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 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 their roles in fungal growth, conidiation, germination, destruxins biosynthesis, environmental stress response and insecticidal virulence. BCAA metabolism affects conidial yields and germination. However, BCAAs cannot recover the conidia germination of MrilvC deficient strain. Further feeding assays with intermediates showed that some conidia of ΔMrilvC start to germinate. Therefore, it is the germination defect that causes the complete failures of conidial penetration and pathogenicity in 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 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.

Spore Germination of the Obligate Biotroph Spongospora subterranea: Transcriptome Analysis Reveals Germination Associated Genes

“…Metabolomics analysis of P. digitatum revealed that central carbon and energy metabolism were upregulated during germination of spores (Che et al, 2020). In line with previously reported works on gene expression during spore germination (Luo et al, 2020;Swarge et al, 2020), important cellular metabolic pathways including fatty acid biosynthesis and amino acid biosynthesis showed significant changes in the germinating spores in comparison with dormant spores (Figure 4 and Table 3). In the lipid fraction, there is a decrease in fatty acid synthase and trehalase activity (Table 2).…”

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

“…In this study, qRT-PCR showed MrilvC expression sharply increased expression at the stage of conidial germination and infection, which is consistent with the impairment of the full virulence on insects treated with direct injection or cuticle infection in our previous study [ 14 ]. BCAAs account for more than 20% of total protein amino acids and are an important constituent of the amino acid pool in organisms [ 29 ].…”

“…The ilvC-deficient (∆MrilvC), and complemented strains (Comp) were constructed from the wild-type (WT) M. robertsii strain ARSEF 23 (ATCC no. MYA-3075) in our previous report [14]. All strains grown on SDAY (4% glucose, 1% peptone, 1% yeast extract powder, and 1.5% agar, w/v) at 28 • C in the dark.…”

“…Previous sequence comparison revealed that those active residues are highly conserved across plant, fungal, and bacterial ILVCs, but the gene or genes responsible for ILVC activity in fungi have not been investigated in any detail. In addition, functional analysis of the ILVC has been carried out in bacteria, plants, and fungi; however, transcriptome analyses for the blocking of BCAA biosynthesis remain unclear [ 6 , 14 ].…”

“…In our previous study, 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 CZA complemented with three BCAAs. Complementation of BCAAs cannot recover the conidial germination for the ilvC -deficient strain [ 14 ]. This study seeks to further characterize ilvC in M. robertsii by phenotypic and comparative analyses of its deletion mutants.…”