Nemat O. Keyhani

Improving mycoinsecticides for insect biological control

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.

Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing components of an arms race between a host and pathogen

Significance Although entomopathogenic fungi and their invertebrate hosts share a >300 million year co-evolutionary history, little is known concerning the biochemical and genetic basis of insect defensive tactics and the countermeasures evolved and evolving by the pathogen to thwart these defenses. Our results provide a molecular mechanism to help explain why some insects are more resistant to broad host-range entomopathogenic fungi. We identify beetle cuticular secretions and a fungal detoxifying enzyme as components of an arms race between insects and the fungal pathogen, suggesting an evolving role for the quinone reductase enzyme as a specific virulence factor for host quinone detoxification. As races have winners and losers, this paper captures a snapshot where the host is leading the race. Entomopathogenic fungi and their insect hosts represent a model system for examining invertebrate-pathogen coevolutionary selection processes. Here we report the characterization of competing components of an arms race consisting of insect protective antimicrobial compounds and evolving fungal mechanisms of detoxification. The insect pathogenic fungus Beauveria bassiana has a remarkably wide host range; however, some insects are resistant to fungal infection. Among resistant insects is the tenebrionid beetle Tribolium castaneum that produces benzoquinone-containing defensive secretions. Reduced fungal germination and growth was seen in media containing T. castaneum dichloromethane extracts or synthetic benzoquinone. In response to benzoquinone exposure, the fungus expresses a 1,4-benzoquinone oxidoreductase, BbbqrA, induced >40-fold. Gene knockout mutants (ΔBbbqrA) showed increased growth inhibition, whereas B. bassiana overexpressing BbbqrA (Bb::BbbqrAO) displayed increased resistance to benzoquinone compared with wild type. Increased benzoquinone reductase activity was detected in wild-type cells exposed to benzoquinone and in the overexpression strain. Heterologous expression and purification of BbBqrA in Escherichia coli confirmed NAD(P)H-dependent benzoquinone reductase activity. The ΔBbbqrA strain showed decreased virulence toward T. castaneum, whereas overexpression of BbbqrA increased mortality versus T. castaneum. No change in virulence was seen for the ΔBbbqrA or Bb::BbbqrAO strains when tested against the greater wax moth Galleria mellonella or the beetle Sitophilus oryzae, neither of which produce significant amounts of cuticular quinones. The observation that artificial overexpression of BbbqrA results in increased virulence only toward quinone-secreting insects implies the lack of strong selection or current failure of B. bassiana to counteradapt to this particular host defense throughout evolution.

A carbon responsive G‐protein coupled receptor modulates broad developmental and genetic networks in the entomopathogenic fungus, Beauveria bassiana

In fungi, G-protein coupled receptors (GPCRs) link ligand/nutrient sensing to growth, mating, developmental/life-stage activation and pathogenesis. A GPCR was characterized from the entomopathogenic fungus, Beauveria bassiana (BbGPCR3), which links nutrient sensing to stress response and development. ΔBbGPCR3 mutants grew slower on various carbohydrates and displayed increased sensitivity to osmotic, oxidative and cell wall stresses. Gene expression profiling revealed a set of heat-shock and antioxidant factors that failed to be induced under oxidative stress and aberrant regulation of compatible solute-forming enzymes and cell wall biosynthesis/remodelling proteins in ΔBbGPCR3 after osmotic stress. Glucose-specific developmental defects included reduced (> 90%) conidiation and reduced dimorphic transition to the production of yeast-like blastospores, effects suppressed in media containing trehalose or glycerol, but not by addition of cyclic AMP. Insect bioassays revealed reduced virulence in topical assays but no effect in intrahaemoceol injection assays, indicating that BbGPCR3 was important in sensing signals during the initial interaction with the host but dispensable for post-penetration events. Comparative gene expression profiling of ΔBbGPCR3 mutants grown in glucose media compared with wild-type/glucose and ΔBbGPCR3/trehalose grown cells revealed sets of genes misregulated and recovered, respectively. These data link BbGPCR3 to broad developmental and genetic networks that include the major MAP kinase pathways.

Bbmsn2 acts as a pH‐dependent negative regulator of secondary metabolite production in the entomopathogenic fungus Beauveria bassiana

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.

Expression of a toll signaling regulator serpin in a mycoinsecticide for increased virulence.

ABSTRACT Serpins are ubiquitously distributed serine protease inhibitors that covalently bind to target proteases to exert their activities. Serpins regulate a wide range of activities, particularly those in which protease-mediated cascades are active. The Drosophila melanogaster serpin Spn43Ac negatively controls the Toll pathway that is activated in response to fungal infection. The entomopathogenic fungus Beauveria bassiana offers an environmentally friendly alternative to chemical pesticides for insect control. However, the use of mycoinsecticides remains limited in part due to issues of efficacy (low virulence) and the recalcitrance of the targets (due to strong immune responses). Since Spn43Ac acts to inhibit Toll-mediated activation of defense responses, we explored the feasibility of a new strategy to engineer entomopathogenic fungi with increased virulence by expression of Spn43Ac in the fungus. Compared to the 50% lethal dose (LD50) for the wild-type parent, the LD50 of B. bassiana expressing Spn43Ac (strain Bb::S43Ac-1) was reduced ∼3-fold, and the median lethal time against the greater wax moth (Galleria mellonella) was decreased by ∼24%, with the more rapid proliferation of hyphal bodies being seen in the host hemolymph. In vitro and in vivo assays showed inhibition of phenoloxidase (PO) activation in the presence of Spn43Ac, with Spn43Ac-mediated suppression of activation by chymotrypsin, trypsin, laminarin, and lipopolysaccharide occurring in the following order: chymotrypsin and trypsin > laminarin > lipopolysaccharide. Expression of Spn43Ac had no effect on the activity of the endogenous B. bassiana-derived cuticle-degrading protease (CDEP-1). These results expand our understanding of Spn43Ac function and confirm that suppression of insect immune system defenses represents a feasible approach to engineering entomopathogenic fungi for greater efficacy.

Involvement of a caleosin in lipid storage, spore dispersal, and virulence in the entomopathogenic filamentous fungus, Beauveria bassiana

Eukaryotic cells store lipids in membrane-encased droplets. The entomopathogenic fungus, Beauveria bassiana, initiates infection via attachment of its spores to the epicuticle or waxy layer of target insects, degrading and assimilating host surface hydrocarbons, carbohydrates and proteins. Caleosins are components of the proteinaceous coat of lipid droplets and a single B. bassiana caleosin homologue, Bbcal1, was identified and characterized. The BbCal1 sequence contained an EF-hand Ca(2+) binding domain and potential hydrophobic stretches similar to those found in plant caleosins, along with a proline knot motif defined by only two proline residues. Targeted gene inactivation of Bbcal1 did not appear to affect spore germination, growth on lipid substrates or stress response, but changes in lipid, vacuole and endoplasmic reticulum/multilamellar vesicle-like structures, and altered cellular lipid profiles were seen in conidia grown on a variety of substrates including potato dextrose agar, olive oil, glyceride trioleate, oleic acid and the alkane, C16 . The ΔBbcal1 mutant produced more compact assemblages of conidia, displayed a reduced and delayed spore dispersal phenotype, and showed decreased virulence in insect bioassays using the greater wax moth, Galleria mellonella. Our data indicate novel functions for caleosins in fungal virulence, spore development and the trafficking and/or turnover of lipid-related structures.

A novel mitochondrial membrane protein, Ohmm, limits fungal oxidative stress resistance and virulence in the insect fungal pathogen Beauveria bassiana

The Hog1 mitogen-activated protein (MAP) kinase regulates environmental stress responses and virulence in the entomopathogenic fungus Beauveria bassiana. To further characterize this pathway, we constructed a subtraction library enriched for genes regulated by Hog1. One targeted gene, encoding a novel membrane protein, Ohmm (oxidative homeostasis membrane-protein-mitochondria), was uniquely identified as being downregulated in the ΔHog1 background during growth under non-stress and osmotic stress conditions, but upregulated under oxidative stress. Ohmm was an experimentally validated flavin-binding protein and targeted to the mitochondria. Deletion of Ohmm resulted in increased oxidative stress resistance, whereas overexpression caused an opposite phenotype. The ΔOhmm showed accumulation of reactive oxygen species with alterations in cell wall composition and compatible solute accumulation evident as compared with the wild type parent. Conidiation was reduced > 80%; however, conidia produced by the ΔOhmm strain germinated significantly faster than wild type cells. Insect bioassays using the greater wax moth revealed increased virulence for the ΔOhmm strain in both topical and intrahemocoel injection assays, indicating a negative effect of the presence of Ohmm with respect to pathogenesis. As predicted from our characterization, deletion of Ohmm in a ΔHog1 background rescued its oxidative sensitivity phenotype, confirming that Ohmm acts downstream of the Hog1 MAP-kinase.

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC50 values differed by up to ~10-fold (5.3×106−4.5×105 conidia/ml) and LT50 values by ~40% (9.8−7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.

A putative methyltransferase, mtrA, contributes to development, spore viability, protein secretion and virulence in the entomopathogenic fungus Beauveria bassiana.

The filamentous fungus, Beauveria bassiana, is a ubiquitously distributed insect pathogen, currently used as an alternative to chemical pesticides for pest control. Conidiospores are the means by which the fungus disseminates in the environment, and these cells also represent the infectious agent most commonly used in field applications. Little, however, is known concerning the molecular basis for maintenance of spore viability, a critical feature for survival and persistence. Here, we report on the role of a putative methyltransferase, BbmtrA, in conidial viability, normal fungal growth and development, and virulence, via characterization of a targeted gene knockout strain. Loss of BbmtrA resulted in pleiotropic effects including reduced germination, growth and conidiation, with growing mycelia displaying greater branching than the WT parent. Conidial viability dramatically decreased over time, with <5 % of the cells remaining viable after 30 days as compared with >80 % of the WT. Reduced production of extracellular proteins was also observed for the ΔBbmtrA mutant, including protease/peptidases, glycoside hydrolases and the hyd1 hydrophobin. The latter was further confirmed by hyd1 gene expression analysis. Insect bioassays using the greater wax moth, Galleria mellonella, further revealed that the ΔBbmtrA strain was attenuated in virulence and failed to sporulate on host cadavers. These data support a global role for mtrA in fungal physiological processes.

The PacC transcription factor regulates secondary metabolite production and stress response, but has only minor effects on virulence in the insect pathogenic fungus Beauveria bassiana

The PacC transcription factor is an important component of the fungal ambient pH-responsive regulatory system. Loss of pacC in the insect pathogenic fungus Beauveria bassiana resulted in an alkaline pH-dependent decrease in growth and pH-dependent increased susceptibility to osmotic (salt, sorbitol) stress and SDS. Extreme susceptibility to Congo Red was noted irrespective of pH, and ΔBbpacC conidia showed subtle increases in UV susceptibility. The ΔBbPacC mutant showed a reduced ability to acidify media during growth due to failure to produce oxalic acid. The ΔBbPacC mutant also did not produce the insecticidal compound dipicolinic acid, however, production of a yellow-colored compound was noted. The compound, named bassianolone B, was purified and its structure determined. Despite defects in growth, stress resistance, and oxalate/insecticidal compound production, only a small decrease in virulence was seen for the ΔBbpacC strain in topical insect bioassays using larvae from the greater waxmoth, Galleria mellonella or adults of the beetle, Tenebrio molitor. However, slightly more pronounced decreases were seen in virulence via intrahemcoel injection assays (G. mellonella) and in assays using T. molitor larvae. These data suggest important roles for BbpacC in mediating growth at alkaline pH, regulating secondary metabolite production, and in targeting specific insect stages.