CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew
Abstract Background The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene P owdery M ildew R esistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery milde...
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| Format: | Article |
| Language: | English |
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BMC
2020-06-01
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| Series: | BMC Plant Biology |
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| Online Access: | http://link.springer.com/article/10.1186/s12870-020-02497-y |
| _version_ | 1828870163322109952 |
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| author | Miguel I. Santillán Martínez Valentina Bracuto Eleni Koseoglou Michela Appiano Evert Jacobsen Richard G. F. Visser Anne-Marie A. Wolters Yuling Bai |
| author_facet | Miguel I. Santillán Martínez Valentina Bracuto Eleni Koseoglou Michela Appiano Evert Jacobsen Richard G. F. Visser Anne-Marie A. Wolters Yuling Bai |
| author_sort | Miguel I. Santillán Martínez |
| collection | DOAJ |
| description | Abstract Background The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene P owdery M ildew R esistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On). Results To study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants. Conclusion CRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations. |
| first_indexed | 2024-12-13T06:05:11Z |
| format | Article |
| id | doaj.art-251910cf83fb4caabf4ccf37d2452ba5 |
| institution | Directory Open Access Journal |
| issn | 1471-2229 |
| language | English |
| last_indexed | 2024-12-13T06:05:11Z |
| publishDate | 2020-06-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Plant Biology |
| spelling | doaj.art-251910cf83fb4caabf4ccf37d2452ba52022-12-21T23:57:14ZengBMCBMC Plant Biology1471-22292020-06-0120111310.1186/s12870-020-02497-yCRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildewMiguel I. Santillán Martínez0Valentina Bracuto1Eleni Koseoglou2Michela Appiano3Evert Jacobsen4Richard G. F. Visser5Anne-Marie A. Wolters6Yuling Bai7Plant Breeding, Wageningen University & ResearchPlant Breeding, Wageningen University & ResearchPlant Breeding, Wageningen University & ResearchPlant Breeding, Wageningen University & ResearchPlant Breeding, Wageningen University & ResearchPlant Breeding, Wageningen University & ResearchPlant Breeding, Wageningen University & ResearchPlant Breeding, Wageningen University & ResearchAbstract Background The development of CRISPR/Cas9 technology has facilitated targeted mutagenesis in an efficient and precise way. Previously, RNAi silencing of the susceptibility (S) gene P owdery M ildew R esistance 4 (PMR4) in tomato has been shown to enhance resistance against the powdery mildew pathogen Oidium neolycopersici (On). Results To study whether full knock-out of the tomato PMR4 gene would result in a higher level of resistance than in the RNAi-silenced transgenic plants we generated tomato PMR4 CRISPR mutants. We used a CRISPR/Cas9 construct containing four single-guide RNAs (sgRNAs) targeting the tomato PMR4 gene to increase the possibility of large deletions in the mutants. After PCR-based selection and sequencing of transformants, we identified five different mutation events, including deletions from 4 to 900-bp, a 1-bp insertion and a 892-bp inversion. These mutants all showed reduced susceptibility to On based on visual scoring of disease symptoms and quantification of relative fungal biomass. Histological observations revealed a significantly higher occurrence of hypersensitive response-like cell death at sites of fungal infection in the pmr4 mutants compared to wild-type plants. Both haustorial formation and hyphal growth were diminished but not completely inhibited in the mutants. Conclusion CRISPR/Cas-9 targeted mutagenesis of the tomato PMR4 gene resulted in mutants with reduced but not complete loss of susceptibility to the PM pathogen On. Our study demonstrates the efficiency and versatility of the CRISPR/Cas9 system as a powerful tool to study and characterize S-genes by generating different types of mutations.http://link.springer.com/article/10.1186/s12870-020-02497-yCRISPR/Cas9Targeted mutagenesisPMR4Powdery mildewSusceptibility gene |
| spellingShingle | Miguel I. Santillán Martínez Valentina Bracuto Eleni Koseoglou Michela Appiano Evert Jacobsen Richard G. F. Visser Anne-Marie A. Wolters Yuling Bai CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew BMC Plant Biology CRISPR/Cas9 Targeted mutagenesis PMR4 Powdery mildew Susceptibility gene |
| title | CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew |
| title_full | CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew |
| title_fullStr | CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew |
| title_full_unstemmed | CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew |
| title_short | CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew |
| title_sort | crispr cas9 targeted mutagenesis of the tomato susceptibility gene pmr4 for resistance against powdery mildew |
| topic | CRISPR/Cas9 Targeted mutagenesis PMR4 Powdery mildew Susceptibility gene |
| url | http://link.springer.com/article/10.1186/s12870-020-02497-y |
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