The Velvet Family of Fungal Regulators Contains a DNA-Binding Domain Structurally Similar to NF-κB

This study reveals an important family of fungal regulatory proteins to be transcription factors that contain a DNA-binding “velvet” domain structurally related to that of mammalian NFkB.

generated (VelB_ΔIL, IL = inserted loop). It was cloned into a modified pET-24d-vector (pETM13), encompassing amino acid residues 44-126 and 240-343 connected by a short linker of eight amino acids with the sequence ASIPPSTA (the corresponding loop in VosA).
The resulting C-terminally His-tagged protein VelB_ΔIL was used for size exclusion chromatography and multi angle light scattering to verify stable hetero-dimer formation.
VelB_ΔIL was expressed in Rosetta 2 (DE3) cells (Merck) at 16°C for 72h using autoinduction medium [1]. After harvesting, the cells were lysed in lysis buffer (35 mM Imidazol, 400 mM NaCl und 35 mM HEPES pH 7.5.) using the Fluidizer (Microfluidics) and a cleared supernatant obtained by centrifugation for 20 min at 4°C and 30,000 ×g. VelB_ΔIL was purified from the supernatant using a two-step purification scheme. Affinity chromatography was applied first using HisTrap columns (GE-Healthcare). The bound protein was washed in 2 M LiCl in Lysis buffer to remove putative DNA bound to VelB, and subsequently eluted with a gradient of Imidazol (35 -400mM) in lysis buffer. The final purification step was performed using an S75 (16/600; GE-Healthcare) equilibrated in 10 mM HEPES pH 7.5 und 150 mM NaCl.
The identical buffer was used for the SEC and MALS experiments. MALS has been performed using a Mini Dawn Treos (Wyatt) and ASTRA version 5.4.1 in line with an Äkta purifier (GE Healthcare). MM (molecular mass) and extinction coefficient have been determined using ProtParam: http://web.expasy.org/protparam/. 2 For the CD-measurements, the individual proteins, stored at -80°C in the buffer (10 mM HEPES pH 7.5 und 150 mM NaCl) and a concentration of 5 mg/ml, were diluted 1:10 in 10 mM sodiumphosphate buffer pH 7.5 before measurement. Secondary structure deduction has been performed using CDNN (200-260 nm) and normalized to 100 [2]. was digested from the plasmid pME4163 with PmeI. Transformation in A. nidulans was performed by polyethylene glycol mediated fusion of protoplasts [3,4].

Genomic DNA extraction and Southern hybridization
Genomic DNA was extracted from vegetative grown cultures by standard protocols as described previously [5]. For Southern analysis, 25 µg genomic DNA were digested with ScaI overnight.
The hybridization was performed according to standard protocols [6] with a non-radioactively labelled probe. The DNA for probe design was amplified from genomic DNA with primers JG493/494.

Viability test
Fresh spores were spread on an agar plate and cultivated for 10 days at 37°C. The spores were harvested and 250 of these old spores were grown on new agar plates for two days at 37°C. The amount of surviving colonies has been compared between all strains.

Statistical analysis
Statistical differences between WT and mutant strains were evaluated with student's unpaired ttest (2-tailed). Mean ±SD are shown. P values < 0.05 were considered significant.

Structure representations have been made using PYMOL (The PyMOL Molecular Graphics
System, Version 1.5.0.4 Schrödinger, LLC) and CHIMERA [8]. The alignments in Fig S7 have been extracted from the respective PDB-file using CHIMERA, missing residues have been manually inserted into the alignment file and the final figure has been made using ESPRIPT [9]