Nitrate-Induced MtCLE34 Gene Lacks the Ability to Reduce Symbiotic Nodule Number and Carries Nonsense Mutation in a Few Accessions of Medicago truncatula

Legume plants form nitrogen-fixing nodules on their roots in symbiosis with soil bacteria rhizobia. The number of symbiotic nodules is controlled by a host plant via a systemic mechanism known as autoregulation of nodulation (AON). The key players of AON are the CLE peptides which are produced in the root in response to rhizobia inoculation and are transported via xylem to the shoot. In the shoot, the CLE peptides are recognized by a CLV1-like receptor kinase, which results in subsequent inhibition of nodule development in the root via a negative feedback mechanism. In addition to the CLE peptides induced by rhizobia, nitrate-induced CLE peptides involved in the control of nodulation have been identified. In Medicago truncatula, the MtCLE34 gene has been described, which was activated by nitrate and in response to rhizobial inoculation. However, this gene contains a premature stop codon in the reference M. truncatula genome of the A17 line, and therefore, it was suggested to be a pseudogene. Here, we analyzed nucleotide sequences of the MtCLE34 gene available from the genomes of different M. truncatula accessions from the Medicago HAPMAP project and found that the majority of M. truncatula accession lines do not carry nonsense mutations in the MtCLE34 gene and should encode functional products. Overexpression of the MtCLE34 gene from the R108 line, which does not have a premature stop codon, did not inhibit nodulation. Therefore, in spite of having high sequence similarity to the nodulation-suppressing CLE genes, the MtCLE34 gene from the R108 line was not able to trigger AON in M. truncatula. Our findings shed light on the evolutionary changes in the CLE proteins in legume plants and can be used in the future to understand which amino acid residues within CLE proteins could be important for their ability to suppress nodulation.