Daniel G. Panaccione

We tested whether ergot alkaloids would affect the interaction of a protist with an ergot alkaloid-producing fungus by studying the response of the plasmodial slime mold Physarum polycephalum to cultures of Aspergillus leporis. Ergot alkaloid profiles were manipulated by culturing A. leporis and its easD knockout derivative on media for 6 or 13 days. Six-day-old, wild-type A. leporis samples contained abundant lysergic acid α-hydroxyethylamide (LAH) that was greatly depleted by 13 days. The intermediate chanoclavine-I was the predominant ergot alkaloid in all samples of the easD knockout. Inoculum of P. polycephalum was placed equidistant between the fungus-colonized agar medium explant, and the preference of the slime mold for either fungal explant was observed. When offered 6-day-old samples of wild-type A. leporis (containing mainly LAH) or the easD knockout (containing mainly chanoclavine-I), plasmodia of P. polycephalum preferred the easD knockout strain over wild type by a significant margin (p = 0.0002). When given the same options at 13 days, there was no longer a preference for the easD knockout over wild type, which by that time had lost more than 90% of its LAH. Our data demonstrate that ergot alkaloids, including LAH and chanoclavine-I, affect the interaction of P. polycephalum with A. leporis.

The agriculturally and pharmaceutically important lysergic acid amides ergonovine and lysergic acid α-hydroxyethylamide (LAH) are synthesized from a lysergyl-alanine precursor. In ergot-alkaloid producing fungi of the family Clavicipitaceae, lysergyl-alanine is assembled and then reduced to ergonovine by a complex of two monomodular nonribosomal peptide synthetases: lysergyl peptide synthetase 2 (Lps2) and Lps3. LAH is the major ergot alkaloid product of these fungi when the Lps2/Lps3 complex interacts with the Bayer-Villiger monooxygenase encoded by easO . An α/β hydrolase fold protein encoded by easP increases LAH accumulation but is not essential for LAH biosynthesis. Lps2 and Lps3 do not occur in the several species of Aspergillus (including A. leporis ) that produce LAH and ergonovine. Instead, ergot alkaloid synthesis clusters of these Aspergillus species encode a novel two-module Lps gene, lpsD . We hypothesized the product of lpsD was functionally equivalent to the two separately encoded, monomodular enzymes of the Clavicipitaceae and tested this hypothesis by introducing lpsD of A. leporis into a strain of Aspergillus fumigatus that had been modified previously to accumulate lysergic acid as substrate. Introduction of lpsD resulted in accumulation of ergonovine as evidenced by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The addition of the A. leporis allele of easO into the lpsD -transformed A. fumigatus strain led to accumulation of LAH. Introduction of a construct containing easP as well as easO into the lpsD -transformed A. fumigatus strain resulted in higher concentrations of LAH than in strains containing only lpsD and easO , consistent with previous studies in the Clavicipitaceae. The data support the hypothesis that ergot alkaloid-producing Aspergillus species independently evolved a single enzyme that serves the purpose of the two monomodular peptide synthetases of the Clavicipitaceae.

Ergot alkaloids derived from lysergic acid have impacted humankind significantly as toxins in agriculture and as the foundations of several pharmaceuticals. Few fungi capable of producing lysergic acid derivatives have been found outside the family Clavicipitaceae. Based on its phylogenetic placement, we hypothesized the recently described fungus Aspergillus aspearensis (Aspergillaceae) would synthesize lysergic acid amides. Cultures of A. aspearensis produced abundant lysergic acid α-hydroxyethylamide (LAH) and lesser amounts of other lysergic acid derivatives. Conidia contained high concentrations of ergot alkaloids, whereas sclerotia contained significantly less. Approximately half of the ergot alkaloids produced were secreted into the culture medium. When spores of A. aspearensis were injected into larvae of the model insect Galleria mellonella, larvae died at a significantly faster rate than control larvae. The fungus produced ergot alkaloids during insect pathogenesis and later produced conidia and sclerotia on cadavers, indicating it can complete its life cycle in an insect. The genome of A. aspearensis contained two complete ergot alkaloid synthesis gene clusters, similar to those of A. leporis; however, unlike its sister species, none of the ergot cluster genes were pseudogenized. Aspergillus aspearensis is a newly discovered source of ergot alkaloids and may be useful for studying and producing these important chemicals.

Ergot alkaloids derived from lysergic acid are important in agriculture, as food and feed contaminants, and in medicine, as the foundation of several pharmaceuticals. The fungus Metarhizium brunneum makes several lysergic acid amides, with lysergic acid α-hydroxyethylamide (LAH) being produced in by far the highest concentration. The multifunctional enzyme lysergyl peptide synthetase 3 (Lps3) has multiple domains that play important roles in lysergic acid amide synthesis. We hypothesized a role for the reductase domain of Lps3 in liberating LAH from an enzyme-bound precursor and tested this hypothesis with CRISPR/Cas9-based gene editing experiments. We transformed M. brunneum with a Cas9/single guide RNA complex and a donor DNA that replaced the tyrosine at the active site of the reductase domain of Lps3 with a phenylalanine. Sanger sequencing of edited and wild-type genes demonstrated successful editing of the reductase domain without non-target mutations in Lps3. High performance liquid chromatography of the edited strain showed a significant reduction of LAH and accumulation of the precursor lysergic acid. The phenotype was similar when the edited allele of lpsC was in a wild-type background or in backgrounds with late pathway genes easO or easP knocked out, except no LAH was detectable when the edit was in the easO knockout background. The data demonstrate that the reductase domain plays a key role or roles in formation of LAH. The abundant lysergic acid accumulating in the mutants, as opposed to later pathway intermediates in LAH biosynthesis (such as lysergyl-alanine), indicated severe debilitation of Lps3. The data indicate a requirement for the reductase domain of Lps3 in synthesis of lysergic acid amides and demonstrate the feasibility of the CRISPR/Cas9-based approach for editing genes in Metarhizium species.

Many morning glories (family Convolvulaceae) contain ergot alkaloids-important bioactive compounds produced exclusively by fungi. The ergot alkaloids of the few investigated morning glories are associated with the presence of a symbiotic Clavicipitaceous fungus. The genus Periglandula (Clavicipitaceae) was erected recently for two epibiotic species of morning glory symbionts. Biochemical and limited sequence data indicate that Ipomoea tricolor, a commonly cultivated morning glory from Mexico, contains a Periglandula species, but no signs of the fungus have ever been detected. Our goal was to isolate and describe this fungus, which we hypothesize represents a new species. Observation of fungal hyphae in evacuated seed coats of I. tricolor and subsequent transfer onto malt extract agar resulted in cultures of the symbiont isolated from the plant. The fungus grew slowly as white hyphae and sometimes aggregated into synnema-like structures, both of which lacked spores. We isolated sufficient DNA to sequence the genome with Illumina technology. Phylogenetic analyses based on multiple genes indicated that the symbiont of I. tricolor was distinct from, but related to, the two described species of Periglandula previously observed in other species of morning glories. Using quantitative polymerase chain reaction (qPCR), the fungus was quantified most abundantly in hypocotyls of I. tricolor, with lesser quantities in stems, cotyledons, and leaves. The fungus was not detected in roots, although ergot alkaloids were abundant in all tissues including roots. We conclude that the symbiotic fungus of I. tricolor is a distinct species of Periglandula and propose the name Periglandula clandestina, sp. nov.

Ergot alkaloid synthesis ( eas ) gene clusters found in several fungi encode biosynthesis of agriculturally and pharmaceutically important ergot alkaloids. Although the biosynthetic genes of the ergot alkaloid pathway have been well characterized, regulation of those genes is unknown. We characterized a gene with sequence similarity to a putative transcription factor and that was found adjacent to the eas cluster of Metarhizium brunneum , a plant symbiont and insect pathogen. Function of the novel gene, easR, was explored by CRISPR-Cas9-derived gene knockouts. To maximize potential for ergot alkaloid accumulation, strains of M. brunneum were injected into larvae of the insect Galleria mellonella . Larvae infected with the wild type contained abundant ergot alkaloids, but those infected with easR knockouts lacked detectable ergot alkaloids. The easR knockout strains had significantly reduced or no detectable mRNA from eas cluster genes in RNAseq and qualitative RT-PCR analyses, whereas the wild-type strain contained abundant mRNA from all eas genes. These data demonstrate that the product of easR is required for ergot alkaloid accumulation and provide evidence that it has a role in the expression of ergot alkaloid biosynthesis genes. Larvae infected with an easR knockout survived significantly longer than those infected with the wild type ( P < 0.0001), indicating a role for EasR, and indirectly confirming a role for ergot alkaloids, in the virulence of M. brunneum to insects. Homologs of easR were found associated with eas clusters of at least 15 other ergot alkaloid-producing fungi, indicating that EasR homologs may contribute to regulation of ergot alkaloid synthesis in additional fungi. IMPORTANCE: Ergot alkaloids produced by several species of fungi are important as contaminants of food and feed in agriculture and also as the foundation of numerous pharmaceuticals prescribed for dementia, migraines, hyperprolactinemia, and several other disorders. Information on control of the ergot alkaloid pathway may contribute to strategies to limit their production in agricultural settings or increase their yield for pharmaceutical production. Our results demonstrate that a previously uncharacterized gene clustered with the ergot alkaloid synthesis genes is required for the sufficient transcription of the ergot alkaloid biosynthesis genes. This observation suggests the gene encodes a factor regulating transcription of those biosynthetic genes.

Ergot alkaloids are fungal natural products with important roles in agriculture and medicine. We used heterologous expression and gene knockout approaches to investigate potential roles for the product of a major facilitator superfamily transporter gene (easT) recently found in an ergot alkaloid biosynthetic gene cluster in Aspergillus leporis. A strain of Aspergillus fumigatus previously engineered to accumulate lysergic acid, but which did not convert the precursor agroclavine to lysergic acid efficiently or secrete lysergic acid well, was chosen as an expression host for easT. Expression of easT in this strain resulted in accumulation of significantly more pathway intermediates but no detectable lysergic acid. Secretion of ergot alkaloids was reduced in the easT-expressing strain. EasT localized to discrete vesicle-like structures in the cytosol of A. fumigatus, with no localization detected in the plasma membrane. When easT was knocked out in A. leporis, accumulation of lysergic acid amides was reduced relative to the wild type. There was no negative effect on secretion of ergot alkaloids in the knockout mutant. The data indicate that easT encodes a product that contributes to accumulation of ergot alkaloids, perhaps by transporting intermediates between cellular compartments, but does not have a significant role in secreting ergot alkaloids.

Ergot alkaloids are chemicals produced by several species of fungi and are notable for their impacts on agriculture and medicine. The ability to make ergot alkaloids is typically encoded by a clustered set of genes that are physically adjacent on a chromosome.

Certain environmental fungi infect animals occasionally or conditionally, whereas others do not. Factors that affect the virulence of these opportunistically pathogenic fungi may have originally evolved to fill some other role for the fungus in its primary environmental niche.