Teh-hui Kao 96-03993 Petunia inflata possesses gametophytic self-incompatibility which is controlled by a polymorphic locus, the S locus. Matching of S alleles carried by the pollen and pasta results In Inhibition of pollen tube growth in the style. The goal of this proposal is to understand how a pistil distinguishes between self and non-self pollen. Because so far only the products of pistil S alleles, S proteins or S RNases, have been identified, the first two objectives of this proposal are the identification of the pollen S gene and its allelic products. In the first objective, four approaches will be used to identify potential candidates for the pollen S gene: S protein affinity chromatography, the yeast two-hybrid protein-protein interaction assay, peptide display library screening, and mRNA differential display. cDNAs for the proteins peptides, and PCR fragments identified will be isolated and examined to determine whether their corresponding genes co-segregate with S alleles and show sequence variation between alleles, two criteria for the pollen S gene. The second objective is to use gain-of-function and loss-of-function approaches to ascertain whether any of those pollen genes that meet the above two criteria is the pollen S gene. The third objective is to use two approaches to ascertain whether allele specific inhibition of pollen lies in selective uptake of self S proteins into pollen tubes. The first approach is to examine whether production of a mutant S3 protein lacking RNase activity in the cytoplasm of S3 pollen of transgenic plants has a dominant negative effect on the self-incompatibility behavior of S3 pollen. The second approach is to examine whether S3 protein is taken up by S2 (non-self) and/or S3 (self) pollen tubes by immunolocalization of microscopic sections of pollen tubes that grow out of cut ends of S3S3 pistils that have been pollinated with S2 and S3 pollen, separately. The use of RNases as recognition molecules is unique to the type of self-incompatibility studied here, thus this proposed research when completed will uncover a new paradigm for cellular recognition in biological systems and expand the scope of the understanding of the biological functions of RNases. On the practical side, once the pollen S gene is identified, one can explore the possibility of restoring the self-incompatibility trait to self-compatible cultivated species to facilitate hybrid seed production. If successful, this will have a very important impact on agriculture.
