Ergot Alkaloid Biosynthesis in the Maize (<i>Zea mays</i>) Ergot Fungus <i>Claviceps gigantea</i>

Biosynthesis of the dihydrogenated forms of ergot alkaloids is of interest because many of the ergot alkaloids used as pharmaceuticals may be derived from dihydrolysergic acid (DHLA) or its precursor dihydrolysergol. The maize (<i>Zea mays</i>) ergot pathogen <i>Claviceps gigantea</i> has been reported to produce dihydrolysergol, a hydroxylated derivative of the common ergot alkaloid festuclavine. We hypothesized expression of <i>C. gigantea cloA</i> in a festuclavine-accumulating mutant of the fungus <i>Neosartorya fumigata</i> would yield dihydrolysergol because the P450 monooxygenase CloA from other fungi performs similar oxidation reactions. We engineered such a strain, and high performance liquid chromatography and liquid chromatography–mass spectrometry analyses demonstrated the modified strain produced DHLA, the fully oxidized product of dihydrolysergol. Accumulation of high concentrations of DHLA in field-collected <i>C. gigantea</i> sclerotia and discovery of a mutation in the gene <i>lpsA</i>, downstream from DHLA formation, supported our finding that DHLA rather than dihydrolysergol is the end product of the <i>C. gigantea</i> pathway

Molecular identification and characterization of endophytes from uncultivated barley

<p><i>Epichloë</i> species (Clavicipitaceae, Ascomycota) are endophytic symbionts of many cool-season grasses. Many interactions between <i>Epichloë</i> and their host grasses contribute to plant growth promotion, protection from many pathogens and insect pests, and tolerance to drought stress. Resistance to insect herbivores by endophytes associated with <i>Hordeum</i> species has been previously shown to vary depending on the endophyte-grass-insect combination. We explored the genetic and chemotypic diversity of endophytes present in wild <i>Hordeum</i> species. We analyzed seeds of <i>Hordeum bogdanii, H. brevisubulatum</i>, and <i>H. comosum</i> obtained from the US Department of Agriculture’s (USDA) National Plant Germplasm System (NPGS), of which some have been reported as endophyte-infected. Using polymerase chain reaction (PCR) with primers specific to <i>Epichloë</i> species, we were able to identify endophytes in seeds from 17 of the 56 Plant Introduction (PI) lines, of which only 9 lines yielded viable seed. Phylogenetic analyses of housekeeping, alkaloid biosynthesis, and mating type genes suggest that the endophytes of the infected PI lines separate into five taxa: <i>Epichloë bromicola, Epichloë tembladerae</i>, and three unnamed interspecific hybrid species. One PI line contained an endophyte that is considered a new taxonomic group, <i>Epichloë</i> sp. HboTG-3 (<i>H. bogdanii</i> Taxonomic Group 3). Phylogenetic analyses of the interspecific hybrid endophytes from <i>H. bogdanii</i> and <i>H. brevisubulatum</i> indicate that these taxa all have an <i>E. bromicola</i> allele but the second allele varies. We verified in planta alkaloid production from the five genotypes yielding viable seed. Morphological characteristics of the isolates from the viable <i>Hordeum</i> species were analyzed for their features in culture and in planta. In the latter, we observed epiphyllous growth and in some cases sporulation on leaves of infected plants.</p

Boyce et al. 2018 Movie S1

<p><b>Movie S1.</b> Living <i>Platypedia putnami</i> with conidial and resting spore infections.</p

Boyce et al. Movie S2

<p><b>Movie S2.</b> Living Brood V <i>Magicicada septendecim</i> with conidial spore infection.</p

Structures of the indole-diterpene biosynthesis loci (<i>IDT/LTM</i>) in sequenced genomes.

<p><i>IDT/LTM</i> genes are indicated by single letters, whereby <i>Q = idtQ</i> or <i>ltmQ</i> (in <i>E. festucae</i>), 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 <i>E. festucae</i> 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 <i>idt/ltmS</i>, 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g003" target="_blank">Figure 3</a>, and in bold for those confirmed in this study.</p

Phylogenies of <i>rpbA</i> from sequenced isolates and other Clavicipitaceae.

<p>The phylogenetic tree is based on nucleotide alignment for a portion of the RNA polymerase II largest subunit gene, <i>rpbA</i>. This tree is rooted with <i>Fusarium graminearum</i> as the outgroup. Epichloae are indicated in green, <i>Claviceps</i> species are indicated in blue, <i>Periglandula</i> species are indicated in red, and <i>Aciculosporium take</i> 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g002" target="_blank">Figure 2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g003" target="_blank">Figure 3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003323#pgen-1003323-g004" target="_blank">Figure 4</a>. 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 <i>perA</i>.</p

GC proportions in genic and repeat DNA of sequenced genomes.^a

a<p>Abbreviations: CDS = coding sequence, GC = proportion of sequence that is G or C, non-Rpt-IG = nonrepetitive intergenic DNA, Rpt = repetitive DNA.</p>b<p>Statistics for <i>P. ipomoeae</i> are tentative because the assembly was filtered by selecting only contigs containing tBLASTx matches to genome sequences from the other Clavicipitaceae.</p

Alkaloid profiles of sequenced isolates.^a

a<p>Strains are abbreviated as follow: <i>Cpu</i> = <i>Claviceps purpurea</i> 20.1, <i>Cfu</i> = <i>C. fusiformis</i> PRL 1980, Cpa = <i>C. paspali</i> RRC-1481, <i>Eam</i> = <i>Epichloë amarillans</i> E57, <i>Ebe</i> = <i>E. brachyelytri</i> E4804, <i>Eel</i> = <i>E. elymi</i> E56, <i>Ef</i>1 = <i>E. festucae</i> Fl1, <i>Ef</i>2 = <i>E. festucae</i> E2368, <i>Egl</i> = <i>E. glyceriae</i> E2772, <i>Et</i>8 = <i>E. typhina</i> E8, <i>Et</i>5 = <i>E. typhina</i> E5819, <i>Nga</i> = <i>N. gansuense</i> E7080, <i>Ngi</i> = <i>N. gansuense</i> var. <i>inebrians</i> E818, <i>Nun</i> = <i>N. uncinatum</i> E167, <i>Pip</i> = <i>P. ipomoeae</i> IasaF13. Symbols: + = present, (+) = intermediate inferred to be synthesized because downstream product is present, − = not predicted and not detected, (−) = predicted but not detected, nt = predicted but not tested, ERA = ergotamine, ERB = ergobalansine, ERC = ergocryptine, ERV = ergovaline. Blank cells indicate compounds not predicted from genotype, and not tested.</p>b<p>Identification of IDT-436 and terpendoles E, I, J, K, M, M, and A are tentative because authentic standards are unavailable.</p

Summary of loline alkaloid-biosynthesis pathway.

<p>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 <i>LOL</i> genes that were newly discovered in the genome sequence of <i>E. festucae</i> E2368.</p

Symbiosis of meadow fescue with <i>Epichloë festucae</i>, a heritable symbiont.

<p>Single optical slice confocal micrographs of <i>E. festucae</i> 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 <i>E. festucae</i> genotype. Vertical (seed) transmission of the symbiont occurs via the asymptomatic inflorescence, whereas the choked inflorescence bears the <i>E. festucae</i> fruiting structure (stroma), which produces sexually derived spores (ascospores) that mediate horizontal transmission.</p