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.

大多数开花植物开花时，雄花和雌花生殖器官(花药和雌蕊)在同一朵花中距离很近。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，随后种子发育。由于植物不能自由移动来选择配偶，它们进化出了各种策略来防止有害的近亲繁殖。自交不亲和(SI)是这样一种策略，雌蕊拒绝遗传上相同的自体花粉，而只接受非自体花粉受精。SI被认为是开花植物取得爆炸性成功的主要因素。两个基本问题是：雌蕊如何区分自体和非自体花粉，以及这种自我/非我识别如何导致对自体花粉的特定排斥？为了实现这一目标，开花植物采用了多种机制。PI一直以矮牵牛为模型来研究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、生化、遗传、基因组和结构方法来研究这种机制，花粉利用这种机制来调节由非自体雌蕊产生的有毒蛋白质的变体的降解，从而允许异交，并保持自体雌蕊产生的变体不受影响，从而允许这些雌蕊破坏自体花粉管。这一结果对利用自我/非我识别的其他生物系统的研究具有更广泛的意义。如果能够在农作物中重组SI机械，以促进杂交种子生产，这将带来巨大的农艺效益。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，并将主办高中生的暑期研究。矮牵牛的授粉结果是由S基因座的多态决定的，花粉S单倍型与雌蕊的S单倍型的匹配会导致花粉管的生长抑制。S核糖核酸酶编码雌蕊决定基因，多个S F-box基因编码花粉决定基因。基于协作的非自我识别模型，每个SLF与它的一些非自我S核糖核酸酶相互作用，介导它们在花粉管中的泛素化和降解，从而打开了非自我受精的大门。没有一种SLF与自身的S核糖核酸酶相互作用，使S核糖核酸酶抑制自交花粉管的生长。所有SLF都被组装成SCF(Skp1-Cullin1-F-Box Protein)复合体，每个复合体都含有自交不亲和(SI)特异的Cullin1(PiCUL1-P或PiCUL1-B)和Skp1(PiSSK1)亚基，以及一个典型的Rbx1。本项目将研究三个主题：SCFSLF-S-核糖核酸酶的相互作用，SCFSLF的性质，以及SCFSLF、SLF和S-核糖核酸酶的调节蛋白。OBJ。I研究了S2-SLF1的两个氨基酸在其与S3-RNase特异性相互作用中的作用；鉴定了稳定SCFS2-SLF1与S3-RNase相互作用所需的蛋白质(S)；通过冷冻-EM确定了SCFS2-SLF1：S3-RNase：X的结构。OBJ。II使用细胞器和细胞质标记物来确定SLF是否定位于花粉管细胞质。OBJ。III研究了PiSSK1、PiCUL1-P和PiCUL1-B特有的氨基酸在SI中的作用，并对SCFS2-SLF1的结构进行了建模。OBJ。IV研究可能参与调节SCFSLF动态(激活复合体和降解SLF)的蛋白质。OBJ。V考察了S定位的PDIL(类蛋白质二硫键异构酶)在SI中的作用。这一奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。