Protein Production and Detection Protocol

For protein production, 25 ml of PcMM or PdMM were inoculated with a final concentration of 10⁶ conidia ml^‐1 of transformants and were incubated at 25°C and 150 rpm for 5 (P. chrysogenum) or 11 days (P. digitatum). Total proteins from supernatants and purified PeAfpA and PdAfpB were separated by SDS‐PAGE (16% polyacrylamide gels) and transferred to Amersham Protran 0.20 µm NC nitrocellulose transfer membrane (GE Healthcare Life Sciences, Chicago, IL, USA) as described (Garrigues et al., 2018 (link)). Protein detection was accomplished using anti‐PeAfpA antibody diluted 1:2,500 (Garrigues et al., 2018 (link)) or anti‐PAFB antibody diluted 1:1,000 (Garrigues et al., 2017 (link)). As secondary antibody, 1 : 20 000 dilution of ECL NA934 horseradish peroxidase donkey anti‐rabbit (GE Healthcare Life Sciences) was used and chemiluminescent detection was performed with ECL^TM Select Western blotting detection reagent (GE Healthcare Life Sciences) using Amersham Imager 680 (GE Healthcare Life Sciences). The experiments were repeated at least twice.