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

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.