Systematic Characterization of bZIP Transcription Factors Required for Development and Aflatoxin Generation by High-Throughput Gene Knockout in Aspergillus flavus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Culture Conditions
2.2. Prediction of bZIPs in A. flavus
2.3. Generation of the Yeast–Escherichia–Agrobacterium Shuttle Vector pUM-GFP
2.4. High-Throughput Construction of the Gene Knockout Vector
2.5. Generation of the Knockout Mutants by ATMT
2.6. Identification of Gene-Deleted Mutants by Double Fluorescence
2.7. Verification of Gene-Deleted Mutants by PCR and Southern Blotting
2.8. Complementation of Null Mutants with Native Genes
2.9. RNA Isolation and Quantitative PCR
2.10. Fungal Growth, Conidial and Sclerotial Production
2.11. Abiotic Stress Conditions
2.12. Aflatoxin Analysis
2.13. Kernel Infection Assay
2.14. Statistical Analysis
3. Results
3.1. Identification of bZIP Transcription Factors in the Aspergillus flavus Genome
3.2. Strategy of the Double Fluorescence Knockout System in A. flavus
3.3. Construction of bZIP Deletion Mutants in A. flavus
3.4. Phenotypic Analyses of the bZIP Transcription Factor Deletion Mutants
3.5. bZIP Transcription Factors Involved in Fungal Growth
3.6. bZIP Transcription Factors Involved in Conidial Production
3.7. bZIP Transcription Factors Involved in Sclerotial Development
3.8. bZIP Transcription Factors Involved in Aflatoxin Production
3.9. bZIP Transcription Factors Related to Oxidative Stress
3.10. bZIP Transcription Factors Related to Cell Wall, Osmotic, Acid and Alkali Stress
3.11. bZIP Genes Required for Pathogenicity
3.12. Hapx Is Important for A. flavus to Adapt to an Excess of Iron
3.13. MetR and Methionine Biosynthesis Is Important for the Development of A. flavus
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Correction Statement
References
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Mutant | Growth | Conidiation | Sclerotia | Aflatoxin | Oxidative Stress | Cell Wall Stress | Osmotic Stress | Acid and Alkali Stress | Pathogenicity |
---|---|---|---|---|---|---|---|---|---|
ΔbZIP1 | Reduced 1 | Increased 2 | None | Reduced | Affected ↓ | No | No | No | Affected 6 |
ΔbZIP2 | Reduced 1 | Reduced 1 | Reduced | Reduced | Affected ↑ | No | No | No | Affected 6 |
ΔbZIP4 | Reduced 2 | Reduced 1 | None | Reduced | Affected ↓ | No | No | Affected 3 | Affected 5,6 |
ΔbZIP5 | Normal | Reduced 1 | Reduced | Reduced | No | No | No | No | No |
ΔbZIP6 | Normal | Normal | Reduced | Normal | No | Affected | No | No | Affected 5 |
ΔAP1 | Normal | Normal | Reduced | Normal | Affected ↓ | No | No | No | Affected 5 |
ΔAtfA | Reduced 2 | Reduced 1 | None | Reduced | Affected ↓ | Affected | No | No | Affected 6 |
ΔAtfB | Reduced 2 | Reduced 1 | Reduced | Reduced | Affected ↓ | No | No | No | No |
ΔCpcA | Reduced 2 | Reduced 1 | Reduced | Normal | No | Affected | No | Affected 4 | No |
ΔFcr3 | Normal | Reduced 1 | Reduced | Normal | No | No | No | No | Affected 5 |
ΔHapX | Normal | Reduced 1 | Increased | Reduced | Affected ↓ | No | Affected | No | No |
ΔJlbA | Reduced 2 | Reduced 1 | Reduced | Reduced | Affected ↑ | No | No | No | No |
ΔLziP | Normal | Reduced 1 | Increased | Normal | Affected ↓ | Affected | No | Affected 4 | Affected 5,6 |
ΔMeaB | Normal | Reduced 1,2 | None | Reduced | Affected ↓ | No | Affected | No | No |
ΔMetR | Reduced 1,2 | Reduced 1,2 | None | Reduced | Affected ↓ | Affected | Affected | Affected 4 | Affected 5,6 |
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Zhao, Q.; Pei, H.; Zhou, X.; Zhao, K.; Yu, M.; Han, G.; Fan, J.; Tao, F. Systematic Characterization of bZIP Transcription Factors Required for Development and Aflatoxin Generation by High-Throughput Gene Knockout in Aspergillus flavus. J. Fungi 2022, 8, 356. https://doi.org/10.3390/jof8040356
Zhao Q, Pei H, Zhou X, Zhao K, Yu M, Han G, Fan J, Tao F. Systematic Characterization of bZIP Transcription Factors Required for Development and Aflatoxin Generation by High-Throughput Gene Knockout in Aspergillus flavus. Journal of Fungi. 2022; 8(4):356. https://doi.org/10.3390/jof8040356
Chicago/Turabian StyleZhao, Qianqian, Hao Pei, Xiaoling Zhou, Kai Zhao, Min Yu, Guomin Han, Jun Fan, and Fang Tao. 2022. "Systematic Characterization of bZIP Transcription Factors Required for Development and Aflatoxin Generation by High-Throughput Gene Knockout in Aspergillus flavus" Journal of Fungi 8, no. 4: 356. https://doi.org/10.3390/jof8040356