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

Young, Carolyn Anne

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

dc.contributor.author	Young, Carolyn Anne
dc.date.accessioned	2010-09-23T01:23:07Z
dc.date.available	NO_RESTRICTION	en_US
dc.date.available	2010-09-23T01:23:07Z
dc.date.issued	2005
dc.description	Content removed due to copyright: Young, C. A., Bryant, M. K., Christensen, M. J., Tapper, B. A., Bryan, G. T., & Scott, B. (2005). Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass. Molecular Genetics and Genomics, 274(1), 13-29.	en_US
dc.description.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.	en_US
dc.identifier.uri	http://hdl.handle.net/10179/1670
dc.language.iso	en	en_US
dc.publisher	Massey University	en_US
dc.rights	The Author	en_US
dc.subject	Neotyphodium lolii	en_US
dc.subject	Plant molecular genetics	en_US
dc.subject.other	Fields of Research::270000 Biological Sciences::270200 Genetics	en_US
dc.title	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	en_US
dc.type	Thesis	en_US
massey.contributor.author	Young, Carolyn Anne
thesis.degree.discipline	Molecular Biology	en_US
thesis.degree.grantor	Massey University	en_US
thesis.degree.level	Doctoral	en_US
thesis.degree.level	Doctoral	en
thesis.degree.name	Doctor of Philosophy (Ph.D.)	en_US