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Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii.

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.

SUBMITTER: Wang Y

PROVIDER: S-EPMC8471054 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description: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.

Project description:G proteins are critical modulators or transducers in various transmembrane signaling systems. They play key roles in numerous biological processes in fungi, including vegetative growth, development of infection-related structures, asexual conidiation, and virulence. However, functions of G proteins in entomopathogenic fungi remain unclear. Here, we characterized the roles of MrGPA1, a G-protein subunit Gαi, in conidiation, stress resistance, and virulence in Metarhizium robertsii. MrGPA1 was localized in the mitochondria. MrGpa1 deletion resulted in a significant reduction (47%) in the conidiation capacity, and reduced expression of several key conidiation-related genes, including fluG, flbD, brlA, wetA, phiA, and stuA. Further, MrGpa1 disruption resulted in decreased fungal sensitivity to UV irradiation and thermal stress, as determined based on conidial germination of ΔMrGpa1 and wild-type (WT) strains. Chemical stress analysis indicated that MrGpa1 contributes to fungal antioxidant capacity and cell wall integrity, but is not involved in tolerance to antifungal drug and osmotic stress. Importantly, insect bioassays involving (topical inoculation and injection) of Galleria mellonella larvae revealed decreased virulence of ΔMrGpa1 strain after cuticle infection. This was accompanied by decreased rates of appressorium formation and reduced expression of several cuticle penetration-related genes. Further assays showed that MrGpa1 regulated intracellular cyclic AMP (cAMP) levels, but feeding with cAMP could not recover the appressorium formation rate of ΔMrGpa1. These observations suggest that MrGpa1 contributes to the regulation of conidiation, UV irradiation, thermal stress response, antioxidant capacity, and cell wall integrity in M. robertsii. This gene is also involved in insect cuticle penetration during infection. These findings raise the possibility of designing powerful strategies for genetic improvement of M. robertsii conidiation capacity and virulence for killing pests.

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Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii.

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