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

大多数开花植物开花时，雄性生殖器官(花药)和雌性生殖器官(雌蕊)位于很近的位置。这种安排将允许花药产生的花粉落在雌蕊顶部，从而导致自花授粉和随之而来的近亲繁殖。近亲交配对任何生物体都是有害的，因为它会降低后代的适合度。开花植物采用自交不亲和性(SI)，允许雌蕊拒绝自交花粉以防止近亲交配，但接受非自体花粉以促进异交。自从查尔斯·达尔文在1876年出版的一本专著中首次记录了这一现象以来，SI吸引了广泛范围的生物学家的兴趣。PI已经确定了一个雌蕊蛋白和17个花粉蛋白，它们是调节矮牵牛的SI的关键因素，矮牵牛是花园矮牵牛的野生近亲。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是如何共同作用的，从而允许雌蕊特异性地拒绝自体花粉。这些结果对许多其他利用自我/非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以用于促进杂交种子的生产，如果实现这一点，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绑定数学和科学计划为高中生提供暑期研究机会。在基于S-核糖核酸酶的矮牵牛自交不亲和性中，S基因座的多态控制授粉结果；花粉S单倍型与雌蕊S单倍型的匹配导致花粉管生长抑制。等电点分析表明，S核糖核酸酶基因控制着雌蕊专一性，并在S_2单倍型中鉴定出17个S位点F-box基因，它们共同控制着花粉专一性。在这个项目中，PI将完成全部17个SLF蛋白与S核糖核酸酶11个等位基因变异之间的187个相互作用关系的确定。这些结果将被用来研究SLF蛋白和S-核糖核酸酶之间差异相互作用的生化基础，并选择SLF/S-核糖核酸酶对进行相互作用的X射线结构研究。分离、测序和组装了含S核糖核酸酶和17个SLF基因的细菌人工染色体克隆，总长约3.1MbP。为了填补S基因座序列的空白，还将分离和测序更多的BAC克隆。等电点分析表明，所有17个SLF蛋白都形成了类似的SCF复合体，但Cullin1和Skp1组分(PiCUL1-P和PiSSK1)是花粉特有的。将检测PiCUL1-P和PiSSK1的敲除和抑制表达的影响，并确定SCFSLF复合体的X射线结构。PI发现SLF蛋白本身受到26S蛋白酶体泛素介导的降解，并确定了可能参与调节SCFSLF复合体动态的蛋白质。我们将研究这些蛋白质的作用。所有的X射线结晶实验都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.