Proteomic investigation of Rhizoctonia solani AG 4 identifies secretome and mycelial proteins with roles in plant cell wall degradation and virulence

Authors: Lakshman, Dilip; Roberts, Daniel; Natarajan, Savithiry - Savi; Garrett, Wesley; ALKHAROUF, NADIM - Towson University; DARWISH, OMAR - Towson University; Khan, Farooq; PAINE, ARNAB - King Abdullah University Of Science And Technology; MITRA, AMITAVA - King Abdullah University Of Science And Technology

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2016
Publication Date: 3/28/2016
Citation: Lakshman, D.K., Roberts, D.P., Natarajan, S.S., Garrett, W.M., Alkharouf, N., Darwish, O., Khan, F.H., Paine, A., Mitra, A. 2016. Proteomic investigation of Rhizoctonia solani AG 4 identifies secretome and mycelial proteins with roles in plant cell wall degradation and virulence. Journal of Agricultural and Food Chemistry. 64:3101-3110.

Interpretive Summary: Rhizoctonia solani AG 4 is a soilborne fungal plant pathogen that causes economically important diseases on crops in the United States and elsewhere in the world. Soilborne plant pathogens, such as R. solani AG 4, are difficult to control due to their longevity in soil, broad host range, ability to outgrow or evade plant defenses, and the logistics, cost, and sometimes poor efficacy of fungicides. Our long-term goal is to determine the molecular basis of pathogenesis of isolates of R. solani AG 4 in an effort to develop more effective control methods for this important pathogen. Here we used a proteomics approach to characterize the proteins that R. solani AG 4 releases into the environment under several growth conditions; these proteins are important in disease incidence as they degrade and kill plant cells in advance of colonization by this pathogen. We also performed a proteomic analysis of intracellular mycelial proteins from this pathogen grown on Potato Dextrose Broth. This investigation is the most comprehensive undertaking to date regarding proteomic profiles of cellular proteins and proteins released into the environment by a R. solani AG 4 isolate. Proteins were categorized to possible cellular locations and functional groups; for some proteins, putative roles were identified in plant cell wall degradation and virulence, as well in ecology and biology. This information will be useful to scientists determining the molecular basis of pathogenesis by this pathogen. Ultimately this information will be useful to scientists developing more effective disease control methods.

Technical Abstract: Rhizoctonia solani AG 4 is a soilborne necrotrophic fungal plant pathogen that causes economically important diseases on agronomic crops worldwide. Our long-term goal is to elucidate the molecular basis of pathogenesis of isolates of R. solani AG 4 in an effort to develop more effective control methods for this important pathogen. Here we used a proteomics approach to characterize the secretome of R. solani AG 4 isolate Rs23A under several growth conditions; the secretome is important in disease incidence as it degrades and kills plant cells in advance of colonization by this pathogen. Extracellular proteins, comprising the secretome of R. solani, grown on glucose only, glucose plus a soybean seedling, and glucose plus citrus pectin, were resolved on 2-D gels. The first dimension isoelectric focusing (IEF) was performed at pH 4-7, while the second dimension separated proteins within the molecular weight range of 6 to 205 kDa. We also performed a proteomic analysis of intracellular mycelial proteins from this pathogen grown on Potato Dextrose Broth following an optimized phosphate soluble protein purification procedure. Intracellular mycelial proteins were resolved using first dimension IEF at pH 4-7 and also at pH 6-11. The second dimension separated proteins within the molecular weight range of 6 to 205 kDa. From over 500 total secretome and soluble intracellular proteins from 2-D gels, 457 protein spots were processed for MALDI-TOF-MS and MALDI-TOF-MS-MS analysis and 318 proteins were positively matched with fungal proteins of known function by comparison with the three available R. solani genome databases specific for anastomosis groups 1-IA, 1-IB, and 3. These proteins were categorized to possible cellular locations and functional groups; for some proteins, putative roles were identified in plant cell wall degradation and virulence, as well as putative roles in mating, ecology, and biology.