BRC-BIO: MediCARGO- Decoding peptide perception during Medicago-Sinorhizobium symbiosis using CRISPR-cas9 As a Reverse Genetics tool

生物活性小分子在优化农业系统方面发挥着重要作用，使植物能够耐受高温等非生物胁迫和致病病原体等生物胁迫，同时能够与有益微生物相互作用。由于我们的星球面临着不确定的环境挑战，寻求了解新型植物小分子作用的研究可以提供急需的解决方案。小信号肽，或肽激素，是基因组编码的生物活性分子，植物天然部署作为信号，以介导对环境的生理反应，如缺乏必需的宏量营养素，包括氮。该项目将使用模式豆科植物，蒺藜苜蓿，它能够与有益细菌相互作用，以帮助在没有添加氮肥的情况下从环境中获得氮。这种能力是通过检测环境中的肽来介导的，尽管其机制知之甚少。更详细地了解这一过程可以为化学合成肽作为氮基肥料的替代品的潜在用途提供一条途径，氮基肥料可能对环境有害。因此，这项工作的成果可以提供一种负担得起的非转基因途径来调节植物生长。该项目还将产生广泛的影响，为少数群体服务机构的一名新教员提供职业发展机会;为研究生和本科生提供指导和研究培训;以及对农民、生产者、本项目的目的是通过对基因冗余的遗传解剖，了解豆科植物在根瘤共生过程中如何感知肽在最常与肽结合相关的受体类别中，即，富含亮氨酸的重复受体样激酶（LRR-RLK）。植物肽信号传导和感知的分子机制知之甚少。几种肽激素已被确定在豆类，然而，它们的同源受体仍然难以捉摸。该项目将使用反向遗传学工具，主要是CRISPR-cas9介导的基因编辑，并利用模式豆科植物M. truncatula，以开发内部lrr-rlk突变体收集。该项目的另一个目标将是开发一个工作协议，以确定肽受体对使用共免疫沉淀，然后质谱。本研究主要研究了M. truncatula lrr-rlks将成为豆类研究界的重要资源，不仅有助于研究肽激素在根瘤共生和氮吸收中的功能，而且有助于研究肽激素与丛枝菌根真菌的相互作用以及对大量营养素缺乏的反应。从长远来看，这些资源将为了解假定的农用化学品在控制植物-微生物相互作用中的作用提供基础知识。本研究以豆科模式植物M.该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Bioactive small molecules play an important role in optimizing agricultural systems, allowing plants to tolerate abiotic stresses like high temperature and biotic stresses like disease-causing pathogens, while enabling interactions with beneficial microbes. As our planet is faced with uncertain environmental challenges, research seeking to understand roles of novel, plant-based small molecules can provide much needed solutions. Small signaling peptides, or peptide hormones, are genome-encoded bioactive molecules that plants naturally deploy as signals to mediate physiological responses to their environment, such as the absence of essential macronutrients, including nitrogen. This project will use the model legume, Medicago truncatula, which is capable of interacting with beneficial bacteria to help acquire nitrogen from the environment in the absence of added nitrogenous fertilizers. This ability is mediated by detection of peptides in the environment, although the mechanism is poorly understood. Understanding this process in more detail could provide an avenue to the potential use of chemically synthesized peptides as alternatives to nitrogen-based fertilizers, which can be harmful to the environment. Thus, the outcomes of this work could provide an affordable, non-GMO route to regulate plant growth. The project will also have broad impact by providing career development opportunities for a new faculty member at a Minority-Serving Institution; mentoring and research training for graduate and undergraduate students; and outreach to farmers, producers, and the general public on factors influencing agricultural sustainability.The objective of this project is to understand how legumes perceive peptides during root nodule symbiosis by genetically dissecting gene redundancy in the receptor class most commonly associated with peptide binding, namely, Leucine-Rich Repeat Receptor Like Kinases (LRR-RLKs). Molecular mechanisms of plant peptide signaling, and perception are poorly understood. Several peptide hormones have been identified in legumes; however, their cognate receptors remain elusive. The project will use reverse genetics tools, primarily CRISPR-cas9 mediated gene editing, and make use of the pre-existing Tnt1 insertion mutant collection in the model legum, M. truncatula, to develop an in-house lrr-rlk mutant collection. Another objective of the project will be to develop a working protocol to identify peptide-receptor pairs using co-immunoprecipitation followed by mass spectrometry. The development and curation of mutant lines in M. truncatula lrr-rlks will serve as a vital resource for the legume research community, facilitating investigation of peptide hormone function not only in root nodule symbiosis and nitrogen uptake, but also in interactions with arbuscular mycorrhizal fungi, and responses to macronutrient deficiency. In the long term, these resources will provide fundamental knowledge for understanding effects of putative agrochemicals in control of plant-microbe interactions. This work using the model legume M. truncatula will have a profound impact on the state of knowledge in this nascent field and uncover roles of symbiotically expressed receptors exclusively present in legumes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.