Molecular analysis of two functional homologues of the S-3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations

The S-3 allele of the S gene has been cloned from Papaver rhoeas cv. Shirley. The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S-1 allele, preceded by an 18 amino acid signal sequence. Expression of the S-3 coding region in Escherichia coli produced a form of the protein, denoted S(3)e, which specifically inhibited S-3 pollen in an in vitro bioassay. The recombinant protein was ca. 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S-1 protein. In contrast to other S proteins identified to date, S-3 protein does not appear to be glycosylated. Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S-1 and S-3 proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the P. rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea. Previously, cross-classification of different populations of P. rhoeas had revealed a number of functionally identical alleles. Probing of western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S-3 allele with antiserum raised to S(3)e, revealed a protein (S(3)s) which was indistinguishable in pi and M(r) from that in the Shirley population. A cDNA encoding S(3)s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level.