CRISPR/Cas9-Mediated Knock-Out of the MtCLE35 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 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 MtCLE35 gene was found to be upregulated by rhizobia and nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. In this study, we obtained several knock-out lines in which the MtCLE35 gene was mutated using the CRISPR/Cas9-mediated system. M. truncatula lines with the MtCLE35 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 MtCLE genes, MtCLE12 and MtCLE13, were reduced in rhizobia-inoculated roots, whereas no significant difference in MtCLE35 gene expression was observed between nitrate-treated and rhizobia-inoculated control roots. Together, these findings suggest the key role of MtCLE35 in the number of nodule numbers under high-nitrate conditions, under which the expression levels of other nodulation-related MtCLE genes are reduced.

Identification and Expression Analysis of the C-TERMINALLY ENCODED PEPTIDE Family in Pisum sativum L.

The C-TERMINALLY ENCODED PEPTIDE(CEP) peptides play crucial roles in plant growth and response to environmental factors. These peptides were characterized as positive regulators of symbiotic nodule development in legume plants. However, little is known about the CEP peptide family in pea. Here, we discovered in pea genome 21 CEP genes (PsCEPs), among which three genes contained additional conserved motifs corresponding to the PIP (PAMP-induced secreted peptides) consensus sequences. We characterized the expression patterns of pea PsCEP genes based on transcriptomic data, and for six PsCEP genes with high expression levels in the root and symbiotic nodules the detailed expression analysis at different stages of symbiosis and in response to nitrate treatment was performed. We suggest that at least three PsCEP genes, PsCEP1, PsCEP7 and PsCEP2, could play a role in symbiotic nodule development, whereas the PsCEP1 and PsCEP13 genes, downregulated by nitrate addition, could be involved in regulation of nitrate-dependent processes in pea. Further functional studies are required to elucidate the functions of these PsCEP genes.