Cloning, characterisation and evolutionary relationships of two pyr4 genes from an Acremonium endophyte of perennial ryegrass : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Genetics at Massey University, Palmerston North, New Zealand
A fragment of the Claviceps purpurea pyr4 gene, encoding the enzyme orotidine-5'-monophosphate decarboxylase (OMPdecarboxylase) was used to screen a genomic library to an isolate (designated Lp1) of an Acremonium sp. which grows as an endophyte in perennial ryegrass (Lolium perenne). Four positive clones, λMC11, λMC12, λMC14 and λMC20 were isolated. Three of these clones, λMC12, λMC14 and λMC20 were overlapping clones from the same locus, while λMC11 was from a different locus. Fragments of these clones which hybridised with C. purpurea pyr4 were sequenced and found to have similarity with pyr4 from other fungi of the Pyrenomycetes and related Deuteromycetes, suggesting that Lp1 has evolved from a sexual Pyrenomycetes species. The pyr4 from λMC12, λMC14 and λMC20 was designated pyr4-1 and that from λMC11 was designated pyr4-2. The predicted ORFs of the two genes were highly conserved and the 5' non-coding nucleotide sequences were the least conserved regions. RT-PCR and northern analysis of total RNA from Lp1 demonstrated that transcripts approximately 1.4 kb in length were produced from the two genes and present at similar levels. Genomic fragments containing pyr4-1 or pyr4-2 were transformed into a strain of Aspergillus nidulans which has a mutation in the pyrG gene (encoding OMPdecarboxylase). Both of the Lp1 pyr4 complemented a pyrG mutation in Aspergillus nidulans, confirming that both pyr4-1 and pyr4-2 encode functional OMPdecarboxylases. Comparisons of pyr4 restriction fragment length polymorphisms (RFLPs) from Lp1 and isolates of Epichloë typhina, E. festucae, A. lolii, A. uncinatum, and three endophyte taxonomic groupings from Festuca arundinacea: FaTG-1 (=A. coenophialum), FaTG-2 and FaTG-3 suggested that pyr4-1 originated from E. typhina, the ryegrass choke pathogen, and pyr4-2 originated from A. lolii, another endophyte from perennial ryegrass. This suggested that Lp1 is an interspecific hybrid, between E. typhina and A. lolii. Comparisons of the variable 5' non-coding nucleotide sequences from pyr4 of Lp1 and other isolates demonstrated that E. typhina, and A. lolii or E. festucae were the most likely ancestors of the two pyr4 found in Lp1. The A. lolii and E. festucae sequences were very similar, suggesting they are closely related. A. lolii has most probably evolved from an E. festucae, and in the process lost the sexual cycle. Analysis of single spore purified isolates of Lp1 revealed that Lp1 was a homokaryon for pyr4. A Southern blot of a CHEF gel of Lp1 and these single spored isolates was hybridised to a pyr4 probe and demonstrated that pyr4-1 and pyr4-2 were present on either two chromosomes of similar size, or one chromosome. The hybridisation that gave rise to Lp1 was concluded to have been a relatively recent event, given the similarity of pyr4-1 and pyr4-2 nucleotide sequences to those of their probable ancestors, and the fact that both genes are expressed and functional. Interspecific hybridisation is probably widespread in the asexual endophytes, and may be an important event in their evolution, and the evolution of other fungal species.