Molecular Methods for Studying the Cryphonectria parasitica – Hypovirus Experimental System

Abstract

The interaction of the filamentous fungal plant pathogen Cryphonectria parasitica with its virulence-attenuating viruses provides a unique platform to explore the molecular biology and genetics of virus-host interactions. Following the development of transformation procedures for this fungus, subsequent advances include infectious cDNA clones of several members of the Hypoviridae and an imminently complete fungal genome project. Presented here are basic protocols for growth of the organism and the extraction of DNA, RNA, and protein. Additionally, two further protocols are provided for investigations of host protein phosphorylation and for viral genome secondary structure.

A Proteomics Approach to Study Soybean and Its Symbiont Bradyrhizobium japonicum –A Review

“…In brief, flash frozen leaf samples are ground in liquid nitrogen and polyphenols; tannins and other interfering substances such as chlorophyll are removed. The processed tissue is resuspended in a chaotropic reagent to extract proteins in the upper phase, and the plant debris is discarded [64][65][66][67][68][69][70]. The total protein set, in the resulting solution, is further quantified using the Lowry method [71].…”

Conserved and variable structural elements in the 5′ untranslated region of two hypoviruses from the filamentous fungus Cryphonectria parasitica

“…Nuss (University of Maryland) that contain full-length cDNA copies of the positive sense strand of CHV1-EP713 (Choi and Nuss, 1992) or CHV1-Euro7, respectively. Detailed protocols for RNA isolation and analysis are described in detail elsewhere (Dawe et al, 2011). Briefly, 1 μg pLDST and pTE7 were linearized with SpeI (New England Biolabs) and transcribed with T7 RNA polymerase (AmpliCap ™ T7 and SP6 kits, Epicenter Technologies) respectively, then purified using an RNeasy Mini Kit (QIAGEN Inc.).…”