Collaborative Research: Elucidating the Role of Rhizobial tRNA-Derived Fragments in Soybean Nodule Development and Symbiotic Nitrogen Fixation

自20世纪60年代的绿色革命以来，氮肥被广泛用于提高作物产量，以满足不断增长的世界人口的需求。然而，过度依赖化肥导致水、土壤和空气污染；因此，可持续农业需要环境友好型氮源。豆科作物，如大豆、普通豆和花生，通过与一组固氮土壤细菌（称为根瘤菌）建立共生关系，有能力利用大气中的氮来生长。共生的相互作用开始于一个叫做根瘤形成的过程，根瘤菌在这个过程中将大气中的氮转化为可被寄主植物利用的氨，这个过程称为共生固氮。大豆生产所需的约50-60%的氮是由共生固氮提供的，因此在美国农业系统中，大豆是玉米两年轮作的重要组成部分。结瘤和固氮都需要在共生伙伴之间协调交换分子信号，根瘤菌中产生的转移RNA衍生的小RNA片段最近被发现是介导根瘤菌-寄主植物通信的一种分子。该项目将研究这些RNA片段如何以及在多大程度上介导结瘤和固氮过程。这些知识将有助于开发新的生物技术工具，以优化豆科植物与根瘤菌的共生关系，促进可持续作物生产。该项目将通过一年一度的“农业科学中的生物信息学”暑期研讨会，为未被充分代表的少数民族本科生和高中生提供实践经验。植物与微生物的相互作用需要跨界小RNA （sRNA）干扰。虽然真核生物srna在植物与真菌相互作用中的作用已经被很好地表征，但srna介导的原核生物与真核生物之间的通讯才刚刚开始引起人们的关注。转移rna衍生的sRNA片段（trf）通常被视为降解产物，因此在以往的研究中很大程度上被忽视。最近，Ma实验室证明了三种根瘤菌tRFs能够通过大豆Argonaute 1引导的mRNA切割来调节影响大豆结瘤的五种基因的表达。该项目将整合基因组、分子和生化工具，阐明根瘤菌tRFs劫持宿主细胞机制来调节结瘤和共生固氮（SNF）的程度，并进一步剖析根瘤菌tRFs调节这些过程的机制，使用大豆-慢生根瘤菌重氮效率共生伙伴作为实验系统。在Aim 1中，将研究根瘤菌tRFs丰度和在发育、成熟和衰老根瘤中可能的靶基因表达的动态；在第二阶段，将确定根瘤菌trf -宿主基因相互作用的机制和相对大小；在Aim 3中，将对结节发展和SNF背后的tRFs子集及其靶基因的功能进行表征；在Aim 4中，将通过基因编辑开发具有根瘤性状定量变异的大豆实验品系，并评估根瘤和单核苷酸多态性效率以及植株生产力。该项目可能揭示trf在不同宿主-微生物相互作用中的潜在作用，将这些发现转化为控制细菌病原体或促进有益相互作用的实践。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。

Since the Green Revolution of the 1960s, nitrogen fertilizers have been widely deployed to boost crop yields to meet the demands of the growing world population. However, excessive reliance on fertilizers leads to water, soil and air pollution; therefore, environmentally friendly sources of nitrogen are needed for sustainable agriculture. Legume crops such as soybean, common bean, and peanuts have the capability to harness atmospheric nitrogen for their growth by establishing symbiotic relationships with a group of nitrogen-fixing soil bacteria known as rhizobia. The symbiotic interactions begin with a process called nodulation that forms root nodules, within which rhizobia convert atmospheric nitrogen into ammonia that can be utilized by host plants – a process called symbiotic nitrogen fixation. Approximately 50-60% of the nitrogen needed for soybean production is provided by symbiotic nitrogen fixation, thus positioning soybean as an important component of the biennial rotation with corn in the US agricultural system. Both nodulation and nitrogen fixation require coordinated exchanges of molecular signals between the symbiotic partners, and transfer RNA-derived small RNA fragments produced in rhizobia have recently been found to be one type of molecule mediating rhizobia-host plant communications. This project will address how and to what extent such RNA fragments mediate the nodulation and nitrogen fixation processes. Such knowledge will facilitate the development of new biotechnological tools for optimizing the legume-rhizobia symbioses for sustainable crop production. This project will provide hands-on experience to underrepresented minority undergraduate and high school students through an annual “Bioinformatics in Agricultural Science” summer workshop.Plant-microbe interactions require cross-kingdom small RNA (sRNA) interference. While the roles of eukaryotic sRNAs in plant-fungus interactions have been well characterized, sRNA-mediated prokaryote-eukaryote communications have just begun to garner attention. Transfer RNA-derived sRNA fragments (tRFs) are often viewed as degradation products and thus have been largely ignored in previous studies. Recently, the Ma laboratory demonstrated that three rhizobial tRFs were able to regulate the expression of five soybean genes affecting nodulation via soybean Argonaute 1-guided mRNA cleavage. This project will integrate genomic, molecular, and biochemical tools to elucidate the extent to which rhizobial tRFs hijack the host cellular machineries to regulate nodulation and symbiotic nitrogen fixation (SNF) and to further dissect mechanisms by which rhizobial tRFs regulate these processes using soybean-Bradyrhizobium diazoefficiens symbiotic partners as an experimental system. In Aim 1, the dynamics of rhizobial tRFs abundance and putative target gene expression in developing, mature, and senescing nodules will be investigated; in Aim 2, the mechanisms of rhizobial tRF-host gene interactions and relative magnitudes will be determined; in Aim 3, the functions of a subset of tRFs and their target genes underlying nodule development and SNF will be characterized; in Aim 4, experimental soybean lines with quantitative variation of nodule traits will be developed through gene-editing and evaluated for nodulation and SNF efficiencies and the plant productivity. This project may shed light on the potential roles of tRFs in diverse host-microbe interactions, towards translation of the discoveries into practice for control of bacterial pathogens or to promote beneficial interactions.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.