CRISPR/Cas9-Mediated Knock-Out of the <i>MtCLE35</i> Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions
Legume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume–rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nit...
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MDPI AG
2023-11-01
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| Series: | International Journal of Molecular Sciences |
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| author | Maria A. Lebedeva Daria A. Dobychkina Lyudmila A. Lutova |
| author_facet | Maria A. Lebedeva Daria A. Dobychkina Lyudmila A. Lutova |
| author_sort | Maria A. Lebedeva |
| collection | DOAJ |
| description | Legume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume–rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate have been shown to control the number of symbiotic nodules. Previously, the <i>MtCLE35</i> gene was found to be upregulated by rhizobia and nitrate treatment in <i>Medicago truncatula</i>, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the <i>MtCLE35</i> gene was mutated using the CRISPR/Cas9-mediated system. <i>M. truncatula</i> lines with the <i>MtCLE35</i> gene knocked out produced increased numbers of nodules in the presence of nitrate in comparison to wild-type plants. Moreover, in the presence of nitrate, the expression levels of two other nodulation-related <i>MtCLE</i> genes, <i>MtCLE12</i> and <i>MtCLE13</i>, were reduced in rhizobia-inoculated roots, whereas no significant difference in <i>MtCLE35</i> gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of <i>MtCLE35</i> in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related <i>MtCLE</i> genes are reduced. |
| first_indexed | 2024-03-09T01:49:43Z |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T01:49:43Z |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-44c5447dc7c84048b41c2216b1c34c612023-12-08T15:17:15ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-11-0124231681610.3390/ijms242316816CRISPR/Cas9-Mediated Knock-Out of the <i>MtCLE35</i> Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate ConditionsMaria A. Lebedeva0Daria A. Dobychkina1Lyudmila A. Lutova2Department of Genetics and Biotechnology, Saint Petersburg State University, Universitetskaya Emb. 7/9, 199034 Saint Petersburg, RussiaDepartment of Genetics and Biotechnology, Saint Petersburg State University, Universitetskaya Emb. 7/9, 199034 Saint Petersburg, RussiaDepartment of Genetics and Biotechnology, Saint Petersburg State University, Universitetskaya Emb. 7/9, 199034 Saint Petersburg, RussiaLegume plants have the ability to establish a symbiotic relationship with soil bacteria known as rhizobia. The legume–rhizobium symbiosis results in the formation of symbiotic root nodules, where rhizobia fix atmospheric nitrogen. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate have been shown to control the number of symbiotic nodules. Previously, the <i>MtCLE35</i> gene was found to be upregulated by rhizobia and nitrate treatment in <i>Medicago truncatula</i>, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the <i>MtCLE35</i> gene was mutated using the CRISPR/Cas9-mediated system. <i>M. truncatula</i> lines with the <i>MtCLE35</i> gene knocked out produced increased numbers of nodules in the presence of nitrate in comparison to wild-type plants. Moreover, in the presence of nitrate, the expression levels of two other nodulation-related <i>MtCLE</i> genes, <i>MtCLE12</i> and <i>MtCLE13</i>, were reduced in rhizobia-inoculated roots, whereas no significant difference in <i>MtCLE35</i> gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of <i>MtCLE35</i> in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related <i>MtCLE</i> genes are reduced.https://www.mdpi.com/1422-0067/24/23/16816autoregulation of nodulation (AON)CLE peptidesrhizobianodulationnitrate |
| spellingShingle | Maria A. Lebedeva Daria A. Dobychkina Lyudmila A. Lutova CRISPR/Cas9-Mediated Knock-Out of the <i>MtCLE35</i> Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions International Journal of Molecular Sciences autoregulation of nodulation (AON) CLE peptides rhizobia nodulation nitrate |
| title | CRISPR/Cas9-Mediated Knock-Out of the <i>MtCLE35</i> Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions |
| title_full | CRISPR/Cas9-Mediated Knock-Out of the <i>MtCLE35</i> Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions |
| title_fullStr | CRISPR/Cas9-Mediated Knock-Out of the <i>MtCLE35</i> Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions |
| title_full_unstemmed | CRISPR/Cas9-Mediated Knock-Out of the <i>MtCLE35</i> Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions |
| title_short | CRISPR/Cas9-Mediated Knock-Out of the <i>MtCLE35</i> Gene Highlights Its Key Role in the Control of Symbiotic Nodule Numbers under High-Nitrate Conditions |
| title_sort | crispr cas9 mediated knock out of the i mtcle35 i gene highlights its key role in the control of symbiotic nodule numbers under high nitrate conditions |
| topic | autoregulation of nodulation (AON) CLE peptides rhizobia nodulation nitrate |
| url | https://www.mdpi.com/1422-0067/24/23/16816 |
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