Molecular, Biochemical, and Structural Studies of the Mechanism of S-RNase-Based Self-Incompatibility in Petunia

矮牵牛基于 S-RNase 的自交不亲和机制的分子、生化和结构研究

• 批准号: 2138062
• 负责人: Teh-hui Kao
• 金额: $ 70万
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2138062&HistoricalAwards=false
• 关键词: Molecular; Biochemical; Structural; Studies; Mechanism

Most flowering plant species produce flowers with male and female reproductive organs (anther and pistil) located in close proximity in the same flowers. This arrangement is conducive to self-fertilization, where pollen from the anther lands on the pistil and germinates, leading to fertilization of the ovule, followed by seed development. As plants cannot move freely to select mates, they have evolved a variety of strategies to prevent deleterious inbreeding. Self-incompatibility (SI) is one such strategy, by which the pistil rejects genetically identical self-pollen and only accepts non-self pollen for fertilization. SI is thought to be a major factor for the explosive success of flowering plants. Two fundamental questions are: how does a pistil distinguish self- and non-self pollen, and how does this self/non-self recognition result in specific rejection of self-pollen? Multiple mechanisms have been adopted by flowering plants to achieve this goal. The PI has been using Petunia inflata as a model to study a SI mechanism found in Solanaceae and two other families. In this project, the PI and his collaborator will use molecular, biochemical, genetic, genomic, and structural approaches to study this mechanism, employed by pollen, to mediate degradation of variants of a toxic protein produced by non-self pistils to allow outcrossing and to leave untouched the variant produced by self-pistils, allowing these pistils to destroy self-pollen tubes. The results have wider implications for the study of other biological systems employing self/non-self recognition. If the SI machinery could be reconstituted in crop plants to facilitate hybrid seed production, this would have tremendous agronomic benefits. The PI will engage a postdoc, and graduate and undergraduate students to prepare them for future professional careers and will host summer research of high school students. The outcome of pollination in Petunia inflata is determined by the polymorphic S-locus; matching of the pollen S-haplotype with either S-haplotype of the pistil results in growth inhibition of pollen tubes. S-RNase encodes the pistil determinant; multiple S-locus F-box (SLF) genes encode the pollen determinant. Based on the collaborative non-self recognition model, each SLF interacts with some of its non-self S-RNases to mediate their ubiquitination and degradation inside pollen tubes, thereby opening the door to non-self fertilization. None of the SLFs interact with their self S-RNase, allowing the S-RNAse to inhibit growth of self-pollen tubes. All SLFs are assembled into SCF (Skp1-Cullin1-F-Box protein) complexes, each containing self-incompatibility (SI)-specific Cullin1 (PiCUL1-P or PiCUL1-B) and Skp1 (PiSSK1) subunits, and a canonical Rbx1. This project will study three themes: SCFSLF-S-RNase interactions, nature of SCFSLF, and regulatory proteins of SCFSLF, SLF and S-RNase. Obj. I examines the role of two amino acids of S2-SLF1 in its specific interaction with S3-RNase; identifies protein(s) that is(are) required to stabilize the interaction between SCFS2-SLF1 and S3-RNase; determines the structure of SCFS2-SLF1:S3-RNase:X by Cryo-EM. Obj. II uses organelle and cytosol markers to determine whether SLFs are localized to the pollen tube cytosol. Obj. III examines the roles of the amino acids unique to PiSSK1, PiCUL1-P, and PiCUL1-B in their function in SI; and models the structure of SCFS2-SLF1. Obj. IV studies the proteins that may be involved in regulating the dynamics of SCFSLF (activation of the complex and degradation of SLFs). Obj. V examines the role of S-locus-localized PDIL (Protein Disulfide Isomerase-Like) in SI.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.

大多数开花植物物种产生的花具有位于相同花中的紧密靠近的雄性和雌性生殖器官（花药和雌蕊）。这种排列有利于自花受精，花药中的花粉落在雌蕊上并萌发，导致胚珠受精，随后是种子发育。由于植物不能自由选择配偶，它们进化出各种策略来防止有害的近亲繁殖。自交不亲和（SI）就是这样一种策略，雌蕊通过这种策略拒绝遗传上相同的自花花粉，只接受非自花花粉进行受精。SI被认为是显花植物爆炸性成功的主要因素。两个基本问题是：雌蕊如何区分自花和非自花花粉，这种自花/非自花的识别又是如何导致对自花花粉的特异性排斥的？开花植物采用多种机制来实现这一目标。PI一直使用矮牵牛作为模型来研究在茄科和其他两个科中发现的SI机制。在这个项目中，PI和他的合作者将使用分子，生物化学，遗传学，基因组学和结构方法来研究花粉所采用的这种机制，以介导非自我雌蕊产生的有毒蛋白质变体的降解，以允许异交，并使自雌蕊产生的变体保持不变，允许这些雌蕊破坏自花粉管。这些结果对研究其他使用自我/非自我识别的生物系统具有更广泛的意义。如果SI机器可以在作物植物中重建以促进杂交种子生产，这将具有巨大的农艺效益。PI将聘请博士后，研究生和本科生为他们未来的职业生涯做准备，并将主持高中生的暑期研究。矮牵牛授粉的结果是由多态性的S-位点;匹配的花粉S-单倍型与任一S-单倍型的雌蕊的结果在生长抑制的花粉管。S-RNase编码雌蕊决定子，多个S-locus F-box（SLF）基因编码花粉决定子。基于协同非自我识别模型，每个SLF与其一些非自我S-RNases相互作用，介导它们在花粉管内的泛素化和降解，从而打开了非自我受精的大门。没有SLF与它们自身的S-RNase相互作用，允许S-RNase抑制自花花粉管的生长。所有的SLF被组装成SCF（Skp 1-Cullin 1-F-Box蛋白）复合物，每个复合物含有自交不亲和（SI）特异性Cullin 1（PiCUL 1-P或PiCUL 1-B）和Skp 1（PiSSK 1）亚基，以及典型的Rbx 1。本项目将研究三个主题：SCFSLF-S-RNase相互作用，SCFSLF的性质，以及SCFSLF，SLF和S-RNase的调节蛋白。目的研究S2-SLF 1的两个氨基酸在与S3-RNase的特异性相互作用中的作用;鉴定稳定SCFS 2-SLF 1与S3-RNase相互作用所需的蛋白质;用冷冻电镜确定SCFS 2-SLF 1：S3-RNase：X的结构。目的II利用细胞器和胞质标记来确定SLF是否定位于花粉管胞质。目的III研究PiSSK 1、PiCUL 1-P和PiCUL 1-B特有的氨基酸在SI中的功能中的作用;并模拟SCFS 2-SLF 1的结构。目的IV研究可能参与调节SCFSLF动力学（复合物的激活和SLF的降解）的蛋白质。目的V考察了S位点定位的PDIL（蛋白质二硫化物异构酶样）在SI中的作用。该奖项反映了NSF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。