Gene Mining for Conserved, Non-Annotated Proteins of Podosphaera xanthii Identifies Novel Target Candidates for Controlling Powdery Mildews by Spray-Induced Gene Silencing
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plants, Microbes and Culture Conditions
2.2. Sequence Analysis, Protein Modeling and Protein Function Prediction
2.3. Isolation of Nucleic Acids and cDNA Synthesis
2.4. In Vitro Production of dsRNA
2.5. dsRNA-Mediated Gene Silencing
2.5.1. Leaf Disc Assay
2.5.2. Cotyledon Infiltration Assay
2.6. Quantitative Reverse Transcription RT-qPCR and qPCR
2.7. Haustorial Counts
2.8. SIGS Assay
2.9. Statistical Analysis
3. Results
3.1. Identification and Selection of CNAPs
3.2. Development of a dsRNA-Mediated Gene-Silencing Assay for P. xanthii
3.3. CNAP948, CNAP1048, CNAP8878, CNAP9066, CNAP10905 and CNAP30520 Are Essential Proteins for P. xanthii
3.4. Efficient Control of Cucurbit Powdery Mildew by SIGS
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sequence ID | Protein Name | Protein Length 1 | Subject ID | Description | E-Value | Identity |
---|---|---|---|---|---|---|
Pxanthii_948 | CNAP948 | 708 | XP_007290516 | Hypothetical protein Marssonina brunnea f. sp. multigermtubi MB_m1 | 0.0 | 80% (571/711) |
Pxanthii_1048 | CNAP1048 | 421 | KAE8449429 | Hypothetical protein Helotiales sp. DMI_Dod_QoI | 0.0 | 81% (342/421) |
Pxanthii_8878 | CNAP8878 | 103 | RKF57155 | Hypothetical protein Oidium neolycopersici | 7 × 10−58 | 81% (83/103) |
Pxanthii_9066 | CNAP9066 | 122 | CCU82200 | Hypothetical protein Blumeria graminis f. sp. hordei DH14 | 1 × 10−59 | 75% (92/122) |
Pxanthii_10905 | CNAP10905 | 74 | ESZ98542 | Hypothetical protein Sclerotinia borealis F-4128 | 8 × 10−31 | 77% (51/66) |
Pxanthii_30520 | CNAP30520 | 80 | TVY46286 | Hypothetical protein Lachnellula occidentalis | 3 × 10−47 | 88% (70/80) |
Score Values 1 | Structural Analogs 2 | Predicted Features 3 | |||||
---|---|---|---|---|---|---|---|
Protein Model | C Score | TM Score | PDB Code | Species | Activity | Ligands | Putative Function 4 |
CNAP948 | −0.63 | 0.781 | 6TQE | Mycobacterium tuberculosis | Efflux transporter | ATP | Efflux transport |
CNAP1048 | −0.82 | 0.873 | 6SNH | Saccharomyces cerevisiae | Glucosyltransferase | Glucose | Glycosylation |
CNAP8878 | −1.22 | 0.689 | 6RFQ | Yarrowia lipolytica | Oxidoreductase | Iron-sulfur | Respiration |
CNAP9066 | −1.49 | 0.710 | 1XME | Thermus thermophilus | Oxidoreductase | Copper | Respiration |
CNAP10905 | 0.37 | 0.624 | 5LQX | Pichia angusta | ATP synthase | ADP | Respiration |
CNAP30520 | 0.80 | 0.839 | 6Y79 | Yarrowia lipolytica | Oxidoreductase | Iron-sulfur | Respiration |
Treatment | Dose of dsRNA (µg mL−1) | Leaf Surface Covered by Powdery Mildew (%) 1 | Disease Reduction (%) 2 |
---|---|---|---|
Water | - | 59.35 ± 3.78 a | - |
Empty vector-dsRNA | 30 | 55.60 ± 4.75 a | 6.32 |
CNAP1048-dsRNA | 30 | 5.62 ± 1.01 ef | 90.53 |
20 | 5.25 ± 0.72 ef | 91.15 | |
10 | 10.04 ± 0.58 cde | 83.08 | |
5 | 21.26 ± 1.28 b | 64.18 | |
CNAP30520-dsRNA | 30 | 3.92 ± 0.62 f | 93.40 |
20 | 3.74 ± 0.46 f | 93.70 | |
10 | 9.11 ± 0.78 cdef | 84.65 | |
5 | 14.83 ± 1.26 c | 75.01 | |
CNAP10905-dsRNA | 30 | 3.41 ± 0.28 f | 94.25 |
20 | 3.53 ± 0.26 f | 94.05 | |
10 | 8.24 ± 1.51 def | 86.12 | |
5 | 12.96 ± 1.49 cd | 78.16 | |
PxTUB2-dsRNA | 30 | 3.86 ± 0.23 f | 93.50 |
20 | 4.51 ± 0.45 ef | 92.40 | |
10 | 11.94 ± 1.89 cd | 79.88 | |
5 | 22.83 ± 0.60 b | 61.53 |
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Ruiz-Jiménez, L.; Polonio, Á.; Vielba-Fernández, A.; Pérez-García, A.; Fernández-Ortuño, D. Gene Mining for Conserved, Non-Annotated Proteins of Podosphaera xanthii Identifies Novel Target Candidates for Controlling Powdery Mildews by Spray-Induced Gene Silencing. J. Fungi 2021, 7, 735. https://doi.org/10.3390/jof7090735
Ruiz-Jiménez L, Polonio Á, Vielba-Fernández A, Pérez-García A, Fernández-Ortuño D. Gene Mining for Conserved, Non-Annotated Proteins of Podosphaera xanthii Identifies Novel Target Candidates for Controlling Powdery Mildews by Spray-Induced Gene Silencing. Journal of Fungi. 2021; 7(9):735. https://doi.org/10.3390/jof7090735
Chicago/Turabian StyleRuiz-Jiménez, Laura, Álvaro Polonio, Alejandra Vielba-Fernández, Alejandro Pérez-García, and Dolores Fernández-Ortuño. 2021. "Gene Mining for Conserved, Non-Annotated Proteins of Podosphaera xanthii Identifies Novel Target Candidates for Controlling Powdery Mildews by Spray-Induced Gene Silencing" Journal of Fungi 7, no. 9: 735. https://doi.org/10.3390/jof7090735