Pollen-Pistil Interactions in Nicotiana

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IBN-0315647McClureBecause plants are sessile, they rely on external agents such as wind or insects to disperse pollen and effect fertilization. Thus, pollen dispersal is relatively non-specific, and plants have evolved elaborate pollen-pistil interactions to control mating. The overall goal of this research is to understand the molecular basis for these interactions. The experimental system is S-RNase-based pollen recognition and rejection in Nicotiana. S-RNase determines the specificity of pollen recognition in this system. The model is that S-RNase is secreted into the extracellular matrix of the pistil where it forms complexes with glycoproteins known to interact with pollen tubes. Binding to the glycoproteins facilitates S-RNase uptake by pollen tubes. Once inside the pollen tube, a newly identified pistil factor (i.e., 4936-factor) is required for S-RNase to cause pollen rejection. Pollen factors also contribute. Some bind directly to the glycoproteins present in S-RNase complexes. Others directly determine compatibility either by inhibiting S-RNase's cytotoxic activity or by preventing such inhibition. This project has three objectives designed to test aspects of this model. First, a comprehensive screen for pistil-expressed sequences will be used to identify candidates for 4936-factor. Candidates will be sequenced, and their expression patterns will be examined by RNA blot analysis. Antibodies will be raised to test for uptake into pollen tubes. The model predicts that 4936-factor will activate S-RNase after uptake into pollen tubes. Second, a yeast two-hybrid screen for pollen and pistil factors that interact with S-RNase binding glycoproteins will be conducted. The model predicts that pollen expresses proteins that will bind to these glycoproteins. Third, immunolocalization experiments will be performed to determine whether RNase uptake occurs normally in plants with a variety of different pollen rejection responses. These experiments may reveal new steps in the model or may help define when factors act during pollen recognition and rejection. This project is linked to undergraduate teaching. Using the arrayed cDNA libraries produced to screen for 4936-factor, students will conduct an EST project to identify sequences expressed in mature pistils. Students will pick clones, analyze sequences, and pool their data to make an EST list. These activities will better prepare them for later research experiences.