title: Hunting down fungal secretomes using liquid-phase IEF prior to high resolution 2-DE.
abstract: 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.
authors: INRA, UMR1202 BIOGECO, F-33612 Cestas, France. vincent@pierroton.inra.fr
authors: Delphine
authors: D
authors: Vincent
authors: Marie-Hélène
authors: MH
authors: Balesdent
authors: Julien
authors: J
authors: Gibon
authors: Stéphane
authors: S
authors: Claverol
authors: Delphine
authors: D
authors: Lapaillerie
authors: Anne-Marie
authors: AM
authors: Lomenech
authors: Françoise
authors: F
authors: Blaise
authors: Thierry
authors: T
authors: Rouxel
authors: Francis
authors: F
authors: Martin
authors: Marc
authors: M
authors: Bonneu
authors: Joëlle
authors: J
authors: Amselem
authors: Victoria
authors: V
authors: Dominguez
authors: Barbara J
authors: BJ
authors: Howlett
authors: Patrick
authors: P
authors: Wincker
authors: Johann
authors: J
authors: Joets
authors: Marc-Henri
authors: MH
authors: Lebrun
authors: Christophe
authors: C
authors: Plomion
mesh_terms: 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
chemical_list: D005656:Fungal Proteins; D010446:Peptide Fragments