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.

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...

Quinones are widely distributed in nature and exhibit diverse biological or pharmacological activities; however, their biosynthetic machineries are largely unknown. The bibenzoquinone oosporein was first identified from the ascomycete insect pathogen Beauveria bassiana >50 y ago. The toxin can also be produced by different plant pathogenic and endophytic fungi with an array of biological activities. Here, we report the oosporein biosynthetic machinery in fungi, a polyketide synthase (PKS) pathway including seven genes for quinone biosynthesis. The PKS oosporein synthase 1 (OpS1) produces orsellinic acid that is hydroxylated to benzenetriol by the hydroxylase OpS4. The intermediate is oxidized either nonenzymatically to 5,5′-dideoxy-oosporein or enzymatically to benzenetetrol by the putative dioxygenase OpS7. The latter is further dimerized to oosporein by the catalase OpS5. The transcription factor OpS3 regulates intrapathway gene expression. Insect bioassays revealed that oosporein is required for fungal virulence and acts by evading host immunity to facilitate fungal multiplication in insects. These results contribute to the known mechanisms of quinone biosynthesis and the understanding of small molecules deployed by fungi that interact with their hosts.

Cordycepin (COR) and pentostatin (PTN) are adenosine analogs with related bioactivity profiles as both mimic adenosine and can inhibit some of the processes that are adenosine dependent. Both COR and PTN are also natural products and were originally isolated from the fungus Cordyceps militaris and the bacterium Streptomyces antibioticus, respectively. Here, we report that not only is PTN produced by C. militaris but that biosynthesis of COR is coupled with PTN production by a single gene cluster. We also demonstrate that this coupling is an important point of metabolic regulation where PTN safeguards COR from deamination by inhibiting adenosine deaminase (ADA) activity. ADA is not inhibited until COR reaches self-toxic levels, at which point ADA derepression occurs allowing for detoxification of COR to 3'-deoxyinosine. Finally, we show that using our biosynthetic insights, we can engineer C. militaris to produce higher levels of COR and PTN.

Fungal pathogens of plants and animals have multifarious effects; they cause devastating damages to agricultures, lead to life-threatening diseases in humans, or induce beneficial effects by reducing insect pest populations. Many virulence factors have been determined in different fungal pathogens; however, the molecular determinants contributing to fungal host selection and adaptation are largely unknown. In this study, we sequenced the genomes of seven ascomycete insect pathogens and performed the genome-wide analyses of 33 species of filamentous ascomycete pathogenic fungi that infect insects (12 species), plants (12), and humans (9). Our results revealed that the genomes of plant pathogens encode more proteins and protein families than the insect and human pathogens. Unexpectedly, more common orthologous protein groups are shared between the insect and plant pathogens than between the two animal group pathogens. We also found that the pathogenicity of host-adapted fungi evolved multiple times, and that both divergent and convergent evolutions occurred during pathogen–host cospeciation thus resulting in protein families with similar features in each fungal group. However, the role of phylogenetic relatedness on the evolution of protein families and therefore pathotype formation could not be ruled out due to the effect of common ancestry. The evolutionary correlation analyses led to the identification of different protein families that correlated with alternate pathotypes. Particularly, the effector-like proteins identified in plant and animal pathogens were strongly linked to fungal host adaptation, suggesting the existence of similar gene-for-gene relationships in fungus–animal interactions that has not been established before. These results well advance our understanding of the evolution of fungal pathogenicity and the factors that contribute to fungal pathotype formation.

Autophagy is a highly conserved process that maintains intracellular homeostasis by degrading proteins or organelles in all eukaryotes. The effect of autophagy on fungal biology and infection of insect pathogens is unknown. here, we report the function of MrATG8, an ortholog of yeast ATG8, in the entomopathogenic fungus Metarhizium robertsii. MrATG8 can complement an ATG8-defective yeast strain and deletion of MrATG8 impaired autophagy, conidiation and fungal infection biology in M. robertsii. compared with the wild-type and gene-rescued mutant, Mratg8Δ is not inductive to form the infection-structure appressorium and is impaired in defense response against insect immunity. in addition, accumulation of lipid droplets (LDs) is significantly reduced in the conidia of Mratg8Δ and the pathogenicity of the mutant is drastically impaired. We also found that the cellular level of a LD-specific perilipin-like protein is significantly lowered by deletion of MrATG8 and that the carboxyl terminus beyond the predicted protease cleavage site is dispensable for MrAtg8 function. To corroborate the role of autophagy in fungal physiology, the homologous genes of yeast ATG1, ATG4 and ATG15, designated as MrATG1, MrATG4 and MrATG15, were also deleted in M. robertsii. in contrast to Mratg8Δ, these mutants could form appressoria, however, the LD accumulation and virulence were also considerably impaired in the mutant strains. Our data showed that autophagy is required in M. robertsii for fungal differentiation, lipid biogenesis and insect infection. The results advance our understanding of autophagic process in fungi and provide evidence to connect autophagy with lipid metabolism.

pH-responsive transcription factor of the PacC/Rim101 family governs adaptation to environment, development and virulence in many fungal pathogens. In this study, we report the functions of a PacC homologue, MrpacC, in an insect pathogenic fungus Metarhizium robertsii. The gene was highly transcribed in the fungus in alkaline conditions, and deletion of MrpacC impaired fungal responses to ambient pH and salt/metal challenges but not osmotic stress. We found that MrpacC is required for fungal full virulence by contributing to penetration of insect cuticles, mycosis of insect cadavers and evasion of host immunity. In MrpacC deletion strains, the chitinase but not protease activity was reduced, which was consistent with the downregulation of groups A and C chitinase genes. Further, the glucosyltransferase genes involved in cell wall remodelling and protein glycosylation were upregulated in ΔMrpacC. MrpacC transcriptional control of chitinase and glucosyltransferase genes was verified both by the presence of PacC consensus binding motif in gene promoter regions and the promoter DNA-binding assays. The results of this study not only advances the understanding of PacC function in fungal development and virulence, but will also facilitate future studies on the mechanism(s) behind the selective control of target genes by PacC.