Biology Reference
In-Depth Information
phytopathology [
5
]. Nevertheless, its application is limited to
obtain a quality protein extract free from non-protein contami-
nants, such as polysaccharides and/or polyphenol compounds,
which can interact in a nonspecifi c way with proteins, affecting
their migration and therefore the resolution in the protein separa-
tion. Taking into account that the presence of such contaminants
is determined partly by the correct combination of plant tissue and
extraction method [
6
,
7
], it is necessary to evaluate the different
protocols that lead to fi nd the best conditions for protein extrac-
tion when a proteomics experiment in general is carried out for the
fi rst time. It is important that the methodology used provides a
high yield in the protein extraction and a high sensitivity in the
staining process, because of the proteins associated to plant defense
mechanisms are generally low abundant, and require the highest
possible sensitivity to be detected with alternative types of staining
different than the commonly used [
8
]. The study about defense
responses in plants against a determined pathogen can be per-
formed comparing the biochemical responses among genotypes
with resistance differences to the disease [
9
]. Thus, the evaluation
of proteins associated with defense can be done in carnation by
comparing those ones that are differentially induced between sus-
ceptible and resistant varieties during the inoculation with the
pathogen of interest, in this case
Fusarium oxysporum
f. sp.
dianthi
(
Fod
), the agent responsible of vascular wilting. This chapter dis-
cusses the conditions for the protein extraction and separation
required to carry out the proteome analysis using gel-based pro-
teomic techniques (one-dimensional electrophoresis (1-DE) and
2-DE), in carnation stems and roots during the infection with
Fod
.
The employed workfl ow is described and discussed, and may be
applied in other plant species.
2
Materials
Mention of specifi c companies or pieces of equipment does not
represent an endorsement by the authors.
2.1 Plant Material
Rooted cuttings of carnation cultivars Candy and Tasman, resistant
and susceptible respectively to the vascular wilting caused by
Fod
,
were used for this study (supplied by the company Grupo Chia,
Colombia).
Fusarium oxysporum
f. sp.
dianthi
race 2 was isolated from stems of
a susceptible carnation plant with typical symptoms of vascular
wilt. Typical conidia morphology was evaluated microscopically
and the specie [
10
] and race [
11
] were verifi ed using molecular
markers.
2.2 Fungal Isolate
