Biology Reference
In-Depth Information
4
Notes
1. Dry mature seeds are stored at 10 °C for long term storage.
2. Any soil can be used if recommended for rice growth and
development.
3. Use clean/sterile equipment and gloves at all times for the
experiments.
4. To get differential levels of expression from both host and
pathogen, preparation of synchronously grown inoculum is
critical. High density infection foci are helpful to get more
interaction events.
5. Rice leaf samples used at 72 h after inoculation with
M. oryzae
were in transition phase of biotrophic and early necrotrophic
stage for minimizing cytoplasmic contamination. Certain pro-
tein markers [Glucose-6-phosphate dehydrogenase (G6PDH),
malate dehydrogenase (MDH), mannosidase, phosphoenol-
pyruvate (PEP) carboxylase, and cytosolic aldolase] can be
used for the purity assessment.
6. The Mg/NP-40 extraction systems produced more resolvable
spots by fractionation of RuBisCO into PEG pellet, which is
about 50 % of total soluble rice leaf protein [
11
].
7. The phenol extraction method can minimize proteolysis and
optimize extraction of membrane proteins. This method results
in the best resolution of proteins on 2-DE gels as removing
non-protein components that interfered with IEF [
12
].
8. Prefractionation of protein samples using PEG prior to 2-DE
can specifi cally fractionate RuBisCO into 15 % PEG pellet.
This method can enrich low-abundance proteins. Advantages
of using PEG are that proteins are fractionated under native
conditions and analysis of 2.7 times more well-separated pro-
teins compared with conventional single-step analysis [
12
].
9. Proteomic studies of secretory proteins have been limited in
any organism, including plants. Moreover, it is yet to be deter-
mined how many proteins are found in the secretome of a
given organism under normal growth and adverse environ-
mental conditions. This
in planta
secretome method serves as
a valuable resource toward developing a near complete secre-
tome of rice-pathogen interaction.
10. The methods, which were based on VIC and gravity-extraction,
have primarily been used to isolate apoplastic fl uid from plant
tissue (mainly leaf) for subsequent preparation of secretory
proteins with little or no contamination from intracellular pro-
teins [
4
]. Both methods suffer from the drawback of low
recovery of secretory proteins and hence require further
improvement in their workfl ow. To improve these problems,
