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

豆科植物和根瘤菌能够形成共生关系。来自大气的二氮（N2）被根瘤菌在植物源性根瘤内转化为植物可利用的氮，作为回报，根瘤菌获得碳化合物以供生长（Baxter等，2021）。豆科植物寄主基因调控可以优化植物的这种关系，而称为根瘤菌自动调节（AON）的系统性负反馈过程允许植物调节根瘤菌形成的根瘤数量，因为根瘤菌的器官发生和根瘤菌的碳支持需要消耗能量（Moreau等，2021）。clavata样肽（CLE）是一种小而可移动的肽，被认为是AON的一个信号。这些肽的表达是对内部（固氮效率）和外部（土壤含氮量）氮的响应，从而抑制结瘤（Lebedeva等，2020）。然而，在环境中，这些值不是恒定的，这意味着短时间的高浓度氮可以阻止结瘤，因此即使可用氮减少，也限制了共生中硝酸盐的益处。该项目的目的是表征一种被忽视的肽，Medicago truncatula CLE34在氮信号整合中的作用，这些信号塑造了植物结瘤和氮利用效率。先前对CLE34的研究是有限的；由于存在一个停止密码子，该肽被描述为一个假基因，没有功能性CLE结构域。然而，大多数其他品种，如短形紫花苜蓿生态型R108，没有这种特征。吉福德实验室之前的研究发现，在与高效根瘤菌相互作用的植物中，以及在高氮土壤中生长的植物中，CLE34的表达水平更高。据推测，由于CLE34水平是由外部（环境）氮和根瘤菌的固氮效率（内部氮）精细调节的，因此它充当了两者的传感器或信号。首先在项目中，确定CLE34是否对内氮、外氮或两者都有响应。这将涉及使用劈裂根实验，其中可以分析不同氮浓度和根瘤菌的存在对根发育的影响。还将测试不同效率的根瘤菌菌株，以确定CLE34是否是效率的标志；从结节中提取mRNA，用qPCR检测CLE34的表达。还将提取蛋白质，并分析可能影响功能和表达条件的翻译后修饰。作为与CLE34可能的相互作用因子，CLE35将在这些实验中与之前发现的能够抑制A17生态型结瘤的CLE35一起测量（Lebedeva et al, 2020）。通过将R108和A17的CLE34启动子和基因与GUS报告基因构建成不同组合的构建体，然后转化到CLE34突变体和野生型植物中，也将检验R108和A17中CLE34上游调控区域的重要性。将确定CLE34的颞部和特殊组织定位。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，我们将确定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肽的关键受体，并存在于茎部中，但尚未确定其是否与CLE34相互作用。这项工作将包括比较野生型和日光突变体之间的CLE34蛋白表达，并利用从植物材料中提取的蛋白在根瘤和茎中进行亲和富集。

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.