Molecular methods for studying the Cryphonectria parasitica - hypovirus experimental system.

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.

Phosphorylation of phosducin-like protein BDM-1 by protein kinase 2 (CK2) is required for virulence and G beta subunit stability in the fungal plant pathogen Cryphonectria parasitica.

Phosducin-like proteins are conserved regulatory components of G-protein signalling pathways, which mediate many physiological processes. Identified throughout eukaryotic genomes, they are thought to serve as regulators of G betagamma assembly. Cryphonectria parasitica, a plant pathogen and causative agent of chestnut blight, contains three G alpha, one G beta, one G gamma subunits and phosducin-like protein BDM-1 that have important roles in pigmentation, sporulation and virulence.

Protein phosphatase type 1 directs chitin synthesis at the bud neck in Saccharomyces cerevisiae.

Yeast chitin synthase III (CSIII) is targeted to the bud neck, where it is thought to be tethered by the septin-associated protein Bni4. Bni4 also associates with the yeast protein phosphatase (PP1) catalytic subunit, Glc7. To identify regions of Bni4 necessary for its targeting functions, we created a collection of 23 deletion mutants throughout the length of Bni4.

GLUT1CBP(TIP2/GIPC1) interactions with GLUT1 and myosin VI: evidence supporting an adapter function for GLUT1CBP.

We identified a novel interaction between myosin VI and the GLUT1 transporter binding protein GLUT1CBP(GIPC1) and first proposed that as an adapter molecule it might function to couple vesicle-bound proteins to myosin VI movement. This study refines the model by identifying two myosin VI binding domains in the GIPC1 C terminus, assigning respective oligomerization and myosin VI binding functions to separate N- and C-terminal domains, and defining a central region in the myosin VI tail that binds GIPC1. Data further supporting the model demonstrate that 1) myosin VI and GIPC1 interactions do not require a mediating protein; 2) the myosin VI binding domain in GIPC1 is necessary for intracellular interactions of GIPC1 with myosin VI and recruitment of overexpressed myosin VI to membrane structures, but not for the association of GIPC1 with such structures; 3) GIPC1/myosin VI complexes coordinately move within cellular extensions of the cell in an actin-dependent and microtubule-independent manner; and 4) blocking either GIPC1 interactions with myosin VI or GLUT1 interactions with GIPC1 disrupts normal GLUT1 trafficking in polarized epithelial cells, leading to a reduction in the level of GLUT1 in the plasma membrane and concomitant accumulation in internal membrane structures.