Expression of 10 S-class SLF-like genes in Nicotiana alata pollen and its implications for understanding the pollen factor of the S locus

Abstract

The S locus of Nicotiana alata encodes a polymorphic series of ribonucleases (S-RNases) that determine the self-incompatibility (SI) phenotype of the style. The pollen product of the S locus (pollen S) in N. alata is unknown, but in species from the related genus Petunia and in self-incompatible members of the Plantaginaceae and Rosaceae, this function has been assigned to an F-box protein known as SLF or SFB. Here we describe the identification of 10 genes (designated DD1-10) encoding SLF-related proteins that are expressed in N. alata pollen. Because our approach to cloning the DD genes was based on sequences of SLFs from other species, we presume that one of the DD genes encodes the N. alata SLF ortholog. Seven of the DD genes were exclusively expressed in pollen and a low level of sequence variation was found in alleles of each DD gene. Mapping studies confirmed that all 10 DD genes were linked to the S locus and that at least three were located in the same chromosomal segment as pollen S. Finally, the different topologies of the phylogenetic trees produced using available SLF-related sequences and those produced using S-RNase sequences suggests that pollen S and the S-RNase have different evolutionary histories.

Figure 1.—

Amino acid sequences encoded by the 10 N. alata DD cDNAs aligned with representative SLF-like sequences from A. hispanicum, P. inflata, and P. mume. Primer binding sites for degenerate primers D2 and D4 are indicated by arrows. The F-box and four conserved motifs identified in L. Wang et al. (2004) are indicated by solid lines. The C-terminal F-box associated domain (FBA 1 and FBA 3) is indicated by the dashed line below the alignment. Accession numbers of sequences from species other than N. alata are AhSLFS₁ (AJ515535), PiSLFS₁ (AY500390), PiSLFS₂ (AY136628), PiSLFS₃ (AY500392), PiS3A134 (AY363975), and PmSFBS₁ (BAD08320).

Figure 2.—

Maximum-parsimony tree (5087 steps) derived from aligned SLF/SFB and SLF-like DNA sequences. The three Petunia sequences identified with pollen S are indicated by boldface type. Bootstrap values (1000 pseudoreplicates) for numbered nodes are 1 = 100%, 2 = 100%, 3 = 96%, 4 = 100%, 5 = 99%, 6 = 100%. Sequences in the tree and their accession numbers are PaSLFS₁₇ (AY766153), PaSLFS₁₉ (AY766154), PhSLFS3A (AY639403), PhSLFS3B (AY639402), PaF1 (AY766155), PiS3A113 (AY363972), PiS1A134 (AY363973), PiS2A134 (AY363974), PiS2A113 (AY363971), AhSLFS₅ (AJ515536), AhSLF-S1E (AJ515535), AhSLF-S4D (AJ515534), AhSLFS4A (AJ515534), PmSFBS₇ (AB092622), PdSFBa (AB092966), MdSFBB9b (AB270794), MdSFBB9a (AB270793), PpFBB4a (AB270797), PpSFBB4b (AB270798), PavSFB1 (PavSFB1), and PbSFBb (AB081648).

Figure 3.—

RT–PCR was carried out using primers specific for each DD gene shown on the right, with pollen cDNA templates of the indicated S genotype shown at the top of the gel. “No reverse transcriptase controls” are indicated by the minus sign and the size of each PCR product in base pairs is shown at the left of each gel.

Figure 4.—

Spatial expression pattern of DD genes. RT–PCR was performed on N. alata tissue indicated at the top of each gel. Primers used were specific for the gene shown at the right, and the cDNA genotype tested is indicated in parentheses. “No reverse transcriptase controls” are indicated by the minus sign, and actin-specific primers were used to confirm the presence of template. PCR product sizes in base pairs are indicated at the left.

Figure 5.—

Linkage of the DD genes to the S locus. (A) Representative Southern blots using probes specific for the indicated DD gene. The source of genomic DNA is shown at the top of each lane and the different alleles are indicated at the right. (B) Maps of dS₃ in each of the indicated PPMs. A solid dot indicates the centromere. All PPMs used in this experiment have the genotype S₆S₆dS₃. (C) Map of the N. alata S locus showing relative placement of the seven DD genes mapped using this approach. Placement of genes in the segment between CP100 and S-RNase with respect to each other is arbitrary.