Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line
Abstract
:1. Introduction
2. Materials and Methods
2.1. Material Used and Experimental Conditions
2.2. Construction of Vector and Rice Transformation
2.3. Genotyping, Screening of Transgene-free Plants, and Off-target Assessment
2.4. Pathogen Inoculation and Disease Scoring
2.5. Phenotyping of Mutant Lines
2.6. Protein Extraction and Digestion
2.7. iTRAQ Labeling and Mass Spectrophotometry Analysis
2.8. Data Analysis
2.9. RT–qPCR-Based Validation of Proteomic Data
2.10. Statistical Analysis
3. Results
3.1. Construction of Vector
3.2. Mutation Frequency, Off-Target Effects, and Segregation of Mutants
3.3. T-DNA-Free Plants in T1 Generation
3.4. Resistance to Pathogen in Pi21 Mutants
3.5. Agronomic Performance Assessment of Wild Type and Mutant Lines
3.6. Proteins Extraction and Quantification
3.7. Functional Networks of DEPs
3.8. Gene Ontology (GO) Annotation of DEPs
3.9. Pathway Enrichment Analysis
3.10. Identification of Hub-Proteins
3.11. RT-qPCR-Based Analysis of CRISPR Mutants
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Mutation Type | |||||||
---|---|---|---|---|---|---|---|
Targets | Bi-Allelic | Homozygous | Heterozygous | Chimeric | WT | Total | |
Pi21T1 | NOP | 7 | 6 | 2 | 2 | 8 | 25 |
MR | 28.00 | 24.00 | 8.00 | 8.00 | 68.00 | ||
Pi21T2 | NOP | 6 | 5 | 4 | 1 | 9 | 25 |
MR | 24.00 | 20.00 | 16.00 | 4.00 | 64.00 | ||
Mean MR | 66.00% |
Generation | Line | PH (cm) | FLL (cm) | FLW (cm) | PN | PL (cm) | GNPP | SSR (%) | GWT (g) |
---|---|---|---|---|---|---|---|---|---|
WT | 105.8 ± 4.3 | 37.36 ± 1.4 | 1.63 ± 0.2 | 10.81 ± 0.7 | 21.36 ± 1.8 | 155 ± 12 | 82.12 ± 5.5 | 29.34 ± 1.1 | |
GN-3 | 102.5 ± 2.3 ns | 36.55 ± 1.6 ns | 1.51 ± 0.3 ns | 10.44 ± 0.4 ns | 21.84 ± 1.9 ns | 158 ± 10 ns | 83.10 ± 4.1 ns | 28.63 ± 1.6 ns | |
T1 | GN-5 | 106.6 ± 3.4 ns | 35.23 ± 1.5 ns | 1.67 ± 0.2 ns | 9.98 ± 0.6 ns | 20.70 ± 2.2 ns | 159 ± 11 ns | 85.33 ± 4.5 ns | 29.93 ± 1.1 ns |
GN-9 | 107.4 ± 3.8 ns | 35.88 ± 1.3 ns | 1.64 ± 0.3 ns | 10.12 ± 0.5 ns | 21.30 ± 1.7 ns | 157 ± 12 ns | 82.03 ± 3.7 ns | 28.43 ± 1.5 ns | |
GN-13 | 108.1 ± 3.6 ns | 36.28 ± 1.6 ns | 1.59 ± 0.4 ns | 10.29 ± 0.3 ns | 21.88 ± 1.5 ns | 156 ± 13 ns | 83.44 ± 4.3 ns | 28.90 ± 1.2 ns | |
WT | 103.5 ± 3.9 | 38.16 ± 1.4 | 1.70 ± 0.3 | 9.94 ± 0.9 | 20.75 ± 1.6 | 157 ± 11 | 84.52 ± 3.9 | 28.75 ± 1.2 | |
GN-3 | 105.2 ± 2.3 ns | 37.21 ± 1.3 ns | 1.62 ± 0.1 ns | 9.70 ± 0.4 ns | 20.99 ± 1.7 ns | 159 ± 10 ns | 84.30 ± 4.1 ns | 29.21 ± 1.5 ns | |
T2 | GN-5 | 104.8 ± 3.6 ns | 34.29 ± 1.9 ns | 1.56 ± 0.2 ns | 9.50 ± 0.6 ns | 20.26 ± 1.9 ns | 158 ± 12 ns | 85.80 ± 3.6 ns | 28.27 ± 1.2 ns |
GN-9 | 108.6 ± 3.8 ns | 35.12 ± 1.3 ns | 1.59 ± 0.3 ns | 9.75 ± 0.5 ns | 21.60 ± 2.1 ns | 156 ± 13 ns | 83.83 ± 4.7 ns | 29.34 ± 1.3 ns | |
GN-13 | 103.3 ± 2.7 ns | 35.50 ± 1.6 ns | 1.54 ± 0.1 ns | 10.50 ± 0.7 ns | 21.40 ± 2.8 ns | 158 ± 12 ns | 82.85 ± 4.9 ns | 28.72 ± 1.4 ns | |
WT | 107.5 ± 4.1 | 36.46 ± 1.3 | 1.58 ± 0.2 | 10.21 ± 0.8 | 21.15 ± 1.5 | 156 ± 13 | 83.12 ± 3.8 | 29.60 ± 1.4 | |
GN-3 | 101.4 ± 2.5 ns | 36.12 ± 1.6 ns | 1.69 ± 0.3 ns | 10.17 ± 0.3 ns | 20.41 ± 1.4 ns | 158 ± 11 ns | 83.65 ± 3.3 ns | 28.93 ± 1.3 ns | |
T3 | GN-5 | 105.9 ± 3.7 ns | 35.91 ± 1.5 ns | 1.62 ± 0.3 ns | 9.72 ± 0.5 ns | 20.55 ± 2.1 ns | 157 ± 12 ns | 85.00 ± 4.4 ns | 29.95 ± 1.2 ns |
GN-9 | 104.6 ± 3.2 ns | 36.10 ± 1.4 ns | 1.67 ± 0.3 ns | 9.60 ± 0.6 ns | 20.95 ± 2.3 ns | 155 ± 14 ns | 85.52 ± 3.5 ns | 28.75 ± 1.5 ns | |
GN-13 | 106.7 ± 2.8 ns | 35.85 ± 1.7 ns | 1.60 ± 0.2 ns | 10.21 ± 0.7 ns | 21.75 ± 2.4 ns | 157 ± 12 ns | 84.28 ± 5.3 ns | 29.29 ± 1.2 ns |
Protein ID | Gene | Annotation | Regulation |
---|---|---|---|
Q6Z9C3 | Os08g0547100 | Probable 6-phosphogluconolactonase 3, chloroplastic | Up |
Q5KQI6 | SNAT1 | Serotonin N-acetyltransferase 1, chloroplastic | Up |
Q69PS6 | OsNTRA | Thioredoxin reductase NTRA | Up |
Q0IZQ2 | Os09g0556500 | Cysteine—tRNA ligase CPS1 homolog, chloroplastic | Up |
Q6ATB2 | CRSH2 | Probable GTP diphosphokinase CRSH2, chloroplastic | Up |
Q7XTK3 | AMI1 | Amidase 1 | Up |
Q6K8R2 | Cht6 | Pathogenesis related (PR)-3 chitinase 6 | Up |
Q67WM9 | OS9 | Protein OS-9 homolog | Up |
Q75T45 | RSOsPR10 | Os12g0555000 protein | Up |
Q10HD0 | RCABP89 | Chlorophyll a-b binding protein, chloroplastic | Up |
Q6ESR4 | DHN1 | Dehydration stress-inducible protein 1 | Up |
Q6K4P5 | Os02g0787800 | diacylglycerol kinase | Up |
Q84ZE8 | OsJ_24279 | Auxin-regulated protein-like | Up |
A0A0P0XMI4 | Os09g0446200 | γ-tubulin complex component | Up |
Q2R351 | LOC_Os11g33120 | Respiratory burst oxidase protein D | Up |
A3BUY8 | OsJ_27981 | PEROXIDASE_4 domain-containing protein | Up |
A0A0P0VAH1 | Os01g0849000 | Os01g0849000 protein | Up |
Q75IS1 | Os05g0162000 | Peroxidase | Up |
Q5U1N1 | prx62 | Peroxidase | Up |
Q8HCQ0 | nad3 | NADH-ubiquinone oxidoreductase chain 3 | Up |
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Nawaz, G.; Usman, B.; Peng, H.; Zhao, N.; Yuan, R.; Liu, Y.; Li, R. Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line. Genes 2020, 11, 735. https://doi.org/10.3390/genes11070735
Nawaz G, Usman B, Peng H, Zhao N, Yuan R, Liu Y, Li R. Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line. Genes. 2020; 11(7):735. https://doi.org/10.3390/genes11070735
Chicago/Turabian StyleNawaz, Gul, Babar Usman, Haowen Peng, Neng Zhao, Ruizhi Yuan, Yaoguang Liu, and Rongbai Li. 2020. "Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line" Genes 11, no. 7: 735. https://doi.org/10.3390/genes11070735
APA StyleNawaz, G., Usman, B., Peng, H., Zhao, N., Yuan, R., Liu, Y., & Li, R. (2020). Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line. Genes, 11(7), 735. https://doi.org/10.3390/genes11070735