⁺1 in this study was able to elucidate these phenomena. It could be shown that the porin NagC, encoded by nagC being part of the nag operon, is not necessary, while NagE is required and is probably responsible for glucose transport through the cell membrane. The intracellular phosphorylation of glucose is obviously mediated by the glucokinase GLK and not by NagF (cytosolic component comprising the three soluble domains EIIA(Nag), HPr(Nag), and EI(Nag)). Our data clearly indicate that the derepression of the nag operon is essential for glucose uptake. The point mutation in NagE is not an essential prerequisite for glucose transport although it increased glucose transport as observed in this study.