Many flowering plants avoid inbreeding through a genetic mechanism termed self-incompatibility. An extremely polymorphic S-locus controls the gametophytic self-incompatibility system that causes pollen rejection (that is, active arrest of pollen tube growth inside the style) when an S-allele carried by haploid pollen matches one of the S-alleles present in the diploid style. The only known product of the S-locus is an S-RNase expressed in the mature style. The pollen component to this cell-cell recognition system is unknown and current models propose that it either acts as a gatekeeper allowing only its cognate S-RNase to enter the pollen tube, or as an inhibitor of non-cognate S-RNases. In the latter case, all S-RNases are presumed to enter pollen tubes; thus, the two models make diametrically opposed predictions concerning the entry of S-RNases into compatible pollen. Here we use immunocytochemical labelling of pollen tubes growing in styles to show accumulation of an S-RNase in the cytoplasm of all pollen-tube haplotypes, thus providing experimental support for the inhibitor model.

Self-incompatibility (SI) in angiosperms is a genetic mechanism that promotes outcrossing through rejection of self-pollen. In the Solanaceae, SI is determined by a multiallelic S locus whose only known product is an S RNase. S RNases show a characteristic pattern of five conserved and two hypervariable regions. These are thought to be involved in the catalytic function and in allelic specificity, respectively. When the Solanum chacoense S12S14 genotype is transformed with an S11 RNase, the styles of plants expressing significant levels of the transgene reject S11 pollen. A previously characterized S RNase, S13, differs from the S11 RNase by only 10 amino acids, four of which are located in the hypervariable regions. When S12S14 plants were transformed with a chimeric S11 gene in which these four residues were substituted with those present in the S13 RNase, the transgenic plants acquired the S13 phenotype. This result demonstrates that the S RNase hypervariable regions control allelic specificity.

'Self-incompatibility (SI) in angiosperms is a genetic mechanism that promotes outcrossing through rejection of selfpollen. In the Solanaceae, SI is determined by a multiallelic S locus whose only known product is an S RNase. S RNases show a characteristic pattern of five conserved and two hypervariable regions. These are thought to be involved in the catalytic function and in allelic specificity, respectively. When the Solanum chacoense Si&, genotype is transformed with an Sll RNase, the styles of plants expressing significant levels of the transgene reject Sll pollen. A previously characterized S RNase, S13, differs from the Sl1 RNase by only 10 amino acids, four of which are located in the hypervariable regions. When S1Si4 plants were transformed with a chimeric Sii gene in which these four residues were substituted with those present in the SI, RNase, the transgenic plants acquired the SI3 phenotype. This result demonstrates that the S RNase hypervariable regions control allelic specificity.

Gametophytic self-incompatibility in plants involves rejection of pollen when pistil and pollen share the same allele at the S locus. This locus is highly multiallelic, but the mechanism by which new functional S alleles are generated in nature has not been determined and remains one of the most intriguing conceptual barriers to a full understanding of self-incompatibility. The S(11) and S(13) RNases of Solanum chacoense differ by only 10 amino acids, but they are phenotypically distinct (i.e., they reject either S(11) or S(13) pollen, respectively). These RNases are thus ideally suited for a dissection of the elements involved in recognition specificity. We have previously found that the modification of four amino acid residues in the S(11) RNase to match those in the S(13) RNase was sufficient to completely replace the S(11) phenotype with the S(13) phenotype. We now show that an S(11) RNase in which only three amino acid residues were modified to match those in the S(13) RNase displays the unprecedented property of dual specificity (i.e., the simultaneous rejection of both S(11) and S(13) pollen). Thus, S(12)S(14) plants expressing this hybrid S RNase rejected S(11), S(12), S(13), and S(14) pollen yet allowed S(15) pollen to pass freely. Surprisingly, only a single base pair differs between the dual-specific S allele and a monospecific S(13) allele. Dual-specific S RNases represent a previously unsuspected category of S alleles. We propose that dual-specific alleles play a critical role in establishing novel S alleles, because the plants harboring them could maintain their old recognition phenotype while acquiring a new one.

Gametophytic self-incompatibility in plants involves rejection of pollen when pistil and pollen share the same allele at the S locus. This locus is highly multiallelic, but the mechanism by which new functional S alleles are generated in nature has not been determined and remains one of the most intriguing conceptual barriers to a full understanding of selfincompatibility. The S 11 and S 13 RNases of Solanum chacoense differ by only 10 amino acids, but they are phenotypically distinct (i.e., they reject either S 11 or S 13 pollen, respectively). These RNases are thus ideally suited for a dissection of the elements involved in recognition specificity. We have previously found that the modification of four amino acid residues in the S 11 RNase to match those in the S 13 RNase was sufficient to completely replace the S 11 phenotype with the S 13 phenotype. We now show that an S 11 RNase in which only three amino acid residues were modified to match those in the S 13 RNase displays the unprecedented property of dual specificity (i.e., the simultaneous rejection of both S 11 and S 13 pollen). Thus, S 12 S 14 plants expressing this hybrid S RNase rejected S 11 , S 12 , S 13 , and S 14 pollen yet allowed S 15 pollen to pass freely. Surprisingly, only a single base pair differs between the dual-specific S allele and a monospecific S 13 allele. Dual-specific S RNases represent a previously unsuspected category of S alleles. We propose that dualspecific alleles play a critical role in establishing novel S alleles, because the plants harboring them could maintain their old recognition phenotype while acquiring a new one. INTRODUCTIONAmong the cell-cell recognition phenomena present in living organisms, self-incompatibility (SI) plays a major evolutionary role because it constitutes an important mechanism for preventing inbreeding. SI is present in hermaphroditic animals such as tunicates (Grosberg, 1988), in fungi (Kronstad and Leong, 1990), and in many Angiosperm families (de Nettancourt, 1977). In the most widespread type of SI, gametophytic SI, the genotype of the haploid pollen determines its own incompatibility phenotype. For the Solanaceae, the gametophytic SI phenotype is specified by a highly multiallelic S locus (de Nettancourt, 1977(de Nettancourt, , 1997 whose only known product is a ribonuclease (S RNase; McClure et al., 1989) expressed in the transmitting tissue of the style (Anderson et al., 1986). Gain-of-function experiments have shown that expression of an S RNase transgene is sufficient to alter the SI phenotype of the pistil but not that of the pollen Murfett et al., 1994;Matton et al., 1997), and thus the identity of the pollen S gene (unknown to date) is likely to be different from that of the S RNase (Kao and McCubbin, 1997). RNase activity, although essential for expression of the SI phenotype , seems not to be involved in the specificity of the cell-cell recognition phenomenon. In closely related S RNases, such specificity has been shown to depend on the amino acid sequence at the two hypervariable ...

The stylar component to gametophytic self-incompatibility in Solanaceae is an S-RNase. Its primary structure has a characteristic pattern of two hypervariable regions, involved in pollen recognition, and five constant regions. Two of the latter (C2 and C3) constitute the active site, while the highly hydrophobic C1 and C5 are believed to be involved in protein stability. We analyzed the role of the C4 region by site-directed mutagenesis. A GGGG mutant, in which the four charged residues in the C4 region were replaced with glycine, did not accumulate the protein to detectable levels in styles, suggestive of a role in protein stability. A R115G mutant, in which a charged amino acid was eliminated to reduce the potential binding affinity, had no effect on the pollen rejection phenotype. This suggests the C4 does not interact with partners such as potential pollen tube receptors facilitating S-RNase uptake. Finally, a K113R mutant replaced a potential ubiquitination target with arginine. However, this RNase acted as the wild type in both incompatible and compatible crosses. The latter crosses rule out the role of the conserved C4 lysine in ubiquitination.

The stylar determinant of gametophytic self-incompatibility (GSI) in Solanaceae, Rosaceae, and Plantaginaceae is an S-RNase encoded by a multiallelic S-locus. The primary structure of S-RNases shows five conserved (C) and two hypervariable (HV) regions, the latter forming a domain implicated in S-haplotype-specific recognition of the pollen determinant to SI. All S-RNases are glycosylated at a conserved site in the C2 region, although previous studies have shown that N-linked glycans at this position are not required for S-haplotype-specific recognition and pollen rejection. Here the incompatibility phenotype of three constructs derived from an originally monoglycosylated S11-RNase of Solanum chacoense, that were designed to explore the role of the HV domain in determining pollen recognition and the role of the N-linked glycan in the C2 region, is reported. In one series of experiments, a second glycosylation site was introduced in the HVa region to test for inhibition of pollen-specific recognition. This modification does not impede pollen rejection, although analysis shows incomplete glycosylation at the new site in the HVa region. A second construct, designed to permit complete glycosylation at the HVa site by suppression of the conserved site in the C2 region, did increase the degree of site occupancy, but, again, glycosylation was incomplete. Plants expressing this construct rejected S 11 pollen and, surprisingly, also rejected S 13 pollen, thus displaying an unusual dual specificity phenotype. This construct differs from the first by the absence of the conserved C2 glycosylation site, and thus the dual specificity is observed only in the absence of the C2 glycan. A third construct, completely lacking glycosylation sites, conferred an ability to reject only S 11 pollen, disproving the hypothesis that lack of a conserved glycan would confer a universal pollen rejection phenotype to the plant.

S-heteroallelic pollen (HAP) grains are usually diploid and contain two different S-alleles. Curiously, HAP produced by tetraploids derived from self-incompatible diploids are typically self-compatible. The two different hypotheses previously advanced to explain the compatibility of HAP are the lack of pollen-S expression and the “competition effect” between two pollen-S gene products expressed in a single pollen grain. To distinguish between these two possibilities, we used a previously described dual-specific S11/13-RNase, termed HVapb-RNase, which can reject two phenotypically distinct pollen (P11 and P13). Since the HVapb-RNase does not distinguish between the two pollen types (it recognizes both), P11P13 HAP should be incompatible with the HVapb-RNase in spite of the competition effect. We show here that P11P13 HAP is accepted by S11S13 styles, but is rejected by the S11/13-RNase, which demonstrates that the pollen-S genes must be expressed in HAP. A model involving tetrameric pollen-S is proposed to explain both the compatibility of P11P13 HAP on S11S13-containing styles and the incompatibility of P11P13 HAP on styles containing the HVapb-RNase.