The pollen determinant of self-incompatibility in Brassica campestris

Abstract

Many flowering plants possess self-incompatibility (SI) systems that prevent inbreeding. In Brassica, SI is controlled by a single polymorphic locus, the S locus. Two highly polymorphic S locus genes, SLG (S locus glycoprotein) and SRK (S receptor kinase), have been identified, both of which are expressed predominantly in the stigmatic papillar cell. We have shown recently that SRK is the determinant of the S haplotype specificity of the stigma. SRK is thought to serve as a receptor for a pollen ligand, which presumably is encoded by another polymorphic gene at the S locus. We previously have identified an S locus gene, SP11 (S locus protein 11), of the S(9) haplotype of Brassica campestris and proposed that it potentially encodes the pollen ligand. SP11 is a novel member of the PCP (pollen coat protein) family of proteins, some members of which have been shown to interact with SLG. In this work, we identified the SP11 gene from three additional S haplotypes and further characterized the gene. We found that (i) SP11 showed an S haplotype-specific sequence polymorphism; (ii) SP11 was located in the immediate flanking region of the SRK gene of the four S haplotypes examined; (iii) SP11 was expressed in the tapetum of the anther, a site consistent with sporophytic control of Brassica SI; and (iv) recombinant SP11 of the S(9) haplotype applied to papillar cells of S(9) stigmas, but not of S(8) stigmas, elicited SI response, resulting in inhibition of hydration of cross-pollen. All these results taken together strongly suggest that SP11 is the pollen S determinant in SI.

Figure 1

Alignment of nucleotide sequences of SP11–9 and SP11–8 (GenBank accession nos. AB022078 and AB035504, respectively). Gaps (hyphens) were introduced to optimize the alignment. The coding region and the polyadenylation signal consensus sequence for each gene are boxed. The sequence for the putative signal peptide is underlined. Conserved nucleotides are indicated by vertical lines. The arrow indicates the conserved location of the intron.

Figure 2

Alignment of predicted amino acid sequences of four allelic variants of SP11 and of PCP-A1. Gaps (hyphens) were introduced to optimize the alignment. The amino acids of the putative signal peptide for each protein are shown in italics; amino acid residues conserved in all four allelic variants of SP11 are shown in bold; and the conserved cysteine residues are boxed. The asterisk (*) indicates the glutamine residue at the N terminus of the recombinant SP11–9 protein used in pollination bioassays. GenBank accession nos. for SP11–12 and SP11–52 are AB035503 and AB035505, respectively.

Figure 3

Genomic organization of the SLG/SRK region of the S locus of B. campestris. The arrows indicate the direction of transcription of each gene. Exons are indicated by solid boxes and introns are indicated by dips.

Figure 4

Expression of SP11. (a) Northern blot analysis. Total RNA from anther, pistil, and leaf tissues was hybridized with an SP11–8 (coding region) probe and a BcPCP-A1 (coding region) probe. The stage numbers correspond to different bud sizes, with 1 = 0–1 mm, 2 = 1–2 mm, 3 = 2–3 mm, 4 = 3–4 mm, 5 = 4–5 mm, 6 = 5–7 mm, 7 = 7–10 mm, and 8 = open flower. EtBr, ethidium bromide staining of the gel before blotting. (b) In situ hybridization. Anther sections derived from flower buds of stages 5 and 7 were hybridized with SP11–8 and BcPCP-A1 antisense riboprobes and their sense riboprobes (negative control).