Protran ba 85

The tetrapeptide AcYPFF and PalmYPFF were synthesized by Lipopharm (Poland). Potassium phosphate buffer was purchased from J. T. Baker^® (USA). Synthetic membranes made of nitrocellulose (Protran BA 85) and polytetrafluoroethylene (PTFE) were purchased from Whatman (USA). Nylon membrane was obtained from GVS Filter Technology (USA). Cuprophan (regenerated cellulose) and polyvinylidene fluoride (PVDF) were purchased from Agilent Technologies (USA) and GemaMedical (Israel), respectively.

To examine dityrosine fluorescence as an indicator of the formation of mature asci, patches of cells grown on YPDA plates were replica-plated to sporulation plates overlaid with a nitrocellulose filter (Protran BA85, Whatman). After 3-day incubation at 30°C, fluorescence was visualized by illuminating the open plates from the top with a hand-held 302-nm ultraviolet (UV) lamp. Images were taken using a Gel Doc XR system (Bio-Rad). Sporulation efficiency was quantitated by microscopic examination of asci formation after 3 days on sporulation plates. Both mature and immature asci were scored. At least 300 cells were counted for every strain. Spore viability was assessed by tetrad dissection. At least 216 spores were scored for every strain.

Lactobacillus strains were streaked onto MRS agar and grown for 48 h under anaerobic conditions at 37 °C. Colonies from each strain were picked and then patched onto a fresh MRS agar plate fitted with a 50-square grid. After 48 h, an 82-mm nitrocellulose membrane (Whatman Protran BA85) was laid onto the plates, then lifted, along with adherent bacterial growth and placed into PBST buffer (1× PBS with 0.05% Tween-20, pH 7.4). Blots were washed 3× in PBST at room temperature (RT). The blot was blocked in PBST + 5% BSA and washed 3× in PBST. Then they were incubated with 2 µg/ml purified nanobody proteins in PBST + 5% BSA for 1 h as described above. Finally, blots were washed in PBST and then incubated with a 1:5000 dilution of HisProbe HRP-conjugated antibody (Thermo Scientific) for 1 h at RT with gentle shaking (50 RPM). After final washing, blots were incubated with ECL Plus per manufacturer’s instructions (Cytiva). For the fluorescent nanobody assay, the blots were incubated with Lc58-RFP (50 µg/ml) and Lj75GFP (25 µg/ml), washed, and then imaged. The blots were imaged with a Gbox Chemi XX6 Imager (Syngene) using the ECL western blot program for non-fluorescent nanobody binding and Texas Red and TurboGFP filters to detect fluorescent nanobody binding. Image analysis was conducted with GeneTools Software.

The secretomes of the Botrytis transformants were analyzed by SDS-PAGE and western-blots using nitrocellulose membranes (Whatman Protran BA 85) and monoclonal anti-GFP or anti-c-myc primary antibodies (Roche) diluted 1:1000. Secondary antibodies consisted of goat anti-mouse IgG conjugated to Horseradish peroxidase (Sigma-Aldrich) and were used at a 1:3000 dilution. The peroxidase was detected with Immobilon Western Chemiluminescent HRP Substrate (Millipore). Quantification of western-blot bands was done with the software Quantity One (Bio-Rad) on the chemiluminescence signal recorded with a Gel Doc XR + system (Bio-Rad).

Proteins separated by standard SDS-PAGE, Phos-tag SDS-PAGE or NuPAGE were transferred to nitrocellulose membrane (Protran BA85, Whatman). The membranes were blocked with 5% skim milk and then incubated with 1 µg/ml anti-phospho-RBM20 (TF1049-02 or TF1510-A), anti-DDDDK-tag polyclonal antibody (MBL) or anti-FLAG monoclonal antibody (M2, Sigma) and 1:1,000-diluted HRP-conjugated anti-rabbit IgG antibody (Amersham or Pierce) or anti-mouse IgG antibody (MBL). Chemiluminescence signals (West Dura, Thermo) were detected by using LAS4000 (GE Healthcare).