CAREER: Deciphering the roles of nodule-specific PLAT domain genes in the nitrogen-fixing symbiosis and host-strain specificity

该奖项的全部或部分资金来自《2021年美国救援计划法案》(公共法律117-2)。尽管氮是地球大气中最丰富的气体，但除非在一个称为固氮的过程中将其还原为氨和其他可利用的生物形式，否则植物和动物无法吸收它。种植世界上大多数农作物的传统方法是使用降低形态的氮作为肥料。虽然它提高了作物产量，但大部分化肥渗入地下水、溪流和海洋，造成严重的生态干扰，包括植物生命过度生长、氧气耗尽和动物死亡。由于过度使用，全球500多个沿海水域现已被宣布为“死亡区”。一组统称为根瘤菌的土壤细菌可以还原(固定)氮。豆科植物通过允许它们选择性地进入新发育的器官(根瘤)，与相容的根瘤菌建立互惠的联合(共生)，从而获得一种内在的肥料来源。不同的豆科植物-根瘤菌组合的固氮效率不同，因此，为了高产，需要在豆科作物上施用氮肥。这就关闭了共生固氮(SNF)。该项目旨在揭示寄主-菌株专一性的新机制，以提高SNF的效率，为作物品种和工程菌株的开发提供信息，这些品种和工程菌可以提高SNF在低投入、可持续农业中的经济潜力。该项目的研究活动将被整合到一个以探究和项目为基础的研究生课程实验室，以及各种教育活动中，包括培训本科生和研究生、博士后研究人员，以及培训德克萨斯州农村地区的贫困初高中女孩。SNF的一个耐人寻味的方面是宿主菌株特异性，这对有效的固氮至关重要，但在遗传和分子水平上仍然知之甚少。由于根际在任何时候都含有多种根瘤菌，因此对于豆科植物的寄主来说，区分朋友和敌人，以及区分高效和低效的朋友，对于最佳固氮来说是至关重要的。拟议的研究建立在这样的假设基础上，即紫花苜蓿MtNPD1基因(根瘤特异性多囊蛋白-1，脂氧合酶，包含α-毒素结构域的蛋白)协调根瘤菌的选择，以维持根瘤的有效固氮。NPD1突变体的宿主-菌株特异性表型表明，MtNPD1可能与某些细菌因子相互作用，促进根瘤内亲和菌株的存活和正常功能。将使用分子、遗传、蛋白质组、基因组和显微方法来破译MtNPD1和这个根瘤特异基因家族的其他四个成员的生物学作用。该项目的主要目标是确定MtNPD1的植物和/或细菌蛋白伙伴，以菌株依赖的方式改进MtNPD1在细胞内的定位，并通过泛基因组分析和NPD1结节中命运相反的菌株之间的基因组文库切换来识别与NPD1基因功能和宿主菌株特异性相关的细菌因子。总之，拟议的工作将加强我们对元胞杆菌如何选择有利的共生伙伴的理解，从而优化具有特定根瘤菌菌株的SNF。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117- 2) Although nitrogen is the most abundant gas in the Earth’s atmosphere, it cannot be assimilated by plants and animals unless it is reduced to ammonium and other bioavailable forms, in a process called nitrogen fixation. The conventional way of growing most of the world’s crops is to apply reduced forms of nitrogen as fertilizer. Although it promotes crop yields, much of the fertilizer leaches out into groundwater, streams, and oceans, causing severe ecological disturbances, including overgrowth of plant life, oxygen depletion, and death of animal life. Due to overuse, more than 500 sites of coastal waters worldwide are now declared ‘dead zones’. A group of soil bacteria collectively known as rhizobia can reduce (fix) nitrogen. Legumes establish mutually beneficial associations (symbioses) with compatible rhizobia, by allowing their selective entry into newly developed organs (root nodules), thus acquiring an internal source of fertilizer. Nitrogen fixation efficiency varies in different legume-rhizobia associations; therefore, for high yields, fertilizer nitrogen needs to be applied on legume crops. This shuts down symbiotic nitrogen fixation (SNF). The project seeks to uncover new mechanisms of host-strain specificity to improve SNF efficiency, informing the development of crop varieties and engineered bacterial strains that can enhance the economic potential of SNF for low-input, sustainable agriculture. Research activities from this project will be integrated into an inquiry- and project-based revamped graduate course-lab, and into various educational activities including training of undergraduate and graduate students, a postdoctoral researcher, and training disadvantaged middle- and high school girls from rural Texas. An intriguing aspect of SNF is host-strain specificity, critical for efficient nitrogen fixation, but, still, poorly understood at the genetic and the molecular level. Because the rhizosphere contains multiple rhizobial strains at any time, it is critical for a legume host to distinguish between friend and foe, and, also, to distinguish between efficient and less efficient friends, for optimal nitrogen fixation. The proposed research builds on the hypothesis that the Medicago truncatula MtNPD1 gene (nodule-specific polycystin-1, lipoxygenase, alpha-toxin domain-containing protein) orchestrates rhizobial selection in order to maintain effective nitrogen fixation in root nodules. The host-strain specific phenotype of the npd1 mutant implies that MtNPD1 may be interacting with certain bacterial factors to promote survival and normal function of compatible strains inside root nodules. An assortment of molecular, genetic, proteomic, genomic, and microscopic approaches will be used to decipher the biological roles of MtNPD1 and the other four members of this nodule-specific gene family. The main goals of the project are to identify plant and/or bacterial protein partners of MtNPD1, refine intracellular MtNPD1 localization in a strain-dependent manner, and identify bacterial factors linked to the NPD1 gene function and host-strain specificity using pan-genome analysis and genomic library switching between strains with contrasting fate in npd1 nodules. Altogether, the proposed work is poised to enhance our understanding of how M. truncatula selects favorable symbiotic partners, thus optimizing SNF with specific rhizobial strains.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.