Trehalase Assay
1.
Harvest the conidia from plates as usual through Miracloth into Oakridge
tubes. Use approximately 90 plates Guy-11, 210 for TPS1 and 70 plates of
NTH1 and TRE1.
2.
Spin at 4000rpm for 20 minutes at 4oC.
3.
Pour off the majority of the supernatant, leaving a small amount around
the pellet.
4.
Resuspend the pellet by vortexing and then pool the pellets and add SDW
upto the top of the Oakridge tube that holds 41ml.
5.
Count the conidia and resuspend at 1x106 and split the amount of liquid
into a sample for each time point.
6.
Plate droplets of the sample onto plastic petri dishes using a Pasteur pipette
and leave at 24oC in the light for e.g. 1 hour.
7.
Re-harvest the droplets from the petri dishes into Oakridge tubes using a
plastic cover slip to scrape off the germinating conidia.
8.
Spin at 6000rpm for 20 minutes at 4oC.
9.
Decant the majority of the supernatant leaving the spores in small amount
of liquid and pool them in one tube per time point and strain. Resuspend
the pooled pellets in 15ml of extraction buffer (50mM Tris–HCl pH 7.0
and EDTA-free protease cocktail tablets (Roche), for 100ml use 5ml 1M
Tris-HCl and 8 tablets). If using the larger bead beater vessel 40ml of
buffer should be used.
10.
Disrupt the conidia using a bead beater with zirconia/silica beads of
0.5mm that have previously been acid washed (1:10 dilution of HCl) and
rinsed several times in distilled water. Add the sample to the small pot and
then add beads until the pot is completely full since the presence of air
bubbles will cause foaming of the sample. Each sample should be beaten
for 7 x 20 second bursts with 1 minute on ice in between each burst. The
larger vessel will require 7 bursts of 40 seconds.
11.
The sample should then be checked for complete disruption under the
microscope and then transferred to an Oakridge tube and left on ice until
all the samples have been beaten.
12.
Centrifuge the samples to pellet the debris at 10,000 rpm for 10 minutes.
13.
Aliquot 60l of each extract supernatant into 5 eppendorffs and add to
three tubes (the positives) 60l of assay buffer (10ml is composed of
50mM Tris pH 7.0 (500l of 1M), 1 tablet of EDTA-free protease cocktail,
40mM trehalose, 10mM CaCl (100l of 1M), 18mM MgCl2 (180l 1M),
1mM ATP, 20M cAMP) to the other two tubes add 60l of assay buffer
that has been made without trehalose. The remainder of the supernatant,
crude extract, should be frozen.
Trehalose add 2ml of 200mM for 10ml use 756.6mg since 1M 378.3 mg/ml
ATP 551.1 mg/ml 1M 10mM 5.51 mg/ml 10ml 55.1 mg add 1ml 10mM
cAMP 1M-351.2 mg/ml 20mM 7 mg/ml add 10l
14.
Incubate the samples at 30oC for 2 hours.
15.
Boil the samples for 5 minutes ensuring they are tightly capped and
clipped. The samples can then be frozen until the glucose assay is
performed.
16.
Spin the samples at 13,000rpm for 5 minutes.
17.
Avoiding the debris remove 100l of the sample and use this for the
glucose assay.
18.
Perform the glucose assay as described in the D-glucose kit using 4ml
disposable cuvettes. The blank for the glucose assay is 100l of water
instead of sample and the blank for the trehalase assay is 50l of buffer
and 50l of water. Three blanks of 50l extraction buffer and 50l of
assay buffer should also be set up.
19.
Protein Assay. The protein assay follows the microassay procedure in the
booklet supplied with the Bio-Rad Protein Assay Dye Reagent. It will
probably be necessary to supply 1:10 dilutions of the crude extract for
assay, further dilution may also be necessary. The assay is performed in
clean 1ml disposable cuvettes.