KSEF RDE: Remodeling Alkaloid Architecture in Forage Grass Endophytes

Several very important cool-season forage and turf grasses rely on naturally symbiotic fungi (Epichloe species; endophytes) for fitness and longevity in the field. For example, the endophytes of tall fescue (TF) and perennial ryegrass (PRG) transmit solely and efficiently via seeds as if they are organelles of the plant, and are necessary for pasture sustainability in large portions of the U.S., Australia and New Zealand. A major endophyte benefit is production of anti-invertebrate alkaloids such as peramine (in TF and PRG) and the especially effective lolines (in TF), but common endophyte strains also produce alkaloids that are toxic to livestock; namely, ergot alkaloids (in TF and PRG) and indole-diterpenes (in PRG). Technologies have been developed to culture and transfer endophytes with desired characteristics - including particular alkaloid profiles - for commercial TF and PRG cultivar development. However, despite over a decade of efforts in the U.S., Japan, Australia and New Zealand, efforts to produce a stable symbiosis of PRG with loline-producing endophytes have not succeeded due to host specificities of the endophytes. As an alternative approach, we have developed novel molecular genetic methods for targeted removal of ergot-alkaloid and indole-diterpene biosynthesis genes, and have done so with no net introduction of genes from sources other than Epichloe species in order to obviate regulatory and public concerns. Furthermore, we have characterized the loline-alkaloid-biosynthesis pathway and genes, and now know that 5-7 genes are sufficient for production of lolines. Our proposal is to refine our methods for targeted genetic alteration of endophytes in order to replace the ergot-alkaloid and indole-diterpene biosynthesis genes with loline-alkaloid biosynthesis genes. Our expected output will be PRG endophytes that are more sustainable in pastures and suitable for livestock than the endophytes in existing PRG cultivars. The technology can also be used for introduction of other beneficial genes into endophytes.