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Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani

     Floral and Nursery Plants Research Unit, USDA-ARS, 10300 Baltimore Avenue, Beltsville, Maryland 20705

Savithiry S. Natarajan
Sukla Lakshman

     USDA-ARS Soybean Genomics and Improvement Laboratory, Building 006, Room 203, BARC West, Beltsville, Maryland 20705

Wesley M. Garrett

     Animal Biosciences and Biotechnology Laboratory, 10300 Baltimore Avenue, Building 200, Beltsville, Maryland 20705

Arun K. Dhar

     Advanced Bionutrition Corp., 6430 Dobbin Road, Columbia, Maryland 21045

Rhizoctonia solani (Teleomorph: Thanatephorus cucumeris, T. praticola) is a basidiomycetous fungus and a major cause of root diseases of economically important plants. Various isolates of this fungus are also beneficially associated with orchids, may serve as biocontrol agents or remain as saprophytes with roles in decaying and recycling of soil organic matter. R. solani displays several hyphal anastomosis groups (AG) with distinct host and pathogenic specializations. Even though there are reports on the physiological and histological basis of Rhizoctonia-host interactions, very little is known about the molecular biology and control of gene expression early during infection by this pathogen. Proteomic technologies are powerful tools for examining alterations in protein profiles. To aid studies on its biology and host pathogen interactions, a two-dimensional (2-D) gel-based global proteomic study has been initiated. To develop an optimized protein extraction protocol for R. solani, we compared two previously reported protein extraction protocols for 2-D gel analysis of R. solani (AG-4) isolate Rs23. Both TCA-acetone precipitation and phosphate solubilization before TCA-acetone precipitation worked well for R. solani protein extraction, although selective enrichment of some proteins was noted with either method. About 450 spots could be detected with the densitiometric tracing of Coomassie blue-stained 2-D PAGE gels covering pH 4–7 and 6.5–205 kDa. Selected protein spots were subjected to mass spectrometric analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Eleven protein spots were positively identified based on peptide mass fingerprinting match with fungal proteins in public databases with the Mascot search engine. These results testify to the suitability of the two optimized protein extraction protocols for 2-D proteomic studies of R. solani.

Key words: MALDI-TOF-MS, proteomics, Rhizoctonia solani, Thanatephorus cucumeris, 2-D PAGE