Mapping MaCFEM85-MsWAK16 Interaction Site

To determine the site required for the interaction of MaCFEM85 with MsWAK16, PCR amplification was performed to generate multiple truncated forms of MaCFEM85. The CFEM domain (MaCFEM85-CFEM; aa residues 19–86) and the C terminal without the CFEM domain (MaCFEM85-C, aa residues 87–170) were inserted into the vector pGBKT7 as the bait protein (Supplementary Table S1). Another five variants were constructed using polypeptide synthesis to mutate cysteine to alanine at positions 26, 30, 43, 52, and 26/30/43/50/52/64/69/85 (ΔCFEM85₂₆, ΔCFEM85₃₀, ΔCFEM85₄₃, ΔCFEM85₅₂, and ΔCFEM85_{8 all}, respectively). These variants were used to perform Y2H experiments with MsWAK16-ED. The transformed yeast cells were assayed for growth on synthetic dropout SD/-Trp-Leu plates and SD/-Trp-Leu-His-Ade plates containing X-α-Galactosidase (X-α-Gal).