Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing components of an arms race between a host and pathogen
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 inEscherichia 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.
Learn more about Dr. Nemat Keyhani!
Department of Microbiology and Cell Science University of FloridaPh.D. (1994) Johns Hopkins University
MCB 4304 Molecular Genetics of Prokaryotes
Fungi and Insects Around Us: Animation Project
Description of Research
Our laboratory performs research in two main areas. (1) Tripartite interactions between Beauveria bassiana, a fungal pathogen of arthropods, their insect hosts, and plant partners with which the fungus forms associations. This project involves fundamental and applied research in host-pathogen interactions and insect biological control. We use a variety of approaches including molecular (gene knockouts, transcriptomics), biochemical (enzyme assays, yeast two-hybrid), and cell biological (microscopy; electron, fluorescent, and Atomic Force) to probe fungal development, insect virulence, and plant interactions. (2) Chemical communication in insects. We are examining proteins involved in olfaction and ligand transport in a number of insects including mosquitoes, the tsetse fly, and ants. In particular, we are interested in exploring the contributions of these proteins within a framework of the biochemistry and evolution of insect social behavior.
Ying, S.-H., Feng, M.-G., and Keyhani, N.O. (2013) A carbon responsive G-protein coupled receptor modulates broad developmental and genetic networks in the entomopathogenic fungus, Beauveria bassiana, Environmental Microbiology, accepted (doi:10.1111/1462-2920.12169).
Ortiz-Urquiza, A. and Keyhani, N.O. (2013) Action on the surface: entomopathogenic fungi versus the insect cuticle, Insects 4:357-374
Ortiz-Urquiza, A., Keyhani, N.O., and Quesada-Moraga, E. (2013) Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae, Biocontrol Science and Technology, accepted (dio:10.1080/09583157.2013.822474)
Pedrini, N., Ortiz-Urquiza, A., Huarte-Bonnet, C. and Keyhani, N.O. (2013) Targeting of insect cuticular lipids by entomopathogenic fungi: hydrocarbon degradation within the context of a host-pathogen interaction, Frontiers in Microbial Chemistry 4:R24
Kamareddine, L., Fan, Y., Osta, M.A., Keyhani, N.O. (2013) Expression of trypsin modulating oostatic factor (TMOF) in an entomopathogenic fungus increases its virulence towards Anopheles gambiae and reduces fecundity in the target mosquito. Parasites and Vectors 6(1): 22.
Wang, X.X., Ji, X.P., Li, J.X., Keyhani, N.O., Feng, M.-G., Ying, S.-H. (2013) A putative α-glucoside transporter gene, BbAGT1, contributes to carbohydrate utilization, growth, conidiation and virulence in the filamentous entomopathogenic fungus Beauveria bassiana. Res. Microbiol. 164: 480-489
Ying, S.-H., Feng, M.-G., and Keyhani, N.O. (2013) Use of uridine auxotrophy (ura3) for markerless transformation of the mycoinsecticide Beauveria bassiana. Appl. Microbiol. Biotech. 97: 3017-3025.
Zhang, L., Wang, J., Xie, X., Keyhani, N.O., Feng, M.-G., and Ying, S.-H. (2013) The autophagy gene BbATG5, involved in the formation of the autophagosome, contributes to cell differentiation and growth but is dispensable for pathogenesis in the entomopathogenic fungus Beauveria bassiana. Microbiology 159: 243-252
Keyhani, N.O. (2012) Using host molecules to increase fungal virulence for biological control of insects. Virulence 3(4): 415-418.
Zhang, S., Widemann, E., Bernard. G, Lesot, A., Pinot, F., Pedrini, N., and Keyhani, N.O. (2012) CYP52X1, representing a new cytochrome P450 subfamily, displays fatty acid hydroxylase activity and contributes to virulence and growth on insect cuticular substrates in the entomopathogenic fungus Beauveria bassiana. J. Biol. Chem. 287: 13477-13486.
Fan, Y., Borovsky, D., Hawkings, C., Ortiz-Urquiza, A., and Keyhani, N.O. (2012) Exploiting host molecules to augment mycoinsecticide virulence. Nature Biotechnol. 30: 35-37
Fan, Y., Pereira, R.M., Kilic, E., Casella, G., and Keyhani, N.O. (2012) Pyrokinin b-neuropeptide affects necrophoretic behavior in fire ants, and expression of b-NP in a mycoinsecticide increases its virulence. PLoS One 7(1): e26924.
Luo, X, Keyhani, N.O., Yu, X., He, Z., Luo, Z., Pei, Y., and Zhang, Y. (2012) The MAP kinase Bbslt2 controls growth conidiation, cell wall integrity, and virulence in the insect pathogenic fungus Beauveria bassiana. Fungal Genet. Biol. 49: 544-555.
Fan, Y., Ortiz-Urquiza, A., Kudia, R., and Keyhani, N.O. (2012) Bbcsa1, a fungal homolog of neuronal calcium sensor-1 regulates extracellular acidification and contributes to virulence in the entomopathogenic fungus Beauveria bassiana. Microbiology, 158: 1843-1851.
Mantilla, J.G., Galeano, N.F., Gaitan, A.L., Christancho, M.A., Keyhani, N.O. and Gongora, C.E. (2012) Comparative transcriptome analysis of the entomopathogenic fungus Beauveria bassiana grown on the coffee berry borer (Hypothenemus hampei). Microbiology, 158: 1826-1842.
Glare, T., Caradus, J., Gelernter, W., Jackson, T., Keyhani, N., Kohl, J., Marrone, P., Morin, L., and Stewart, A. Have biopesticides come of age? (2012) Trends in Biotechnology 30: 250-258.
Zhang, S., Xia, Y., Kim, B., and Keyhani, N.O. (2011) Two hydrophobins are involved in fungal spore coat rodlet layer assembly and each play distinct roles in surface interactions, development, and pathogenesis in the entomopathogenic fungus Beauveria bassiana, Mol. Micobiol. 80: 811-826
Zhang, S., Xia, Y., and Keyhani, N.O. (2011) Contribution of the gas1 gene of the entomopathogenic fungus Beauveria bassiana,encoding a putative GPI-anchored b-glucanosyltransferase to conidial thermotolerance and virulence. Appl. Environ. Microbiol. 77: 2676-2684
Fan, Y., Zhang, S., Kruer-Zerhusen, N., and Keyhani, N.O. (2011) High-throughput insertion mutagenesis and screening of clones of the entomopathogenic fungus Beauveria bassiana. J Invert Pathology 106: 274-279
Kirkland, B.H. and Keyhani, N.O. (2011) Expression and purification of a functionally active Class I fungal hydrophobin from the entomopathogenic fungus Beauveria bassiana in E. coli. J Indust Micro & Biotech 38: 327-335
Pedrini, N., Zhang, S., Juarez, M.P., Keyhani, N.O. (2010) Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana. Microbiology 156: 2549-2557
Zhang, S., Fan, Y., Xia, Y. X., and Keyhani, N.O. (2010) Sulfonylurea resistance as a new selectable marker for the entomopathogenic fungus Beauveria bassiana. Appl Microbiol and Biotech 87:1151-1156
Bidochka, M.J., Clark, D.C., Lewis, M.W., and Keyhani, N.O. (2010) Could insect phagocytic avoidance by entomogenous fungi have evolved via selection against soil amoeboid predators? Microbiology 156:2164-2171
de Crecy, E., Jaronski, S., Lyons, B., Lyons, T.J., and Keyhani, N.O. (2009) Directed evolution of a filamentous fungus for thermotolerance. BMC Biotechnology 9(1):74
Lewis, M.W., Robalino, I.V., and Keyhani, N.O. (2009) Uptake of the fluorescent probe FM4-64 by hyphae and hemolymph-derived in vivo hyphal bodies of the entomopathogenic fungus Beauveria bassiana. Microbiology 155: 3110-3120
Wanchoo, A., Lewis, M.W., and Keyhani, N.O. (2009) Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and hemolymph derived cells of the entomopathogenic fungus Beauveria bassiana. Microbiology 155: 3121-3133
Kurtti, T. and Keyhani, N.O. (2008) Intracellular infection of tick cell lines by the entomopathogenic fungus Metarhizium anisopliae. Microbiology 154: 1700-1709