Molecular analysis of two functional homologues of the S3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations.

Abstract

The S3 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 S1 allele, preceded by an 18 amino acid signal sequence. Expression of the S3 coding region in Escherichia coli produced a form of the protein, denoted S3e, which specifically inhibited S3 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 S1 protein. In contrast to other S proteins identified to date, S3 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 S1 and S3 proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the S1 and S3 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 S3 allele with antiserum raised to S3e, revealed a protein (S3s) which was indistinguishable in pI and Mr from that in the Shirley population. A cDNA encoding S3s 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.