1 10 phenanthroline

The (−) mating type M. circinelloides f. lusitanicus CBS277.49 (syn. Mucor racemosus ATCC 1216b) and its derived leucine auxotroph R7B were used as the wild type strains according to their auxotrophies. Strain MU402 is a uracil and leucine auxotroph derived from R7B used for the generation of deletion mutants^{31 (link)}. The M. circinelloides f. lusitanicus strain of the (+) mating type NRRL3631 was used in virulence assays as an avirulent control^{13 (link)}. Cultures were grown at 26 °C in complete YPG medium or in MMC medium as described previously^{31 (link)}. The pH was adjusted to 4.5 and 3.2 for mycelial and colonial growth, respectively. Transformation was carried out by electroporation of protoplasts as described previously^{45 (link)}. The phenotypic growth analysis was performed in minimal media YNB pH 3.2 and iron was depleted with 50 μM of 1,10-Phenanthroline (Sigma-Aldrich). For expression analysis L15 cell culture medium (Sigma-Aldrich) was used and iron was depleted adding 10 μM of 1,10-Phenanthroline (iron-depleted medium by a synthetic iron chelator)^{46 (link)} or 20% of FBS (Sigma-Aldrich) (iron-depleted medium mimicking iron chelating in the host). In both growth and expression analyses, FeCl₃ was added at 350 μM to obtain a high iron medium. Media were supplemented with uridine (200 mg/l) or leucine (20 mg/l) when required for auxotrophy.

APN inhibitors bestatin (TargetMol, USA), 2,2′-dipyridyl, and 1,10-phenanthroline (Sigma, USA) were each dissolved in water at a concentration of 200 mM. The inhibitors were then diluted to various concentrations for use at 300 μM (bestatin), 250 μM (2,2′-dipyridyl), and 15 μM (1,10-phenanthroline) as previously described^{36 (link)}. IPI-2I cells cultured in 24-well plates were pre-incubated with inhibitors, rabbit polyclonal antibody against APN, or anti-Flag rabbit polyclonal antibody (control) for 1 h and subsequently infected with PDCoV [multiplicity of infection (MOI) = 2] for 1 h. The cells were further washed three times with Dulbecco’s modified Eagle medium and maintained with inhibitors, APN antibody, or control antibody in cell culture containing 2.5 μg/ml trypsin. At 24 h post infection, cells were collected for IFA, and PDCoV titers in LLC-PK1 cells were determined by 50% tissue culture infective dose (TCID₅₀).

Total protein was extracted from the infiltrated leaves of N. benthamiana by grinding in a lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, 5% glycerol) containing protease inhibitor cocktail (AEBSF, Bestatin, E-64, Leupeptin, Pepstatin A, and 1,10-Phenanthroline; MilliporeSigma, Burlington, MA, USA). The homogenate was incubated on ice for 1 h with gentle shaking on the rocker and then clarified by centrifugation at 3000× g for 10 min at 4 °C. The supernatant (600 μL) was immunoprecipitated using GFP-Trap^® or RFP-Trap^® (ChromoTek GmbH, Planegg-Martinsried, Germany)—anti-green fluorescent protein (GFP) or anti-red fluorescent protein (RFP) antibody-conjugated agarose beads (50 µL)—by incubating for 2 h on ice with gentle shaking. The supernatant described at this step was also used as an ‘Input’ sample for reverse transcription-PCR (RT-PCR) or immunoblot analysis. The binding reaction was washed three times as per the manufacturer’s instruction by centrifugation at 3000× g for 2 min at 4°C. The pellet of the agarose beads collected at this step was used as ‘Elute’ for further analysis. For the immunoblot analysis, total protein was extracted as described below. For RT-PCR analysis, total RNA was extracted using the TRIzol™ reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s procedure. RNA extracts were resuspended in 50 μL of RNase-free water.