Extraction of proteins for two-dimensional gel electrophoresis and proteomic analysis from an endophytic fungus

Abstract

Piriformospora indica is a mycorrhiza like fungus of the order Sebacinales,which colonizes root and forms symbiosis with almost all terrestrial plants on earth. Information about the P. indica quantitative proteomics is limited.This limits our understanding of its multitrophic interaction with plants. A quantitative proteomic analysis of the fungus vis a vis its interaction with rhizospheric bacteria and the plant can reflect on the underlying proteins/enzymes. We describe a protocol for efficient extraction of cellular proteins from the fungus for two-dimensional gel electrophoresis following interaction with Azotobacter chroococcum in axenic culture. Polysaccharides and abundant salts present in Hill and Kaefer broth hinders extraction of protein from fungal biomass. Good quantity and quality of proteins are required for two-dimensional gel electrophoresis and proteomic analysis. We could use the extracts for proteomic analysis following a high resolution of 2-DE reference map of the fungus after interaction with A. chroococcum.

Bacterial endophytes: Molecular interactions with their hosts

“…Using a direct lysis strategy, total protein content can directly be extracted from endosphere to study the different metabolite production by endophytes. In the indirect lysis method, total protein content can be extracted from pre-isolated endophytes [58]. Furthermore, total proteins of host plants in the presence and absence of endophytes can also be compared to identify actual specific proteins involved in interactions between the two partners.…”

Mass Spectrometry-Based Technology and Workflows for Studying the Chemistry of Fungal Endophyte Derived Bioactive Compounds

“…Protein extraction and their analysis was done using 2-DE for tracing the underlying proteins/enzymes involved in the interaction of fungal endophyte Piriformospora indica DSM 11827 with rhizobacterium Azotobacter chroococcum and with the terrestrial plants. The study reflected the effect of bacterium on the fungal endophyte physiology and growth . LC-MS based metabolomics have elucidated the potential of fungal endophyte Geomyces sp.…”

“…The study reflected the effect of bacterium on the fungal endophyte physiology and growth. 178 LC-MS based metabolomics have elucidated the potential of fungal endophyte Geomyces sp. of plant Nerium indicum to produce vincamine and vincamine analogues.…”