Biology Reference
In-Depth Information
metabolites, ions, and a large variety of hydrolytic enzymes in the
ECS for survival [
4
,
5
]. ECS is also a place where pathogens secrete
proteins, such as cell wall hydrolytic enzymes, peptidases, toxins,
oxidation/reduction, and apoplastic elicitors or effectors, and that
play a crucial role in infection and pathogenicity [
6
-
8
].
To understand their interaction mechanism, the genome-geared
high-throughput technologies, such as transcriptomics, proteomics,
and metabolomics, have been conducted [
5
,
9
,
10
]. Plant proteomic
studies have been performed during its growth and development,
and against biotic and abiotic stresses. However, little report is avail-
able on
in planta
secretome investigation of rice leaves infected with
pathogen. The success of the 2-DE technology for proteome analy-
sis will rely on the ability to display more rare proteins and insoluble
proteins. In this chapter, we have reported to provide step-by-step
protocol for protein analyses of rice and pathogen during rice-
pathogen interaction through phenol, PEG fractionation, and
secreted protein extraction method.
2
Materials
1.
M. oryzae
(compatible, incompatible race).
2. Petri dish.
3. Sterile glass beaker.
4. Rice bran agar medium (25 g/L rice bran powder, 1 g/L sucrose,
and 20 g/L agar).
2.1 Preparation of
Magnaporthe oryzae
5. Incubator (28 °C).
2.2 Preparation of
Xanthomonas oryzae
1.
Xanthomonas oryzae
pv.
oryzae
(compatible, incompatible races).
2. Petri dish.
3. PSA plate; 1 % w/v peptone (Becton and Dickinson, Franklin
Lakes, NJ, USA), 1 % w/v sucrose, and 1.5 % w/v agar.
4. PS liquid medium; 1 % w/v peptone, 1 % w/v sucrose.
5. 0.01 % Tween-20 (Amersco, Solon, OH, USA).
6. Incubator (30 °C).
1. Dry mature seeds of rice (
Oryzae sativa
L.) (
see
Note 1
).
2. Washing solution: 70 % ethanol in distilled or Milli Q water.
3. Sterilizing solution: 3 % sodium hypochlorite (Junsei Chemical,
Tokyo, Japan) in distilled or Milli Q water.
4. Humidity growth chamber (Daehan, Seoul, Korea) with white
fl uorescent light (wavelength 390-500 nm, 150
2.3 Preparation
of Host Plant
mol/m/
2
/s,
16 h light/8 h dark cycles) and 70 % relative humidity at 28 °C.
5. Soil (Pot mix for rice) (
see
Note 2
).
μ
