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.

Glycoside hydrolase family 18 (GH18) proteins can hydrolyze the β-1,4-glycosidic bonds of chitin, which is a common structure component of insect exoskeletons and fungal cell walls. In this study, 36 GH18 genes were identified and subjected to bioinformatic analysis based on the genomic data of Glycine max. They were distributed in 16 out of 20 tested soybean chromosomes. According to the amino acid sequences, they can be further divided into five subclades. Class III chitinases (22 members) and class V chitinases (6 members) are the major two subclades. The amino acid size of soybean GH18 proteins ranges from 173 amino acids (aa) to 820 aa and the molecular weight ranges from 19.46 kDa to 91.01 kDa. From an evolutionary perspective, soybean GH18 genes are closely related to Medicago (17 collinear loci with soybean) and Lotus (23 collinear loci with soybean). Promoter analysis revealed that GH18 genes could be induced by environmental stress, hormones, and embryo development. GmGH18-15, GmGH18-24, and GmGH18-33 were screened out due to their nodulation specific expression and further verified by RT-qPCR. These results provide an elaborate reference for the further characterization of specific GH18 genes, especially during nodule formation in soybean.