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Weiguo Fang - One of the best experts on this subject based on the ideXlab platform.

  • a novel cascade allows Metarhizium robertsii to distinguish cuticle and hemocoel microenvironments during infection of insects
    PLOS Biology, 2021
    Co-Authors: Xing Zhang, Yamin Meng, Yizhou Huang, Dan Zhang, Weiguo Fang
    Abstract:

    Pathogenic fungi precisely respond to dynamic microenvironments during infection, but the underlying mechanisms are not well understood. The insect pathogenic fungus Metarhizium robertsii is a representative fungus in which to study broad themes of fungal pathogenicity as it resembles some major plant and mammalian pathogenic fungi in its pathogenesis. Here we report on a novel cascade that regulates response of M. robertsii to 2 distinct microenvironments during its pathogenesis. On the insect cuticle, the transcription factor COH2 activates expression of cuticle penetration genes. In the hemocoel, the protein COH1 is expressed due to the reduction in epigenetic repression conferred by the histone deacetylase HDAC1 and the histone 3 acetyltransferase HAT1. COH1 interacts with COH2 to reduce COH2 stability, and this down-regulates cuticle penetration genes and up-regulates genes for hemocoel colonization. Our work significantly advances the insights into fungal pathogenicity in insects.

  • Metarhizium robertsii ammonium permeases mepc and mep2 contribute to rhizoplane colonization and modulates the transfer of insect derived nitrogen to plants
    PLOS ONE, 2019
    Co-Authors: Soumya Moonjely, Weiguo Fang, Xing Zhang, Michael J Bidochka
    Abstract:

    The endophytic insect pathogenic fungi (EIPF) Metarhizium promotes plant growth through symbiotic association and the transfer of insect-derived nitrogen. However, little is known about the genes involved in this association and the transfer of nitrogen. In this study, we assessed the involvement of six Metarhizium robertsii genes in endophytic, rhizoplane and rhizospheric colonization with barley roots. Two ammonium permeases (MepC and Mep2) and a urease, were selected since homologous genes in arbuscular mycorrhizal fungi were reported to play a pivotal role in nitrogen mobilization during plant root colonization. Three other genes were selected on the basis on RNA-Seq data that showed high expression levels on bean roots, and these encoded a hydrophobin (Hyd3), a subtilisin-like serine protease (Pr1A) and a hypothetical protein. The root colonization assays revealed that the deletion of urease, hydrophobin, subtilisin-like serine protease and hypothetical protein genes had no impact on endophytic, rhizoplane and rhizospheric colonization at 10 or 20 days. However, the deletion of MepC resulted in significantly increased rhizoplane colonization at 10 days whereas ΔMep2 showed increased rhizoplane colonization at 20 days. In addition, the nitrogen transporter mutants also showed significantly higher 15N incorporation of insect derived nitrogen in barley leaves in the presence of nutrients. Insect pathogenesis assay revealed that disruption of MepC, Mep2, urease did not reduce virulence toward insects. The enhanced rhizoplane colonization of ΔMep2 and ΔMepC and insect derived nitrogen transfer to plant hosts suggests the role of MepC and Mep2 in Metarhizium-plant symbiosis.

  • Metarhizium robertsii ammonium permeases (MepC and Mep2) contribute to rhizoplane colonization and modulates the transfer of insect derived nitrogen to plants.
    'Public Library of Science (PLoS)', 2019
    Co-Authors: Soumya Moonjely, Weiguo Fang, Xing Zhang, Michael J Bidochka
    Abstract:

    The endophytic insect pathogenic fungi (EIPF) Metarhizium promotes plant growth through symbiotic association and the transfer of insect-derived nitrogen. However, little is known about the genes involved in this association and the transfer of nitrogen. In this study, we assessed the involvement of six Metarhizium robertsii genes in endophytic, rhizoplane and rhizospheric colonization with barley roots. Two ammonium permeases (MepC and Mep2) and a urease, were selected since homologous genes in arbuscular mycorrhizal fungi were reported to play a pivotal role in nitrogen mobilization during plant root colonization. Three other genes were selected on the basis on RNA-Seq data that showed high expression levels on bean roots, and these encoded a hydrophobin (Hyd3), a subtilisin-like serine protease (Pr1A) and a hypothetical protein. The root colonization assays revealed that the deletion of urease, hydrophobin, subtilisin-like serine protease and hypothetical protein genes had no impact on endophytic, rhizoplane and rhizospheric colonization at 10 or 20 days. However, the deletion of MepC resulted in significantly increased rhizoplane colonization at 10 days whereas ΔMep2 showed increased rhizoplane colonization at 20 days. In addition, the nitrogen transporter mutants also showed significantly higher 15N incorporation of insect derived nitrogen in barley leaves in the presence of nutrients. Insect pathogenesis assay revealed that disruption of MepC, Mep2, urease did not reduce virulence toward insects. The enhanced rhizoplane colonization of ΔMep2 and ΔMepC and insect derived nitrogen transfer to plant hosts suggests the role of MepC and Mep2 in Metarhizium-plant symbiosis

  • Metarhizium robertsii produces indole 3 acetic acid which promotes root growth in arabidopsis and enhances virulence to insects
    Microbiology, 2017
    Co-Authors: Xinggang Liao, Weiguo Fang, Brian Lovett, Raymond J St Leger
    Abstract:

    The plant root colonizing insect-pathogenic fungus Metarhizium robertsii has been shown to boost plant growth, but little is known about the responsible mechanisms. Here we show that M. robertsii promotes lateral root growth and root hair development of Arabidopsis seedlings in part through an auxin [indole-3-acetic acid (IAA)]-dependent mechanism. M. robertsii, or its auxin-containing culture filtrate promoted root proliferation, activated IAA-regulated gene expression and rescued the root hair defect of the IAA-deficient rhd6 Arabidopsis mutant. Substrate feeding assays suggest that M. robertsii possesses tryptamine (TAM) and indole-3-acetamide tryptophan (Trp)-dependent auxin biosynthetic pathways. Deletion of Mrtdc impaired M. robertsii IAA production by blocking conversion of Trp to TAM but the reduction was not sufficient to affect plant growth enhancement. We also show that M. robertsii secretes IAA on insect cuticle. ∆Mrtdc produced fewer infection structures and was less virulent to insects than the wild-type, whereas M. robertsii spores harvested from culture media containing IAA were more virulent. Furthermore, exogenous application of IAA increased appressorial formation and virulence. Together, these results suggest that auxins play an important role in the ability of M. robertsii to promote plant growth, and the endogenous pathways for IAA production may also be involved in regulating entomopathogenicity. Auxins were also produced by other Metarhizium species and the endophytic insect pathogen Beauveria bassiana suggesting that interplay between plant- and fungal-derived auxins has important implications for plant–microbe–insect interactions.

  • deletion of a histone acetyltransferase leads to the pleiotropic activation of natural products in Metarhizium robertsii
    Organic Letters, 2017
    Co-Authors: Aili Fan, Weiguo Fang, Guohong Zeng, Zhiguo Liu, Peng Zhang, Wen-bing Yin
    Abstract:

    Histone deacetylation normally decreases the gene expression in organisms. By genome-wide deletions of epigenetic regulators in entomopathogenic fungus Metarhizium robertsii, unexpected activations of orphan secondary metabolite genes have been found upon the disruption of a histone acetyltransferase (HAT) gene Hat1. This led to the characterization of 11 new natural products, including eight isocoumarin derivatives meromusides A–H and two nonribosomal peptides meromutides A and B. Therefore, disruption of HAT represents a new approach to mine chemical diversity from fungi.

Michael J Bidochka - One of the best experts on this subject based on the ideXlab platform.

  • Metarhizium robertsii ammonium permeases mepc and mep2 contribute to rhizoplane colonization and modulates the transfer of insect derived nitrogen to plants
    PLOS ONE, 2019
    Co-Authors: Soumya Moonjely, Weiguo Fang, Xing Zhang, Michael J Bidochka
    Abstract:

    The endophytic insect pathogenic fungi (EIPF) Metarhizium promotes plant growth through symbiotic association and the transfer of insect-derived nitrogen. However, little is known about the genes involved in this association and the transfer of nitrogen. In this study, we assessed the involvement of six Metarhizium robertsii genes in endophytic, rhizoplane and rhizospheric colonization with barley roots. Two ammonium permeases (MepC and Mep2) and a urease, were selected since homologous genes in arbuscular mycorrhizal fungi were reported to play a pivotal role in nitrogen mobilization during plant root colonization. Three other genes were selected on the basis on RNA-Seq data that showed high expression levels on bean roots, and these encoded a hydrophobin (Hyd3), a subtilisin-like serine protease (Pr1A) and a hypothetical protein. The root colonization assays revealed that the deletion of urease, hydrophobin, subtilisin-like serine protease and hypothetical protein genes had no impact on endophytic, rhizoplane and rhizospheric colonization at 10 or 20 days. However, the deletion of MepC resulted in significantly increased rhizoplane colonization at 10 days whereas ΔMep2 showed increased rhizoplane colonization at 20 days. In addition, the nitrogen transporter mutants also showed significantly higher 15N incorporation of insect derived nitrogen in barley leaves in the presence of nutrients. Insect pathogenesis assay revealed that disruption of MepC, Mep2, urease did not reduce virulence toward insects. The enhanced rhizoplane colonization of ΔMep2 and ΔMepC and insect derived nitrogen transfer to plant hosts suggests the role of MepC and Mep2 in Metarhizium-plant symbiosis.

  • Metarhizium robertsii ammonium permeases (MepC and Mep2) contribute to rhizoplane colonization and modulates the transfer of insect derived nitrogen to plants.
    'Public Library of Science (PLoS)', 2019
    Co-Authors: Soumya Moonjely, Weiguo Fang, Xing Zhang, Michael J Bidochka
    Abstract:

    The endophytic insect pathogenic fungi (EIPF) Metarhizium promotes plant growth through symbiotic association and the transfer of insect-derived nitrogen. However, little is known about the genes involved in this association and the transfer of nitrogen. In this study, we assessed the involvement of six Metarhizium robertsii genes in endophytic, rhizoplane and rhizospheric colonization with barley roots. Two ammonium permeases (MepC and Mep2) and a urease, were selected since homologous genes in arbuscular mycorrhizal fungi were reported to play a pivotal role in nitrogen mobilization during plant root colonization. Three other genes were selected on the basis on RNA-Seq data that showed high expression levels on bean roots, and these encoded a hydrophobin (Hyd3), a subtilisin-like serine protease (Pr1A) and a hypothetical protein. The root colonization assays revealed that the deletion of urease, hydrophobin, subtilisin-like serine protease and hypothetical protein genes had no impact on endophytic, rhizoplane and rhizospheric colonization at 10 or 20 days. However, the deletion of MepC resulted in significantly increased rhizoplane colonization at 10 days whereas ΔMep2 showed increased rhizoplane colonization at 20 days. In addition, the nitrogen transporter mutants also showed significantly higher 15N incorporation of insect derived nitrogen in barley leaves in the presence of nutrients. Insect pathogenesis assay revealed that disruption of MepC, Mep2, urease did not reduce virulence toward insects. The enhanced rhizoplane colonization of ΔMep2 and ΔMepC and insect derived nitrogen transfer to plant hosts suggests the role of MepC and Mep2 in Metarhizium-plant symbiosis

  • carbon translocation from a plant to an insect pathogenic endophytic fungus
    Nature Communications, 2017
    Co-Authors: Scott W Behie, Larissa Barelli, Camila C Moreira, Irina Sementchoukova, Paul M Zelisko, Michael J Bidochka
    Abstract:

    Metarhizium robertsii is a common soil fungus that occupies a specialized ecological niche as an endophyte and an insect pathogen. Previously, we showed that the endophytic capability and insect pathogenicity of Metarhizium are coupled to provide an active method of insect-derived nitrogen transfer to a host plant via fungal mycelia. We speculated that in exchange for this insect-derived nitrogen, the plant would provide photosynthate to the fungus. By using 13CO2, we show the incorporation of 13C into photosynthate and the subsequent translocation of 13C into fungal-specific carbohydrates (trehalose and chitin) in the root/endophyte complex. We determined the amount of 13C present in root-associated fungal biomass over a 21-day period by extracting fungal carbohydrates and analysing their composition using nuclear magnetic resonance (NMR) spectroscopy. These findings are evidence that the host plant is providing photosynthate to the fungus, likely in exchange for insect-derived nitrogen in a tripartite, and symbiotic, interaction.

  • plant tissue localization of the endophytic insect pathogenic fungi Metarhizium and beauveria
    Fungal Ecology, 2015
    Co-Authors: Scott W Behie, Samantha J Jones, Michael J Bidochka
    Abstract:

    Abstract Endophytic fungi may display preferential tissue colonization within their plant hosts. Here we tested if the endophytic, insect pathogenic fungi (EIPF) Metarhizium and Beauveria showed preferential localization within plant tissues, in the field and under laboratory conditions. In the field, plants were sampled from three separate sites (Brock University, St. Catharines, Ontario; Pelham, Ontario; and Torngat Mountains National Park, Newfoundland, Canada) and EIPF were isolated from plant roots, the hypocotyl, and stem and leaves. Two genera of EIPF, Metarhizium spp. and Beauveria bassiana, were isolated from plants sampled, as well as the nematophagous fungus, Pochonia chlamydosporium. Metarhizium spp. were almost exclusively found in roots, whereas B. bassiana and P. chlamydosporium were found throughout the plant. The Metarhizium species were identified by RFLP and 95 % were Metarhizium robertsii, 4.3 % were M. brunneum, and 0.7 % were M. guizhouense. Lab studies with M. robertsii and B. bassiana reflected observations found in the field, that is, Metarhizium was restricted to the roots of plants while B. bassiana was found throughout the plant. Insect infection by these EIPF is preferential with respect to above and below ground insects, and the present study correlates above and below ground insect infections with endophytic colonization by these EIPF.

  • the insect pathogenic fungus Metarhizium robertsii clavicipitaceae is also an endophyte that stimulates plant root development
    American Journal of Botany, 2012
    Co-Authors: Ramanpreet K Sasan, Michael J Bidochka
    Abstract:

     Premise of the study: The soil - inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. Metarhizium robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass ( Panicum virgatum) and haricot beans ( Phaseolus vulgaris) by M. robertsii were examined after inoculation with fungal conidia.  Methods: We used light and confocal microscopy to ascertain the plant endophytic association with GFP-expressing M. robertsii. Root lengths, root hair density, and lateral roots emerged were also observed.  Key results: Initially, M. robertsii conidia adhered to, germinated on, and colonized roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M. robertsii on the root (within 1 – 2 d). Plants inoculated with M. robertsii Δ MAD2 (plant adhesin gene) took signifi cantly longer to show root hair proliferation than the wild type. Cell free extracts of M. robertsii did not stimulate root hair proliferation. Longer-term (60 d) associations showed that M. robertsii endophytically colonized cortical cells within bean roots. Metarhizium appeared as a mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant.  Conclusions: These results suggest that M. robertsii is not only rhizosphere competent but also displays a benefi cial endophytic association with plant roots that results in the proliferation of root hairs.

I M Dubovskiy - One of the best experts on this subject based on the ideXlab platform.

  • bacterial and fungal infections induce bursts of dopamine in the haemolymph of the colorado potato beetle leptinotarsa decemlineata and greater wax moth galleria mellonella
    Journal of Invertebrate Pathology, 2018
    Co-Authors: Ekaterina A Chertkova, I M Dubovskiy, Ekaterina V Grizanova
    Abstract:

    Dopamine (DA) is known as a hormone neurotrasnmitter molecule involved in several stress reactions in both vertebrates and invertebrates. Following infections with the fungi Metarhizium robertsii or Beauveria bassiana and the bacterium Bacillus thuringiensis, dopamine the concentration was measured at different time points in the haemolymph of the Colorado potato beetle, Leptinotarsa decemlineata and the larvae of the greater wax moth Galleria mellonella. The infection with M. robertsii increased (4 to 12-fold) DA concentrations in the haemolymph of the potato beetle larvae and the oral infection by B. thuringiensis also lead to a 30 and 45-fold increase. During infection of the greater wax moth larvae with Beauveria bassiana and B. thuringiensis DA increased 4 to 20-fold and about 2 to 2,5-fold respectively, compared to non-infected insects. The relative DA concentrations varied between the two insects and depended on the pathogens and post infection time.

  • immunological mechanisms of synergy between fungus Metarhizium robertsii and bacteria bacillus thuringiensis ssp morrisoni on colorado potato beetle larvae
    Journal of Insect Physiology, 2017
    Co-Authors: O. N. Yaroslavtseva, I M Dubovskiy, V P Khodyrev, B A Duisembekov, Vadim Yu Kryukov, V V Glupov
    Abstract:

    The synergistic effect between the entomopathogenic fungus Metarhizium robertsii and a sublethal dose of the bacterium Bacillus thuringiensis ssp. morrisoni var. tenebrionis was studied in terms of immune defense reactions and detoxification system activity of the Colorado potato beetle, Leptinotarsa decemlineata, fourth instar larvae. Bacterial infection led to more rapid germination of fungal conidia on integuments. We found a significant decrease of cellular immunity parameters, including total hemocyte count and encapsulation response, under the influence of bacteria. Phenoloxidase activity in integuments was increased under bacteriosis, mycosis and combined infection compared to controls. However, phenoloxidase activity in the hemolymph was enhanced under bacteriosis alone, and it was decreased under combined infection. Activation of both nonspecific esterases and glutathione-S-transferases in the hemolymph was shown at the first day of mycosis and third day of bacteriosis. However, inhibition of detoxification enzymes was detected under combined infection. The suppression of cellular immunity and detoxification reactions in Colorado potato beetle larvae with a sublethal dose of bacteria is discussed as a reason for synergy between B. thuringiensis and M. robertsii.

  • immune physiological aspects of synergy between avermectins and the entomopathogenic fungus Metarhizium robertsii in colorado potato beetle larvae
    Journal of Invertebrate Pathology, 2016
    Co-Authors: Oksana G Tomilova, O. N. Yaroslavtseva, I M Dubovskiy, B A Duisembekov, Vadim Yu Kryukov, Natalia A Kryukova, M V Tyurin, Valery Skorokhod, V V Glupov
    Abstract:

    Abstract The interaction between the entomopathogenic fungus Metarhizium robertsii and natural avermectin metabolites of the actinomycete Streptomyces avermitilis were investigated on Colorado potato beetle larvae. A synergy in the mortality of larvae was detected after simultaneous treatment with half-lethal doses of avermectins (commercial name actarophit) 0.005% and fungus (5 × 10 5  conidia/ml). The treatment with avermectins led to rapid fungal colonization of the hemolymph. The defense strategies of insects infected by fungus and treated with avermectins and untreated insects were compared to investigate the mechanisms of this synergy. We have shown an increase in hemocytes, especially immunocompetent cells – plasmatocytes and granular cells in the initial stages of mycosis (third day post inoculation). In contrast, avermectins suppressed cellular immunity in hemolymph. Specifically, avermectins dramatically decreased the count of granular cells in larvae infected and uninfected with fungus. Apoptosis inducement and hemocyte necrosis under the influence of avermectins has been shown in vitro as one of the possible reasons for hemocyte mortality. In addition, avermectins enhanced the activity of phenoloxidases in integuments and hemolymph and increased the activity of glutathione-S-transferases activity in the fat body and hemolymph of infected and uninfected larvae, thereby intensifying the development of fungal infection by M. robertsii in Colorado potato beetle larvae. The combination of fungal infection and avermectins constitutes a new perspective for developing multicomponent bioinsecticides.

  • comparative analysis of the immune reactions in colorado potato beetle larvae under development of mycoses caused by Metarhizium robertsii m brunneum and m pemphigi
    Journal of Evolutionary Biochemistry and Physiology, 2016
    Co-Authors: M V Tyurin, O. N. Yaroslavtseva, I M Dubovskiy, Yu V Kryukov, E A Elisaphenko, V V Glupov
    Abstract:

    A comparative investigation of humoral and cellular immune response in larvae of Colorado potato beetle Leptinotarsa decemlineata was conducted under development of mycoses caused by entomopatho- genic fungi Metarhizium robertsii, M. brunneum and M. pemphigi. The larvae were found highly suscep- tible to M. robertsii, M. brunneum and less susceptible to M. pemphigi. The susceptibility to the fungi was not correlated with the rate of conidia germination in epicuticular extracts of larvae. A non-specific for Colorado beetle pathogen M. pemphigi did not cause significant changes in the immune response and did not result in colonization of larvae hemocoel. Infection with M. robertsi and M. brunneum led to an increase in total hemocyte count at the initial stages of mycoses (day 2) followed by a sharp decrease on day 3. The strongest decrease was observed for the immunocompetent cells - plasmatocytes and granu- locytes. Enhanced phenoloxidase activity in hemolymph and cuticle was found on days 2 and 3 after in- fection. These changes in immune reactions correlated with the level of virulence of the strains. Thus, the immune response in Colorado potato beetle larvae is an important factor determining differences in the development of mycoses caused by different Metarhizium species.

Chengshu Wang - One of the best experts on this subject based on the ideXlab platform.

  • mrhex1 is required for woronin body formation fungal development and virulence in Metarhizium robertsii
    Journal of Fungi, 2020
    Co-Authors: Guirong Tang, Yanfang Shang, Chengshu Wang
    Abstract:

    The Woronin body (WB) is a peroxisome-derived dense-core vesicle, a self-assembling hexagonal crystal of a single protein Hex1. This organelle is specific to the ascomycete fungi belonging to the Pezizomycotina subphylum by functioning in sealing septal pores in response to mycelium damage and the control of cell heterogeneity. We retrieved all available Hex1-domain containing proteins of different fungi from the GenBank database and found considerable length variations among 460 obtained Hex1 proteins. However, a highly conserved Hex1 domain containing 75 amino acid residues with a specific S/A-R/S-L consensus motif for targeting peroxisome is present at the carboxy-terminus of each protein. A homologous Hex1 gene, named MrHex1, was deleted in the entomopathogenic fungus Metarhizium robertsii. It was found that MrHex1 was responsible for WB formation in M. robertsii and involved in sealing septal pores to maintain cell integrity and heterogeneity. Different assays indicated that, relative to the wild-type (WT) strain, ∆Mrhex1 demonstrated a growth defect on a solid medium and substantial reductions of conidiation, appressorium formation and topical infectivity against insect hosts. However, there was no obvious virulence difference between WT and mutants during injection of insects. We also found that ∆MrHex1 could tolerate different stress conditions like the WT and the gene-rescued mutant of M. robertsii, which is in contrast to the reports of the stress-response defects of the Hex1 null mutants of other fungal species. In addition to revealing the phenotypic/functional alterations of the Hex1 deletion mutants between different pathotype fungi, the results of this study may benefit the understanding of the evolution and WB-control of fungal entomopathogenicity.

  • tryptamine accumulation caused by deletion of mrmao 1 in Metarhizium genome significantly enhances insecticidal virulence
    PLOS Genetics, 2020
    Co-Authors: Xiwen Tong, Chengshu Wang, Pengcheng Yang, Y. L. Wang, Le Kang
    Abstract:

    Metarhizium is a group of insect-pathogenic fungi that can produce insecticidal metabolites, such as destruxins. Interestingly, the acridid-specific fungus Metarhizium acridum (MAC) can kill locusts faster than the generalist fungus Metarhizium robertsii (MAA) even without destruxin. However, the underlying mechanisms of different pathogenesis between host-generalist and host-specialist fungi remain unknown. This study compared transcriptomes and metabolite profiles to analyze the difference in responsiveness of locusts to MAA and MAC infections. Results confirmed that the detoxification and tryptamine catabolic pathways were significantly enriched in locusts after MAC infection compared with MAA infection and that high levels of tryptamine could kill locusts. Furthermore, tryptamine was found to be capable of activating the aryl hydrocarbon receptor of locusts (LmAhR) to produce damaging effects by inducing reactive oxygen species production and immune suppression. Therefore, reducing LmAhR expression by RNAi or inhibitor (SR1) attenuates the lethal effects of tryptamine on locusts. In addition, MAA, not MAC, possessed the monoamine oxidase (Mao) genes in tryptamine catabolism. Hence, deleting MrMao-1 could increase the virulence of generalist MAA on locusts and other insects. Therefore, our study provides a rather feasible way to design novel mycoinsecticides by deleting a gene instead of introducing any exogenous gene or domain.

  • clarification of swainsonine biosynthesis by a multi branched pathway and non accumulation of mycotoxin in plants after fungal colonization
    Social Science Research Network, 2019
    Co-Authors: Feifei Luo, Guirong Tang, Song Hong, Bo Chen, Ying Yin, Huizhan Zhang, Chengshu Wang
    Abstract:

    The indolizidine alkaloid swainsonine (SW) is a deadly mycotoxin to livestock. The genetic mechanism of SW biosynthesis remains obscure for decades. Here we report that SW is produced via a multi-branched pathway by a hybrid nonribosomal peptide/polyketide synthase (NRPS-PKS) gene cluster in the endophytic entomopathogenic fungus Metarhizium robertsii. The precursor pipecolic acid can be redundantly converted from lysine by both clustered and unclustered genes. The hybrid enzyme that begins with a NRPS module produces three intermediates with and without domain skipping. Intriguingly, the biosynthetic process is coupled with the trans to cis non-enzymatic epimerization of C1-OH for both hydroxyl- and dihydroxyl-indolizidine intermediates. We also found that SW production is dispensable for fungal colonization of plants and the infection of insect hosts. Our finding that SW does not accumulate in planta after Metarhizium colonization provides a notable indication of safety when using this fungus to control insect pests.

  • mrpacc regulates sporulation insect cuticle penetration and immune evasion in Metarhizium robertsii
    Environmental Microbiology, 2015
    Co-Authors: Wei Huang, Yanfang Shang, Peilin Chen, Chengshu Wang
    Abstract:

    Summary pH-responsive transcription factor of the PacC/Rim101 family governs adaptation to environment, development and virulence in many fungal pathogens. In this study, we report the functions of a PacC homologue, MrpacC, in an insect pathogenic fungus Metarhizium robertsii. The gene was highly transcribed in the fungus in alkaline conditions, and deletion of MrpacC impaired fungal responses to ambient pH and salt/metal challenges but not osmotic stress. We found that MrpacC is required for fungal full virulence by contributing to penetration of insect cuticles, mycosis of insect cadavers and evasion of host immunity. In MrpacC deletion strains, the chitinase but not protease activity was reduced, which was consistent with the downregulation of groups A and C chitinase genes. Further, the glucosyltransferase genes involved in cell wall remodelling and protein glycosylation were upregulated in ΔMrpacC. MrpacC transcriptional control of chitinase and glucosyltransferase genes was verified both by the presence of PacC consensus binding motif in gene promoter regions and the promoter DNA-binding assays. The results of this study not only advances the understanding of PacC function in fungal development and virulence, but will also facilitate future studies on the mechanism(s) behind the selective control of target genes by PacC.

  • mrskn7 controls sporulation cell wall integrity autolysis and virulence in Metarhizium robertsii
    Eukaryotic Cell, 2015
    Co-Authors: Yanfang Shang, Yixiong Chen, Peilin Chen, Chengshu Wang
    Abstract:

    Two-component signaling pathways generally include sensor histidine kinases and response regulators. We identified an ortholog of the response regulator protein Skn7 in the insect-pathogenic fungus Metarhizium robertsii, which we named MrSkn7. Gene deletion assays and functional characterizations indicated that MrSkn7 functions as a transcription factor. The MrSkn7 null mutant of M. robertsii lost the ability to sporulate and had defects in cell wall biosynthesis but was not sensitive to oxidative and osmotic stresses compared to the wild type. However, the mutant was able to produce spores under salt stress. Insect bioassays using these spores showed that the virulence of the mutant was significantly impaired compared to that of the wild type due to the failures to form the infection structure appressorium and evade host immunity. In particular, deletion of MrSkn7 triggered cell autolysis with typical features such as cell vacuolization, downregulation of repressor genes, and upregulation of autolysis-related genes such as extracellular chitinases and proteases. Promoter binding assays confirmed that MrSkn7 could directly or indirectly control different putative target genes. Taken together, the results of this study help us understand the functional divergence of Skn7 orthologs as well as the mechanisms underlying the development and control of virulence in insect-pathogenic fungi.

Bo Huang - One of the best experts on this subject based on the ideXlab platform.

  • Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii
    'MDPI AG', 2021
    Co-Authors: Yulong Wang, Shihong Liu, Xuebing Yin, Xiangyun Xie, Bo Huang
    Abstract:

    Ketol-acid reductoisomerase (ILVC) is the second enzyme in the branched-chain amino acid (BCAA) biosynthesis, which regulates many physiological activities in a variety of organisms from bacteria to fungi and plants. In this work, function mechanisms of ILVC in Metarhizium robertsii Metchnikoff (Hypocreales: Clavicipitaceae) were explored with site-directed mutagenesis, reductase activity assays and transcriptomics analysis. The reductase activity assays showed that ILVC from phytopathogenic fungi exhibited significantly higher activities than those from entomopathogenic fungi but lower than those from yeast. Site-directed mutagenesis and enzymatic activities of MrILVC with different active-site mutants (Arg-113, Ser-118, Asp-152, Asp-260, and Glu-264) confirmed that active sites of MrILVC are conserved with plant and bacterial ILVCs. Deleting MrilvC causes the complete failures of vegetative growth and conidial germination, feeding with branched-chain amino acids (BCAAs) recovers the fungal growth but not conidial germination, while both characteristics are restored when supplemented with yeast extract. Compared to ΔMrilvC cultured in czapek agar (CZA), plenty of genes involved in the biosynthesis of antibiotics and amino acids were up- or down-regulated in the wild type or ΔMrilvC feeding with either BCAAs or yeast extract. Further analysis showed some genes, such as catalase A, participate in mycelial growth and conidial germination was down-regulated in ΔMrilvC from CZA, revealing that MrILVC might control the fungal development by gene regulation and BCAAs or yeast extract could play partial roles of MrILVC. This study will advance our understanding of ILVC function mechanisms in fungi

  • Integration of dsRNA against host immune response genes augments the virulence of transgenic Metarhizium robertsii strains in insect pest species
    'Wiley', 2021
    Co-Authors: Yulong Wang, Zhangxun Wang, Xiangyun Xie, Li Qin, Bo Huang
    Abstract:

    Summary The slow lethality of fungal biopesticides to insects restrains their widespread application as a strategy of pest control. In this study, unary, binary and ternary transgenic Metarhizium robertsii were created by integrating genes that encode the scorpion neurotoxin BjαIT, the cuticle‐degrading protease Pr1A, and a double‐stranded RNA (dsRNA) that targets host gnbp3, individually or collectively under a constitutive promoter to enhance virulence. Compared with the parental wild type, all unary transgenic strains had increased virulence against four insect species, Tenebrio molitor, Locusta migratoria, Plutella xylostella and Galleria mellonella, whereas the binary transgenic strain expressing both pr1A and BjαIT had increased virulence to T. molitor and L. migratoria, with no change in virulence against P. xylostella and G. mellonella. Importantly, all ternary transgenic strains simultaneously expressing pr1A, BjαIT, and the dsRNA specific to host gnbp3 exhibited the highest increase in insect‐specific virulence. This finding highlights a novel strategy for genetic engineering of dsRNAs that target genes associated with the host immune response alongside virulence genes to maximize fungal virulence and lethality against insect pests

  • The polyubiquitin gene MrUBI4 is required for conidiation, conidial germination, and stress tolerance in the filamentous fungus Metarhizium robertsii
    MDPI AG, 2019
    Co-Authors: Zhangxun Wang, Hong Zhu, Yuran Cheng, Yuanyuan Jiang, Bo Huang
    Abstract:

    The polyubiquitin gene is a highly conserved open reading frame that encodes different numbers of tandem ubiquitin repeats from different species, which play important roles in different biological processes. Metarhizium robertsii is a fungal entomopathogen that is widely applied in the biological control of pest insects. However, it is unclear whether the polyubiquitin gene is required for fungal development, stress tolerance, and virulence in the entomopathogenic fungus. In the present study, the polyubiquitin gene (MrUBI4, MAA_02160) was functionally characterized via gene deletion in M. robertsii. Compared to the control strains, the MrUBI4 deletion mutant showed delayed conidial germination and significantly decreased conidial yields (39% of the wild-type 14 days post-incubation). Correspondingly, the transcript levels of several genes from the central regulatory pathways associated with conidiation, including brlA, abaA, and wetA, were significantly downregulated, which indicated that MrUBI4 played an important role in asexual sporulation. Deletion of MrUBI4 especially resulted in increased sensitivity to ultraviolet (UV) and heat-shock stress based on conidial germination analysis between mutant and control strains. The significant increase in sensitivity to heat-shock was accompanied with reduced transcript levels of genes related to heat-shock protein (hsp), trehalose, and mannitol accumulation (tps, tpp, nth, and mpd) in the MrUBI4 deletion mutant. Deletion of MrUBI4 has no effect on fungal virulence. Altogether, MrUBI4 is involved in the regulation of conidiation, conidial germination, UV stress, and heat-shock response in M. robertsii

  • MrSVP, a secreted virulence-associated protein, contributes to thermotolerance and virulence of the entomopathogenic fungus Metarhizium robertsii
    BMC, 2019
    Co-Authors: Tian Xie, Zhangxun Wang, Yulong Wang, Qilin Zhang, Tingting Zhang, Bo Huang
    Abstract:

    Abstract Background Metarhizium robertsii, a widely distributed insect pathogen, is presently used as a natural alternative to chemical insecticides. Unfortunately, its worldwide commercial use has been restricted by a short shelf life and inconsistencies in virulence. In our previous study, a gene (GenBank accession number EFZ01626) was found to be significantly upregulated in heat-treated conidia. In the present study, this gene was characterized via gene disruption and complementation strategies. Results The gene (amplified by rapid amplification of cDNA ends PCR) was 1219 bp long and contained an open reading frame (ORF) of 777 bp. It encoded a protein of 234 amino acid residues with a 26-residue signal peptide. Bioinformatics analyses did not identify conserved functional domains; therefore, it was assumed to be a secreted virulence-associated protein according to its signal peptide and bioassay results. We found that the conidial germination rate of the ΔMrSVP mutant fungi dramatically decreased after heat shock treatment in a thermotolerance test. In addition, transcription levels of all tested heat shock–related genes were significantly lower in the mutant than in the wild type. We also demonstrated that the mean lethal time to death (LT50) of ΔMrSVP significantly increased relative to the wild type in insect bioassays (both topical inoculation and injection) involving Galleria mellonella. Moreover, similar rates of appressorium formation between ΔMrSVP and the wild type—and the significantly different expression of virulence-related genes such as acid trehalase and sucrose nonfermenting protein kinase in the haemocoel after injection—revealed that MrSVP is required for virulence in the insect haemocoel. Conclusions Overall, our data suggest that the Mrsvp gene contributes to thermotolerance and virulence of M. robertsii. Furthermore, this gene is deeply involved in the mycosis of insect cadavers and in immune escape rather than insect cuticle penetration during infection

  • Differential Functions of Two Metalloproteases, Mrmep1 and Mrmep2, in Growth, Sporulation, Cell Wall Integrity, and Virulence in the Filamentous Fungus Metarhizium robertsii
    'Frontiers Media SA', 2018
    Co-Authors: Rong Zhou, Zhangxun Wang, Xiazhi Zhou, Ali Fan, Bo Huang
    Abstract:

    The Metarhizium genus of filamentous entomopathogenic fungi plays a pivotal role in regulating insect populations. Metalloproteases (MEPs) are a widely distributed and diverse family of hydrolytic enzymes that are important toxicity factors in the interactions between fungi and their hosts. Herein, we characterized two MEPs, Mrmep1 and Mrmep2, in Metarhizium robertsii using gene deletion. Growth rates of the resulting ΔMrmep1 and ΔMrmep2 mutants decreased by 16.2 and 16.5%, respectively, relative to the wild-type (WT) strain. Both mutants were less sensitive to cell wall-perturbing agents, sodium dodecyl sulfate and Congo red than the WT strain, whereas did not show any obvious changes in fungal sensitivity to ultraviolet B irradiation or heat stress. The conidial yield of ΔMrmep1, ΔMrmep2, and ΔMrmep1ΔMrmep2 mutants decreased by 56.0, 23, and 53%, respectively. Insect bioassay revealed that median lethal time values against Galleria mellonella increased by 25.5% (ΔMrmep1), 19% (ΔMrmep2), and 28.8% (ΔMrmep1ΔMrmep2) compared with the WT strain at a concentration of 1 × 107 conidia mL-1, suggesting attenuated fungal virulence in the ΔMrmep1, ΔMrmep2, and ΔMrmep1ΔMrmep2 strains. During fungal infection, transcription levels of Mrmep1 was 1.6-fold higher than Mrmep2 at 36 h post inoculation. Additionally, transcription levels of gallerimycin gene were 1.2-fold, 2.18-fold, and 2.5-fold higher in insects infected with the ΔMrmep1, ΔMrmep2, or ΔMrmep1ΔMrmep2 mutant than those infected with the WT strain, respectively. Our findings suggest that Mrmep1 and Mrmep2 are differentially contributed to the growth, sporulation, cell wall integrity, and virulence of M. robertsii