Biology Reference
In-Depth Information
Chapter 39
Protein Extraction and Gel-Based Separation Methods
to Analyze Responses to Pathogens in Carnation
(
Dianthus caryophyllus
L)
Harold Duban Ardila , Raquel González Fernández , Blanca Ligia Higuera ,
Inmaculada Redondo , and Sixta Tulia Martínez
Abstract
We are currently using a 2-DE-based proteomics approach to study plant responses to pathogenic fungi by
using the carnation (
Dianthus caryophyllus
L)-
Fusarium oxysporum
f. sp.
dianthi
pathosystem. It is clear
that the protocols for the fi rst stages of a standard proteomics workfl ow must be optimized to each biologi-
cal system and objectives of the research. The optimization procedure for the extraction and separation of
proteins by 1-DE and 2-DE in the indicated system is reported. This strategy can be extrapolated to other
plant-pathogen interaction systems in order to perform an evaluation of the changes in the host protein
profi le caused by the pathogen and to identify proteins which, at early stages, are involved or implicated in
the plant defense response.
Key words
Carnation, Fusarium, Plant defense, Plant-pathogen interaction proteomics
1
Introduction
Carnation (
Dianthus caryophyllus
L) is one of the most important
products in commercial fl owers production worldwide. The
researches carried out on this plant have been mainly focused on
the molecular study of the processes associated to the biosynthesis
of pigments and senescence [
1
,
2
]. However, the studies at a bio-
chemical level for this species have been too limited and there are
not any established protocols that allow analysis at a proteomic
level. Protein analysis using two-dimensional electrophoresis
(2-DE) is one of the most used tools for the analysis of proteomes
in plants and has allowed the study of a large number of biological
processes such as the interactions with pathogen microorganisms
[
3
,
4
]. In recent years, the descriptive analysis of proteins involved
in interactions with pathogens, using 2-DE and subsequent mass
spectrometry (MS) identifi cation, has become an important tool in
