Loline Biosynthesis Gene Expression by Epichloë Fungi Grown under In Vivo and In Vitro Conditions

It is known that the fungus, Epichloë coenophiala, produces alkaloids, such as lolines, in response to physical damage to its host. I explored presence of the lol-cluster genes (Lol A1, A2, C2, D2, E2, F2, O2, T2, P2, U2) in cultured Epichloë isolates and the inducibility of lol cluster gene expression within Tall Fescue grass (Schedonorus arundinaceus). Isolated cultures of the fungus were procured from Rutgers University and University of Kentucky (UK); additionally, cultures were isolated at Hope College (HC) to be used in the on-site study. RNA was extracted using the Quiagen RNeasy kit following the manufacturer’s protocol. Samples were analyzed using qPCR, to test for expression of specific lol-cluster genes, as well as B-tubulin as a control. Previous studies had only looked for the expression of Lol C in vitro, and had not shown positive results. Ours is the first study to show expression in vitro of lol-cluster genes. Lol A2, E2 and T2 expressed in samples from HC and UK cultures as well as samples drawn in vivo from Tall Fescue grass. Samples from Tall Fescue were collected before it was physically damaged, and then again one week after physical damage. The in vivo Tall Fescue grass samples indicated the inducibility of A2 and O2 (up regulation) and E2 and T2 (down regulation). These indications of inducibility merit further study as lolines can influence preference and performance of herbivores of Tall Fescue