Dataset Information

Molecular evolution and transcriptional profile of GH3 and GH20 ?-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae.

ABSTRACT: Cell walls are involved in manifold aspects of fungi maintenance. For several fungi, chitin synthesis, degradation and recycling are essential processes required for cell wall biogenesis; notably, the activity of ?-N-acetylglucosaminidases (NAGases) must be present for chitin utilization. For entomopathogenic fungi, such as Metarhizium anisopliae, chitin degradation is also used to breach the host cuticle during infection. In view of the putative role of NAGases as virulence factors, this study explored the transcriptional profile and evolution of putative GH20 NAGases (MaNAG1 and MaNAG2) and GH3 NAGases (MaNAG3 and MaNAG4) identified in M. anisopliae. While MaNAG2 orthologs are conserved in several ascomycetes, MaNAG1 clusters only with Aspergilllus sp. and entomopathogenic fungal species. By contrast, MaNAG3 and MaNAG4 were phylogenetically related with bacterial GH3 NAGases. The transcriptional profiles of M. anisopliae NAGase genes were evaluated in seven culture conditions showing no common regulatory patterns, suggesting that these enzymes may have specific roles during the Metarhizium life cycle. Moreover, the expression of MaNAG3 and MaNAG4 regulated by chitinous substrates is the first evidence of the involvement of putative GH3 NAGases in physiological cell processes in entomopathogens, indicating their potential influence on cell differentiation during the M. anisopliae life cycle.

SUBMITTER: Oliveira ES

PROVIDER: S-EPMC6415606 | biostudies-literature | 2018 Oct-Dec

REPOSITORIES: biostudies-literature

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Molecular evolution and transcriptional profile of GH3 and GH20 β-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae.

Oliveira Eder Silva de ES Junges Ângela Â Sbaraini Nicolau N Andreis Fábio Carrer FC Thompson Claudia Elizabeth CE Staats Charley Christian CC Schrank Augusto A

Genetics and molecular biology 20181001 4

Cell walls are involved in manifold aspects of fungi maintenance. For several fungi, chitin synthesis, degradation and recycling are essential processes required for cell wall biogenesis; notably, the activity of β-N-acetylglucosaminidases (NAGases) must be present for chitin utilization. For entomopathogenic fungi, such as Metarhizium anisopliae, chitin degradation is also used to breach the host cuticle during infection. In view of the putative role of NAGases as virulence factors, this study ...[more]

PMID: 30534852

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Project description:Fungal chitin metabolism involves diverse processes such as metabolically active cell wall maintenance, basic nutrition, and different aspects of virulence. Chitinases are enzymes belonging to the glycoside hydrolase family 18 (GH18) and 19 (GH19) and are responsible for the hydrolysis of β-1,4-linkages in chitin. This linear homopolymer of N-acetyl-β-D-glucosamine is an essential constituent of fungal cell walls and arthropod exoskeletons. Several chitinases have been directly implicated in structural, morphogenetic, autolytic and nutritional activities of fungal cells. In the entomopathogen Metarhizium anisopliae, chitinases are also involved in virulence. Filamentous fungi genomes exhibit a higher number of chitinase-coding genes than bacteria or yeasts. The survey performed in the M. anisopliae genome has successfully identified 24 genes belonging to glycoside hydrolase family 18, including three previously experimentally determined chitinase-coding genes named chit1, chi2 and chi3. These putative chitinases were classified based on domain organization and phylogenetic analysis into the previously described A, B and C chitinase subgroups, and into a new subgroup D. Moreover, three GH18 proteins could be classified as putative endo-N-acetyl-β-D-glucosaminidases, enzymes that are associated with deglycosylation and were therefore assigned to a new subgroup E. The transcriptional profile of the GH18 genes was evaluated by qPCR with RNA extracted from eight culture conditions, representing different stages of development or different nutritional states. The transcripts from the GH18 genes were detected in at least one of the different M. anisopliae developmental stages, thus validating the proposed genes. Moreover, not all members from the same chitinase subgroup presented equal patterns of transcript expression under the eight distinct conditions studied. The determination of M. anisopliae chitinases and ENGases and a more detailed study concerning the enzymes' roles in morphological or nutritional functions will allow comprehensive insights into the chitinolytic potential of this highly infective entomopathogenic fungus.

Project description:Many fungus-growing ants engage in a defensive symbiosis with antibiotic-producing ectosymbiotic bacteria in the genus Pseudonocardia, which help protect the ants' fungal mutualist from a specialized mycoparasite, Escovopsis. Here, using germfree ant rearing and experimental pathogen infection treatments, we evaluate if Acromyrmex ants derive higher immunity to the entomopathogenic fungus Metarhizium anisopliae from their Pseudonocardia symbionts. We further examine the ecological dynamics and defensive capacities of Pseudonocardia against M. anisopliae across seven different Acromyrmex species by controlling Pseudonocardia acquisition using ant-nonnative Pseudonocardia switches, in vitro challenges, and in situ mass spectrometry imaging (MSI). We show that Pseudonocardia protects the ants against M. anisopliae across different Acromyrmex species and appears to afford higher protection than metapleural gland (MG) secretions. Although Acromyrmex echinatior ants with nonnative Pseudonocardia symbionts receive protection from M. anisopliae regardless of the strain acquired compared with Pseudonocardia-free conditions, we find significant variation in the degree of protection conferred by different Pseudonocardia strains. Additionally, when ants were reared in Pseudonocardia-free conditions, some species exhibit more susceptibility to M. anisopliae than others, indicating that some ant species depend more on defensive symbionts than others. In vitro challenge experiments indicate that Pseudonocardia reduces Metarhizium conidiospore germination area. Our chemometric analysis using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) reveals that Pseudonocardia-carrying ants produce more chemical signals than Pseudonocardia-free treatments, indicating that Pseudonocardia produces bioactive metabolites on the Acromyrmex cuticle. Our results indicate that Pseudonocardia can serve as a dual-purpose defensive symbiont, conferring increased immunity for both the obligate fungal mutualist and the ants themselves. IMPORTANCE In some plants and animals, beneficial microbes mediate host immune response against pathogens, including by serving as defensive symbionts that produce antimicrobial compounds. Defensive symbionts are known in several insects, including some leaf-cutter ants where antifungal-producing Actinobacteria help protect the fungal mutualist of the ants from specialized mycoparasites. In many defensive symbioses, the extent and specificity of defensive benefits received by the host are poorly understood. Here, using "aposymbiotic" rearing, symbiont switching experiments, and imaging mass spectrometry, we explore the ecological and chemical dynamics of the model defensive symbiosis between Acromyrmex ants and their defensive symbiotic bacterium Pseudonocardia. We show that the defensive symbiont not only protects the fungal crop of Acromyrmex but also provides protection from fungal pathogens that infect the ant workers themselves. Furthermore, we reveal that the increased immunity to pathogen infection differs among strains of defensive symbionts and that the degree of reliance on a defensive symbiont for protection varies across congeneric ant species. Taken together, our results suggest that Acromyrmex-associated Pseudonocardia have evolved broad antimicrobial defenses that promote strong immunity to diverse fungal pathogens within the ancient fungus-growing ant-microbe symbiosis.

Project description:Entomopathogenic fungi can regulate insect populations and function as crucial biological control agents against insect pests, but their impacts on nontarget microorganisms are poorly understood. In this study, we investigated the potential of the fungal strain Metarhizium anisopliae CQMa421 to control rice planthoppers under field conditions and its effects on rice microbiota. This fungus suppressed rice planthoppers during this period, and its control efficiency was more than 60% 7 days after application and did not significantly differ from that of the chemical treatment except in 2019. Both treatments showed a smaller population of rice planthoppers than the controls. After application, M. anisopliae was maintained on rice plants for approximately 14 days, showing a decreasing trend over time. Furthermore, the results showed that the bacterial and fungal richness (operational taxonomic units) and diversity (Shannon index) did not significantly differ between the fungal treatment and the controls after application. The major bacterial taxa of Proteobacteria (including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria), Actinobacteria, Bacteroidetes, and Cyanobacteria accounted for more than 80% of the bacterial community after fungal application, and the major fungal taxa Ascomycota (including Eurotiomycetes, Dothideomycetes, and Sordariomycetes) and Basidiomycota (including Ustilaginomycetes) represented more than 90% of the fungal community. However, the microbial communities of the rice phyllosphere did not significantly change after entomopathogenic-agent application, indicating that the indigenous microbial communities may adapt to fungal insecticide application. Taken together, the results suggest that this fungal agent has good potential for rice planthopper control with no substantial effects on rice microbial communities.IMPORTANCE Entomopathogenic fungi may be used as crucial biocontrol agents for the control of insect pests, but few effective fungal strains have been reported for the control of the rice planthopper, a major pest of rice. More importantly, the impacts of fungal insecticide application on nontarget microorganisms have not been well evaluated, especially under field conditions. Therefore, in this study, we investigated the effects of the fungal strain M. anisopliae CQMa421 on rice planthopper populations from 2017 to 2019 and evaluated its potential impacts on the microbiota of rice plants after application. The results suggested that this fungal agent has good potential for use in the control of rice planthoppers with no significant effects on rice microbial communities, representing an alternative strategy for the control of rice pests.

Project description:BackgroundThe entomopathogenic fungus Metarhizium anisopliae is a candidate for the integrated management of the disease vector mosquito Aedes aegypti. Metarhizium anisopliae is pathogenic and virulent against Ae. aegypti larvae; however, its half-life is short without employing adjuvants. Here, we investigated the use of neem oil to increase virulence and persistence of the fungus under laboratory and simulated field conditions.MethodsNeem was mixed with M. anisopliae and added to recipients. Larvae were then placed in recipients at 5-day intervals for up to 50 days. Survival rates were evaluated 7 days after exposing larvae to each treatment. The effect of neem on conidial germination following exposure to ultraviolet radiation was evaluated under laboratory conditions. Statistical tests were carried out using ANOVA and regression analysis.ResultsLaboratory bioassays showed that the fungus alone reduced survival to 30% when larvae were exposed to the treatment as soon as the suspension had been prepared (time zero). A mixture of fungus + neem resulted in 11% survival at time zero. The combination of fungus + neem significantly reduced larval survival rates even when suspensions had been maintained for up to 45 days before adding larvae. For simulated-field experiments 1% neem was used, even though this concentration is insecticidal, resulting in 20% survival at time zero. However, this toxic effect was reduced over time. When used alone under simulated-field conditions the fungus rapidly lost virulence. The formulation fungus + neem effectively maintained fungal virulence, with larval survival rates significantly reduced for up to 45 days after preparation of the suspensions. The effective half-life of the fungus or neem when used separately was 6 and 13 days, respectively. The half-life of fungus formulated in 1% neem was 34 days. Conidia suspended in neem maintained high levels of germination even following a 2-h exposure to ultraviolet radiation.ConclusionsA combination of the entomopathogenic fungus M. anisopliae with neem oil effectively increases the half-life and virulence of the fungus when tested against Ae. aegypti larvae, even under simulated field conditions. Neem oil also protected the fungus from the damaging effects of ultraviolet radiation.

Dataset Information

Molecular evolution and transcriptional profile of GH3 and GH20 ?-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae.

Publications

Molecular evolution and transcriptional profile of GH3 and GH20 β-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae.

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