Infections mediated by broad host range entomopathogenic fungi represent seminal observations that led to one of the first germ theories of disease and are a classic example of a co-evolutionary arms ...race between a pathogen and target hosts. These fungi are able to parasitize susceptible hosts via direct penetration of the cuticle with the initial and potentially determining interaction occurring between the fungal spore and the insect epicuticle. Entomogenous fungi have evolved mechanisms for adhesion and recognition of host surface cues that help direct an adaptive response that includes the production of: (a) hydrolytic, assimilatory, and/or detoxifying enzymes including lipase/esterases, catalases, cytochrome P450s, proteases, and chitinases; (b) specialized infectious structures, e.g., appressoria or penetrant tubes; and (c) secondary and other metabolites that facilitate infection. Aside from immune responses, insects have evolved a number of mechanisms to keep pathogens at bay that include: (a) the production of (epi) cuticular antimicrobial lipids, proteins, and metabolites; (b) shedding of the cuticle during development; and (c) behavioral-environmental adaptations such as induced fever, burrowing, and grooming, as well as potentially enlisting the help of other microbes, all intended to stop the pathogen before it can breach the cuticle. Virulence and host-defense can be considered to be under constant reciprocal selective pressure, and the action on the surface likely contributes to phenomena such as strain variation, host range, and the increased virulence often noted once a (low) virulent strain is "passaged" through an insect host. Since the cuticle represents the first point of contact and barrier between the fungus and the insect, the "action on the surface" may represent the defining interactions that ultimately can lead either to successful mycosis by the pathogen or successful defense by the host. Knowledge concerning the molecular mechanisms underlying this interaction can shed light on the ecology and evolution of virulence and can be used for rational design strategies at increasing the effectiveness of entomopathogenic fungi for pest control in field applications.
Broad host range insect pathogenic fungi penetrate through the host cuticle, necessitating an ability to confront and overcome surface lipids and other molecules that often include antimicrobial ...compounds. In this context, induction of lipid assimilatory pathways by exogenous substrates is crucial for successful infection to occur, and lipid growth substrates can have significant effects on the virulence of fungal infectious propagules, e.g. conidia. The production of lipases is a critical part of the cuticle-degrading repertoire and pathways involved in triglyceride metabolism and phospholipid homeostasis have been shown to contribute to host invasion. Mobilization of endogenous lipid stores via the activities of the caleosin and perilipin lipid storage-turnover proteins, have been linked to diverse processes including formation of penetration structures, e.g. germ tubes and appressoria, spore properties and dispersal, and the ability to respond to lipid growth substrates and virulence. Here, we summarize recent advances in our understanding of lipid assimilation and mobilization pathways in the ability of entomogenous fungi to infect and use host substrates. Host surface and internal lipids can alternatively act as antifungal barriers, inducers of pathogenesis-related pathways, and/or as fungal growth substrates. Lipids and lipid assimilation can be considered as forming a co-evolutionary web between the insect host and entomogenous fungi.
Ambrosia beetles and their microbial communities, housed in specialized structures termed mycangia, represent one of the oldest and most diverse systems of mutualism and parasitism described thus ...far. Comprised of core filamentous fungal members, but also including bacteria and yeasts, the mycangia represent a unique adaptation that allows beetles to store and transport their source of nutrition. Although perhaps the most ancient of “farmers,” the nature of these interactions remains largely understudied, with the exception of a handful of emerging pathosystems, where the fungal partner acts as a potentially devastating tree pathogen. Such virulence is often seen during “invasions,” where (invasive) beetles carrying the fungal symbiont/plant pathogen expand into new territories and presumably “naïve” trees. Here, we summarize recent findings on the phylogenetic relationships between beetles and their symbionts and advances in the developmental and genetic characterization of the mechanisms that underlie insect-fungal-plant interactions. Results on genomic, transcriptomic, and metabolomic aspects of these relationships are described. Although many members of the fungal
Raffaelea
-beetle symbiont genera are relatively harmless to host trees, specialized pathosystems including wilt diseases of laurel and oak, caused by specific subspecies (
R. lauricola
and
R. quercus
, in the USA and East Asia, respectively), have emerged as potent plant pathogens capable of killing healthy trees. With the development of genetic tools, coupled to biochemical and microscopic techniques, the ambrosia beetle-fungal symbiont is establishing itself as a unique model system to study the molecular determinants and mechanisms that underlie the convergences of symbioses, mutualism, parasitism, and virulence.
Key points
• Fungal-beetle symbioses are diverse and ancient examples of microbial farming.
• The mycangium is a specialized structure on insects that houses microbial symbionts.
• Some beetle symbiotic fungi are potent plant pathogens vectored by the insect.
Ambrosia beetles require their fungal symbiotic partner as their cultivated (farmed) food source in tree galleries. While most fungal-beetle partners do not kill the host trees they inhabit, since ...their introduction (invasion) into the United states around ~2002, the invasive beetle Xyleborus glabratus has vectored its mutualist partner (but plant pathogenic) fungus, Harringtonia lauricola, resulting in the deaths of over 300 million trees. Concerningly, indigenous beetles have been caught bearing H. lauricola. Here, we show colonization of the mycangia of the indigenous X. affinis ambrosia beetle by H. lauricola. Mycangial colonization occurred within 1 h of feeding, with similar levels seen for H. lauricola as found for the native X. affinis-R. arxii fungal partner. Fungal mycangial occupancy was stable over time and after removal of the fungal source, but showed rapid turnover when additional fungal cells were available. Microscopic visualization revealed two pre-oral mycangial pouches of ~100-200 × 25-50 μm/each, with narrow entry channels of 25-50 × 3-10 μm. Fungi within the mycangia underwent a dimorphic transition from filamentous/blastospore growth to yeast-like budding with alterations to membrane structures. These data identify the characteristics of ambrosia beetle mycangial colonization, implicating turnover as a mechanism for host switching of H. lauricola to other ambrosia beetle species.
The Ascomycete fungal insect pathogens, Beauveria and Metarhizium spp. have emerged as model systems with which to probe diverse aspects of fungal growth, stress response, and pathogenesis. Due to ...the availability of genomic resources and the development of robust methods for genetic manipulation, the last 5 years have witnessed a rapid increase in the molecular characterization of genes and their pathways involved in stress response and signal transduction in these fungi. These studies have been performed mainly via characterization of gene deletion/knockout mutants and have included the targeting of general proteins involved in stress response and/or virulence, e.g. catalases, superoxide dismutases, and osmolyte balance maintenance enzymes, membrane proteins and signaling pathways including GPI anchored proteins and G-protein coupled membrane receptors, MAPK pathways, e.g. (i) the pheromone/nutrient sensing, Fus3/Kss1, (ii) the cell wall integrity, Mpk1, and (iii) the high osmolarity, Hog1, the PKA/adenyl cyclase pathway, and various downstream transcription factors, e.g. Msn2, CreA and Pac1. Here, we will discuss current research that strongly suggests extensive underlying contributions of these biochemical and signaling pathways to both abiotic stress response and virulence.
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.
Summary
The entomogenous filamentous fungus, Beauveria bassiana expresses two hydrophobin genes, hyd1 and hyd2, hypothesized to be involved in cell surface hydrophobicity, adhesion, virulence, and to ...constitute the protective spore coat structure known as the rodlet layer. Targeted gene inactivation of hyd1 resulted in seemingly ‘bald’ conidia that contained significantly altered surface fascicles or bundles. These cells displayed decreased spore hydrophobicity, loss of water mediated dispersal, changes in surface carbohydrate epitopes and β‐1,3‐glucan distribution, lowered virulence in insect bioassays, but no effect on adhesion. In contrast, Δhyd2 mutants retained distorted surface bundles, but truncated/incomplete rodlets could be seen within the bundles. Δhyd2 conidia displayed both decreased cell surface hydrophobicity and adhesion, but the mutant was unaffected in virulence. The double Δhyd1Δhyd2 mutant was distinct from the single mutants, lacking both bundles and rodlets, and displaying additively decreased cell surface hydrophobicity, reduced cell attachment and lowered virulence than the Δhyd1 mutant. Epitope tagged constructs of the proteins were used to examine the expression and distribution of the proteins and to demonstrate the continued presence of Hyd2 in the Δhyd1 strain and vice versa. The implications of our results with respect to fascicle and rodlet assembly on the spore surface are discussed.
The regulatory network and biological functions of the fungal secondary metabolite oosporein have remained obscure. Beauveria bassiana has evolved the ability to parasitize insects and out-compete ...microbial challengers for assimilation of host nutrients. A novel zinc finger transcription factor, BbSmr1 (B. bassiana secondary metabolite regulator 1), was identified in a screen for oosporein overproduction. Deletion of Bbsmr1 resulted in up-regulation of the oosporein biosynthetic gene cluster (OpS genes) and constitutive oosporein production. Oosporein production was abolished in double mutants of Bbsmr1 and a second transcription factor, OpS3, within the oosporein gene cluster (ΔBbsmr1ΔOpS3), indicating that BbSmr1 acts as a negative regulator of OpS3 expression. Real-time quantitative PCR and a GFP promoter fusion construct of OpS1, the oosporein polyketide synthase, indicated that OpS1 is expressed mainly in insect cadavers at 24–48 h after death. Bacterial colony analysis in B. bassiana-infected insect hosts revealed increasing counts until host death, with a dramatic decrease (∼90%) after death that correlated with oosporein production. In vitro studies verified the inhibitory activity of oosporein against bacteria derived from insect cadavers. These results suggest that oosporein acts as an antimicrobial compound to limit microbial competition on B. bassiana-killed hosts, allowing the fungus to maximally use host nutrients to grow and sporulate on infected cadavers.
Significant progress has been made in the biochemical and genetic characterization of the host-pathogen interaction mediated by insect pathogenic fungi, with the most widely studied being the ...Ascomycetes (Hypocrealean) fungi,
Metarhizium robertsii
and
Beauveria bassiana
. However, few studies have examined the consequences and effects of host (insect) microbes, whether compatible or antagonistic, on the development and survival of entomopathogenic fungi. Host microbes can act on the insect cuticular surface, within the gut, in specialized insect microbe hosting structures, and within cells, and they include a wide array of facultative and/or obligate exosymbionts and endosymbionts. The insect microbiome differs across developmental stages and in response to nutrition (e.g., different plant hosts for herbivores) and environmental conditions, including exposure to chemical insecticides. Here, we review recent advances indicating that insect-pathogenic fungi have evolved a spectrum of strategies for exploiting or suppressing host microbes, including the production of antimicrobial compounds that are expressed at discrete stages of the infection process. Conversely, there is increasing evidence that some insects have acquired microbes that may be specialized in the production of antifungal compounds to combat infection by (entomopathogenic) fungi. Consideration of the insect microbiome in fungal insect pathology represents a new frontier that can help explain previously obscure ecological and pathological aspects of the biology of entomopathogenic fungi. Such information may lead to novel approaches to improving the efficacy of these organisms in pest control efforts.
is a worldwide generalist pest with remarkable adaptations to environments and stresses, including developmental stage-related behavioral and physiological adaptations, such as diverse feeding ...preferences, mate seeking, and pesticide resistance. Insects' odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are essential for the chemical recognition during behavioral responses or other physiological processes. The genome-wide identification and the gene expression patterns of all these identified
and
across developmental stage-related
have not been reported. Here, we screened for genome-wide
and
, and analyzed the gene expression patterns of
and
repertoires across all developmental stages and sexes. We found 33
and 22
in the
genome. The majority of the
genes were most highly expressed in the adult male or female stages, while more
genes were highly expressed in the larval or egg stages, indicating their function complementation. The gene expression patterns of
and
revealed strong correlations with their respective phylogenic trees, indicating a correlation between function and evolution. In addition, we analyzed the chemical-competitive binding of a widely expressed protein,
31, to host plant odorants, sex pheromones, and insecticides. Further ligands binding assay revealed a broad functional related binding spectrum of
31 to host plant odorants, sex pheromones, and insecticides, suggesting its potential function in food, mate seeking, and pesticide resistance. These results provide guidance for future research on the development of behavioral regulators of
.
or other environmentally friendly pest-control strategies.
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