Hunting down fungal secretomes using liquid-phase IEF prior to high resolution 2-DE.
The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1-DE and 2-DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2-DE. Compared with the previously published protocols for which only dozens of 2-D spots were recovered from fungal secretome samples, up to approximately 2000 2-D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, beta-glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid-phase IEF to any fungal extracts.
Vincent
Delphine
D
INRA, UMR1202 BIOGECO, F-33612 Cestas, France. vincent@pierroton.inra.fr
Balesdent
Marie-Hélène
MH
Gibon
Julien
J
Claverol
Stéphane
S
Lapaillerie
Delphine
D
Lomenech
Anne-Marie
AM
Blaise
Françoise
F
Rouxel
Thierry
T
Martin
Francis
F
Bonneu
Marc
M
Amselem
Joëlle
J
Dominguez
Victoria
V
Howlett
Barbara J
BJ
Wincker
Patrick
P
Joets
Johann
J
Lebrun
Marc-Henri
MH
Plomion
Christophe
C
D001203:Ascomycota / Q000737:chemistry; D003956:Dialysis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005612:Freeze Drying; D005656:Fungal Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D007525:Isoelectric Focusing / Q000379:methods*; D055399:Laccaria / Q000737:chemistry; D025282:Mycelium / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
D005656:Fungal Proteins; D010446:Peptide Fragments