ARS | Publication request: Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani

Submitted to: Mycopathologia
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 30, 2008
Publication Date: September 1, 2008
Citation: Lakshman, Dilip K., Natarajan, Savithiry S., Garrett, Wesley M., Lakshman, Sukla, Dhar, Arun K. 2008. Optimized protein extraction methods for proteomic analysis of Rhizoctonia solani. Mycopathologia.

Interpretive Summary: Root rot diseases account for the largest percentage of loss in commercial ornamental production and the fungus Rhizoctonia solani is a major cause of root diseases. In addition to important plant pathogens of agricultural crops, isolates of this fungus are beneficially associated with orchids, may serve as biocontrol agents and remain as saprophytes with roles in decaying and recycling of soil organic matter. The pathogen was once successfully controlled by methyl bromide - a pesticide recently banned due to environmental concerns. On the other hand, control of the pathogen is difficult using conventional pesticides, mostly due to lack of sufficient knowledge of its biology and pathology. 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 during infection by this pathogen. Increased understanding of the disease interaction will aid in protection of plants against the pathogen. To our knowledge, there is no investigation so far on the pathogenic mechanism of Rhizoctonia solani at the global protein level. In this vein, we investigated and optimized two protein extraction protocols and resolved several Rhizoctonia solani proteins by two-dimensional gel electrophoresis. In demonstration of the suitability of this technique for global proteomic studies, we identified six gel-separated proteins by a process called peptide mass fingerprint matching with fungal proteins in public databases. These results testify to the suitability of the optimized protein extraction and separation protocols for proteomic studies of Rhizoctonia solani and its pathogenic interactions.

Technical Abstract: Rhizoctonia solani (Teleomorph: Thanatephorus cucumeris) is a basidiomycetous fungus which includes important plant pathogens, saprophytes and mycorrhizae. R. solani displays several hyphal anastomosis groups (AGs) with distinct host plant specializations. In order to facilitate 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 prior to TCA-acetone precipitation worked well for R. solani protein extraction, although selective enrichment of some proteins was noted using either method. Using either method, over 500 spots were detectable in Coomassie Brilliant Blue-stained 2-D protein gels covering pH 4-7 and 6.5 ¿ 205 kDa. Selected protein spots were subjected to mass spectrometric analysis, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Six proteins were identified based on peptide mass fingerprinting match with fungal proteins in public databases using 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.