The Functional Association of <i>ACQOS/VICTR</i> with Salt Stress Resistance in <i>Arabidopsis thaliana</i> Was Confirmed by CRISPR-Mediated Mutagenesis
Clustered regularly interspaced palindromic repeat (CRISPR)-mediated mutagenesis has become an important tool in plant research, enabling the characterization of genes via gene knock-out. CRISPR genome editing tools can be applied to generate multi-gene knockout lines. Typically, multiple single-str...
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| Autores principales: | , , , |
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| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
MDPI AG
2021
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/2bbdd92e8b604df882798025a1a15334 |
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| Sumario: | Clustered regularly interspaced palindromic repeat (CRISPR)-mediated mutagenesis has become an important tool in plant research, enabling the characterization of genes via gene knock-out. CRISPR genome editing tools can be applied to generate multi-gene knockout lines. Typically, multiple single-stranded, single guide RNAs (gRNAs) must be expressed in an organism to target multiple genes simultaneously; however, a single gRNA can target multiple genes if the target genes share similar sequences. A gene cluster comprising ACQUIRED OSMOTOLERANCE (<i>ACQOS</i>; <i>AT5G46520</i>) and neighboring nucleotide-binding leucine-rich repeats (<i>NLR</i>s; <i>AT5G46510</i>) is associated with osmotic tolerance. To investigate the role of <i>ACQOS</i> and the tandemly arranged NLR in osmotic tolerance, we introduced small insertion/deletion mutations into two target genes using a single gRNA and obtained transformant plant lines with three different combinations of mutant alleles. We then tested our mutant lines for osmotic tolerance after a salt-stress acclimation period by determining the chlorophyll contents of the mutant seedlings. Our results strongly suggest that <i>ACQOS</i> is directly associated with salt resistance, while the neighboring NLR is not. Here, we confirmed previous findings suggesting the involvement of <i>ACQOS</i> in salt tolerance and demonstrated the usefulness of CRISPR-mediated mutagenesis in validating the functions of genes in a single genetic background. |
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