Plant-Symbiotic Fungi as Chemical Engineers: Multi-Genome Analysis of the Clavicipitaceae Reveals Dynamics of Alkaloid Loci

Christopher L Schardl,Eckhard Leistner,Ulrike Steiner,Daniel R Harris,Jerzy W Jaromczyk,Christine R Voisey,Padmaja Nagabhyru,Chunjie Li,Zheng Zeng,Daniel G Panaccione,Uljana Hesse,Charles T Bullock,Eiji Tanaka,Paul Tudzynski,Jan Schmid,Stefan G Amyotte,Bruce A Roe,Patrick J Calie,Richard D Johnson,Ulrich Güldener,Mark L Farman,Elissaveta G Arnaoudova,Kalina Andreeva,Caroline Machado,Damien J Fleetwood,Juan Pan,Adrian Leuchtmann,Barry Scott,Birgitt Oeser,Ella V Wilson,Anna Gordon,Jolanta Jaromczyk,Randy D Dinkins,Neil Moore,Miao Liu,Jinze Liu,Jennifer L Wiseman,Simona Florea,Johanna E Takach,Zhiqiang An,Nikki D Charlton,Li Chen,Anthony E Glenn,Carolyn A Young,Walter Hollin,Ruriko Yoshida,Anar Khan ,David Haws ,James D Webb ,Kathryn Schweri ,Kazuo Sugawara ,Murray P Cox ,Donal M O’sullivan ,Wade J Mace ,Jin Ge Liu

doi:10.1371/journal.pgen.1003323

Abstract

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.

Highlights

Alkaloids play key roles in plant ecology by targeting the central and peripheral nervous systems of invertebrate and vertebrate animals, affecting their behavior, eliciting toxicoses, and reducing herbivory [1]
Genome sequences Clusters of genes have been identified for the four alkaloid biosynthesis classes [16,41,42,43], but in the absence of complete genome sequences it was unknown if the clusters had been fully characterized for any known producers in the Clavicipitaceae
Adding mate-pair pyrosequencing of C. purpurea DNA resulted in a 186-supercontig assembly of 32.1 Mb, and adding end-sequencing of fosmid clones E. festucae Fl1 DNA resulted in a 170-supercontig assembly of 34.9 Mb

Summary

Introduction

Alkaloids play key roles in plant ecology by targeting the central and peripheral nervous systems of invertebrate and vertebrate animals, affecting their behavior, eliciting toxicoses, and reducing herbivory [1]. Alkaloids are very common in plants as well as certain plant-associated fungi, those in the family Clavicipitaceae Plant parasites such as Claviceps species often produce high levels of ergot alkaloids or indole-diterpenes, probably to defend their resting and overwintering structures (commonly called ergots) [2,3]. The major clade of plantassociated Clavicipitaceae [6] includes mutualistic symbionts as well as plant pathogens, many of which produce alkaloids with diverse neurotropic effects on vertebrate and invertebrate animals with important implications for human health, agriculture and food security [7,8]. Most species of plant-associated Clavicipitaceae grow in or on grasses, but the group includes systemic parasites of sedges or other plants, and heritable symbionts of morning glories [9]. Epichloae have diverse alkaloid profiles, and in addition to ergot alkaloids or indole-diterpenes, many produce lolines or peramine, which help to protect their grass hosts from insects [15,16] and possibly other invertebrates [17]

Methods

Results

Conclusion