Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii
Abstract
:1. Introduction
2. Materials and Methods
2.1. Phylogenetic Analysis
2.2. Fungal Strain and Maintenance
2.3. Site-Directed Mutagenesis, Protein Expression and Purification
2.4. Reductase Activity Assays
2.5. The Sensitivity to Herbicides
2.6. Quantitative RT-PCR Analysis of MrilvC
2.7. Phenotypic Assays
2.8. Transcriptomics Analysis
2.9. Statistical Analysis
3. Results
3.1. Phylogenetic Analysis of ILVC from Eukaryotes
3.2. Enzymatic Activity of ILVC from Different Fungi and MrILVC with Active-Site Mutant
3.3. Deletion of MrILVC Increased Tolerance to AHAS Inhibitors
3.4. ILVC Contributes to Mycelial Growth and Conidial Germination
3.5. RNA-Seq Analysis of ΔMrilvC on Different Medium
3.6. MrILVC Affects Expression of Genes Involved in Mycelial Growth and Conidial Germination
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Neinast, M.; Murashige, D.; Arany, Z. Branched Chain Amino Acids. Annu. Rev. Physiol. 2019, 81, 139–164. [Google Scholar] [CrossRef]
- Steyer, J.T.; Downes, D.J.; Hunter, C.C.; Migeon, P.A.; Todd, R.B. Duplication and functional divergence of branched-chain amino acid biosynthesis genes in Aspergillus nidulans. mBio 2021, 12, e00768-21. [Google Scholar] [CrossRef]
- Mccourt, J.A.; Duggleby, R.G. Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids. Amino Acids 2006, 31, 173–210. [Google Scholar] [CrossRef] [PubMed]
- Binder, S. Branched-chain amino acid metabolism in Arabidopsis thaliana. Arabidopsis Book Am. Soc. Plant Biol. 2010, 8, e0137. [Google Scholar]
- Tan, S.; Evans, R.; Singh, B. Herbicidal inhibitors of amino acid biosynthesis and herbicide-tolerant crops. Amino Acids 2006, 30, 195–204. [Google Scholar] [CrossRef]
- Liu, X.; Wang, J.; Xu, J.; Shi, J. FgIlv5 is required for branched-chain amino acid biosynthesis and full virulence in Fusarium graminearum. Microbiology 2014, 160, 692–702. [Google Scholar] [CrossRef]
- Duggleby, R.G.; McCourt, J.A.; Guddat, L.W. Structure and mechanism of inhibition of plant acetohydroxyacid synthase. Plant Physiol. Biochem. 2008, 46, 309–324. [Google Scholar] [CrossRef] [PubMed]
- Shimizu, M.; Fujii, T.; Masuo, S.; Takaya, N. Mechanism of de novo branched-chain amino acid synthesis as an alternative electron sink in hypoxic Aspergillus nidulans cells. Appl. Environ. Microb. 2010, 76, 1507–1515. [Google Scholar] [CrossRef] [Green Version]
- Lee, Y.T.; Cui, C.J.; Chow, E.; Pue, N.; Lonhienne, T.; Wang, J.G.; Fraser, J.A.; Guddat, L.W. Sulfonylureas have antifungal activity and are potent inhibitors of Candida albicans acetohydroxyacid synthase. J. Med. Chem. 2013, 56, 210–219. [Google Scholar] [CrossRef] [PubMed]
- Garcia, M.; Chua, S.; Low, Y.S.; Lee, Y.T.; Guddat, L.W. Commercial AHAS-inhibiting herbicides are promising drug leads for the treatment of human fungal pathogenic infections. Proc. Natl. Acad. Sci. USA 2018, 115, E9649–E9658. [Google Scholar] [CrossRef] [Green Version]
- Tyagi, R.; Lee, Y.-T.; Guddat, L.W.; Duggleby, R.G. Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site. FEBS J. 2005, 272, 593–602. [Google Scholar] [CrossRef]
- Wong, S.H.; Lonhienne, T.; Winzor, D.J.; Schenk, G.; Guddat, L.W. Bacterial and plant ketol-acid reductoisomerases have different mechanisms of induced fit during the catalytic cycle. J. Mol. Biol. 2012, 424, 168–179. [Google Scholar] [CrossRef]
- Ahn, H.J.; Su, J.E.; Yoon, H.J.; Lee, B.I.; Cho, H.; Suh, S.W. Crystal structure of class i acetohydroxy acid isomeroreductase from Pseudomonas aeruginosa. J. Mol. Biol. 2003, 328, 505–515. [Google Scholar] [CrossRef]
- Luo, F.; Zhou, H.; Zhou, X.; Xie, X.; Li, Y.; Hu, F.; Huang, B. The intermediates in branched-chain amino acid biosynthesis are indispensable for conidial germination of the insect-pathogenic fungus Metarhizium robertsii. Appl. Environ. Microbiol. 2020, 86, e01682-20. [Google Scholar] [CrossRef]
- Kim, G.; Shin, D.; Lee, S.; Yun, J.; Lee, S. Crystal structure of ilvc, a ketol-acid reductoisomerase, from Streptococcus Pneumoniae. Crystals 2019, 9, 551. [Google Scholar] [CrossRef] [Green Version]
- Wang, Z.; Meng, H.; Zhuang, Z.; Chen, M.; Bo, H. Molecular cloning of a novel subtilisin-like protease (Pr1A) gene from the biocontrol fungus Isaria farinosa. Appl. Entomol. Zool. 2013, 48, 477–487. [Google Scholar] [CrossRef]
- Brinkmann-Chen, S.; Flock, T.; Cahn, J.; Snow, C.D.; Brustad, E.M.; Mcintosh, J.A.; Meinhold, P.; Zhang, L.; Arnold, F.H. General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH. Proc. Natl. Acad. Sci. USA 2013, 110, 10946–10951. [Google Scholar] [CrossRef] [Green Version]
- Li, K.-H.; Yu, Y.-H.; Dong, H.-J.; Zhang, W.-B.; Ma, J.-C.; Wang, H.-H. Biological functions of ilvc in branched-chain fatty acid synthesis and diffusible signal factor family production in Xanthomonas campestris. Front. Microbiol. 2017, 8, 2486. [Google Scholar] [CrossRef]
- Wang, Z.; Feng, J.; Jiang, Y.; Xu, X.; Xu, L.; Zhou, Q.; Huang, B. MrPEX33 is involved in infection-related morphogenesis and pathogenicity of Metarhizium robertsii. Appl. Microbiol. Biotech. 2021, 3, 1079–1090. [Google Scholar] [CrossRef]
- Wang, Y.; Xie, X.; Qin, L.; Yu, D.; Wang, Z.; Huang, B. Integration of dsRNA against host immune response genes augments the virulence of transgenic Metarhizium robertsii strains in insect pest species. Microb. Biotech. 2021, 14, 1433–1444. [Google Scholar] [CrossRef]
- Wang, Y.; Zhou, Q.; Zhang, H.; Qin, L.; Huang, B. Immunotranscriptome analysis of Plutella xylostella reveals differences in innate immune responses to low- and high-virulence Beauveria bassiana strain challenges. Pest Manag. Sci. 2021, 77, 1070–1080. [Google Scholar] [CrossRef]
- Mortazavi, A.; Williams, B.A.; McCue, K.; Schaeffer, L.; Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 2008, 5, 621–628. [Google Scholar] [CrossRef]
- Audic, S.p.; Claverie, J.M. The significance of digital gene expression profiles. Genome Res. 1997, 7, 986–995. [Google Scholar] [CrossRef]
- Bello, M.H.; Barrera-Perez, V.; Morin, D.; Epstein, L. The Neurospora crassa mutant NcDEgt-1 identifies an ergothioneine biosynthetic gene and demonstrates that ergothioneine enhances conidial survival and protects against peroxide toxicity during conidial germination. Fungal Genet. Biotech. 2012, 49, 160–172. [Google Scholar] [CrossRef]
- Baltussen, T.J.H.; Zoll, J.; Verweij, P.E.; Melchers, W.J.G. Molecular mechanisms of conidial germination in Aspergillus. Microbiol. Mol. Biol. Rev. 2020, 84, e00049-19. [Google Scholar] [CrossRef]
- Jin, D.; Sun, B.; Zhao, W.; Ma, J.; Zhou, Q.; Han, X.; Mei, Y.; Fan, Y.; Pei, Y. Thiamine-biosynthesis genes Bbpyr and Bbthi are required for conidial production and cell wall integrity of the entomopathogenic fungus Beauveria bassiana. J. Invertebr. Pathol. 2021, 184, 107639. [Google Scholar] [CrossRef]
- Zhang, E.; Cao, Y.; Xia, Y. Ethanol dehydrogenase I contributes to growth and sporulation under low oxygen condition via detoxification of acetaldehyde in Metarhizium acridum. Front. Microbiol. 2018, 9, 1932. [Google Scholar] [CrossRef]
- Hernandez, C.E.M.; Guerrero, I.E.P.; Hernandez, G.A.G.; Solis, E.S.; Guzman, J.C.T. Catalase overexpression reduces the germination time and increases the pathogenicity of the fungus Metarhizium anisopliae. Appl. Microbiol. Biotech. 2010, 87, 1033–1044. [Google Scholar] [CrossRef]
- Garault, P.; Letort, C.; Juillard, V.; Monnet, V. Branched-chain amino acid biosynthesis is essential for optimal growth of Streptococcus thermophilus in milk. Appl. Environ. Microbiol. 2000, 66, 5128–5133. [Google Scholar] [CrossRef] [Green Version]
- Paris, S.; Wysong, D.; Debeaupuis, J.-P.; Shibuya, K.; Philippe, B.; Diamond, R.D.; Latgé, J.-P. Catalases of Aspergillus fumigatus. Infect. Immun. 2003, 71, 3551–3562. [Google Scholar] [CrossRef] [Green Version]
Pathway ID | Pathway Name | Gene Number | |||
---|---|---|---|---|---|
CZA/WT | CZA + AA/CZA | CZA + Yeast/CZA | CZA + Yeast/CZA + AA | ||
ko01230 | Biosynthesis of amino acids | 116 | 95 | 106 | - |
ko00260 | Glycine, serine, and threonine metabolism | 91 | 76 | 82 | 54 |
ko01130 | Biosynthesis of antibiotics | 267 | 221 | 259 | - |
ko00970 | Aminoacyl-tRNA biosynthesis | 51 | 39 | 47 | - |
ko01210 | 2-Oxocarboxylic acid metabolism | 45 | 35 | - | - |
ko00220 | Arginine biosynthesis | 26 | 22 | 22 | - |
ko00360 | Phenylalanine metabolism | 62 | 60 | 56 | 44 |
ko00910 | Nitrogen metabolism | 28 | 24 | 26 | - |
ko00410 | beta-Alanine metabolism | 54 | 44 | - | - |
ko00650 | Butanoate metabolism | 36 | 29 | - | - |
ko00630 | Glyoxylate and dicarboxylate metabolism | 43 | - | - | - |
ko00920 | Sulfur metabolism | 24 | - | 51 | - |
ko00310 | Lysine degradation | 47 | - | 40 | - |
ko00350 | Tyrosine metabolism | 63 | 55 | - | |
ko00740 | Riboflavin metabolism | 25 | - | 27 | - |
ko00750 | Vitamin B6 metabolism | 13 | - | - | - |
ko00290 | Valine, leucine, and isoleucine biosynthesis | 19 | 17 | - | - |
ko00380 | Tryptophan metabolism | - | 63 | 67 | 48 |
ko00400 | Phenylalanine, tyrosine, and tryptophan biosynthesis | - | 22 | 23 | - |
ko00250 | Alanine, aspartate, and glutamate metabolism | - | 41 | 46 | - |
ko00340 | Histidine metabolism | - | 21 | - | - |
ko00254 | Aflatoxin biosynthesis | - | 21 | - | - |
ko00603 | Glycosphingolipid biosynthesis—globo and isoglobo series | - | 6 | - | - |
ko02010 | ABC transporters | - | 28 | - | - |
ko00561 | Glycerolipid metabolism | - | 31 | 37 | - |
ko00052 | Galactose metabolism | - | 22 | - | - |
ko00330 | Arginine and proline metabolism | - | 41 | - | - |
ko00460 | Cyanoamino acid metabolism | - | 27 | 36 | - |
ko00300 | Lysine biosynthesis | - | 14 | - | - |
ko00520 | Amino sugar and nucleotide sugar metabolism | - | - | - | 109 |
ko00072 | Synthesis and degradation of ketone bodies | - | - | 11 | - |
ko00450 | Selenocompound metabolism | - | - | 12 | - |
ko01200 | Carbon metabolism | - | - | 96 | - |
ko00240 | Pyrimidine metabolism | - | - | 31 | - |
Gene Name | FPKM | |||
---|---|---|---|---|
WT | CZA + Yeast | CZA + AA | CZA | |
hypothetical protein | 214.29 | 706.56 | 103.95 | 20.36 |
catalase A | 99.73 | 59.86 | 19.71 | 4.41 |
DNA-binding WRKY domain-containing protein | 46.66 | 55.6 | 21.36 | 5.12 |
bacterial-type extracellular deoxyribonuclease | 55.33 | 21.65 | 8.84 | 3.24 |
hypothetical protein MAA_10685 | 22.67 | 44.16 | 8.14 | 0.1 |
macrophomate synthase | 32.71 | 31.71 | 6.15 | 0.08 |
Cytochrome P450 CYP684F1 | 30.19 | 11.43 | 1.82 | 0.16 |
NAD(P)-binding domain protein | 8.44 | 30.33 | 3.3 | 0.17 |
hypothetical protein | 6.03 | 31.47 | 2.19 | 0.33 |
lanosterol synthase | 8.76 | 30.28 | 2.79 | 2.82 |
HypA | 21.51 | 12.53 | 6.18 | 0.34 |
D-isomer specific 2-hydroxyacid dehydrogenase | 15.77 | 8.56 | 0.82 | 0.24 |
methyltransferase | 16.66 | 11.94 | 4.87 | 0.92 |
SAM-dependent methyltransferase | 16.22 | 10.06 | 2.6 | 1.32 |
NAD(P)-binding domain protein | 10.49 | 18.05 | 4.5 | 1.94 |
hypothetical protein | 5.59 | 18.58 | 2.04 | 0.37 |
bacterial-type extracellular deoxyribonuclease | 12.56 | 14.15 | 5.36 | 2.05 |
membrane copper amine oxidase | 9.93 | 9.4 | 3.94 | 1.79 |
NADP-dependent alcohol dehydrogenase C | 6.71 | 7.22 | 2.98 | 0 |
Cytochrome P450 CYP68N3 | 1.6 | 2.4 | 0.21 | 0 |
Gene Name | FPKM | |||
---|---|---|---|---|
WT | CZA + Yeast | CZA + AA | CZA | |
sarcosine oxidase | 2856.28 | 3610.63 | 1698.84 | 765.77 |
tryptophan synthase beta subunit-like PLP-dependent enzyme | 220.33 | 216.52 | 115.84 | 5.72 |
hypothetical protein | 258 | 238.66 | 152.01 | 20.68 |
alcohol dehydrogenase superfamily, zinc-type | 265.12 | 239.15 | 182.46 | 42.76 |
catalase A | 99.73 | 59.86 | 19.71 | 4.41 |
macrophomate synthase | 32.71 | 31.71 | 6.15 | 0.08 |
hypothetical protein | 62.78 | 59.9 | 38.78 | 6.99 |
aldehyde dehydrogenase | 103.97 | 90.51 | 74.29 | 30.37 |
Chitinase | 72.67 | 31.01 | 15.75 | 0.3 |
bacterial-type extracellular deoxyribonuclease | 55.33 | 21.65 | 8.84 | 3.24 |
glutamate decarboxylase | 149.42 | 125.48 | 112.82 | 49.62 |
Amine oxidase | 38.32 | 37.46 | 25.08 | 3.05 |
hypothetical protein | 184.05 | 33.08 | 22.57 | 2.77 |
Cytochrome P450 CYP684F1 | 30.19 | 11.43 | 1.82 | 0.16 |
hypothetical protein | 31.27 | 19.17 | 10.99 | 0 |
hypothetical protein | 201.58 | 43.19 | 35.55 | 14 |
methyltransferase | 16.66 | 11.94 | 4.87 | 0.92 |
aldose 1-epimerase | 33.08 | 29.01 | 22.51 | 6.58 |
HypA | 21.51 | 12.53 | 6.18 | 0.34 |
membrane copper amine oxidase | 9.93 | 9.4 | 3.94 | 1.79 |
alpha/beta hydrolase fold-3 | 40.18 | 35.92 | 30.48 | 13.89 |
hypothetical protein | 18.13 | 6.68 | 3.1 | 0.36 |
cytochrome P450 CYP5321A1 | 54.37 | 34.38 | 32.75 | 1.61 |
peroxisomal copper amine oxidase | 2.92 | 2.07 | 1.96 | 0.9 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Wang, Y.; Liu, S.; Yin, X.; Yu, D.; Xie, X.; Huang, B. Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii. J. Fungi 2021, 7, 737. https://doi.org/10.3390/jof7090737
Wang Y, Liu S, Yin X, Yu D, Xie X, Huang B. Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii. Journal of Fungi. 2021; 7(9):737. https://doi.org/10.3390/jof7090737
Chicago/Turabian StyleWang, Yulong, Shihong Liu, Xuebing Yin, Deshui Yu, Xiangyun Xie, and Bo Huang. 2021. "Functional Analysis of Keto-Acid Reductoisomerase ILVC in the Entomopathogenic Fungus Metarhizium robertsii" Journal of Fungi 7, no. 9: 737. https://doi.org/10.3390/jof7090737