Metarhizium spp. are being used as environmentally friendly alternatives to chemical insecticides, as model systems for studying insect-fungus interactions, and as a resource of genes for biotechnology. We present a comparative analysis of the genome sequences of the broad-spectrum insect pathogen Metarhizium anisopliae and the acridid-specific M. acridum. Whole-genome analyses indicate that the genome structures of these two species are highly syntenic and suggest that the genus Metarhizium evolved from plant endophytes or pathogens. Both M. anisopliae and M. acridum have a strikingly larger proportion of genes encoding secreted proteins than other fungi, while ∼30% of these have no functionally characterized homologs, suggesting hitherto unsuspected interactions between fungal pathogens and insects. The analysis of transposase genes provided evidence of repeat-induced point mutations occurring in M. acridum but not in M. anisopliae. With the help of pathogen-host interaction gene database, ∼16% of Metarhizium genes were identified that are similar to experimentally verified genes involved in pathogenicity in other fungi, particularly plant pathogens. However, relative to M. acridum, M. anisopliae has evolved with many expanded gene families of proteases, chitinases, cytochrome P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degradation, detoxification, and toxin biosynthesis that may facilitate its ability to adapt to heterogenous environments. Transcriptional analysis of both fungi during early infection processes provided further insights into the genes and pathways involved in infectivity and specificity. Of particular note, M. acridum transcribed distinct G-protein coupled receptors on cuticles from locusts (the natural hosts) and cockroaches, whereas M. anisopliae transcribed the same receptor on both hosts. This study will facilitate the identification of virulence genes and the development of improved biocontrol strains with customized properties.

The ascomycete fungus Beauveria bassiana is a pathogen of hundreds of insect species and is commercially produced as an environmentally friendly mycoinsecticide. We sequenced the genome of B. bassiana and a phylogenomic analysis confirmed that ascomycete entomopathogenicity is polyphyletic, but also revealed convergent evolution to insect pathogenicity. We also found many species-specific virulence genes and gene family expansions and contractions that correlate with host ranges and pathogenic strategies. These include B. bassiana having many more bacterial-like toxins (suggesting an unsuspected potential for oral toxicity) and effector-type proteins. The genome also revealed that B. bassiana resembles the closely related Cordyceps militaris in being heterothallic, although its sexual stage is rarely observed. A high throughput RNA-seq transcriptomic analysis revealed that B. bassiana could sense and adapt to different environmental niches by activating well-defined gene sets. The information from this study will facilitate further development of B. bassiana as a cost-effective mycoinsecticide.

BackgroundSpecies in the ascomycete fungal genus Cordyceps have been proposed to be the teleomorphs of Metarhizium species. The latter have been widely used as insect biocontrol agents. Cordyceps species are highly prized for use in traditional Chinese medicines, but the genes responsible for biosynthesis of bioactive components, insect pathogenicity and the control of sexuality and fruiting have not been determined.ResultsHere, we report the genome sequence of the type species Cordyceps militaris. Phylogenomic analysis suggests that different species in the Cordyceps/Metarhizium genera have evolved into insect pathogens independently of each other, and that their similar large secretomes and gene family expansions are due to convergent evolution. However, relative to other fungi, including Metarhizium spp., many protein families are reduced in C. militaris, which suggests a more restricted ecology. Consistent with its long track record of safe usage as a medicine, the Cordyceps genome does not contain genes for known human mycotoxins. We establish that C. militaris is sexually heterothallic but, very unusually, fruiting can occur without an opposite mating-type partner. Transcriptional profiling indicates that fruiting involves induction of the Zn2Cys6-type transcription factors and MAPK pathway; unlike other fungi, however, the PKA pathway is not activated.ConclusionsThe data offer a better understanding of Cordyceps biology and will facilitate the exploitation of medicinal compounds produced by the fungus.

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Much remains unknown regarding speciation. Host-pathogen interactions are a major driving force for diversification, but the genomic basis for speciation and host shifting remains unclear. The fungal genus Metarhizium contains species ranging from specialists with very narrow host ranges to generalists that attack a wide range of insects. By genomic analyses of seven species, we demonstrated that generalists evolved from specialists via transitional species with intermediate host ranges and that this shift paralleled insect evolution. We found that specialization was associated with retention of sexuality and rapid evolution of existing protein sequences whereas generalization was associated with protein-family expansion, loss of genome-defense mechanisms, genome restructuring, horizontal gene transfer, and positive selection that accelerated after reinforcement of reproductive isolation. These results advance understanding of speciation and genomic signatures that underlie pathogen adaptation to hosts.peciation is a central component of biological diversification and is increasingly viewed as a continuum or process rather than an event. However, failure to identify transitional species has hindered progress in understanding genomic patterns of divergence along the speciation continuum (1). Plant or animal pathogenic fungi are genetically tractable models for the study of speciation due to their diverse lifestyles and the occurrence of sibling species that differ from each other principally in host specificity (2, 3). However, fundamental questions remain, including whether generalization or specialization to particular hosts is the ancestral condition, whether we can identify the existence of transitional forms, and what are the underlying molecular mechanisms driving speciation (4).We exploited the ascomycete genus Metarhizium, a radiating lineage of insect pathogens that are frequently used as biological insecticides (5, 6) and for genomic studies into the nature of adaptive differences by which novel pathogens emerge and form new species. Besides the previously sequenced Metarhizium robertsii (abbreviated as MAA) and Metarhizium acridum (MAC) (7), five new species were sequenced: Metarhizium album (MAM), Metarhizium majus (MAJ), Metarhizium guizhouense (MGU), Metarhizium brunneum (MBR), and Metarhizium anisopliae (MAN) (Dataset S1, Table S1). MAM is specific for hemipteran insects (8) whereas MAJ and MGU have intermediate host ranges as they are predominately associated with coleopteran insects but can also infect lepidopterans (9). Like MAA, MBR and MAN are generalists parasitizing a broad range of insects representing more than seven orders (10,11). Generalist species such as MAA and MBR can also colonize the roots of plants (12), consistent with increased phenotypic flexibility.Our analyses revealed that the evolutionary trajectory of Metarhizium spp. was from specialists via intermediate host range species to generalists that coincided with host insect diversification and availability. This host adaptati...

Metarhizium anisopliae is a fungus of considerable metabolic and ecological versatility, being a potent insect pathogen that can also colonize plant roots. The mechanistic details of these interactions are unresolved. We provide evidence that M. anisopliae adheres to insects and plants using two different proteins, MAD1 and MAD2, that are differentially induced in insect hemolymph and plant root exudates, respectively, and produce regional localization of adhesive conidial surfaces. Expression of Mad1 in Saccharomyces cerevisiae allowed this yeast to adhere to insect cuticle. Expression of Mad2 caused yeast cells to adhere to a plant surface. Our study demonstrated that as well as allowing adhesion to insects, MAD1 at the surface of M. anisopliae conidia or blastospores is required to orientate the cytoskeleton and stimulate the expression of genes involved in the cell cycle. Consequently, the disruption of Mad1 in M. anisopliae delayed germination, suppressed blastospore formation, and greatly reduced virulence to caterpillars. The disruption of Mad2 blocked the adhesion of M. anisopliae to plant epidermis but had no effects on fungal differentiation and entomopathogenicity. Thus, regulation, localization, and specificity control the functional distinction between Mad1 and Mad2 and enable M. anisopliae cells to adapt their adhesive properties to different habitats.As judged by species number, the ascomycetes are the most successful fungi, comprising some 75% of known species (30). They include many pathogens of plants and animals, and ascomycetes are also the principal inhabitants of soils containing solid resources such as plant and animal remains. Many economically important pathogenic fungi have the ability to be free living in soil and elsewhere, but there is limited knowledge of the range of ecological niches that they can occupy in the absence of a host (16). For example, the commercially important biocontrol agent Metarhizium anisopliae is a ubiquitous pathogen of insects and a well-established model organism for the study of insect-microbe interactions (2, 26). However, only recently was it reported to colonize the rhizosphere (the layer of soil influenced by root metabolism) and adhere to plant root surfaces (15). M. anisopliae is believed to exert a considerable influence within this ecological niche by repelling and killing soil insects (15).A major goal of evolutionary and ecological genetics is to identify genes that adapt organisms to their ecological niches and to understand the interaction between their products and the environment. However, genes directly involved in ecological attributes are hard to identify (4). Most forms of fungal adaptation at the molecular level, such as circadian rhythms (20), have focused on the traditional model systems used for genetic analysis such as yeasts and Neurospora crassa. However, many ecologically important lifestyles such as pathogenicity are not represented by these models. Acquiring a detailed understanding of each species in every ecosystem is unrealist...

The caterpillar fungus Ophiocordyceps sinensis (best known as Cordyceps sinensis) mummifies ghost moth larvae exclusively in Tibetan Plateau alpine ecosystems. Touted as "Himalayan Viagra", the fungus is highly prized due to its medical benefits and dwindling supplies. Attempts to culture the sexual fruiting-body have failed and the huge market demand has led to severe devastation of local ecosystems and to the fungus heading towards extinction. By genome sequencing, we establish that unlike related insect pathogens O. sinensis contains two compatible mating-type genes in its genome and is self-fertile, i.e. homothallic. However, sexual processes are only initiated under native environmental conditions. O. sinensis resembles biotrophic plant pathogens in having a genome shaped by retrotransposon-driven expansions. The resulting changes in gene content suggest that O. sinensis has a biphasic pathogenic mechanism beginning with stealth pathogenesis in early host instars. O. sinensis is the first psychrophilic fungus sequenced and is adapted to extreme cold with putative antifreeze proteins and mechanisms for increasing lipid accumulation and fatty acid unsaturation. We hypothesize that for the inbreeding O. sinensis the massive proliferation of retrotransposons provides a tradeoff between the advantages of increased genetic variation independent of sexual recombination and deletion of genes dispensable for its specialized pathogenic lifestyle.Ophiocordyceps sinensis, genome expansion, homothallism, biotrophic parasitism, psychrophile Citation:Hu X, Zhang Y J, Xiao G H, et al. Genome survey uncovers the secrets of sex and lifestyle in caterpillar fungus.

Metarhizium anisopliae infects mosquitoes through the cuticle and proliferates in the hemolymph. To allow M. anisopliae to combat malaria in mosquitoes with advanced malaria infections, we produced recombinant strains expressing molecules that target sporozoites as they travel through the hemolymph to the salivary glands. Eleven days after a Plasmodium-infected blood meal, mosquitoes were treated with M. anisopliae expressing salivary gland and midgut peptide 1 (SM1), which blocks attachment of sporozoites to salivary glands; a single-chain antibody that agglutinates sporozoites; or scorpine, which is an antimicrobial toxin. These reduced sporozoite counts by 71%, 85%, and 90%, respectively. M. anisopliae expressing scorpine and an [SM1]8:scorpine fusion protein reduced sporozoite counts by 98%, suggesting that Metarhizium-mediated inhibition of Plasmodium development could be a powerful weapon for combating malaria.