부산대학교 도서관

Simple Summary: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has become an important research tool for targeted mutagenesis in plants. However, the construction of mutants in the model legume Medicago truncatula remains challenging as it requires the combination of an efficient CRISPR/Cas9-mediated gene editing system for explants and specific tissue culture techniques for regeneration to whole plants. Here, we describe the construction of M. truncatula mutants using the Agrobacterium tumefaciens-mediated transformation of leaf explants. Regeneration to whole plants was performed at high efficiency (80%), and a gene editing efficiency of up to 70% was reached in the obtained plants. CRISPR/Cas9-mediated gene editing was achieved for three M. truncatula genes. Three mutant lines with knockout mutations in the LysM receptor kinase gene MtLYK10 were further characterized. Inoculation experiments with nitrogen-fixing Sinorhizobium meliloti bacteria indicated that root nodule formation was impaired in the constructed MtLYK10 mutants. In conclusion, we show that the described CRISPR/Cas9 system is suitable for efficient gene editing in M. truncatula and that MtLYK10 plays a role in nodule symbiosis. CRISPR/Cas9 systems are commonly used for plant genome editing; however, the generation of homozygous mutant lines in Medicago truncatula remains challenging. Here, we present a CRISPR/Cas9-based protocol that allows the efficient generation of M. truncatula mutants. Gene editing was performed for the LysM receptor kinase gene MtLYK10 and two major facilitator superfamily transporter genes. The functionality of CRISPR/Cas9 vectors was tested in Nicotiana benthamiana leaves by editing a co-transformed GUSPlus gene. Transformed M. truncatula 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 MtLYK10 knockout mutations were propagated, and three independent homozygous mutant lines were further characterized. No off-target mutations were identified in these lyk10 mutants. Finally, the lyk10 mutants and wild-type plants were compared with respect to the formation of root nodules induced by nitrogen-fixing Sinorhizobium meliloti bacteria. Nodule formation was considerably delayed in the three lyk10 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 lyk10 mutants generated with the developed CRISPR/Cas9 protocol indicated a role of MtLYK10 in nodule formation. [ABSTRACT FROM AUTHOR]