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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要在许多开花植物物种中，雄性生殖器官（花药）负责培育花粉粒（含有精子，植物的雄性种系）及其祖细胞，积累两类小细胞 RNA，即减数分裂前 (21-nt) 和减数分裂 (24-nt) 定相小干扰 RNA (phasiRNA)。水稻或玉米中 phasiRNA 生物发生途径的扰动导致环境敏感的雄性不育。这些植物 phasiRNA 和动物 PIWI 相互作用之间有许多有趣的相似之处 RNA (piRNA)，也被证明对男性生育能力至关重要。然而，我们的差距还很大了解植物生殖 phasiRNA 如何发挥调节发育过程的作用对于繁殖很重要。我们的长期目标是了解其机制和功能基础植物雄性生殖发育依赖这些小RNA。我们和合作者已经描述了许多植物物种的生殖 phasiRNA 途径。我们已经表明，21-nt、减数分裂前 phasiRNA 的生物发生是在花药表皮中启动的，但它们积累在绒毡层中，远端三层细胞，表明跨细胞层的运动。然而，这很大程度上是未知 21-nt phasiRNA 及其生物发生成分在整个花药中的定位开发，以及 21-nt phasiRNA 的目标是什么以及其精确的功能。我们的强初步数据使我们重点关注三个具体目标，以表征 21-nt、减数分裂前生殖玉米中的 phasiRNA，植物生殖生物学、基因组学和遗传学的模型。目标是：（1）使用减数分裂前 phasiRNA 生物发生中关键参与者的时空定位特征先进的成像和单细胞 RNA 测序技术；该目标将生成 21-nt 的时空图 phasiRNA、其前体、microRNA 触发器和生物发生蛋白在玉米花药发育中的作用。 (2) 通过鉴定新的减数分裂前 phasiRNA 的生物发生及其发育作用使用生化方法、使用公开可用的突变体加上 CRISPR-Cas9 的生物发生因子- 生成突变体以了解减数分裂前 phasiRNA 是否是雄性生殖所必需的开发，然后将功能分配给该途径的新组件。（三）明确监管减数分裂前 phasiRNA 介导的机制，使用已建立的和新的分子方法来识别 21-nt phasiRNA 的内源靶标并检查它们如何受到 phasiRNA 的调节。拟议的研究将通过表征生物发生对小 RNA 生物学产生广泛影响以及减数分裂前植物小RNA在玉米花药发育中的功能。实验将提供关键深入了解它们的产生地点和方式，以及它们在花药细胞分化和雄性中的作用生育能力。将这项工作的结果与正在进行的哺乳动物 piRNA 研究的结果进行比较可能会揭示小RNA途径在生殖和性腺发育中的利用的共性。