A Complex Ergovaline Gene Cluster in Epichloë Endophytes of Grasses

Abstract

Clavicipitaceous fungal endophytes of the genera Epichloë and Neotyphodium form symbioses with grasses of the subfamily Pooideae, in which they can synthesize an array of bioprotective alkaloids. Some strains produce the ergopeptine alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyteinfected grasses. Cloning and analysis of a nonribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii revealed a putative gene cluster for ergovaline biosynthesis containing a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Despite conservation of gene sequence, gene order is substantially different between the N. lolii, C. purpurea, and A. fumigatus ergot alkaloid gene clusters. Southern analysis indicated that the N. lolii cluster was linked with previously identified ergovaline biosynthetic genes dmaW and lpsA. The ergovaline genes are closely associated with transposon relics, including retrotransposons and autonomous and nonautonomous DNA transposons. All genes in the cluster were highly expressed in planta, but expression was very low or undetectable in mycelia from axenic culture. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.

Endophytes: exploiting biodiversity for the improvement of natural product-based drug discovery

“…Attendant with this, the eas genes proved all to be expressed in planta, while giving no evidence for expression under axenic culture conditions. These results suggest that specific plant environment may be required for the induction of ergopeptine biosynthetic genes in the fungal endophyte (Fleetwood et al 2007).…”

“…Firstly, the association of the genes composing both these endophytic clusters with the transposon relics appears to be of considerable importance (Young et al 2006;Fleetwood et al 2007). The abundance of these relic sequences adds to the evolutionary potential of both clusters.…”

“…Secondly, all ten genes identified at the LTM locus, as well as each gene Figure 14. Comparative eas gene order among C. purpurea (Cp), N. lolii (Nl) and A. fumigatus (Af); black arrows, genes proposed to be required for ergot alkaloid synthesis, but not yet identified for N. lolii; light grey arrows, genes found in N. lolii and C. purpurea, but not in A. fumigatus; dark grey arrows, genes found only in A. fumigatus; N. lolii dmaW, cloA and lpsA are not shown, as their locations relative to EAS cluster are not known (Fleetwood et al 2007). in EAS cluster, have been shown to be highly expressed in planta, but only to a minimal extent or not at all Á in axenic mycelial cultures, strongly suggesting that plant signalling is indispensable to induce their expression (Young et al 2006;Fleetwood et al 2007). However, the specific host stimuli required for the activation of the fungal genes remain to be identified.…”

“…Subsequent complementation experiments resulted in the ultimate confirmation of the postulated role of DMATS in the biosynthetic process in question (Wang et al 2004). Analogous experimental procedure allowed for the characterization of two consecutive genes, displaying the capacity to encode nonribosomal peptide synthetases (NRPS), namely: lpsA (Pannacione et al 2001,2003) and lpsB Fleetwood et al 2007), playing a vital role in the ergopeptine formation (Figure 13b) (Riederer et al 1996;Walzel et al 1997;Correia et al 2003). The ultimate objective: to determine whether ergot alkaloid biosynthetic genes were clustered in Neotyphodium lolii was pursued by using chromosome walking and Southern blot analysis and successfully attained (Fleetwood et al 2007).…”

Intact-cell MALDI-TOF mass spectrometry analysis of peptaibol formation by the genus Trichoderma/Hypocrea: can molecular phylogeny of species predict peptaibol structures?

“…Two NRPS clusters and operons were positionally conserved with respect to B. subtilis, and several transposase sequences are contained in it. As for fungi, transposon relics have so far only been found in the ergovaline biosynthetic clusters of the endophyte Epichloe (Fleetwood et al, 2007). Interestingly in Trichoderma, similar genes are found 59 and 39 of the atroviridine synthetase of H. atroviridis and of a putative harzianine synthetase in H. jecorina which encode SF1 and SF4 peptaibol types, respectively (H. von Döhren, unpublished data).…”