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

项目总结 在许多开花植物物种中，孕育花粉粒的雄性生殖器官(花药) (含有精子，植物的雄性生殖系)及其祖细胞，积累了两类小的 RNAs，即减数分裂前(21-nT)和减数分裂(24-nT)相的小干扰RNA(PhasiRNAs)。 水稻或玉米中phasiRNA生物发生途径的扰动导致环境敏感型雄性 不孕不育。在这些植物phasiRNA和动物PIWI相互作用之间有许多有趣的相似之处 RNA(PiRNAs)，这也被证明是男性生育所必需的。然而，我们有一个很大的差距， 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 植物雄性生殖发育对这些小RNA的依赖。 我们和合作者已经描述了许多植物物种中的生殖phasiRNA途径。我们 已经表明，21-NT，减数分裂前的phasiRNAs的生物发生是在花药表皮启动的，但它们 在绒毛层中积累，远端有三层细胞，表明细胞层之间的运动。然而，它在很大程度上 未知21-NT的phasiRNAs及其生物发生成分在整个花药中的定位 发展，以及目标是什么，因此21-nT phasiRNA的确切功能。我们的强者 初步数据使我们将重点放在三个具体目标上，以表征21-NT，减数分裂前生殖 玉米中的phasiRNAs，植物生殖生物学、基因组学和遗传学的模型。目标是：(1) 描述减数分裂前phasiRNA生物发生中关键角色的时空定位，使用 先进的成像和单细胞rna-seq技术；这一目标将产生21-nT的时空图 在玉米花药发育中，phasiRNAs、其前体、microRNA触发器和生物发生蛋白。(2) 通过对新基因的鉴定，破译减数分裂前phasiRNAs的生物发生及其发育作用 使用生化方法的生物发生因子，使用公开可用的突变体加上CRISPR-Cas9- 产生突变体以了解减数分裂前的phasiRNA是否对雄性生殖是必要的 开发，然后将功能分配给该途径的新组件。以及(3)阐明调控机制 减数分裂前αRNA介导的机制，使用已有的和新的分子方法来鉴定 21-NT phasiRNAs的内源靶点，并研究它们是如何被phasiRNAs调控的。 这项研究将通过描述小RNA的生物发生过程对其生物学产生广泛的影响 以及减数分裂前植物小RNA在玉米花药发育中的作用。这些实验将提供关键 洞察它们在哪里和如何产生，以及它们在花药细胞分化和雄性中所扮演的角色 生育能力。将这项工作的结果与正在进行的哺乳动物piRNAs研究进行比较，可能会揭示 小RNA途径在生殖和性腺发育中利用的共性。

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.