CRISPR/Cas9-Mediated Generation of Mutant Lines in Indicates a Symbiotic Role of during Nodule Formation.

CRISPR/Cas9 systems are commonly used for plant genome editing; however, the generation of homozygous mutant lines in remains challenging. Here, we present a CRISPR/Cas9-based protocol that allows the efficient generation of mutants. Gene editing was performed for the LysM receptor kinase gene and two major facilitator superfamily transporter genes. The functionality of CRISPR/Cas9 vectors was tested in leaves by editing a co-transformed gene. Transformed 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 knockout mutations were propagated, and three independent homozygous mutant lines were further characterized. No off-target mutations were identified in these mutants. Finally, the mutants and wild-type plants were compared with respect to the formation of root nodules induced by nitrogen-fixing bacteria. Nodule formation was considerably delayed in the three 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 mutants generated with the developed CRISPR/Cas9 protocol indicated a role of in nodule formation.