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，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，植物自然地将其作为信号来介导对其环境的生理反应，例如缺乏必需的大量营养素（包括氮）。该项目将使用豆科植物模型——蒺藜苜蓿，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肽来介导的，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氮基肥料的替代品提供一条途径，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经济实惠的非转基因途径来调节植物生长。该项目还将通过为少数族裔服务机构的新教员提供职业发展机会而产生广泛影响；研究生和本科生的指导和研究培训；并就影响农业可持续性的因素向农民、生产者和公众进行宣传。该项目的目标是通过对最常与肽结合相关的受体类中的基因冗余进行遗传分析，了解豆科植物在根瘤共生期间如何感知肽，即，富含亮氨酸的重复受体样激酶 (LRR-RLK)。人们对植物肽信号传导和感知的分子机制知之甚少。豆类中已鉴定出多种肽激素；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，并利用模型豆类 M. truncatula 中预先存在的 Tnt1 插入突变体集合来开发内部 lrr-rlk 突变体集合。该项目的另一个目标是开发一个工作方案，通过免疫共沉淀和质谱分析来鉴定肽-受体对。 M. truncatula lrr-rlks 突变系的开发和培育将成为豆科植物研究界的重要资源，促进肽激素功能的研究，不仅在根瘤共生和氮吸收方面，而且在与丛枝菌根真菌的相互作用中，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-微生物相互作用方面的作用提供基础知识。这项使用豆科植物 M. truncatula 模型的工作将对这一新兴领域的知识状况产生深远的影响，并揭示豆类中专门存在的共生表达受体的作用。该奖项反映了 NSF 的法定使命，并通过评估被认为值得支持利用基金会的智力优势和更广泛的影响审查标准。

项目成果

Gene editing to improve legume-rhizobia symbiosis in a changing climate

基因编辑改善气候变化中的豆科植物-根瘤菌共生

• DOI: 10.1016/j.pbi.2022.102324
• 发表时间: 2023-02
• 期刊: Current Opinion in Plant Biology
• 影响因子: 9.5
• 作者: Jain, Divya;Jones, Lauren;Roy, Sonali
• 通讯作者: Roy, Sonali

Running a research group in the next generation: combining sustainable and reproducible research with values-driven leadership

管理下一代研究小组：将可持续和可重复的研究与价值观驱动的领导力相结合

• DOI: 10.1093/jxb/erac407
• 发表时间: 2023-01-01
• 期刊: Journal of Experimental Botany
• 影响因子: 6.9
• 作者: Jacqueline Monaghan; Siobhan M
• 通讯作者: Siobhan M