Biology Reference
In-Depth Information
8. Rehydration buffer: 8 M (w/v) urea (Amresco, Solon, OH,
USA), 2 % (w/v) CHAPS (Amresco, Solon, OH, USA), 0.002 %
(w/v) bromophenol blue (Amresco, Solon, OH, USA), 20 mM
dithiothreitol (DTT; Amresco, Solon, OH, USA), 0.5 % (v/v)
pharmalyte (pH 5-8; GE Healthcare Bio-Sciences AB, Uppsala,
Sweden).
3
Methods
3.1 Preparation of
Synchronous Culture
Inoculum and
Infection of M. oryzae
Fungal Pathogens
1. Fungal conidia were spread on the rice bran agar medium and
grew for 3 days.
2. Aerial mycelia were removed by a spreader and incubated
under infl orescent light for another 3 days to get synchro-
nously produced conidia for inoculation.
3. Four-week-old rice seedlings were inoculated with conidia sus-
pension (1 × 10
6
conidia/mL) of incompatible or compatible
races of
M. oryzae
using an air sprayer (
see
Note 4
).
4. Inoculated plants were kept in a humidity chamber at 28 °C.
5. Leaves were harvested at 72 h post-inoculation (PI) and processed
immediately for isolation of proteins (
see
Note 5
).
6. For transcript profi ling, leaf samples were collected at 12, 48,
and 72 h PI, frozen in liquid nitrogen, and stored at −70 °C.
1. Inoculate
X. oryzae
into 5 mL PS liquid medium from glycerol
stock or PSA agar medium culture.
2. Culture cells of
X. oryzae
in PS liquid medium at 30 °C incu-
bator with shaking at 150 ×
g
overnight.
3. Transfer the 5 mL cultured cells into 500 mL fresh PS liquid
medium and cultured for 6-8 h until OD 0.8-1.0.
4. Collect and wash cells of
X. oryzae
with distilled water twice.
5. Dilute cells to 1 × 10
8
cfu/mL with 0.01 % tween-20 in distilled
water.
3.2 Infection of
Xanthomonas oryzae
6. Cut leaf tip (appox. 2 cm) with sterilized scissors and immerse
leaf into the
X. oryzae
solutions for 30 min.
7. Put into the humidity chamber for disease development.
3.3 Total Protein
Extraction: Phenol
Method
This method is depicted as a fl ow chart in Fig.
1
.
1. Before use, mix equal volume of Mg/NP-40 extraction
buffer (
see
Note 6
) and phenol (phenol extraction solution)
(
see
Note 7
).
2. Add 10 mL phenol extraction solution to a 50 mL Nalgene
centrifuge tube containing 2 g of fi nely powdered tissue sample.
Mix gently at room temperature (RT) for 10 min.
