Figure 1.
Symbiosis of meadow fescue with Epichloë festucae, a heritable symbiont.
Single optical slice confocal micrographs of E. festucae expressing enhanced cyan-fluorescent protein were overlain with DIC bright field images of (A) ovules (bar = 100 µm), (B) embryos (bar = 200 µm), and (C) shoot apical meristem and surrounding new leaves (bar = 200 µm). (D) Asymptomatic (left) and “choked” (right) inflorescences simultaneously produced on a single grass plant infected with a single E. festucae genotype. Vertical (seed) transmission of the symbiont occurs via the asymptomatic inflorescence, whereas the choked inflorescence bears the E. festucae fruiting structure (stroma), which produces sexually derived spores (ascospores) that mediate horizontal transmission.
Figure 2.
Ergot alkaloids and summary of biosynthesis pathway.
(A) Ergoline alkaloid biosynthesis pathways in the Clavicipitaceae. Arrows indicate one or more steps catalyzed by products of genes indicated. Arrows and genes in blue indicate steps in synthesis of the first fully cyclized intermediate (skeleton). Variation in the easA gene (underlined) determines whether the ergoline skeleton is festuclavine or agroclavine. Arrows and genes in red indicate steps in decoration of the skeleton to give the variety of ergolines in the Clavicipitaceae. Asterisks indicate genes newly discovered in the genome sequences of C. paspali, N. gansuense var. inebrians and P. ipomoeae. (B) Ergopeptines produced by strains in this study.
Figure 3.
Summary of indole-diterpene biosynthesis pathway.
Arrows indicate one or more steps catalyzed by products of the genes indicated, where each idt/ltm gene is designated by its final letter (G = idtG/ltmG, etc.). Arrows and genes in blue indicate steps in synthesis of the first fully cyclized intermediate (paspaline). Arrows and genes in red indicate steps in decoration of paspaline to give the variety of indole-diterpenes in the Clavicipitaceae. Structures shown in gray are not yet verified.
Figure 4.
Summary of loline alkaloid-biosynthesis pathway.
Arrows indicate one or more steps catalyzed by products of the genes indicated. Arrows and genes in blue indicate steps in synthesis of the first fully cyclized intermediate (NANL). Arrows and genes in red indicate steps in modification of NANL to give the variety of lolines found in the epichloae. Asterisks indicate LOL genes that were newly discovered in the genome sequence of E. festucae E2368.
Figure 5.
Phylogenies of rpbA from sequenced isolates and other Clavicipitaceae.
The phylogenetic tree is based on nucleotide alignment for a portion of the RNA polymerase II largest subunit gene, rpbA. This tree is rooted with Fusarium graminearum as the outgroup. Epichloae are indicated in green, Claviceps species are indicated in blue, Periglandula species are indicated in red, and Aciculosporium take is in black. Species for which genomes were sequenced in this study are shown in bold type, and asterisks indicate plant-associated fungi. Alkaloids listed are the major pathway end-products predicted from the genome sequences, abbreviated as shown in Figure 2, Figure 3, Figure 4. Other abbreviations: (−) = some genes or remnants present, but not predicted to make alkaloids of this class, – = no genes present for this alkaloid class, EA = ergot alkaloids may be produced; IDT = indole-diterpenes may be produced, (ΔR*) = deletion of terminal reductase domain of perA.
Table 1.
Genome sequencing statistics for plant-associated Clavicipitaceae.a
Table 2.
Genic and repeat DNA contents of sequenced genomes.a
Table 3.
GC proportions in genic and repeat DNA of sequenced genomes.a
Table 4.
Alkaloid profiles of sequenced isolates.a
Figure 6.
Structures of the ergot alkaloid biosynthesis loci (EAS) in sequenced genomes.
Tracks from top to bottom of each map represent the following: genes, repeats, MITEs, and graphs of AT (red) and GC (blue) contents. Each gene is represented by one or more boxes representing the coding sequences in exons, and an arrow indicating the direction of transcription. Double-slash marks (//) indicate sequence gaps within scaffolds of the assembled E. festucae genome sequences. Closed circles indicate telomeres, and distances from the telomere on the E. festucae map are indicated in kilobasepairs (kb). Cyan bars beneath each map represent repeat sequences, and are labeled with names or numbers to indicate relationships between repeats in the different species. Vertical bars beneath the repeat maps indicate MITEs. Gene names are abbreviated A through P for easA through easP, W for dmaW, and clo for cloA. Genes for synthesis of the ergoline ring system (skeleton) are shown in dark blue for the steps to chanoclavine-I (W, F, E, and C), and in light blue (D, A, and G) for steps to agroclavine. Genes for subsequent chemical decorations are shown in red (clo, H, O, P, lpsA, lpsB, and lpsC). Identifiable genes flanking the clusters are indicated in gray, and unfilled arrows indicate pseudogenes. The major pathway end-products for each strain are listed below each species name, abbreviated as indicated in Figure 2, and in bold for those confirmed in this study. Note that LAH is a reported product of C. paspali, but the sequenced strain is predicted not to synthesize it due to a defective easE gene.
Table 5.
Alkaloid biosynthesis genes in sequenced isolates.a
Figure 7.
Structures of the indole-diterpene biosynthesis loci (IDT/LTM) in sequenced genomes.
IDT/LTM genes are indicated by single letters, whereby Q = idtQ or ltmQ (in E. festucae), and so forth. Tracks from top to bottom of each map represent the following: genes, repeats, MITEs, and graphs of AT (red) and GC (blue) contents. Each gene is represented by a filled arrow indicating its direction of transcription. Closed circles indicate telomeres, and distances from the telomere on the E. festucae map are indicated in kilobasepairs (kb). Cyan bars representing repeat sequences are labeled with names or numbers to indicate relationships between repeats in the different species. Vertical bars beneath the repeat maps indicate MITEs. Genes for the first fully cyclized intermediate, paspaline, are indicated in blue, those for subsequent chemical decorations are shown in red, and idt/ltmS, with undetermined function, is in purple. Identifiable genes flanking the clusters are indicated in gray, and unfilled arrows indicate pseudogenes. The major pathway end-product for each strain is listed at the right of its map, abbreviated as indicated in Figure 3, and in bold for those confirmed in this study.
Figure 8.
Relationships of ltmE and ltmJ with other LTM genes.
Filled boxes indicate coding sequences of exons. Gray polygons indicate closest BLASTp matches to inferred polypeptide sequences for each exon, and are labeled with percent amino-acid identities.
Figure 9.
Loline alkaloid biosynthesis loci (LOL) in epichloae and the homologous loci in other Clavicipitaceae.
LOL genes are indicated by single letters, whereby F = lolF, C = lolC, and so forth. Features are indicated as in Figure 7. Double-slash marks (//) indicate sequence gaps within scaffolds of the assembled E. festucae E2368 genome sequence. Genes for the first fully cyclized intermediate, NANL, are indicated in blue, and those for subsequent chemical decorations are shown in red. The major pathway end-product for each strain is listed at the right of its map, abbreviated as indicated in Figure 4, and in bold for those confirmed in this study.
Figure 10.
Peramine biosynthesis loci (PER) in epichloae and the homologous loci in other Clavicipitaceae.
On each map perA is color-coded blue for a complete gene and as an open box for perA-ΔR*. Domains of perA are indicated as A (adenylation), T (thiolation), C (condensation), M (N-methylation) and R* (reduction). Subscripts indicate postulated specificity of adenylation domains for 1-pyrroline-5-carboxylate (AP) and arginine (AR) [16]. Other features are indicated as in Figure 7.
Figure 11.
Fine-mapping of repeats in two regions of the EAS clusters of epichloae.
(A) The easE-easF-easG regions. (B) The dmaW-cloA-easC-easD regions. Genes are colored as in Figure 6. AT-rich repeats are in gray, and named or numbered to indicate relationships between repeats in the different species. MITEs are indicated by labeled vertical black bars. In some cases, the gene cluster orientation is different from those shown in Figure 6 to facilitate gene alignment. The Waru element is an autonomous parent element of MITE 8m.
Figure 12.
Relative repeat contents in specialized metabolite clusters of Epichloë festucae.
Log-ratios of repeat sequences (Rpt) to coding sequences (CDS) are shown in order of increasing proportions of repeats. Open boxes represent clusters that are apparently nonfunctional due to inactivation of signature genes.