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. infectoria

precipitates 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 mM KCl. 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 in A. infectoria, we designed several degenerate primers (Table 1)

based on the conserved regions of the Drechslera tritici-repentis and A. brassicicola

PKS, scytalone dehydratase, trihydroxynaphthalene reductase and vermelone

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 cycles of 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 and D. 150 MHz 13C CPMAS solid-state NMR

spectrum. E. ESR spectra of A. infectoria conidia 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

(http://genome.jgi-psf.org/Altbr1/Altbr1.home.html) and D. tritici-repentis

(http://www.broadinstitute.org). These genomes were considered appropriate for this

analysis since we showed previously that A. infectoria FKS1 (Accession number:

JF742672) and eight chitin synthases CHSA 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.

Reference List

1. Beltrán-García MJ, Prado FM, Oliveira MS, Ortiz-Mendoza D, Scalfo AC,

Pessoa AJr, Medeiros MH, White JF, Di Mascio P. 2014. Singlet molecular oxygen

generation by light-activated DHN-melanin of the fungal pathogen Mycosphaerella

fijiensis in black Sigatoka disease of bananas. PLoS One 9:e91616.

2. Rajagopal K, Kathiravan G, Karthikeyan S. 2011. Extraction and characterization

of melanin from Phomopsis: A phellophytic fungi isolated from Azadirachta indica A.

Juss. African J. Microb. Res. 5:762–766.

3. Metz G, Wu XL, Smith SO. 1994. Ramped-amplitude cross polarization in magic-

angle-spinning NMR. J. Magn. Reson. Ser. A 110:219–227.

4. Fung BM, Khitrin AK, Ermolaev K. 2000. An improved broadband decoupling

sequence for liquid crystals and solids. J. Magn. Reson. 142:97–101.

5. Chatterjee S, Prados-Rosales R, Frases S, Itin B, Casadevall A, Stark RE. 2012.

Using solid-state NMR to monitor the molecular consequences of Cryptococcus

neoformans melanization with different catecholamine precursors. Biochemistry

51:6080–6088.

6. Chatterjee S, Prados-Rosales R, Tan S, Itin B, Casadevall A, Stark RE. 2014.

Demonstration of a common indole-based aromatic core in natural and synthetic

eumelanins by solid-state NMR. Org. Biomol. Chem. 12:6730–6.

7. Morcombe CR, Zilm KW. 2003. Chemical shift referencing in MAS solid state

NMR. J. Magn. Reson. 162:479–86.

8. Ranguelova K, Girotto S, Gerfen GJ, Yu S, Suarez J, Metlitsky L, Magliozzo

RS. 2007. Radical sites in M. tuberculosis KatG identified using EPR spectroscopy, the

3-D crystal structure and electron transfer couplings. J. Biol. Chem. 282:6255–6264.

9. Nosanchuk JD, Casadevall A. 1997. Cellular charge of Cryptococcus neoformans:

contributions from the capsular polysaccharide, melanin, and monoclonal antibody

binding. Infect. Immun. 65:1836–1841.

10. Fernandes C, Anjos J, Walker LA, Silva BM, Cortes L, Mota M, Munro CA,

Gow NA, Gonçalves T. 2014. Modulation of Alternaria infectoria cell wall chitin and

glucan synthesis by cell wall synthase inhibitors. Antimicrob. Agents Chemother.

58:2894–2904.