Biochemical and Genetic Basis of Ergot Alkaloid Diversification

In support of Obiective l: la) Provide samples of Neotyphodium sp. Lpl endophyte-infected and endophyte-free perennial ryegrass pseudostems, leaf blades, and seeds from which collaborators at WVU will extract ergot alkaloids. lb) Perform, on sub samples of the tissues listed in item la, quantitative real-time PCR analyses of five genes in the ergot alkaloid pathway, as well as appropriate control genes. In support of Objective 2: 2a) Introduce Neotyphodium sp. Lpl transformants (provided by WVU collaborators and containing constructs designed for over-expressing easA or for interfering with easA transcript accumulation) into endophyte-free perennial ryegrass and cultivate infected plants. Samples of plant material will be returned to WVU collaborators for ergot alkaloid analysis. 2b) With samples from modified endophyte-infected plants described in item 2a and samples from control plants containing wild-type endophyte, perform quantitative real-time PCR experiments to quantify easA mRNA and dmaW mRNA relative to mRNA from appropriate control genes.