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The Intermediates in Branched-Chain Amino Acid Biosynthesis Are Indispensable for Conidial Germination of the Insect-Pathogenic Fungus Metarhizium robertsii.

ABSTRACT: Metarhizium spp. are well-known biocontrol agents used worldwide to control different insect pests. Keto-acid reductoisomerase (ILVC) is a key enzyme for branched-chain amino acid (BCAA) biosynthesis, and it regulates many physiological activities. However, its functions in insect-pathogenic fungi are poorly understood. In this work, we identified MrilvC in M. robertsii and dissected its roles in fungal growth, conidiation, germination, destruxin biosynthesis, environmental stress response, and insecticidal virulence. BCAA metabolism affects conidial yields and germination. However, BCAAs cannot recover the conidial germination of an MrilvC-deficient strain. Further feeding assays with intermediates showed that some conidia of the ΔMrilvC mutant start to germinate. Therefore, it is the germination defect that causes the complete failures of conidial penetration and pathogenicity in the ΔMrilvC mutant. In conclusion, we found intermediates in BCAA biosynthesis are indispensable for Metarhizium robertsii conidial germination. This study will advance our understanding of the fungal germination mechanism.IMPORTANCE Branched-chain amino acid (BCAA) metabolism plays a significant role in many biological activities beyond protein synthesis. Spore germination initiates the first stage of vegetative growth, which is critical for the virulence of pathogenic fungi. In this study, we demonstrated that the keto-acid reductoisomerase MrILVC, a key enzyme for BCAA biosynthesis, from the insect-pathogenic fungus Metarhizium robertsii is associated with conidial germination and fungal pathogenicity. Surprisingly, the germination of the ΔMrilvC mutant was restored when supplemented with the intermediates of BCAA metabolism rather than three BCAAs. The result was significantly different from that of plant-pathogenic fungi. Therefore, this report highlights that the intermediates in BCAA biosynthesis are indispensable for conidial germination of M. robertsii.

SUBMITTER: Luo F

PROVIDER: S-EPMC7531975 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Conidial maturation, which is crucial for conidial quality, is controlled by the asexual development activator WetA and the downstream, velvety protein VosA in Aspergillus. Their orthologs have proved functional in conidial quality control of Beauveria bassiana, as seen in Aspergillus, but are functionally unexplored, in Metarhizium robertsii, another hypocrealean insect pathogen. Here, WetA and VosA prove essential and nonessential for M. robertsii's life cycle, respectively. Disruption of wetA increased hyphal sensitivity to oxidative stress and Congo red-induced cell wall stress, but had little impact on radial growth. The ΔwetA mutant was severely compromised in conidiation capacity and conidial quality, which was featured by slower germination, decreased UV resistance, reduced hydrophobicity, and deformed hydrophobin rodlet bundles that were assembled onto conidial coat. The mutant's virulence was greatly attenuated via normal infection due to a blockage of infection-required cellular processes. All examined phenotypes were unaffected for the ΔvosA mutant. Intriguingly, mannitol was much less accumulated in the 7- and 15-day-old cultures of ΔwetA and ΔvosA than of control strains, while accumulated trehalose was not detectable at all, revealing little a link of intracellular polyol accumulation to conidial maturation. Transcriptomic analysis revealed differential regulation of 160 genes (up/down ratio: 72:88) in ΔwetA. These genes were mostly involved in cellular component, biological process, and molecular function but rarely associated with asexual development. Conclusively, WetA plays a relatively conserved role in M. robertsii's spore surface structure, and also a differentiated role in some other cellular processes associated with conidial maturation. VosA is functionally redundant in M. robertsii unlike its ortholog in B. bassiana. IMPORTANCE WetA and VosA regulate conidiation and conidial maturation required for the life cycle of Beauveria bassiana, like they do in Aspergillus, but remain functionally unexplored in Metarhizium robertsii, another hypocrealean pathogen considered to have evolved insect pathogenicity ~130 million years later than B. bassiana. This study reveals a similar role of WetA ortholog in asexual development, conidial maturation, and insect pathogenicity, and also its distinctive role in mediating some other conidial maturation-related cellular events, but has functional redundancy of VosA in M. robertsii. The maturation process vital for conidial quality proves dependent on a role of WetA in spore wall assembly but is independent of its role in intracellular polyol accumulation. Transcriptomic analysis reveals a link of WetA to 160 genes involved in cellular component, biological process, and molecular function. Our study unveils that M. robertsii WetA or VosA is functionally differential or different from those learned in B. bassiana and other ascomycetes.

Project description: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 (?MrKu70) 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 ?MrKu70 was 93% compared to 7% in the wild-type strain. Since ?MrKu70 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.

Project description:RNA interference (RNAi) is a gene-silencing mechanism that plays an important role in gene regulation in a number of eukaryotic organisms. Two core components, Dicer and Argonaute, are central in the RNAi machinery. However, the physiological roles of Dicer and Argonaute in the entomopathogenic fungus Metarhizium robertsii have remained unclear. Here, the roles of genes encoding Dicer (M. robertsiidcl1 [Mrdcl1] and Mrdcl2) and Argonaute (Mrago1 and Mrago2) proteins in M. robertsii were investigated. The results showed that the Dicer-like protein MrDCL2 and Argonaute protein MrAGO1 are the major components of the RNAi process occurring in M. robertsii The Dicer and Argonaute genes were not involved in the regulation of growth and diverse abiotic stress response in M. robertsii under the tested conditions. Moreover, our results showed that the Dicer and Argonaute gene mutants demonstrated reduced abilities to produce conidia, compared to the wild type (WT) and the gene-rescued mutant. In particular, the conidial yields in the Δdcl2 and Δago1 mutants were reduced by 55.8% and 59.3%, respectively, compared with those from the control strains. Subsequently, for the WT and Δdcl2 mutant strains, digital gene expression (DGE) profiling analysis of the stage of mycelium growth and conidiogenesis revealed that modest changes occur in development or metabolism processes, which may explain the reduction in conidiation in the Δdcl2 mutant. In addition, we further applied high-throughput sequencing technology to identify small RNAs (sRNAs) that are differentially expressed in the WT and the Δdcl2 mutant and found that 4 known microRNA-like small RNAs (milRNAs) and 8 novel milRNAs were Mrdcl2 dependent in M. robertsiiIMPORTANCE The identification and characterization of components in RNAi have contributed significantly to our understanding of the mechanism and functions of RNAi in eukaryotes. Here, we found that Dicer and Argonaute genes play an important role in regulating conidiation in M. robertsii Our study also demonstrates that diverse small RNA pathways exist in M. robertsii The study provides a theoretical platform for exploration of the functions of Dicer and Argonaute genes involved in RNAi in fungi.

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The Intermediates in Branched-Chain Amino Acid Biosynthesis Are Indispensable for Conidial Germination of the Insect-Pathogenic Fungus Metarhizium robertsii.

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