SUPPLEMENTARY RESULTS
Supplementary Methods
Fungal growth for pigment characterization of the accumulated pigments
The extracted melanin-derived pigment was purified for further melanin charcaterization (UV-visible and IR). Since the pigment extracted from A. infectoriaprecipitates in HCl, but does not form a pellet upon centrifugation, we introduced slight modifications to a procedure described by others (1, 2). The freeze-dried mycelial mats were ground with a mortar and pestle and then extracted with 1 M NaOH at 121 ºC for 30 min. The cell extract was centrifuged at 16,000 g and the supernatant was air dried at 60 ºC. Then, concentrated HCl was added very carefully to avoid the deleterious effects in melanin structure caused by heating from exothermic reaction. This results in a white precipitate that was recovered by centrifugation at 16,000 rpm for 2 min and discarded. The supernatants containing melanin-derived pigments were further air dried, then treated with ethanol and chloroform to remove lipids and finally suspended in water (Milli Q). The pigments were not soluble in these solvents. Then the samples were lyophilized prior to FTIR.
Ultraviolet-visible and infrared spectroscopy
The isolated and purified melanin pigments from A. alternata and A. infectoria mycelia dissolved in 1 ml of water (MilliQ) and the ultraviolet-visible spectra were recorded in a Spectra Max Plus384 spectrophotometer (Molecular Devices, LLC) from 250–600 nm. The infrared spectra of the pigments were recorded on a ThermoNicolet IR300 Fourier transform infrared spectrometer, equipped with a deuterated triglycine sulfate (DTGS) detector and a KBr beam splitter. Thermo Scientific Nicolet Smart Orbit diamond ATR accessory was also used with a resolution of 1 cm-1.
Zeta potential measure
The melanin ghosts were washed twice with 1 mMKCl. The measurements were taken with a Zeta Plus zeta potential analyzer Zeta Plus (Brookhaven Instruments Corporation, Holtsville, NY, USA).
Nuclear Magnetic Resonance (NMR) study
We conducted solid-state 13C NMR measurements using a Varian (Agilent Technologies, Santa Clara, CA) DirectDrive1 (VNMRs) NMR spectrometer operating at a 1H frequency of 600 MHz (150 MHz 13C frequency) and a temperature set to 25 °C. The 13C cross-polarization (CP) experiments were carried out on ~ 3 mg of powdered sample at a magic-angle spinning (MAS) frequency of 20 kHz (±20 Hz) using a 1.6-mm HXY fastMAS probe. Ramped-amplitude 13C CPMAS (RAMP-CP) measurements were performed to identify the carbon-containing chemical moieties of the melanin ghosts (3). We used a 1.5 ms cross-polarization (CP) period and a 3-sec recycle delay for the RAMP-CP measurements. The SPINAL method (4) was implemented to achieve high-power heteronuclear 1H decoupling of ~ 140 kHz, and ~26000 transients were acquired to generate a 13C spectrum of the natural-abundance pigment. The reproducibility of the spectroscopic measurements was assessed obtaining duplicate 13C spectra at two MAS frequencies (10 and 20 kHz). Detailed experimental parameters for CPMAS measurements on fungal melanins have been reported elsewhere (5, 6).1D 13C spectrum was processed with 150 Hz of line broadening; chemical shifts were referenced externally to the ethylene (-CH2-) group of adamantane (Sigma) resonating at 38.48 ppm (7).
Electron Paramagnetic Resonance (EPR) spectra
X-band (9 GHz) measurements were made on a Varian E-line spectrometer at 77K using a liquid nitrogen finger dewar inserted into a TE102 resonator. Typical instrumental parameters were as follows: frequency, 9.1 GHz; power, 0.2 mW; modulation amplitude, 3.2 G; scan time 2 min; time constant, 0.5 s; number of scans averaged, 8 to 16. D-band (130 GHz) EPR spectra were performed on a spectrometer assembled at Albert Einstein College of Medicine, as previously described (8). The magnetic field was generated using a 7 T Magnex superconducting magnet equipped with a 0.5 T sweep/active shielding coil. Field swept spectra were obtained in the two pulse echo-detected mode with the following parameters: frequency, 130.001 GHz; temperature, 7 K; repetition rate, 15 Hz; 90 degree pulse, 40 ns; time τ between pulses, 150 ns. The temperature of the sample was maintained to an accuracy of approximately ±0.3 K using an Oxford Spectrostat continuous flow cryostat and ITC503 temperature controller. The magnetic field at both X-band and D-band was calibrated to an accuracy of 3 gauss using a sample of manganese doped into MgO.
Identification of partial sequences of genes involved in DHN-melanin synthesis
Liquid cultures of A. infectoria were harvested by centrifugation and DNA extractions were performed using a ZR Fungal/ Bacterial Miniprep DNA kit (Zymo Research). To identify the partial sequences of four genes encoding potential enzymes involved in melanin synthesis inA. infectoria,we designed several degenerate primers (Table 1) based on the conserved regions of the Drechsleratritici-repentis and A. brassicicolaPKS,scytalone dehydratase, trihydroxynaphthalene reductase andvermelone dehydratase. Partial fragments of these genes were obtained by PCR amplifications with DNA polymerase DyNA (Fisher Scientific) and 1.5 mM MgCl2 under the following conditions: 94°C for 5 min; 35 cyclesof 94°C for 1 min, 48°C for 1 min, and 72°C for 1 min; and an additional extension for 7 min at 72°C at the end of the program. The PCR products were visualized in a 1% agarose gel, and DNA fragments with the expected sizes were extracted from the gel and purified for further sequencing.
Supplementary results
Spectral and physical characteristics of A. infectoria DHN-melanin
The nature of the alkali-extracted pigment was further confirmed by its spectral properties. The UV-visible absorbance (250 – 600 nm) spectrum of the purified pigment showed a strong absorbance in the UV region with a small shoulder at 260 – 280 nm (Fig. S1A) suggesting the presence of phenol groups. No absorption occurred in the visible region. The FTIR spectrum of A. infectoria pigment revealed characteristic absorption peaks similar to the A. alternata spectrum (Fig. S1B). The solid-state 13C NMR spectrum of the A. infectoria melanin ghosts (Fig. S1D) showed resonances corresponding long-chain methylene groups ((CH2)n, 20-40 ppm), oxygenated aliphatic carbons (CHnO, 50-105 ppm), aromatic and/or multiply-bonded carbons (110-160 ppm) and carboxyls or amides (COO or CONH, 170-173 ppm) (Figure S1D). Overall, the solid-state 13C NMR spectra of these melanized cells indicate the formation of chemically heterogeneous aromatic-based pigments that are associated with chemically resistant aliphatic moieties that could survive exhaustive degradative treatments. The zeta potential measurements in isolated ghost from A. infectoria conidia and hyphae were almost identical (Fig. S1C). Of note, these values are in agreement with that of Saccharomyces cerevisiae melanins (-8.37 ± 2.91 mV) (9).The ESR signal shows a narrow single peak located at approximately 3252 Gauss which is defined as the characteristic of all melanins.
Fig. S1.A. UV-visible spectra, and B. FTIR spectra of A. infectoria melanin.C. Zeta potentials of conidial and hyphae andD. 150 MHz 13C CPMAS solid-state NMR spectrum.E. ESR spectra of A. infectoriaconidia and hyphae and conidial and hyphal melanin derived from A. infectoria. ESR spectra were obtained by suspending conidia, hyphae or melanin isolated from conidia or hyphae in water.
Identification of partial sequences of genes involved in DHN-melanin synthesis
Because the A. infectoria genome is not complete, we compared gene sequences of the enzymes involved in the melanin pathway with those in the genomes of A. brassicicola ( and D. tritici-repentis ( These genomes were considered appropriate for this analysis since we showed previously that A. infectoriaFKS1 (Accession number: JF742672) and eight chitin synthasesCHSA to CHSH (Accession numbers JX436211 to JX436224, JX443517, and JX443518) shares high nucleotide homology with the orthologues from other species (10). We designed degenerate primers that were used to amplify nucleotide fragments from A. infectoria (Table 1). The sequences obtained and the respective orthologues in A. brassicicola and D. tritici-repentis are represented in Supplementary results. Curiously, D. tritici-repentis possess another set of genes potentially involved in DHN-melanin synthesis.
Fig. S2. A. Biosynthetic pathway leading to the formation of melanin from acetate and identification of the enzymes involved (a, polyketide synthase; b, T4HN reductase; c, scytalone dehydratase; d, T3HN reductase; e, vermelone dehydratase; f, several candidate enzymes for this step, including phenoloxidases such as tyrosinase and laccases, peroxidases or catalases). B. Representation of the amplified genomic regions of A. infectoria and comparison with the respective ORFs encoding putative chitin synthases and respective transduction to protein in A. brassicicola and D. tritici-repentis.
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