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1.
J Agric Food Chem ; 67(33): 9265-9276, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31361479

RESUMO

Fungal infections significantly alter the emissions of volatile organic compounds (VOCs) by plants, but the mechanisms for VOCs affecting fungal infections of plants remain largely unknown. Here, we found that infection by Botrytis cinerea upregulated linalool production by strawberries and fumigation with linalool was able to inhibit the infection of fruits by the fungus. Linalool treatment downregulated the expression of rate-limiting enzymes in the ergosterol biosynthesis pathway, and this reduced the ergosterol content in the fungi cell membrane and impaired membrane integrity. Linalool treatment also caused damage to mitochondrial membranes by collapsing mitochondrial membrane potential and also downregulated genes involved in adenosine triphosphate (ATP) production, resulting in a significant decrease in the ATP content. Linalool treatment increased the levels of reactive oxygen species (ROS), in response to which the treated fungal cells produced more of the ROS scavenger pyruvate. RNA-Seq and proteomic analysis data showed that linalool treatment slowed the rates of transcription and translation.


Assuntos
Botrytis/efeitos dos fármacos , Fragaria/metabolismo , Frutas/microbiologia , Monoterpenos/metabolismo , Doenças das Plantas/microbiologia , Compostos Orgânicos Voláteis/metabolismo , Trifosfato de Adenosina/metabolismo , Botrytis/crescimento & desenvolvimento , Fragaria/química , Fragaria/microbiologia , Frutas/química , Frutas/metabolismo , Interações Hospedeiro-Patógeno , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Monoterpenos/farmacologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteômica , Espécies Reativas de Oxigênio/metabolismo , Compostos Orgânicos Voláteis/farmacologia
2.
Fungal Genet Biol ; 131: 103244, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31228645

RESUMO

Metarhizium robertsii is a versatile fungus with multifactorial lifestyles, and it is an emerging fungal model for investigating the mechanisms of multiple lifestyle transitions that involve trans-kingdom host jumping. Penetration of the insect cuticle is the necessary step for the transition from saprophytic or symbiotic to pathogenic lifestyle. Previously, we found the transcription factor AFTF1 plays an important role in cuticle penetration, which is precisely regulated by Fus3-MAPK, Slt2-MAPK, and the membrane protein Mr-OPY2. Here, we identified a transcription factor (MrSt12) that directly regulated the transcription of Aftf1 by physically interacting with the cis-acting element (ATGAAACA) in the promoter of Aftf1. The deletion mutant of MrSt12 failed to form the infection structure appressorium and was thus nonpathogenic. We further found that the regulation of Aftf1 by MrSt12 was directly controlled by the Fus3-MAPK. In conclusion, we found a new signaling cascade containing Fus3-MAPK, MrSt12, and AFTF1, which regulates cuticle penetration by M. robertsii.

3.
Proc Natl Acad Sci U S A ; 116(16): 7982-7989, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30948646

RESUMO

The emergence of new pathogenic fungi has profoundly impacted global biota, but the underlying mechanisms behind host shifts remain largely unknown. The endophytic insect pathogen Metarhizium robertsii evolved from fungi that were plant associates, and entomopathogenicity is a more recently acquired adaptation. Here we report that the broad host-range entomopathogen M. robertsii has 18 genes that are derived via horizontal gene transfer (HGT). The necessity of degrading insect cuticle served as a major selective pressure to retain these genes, as 12 are up-regulated during penetration; 6 were confirmed to have a role in penetration, and their collective actions are indispensable for infection. Two lipid-carrier genes are involved in utilizing epicuticular lipids, and a third (MrNPC2a) facilitates hemocoel colonization. Three proteases degraded the procuticular protein matrix, which facilitated up-regulation of other cuticle-degrading enzymes. The three lipid carriers and one of the proteases are present in all analyzed Metarhizium species and are essential for entomopathogenicity. Acquisition of another protease (MAA_01413) in an ancestor of broad host-range lineages contributed to their host-range expansion, as heterologous expression in the locust specialist Metarhizium acridum enabled it to kill caterpillars. Our work reveals that HGT was a key mechanism in the emergence of entomopathogenicity in Metarhizium from a plant-associated ancestor and in subsequent host-range expansion by some Metarhizium lineages.

4.
PLoS Genet ; 14(6): e1007472, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29958281

RESUMO

The ecological importance of the duplication and diversification of gene clusters that synthesize secondary metabolites in fungi remains poorly understood. Here, we demonstrated that the duplication and subsequent diversification of a gene cluster produced two polyketide synthase gene clusters in the cosmopolitan fungal genus Metarhizium. Diversification occurred in the promoter regions and the exon-intron structures of the two Pks paralogs (Pks1 and Pks2). These two Pks genes have distinct expression patterns, with Pks1 highly expressed during conidiation and Pks2 highly expressed during infection. Different upstream signaling pathways were found to regulate the two Pks genes. Pks1 is positively regulated by Hog1-MAPK, Slt2-MAPK and Mr-OPY2, while Pks2 is positively regulated by Fus3-MAPK and negatively regulated by Mr-OPY2. Pks1 and Pks2 have been subjected to positive selection and synthesize different secondary metabolites. PKS1 is involved in synthesis of an anthraquinone derivative, and contributes to conidial pigmentation, which plays an important role in fungal tolerance to UV radiation and extreme temperatures. Disruption of the Pks2 gene delayed formation of infectious structures and increased the time taken to kill insects, indicating that Pks2 contributes to pathogenesis. Thus, the duplication of a Pks gene cluster and its subsequent functional diversification has increased the adaptive flexibility of Metarhizium species.

5.
Environ Microbiol ; 20(3): 1158-1169, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29411499

RESUMO

It is commonly observed that microorganisms subjected to a mild stress develop tolerance not only to higher doses of the same stress but also to other stresses - a phenomenon called cross protection. The mechanisms for cross protection have not been fully revealed. Here, we report that heat shock induced cross protection against UV, oxidative and osmotic/salt stress conditions in the cosmopolitan fungus Metarhizium robertsii. Similarly, oxidative and osmotic/salt stresses also induced cross protection against multiple other stresses. We found that oxidative and osmotic/salt stresses produce an accumulation of pyruvate that scavenges stress-induced reactive oxygen species and promotes fungal growth. Thus, stress-induced pyruvate accumulation contributes to cross protection. RNA-seq and qRT-PCR analyses showed that UV, osmotic/salt and oxidative stress conditions decrease the expression level of pyruvate consumption genes in the trichloroacetic acid cycle and fermentation pathways leading to pyruvate accumulation. Our work presents a novel mechanism for cross protection in microorganisms.

6.
Nat Commun ; 8(1): 1565, 2017 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-29146899

RESUMO

Metarhizium robertsii is a versatile fungus with saprophytic, plant symbiotic and insect pathogenic lifestyle options. Here we show that M. robertsii mediates the saprophyte-to-insect pathogen transition through modulation of the expression of a membrane protein, Mr-OPY2. Abundant Mr-OPY2 protein initiates appressorium formation, a prerequisite for infection, whereas reduced production of Mr-OPY2 elicits saprophytic growth and conidiation. The precise regulation of Mr-OPY2 protein production is achieved via alternative transcription start sites. During saprophytic growth, a single long transcript is produced with small upstream open reading frames in its 5' untranslated region. Increased production of Mr-OPY2 protein on host cuticle is achieved by expression of a transcript variant lacking a small upstream open reading frame that would otherwise inhibit translation of Mr-OPY2. RNA-seq and qRT-PCR analyses show that Mr-OPY2 is a negative regulator of a transcription factor that we demonstrate is necessary for appressorial formation. These findings provide insights into the mechanisms regulating fungal lifestyle transitions.


Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Metarhizium/genética , Sítio de Iniciação de Transcrição , Regiões 5' não Traduzidas/genética , Animais , Interações Hospedeiro-Patógeno , Insetos/microbiologia , Metarhizium/patogenicidade , Plantas/microbiologia , Fatores de Transcrição/genética , Virulência/genética
7.
MBio ; 8(5)2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28874474

RESUMO

Heat tolerance is well known to be key to fungal survival in many habitats, but our mechanistic understanding of how organisms adapt to heat stress is still incomplete. Using Metarhizium robertsii, an emerging model organism for assessing evolutionary processes, we report that pyruvate is in the vanguard of molecules that scavenge heat-induced reactive oxygen species (ROS). We show that, as well as inducing a rapid burst of ROS production, heat stress also downregulates genes for pyruvate consumption. The accumulating pyruvate is the fastest acting of several M. robertsii ROS scavengers, efficiently reducing protein carbonylation, stabilizing mitochondrial membrane potential, and promoting fungal growth. The acetate produced from pyruvate-ROS reactions itself causes acid stress, tolerance to which is regulated by Hog1 mitogen-activated protein kinase. Heat stress also induces pyruvate accumulation in several other fungi, suggesting that scavenging of heat-induced ROS by pyruvate is widespread.IMPORTANCE Heat is a dangerous challenge for most organisms, as it denatures proteins and induces the production of ROS that inactivate proteins, lipid membranes, and DNA. How organisms respond to this stress is not fully understood. Using the experimentally tractable insect pathogen Metarhizium robertsii as a model organism, we show for the first time that heat stress induces pyruvate production and that this functions as the first line of defense against heat-induced ROS. Heat stress also induces rapid pyruvate accumulation in other fungi, suggesting that pyruvate is a common but unappreciated defense against stress.


Assuntos
Resposta ao Choque Térmico , Metarhizium/genética , Metarhizium/metabolismo , Ácido Pirúvico/metabolismo , Acetatos/metabolismo , Regulação para Baixo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/genética , Fungos/metabolismo , Resposta ao Choque Térmico/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo
8.
Microbiology ; 163(7): 980-991, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28708056

RESUMO

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.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismo , Insetos/microbiologia , Metarhizium/metabolismo , Metarhizium/patogenicidade , Animais , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Metarhizium/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/microbiologia , Virulência
9.
Sci Rep ; 7(1): 3433, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28611355

RESUMO

The continued success of malaria control efforts requires the development, study and implementation of new technologies that circumvent insecticide resistance. We previously demonstrated that fungal pathogens can provide an effective delivery system for mosquitocidal or malariacidal biomolecules. Here we compared genes from arthropod predators encoding insect specific sodium, potassium and calcium channel blockers for their ability to improve the efficacy of Metarhizium against wild-caught, insecticide-resistant anophelines. Toxins expressed under control of a hemolymph-specific promoter increased fungal lethality to mosquitoes at spore dosages as low as one conidium per mosquito. One of the most potent, the EPA approved Hybrid (Ca++/K+ channel blocker), was studied for pre-lethal effects. These included reduced blood feeding behavior, with almost 100% of insects infected with ~6 spores unable to transmit malaria within 5 days post-infection, surpassing the World Health Organization threshold for successful vector control agents. Furthermore, recombinant strains co-expressing Hybrid toxin and AaIT (Na+ channel blocker) produced synergistic effects, requiring 45% fewer spores to kill half of the mosquitoes in 5 days as single toxin strains. Our results identify a repertoire of toxins with different modes of action that improve the utility of entomopathogens as a technology that is compatible with existing insecticide-based control methods.

10.
Environ Microbiol ; 19(10): 3896-3908, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28447400

RESUMO

Metarhizium robertsii occupies a wide array of ecological niches and has diverse lifestyle options (saprophyte, insect pathogen and plant symbiont), that renders it an unusually effective model for studying genetic mechanisms for fungal adaptation. Here over 20,000 M. robertsii T-DNA mutants were screened in order to elucidate genetic mechanism by which M. robertsii replicates and persists in diverse niches. About 287 conidiation, colony sectorization or pathogenicity loci, many of which have not been reported in other fungi were identified. By analysing a series of conidial pigmentation mutants, a new fungal pigmentation gene cluster, which contains Mr-Pks1, Mr-EthD and Mlac1 was identified. A conserved conidiation regulatory pathway containing Mr-BrlA, Mr-AbaA and Mr-WetA regulates expression of these pigmentation genes. During conidiation Mr-BlrA up-regulates Mr-AbaA, which in turn controls Mr-WetA. It was found that Hog1-MAPK regulates fungal conidiation by controlling the conidiation regulatory pathway, and that all three pigmentation genes exercise feedback regulation of conidiation. This work provided the foundation for deeper understanding of the genetic processes behind M. robertsii adaptive phenotypes, and advances our insights into conidiation and pigmentation in this fungus.


Assuntos
DNA Bacteriano/genética , Metarhizium/genética , Metarhizium/patogenicidade , Pigmentação/genética , Esporos Fúngicos/genética , Animais , Agentes de Controle Biológico , DNA Fúngico/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Insetos/microbiologia , Sistema de Sinalização das MAP Quinases/genética , Família Multigênica/genética , Esporos Fúngicos/metabolismo , Virulência/genética
11.
Sci Rep ; 7: 44527, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28300148

RESUMO

The fungal kingdom potentially has the most complex chitin synthase (CHS) gene family, but evolution of the fungal CHS gene family and its diversification to fulfill multiple functions remain to be elucidated. Here, we identified the full complement of CHSs from 231 fungal species. Using the largest dataset to date, we characterized the evolution of the fungal CHS gene family using phylogenetic and domain structure analysis. Gene duplication, domain recombination and accretion are major mechanisms underlying the diversification of the fungal CHS gene family, producing at least 7 CHS classes. Contraction of the CHS gene family is morphology-specific, with significant loss in unicellular fungi, whereas family expansion is lineage-specific with obvious expansion in early-diverging fungi. ClassV and ClassVII CHSs with the same domain structure were produced by the recruitment of domains PF00063 and PF08766 and subsequent duplications. Comparative analysis of their functions in multiple fungal species shows that the emergence of ClassV and ClassVII CHSs is important for the morphogenesis of filamentous fungi, development of pathogenicity in pathogenic fungi, and heat stress tolerance in Pezizomycotina fungi. This work reveals the evolution of the fungal CHS gene family, and its correlation with fungal morphogenesis and adaptation to ecological niches.


Assuntos
Quitina Sintase/genética , Evolução Molecular , Filogenia , Adaptação Fisiológica/genética , Ascomicetos/genética , Ecossistema , Proteínas Fúngicas/genética , Morfogênese/genética
12.
Org Lett ; 19(7): 1686-1689, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28301168

RESUMO

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.


Assuntos
Metarhizium , Produtos Biológicos , Histona Acetiltransferases , Estrutura Molecular , Esporos Fúngicos
13.
Genome Biol Evol ; 9(2): 311-322, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-26957028

RESUMO

The fungal kingdom displays an extraordinary diversity of lifestyles, developmental processes, and ecological niches. The MAPK (mitogen-activated protein kinase) cascade consists of interlinked MAPKKK, MAPKK, and MAPK, and collectively such cascades play pivotal roles in cellular regulation in fungi. However, the mechanism by which evolutionarily conserved MAPK cascades regulate diverse output responses in fungi remains unknown. Here we identified the full complement of MAPK cascade components from 231 fungal species encompassing 9 fungal phyla. Using the largest data set to date, we found that MAPK family members could have two ancestors, while MAPKK and MAPKKK family members could have only one ancestor. The current MAPK, MAPKK, and MAPKKK subfamilies resulted from duplications and subsequent subfunctionalization during the emergence of the fungal kingdom. However, the gene structure diversification and gene expansion and loss have resulted in significant diversity in fungal MAPK cascades, correlating with the evolution of fungal species and lifestyles. In particular, a distinct evolutionary trajectory of MAPK cascades was identified in single-celled fungi in the Saccharomycetes. All MAPK, MAPKK, and MAPKKK subfamilies expanded in the Saccharomycetes; genes encoding MAPK cascade components have a similar exon-intron structure in this class that differs from those in other fungi.


Assuntos
Evolução Molecular , Sistema de Sinalização das MAP Quinases/genética , Filogenia , Saccharomyces/genética , Genes Fúngicos , Polimorfismo Genético , Saccharomyces/classificação , Saccharomyces/enzimologia , Saccharomyces/crescimento & desenvolvimento
14.
Environ Microbiol ; 18(3): 1048-62, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26714892

RESUMO

Metarhizium robertsii has been used as a model to study fungal pathogenesis in insects, and its pathogenicity has many parallels with plant and mammal pathogenic fungi. MAPK (Mitogen-activated protein kinase) cascades play pivotal roles in cellular regulation in fungi, but their functions have not been characterized in M. robertsii. In this study, we identified the full complement of MAPK cascade components in M. robertsii and dissected their regulatory roles in pathogenesis, conidiation and stress tolerance. The nine components of the Fus3, Hog1 and Slt2-MAPK cascades are all involved in conidiation. The Fus3- and Hog1-MAPK cascades are necessary for tolerance to hyperosmotic stress, and the Slt2- and Fus3-MAPK cascades both mediate cell wall integrity. The Hog1 and Slt2-MAPK cascades contribute to pathogenicity; the Fus3-MAPK cascade is indispensable for fungal pathogenesis. During its life cycle, M. robertsii experiences multiple microenvironments as it transverses the cuticle into the haemocoel. RNA-seq analysis revealed that MAPK cascades collectively play a major role in regulating the adaptation of M. robertsii to the microenvironmental change from the cuticle to the haemolymph. The three MAPKs each regulate their own distinctive subset of genes during penetration of the cuticle and haemocoel colonization, but they function redundantly to regulate adaptation to microenvironmental change.


Assuntos
Insetos/microbiologia , Metarhizium/patogenicidade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pressão Osmótica , Esporos Fúngicos/crescimento & desenvolvimento , Animais , Parede Celular/metabolismo , Virulência
15.
Sci Rep ; 4: 7345, 2014 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-25475694

RESUMO

Locusts and grasshoppers (acridids) are among the worst pests of crops and grasslands worldwide. Metarhizium acridum, a fungal pathogen that specifically infects acridids, has been developed as a control agent but its utility is limited by slow kill time and greater expense than chemical insecticides. We found that expression of four insect specific neurotoxins improved the efficacy of M. acridum against acridids by reducing lethal dose, time to kill and food consumption. Coinoculating recombinant strains expressing AaIT1(a sodium channel blocker) and hybrid-toxin (a blocker of both potassium and calcium channels), produced synergistic effects, including an 11.5-fold reduction in LC50, 43% reduction in LT50 and a 78% reduction in food consumption. However, specificity was retained as the recombinant strains did not cause disease in non-acridids. Our results identify a repertoire of toxins with different modes of action that improve the utility of fungi as specific control agents of insects.


Assuntos
Melhoramento Genético/métodos , Gafanhotos/microbiologia , Gafanhotos/fisiologia , Metarhizium/fisiologia , Neurotoxinas/genética , Controle Biológico de Vetores/métodos , Animais , Neurotoxinas/metabolismo , Taxa de Sobrevida
16.
PLoS One ; 9(9): e107657, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25222118

RESUMO

Systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. Metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. Thus, M. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the "model" fungi Saccharomyces cerevisiae and Neurospora crassa; a systematic genetic analysis of M. robertsii will benefit studies in other fungi. In order to systematically disrupt genes in M. robertsii, we developed a high-throughput gene disruption methodology, which includes two technologies. One is the modified OSCAR-based, high-throughput construction of gene disruption plasmids. This technology involves two donor plasmids (pA-Bar-OSCAR with the herbicide resistance genes Bar and pA-Sur-OSCAR with another herbicide resistance gene Sur) and a recipient binary plasmid pPK2-OSCAR-GFP that was produced by replacing the Bar cassette in pPK2-bar-GFP with a ccdB cassette and recombination recognition sites. Using this technology, a gene disruption plasmid can be constructed in one cloning step in two days. The other is a highly efficient gene disruption technology based on homologous recombination using a Ku70 deletion mutant (ΔMrKu70) as the recipient strain. The deletion of MrKu70, a gene encoding a key component involved in nonhomologous end-joining DNA repair in fungi, dramatically increases the gene disruption efficiency. The frequency of disrupting the conidiation-associated gene Cag8 in ΔMrKu70 was 93% compared to 7% in the wild-type strain. Since ΔMrKu70 is not different from the wild-type strain in development, pathogenicity and tolerance to various abiotic stresses, it can be used as a recipient strain for a systematic gene disruption project to characterize the whole suite of genes involved in the biological processes of M. robertsii.


Assuntos
Reparo do DNA por Junção de Extremidades/genética , Genoma Fúngico , Metarhizium/genética , Antígenos Nucleares/genética , Proteínas de Ligação a DNA/genética , Genótipo , Autoantígeno Ku , Metarhizium/patogenicidade , Neurospora crassa/genética , Plasmídeos/genética , Saccharomyces cerevisiae/genética , Esporos Fúngicos/genética
17.
Appl Microbiol Biotechnol ; 98(16): 7089-96, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24805846

RESUMO

Metarhizium species have recently been found to be plant rhizosphere associates as well as insect pathogens. Because of their abundance, rhizospheric Metarhizium could have enormous environmental impact, with co-evolutionary implications. Here, we tested the hypothesis that some Metarhizium spp. are multifactorial plant growth promoters. In two consecutive years, corn seeds were treated with entomopathogenic Metarhizium spp. and field tested at the Beltsville Facility in Maryland. Seed treatments included application of green fluorescent protein (GFP)-tagged strains of Metarhizium brunneum, Metarhizium anisopliae, Metarhizium robertsii, and M. robertsii gene disruption mutants that were either avirulent (Δmcl1), unable to adhere to plant roots (Δmad2), or poorly utilized root exudates (Δmrt). Relative to seeds treated with heat-killed conidia, M. brunneum, M. anisopliae, and M. robertsii significantly increased leaf collar formation (by 15, 14, and 13 %), stalk length (by 16, 10, and 10 %), average ear biomass (by 61, 56, and 36 %), and average stalk and foliage biomass (by 46, 36, and 33 %). Their major impact on corn yield was during early vegetative growth by allowing the plants to establish earlier and thereby potentially outpacing ambient biotic and abiotic stressors. Δmcl1 colonized roots and promoted plant growth to a similar extent as the parent wild type, showing that Metarhizium populations are plant growth promoters irrespective of their role as insect pathogens. In contrast, rhizospheric populations and growth promotion by Δmrt were significantly reduced, and Δmad2 failed to colonize roots or impact plant growth, suggesting that colonization of the root is a prerequisite for most, if not all, of the beneficial effects of Metarhizium.


Assuntos
Aderência Bacteriana , Metarhizium/fisiologia , Desenvolvimento Vegetal , Raízes de Plantas/microbiologia , Zea mays/microbiologia , Zea mays/fisiologia , Biomassa , Técnicas de Inativação de Genes , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Maryland , Metarhizium/genética , Metarhizium/crescimento & desenvolvimento , Coloração e Rotulagem
18.
PLoS Pathog ; 10(4): e1004009, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24722668

RESUMO

Metarhizium robertsii is a plant root colonizing fungus that is also an insect pathogen. Its entomopathogenicity is a characteristic that was acquired during evolution from a plant endophyte ancestor. This transition provides a novel perspective on how new functional mechanisms important for host switching and virulence have evolved. From a random T-DNA insertion library, we obtained a pathogenicity defective mutant that resulted from the disruption of a sterol carrier gene (Mr-npc2a). Phylogenetic analysis revealed that Metarhizium acquired Mr-npc2a from an insect by horizontal gene transfer (HGT). Mr-NPC2a binds to cholesterol, an animal sterol, rather than the fungal sterol ergosterol, indicating it retains the specificity of insect NPC2 proteins. Mr-NPC2a is an intracellular protein and is exclusively expressed in the hemolymph of living insects. The disruption of Mr-npc2a reduced the amount of sterol in cell membranes of the yeast-like hyphal bodies that facilitate dispersal in the host body. These were consequently more susceptible to insect immune responses than the wild type. Transgenic expression of Mr-NPC2a increased the virulence of Beauveria bassiana, an endophytic insect-pathogenic fungus that lacks a Mr-NPC2a homolog.


Assuntos
Proteínas de Transporte/biossíntese , Proteínas Fúngicas , Transferência Genética Horizontal/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Metarhizium/fisiologia , Mariposas/microbiologia , Filogenia , Animais , Beauveria/genética , Beauveria/metabolismo , Proteínas de Transporte/genética , Colesterol/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Mariposas/metabolismo
19.
Proc Natl Acad Sci U S A ; 111(4): 1343-8, 2014 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-24474758

RESUMO

Locusts are infamous for their ability to aggregate into gregarious migratory swarms that pose a major threat to food security. Aggregation is elicited by an interplay of visual, tactile, and chemical stimuli, but the aggregation pheromone in feces is particularly important. Infection by the microsporidian parasite Paranosema (Nosema) locustae is known to inhibit aggregation of solitary Locusta migratoria manilensis and to induce gregarious locusts to shift back to solitary behavior. Here we suggest that P. locustae achieves this effect by acidifying the hindgut and modulating the locust immune response, which suppresses the growth of the hindgut bacteria that produce aggregation pheromones. This in turn reduces production of the neurotransmitter serotonin that initiates gregarious behavior. Healthy L. migratoria manilensis exposed to olfactory stimuli from parasite-infected locusts also produced significantly less serotonin, reducing gregarization. P. locustae also suppresses biosynthesis of the neurotransmitter dopamine that maintains gregarization. Our findings reveal the mechanisms by which P. locustae reduces production of aggregation pheromone and blocks the initiation and maintainence of gregarious behavior.


Assuntos
Comportamento Animal , Gafanhotos/microbiologia , Animais , Bactérias/isolamento & purificação , Contagem de Colônia Microbiana , Dopamina/biossíntese , Fezes/química , Estudo de Associação Genômica Ampla , Gafanhotos/genética , Gafanhotos/metabolismo , Microsporídios , Feromônios/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Volatilização
20.
Appl Microbiol Biotechnol ; 98(2): 777-83, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24265026

RESUMO

Temperature extremes are an important adverse factor limiting the effectiveness of microbial pest control agents. They reduce virulence and persistence in the plant root-colonizing insect pathogen Metarhizium robertsii. Small heat shock proteins have been shown to confer thermotolerance in many organisms. In this study, we report on the cloning and characterization of a small heat shock protein gene hsp25 from M. robertsii. hsp25 expression was upregulated when the fungus was grown at extreme temperatures (4, 35, and 42 °C) or in the presence of oxidative or osmotic agents. Expression of hsp25 in Escherichia coli increased bacterial thermotolerance confirming that hsp25 encodes a functional heat shock protein. Overexpressing hsp25 in M. robertsii increased fungal growth under heat stress either in nutrient-rich medium or on locust wings and enhanced the tolerance of heat shock-treated conidia to osmotic stress. In addition, overexpression of hsp25 increased the persistence of M. robertsii in rhizospheric soils in outdoor microcosms, though it did not affect survival in bulk soil, indicating that M. robertsii's survival in soil is dependent on interactions with plant roots.


Assuntos
Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Metarhizium/fisiologia , Metarhizium/efeitos da radiação , Viabilidade Microbiana/efeitos dos fármacos , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/genética , Metarhizium/genética , Microbiologia do Solo
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