Padmaja Nagabhyru

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

The epichloae: alkaloid diversity and roles in symbiosis with grasses

Epichloae (Epichloë and Neotyphodium species; Clavicipitaceae) are fungi that live in systemic symbioses with cool-season grasses, and many produce alkaloids that are deterrent or toxic to herbivores. The epichloae colonize much of the aerial plant tissues, and most benignly colonize host seeds to transmit vertically. Of their four chemical classes of alkaloids, the ergot alkaloids and indole-diterpenes are active against mammals and insects, whereas peramine and lolines specifically affect insects. Comparative genomic analysis of Clavicipitaceae reveals a distinctive feature of the epichloae, namely, large repeat blocks in their alkaloid biosynthesis gene loci. Such repeat blocks can facilitate gene losses, mutations, and duplications, thus enhancing diversity of alkaloid structures within each class. We suggest that alkaloid diversification is selected especially in the vertically transmissible epichloae.

Currencies of Mutualisms: Sources of Alkaloid Genes in Vertically Transmitted Epichloae

The epichloae (Epichloë and Neotyphodium species), a monophyletic group of fungi in the family Clavicipitaceae, are systemic symbionts of cool-season grasses (Poaceae subfamily Poöideae). Most epichloae are vertically transmitted in seeds (endophytes), and most produce alkaloids that attack nervous systems of potential herbivores. These protective metabolites include ergot alkaloids and indole-diterpenes (tremorgens), which are active in vertebrate systems, and lolines and peramine, which are more specific against invertebrates. Several Epichloë species have been described which are sexual and capable of horizontal transmission, and most are vertically transmissible also. Asexual epichloae are mainly or exclusively vertically transmitted, and many are interspecific hybrids with genomic contributions from two or three ancestral Epichloë species. Here we employ genome-scale analyses to investigate the origins of biosynthesis gene clusters for ergot alkaloids (EAS), indole-diterpenes (IDT), and lolines (LOL) in 12 hybrid species. In each hybrid, the alkaloid-gene and housekeeping-gene relationships were congruent. Interestingly, hybrids frequently had alkaloid clusters that were rare in their sexual ancestors. Also, in those hybrids that had multiple EAS, IDT or LOL clusters, one cluster lacked some genes, usually for late pathway steps. Possible implications of these findings for the alkaloid profiles and endophyte ecology are discussed.

Regulation of a Chemical Defense against Herbivory Produced by Symbiotic Fungi in Grass Plants

Neotyphodium uncinatum and Neotyphodium siegelii are fungal symbionts (endophytes) of meadow fescue (MF; Lolium pratense), which they protect from insects by producing loline alkaloids. High levels of lolines are produced following insect damage or mock herbivory (clipping). Although loline alkaloid levels were greatly elevated in regrowth after clipping, loline-alkaloid biosynthesis (LOL) gene expression in regrowth and basal tissues was similar to unclipped controls. The dramatic increase of lolines in regrowth reflected the much higher concentrations in young (center) versus older (outer) leaf blades, so LOL gene expression was compared in these tissues. In MF-N. siegelii, LOL gene expression was similar in younger and older leaf blades, whereas expression of N. uncinatum LOL genes and some associated biosynthesis genes was higher in younger than older leaf blades. Because lolines are derived from amino acids that are mobilized to new growth, we tested the amino acid levels in center and outer leaf blades. Younger leaf blades of aposymbiotic plants (no endophyte present) had significantly higher levels of asparagine and sometimes glutamine compared to older leaf blades. The amino acid levels were much lower in MF-N. siegelii and MF-N. uncinatum compared to aposymbiotic plants and MF with Epichloë festucae (a closely related symbiont), which lacked lolines. We conclude that loline alkaloid production in young tissue depleted these amino acid pools and was apparently regulated by availability of the amino acid substrates. As a result, lolines maximally protect young host tissues in a fashion similar to endogenous plant metabolites that conform to optimal defense theory.Many cool-season grasses (Poaceae, subfam. Pooideae) possess seedborne fungal symbionts, the epichloae, known for their bioprotective properties, and especially for production of anti-insect alkaloids such as lolines. Asexual epichloae (Neotyphodium species) are primarily or entirely transmitted vertically, whereas the sexual structures (stromata) of the related Epichloë species give rise to horizontally transmissible spores (ascospores). In certain grass-Neotyphodium species symbiota, levels of lolines are extremely high and apparently limited by availability of precursor amino acids, whereas sexual epichloae generally produce much lower levels. This may reflect the inherent conflict between the vertical and horizontal transmission; although the plant and seeds may be protected by the alkaloids, the sexual cycle depends on anthomyiid flies for cross-fertilization. Given this insect role, we predicted that loline biosynthesis would be down-regulated in the stromata relative to the corresponding asymptomatic tissues (inflorescences) of the same symbiota. This prediction was substantiated, and RNA-seq and RT-qPCR analysis indicated that the loline biosynthesis genes are dramatically upregulated in asymptomatic inflorescences compared to stromata. The fundamental difference between asexual and sexual epichloae in regulation of loline alkaloid levels is in keeping with evolutionary trends for greater host control on metabolism of their vertically transmitted symbionts compared to contagious symbionts.

Ether bridge formation in loline alkaloid biosynthesis

Lolines are potent insecticidal agents produced by endophytic fungi of cool-season grasses. These alkaloids are composed of a pyrrolizidine ring system and an uncommon ether bridge linking carbons 2 and 7. Previous results indicated that 1-aminopyrrolizidine was a pathway intermediate. We used RNA interference to knock down expression of lolO, resulting in the accumulation of an alkaloid identified as exo-1-acetamidopyrrolizidine based on high-resolution MS and NMR. Genomes of endophytes differing in alkaloid profiles were sequenced, revealing that those with mutated lolO accumulated exo-1-acetamidopyrrolizidine but no lolines. Heterologous expression of wild-type lolO complemented a lolO mutant, resulting in the production of N-acetylnorloline. These results indicated that the non-heme iron oxygenase, LolO, is required for ether bridge formation, probably through oxidation of exo-1-acetamidopyrrolizidine.

Enzymes from Fungal and Plant Origin Required for Chemical Diversification of Insecticidal Loline Alkaloids in Grass- Epichloë Symbiota

The lolines are a class of bioprotective alkaloids that are produced by Epichloë species, fungal endophytes of grasses. These alkaloids are saturated 1-aminopyrrolizidines with a C2 to C7 ether bridge, and are structurally differentiated by the various modifications of the 1-amino group: -NH2 (norloline), -NHCH3 (loline), -N(CH3)2 (N-methylloline), -N(CH3)Ac (N-acetylloline), -NHAc (N-acetylnorloline), and -N(CH3)CHO (N-formylloline). Other than the LolP cytochrome P450, which is required for conversion of N-methylloline to N-formylloline, the enzymatic steps for loline diversification have not yet been established. Through isotopic labeling, we determined that N-acetylnorloline is the first fully cyclized loline alkaloid, implying that deacetylation, methylation, and acetylation steps are all involved in loline alkaloid diversification. Two genes of the loline alkaloid biosynthesis (LOL) gene cluster, lolN and lolM, were predicted to encode an N-acetamidase (deacetylase) and a methyltransferase, respectively. A knockout strain lacking both lolN and lolM stopped the biosynthesis at N-acetylnorloline, and complementation with the two wild-type genes restored production of N-formylloline and N-acetylloline. These results indicated that lolN and lolM are required in the steps from N-acetylnorloline to other lolines. The function of LolM as an N-methyltransferase was confirmed by its heterologous expression in yeast resulting in conversion of norloline to loline, and of loline to N-methylloline. One of the more abundant lolines, N-acetylloline, was observed in some but not all plants with symbiotic Epichloë siegelii, and when provided with exogenous loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylloline, suggesting that a plant acetyltransferase catalyzes N-acetylloline formation. We conclude that although most loline alkaloid biosynthesis reactions are catalyzed by fungal enzymes, both fungal and plant enzymes are responsible for the chemical diversification steps in symbio.

Are loline alkaloid levels regulated in grass endophytes by gene expression or substrate availability

Many cool-season grasses (Poaceae, subfam. Pooideae) possess seedborne fungal symbionts, the epichloae, known for their bioprotective properties, and especially for production of anti-insect alkaloids such as lolines. Asexual epichloae (Neotyphodium species) are primarily or entirely transmitted vertically, whereas the sexual structures (stromata) of the related Epichloë species give rise to horizontally transmissible spores (ascospores). In certain grass-Neotyphodium species symbiota, levels of lolines are extremely high and apparently limited by availability of precursor amino acids, whereas sexual epichloae generally produce much lower levels. This may reflect the inherent conflict between the vertical and horizontal transmission; although the plant and seeds may be protected by the alkaloids, the sexual cycle depends on anthomyiid flies for cross-fertilization. Given this insect role, we predicted that loline biosynthesis would be down-regulated in the stromata relative to the corresponding asymptomatic tissues (inflorescences) of the same symbiota. This prediction was substantiated, and RNA-seq and RT-qPCR analysis indicated that the loline biosynthesis genes are dramatically upregulated in asymptomatic inflorescences compared to stromata. The fundamental difference between asexual and sexual epichloae in regulation of loline alkaloid levels is in keeping with evolutionary trends for greater host control on metabolism of their vertically transmitted symbionts compared to contagious symbionts.

Molecular identification and characterization of endophytes from uncultivated barley

ABSTRACT Epichloë species (Clavicipitaceae, Ascomycota) are endophytic symbionts of many cool-season grasses. Many interactions between Epichloë 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 Hordeum 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 Hordeum species. We analyzed seeds of Hordeum bogdanii, H. brevisubulatum, and H. comosum 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 Epichloë 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: Epichloë bromicola, Epichloë tembladerae, and three unnamed interspecific hybrid species. One PI line contained an endophyte that is considered a new taxonomic group, Epichloë sp. HboTG-3 (H. bogdanii Taxonomic Group 3). Phylogenetic analyses of the interspecific hybrid endophytes from H. bogdanii and H. brevisubulatum indicate that these taxa all have an E. bromicola 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 Hordeum 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.

Epichloë festucae endophytic growth in florets, seeds, and seedlings of perennial ryegrass (Lolium perenne)

ABSTRACT Many symbiotic Epichloë species are seed-transmitted in their grass hosts. For a detailed investigation of Epichloë festucae colonization throughout the life cycle of its host, the authors transformed strain Fl1 with a fungal-active gene for enhanced cyan-fluorescent protein (eCFP), introduced it into perennial ryegrass (Lolium perenne), and used confocal microscopy to track its growth in the shoot apex, floral primordium, floral organs, seeds, and seedlings. Hyphae intercellularly colonized leaf sheaths, blades, true stems, and leaf primordia, and among floral primordia the endophyte exhibited different levels of colonization. In preanthesis florets, E. festucae colonized the pistil and stamen, but not pollen grains, and ramified throughout the ovule nucellus, but not the integument or embryo sac. Generally, only a single hypha was observed extended from the ovary placenta into the ovule. Within 4 d after anthesis, fungal hyphae had ramified throughout the developing seed and embryo. As the embryo matured, fungal hyphae became abundant between the testa and aleurone layer, and around the shoot apex and radical of the embryonic axis. During germination, hyphae accumulated in the mesocotyl and invaded the newly formed shoot apex near the meristem. In this host-fungus symbiosis, transmission to seedlings averaged 41% in 2010 and 76% in 2011. Each year, the frequency of ovary infection was similar to the frequency of infecting embryos and seedlings, indicating that colonization of the ovary and embryo was required for seed transmission.