The biogenesis and functions of pre-meiotic small RNAs in male reproductive development in maize

减数分裂前小RNA在玉米雄性生殖发育中的生物发生和功能

• 批准号: 10718996
• 负责人: Kirk Czymmek
• 金额: $ 63.56万
• 依托单位: DONALD DANFORTH PLANT SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10718996
• 关键词: Angiosperms; Animals; Biochemical; Biogenesis; Biological Models; Biology; CRISPR/Cas technology; Cell Differentiation process; Cells; Chromosomes; DNA Methylation; DNA Polymerase II; DNA Transposable Elements; Data; Defect; Dependence; Development; Developmental Process; Distal; Drosophila genus; Drug or chemical Tissue Distribution; Environment; Epidermis; Evolution; Exhibits; Failure; Family; Fertility; Genetic; Genome; Genome Stability; Genomics; Goals; Grain; Image; Individual; Maize; Male Sterility; Male Sterilizations; Mammals; Maps; Mediating; Meiosis; Messenger RNA; MicroRNAs; Minor; Modeling; Molecular; Movement; Mus; Mutation; Nature; Nematoda; Organ; Oryza sativa; Outcomes Research; Paper; Pathway interactions; Phase; Phenotype; Plant Model; Plants; Poaceae; Pollen; Production; Proteins; RNA; RNA Precursors; Reporting; Reproduction; Reproductive Biology; Research; Rice; Role; Signal Transduction; Small Interfering RNA; Small RNA; Somatic Cell; Sterility; Techniques; Testis; Transcript; Untranslated RNA; Work; Zea mays; chromatin modification; experimental study; genome-wide; gonad development; insight; male; male fertility; mutant; novel; piRNA; pluripotency; reproductive; reproductive development; reproductive organ; single-cell RNA sequencing; spatiotemporal; sperm cell; stem cells; transcription factor

PROJECT SUMMARY In numerous flowering plant species, the male reproductive organs (anthers), which nurture pollen grains (containing the sperm, the male germline of plants) and their progenitor cells, accumulate two classes of small RNAs, i.e., the pre-meiotic (21-nt) and meiotic (24-nt) phased, small interfering RNAs (phasiRNAs). Perturbation of the phasiRNA biogenesis pathways in rice or maize causes environmentally-sensitive male sterility. There are many interesting parallels between these plant phasiRNAs and animal PIWI-interacting RNAs (piRNAs), which have also been shown essential for male fertility. However, there is a major gap in our understanding of how the plant reproductive phasiRNAs function to regulate developmental processes important for reproduction. Our long-term goal is to understand mechanistic and functional basis for the dependence on these small RNAs in plant male reproductive development. We and collaborators have described the reproductive phasiRNA pathways in many plant species. We have shown that biogenesis of the 21-nt, pre-meiotic phasiRNAs is initiated in the anther epidermis, but they accumulate in the tapetum, three layers of cells distal, indicating movement across cell layers. Yet, it is largely unknown where the 21-nt phasiRNAs and their biogenesis components localize throughout anther development, as well as what are the targets and thus precise functions of the 21-nt phasiRNAs. Our strong preliminary data has led to our focus on three Specific Aims to characterize 21-nt, pre-meiotic reproductive phasiRNAs in maize, a model for plant reproductive biology, genomics, and genetics. The aims are: (1) Characterize the spatiotemporal localization of key players in pre-meiotic phasiRNA biogenesis, using advanced imaging and single-cell RNA-seq techniques; this aim will generate a spatiotemporal map of 21-nt phasiRNAs, their precursors, microRNA trigger, and biogenesis proteins, in developing maize anthers. (2) Decipher the biogenesis of pre-meiotic phasiRNAs and their developmental roles, via the identification of novel biogenesis factors using biochemical approaches, using publicly available mutants plus CRISPR-Cas9- generated mutants to understand whether the pre-meiotic phasiRNAs are necessary for male reproductive development, and then assign functions to novel components of the pathway. And (3) elucidate the regulatory mechanisms mediated by pre-meiotic phasiRNAs, using established and new molecular approaches to identify the endogenous targets of 21-nt phasiRNAs and examine how they are regulated by the phasiRNAs. The proposed research will have a broad impact on small RNA biology by characterizing the biogenesis and functions of pre-meiotic plant small RNAs in maize anther development. The experiments will provide key insights into where and how they are produced, and what their roles are in anther cell differentiation and male fertility. Comparison of the results of this work with ongoing studies into mammalian piRNAs may shed light on the commonalities in the utilization of small RNA pathways in reproduction and gonad development.

项目摘要 在众多开花植物物种中，雄性繁殖器官（花药），培育花粉颗粒 （包含精子，植物的雄性种系）及其祖细胞，积累了两类小的小 rNA，即，酸性前（21-nt）和减数分裂（24-nt）逐步分阶段，小干扰RNA（phasirnas）。 水稻或玉米中phasirna生物发生途径的扰动会导致对环境敏感的雄性 不育。这些植物phasirnas和动物piwi相互作用之间有许多有趣的相似之处 RNA（piRNA），这也对男性生育至关重要。但是，我们的 了解植物生殖Phasirnas如何调节发育过程 对繁殖很重要。我们的长期目标是了解机械和功能基础 植物男性生殖发育中对这些小的RNA的依赖。 我们和合作者描述了许多植物物种中生殖的Phasirna途径。我们 已经表明，在花药表皮中启动了21-NT的生物发生，但它们是 堆积在木薯中，三层细胞远端，表明跨细胞层运动。但是，这很大程度上 未知的21-NT PhasirNA及其生物发生成分在整个花药中定位 开发以及21-nt phasirnas的目标和精确功能是什么。我们的坚强 初步数据已导致我们专注于三个特定目标，以表征21-nt，尿生殖前的生殖 玉米中的Phasirnas，这是一种植物生殖生物学，基因组学和遗传学的模型。目的是：（1） 表征主要参与者的时空定位，使用前自然phasirna生物发生，使用 高级成像和单细胞RNA-seq技术；这个目标将产生21-nt的时空图 Phasirnas，它们的前体，microRNA触发和生物发生蛋白在发展玉米花药中。 （2） 通过鉴定新生生物的生物发生及其发育作用 使用生化方法的生物发生因子，使用公共可用的突变体加CRISPR-CAS9- 生成的突变体，以了解雄性生殖是否需要的舒适前phasirnas 开发，然后将功能分配给途径的新组件。 （3）阐明调节 固定型phasirnas介导的机制，采用已建立和新的分子方法来鉴定 21-nt phasirnas的内源性靶标，并检查它们如何受到phasirnas的调节。 拟议的研究将通过表征生物发生对小RNA生物学产生广泛的影响 玉米花药发育中脂源植物小的RNA的功能。实验将提供钥匙 深入了解它们的生产地点和方式，以及它们在花药细胞分化和男性中的作用 生育能力。比较这项工作的结果与正在进行的哺乳动物PIRNA的研究可能会揭示 在繁殖和性腺发育中利用小的RNA途径的共同点。