Molecular characterization and evolution of self-incompatibility genes in Arabidopsis thaliana: the case of the Sc haplotype

Abstract

The switch from an outcrossing mode of mating enforced by self-incompatibility to self-fertility in the Arabidopsis thaliana lineage was associated with mutations that inactivated one or both of the two genes that comprise the self-incompatibility (SI) specificity-determining S-locus haplotype, the S-locus receptor kinase (SRK) and the S-locus cysteine-rich (SCR) genes, as well as unlinked modifier loci required for SI. All analyzed A. thaliana S-locus haplotypes belong to the SA, SB, or SC haplotypic groups. Of these three, the SC haplotype is the least well characterized. Its SRKC gene can encode a complete open-reading frame, although no functional data are available, while its SCRC sequences have not been isolated. As a result, it is not known what mutations were associated with inactivation of this haplotype. Here, we report on our analysis of the Lz-0 accession and the characterization of its highly rearranged SC haplotype. We describe the isolation of its SCRC gene as well as the subsequent isolation of SCRC sequences from other SC-containing accessions and from the A. lyrata S36 haplotype, which is the functional equivalent of the A. thaliana SC haplotype. By performing transformation experiments using chimeric SRK and SCR genes constructed with SC- and S36-derived sequences, we show that the SRKC and SCRC genes of Lz-0 and at least a few other SC-containing accessions are nonfunctional, despite SCRC encoding a functional full-length protein. We identify the probable mutations that caused the inactivation of these genes and discuss our results in the context of mechanisms of S-locus inactivation in A. thaliana.

Figure 1

Comparison of S-haplotype structure in Lz-0 (A), C24 (B), Col-0 (C), and Cvi-0 (D). Numbers in parentheses to the right of the accession name denote the lengths of each of the three cloned segments of the Lz-0 pseudo-S-haplotype, and the PUB8-ARK3 regions of C24, Col-0, and Cvi-0. The gray bars above the map in A indicate the locations of the 2.6-kb 3′ SRKC sequence repeats (see text for details). The dashed lines between A and B link regions of high sequence similarity found in the Lz-0 S haplotype and the recombinant SA-SC haplotype of C24. Transposon sequences were identified using RepBase (Kohany et al. 2006).

Figure 2

Amino-acid sequences of various SCR variants. (A) SCRC variants isolated from various SC-containing A. thaliana accessions are aligned with their functional A. lyrata equivalent, AlSCR36. (B) The Lz-0 SCRC variant is aligned with the SCRA variant of Col-0 along with the corresponding A. lyrata and A. halleri sequences (AlSCR37 and AhSCR-A), and the SCRB variant of Cvi-0 along with the corresponding A. lyrata AlSCR16 sequence. In each alignment, the eight invariant cysteines and one invariant glycine characteristic of SCR proteins are indicated by asterisks above the AtSCRC-Lz sequence, and dots indicate identical amino acids. Signal sequences are shown in boldface type and primer-encoded sequences are represented by dashes at the N- and C termini. Amino-acid positions that differ in the AtSCRC variants relative to AlSCR36 are shaded in the AtSCRC-Lz sequence. The threonine residue in AtSCRC-Lz that is substituted for an isoleucine in AtSCRC-Mr is underlined. The “#” sign in the SCRC variant of the Pro-0 accession indicates the position of a premature stop codon.

Figure 3

DNA gel blot analysis of EcoRI-digested genomic DNA isolated from various A. thaliana accessions. The blot was hybridized with a probe corresponding to Lz-0 SCRC sequences. Molecular length markers are indicated on the left.

Figure 4

Expression of endogenous and transgenic SRK and SCR genes and presence of an SI modifier in Lz-0. (A) RT–PCR of AtSRKC-Lz transcripts in Lz-0 stigmas and of AtSCRC-Lz transcripts in young floral buds. The SRKC-Lz panel shows RT–PCR of AtSRKC-Lz transcripts from floral bud RNA using a forward primer derived from exon 1 in combination with a reverse primer derived from exon 3 (1), or exon 5 (2), or exon 7 (3): the resulting amplicons had the expected sizes for intronless fragments (579 bp, 1028 bp, and 1600 bp, respectively). The SCRC-Lz panel shows RT–PCR of AtSCRC-Lz transcripts from floral bud RNA (1), a no-reverse transcriptase control (2), PCR of genomic DNA (3), and RT–PCR of floral bud RNA from untransformed Lz-0 plants (4). (B) Immunoblot analysis of SRK protein in stigma extracts from a self-fertile Lz-0[AtS1pr::YFP:SRKb-SCRb] transformant and from self-incompatible C24[AtS1pr::YFP:SRKb-SCRb] and Kas[AtS1pr::YFP:SRKb-SCRb] transformants. The “U” lane contains proteins from the stigmas of untransformed Lz-0 plants. The SRKb protein was tagged with yellow fluorescent protein and visualized with anti-GFP antibodies. Antiactin antibodies were used to detect actin as a loading control. Note the equivalent amounts of SRKb in self-compatible and self-incompatible plants. The asterisk indicates the full-length SRK protein; the circle indicates the eSRK, a soluble form of the SRK ectodomain produced from an alternative SRK transcript corresponding to exon 1 and terminating within intron 1. (C) Pollination phenotype of Lz-0[AtS1pr::YFP:SRKb-SCRb] (designated p594) transformants. The absence of pollen tubes indicates an incompatible pollination while the growth of many pollen tubes indicates a compatible pollination.

Figure 5

Diagrams of the SRK (A) and SCR (B) transgenes used in this study.

Figure 6

Pollination analysis of C24 plants transformed with various SRKC-Lz and SCRC-Lz transgenes. Assays were performed by pollinating stigmas from the transformants shown in the first column with pollen from transformants shown in the top row. Shaded blocks show control pollinations of stigmas from the AtS1pr::AleSRK36::AlSRKb and AtS1pr::Ale/tmSRK36::AlSRKb tester lines with pollen from the BrSCR8pr::AlSCR36::ocs tester line. +++, compatible pollination (typically >50 pollen tubes per pollinated stigma); 0, incompatible pollination (typically <5 pollen tubes per pollinated stigma). The numbers in parentheses indicate the number of independent transformants exhibiting the indicated pollination phenotype of the total number of transformants analyzed. [+++] indicates compatible pollinations obtained by performing pollinations between six randomly chosen plants of each genotype.