Fungal pectin assay
The fungal pectin assay is an in vitro assay that observes the effects of PvPGIP proteins on fungal growth. S. cerevisiaecontaining plasmids with PvPGIP2, preOST1-PvPGIP2, or a negative control containing an empty vector were grown for 16 hours in -Trp Dropout YNB. The fungi used were B. cinerea isolate ECC-0165 obtained fromPrunus persica in Fresno, CA and A. niger isolate ATCC 16888 obtained from ARS Culture Collection (NRRL). A plug from an actively growing colony was transferred to fresh potato flake agar plates that were sealed with Parafilm and incubated on the lab bench or not sealed and incubated in a drawer for A. niger and B. cinerea , respectively. After sufficient sporulation, sterile DI water was added to plates, and a cell spreader was used to scrape the surface of the colony. The liquid was then poured through several layers of cheesecloth to exclude hyphal fragments. The spore concentration was quantified with a hemocytometer, then the suspension was diluted to a concentration of 2.5 × 105 spores per mL. The suspension was aliquoted into 2 mL microcentrifuge tubes with screw caps and stored at 4oC until use. Two different growth media were used in this assay: potato flake agar for A. niger and potato dextrose agar (PDA) for B. cinerea . The potato flake medium contains 4g potato flakes (Bob’s Red Mill Potato Flakes), 1g citrus pectin (Fischer Scientific, catalog #AAJ6102122), and 2.5g agarose (VWR, catalog #97062-250) into 200mL of the medium. The PDA medium contains 4.8g potato dextrose broth (VWR, catalog #90003-494), 1g citrus pectin, and 2.5g agarose in 200mL of the medium. The medium was autoclaved and 150µL was pipetted into each well of a 96 well plate, with the exception of the edge-most wells due to evaporation. 60µL of the yeast in YNB was added to each plate along with 20µL of the fungal inoculum. The yeast and inoculum were added directly on top of the wells. These plates were left in a dark room at 20°C for 4 days and observed. The wells were visually compared for their fungal growth and the OD was measured at 630nm in a plate reader using the Gen5TM Data Analysis Software (BioTek Instruments). The OD correlates directly to fungal dry weight. Another fungal pectin assay was performed where the growth radius of the resultant fungal colonies was compared. The same media, potato flake agar, was made with the same protocol as above but poured into petri dishes instead of 96 well plates. The yeast and fungal inoculum are also prepared the same way. Once the plates solidified, 200µL of natamycin, yeast, or water was spread evenly onto the plates and allowed to dry. 2µL of the A. niger fungal inoculum was then spotted into four locations per plate and growth was observed over several days.