A complex ergovaline gene cluster in epichloe 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 endophyte-infected 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.

FIG. 1.

Physical map and clones covering the N. lolii EAS locus. Putative genes are shown as arrows indicating the direction of transcription. Introns are shown as gaps. The solid line above lpsB denotes the ps12 sequence used to probe genomic libraries. “Retro” denotes two highly degenerate nested long terminal repeat retrotransposons. Regions containing putative nonautonomous transposable elements are expanded. SSR, simple sequence repeat (eight times 10 bp).

FIG. 2.

Comparative eas gene order among N. lolii, C. purpurea, and A. fumigatus. Cp, C. purpurea; Nl, N. lolii; Af, A. fumigatus. Open arrows, genes shared between each organism; black arrows, genes proposed to be required for ergot alkaloid synthesis but not yet identified for N. lolii; light gray arrows, genes found in N. lolii and C. purpurea but not A. fumigatus; dark gray arrows, genes found only in A. fumigatus. N. lolii dmaW, cloA, and lpsA are not shown, as their locations relative to the EAS cluster are not known.

FIG. 3.

Sequence diversity at dmaW loci. Nc1, N. coenophialum dmaW locus 1 (accession number AY259838); Nc2, N. coenophialum dmaW locus 2 (accession number AY259839); Lp1, Neotyphodium sp. strain Lp1 dmaW locus (accession number AY259837). LTR, long terminal repeat.

FIG. 4.

Southern blot analysis indicating linkage of lpsB, lpsA, and dmaW. N. lolii Lp19 (Nl) and E. festucae Fl1 (Ef) genomic DNA was digested with NotI, separated by CHEF pulsed-field gel electrophoresis, and transferred to a nylon membrane. The blot was hybridized with ³²P-labeled probes to lpsB (pDF2), lpsA (amplified with primers lpsA-F and lpsA-R), and dmaW (amplified with primers dmaWq-F and dmaWq-R). The lpsA probe contains a NotI site and hence hybridizes to two bands.

FIG. 5.

Targeted gene replacement of lpsB. (A) Diagrammatic representation of homologous recombination at lpsB. The wild-type (WT) gene locus and targeted replacement construct (KO) are shown. The solid line above lpsB represents the PCR probe amplified with primers KOprobe-F and KOprobe-R, used for the results shown in panel B. E, EcoRI site. (B) Southern blot analysis. Wild-type and lpsB mutant DFM3 (KO) genomic DNA was digested with EcoRI and transferred to a nylon membrane, which was hybridized with the probe shown in panel A.

FIG. 6.

Extracted ion chromatograms from LC-MS/MS of extracts of perennial ryegrass plants infected with wild-type E. festucae Fl1 (WT), lpsB mutant DFM3 (KO), or DFM3 transformed with pDF1 (Complement), showing accumulation of lysergic acid (shown above; peak a) and isolysergic acid (peak b) (m/z 268 to 223), lysergyl-alanine (peak c) and isolysergyl-alanine (peak d) (m/z 340 to 223), and ergovaline (peak e) and ergovalinine (peak f) (m/z 340 to 223). Relative abundance values are on the same (arbitrary) scale for each extract.

FIG. 7.

Expression analysis of genes in the EAS cluster. RT-PCR was performed with RNA extracted from E. festucae Fl1 grown axenically in PD medium (A) and L. perenne infected with E. festucae Fl1 (B). cDNA prepared from cultured mycelium was diluted 1/100 to compensate for the biomass differences in planta.