9316152 McClure In solanaceous plants with gametophytic self-incompatibility (GSI), S-allele pollen recognition and rejection are controlled by a single genetic locus, the S-locus. Pollen is rejected if its single S-allele matches either S-allele in the pistil. Information is communicated about the allelic constitution of the S-locus in the sporophyte and in the gametophyte. The products of the S-locus are the carriers of this information. The S-locus products expressed in the pistil comprise a series of allelic ribonucleases, the S-RNases. It has been shown that each S- allele encodes a different S-RNase. However, it is not known how these differences lead to S-allele specific recognition. No S-allele specific product has yet been identified in the male gametophyte (i.e., pollen-S). This proposal seeks to develop an understanding of the molecular basis for S-allele specific recognition and rejection. The first step will be to develop a system in which S-allele specific recognition and rejection can be manipulated. Cloned S-RNases from Nicotiana alata will be used to introduce new allelic specificity into transgenic plants. Using the expression system, it will be possible to manipulate cloned S-RNase sequences in vitro and assess the effects on pollination behavior in vivo. The choice of plant materials for use as transgene recipient is very important. The transformation efficiency of N. alata is too low to generate the necessary number of transformants. Therefore, transgenes will be introduced into a plant such as Nicotiana plumbaginifolia and crossed into N. alata or into a transformable F1 hybrid capable of S-allele specific pollen rejection. In either case, the inherent specificity of the SI reaction provides excellent controls. Dr. McClure will look for the introduction of, or modification of, a new specificity in the transgenic plants. Identification of the pollen-S product is a major goal of this proposal. A combined genetic and molecul ar biological approach is proposed. A large F2 population segregating for two S- alleles has been generated. Plants homozygous for a given S- allele will be pooled. Using these pooled materials, subtractive hybridization and differential mRNA display will be used to identify a putative pollen-S cDNA. GSI has the potential to help understand plant cell-cell communication. To realize its potential, GSI research must move into an analytical phase. This proposal aims to accomplish this by developing a system for direct analysis of the allelic specificity function of S-RNases, and identifying the product responsible for allele specific recognition in pollen. ***
