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

大多数开花植物产生的花与男性生殖器官，花药，和女性生殖器官，雌蕊，位于靠近。 这种排列方式使花药产生的花粉落在雌蕊的顶部，导致自花授粉和随后的近亲繁殖。 近亲繁殖对任何生物体都是有害的，因为它会导致后代适应性的降低。 开花植物采用自交不亲和性（SI），这使得雌蕊拒绝自交花粉以防止近亲繁殖，但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吸引了广泛的生物学家的兴趣。 PI已经鉴定了一种雌蕊蛋白和17种花粉蛋白，它们是介导矮牵牛（Petuniainflata）SI的关键分子，矮牵牛是花园矮牵牛的野生亲戚。 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的组合来确定17种花粉蛋白和单一雌蕊蛋白如何共同作用，使雌蕊特异性地拒绝自花花粉。 这些结果对许多其他采用自我/非自我识别的生物系统的研究具有更广泛的意义。 这些蛋白质的基因可用于促进杂交种子的生产，如果实现，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约束数学和科学计划为高中生提供夏季研究机会。在矮牵牛基于S-RNase的自交不亲和性中，多态性S-位点控制授粉的结果;花粉S-单倍型与雌蕊的任一S-单倍型的匹配导致花粉管的生长抑制。 PI已经表明S-RNase基因控制雌蕊特异性，并在S2单倍型中鉴定了17个共同控制花粉特异性的S-位点F-box（SLF）基因。 在本项目中，PI将完成所有17种SLF蛋白与S-RNase的11种等位基因变体之间的187种相互作用关系的确定。 这些结果将用于研究SLF蛋白和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）克隆集体含有S-RNase和17个SLF基因已被分离，测序，并组装，共约3.1 Mbp。 为了填补S基因座序列中的缺口，将分离并测序额外的BAC克隆。 PI已经表明，所有17个SLF蛋白形成类似的SCF复合物，但Cullin 1和Skp 1组分（PiCUL 1-P和PiSSK 1）是花粉特异性的。 将检查PiCUL 1-P和PiSSK 1的敲除和抑制表达的效果，并将确定SCFSLF复合物的X射线结构。 PI发现SLF蛋白本身受到26 S蛋白酶体的泛素介导的降解，并鉴定了可能参与调节SCFSLF复合物动力学的蛋白质。 这些蛋白质的作用将被研究。 所有X射线结晶实验将在合作者的实验室中进行。

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.