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

BRC-BIO：MediCARGO - 使用 CRISPR-cas9 作为反向遗传学工具解码苜蓿-中华根瘤菌共生期间的肽感知

• 批准号: 2217830
• 负责人: Sonali Roy
• 金额: $ 49.71万
• 依托单位: Tennessee State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2217830&HistoricalAwards=false
• 关键词: BRC; BIO; MediCARGO; Decoding; peptide

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.

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，试图了解以植物为基础的新型小分子的作用的研究可以提供急需的解决方案。小信号肽或多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部署，以调节对环境的生理反应，例如缺乏必要的常量营养素，包括氮。该项目将使用模式豆科植物紫花苜蓿，它能够与有益细菌相互作用，在没有添加氮肥的情况下帮助从环境中获取氮。这种能力是通过检测环境中的多肽来调节的，尽管其机制尚不清楚。更详细地了解这一过程可能为将化学合成的多肽用作氮基肥料的替代品提供一条途径，因为氮基肥料可能对环境有害。因此，这项工作的结果可以提供一种负担得起的、非转基因的方式来调节植物的生长。该项目还将产生广泛的影响，为少数族裔服务机构的一名新教师提供职业发展机会；为研究生和本科生提供指导和研究培训；以及就影响农业可持续发展的因素向农民、生产者和普通公众进行宣传。该项目的目标是通过从基因上解剖与多肽结合最常见的受体类，即富亮氨酸重复序列类似受体蛋白(LRR-RLKs)中的基因冗余，了解豆科植物在根瘤共生过程中如何感知多肽。植物多肽信号和感知的分子机制还知之甚少。已经在豆类中发现了几种多肽激素；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，并利用模式Legum中已有的Tnt1插入突变体集合，M.truncatula，以开发内部的LRR-rlk突变体集合。该项目的另一个目标将是开发一种工作方案，利用免疫共沉淀和质谱仪鉴定多肽-受体对。突变株系的建立和培育将成为豆科植物研究领域的重要资源，不仅有助于研究多肽激素在根瘤共生和氮素吸收中的作用，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，这些资源将为理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。这项使用豆类模式M.truncatula的工作将对这一新兴领域的知识状态产生深远影响，并揭示仅存在于豆类中的共生表达受体的作用。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。