A. pullulans growth modalities, transformation, and targeted integration of the HYG ^R transgene. (A) A. pullulans is polymorphic and has different morphologies when grown on CSM and YPD. Daughter cells born from budding mother cells or hyphae (red arrows) can go on to bud themselves (black arrow), to grow isotopically (blue arrow), or to elongate and form hyphae (purple arrow). Scale bar, 5 µm. (B) Schematic showing the integration vector designed to replace the native URA3 gene with a Hygromycin resistance (HYG^R) cassette. (C) Examples of selected transformants patched onto plates with YPD supplemented with Hygromycin B (Hyg. B) or plates lacking uracil (-Ura). ura3::HYG^R transformants fail to grow without uracil, while other transformants do (red arrows). (D) Schematic showing the steps in the transformation protocol. Notes in green indicate protocol modifications optimized for A. pullulans through the work shown here.