CRISPR/Cas9-Mediated Generation of Mutant Lines in <i>Medicago truncatula</i> Indicates a Symbiotic Role of <i>MtLYK10</i> during Nodule Formation
CRISPR/Cas9 systems are commonly used for plant genome editing; however, the generation of homozygous mutant lines in <i>Medicago truncatula</i> remains challenging. Here, we present a CRISPR/Cas9-based protocol that allows the efficient generation of <i>M. truncatula</i> mut...
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MDPI AG
2024-01-01
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| author | Chun-Xiao Zhang Ru-Jie Li Laura Baude Didier Reinhardt Zhi-Ping Xie Christian Staehelin |
| author_facet | Chun-Xiao Zhang Ru-Jie Li Laura Baude Didier Reinhardt Zhi-Ping Xie Christian Staehelin |
| author_sort | Chun-Xiao Zhang |
| collection | DOAJ |
| description | CRISPR/Cas9 systems are commonly used for plant genome editing; however, the generation of homozygous mutant lines in <i>Medicago truncatula</i> remains challenging. Here, we present a CRISPR/Cas9-based protocol that allows the efficient generation of <i>M. truncatula</i> mutants. Gene editing was performed for the LysM receptor kinase gene <i>MtLYK10</i> and two major facilitator superfamily transporter genes. The functionality of CRISPR/Cas9 vectors was tested in <i>Nicotiana benthamiana</i> leaves by editing a co-transformed <i>GUSPlus</i> gene. Transformed <i>M. truncatula</i> leaf explants were regenerated to whole plants at high efficiency (80%). An editing efficiency (frequency of mutations at a given target site) of up to 70% was reached in the regenerated plants. Plants with <i>MtLYK10</i> knockout mutations were propagated, and three independent homozygous mutant lines were further characterized. No off-target mutations were identified in these <i>lyk10</i> mutants. Finally, the <i>lyk10</i> mutants and wild-type plants were compared with respect to the formation of root nodules induced by nitrogen-fixing <i>Sinorhizobium meliloti</i> bacteria. Nodule formation was considerably delayed in the three <i>lyk10</i> mutant lines. Surprisingly, the size of the rare nodules in mutant plants was higher than in wild-type plants. In conclusion, the symbiotic characterization of <i>lyk10</i> mutants generated with the developed CRISPR/Cas9 protocol indicated a role of <i>MtLYK10</i> in nodule formation. |
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| last_indexed | 2024-03-08T11:04:50Z |
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| spelling | doaj.art-e5a48f68e817449dbbdd2bfa1049575a2024-01-26T15:07:14ZengMDPI AGBiology2079-77372024-01-011315310.3390/biology13010053CRISPR/Cas9-Mediated Generation of Mutant Lines in <i>Medicago truncatula</i> Indicates a Symbiotic Role of <i>MtLYK10</i> during Nodule FormationChun-Xiao Zhang0Ru-Jie Li1Laura Baude2Didier Reinhardt3Zhi-Ping Xie4Christian Staehelin5State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, ChinaState Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, ChinaDepartment of Biology, University of Fribourg, 1700 Fribourg, SwitzerlandDepartment of Biology, University of Fribourg, 1700 Fribourg, SwitzerlandState Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, ChinaState Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, ChinaCRISPR/Cas9 systems are commonly used for plant genome editing; however, the generation of homozygous mutant lines in <i>Medicago truncatula</i> remains challenging. Here, we present a CRISPR/Cas9-based protocol that allows the efficient generation of <i>M. truncatula</i> mutants. Gene editing was performed for the LysM receptor kinase gene <i>MtLYK10</i> and two major facilitator superfamily transporter genes. The functionality of CRISPR/Cas9 vectors was tested in <i>Nicotiana benthamiana</i> leaves by editing a co-transformed <i>GUSPlus</i> gene. Transformed <i>M. truncatula</i> leaf explants were regenerated to whole plants at high efficiency (80%). An editing efficiency (frequency of mutations at a given target site) of up to 70% was reached in the regenerated plants. Plants with <i>MtLYK10</i> knockout mutations were propagated, and three independent homozygous mutant lines were further characterized. No off-target mutations were identified in these <i>lyk10</i> mutants. Finally, the <i>lyk10</i> mutants and wild-type plants were compared with respect to the formation of root nodules induced by nitrogen-fixing <i>Sinorhizobium meliloti</i> bacteria. Nodule formation was considerably delayed in the three <i>lyk10</i> mutant lines. Surprisingly, the size of the rare nodules in mutant plants was higher than in wild-type plants. In conclusion, the symbiotic characterization of <i>lyk10</i> mutants generated with the developed CRISPR/Cas9 protocol indicated a role of <i>MtLYK10</i> in nodule formation.https://www.mdpi.com/2079-7737/13/1/53genome editingLysM receptor kinaseplant tissue culturesymbiosis |
| spellingShingle | Chun-Xiao Zhang Ru-Jie Li Laura Baude Didier Reinhardt Zhi-Ping Xie Christian Staehelin CRISPR/Cas9-Mediated Generation of Mutant Lines in <i>Medicago truncatula</i> Indicates a Symbiotic Role of <i>MtLYK10</i> during Nodule Formation Biology genome editing LysM receptor kinase plant tissue culture symbiosis |
| title | CRISPR/Cas9-Mediated Generation of Mutant Lines in <i>Medicago truncatula</i> Indicates a Symbiotic Role of <i>MtLYK10</i> during Nodule Formation |
| title_full | CRISPR/Cas9-Mediated Generation of Mutant Lines in <i>Medicago truncatula</i> Indicates a Symbiotic Role of <i>MtLYK10</i> during Nodule Formation |
| title_fullStr | CRISPR/Cas9-Mediated Generation of Mutant Lines in <i>Medicago truncatula</i> Indicates a Symbiotic Role of <i>MtLYK10</i> during Nodule Formation |
| title_full_unstemmed | CRISPR/Cas9-Mediated Generation of Mutant Lines in <i>Medicago truncatula</i> Indicates a Symbiotic Role of <i>MtLYK10</i> during Nodule Formation |
| title_short | CRISPR/Cas9-Mediated Generation of Mutant Lines in <i>Medicago truncatula</i> Indicates a Symbiotic Role of <i>MtLYK10</i> during Nodule Formation |
| title_sort | crispr cas9 mediated generation of mutant lines in i medicago truncatula i indicates a symbiotic role of i mtlyk10 i during nodule formation |
| topic | genome editing LysM receptor kinase plant tissue culture symbiosis |
| url | https://www.mdpi.com/2079-7737/13/1/53 |
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