Lsm780 instrument

Experiments were performed as described previously^{21 (link)}. Anti-BiP (ab21685), anti-β-actin (ab6276; Abcam, Cambridge, UK), anti-Myc (sc-40), and anti-aldolase A1 (sc-12059; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies were purchased from commercial sources. Surface biotinylation was performed using 0.3 mg/mL EZ-Link Sulfo-NHS-SS-Biotin and NeutrAvidin (Thermo Fisher Scientific). Immunoblotting was performed using primary antibodies at a 1:1000 dilution, followed by the corresponding anti-isotype secondary antibodies (Santa Cruz Biotechnology) at a 1:2000 dilution. Signals were visualized using the SuperSignal West Pico Kit (Thermo Fisher Scientific). For immunofluorescence experiments, blocking buffer containing 10% donkey serum and 1% bovine serum albumin in phosphate-buffered saline was used, and the dilutions used for primary and fluorophore-tagged secondary antibodies were 1:100 and 1:2000, respectively. Confocal images were obtained using a Carl Zeiss LSM780 instrument, and ZEN software was used for image processing.

For fluorescence and light microscopic analysis, 50 µl AMM on glass coverslips in a wet chamber was inoculated with 2 × 10⁴ conidia. Samples were analyzed after 0, 4, 6, 10, and 24 h using a Zeiss Axio Imager.M2 (Zeiss). Images were taken with an AxioCam MRm and analyzed by the use of AxioVision SE64 Rel. 4.9.1 imaging software (Zeiss). For confocal scanning laser microscopy, a Zeiss LSM 780 instrument was used along with an Airyscan detector where noted. Images were processed using the ZEN software package from Zeiss.
For scanning electron microscopy (SEM) analysis, resting conidia from mycelia grown on AMM agar plates for 5 days were collected using an electrically conductive and adhesive tag (Leit-Tab; Plano GmbH). Samples for resting conidia were fixed for 24 h in a desiccator containing a solution of 25% (vol/vol) glutaraldehyde, whereas swollen conidia were fixed in 2.5% (vol/vol) glutaraldehyde. Further preparation and scanning electron microscopy were carried out as previously described (56 (link)).

Experiments were performed as described previously^{20 (link)}. Anti-BiP (ab21685), anti-β-actin (ab6276, Abcam, Cambridge, UK), anti-Myc (sc-40), anti-aldolase A1 (sc-12059, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GOLGB1 (HPA011008), and anti-FLAG (F3165, Sigma–Aldrich, St. Louis, MI, USA) antibodies were purchased from commercial sources. Coimmunoprecipitation was performed using EZview Red Anti-FLAG M2 and anti-c-Myc Affinity Gel (Sigma). Surface biotinylation was performed using 0.3 mg/mL EZ-Link Sulfo-NHS-SS-Biotin and NeutrAvidin (Thermo Fisher Scientific). Immunoblotting was performed using primary antibodies at a 1:1000 dilution, followed by corresponding anti-isotype secondary antibodies (Santa Cruz Biotechnology) at a 1:2000 dilution. Signals were visualized using the SuperSignal West-Pico Kit (Thermo Fisher Scientific). For immunofluorescence, blocking buffer containing 10% donkey serum and 1% bovine serum albumin in phosphate buffered saline was used, and dilutions of primary and fluorophore-tagged secondary antibodies were 1:100 and 1:2000, respectively. Confocal images were obtained with a Carl Zeiss LSM780 instrument; ZEN software was used for image processing.