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

Yulong Wang,Shihong Liu,Bo Huang,Xiangyun Xie,Deshui Yu,Xuebing Yin

doi:10.3390/jof7090737

Yulong Wang, Shihong Liu + Show 4 more

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https://doi.org/10.3390/jof7090737

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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.

Highlights

Valine, leucine, and isoleucine form the small group of branched chain amino acids (BCAAs), which are synthesized in bacteria, plants, and fungi, but not in animals [1,2]
Site-directed mutagenesis and activity assays showed that plant and bacterial ILVCs have evolved different mechanisms of induced fit to prepare the active site for catalysis [9,12]
We found that ILVC is associated with conidial germination and fungal pathogenicity in the insect–pathogenic fungus Metarhizium robertsii Metchnikoff (Hypocreales: Clavicipitaceae), formerly classified as Metarhizium anisopliae, and the ilvC-deleted mutant failed to germinate on czapek agar (CZA) complemented with three BCAAs

Summary

Introduction

Leucine, and isoleucine form the small group of branched chain amino acids (BCAAs), which are synthesized in bacteria, plants, and fungi, but not in animals [1,2]. The biosynthesis of these amino acids shows high similarity between organisms of the different kingdoms of life, of which valine and isoleucine synthesis are carried out by the same enzymes, and leucine is created from α-ketoisovalerate (a transamination precursor of valine) [3]. The conservation in fungi but absence in mammals of the BCAA biosynthetic pathway makes it as the target for herbicides, fungicides, and antimicrobial compounds [5,6]. BCAA enzyme inhibitors have shown antimicrobial effects on some harmful pathogenic microorganisms, including Fusarium graminearum Petch (Hypocreales: Nectriaceae), Pseudomonas aeruginosa Migula (Pseudomonadales: Pseudomonadaceae) and Candida albicans Berkhout (Saccharomycetales: Saccharomycetaceae), but whether they have inhibitory effects on pest pathogenic fungi are still uncharted [6,9,10]

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