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

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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.