Identifying the role of the CLE34 peptide as a nitrogen sensor in the autoregulation of nodulation in Medicago truncatula

Legumes and rhizobial bacteria are capable of forming a symbiotic relationship. Dinitrogen (N2) from the atmosphere is converted by the rhizobia into a form of nitrogen usable by the plant within plant-derived root nodules, and in return, the rhizobia gains carbon compounds for growth (Baxter et al, 2021). Legume host gene regulation can allow optimisation of this relationship for the plant and a systemic negative feedback process called autoregulation of nodulation (AON) allows the plant to regulate the number of nodules it forms with rhizobia bacteria, as organogenesis of nodules and the carbon support of the rhizobia costs energy (Moreau et al, 2021).

CLAVATA-like (CLE) peptides, which are small and often mobile, have been implicated as a signal in AON. The peptides are expressed in response to the internal (nitrogen-fixing efficiency) and external (soil nitrogen content) nitrogen, leading to inhibition of nodulation (Lebedeva et al, 2020). However, in the environment, these values are not constant, meaning a short high concentration of nitrogen can halt nodulation, therefore limiting nitrate benefits from symbiosis even if the nitrogen available then decreases.

This aim of this project is to characterise the role of an overlooked peptide, Medicago truncatula CLE34 in the integration of nitrogen signals that shape plant nodulation and nitrogen-use efficiency. Previous work on CLE34 has been limited; the peptide has been described as a pseudogene without a functional CLE domain in Medicago truncatula ecotype A17 due to the presence of a stop codon. However most other accessions such as Medicago truncatula ecotype R108 do not have this.

Previous work by the Gifford lab has found CLE34 expressed at higher levels in plants that are interacting with high-efficiency rhizobia, and also when grown in high nitrogen soil. It is hypothesised that, as CLE34 levels are finely tuned by both external (environmental) nitrogen, as well as nitrogen-fixation efficiency (internal nitrogen) by rhizobia, it acts as a sensor or signal of both.

Firstly in the project, whether CLE34 responds to the internal nitrogen, external nitrogen or both will be identified. This will involve the use of split root experiments, where the impact of different nitrogen concentrations and presence of rhizobia on root development can be analysed. Rhizobia strains of differing efficiency will also be tested to identify whether CLE34 is a marker of efficiency; mRNA will be extracted from the nodules and CLE34 expression measured with qPCR. Protein will also be extracted and peptides will be analysed for post-translational modifications that may impact function and expression conditions. As a possible interactor with CLE34, CLE35 will be measured alongside during these experiments which has previously found to be able to inhibit nodulation in the A17 ecotype (Lebedeva et al, 2020).

The importance of the upstream regulatory regions of CLE34 in R108 and A17 wiil also be tested by generating constructs with different combinations of the CLE34 promoters and genes from R108 and A17 with the GUS reporter, then transformed into cle34 mutants and wild-type plants. Temporal and special tissue localisation of CLE34 willl be determined. The CLE34 gene variants in R108 and A17 will also be overexpressed and silenced in R108 to see the impact on nodulation in R108.

Finally, the interaction between CLE34 and its corresponding receptor will be identified. SUNN has been identified as a key receptor for other CLE peptides involved in AON and is present in the shoot, however has yet to be determined if this interacts with CLE34. This work will involve comparing CLE34 protein expression between wildtype and sunn mutants and use of affinity enrichment of protein extracted from plant material in both the nodules and the shoot.