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.

Entomopathogenic fungi can produce a series of chitinases, some of which act synergistically with proteases to degrade insect cuticle. However, chitinase involvement in insect fungus pathogenesis has not been fully characterized. In this paper, an endochitinase, Bbchit1, was purified to homogeneity from liquid cultures of Beauveria bassiana grown in a medium containing colloidal chitin. Bbchit1 had a molecular mass of about 33 kDa and pI of 5.4. Based on the N-terminal amino acid sequence, the chitinase gene, Bbchit1, and its upstream regulatory sequence were cloned. Bbchit1 was intronless, and there was a single copy in B. bassiana. Its regulatory sequence contained putative CreA/Crel carbon catabolic repressor binding domains, which was consistent with glucose suppression of Bbchit1. At the amino acid level, Bbchit1 showed significant similarity to a Streptomyces avermitilis putative endochitinase, a Streptomyces coelicolor putative chitinase, and Trichoderma harzianum endochitinase Chit36Y. However, Bbchit1 had very low levels of identity to other chitinase genes previously isolated from entomopathogenic fungi, indicating that Bbchit1 was a novel chitinase gene from an insect-pathogenic fungus. A gpd-Bbchit1 construct, in which Bbchit1 was driven by the Aspergiullus nidulans constitutive promoter, was transformed into the genome of B. bassiana, and three transformants that overproduced Bbchit1 were obtained. Insect bioassays revealed that overproduction of Bbchit1 enhanced the virulence of B. bassiana for aphids, as indicated by significantly lower 50% lethal concentrations and 50% lethal times of the transformants compared to the values for the wild-type strain.

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.

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Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.