The Global Regulatory Protein VelA Is Required for Symbiosis Between the Endophytic Fungus Epichloë festucae and Lolium perenne.

Epichloë species fungi form bioprotective endophytic symbioses with many cool-season grasses, including agriculturally important forage grasses. Despite its importance, relatively little is known about the molecular details of the interaction and the regulatory genes involved. The conserved velvet-domain protein VelA (or VeA) is a global regulator of a number of cellular and developmental functions in fungi. In this study, the E. festucae velA gene was functionally characterized in vitro and during interaction with perennial ryegrass. The velA gene is required in E. festucae for resistance to osmotic and cell wall-damaging stresses, repression of conidiation, and normal hyphal morphology during nutrient-limited in-vitro conditions. Expression of velA in E. festucae is light- and nitrogen-dependent and is tissue-specific in mature infected plants. In-planta studies showed that velA is required in E. festucae for a compatible interaction. Inoculating seedlings with mutant ΔvelA induced callose deposition and H2O2 production, and a high level of seedling death was observed. In surviving plants infected with ΔvelA mutant fungi, plants were stunted and we observed increased biomass and invasion of vascular bundles. Overall, this work characterizes a key fungal regulatory factor in this increasingly important model symbiotic association.

Efficient targeted mutagenesis in Epichloë festucae using a split marker system.

A split-marker system for targeted gene deletion was developed for the model grass endophytic fungus Epichloë festucae. Compared to the conventional system that yields up to 25% homologous recombinants, the method resulted in 33-74% targeted deletions in E. festucae using as little as 1.5kb of targeting sequence.

Agrobacterium-mediated transformation of white clover using direct shoot organogenesis.

White clover (Trifolium repens L.) plants from the cultivars Grasslands Huia and Grasslands Tahora have been transformed using Agrobacterium-mediated T-DNA transfer. Transgenic plants regenerated directly from cells of the cotyledonary axil. To transform white clover, shoot tips from 3 day old seedlings were co-cultivated with A. tumefaciens strain LBA4404 carrying the plasmid vector pPE64. This vector contains the neomycin phosphotransferase II gene (nptII) and ß-glucuronidase reporter gene (gus) both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 42 days after transfer onto selective media. Integration of the nptII and gus genes into the white clover genome was confirmed using Southern blotting, and histochemical analysis indicated that the gus gene was expressed in a variety of tissues. In reciprocal crosses between a primary transformant and a non-transformed plant the introduced gus gene segregated as a single dominant Mendelian trait.