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.