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

大多数开花植物的花是由雄性生殖器官花药和雌性生殖器官雌蕊紧密相连形成的。这种排列将使花药产生的花粉落在雌蕊的顶部，从而导致自花授粉和随后的近亲繁殖。近亲繁殖对任何生物都是有害的，因为它会降低后代的适应性。开花植物采用自交不亲和（self incompatibility， SI），雌蕊拒绝自交花粉以防止近亲繁殖，而接受非自交花粉以促进异交。自从查尔斯·达尔文在1876年出版的专著中首次记录了这一现象以来，SI就吸引了广泛的生物学家的兴趣。研究人员鉴定出1个雌蕊蛋白和17个花粉蛋白，它们是矮牵牛（Petunia inflata）野生亲缘植物中介导SI的关键蛋白。在这个项目中，PI和他的合作者将使用分子、生化、遗传、基因组和结构方法的组合来确定17种花粉蛋白和单个雌蕊蛋白如何协同工作，从而使雌蕊特异性地拒绝自花花粉。这一结果对许多其他生物系统的自我/非自我识别的研究具有更广泛的意义。这些蛋白质的基因可以用来促进杂交种子的生产，如果完成，将提供巨大的农艺效益。PI将让研究生和本科生参与这个项目，为他们未来的研究生涯做好准备，并将通过宾夕法尼亚州立大学的向上数学和科学项目为高中生提供暑期研究机会。在矮牵牛花s - rase自交不亲和中，多态s位点控制授粉结果；花粉s -单倍型与雌蕊任一s -单倍型的匹配均会抑制花粉管的生长。PI研究表明，S-RNase基因控制雌蕊特异性，并在s2单倍型中鉴定出17个S-locus F-box （SLF）基因共同控制花粉特异性。在本项目中，PI将完成所有17个SLF蛋白与S-RNase的11个等位变异之间187个相互作用关系的测定。这些结果将用于研究SLF蛋白与S-RNase之间差异相互作用的生化基础，并选择SLF/S-RNase对进行相互作用的x射线结构研究。细菌人工染色体（BAC）克隆共包含S-RNase和17个SLF基因，已被分离、测序和组装，总长度约3.1 Mbp。为了填补s位点序列的空白，将对额外的BAC克隆进行分离和测序。PI表明，所有17种SLF蛋白形成相似的SCF复合物，但Cullin1和Skp1组分（PiCUL1-P和PiSSK1）是花粉特异性的。将检测敲除和抑制PiCUL1-P和PiSSK1表达的效果，并确定SCFSLF复合物的x射线结构。PI发现SLF蛋白本身会受到26S蛋白酶体泛素介导的降解，并确定了可能参与调节SCFSLF复合物动力学的蛋白质。我们将研究这些蛋白质的作用。所有的x射线结晶实验将在合作者的实验室进行。

Most flowering plants produce flowers with the male reproductive organ, the anther, and female reproductive organ, the pistil, located in close proximity. This arrangement would allow the pollen produced by the anther to land on the top of the pistil to result in self-pollination and consequent inbreeding. Inbreeding is deleterious to any organism, as it causes reduced fitness in progeny. Flowering plants have adopted self-incompatibility (SI), which allows the pistil to reject self-pollen to prevent inbreeding, but accept non-self pollen to promote outcrossing. Since Charles Darwin first documented this phenomenon in a monograph published in 1876, SI has attracted the interest of a wide spectrum of biologists. The PI has identified a pistil protein and 17 pollen proteins that are key players in mediating SI in Petunia inflata, a wild relative of garden petunia. In this project, the PI and his collaborator will use a combination of molecular, biochemical, genetic, genomic, and structural approaches to determine how the 17 pollen proteins and a single pistil protein work together to allow the pistil to specifically reject self-pollen. The results have wider implications for the study of many other biological systems employing self/non-self recognition. The genes for these proteins could be used to facilitate hybrid-seed production, which, if accomplished, would provide tremendous agronomic benefits. The PI will engage graduate and undergraduate students in this project to prepare them for future research careers, and will offer summer research opportunity for high school students through Penn State's Upward Bound Math and Science Program. In S-RNase-based self-incompatibility in Petunia inflata, the polymorphic S-locus controls the outcome of pollination; matching of the pollen S-haplotype with either S-haplotype of the pistil results in growth inhibition of pollen tubes. The PI has shown that the S-RNase gene controls pistil specificity and identified 17 S-locus F-box (SLF) genes in S2-haplotype that collectively control pollen specificity. In this project, the PI will complete the determination of 187 interaction relationships between all 17 SLF proteins and 11 allelic variants of S-RNase. These results will be used to study the biochemical basis of differential interactions between SLF proteins and S-RNases, and select SLF/S-RNase pairs for X-ray structural study of the interactions. Bacterial Artificial Chromosome (BAC) clones collectively containing the S-RNase and 17 SLF genes have been isolated, sequenced, and assembled, totaling ~3.1 Mbp. To fill gaps in the S-locus sequence, additional BAC clones will be isolated and sequenced. The PI has shown that all 17 SLF proteins form similar SCF complexes, but the Cullin1 and Skp1 components (PiCUL1-P and PiSSK1) are pollen-specific. The effect of knockout and suppressed expression of PiCUL1-P and PiSSK1 will be examined, and the X-ray structure of the SCFSLF complex will be determined. The PI has found that SLF proteins themselves are subject to ubiquitin-mediated degradation by the 26S proteasome, and identified proteins that may be involved in regulating the dynamics of the SCFSLF complex. The role of these proteins will be examined. All X-ray crystallization experiments will be performed in the lab of the collaborator.