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

豆类和根瘤菌能够形成共生关系。来自大气的氮素(N2)由根瘤菌转化为植物在植物来源的根瘤中可用的一种氮，反过来，根瘤菌获得生长所需的碳化合物(Baxter等人，2021)。豆类寄主基因的调节可以优化植物的这种关系，而被称为结瘤自动调节(AON)的系统负反馈过程允许植物调节它与根瘤菌形成的根瘤的数量，因为根瘤的器官发生和根瘤菌的碳支持需要能量(Moreau等人，2021)。棍棒样多肽(CLE)是一种小的且通常可移动的多肽，已被认为是AON的信号。这些多肽是对内部(固氮效率)和外部(土壤含氮量)氮的响应而表达的，从而导致抑制结瘤(Lebedeva等人，2020)。然而，在环境中，这些值并不是恒定的，这意味着短期的高浓度氮素可以阻止结瘤，因此限制了共生的硝酸盐受益，即使可利用的氮素随后减少。本项目的目的是表征被忽视的多肽，紫花苜蓿CLE34在整合形成植物结瘤和氮素利用效率的氮信号中的作用。以前对CLE34的研究一直是有限的；由于终止密码子的存在，该肽被描述为在元胞紫花苜蓿生态型A17中没有功能CLE结构域的假基因。然而，其他大多数材料，如元胞紫花苜蓿生态型R108，都不具有这种特性。吉福德实验室之前的工作发现，CLE34在与高效根瘤菌相互作用的植物中表达水平较高，在高氮土壤中也是如此。假设，由于CLE34水平受外部(环境)氮和根瘤菌的固氮效率(内部氮)的微调，它作为两者的传感器或信号。首先，在项目中，CLE34对内部氮、外部氮或两者都有反应。这将涉及使用分根实验，其中可以分析不同的氮浓度和根瘤菌的存在对根发育的影响。不同效率的根瘤菌也将被测试，以确定CLE34是否是效率的标志；将从根瘤中提取mRNA，并用qPCR测量CLE34的表达。还将提取蛋白质，并分析多肽，以寻找可能影响功能和表达条件的翻译后修饰。作为CLE34的一个可能的相互作用因子，CLE35将在这些实验中与先前发现能够抑制A17生态型中的结瘤的基因一起被测量(Lebedeva等，2020)。CLE34的上游调节区在R108和A17中的重要性也被测试，方法是用不同组合的CLE34启动子以及来自R108和A17的基因与GUS报告基因一起生成构建体，然后转化到cle34突变体和野生型植物中。CLE34的时间和特殊组织定位将被确定。R108和A17中的CLE34基因变异也将在R108中过表达和沉默，以观察对R108结瘤的影响。最后，将确定CLE34与其相应受体之间的相互作用。Sunn已被确定为参与Aon的其他CLE多肽的关键受体，并存在于芽中，但尚未确定这是否与CLE34相互作用。这项工作将包括比较野生型和sunn突变体之间的CLE34蛋白表达，并利用从植物材料中提取的蛋白质在根瘤和地上部中进行亲和浓缩。

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.