A mutualistic fungal symbiont of perennial ryegrass contains two different pyr4 genes, both expressing orotidine-5′-monophosphate decarboxylase

Abstract

A fragment of the Claviceps purpurea pyr4 gene, encoding orotidine-5′-monophosphate decarboxylase (OMP decarboxylase), was used to screen a genomic library from an isolate of a fungus, Acremonium sp. (designated Lpl), which grows as an endophyte in perennial ryegrass ( Lolium perenne). Three positive clones, λMC11, λMC12 and λMC14, were isolated. Two of these clones, λMC12 and λMC14, were overlapping clones from the same locus, while λMCll 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 Pyrenomycete fungi. The pyr4 gene from λMC12 and λMC14 was designated pyr4-1 and that from λMC11 was designated pyr4-2. The predicted ORFs of the two genes were highly conserved, with 97.5% identity at the nucleotide level, the 5′ non-coding sequences were the least conserved with 88.5% identity and the 3′ non-coding sequences had 93.0% identity. RT-PCR analysis of total RNA from Lpl demonstrated that transcripts from the two genes were present at similar levels, and hybridisation of pyr4-1 to Northern blots of total RNA from Lpl showed that full-length transcripts were being produced. Genomic fragments containing pyr4 were transformed into a strain of Aspergillus nidulans which has a mutation in pyrG (encoding OMP decarboxylase). Both pyr4-1 and pyr4-2 complemented the pyrG mutation in A. nidulans, indicating that both encode functional OMP decarboxylases. It has been proposed [Schardl et al., Genetics 136 (1994) 1307–1317] that the two pyr4 in Lpl arose by interspecific hybridisation, most likely between the ryegrass choke pathogen, Epichloë typhina, and another endophyte from perennial ryegrass, Acremonium lolii. Analysis by PCR amplification and direct sequencing of the variable 5′ non-coding regions of pyr4, from possible ancestors to Lpl supports this hypothesis. Comparisons of these sequences to the 5′ non-coding sequences from pyr4-1 and pyr4-2 demonstrated that E. typhina and A. lolii were the most likely ancestors of the two pyr4 found in Lpl.