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，氮肥已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的氮源。豆类作物如大豆、菜豆和花生通过与一组称为根瘤菌的固氮土壤细菌建立共生关系，具有利用大气氮用于其生长的能力。共生相互作用开始于一个形成根瘤的过程，在这个过程中，根瘤菌将大气中的氮转化为可被宿主植物利用的氨--这一过程称为共生固氮。大豆生产所需的氮约50-60%由共生固氮提供，因此大豆是美国农业系统中与玉米两年轮作的重要组成部分。根瘤菌的固氮和固氮作用都需要共生体之间的分子信号协调交换，而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-宿主植物通讯的一种分子。这个项目将解决如何以及在何种程度上这样的RNA片段介导的固氮和固氮过程。这些知识将有助于开发新的生物技术工具，优化豆类-根瘤菌共生体，促进可持续作物生产。该项目将通过每年一度的“农业科学中的生物信息学”暑期讲习班，为代表性不足的少数民族本科生和高中生提供实践经验。虽然真核生物sRNA在植物-真菌相互作用中的作用已经得到了很好的表征，但sRNA介导的原核生物-真核生物通信才刚刚开始受到关注。转移RNA衍生的sRNA片段（tRFs）通常被视为降解产物，因此在以前的研究中很大程度上被忽略。最近，Ma实验室证明，三种根瘤菌tRF能够通过大豆Argonaute 1引导的mRNA切割来调节影响结瘤的五种大豆基因的表达。该项目将整合基因组，分子和生物化学工具，以阐明根瘤菌tRFs劫持宿主细胞机制，以调节固氮和共生固氮（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-Bradyrhizobium diazoefficiens共生伙伴作为实验系统。在目标1中，将研究根瘤菌tRFs丰度的动态和假定的靶基因在发育、成熟和衰老的根瘤中的表达;在目标2中，将确定根瘤菌tRFs-宿主基因相互作用的机制和相对大小;在目标3中，将描述tRFs子集及其靶基因在根瘤发育和SNF中的功能;在目标4中，将通过基因编辑开发具有结节性状数量变异的实验大豆系，并评估结瘤和SNF效率以及植物生产力。该项目可能揭示tRFs在不同宿主微生物相互作用中的潜在作用，将发现转化为控制细菌病原体的实践或促进有益的相互作用。该奖项反映了NSF的法定使命，并被认为值得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来支持。

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.