Intact spore MALDI-TOF mass spectrometry and proteomic analysis of Puccinia pathogenic fungi.
The aim of this work was to develop a method for the identification of pathogens causing rust diseases of crops using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of intact cells or spores (IC/IS). All optimizations were performed with Puccinia triticina, the causal agent of wheat leaf rust. Experiments included selection of washing solvents for spores, finding of an optimal concentration of spores in suspension and the most suitable matrix system as well as an evaluation of different sample preparation techniques. The best results were obtained when the spores were washed with acetonitrile/0.1% (v/v) trifluoroacetic acid, 7:3, v/v. A mixture of ferulic and sinapinic acids (5:15mgml(-1)) dissolved in acetonitrile/2.5% (v/v) trifluoroacetic acid, 7:3, v/v, was found optimal for the deposition of samples (50μg spores per μl) by two-layer volume technique. The optimized protocol was subsequently applied to other Puccinia species (Puccinia graminis, Puccinia striiformis and Puccinia coronata). Together with the use of the software BIOSPEAN, not only different species but also various pathotypes of the same species, which differ in their virulence, could be discriminated. There were 108 and 29 proteins identified from P. striiformis and P. graminis spores, respectively, after an acidic extraction in the matrix solvent mimicking the sample preparation for MALDI. Besides the presence of ribosomal proteins, histones, regulatory proteins and enzymes, also extracellular proteins participating in the pathogenesis were found. Finally, for both species, several proteins were assigned to signals in typical mass spectrometric profiles and suggested as diagnostic markers.
Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, CZ-783 71 Olomouc, Czech Republic.
Jana
J
Beinhauer
Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, CZ-783 71 Olomouc, Czech Republic.
Martin
M
Raus
Department of Genetics and Plant Breeding Methods, Crop Research Institute, Drnovská 507/73, CZ-161 06 Praha 6, Ruzyně, Czech Republic.
Alena
A
Hanzalová
Selgen a. s., Breeding Station Stupice, Stupice 24, 250 84 Sibřina, Czech Republic.
Pavel
P
Horčička
Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, CZ-783 71 Olomouc, Czech Republic. Electronic address: marek.sebela@upol.cz.
Marek
M
Šebela
D000097:Acetonitriles / Q000737:chemistry; D001487:Basidiomycota / Q000737:chemistry*; D003373:Coumaric Acids / Q000737:chemistry; D018556:Crops, Agricultural / Q000382:microbiology; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification*; D006657:Histones / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000382:microbiology; D040901:Proteomics; D012269:Ribosomal Proteins / Q000235:genetics / Q000302:isolation & purification*; D012997:Solvents / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013172:Spores, Fungal / Q000737:chemistry*; D014269:Trifluoroacetic Acid / Q000737:chemistry; D014908:Triticum / Q000382:microbiology*
D000097:Acetonitriles; D003373:Coumaric Acids; D005656:Fungal Proteins; D006657:Histones; D012269:Ribosomal Proteins; D012997:Solvents; C073734:sinapinic acid; C004999:ferulic acid; D014269:Trifluoroacetic Acid; C032159:acetonitrile
Biotyping; Intact cell/spore mass spectrometry; MALDI-TOF; Pathotype; Protein identification; Rust