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.

Beauveria bassiana is an economically important insect-pathogenic fungus which is widely used as a biocontrol agent to control a variety of insect pests. However, its insecticide efficacy in the field is often influenced by adverse environmental factors. Thus, understanding the genetic regulatory processes involved in the response to environmental stress would facilitate engineering and production of a more efficient biocontrol agent. Here, a mitogen-activated protein kinase (MAPK)-encoding gene, Bbhog1, was isolated from B. bassiana and shown to encode a functional homolog of yeast HIGH-OSMOLARITY GLYCEROL 1 (HOG1). A Bbhog1 null mutation was generated in B. bassiana by targeted gene replacement, and the resulting mutants were more sensitive to hyperosmotic stress, high temperature, and oxidative stress than the wild-type controls. These results demonstrate the conserved function of HOG1 MAPKs in the regulation of abiotic stress responses. Interestingly, ⌬Bbhog1 mutants exhibited greatly reduced pathogenicity, most likely due to a decrease in spore viability, a reduced ability to attach to insect cuticle, and a reduction in appressorium formation. The transcript levels of two hydrophobin-encoding genes, hyd1 and hyd2, were dramatically decreased in a ⌬Bbhog1 mutant, suggesting that Bbhog1 may regulate the expression of the gene associated with hydrophobicity or adherence.Mycoinsecticides are important insect pest control agents (10,19,41). Unlike entomopathogenic bacteria and viruses that invade insects through their digestive tracks, fungal pathogens penetrate the host integument and are considered the only group of microbial biocontrol agents active against sucking-type insect pests (21, 49). However, low killing speed and sensitivity to adverse environment factors such as desiccation, high temperature, and UV radiation limit the widespread use of entomopathogenic fungi (6,22,40). Thus, an understanding of the regulatory processes involved in response to environment stress is essential for commercial development and improvement of these biocontrol fungi.Mitogen-activated protein kinases (MAPKs), a family of serine-threonine protein kinases, are widespread in eukaryotic cells and play crucial roles in transduction of a variety of extracellular signals and regulation of various development and differentiation processes (37, 45). MAPKs are usually activated by MAPK kinases, which are in turn activated by MAPK kinase kinases. These MAPK kinase kinase-MAPK kinase-MAPK cascades are conserved in eukaryotic cells and have been studied extensively in many organisms (45). In Saccharomyces cerevisiae, at least five MAPK pathways have been identified. These pathways are designated the FUS3, KSS1, HOG1, SLT2, and SMK1 MAPK cascades, and they are involved in mating, filamentous growth, the high-osmolarity response, cell integrity, and ascospore formation, respectively (20). Recent studies showed that homologs of HOG1 are involved in responses to osmotic stress (12,29,44,58), oxidative stress (28, 47), heat shock (28), and ...

The desire for decreased reliance on chemical pesticides continues to fuel interest in alternative means for pest control including the use of naturally occurring microbial insect pathogens. Insects, as vectors of disease causing agents or as agricultural pests, are responsible for millions of deaths and significant economic losses worldwide, placing stresses on productivity (GDP) and human health and welfare. In addition, alterations in climate change are likely to affect insect ranges, expanding their access to previously constrained geographic areas, a potentially worrisome outcome. Metarhizium anisopliae and Beauveria bassiana, two cosmopolitan fungal pathogens of insects found in almost all ecosystems, are the most commonly applied mycoinsecticides for a variety of insect control purposes. The availability of the complete genomes for both organisms coupled to robust technologies for their transformation has led to several advances in engineering these fungi for greater efficacy and/or utility in pest control applications. Here, we will provide an overview of the fungal-insect and fungal-plant interactions that occur and highlight recent advances in the genetic engineering of these fungi. The latter work has resulted in the development of strains displaying (1) increased resistance to abiotic stress, (2) increased cuticular targeting and degradation, (3) increased virulence via expression of insecticidal protein/peptide toxins, (4) the ability to block transmission of disease causing agents, and (5) the ability to target specific insect hosts, decrease host fecundity, and/or alter insect behaviors.

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For most organisms, carbon and nitrogen uptake are essential for growth, development and, where applicable, pathogenesis. The role of the carbon catabolite repressor transcription factor homologue BbcreA in the entomopathogenic fungus Beauveria bassiana was investigated. Deletion of BbcreA resulted in pleiotropic effects, including nutrient toxicity, leading to a novel cell lytic phenotype. Fungal growth in rich media and minimal media containing select amino acids/peptides was severely compromised, with microscopic examination revealing conidial-base germ tube degeneration and cell lysis occurring during growth, a phenomenon exacerbated at higher temperatures (32°C). Depending upon nutrient conditions, growth, pigment and aerial mycelium production, sporulation and dimorphic transition to blastospore production were also impaired in the ΔBbcreA strain. Although loss of BbcreA resulted in de-repression of secreted protease and lipase, enzymes critical in mediating pathogenesis, insect bioassays indicated severe defects in virulence using both topical and intra-haemocoel injection assays, with eruption and subsequent sporulation on host cadavers greatly reduced in the mutant. These data suggest that BbcreA functions as more than a carbon repressor and plays important roles in nutrient utilization, cell homeostasis and virulence. In particular, BbcreA is required for proper assimilation of select amino acids and peptides, including asparagine, arginine and proline.

Summary Laccases are widely present in bacteria, fungi, plants and invertebrates and involved in a variety of physiological functions. Here, we report that Beauveria bassiana, an economic important entomopathogenic fungus, secretes a laccase 2 (BbLac2) during infection that detoxifies insect immune response‐generated reactive oxygen species (ROS) and interferes with host immune phenoloxidase (PO) activation. BbLac2 is expressed in fungal cells during proliferation in the insect haemocoel and can be found to distribute on the surface of haemolymph‐derived in vivo fungal hyphal bodies or be secreted. Targeted gene‐knockout of BbLac2 increased fungal sensitivity to oxidative stress, decreased virulence to insect, and increased host PO activity. Strains overexpressing BbLac2 showed increased virulence, with reduced host PO activity and lowered ROS levels in infected insects. In vitro assays revealed that BbLac2 could eliminate ROS and oxidize PO substrates (phenols), verifying the enzymatic functioning of the protein in detoxification of cytotoxic ROS and interference with the PO cascade. Moreover, BbLac2 acted as a cell surface protein that masked pathogen associated molecular patterns (PAMPs), enabling the pathogen to evade immune recognition. Our data suggest a multifunctional role for fungal pathogen‐secreted laccase 2 in evasion of insect immune defenses.

Fungal secondary metabolites are chemical compounds important for development, environmental adaptation and for potential biotechnological and pharmaceutical applications. Oosporein, a red-pigmented benzoquinone, produced by many fungal insect pathogenic Beauveria spp., shows remarkable functional diversity, displaying antimicrobial, antiviral and even anti-proliferative activities. A homologue of the msn2/seb1 transcription factor was identified in a Beauveria bassiana random T-DNA insertion library. Targeted gene-knockout of Bbmsn2 resulted in reduced growth and increased sensitivity to Calcofluor White, H2 O2 and Congo Red. However, when normalized to growth at 26°C, the ΔBbmsn2 mutant was more tolerant to high temperature (32°C) than the wild type parent. The ΔBbmsn2 mutant also displayed a pH-dependent growth phenotype, with little growth seen at pH < 5.0 but, better growth at alkaline conditions (pH > 8.0). Unexpectedly, a pH-dependent deregulation of a red pigment, identified as oosporein, was seen in the ΔBbmsn2 mutant. The ΔBbmsn2 strain was impaired in virulence in both topical and intrahaemocoel injection bioassays against Galleria mellonella. ΔBbmsn2 proliferation in the host haemolymph and conidiation on the host cadaver was reduced. These data indicate that Bbmsn2 acts as a negative regulator of oosporein production and contributes to virulence and growth in response to external pH in B. bassiana.