The indole-diterpene gene cluster from the ryegrass endophyte, Neotyphodium lolii, is required for the biosynthesis of lolitrem B, a bioprotective alkaloid : this thesis is presented as a partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph. D.) in Molecular Biology at Massey University, Palmerston North, New Zealand

Abstract

Lolitrems are indole-diterpene alkaloids produced by Epichloë and Neotyphodium endophytes in association with their host grass Lolium perenne. Some indole-diterpene (ID) alkaloids are proposed to have insecticidal properties, but lolitrem B is known as the causative agent of the animal syndrome ryegrass staggers. Lolitrems are preferentially synthesised in planta. which suggests that the genes required for lolitrem biosynthesis are symbiotically expressed. The lolitrem biosynthesis pathway has been proposed as a metabolic grid based on the identification of likely intermediates from endophyte-infected ryegrass. Closely related ID compounds are expected to serve as substrates for the same enzyme, but until recently these steps had not been validated. The identification and characterisation of a Petticillium paxilli gene cluster required for the synthesis of the ID paxilline has identified key enzymes required for the production of the ID backbone. Based on the similarity of lolitrem B to paxilline it was proposed that these two biosynthesis pathways would share orthologous early steps but later steps to convert paxilline to the more complex lolitrem B would require additional enzymes. The lolitrem biosynthesis genes (ltm) were isolated using degenerate PCR and from candidate genes identified as ESTs in cDNA libraries. Ten ltm genes were identified that had functions consistent with those required for lolitrem B biosynthesis. The 10 ltm genes were contained on three gene clusters that are separated by repetitive AT-rich sequences that contain remnants of retrotransposons. The ltm clusters 1 and 2 contain eight genes, seven of which are orthologues of the characterised P. paxilli paxilline biosynthesis gene cluster (pax). Functional characterisation of ltmM an FAD-dependent monooxygenase and ltmC a prenyl transferase confirmed these two genes were required for ID biosynthesis and were orthologues of paxM and paxC, respectively. All 10 ltm genes have similar expression profiles and were highly expressed in planta where the production of lolitrem B is most prevalent. The taxonomic distribution of the ltm genes has established which endophyte strains are likely to produce ID compounds. This work provides the basis for elucidation of the lolitrem biochemical pathway and opens the way for determining how the plant regulates the synthesis of this important group of bioprotective molecules.