S-locus Glycoprotein Gene Research Articles

Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea.

In Brassica oleracea, the pollen-stigma interaction of self-incompatibility is controlled by a single genetically defined locus designated S. Molecular studies have identified two genes that are tightly linked to the classically defined S locus: The S-Locus Glycoprotein (SLG) gene and the S-Receptor Kinase (SRK) gene. In previous RFLP linkage analyses with probes specific for SLG and SRK, we were unable to identify any recombination events between SLG, SRK, and self-incompatibility phenotype. In this paper, we use pulsed-field gel electrophoresis (PFGE) in conjunction with DNA blot analysis to characterize the S-locus region from two highly divergent self-incompatibility genotypes, S2 and S6. We establish the physical linkage of SLG and SRK in each genotype, and demonstrate that the two genes are separated by a maximum distance of 220 kb in the S6 genotype and 350 kb in the S2 genotype. Furthermore, a comparison of the data from the two genotypes reveals that a high level of polymorphism exists across the entire S-locus region.

Use of the polymerase chain reaction to isolate an S-locus glycoprotein cDNA introgressed from Brassica campestris into B. napus ssp. oleifera.

A self-incompatible canola-quality Brassica napus ssp. oleifera line (W1) was generated by introgressing the S-locus from a self-incompatible B. campestris plant into the Westar cultivar. Using the polymerase chain reaction (PCR) with primers derived from conserved regions in S-locus glycoprotein (SLG) alleles, the central region of the active SLG gene (910) was obtained. The remaining portions of the cDNA for this 910 gene were subsequently cloned using the PCR-rapid amplification of cDNA ends (RACE) procedure. Sequence analysis revealed that the 910 cDNA show a high degree of sequence similarity to SLG alleles associated with Class I self-incompatible lines. The 910 gene was found to be absent in the original self-compatible cv. Westar (B. napus) and segregated with self-incompatibility in a mixed population generated from a cross between self-incompatible W1 and self-compatible Westar. RNA blot analysis indicated that high levels of 910 mRNAs were present in the stigma as buds approached anthesis. Thus, the SLG allele of W1 transferred from B. campestris via backcrosses to a line of cv. Westar has been identified.

Expression of S-locus glycoprotein genes from Brassica oleracea and B. campestris in transgenic plants of self-compatible B. napus cv Westar

Self-compatible Brassica napus var ‘Westar’ was transformed with SLG, the S-locus-derived gene that encodes S-locus-specific glycoproteins (SLSG). Four allelic variants of SLG isolated from self-incompatible B. oleracea and B. campestris strains homozygous for different S alleles were used. We show that the transgenic plants synthesized SLSG with the same apparent charge, molecular weight, and antigenic properties as that produced by the corresponding self-incompatible strains from which the cloned SLG genes were isolated. In addition, transgene-encoded SLSG was detected specifically in the papillar cells of the stigma, and was correctly targeted to the papillar cell wall. However, SLSG was produced at reduced levels in transgenic plants relative to self-incompatible strains. The introduction of the SLG genes did not confer a self-incompatibility phenotype on the ‘Westar’ cultivar.

An S5 self-incompatibility allele-specific cDNA sequence from Brassica oleracea shows high homology to the SLR2 gene

The sequence is reported of a cDNA molecule homologous to an mRNA from stigma tissue of Brassica oleracea plants homozygous for the S5 self-incompatibility allele. This cDNA is closely related to a previously published sequence designated SLR2, which was obtained from the same stigma cDNA library and is also related to the SLR1 gene, a cDNA for which has also been obtained from this library. Various B. oleracea lines differing in S alleles and of different varieties have been screened for the presence of particular S gene family sequences using a method involving hybridization of sequence-specific oligonucleotide probes to PCR products. The results indicate that a gene homologous to the sequence presented here is absent from a line lacking the S5 allele, though present in other lines containing the S5 allele, regardless of their genetic background. This finding suggests that the sequence represents a transcript of the SLG (S locus glycoprotein) gene. A similar approach has confirmed that a cDNA derived from a Brassica line containing the S29 allele is also S allele-specific. The predicted amino acid sequences derived from a number of S gene family sequences are compared using numerical methods and possible evolutionary relationships between them are discussed.

A Brassica S locus gene promoter directs sporophytic expression in the anther tapetum of transgenic Arabidopsis

The S locus glycoprotein (SLG) gene of Brassica encodes stigmatic glycoproteins that are implicated in the pollenstigma interaction of self-incompatibility. We have transformed the related plant Arabidopsis thaliana with a chimaeric gene consisting of the promoter region of an SLG gene fused to the reporter gene β-glucuronidase (GUS). In transgenic plants the gene was expressed in two cell types of the flower. In stigmas, the timing and distribution of GUS activity was similar to that previously described for SLG expression in Brassica. In anthers, expression was detected at an earlier stage of flower development with GUS activity restricted to the tapetal cell layer. The novel finding of SLG-promoter activity in the anther supports the hypothesis that sporophytic control of self-incompatibility is a result of SLG-gene expression in the tapetum.

A Brassica S-locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana

The S-locus glycoprotein gene of Brassica is derived from the genetic locus that controls the self-incompatibility response and the specific recognition between pollen and stigma. The promoter of this gene was used to direct expression of the diphtheria toxin A chain gene and the Escherichia coli β-glucuronidase gene in transgenic Nicotiana tabacum. Expression of the promoter in cells of the pistil and in pollen suggests that a single gene may direct the self-incompatibility response in the two interacting cell types. Additionally, the fusion genes were expressed gametophytically in the heterologous host species, Nicotiana, rather than sporophytically as expected for Brassica. Thus, although the genes involved in self-incompatibility in Brassica and Nicotiana are not homologous in their coding regions, signals for expression of these genes are apparently conserved between the two genera. Our analysis of toxic gene fusion transformants shows that genetic ablation is useful for probing developmental processes and for studying temporal and spatial patterns of gene expression in plants.