Investigating the role of the S locus F-box protein SLF in RNase-mediated self-incompatibility

Most plants produce flowers with both male and female parts (stamen and pistil respectively). This can result in the pollen produced by the stamens being deposited on the stigma of the same flower leading to 'self' seed. This self seed if produced over several generations can cause the plants to become weakened through inbreeding. In a similar way incest is generally considered a bad thing in human populations because inbreeding can result in genetic defects. Plants have evolved some elaborate schemes to avoid self seed being produced. These include single sex flowers or the shedding of pollen at a different time to the stigma being receptive. A more subtle approach involves a biochemical mechanism that allows the plants to recognise genetically related pollen. In this system the pollen carries a genetic label that identifies it as coming from the same plant. Over the past 20 years scientists have made significant advances in understanding the molecular basis of this pollen recognition process in five plant families. Three of these families, including plants as diverse as tomatoes, snapdragons and cherries, appear to share the same mechanism. This has led evolutionary biologists to speculate that this particular mechanism may be one of the most ancient. The mechanism involves an enzyme made in the pistil that has the capacity to degrade RNA called a ribonuclease. It is believed that self pollen is prevented from growing through the pistil due to the degradation of its RNA leading to the inability of the pollen to make proteins and grow. We know a lot about the genes that code for these ribonucleases and our own research group has identified such genes in petunia and cherry. The mechanism also involves a label in the pollen and this has recently been identified in snapdragon and cherry as a protein involved in the degradation of other proteins. This may provide an explanation for why the ribonuclease is only active in self pollen, since the enzyme itself may be degraded when genetically unrelated pollen is used. To understand this process further we propose to study the two proteins (the ribonuclease in the pistil and the protein degrading factor in the pollen) in two species we are familiar with, petunia and cherry. We will study the protein interactions in a simple micro organism, Baker's yeast that has been developed to allow a simple test for whether proteins are binding to each other. We suspect that other proteins are involved and this yeast system will allow us to screen for such proteins. We will then study the same proteins in genetically modified plants to test whether the interaction occurs there also. Ultimately this research will provide a more complete understanding of this important process in plant reproduction and evolution.

Self-incompatibility (SI) is a genetically controlled recognition mechanism that prevents inbreeding in a wide range of flowering plants. In RNase-based SI, stylar ribonucleases (S-RNases) control the rejection of 'self' pollen and S-locus F-box (SLF) genes have recently been indicated as good candidates for the pollen-S gene, implicating ubiquitination in the SI mechanism. Ubiquitination is emerging as a post-translational modification of major importance in regulating protein activity. This proposal describes an approach to clarify the role of the SLF protein using the model species Petunia hybrida. Until recently, the SLF protein was thought to inactivate S-RNases by targeting them to the proteasome for degradation. However, our recent characterisation of pollen-part mutants in Prunus avium has shown that this model is incorrect. In contrast, it suggests that the F-box protein provides specificity to the inactivation of S-RNases carried out by a general inactivation mechanism in pollen tubes, by protecting 'self' S-RNases from degradation. We will test our prediction that RNAi silencing of the SLF gene results in self-compatibility in Petunia hybrida. The F-box domain suggests the SLF protein acts as part of an E3 ubiquitin ligase complex in targeted protein degradation via the 26S proteasome, but the target has not been identified; it may also have an unexpected function. We will identify proteins interacting with the SLF proteins of both Petunia hybrida and Prunus avium, using a yeast two-hybrid assay. Putative interactions will be verified in vivo using the novel bimolecular fluorescence complementation (BiFC) assay. Targeted silencing of genes encoding proteins found to interact with SLF will be used to investigate the role of these proteins. In addition, existing mutants with a defective SI response due to 'modifier' loci will be analysed. This will help elucidate the role of the SLF gene and the biochemical nature of RNase-mediated SI.