Symbiotic Fungi: Principles and Practice (Soil Biology)

244 E. Dumas-Gaudot et al.
TEMED N,N,N 0 ,N 0 -Tetramethylethylenediamine
Tris Tris-[hydroxymethyl] aminomethane
US DOE US Department of Energy
15.1 Introduction
The achievement of genome sequencing programs launched for numerous organisms,
including those of several plant, bacteria and fungal species (http://www.ncbi.nih.gov/,
http://www.energy.gov/), together with the increase in available ESTs corresponding
to various biological situations of plants, bacterial and fungal models, have opened the
way for functional genomic analyses. Among them, proteomics looks very promising,
as it gives a direct access to the gene effectors, i.e., proteins.
Proteomics, according to its first definition by Wilkins et al. (1996), designs
the new strategies aimed at researching global protein expression in different
organisms. Thanks to the great improvements in protein separation methods,
development of mass spectrometry techniques and advances in bioinformatic
tools, it has become more and more popular during the last decade. Proteomics
relies to a major extent on experimental analyses to identify and elucidate
proteins. One of the major experimental methods used in protein identification
is two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry
(MS). 2-DE/MS systems have the ability to identify a large number of proteins from
a single sample. Studies have shown that 2-DE/MS systems could identify somewhere
in the region of thousands of proteins per sample (Harry et al. 2000; Görg
et al. 2000). Their possible use for revealing protein modifications in response to
root colonisation by arbuscular mycorrhizal (AM) fungi has already been reported
several times (Bestel-Corre et al. 2004a; Canovas et al. 2004; Jorrin et al. 2006).
Surprisingly, to date, for the other well-known symbiosis, i.e., the ectomycorrhizal
interaction, use appears much more restricted (listed in Canovas et al.
2004). This situation could change very quickly, due to the fact that genomes of
Populus trichocarpa, the first perennial plant to be tackled, and its microcosmassociated
partners are being fully sequenced (Martin et al. 2004). While the
general protocols for successfully performing plant and/or microbe proteomics
have been largely described (Chen et al. 2005; Giavalisco et al. 2003; Grinyer
et al. 2004; Hajheidari et al. 2005; Rose et al. 2004; Saravanan and Rose 2004), the
aim of this chapter will be to address the specific points that have to be considered
when applying a proteomic approach to the study of mycorrhizal symbioses.
AM fungi colonize the vast majority of plant species, yet unlike the ectomycorrhizal
fungi, are incapable of growth without a symbiotic host. Therefore, a first
challenge will deal with the fact that the fungal partner is an uncultivable microorganism.
The second relates to the lack of genomic sequences in database, a hitch
that will soon be bypassed (Lammers et al. 2004). The general strategy we are using
routinely in our laboratory is presented on Fig. 15.1.