Biology Reference
In-Depth Information
The use of proteomics also allows location-specifi c analyses (i.e.,
subproteomes at the level of organelles, cell membranes, cell wall,
secretory proteins), the study of posttranslational modifi cations [
7
],
as well as the study of interactions of host-pathogen [
8
] or host-
pathogen-biocontrol agents [
9
,
10
].
Proteomics involves the combined application of (a) advanced
gel-based separation, such as one- and two-dimensional electro-
phoresis (1-DE and 2-DE), or gel-free based in liquid chromatog-
raphy (LC) techniques; (b) identifi cation techniques based on
mass-spectrometry (MS) analysis; and (c) bioinformatics tools to
characterize the proteins in complex biological mixtures [
3
,
4
].
Different fi elds can be defi ned in proteomics, including
descriptive and differential expression proteomics. In the case of
fungi, a new area can also be defi ned as secretomics (the secretome
is defi ned as the combination of native proteins and cell machinery
involved in their secretion), since many fungi secrete an arsenal of
proteins to accommodate their saprotrophic lifestyle, such as pro-
teins implicated in the adhesion to the plant surface, host-tissue
penetration, and invasion effectors, together with other virulence
factors [
11
]. Over the last years, there has been a great advance in
fungal proteomic research due to the availability of powerful pro-
teomics technologies and the increasing number of fungal genome
sequencing projects. Currently, more than 20 plant pathogenic
fungal genomes have been sequenced (Broad Institute Database,
http://broadinstitute.org/science/project/fungal-
genomeinitiative
)
, and excellent reviews of fungal proteomics
methodologies have been recently published [
3
,
4
,
12
]. The work-
fl ow of a fungal gel-based proteomics experiment includes, among
others, the following steps (Fig.
1
): experimental design, fungal
growth, sampling, sample preparation, protein extraction, separa-
tion, MS analysis, protein identifi cation, statistical analysis of data,
quantifi cation, and data analysis, management, and storage.
Most plant pathogenic fungi are fi lamentous and can be con-
sidered, just like plants, as recalcitrant biological material due to
their robust cell wall accounting for 50-60 % of polysaccharides
(glucans), 20-30 % of glycoproteins (mannoproteins), and 10-20 %
of chitin [
13
]. Protein sample preparation is a critical step. The cell
breakdown and later protein extraction are diffi cult because of the
presence of a cell wall that makes up the majority of the cell mass
[
13
]. Cell disruption can be performed using mechanical lysis via
glass beads [
14
-
16
], with a cell mill [
17
] or by sonication [
18
-
20
].
These methods are more effi cient than those based on chemical or
enzyme extraction [
21
]. An alternative approach to avoid the dif-
fi culty of lysing the fungal cell wall might be the generation of
protoplasts (cells whose wall has been completely or partially
removed using either mechanical or enzymatic means) [
22
]. The
most widely used method for cell disruption is pulverizing the
mycelium in liquid nitrogen using a mortar and pestle [
3
,
4
].
