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中发挥着重要作用，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，寻求理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，植物自然地将其作为信号来调节对其环境的生理反应，例如缺乏必需的宏量营养素，包括氮。该项目将使用模型豆科植物，Medicago truncatula，它能够与有益细菌相互作用，帮助在没有添加氮肥的情况下从环境中获取氮。这种能力是通过检测环境中的多肽介导的，尽管其机制尚不清楚。更详细地了解这一过程可以为化学合成肽作为对环境有害的氮基肥料的替代品的潜在用途提供一条途径。因此，这项工作的结果可能提供一种经济实惠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，为少数民族服务机构的新教员提供职业发展机会；研究生和本科生的指导和研究训练；向农民、生产者和公众宣传影响农业可持续性的因素。该项目的目的是通过基因剖析与肽结合最相关的受体类（即富含亮氨酸的重复受体样激酶（LRR-RLKs））的基因冗余，了解豆科植物在根瘤共生过程中如何感知肽。植物多肽信号的分子机制和感知尚不清楚。在豆科植物中发现了几种肽激素；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，并利用模型豆科植物M. truncatula中已有的Tnt1插入突变体收集，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种工作方案，利用共免疫沉淀和质谱法鉴定肽受体对。突变株的培育和培育将为豆科研究提供重要资源，不仅有助于研究多肽激素在根瘤共生和氮吸收中的作用，还有助于研究其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与微生物相互作用方面的作用提供基础知识。这项使用模型豆科植物M. truncatula的工作将对这一新兴领域的知识状态产生深远的影响，并揭示仅存在于豆科植物中的共生表达受体的作用。该奖项反映了美国国家科学基金会的法定使命，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.