Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani

Dilip K. Lakshman, Savithiry S. Natarajan, Sukla Lakshman, Wesley M. Garrett, Arun K. Dhar

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

Rhizoctonia solani (Teleomorph: Thanate-phorus 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 solubiliza-tion 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/ioniza-tion 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 2D proteomic studies of R. solani.

Original languageEnglish (US)
Pages (from-to)867-875
Number of pages9
JournalMycologia
Volume100
Issue number6
DOIs
StatePublished - Nov 2008
Externally publishedYes

Keywords

  • 2-D PAGE
  • MALDI-TOFS-MS
  • Proteomics
  • Rhizoctonia solani
  • Thanatephorus cucumeris

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Physiology
  • Molecular Biology
  • Genetics
  • Cell Biology

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  • Cite this

    Lakshman, D. K., Natarajan, S. S., Lakshman, S., Garrett, W. M., & Dhar, A. K. (2008). Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani. Mycologia, 100(6), 867-875. https://doi.org/10.3852/08-065