S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of Petunia Pollen.

Self-incompatibility (SI) in Petunia is regulated by a polymorphic S-locus. For each S-haplotype, the S-locus contains a pistil-specific S-RNase gene and multiple pollen-specific S-locus F-box (SLF) genes. Both gain-of-function and loss-of-function experiments have shown that S-RNase alone regulates pistil specificity in SI. Gain-of-function experiments on SLF genes suggest that the entire suite of encoded proteins constitute the pollen specificity determinant. However, clear-cut loss-of-function experiments must be performed to determine if SLF proteins are essential for SI of pollen. Here, we used CRISPR/Cas9 to generate two frame-shift indel alleles of S2 -SLF1 (SLF1 of S2 -haplotype) in S2S3 plants of P. inflata and examined the effect on the SI behavior of S2 pollen. In the absence of a functional S2-SLF1, S2 pollen was either rejected by or remained compatible with pistils carrying one of eight normally compatible S-haplotypes. All results are consistent with interaction relationships between the 17 SLF proteins of S2 -haplotype and these eight S-RNases that had been determined by gain-of-function experiments performed previously or in this work. Our loss-of-function results provide definitive evidence that SLF proteins are solely responsible for SI of pollen, and they reveal their diverse and complex interaction relationships with S-RNases to maintain SI while ensuring cross-compatibility.

CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata.

KEY MESSAGE: Function of Petunia PiSSK1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S 2-haplotype and S 3-haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCFSLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1 and examined the SI behavior of a T 0 plant (S 2 S 3) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatible S-genotypes, but fully compatible with pistils of an S 3 S 3 transgenic plant in which production of S3-RNase was completely suppressed by an antisense S 3-RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.

Use of Domain-Swapping to Identify Candidate Amino Acids Involved in Differential Interactions between Two Allelic Variants of Type-1 S-Locus F-Box Protein and S3-RNase in Petunia inflata.

Petunia inflata possesses a self-incompatibility (SI) mechanism, which involves S-RNase and multiple S-locus F-box (SLF) genes at the polymorphic S-locus. For a given S-haplotype, each SLF is thought to interact with some of its non-self S-RNases, but not with its self S-RNase. In this work, we studied an allelic pair of SLF1, S2-SLF1 and S3-SLF1, which differ in 44 amino acids and show differential interactions with S3-RNase. We first used an in vivo transgenic assay to determine whether four chimeric proteins of S2-SLF1 and S3-SLF1, each with one of the three functional domains swapped, interact with S3-RNase. The results narrowed the candidate amino acids for specific interaction of S2-SLF1 with S3-RNase to the 16 in domain FD3. We then examined seven additional chimeric proteins by dividing FD3 into two subdomains and four mini-domains (A, B, C and D). The results further narrowed the candidate amino acids to four in mini-domain A and four in mini-domain D. Molecular modeling of interactions between S3-RNase and S2-SLF1 revealed that three of these eight are at the interaction surface, and all three are conserved in S1-SLF1 and S6a-SLF1, both of which interact with S3-RNase based on the in vivo transgenic assay. Three of the chimeric proteins were used for the in vivo transgenic assay to determine whether FD3 alone contains the amino acids required for S2-SLF1 to interact with S7-RNase and S13-RNase. The results revealed the diversity and complexity of interactions between SLF proteins and S-RNases.

All 17 S-locus F-box proteins of the S2 - and S3 -haplotypes of Petunia inflata are assembled into similar SCF complexes with a specific function in self-incompatibility.

The collaborative non-self-recognition model for S-RNase-based self-incompatibility predicts that multiple S-locus F-box proteins (SLFs) produced by pollen of a given S-haplotype collectively mediate ubiquitination and degradation of all non-self S-RNases, but not self S-RNases, in the pollen tube, thereby resulting in cross-compatible pollination but self-incompatible pollination. We had previously used pollen extracts containing GFP-fused S2 -SLF1 (SLF1 with an S2 -haplotype) of Petunia inflata for co-immunoprecipitation (Co-IP) and mass spectrometry (MS), and identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (a conventional Rbx1) as components of the SCF(S) (2-) (SLF) (1) complex. Using pollen extracts containing PiSSK1:FLAG:GFP for Co-IP/MS, we identified two additional SLFs (SLF4 and SLF13) that were assembled into SCF(SLF) complexes. As 17 SLF genes (SLF1 to SLF17) have been identified in S2 and S3 pollen, here we examined whether all 17 SLFs are assembled into similar complexes and, if so, whether these complexes are unique to SLFs. We modified the previous Co-IP/MS procedure, including the addition of style extracts from four different S-genotypes to pollen extracts containing PiSSK1:FLAG:GFP, to perform four separate experiments. The results taken together show that all 17 SLFs and an SLF-like protein, SLFLike1 (encoded by an S-locus-linked gene), co-immunoprecipitated with PiSSK1:FLAG:GFP. Moreover, of the 179 other F-box proteins predicted by S2 and S3 pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co-immunoprecipitated with PiSSK1:FLAG:GFP. These results suggest that SCF(SLF) complexes have evolved specifically to function in self-incompatibility.

Pollen S-locus F-box proteins of Petunia involved in S-RNase-based self-incompatibility are themselves subject to ubiquitin-mediated degradation.

Many flowering plants show self-incompatibility, an intra-specific reproductive barrier by which pistils reject self-pollen to prevent inbreeding and accept non-self pollen to promote out-crossing. In Petunia, the polymorphic S-locus determines self/non-self recognition. The locus contains a gene encoding an S-RNase, which controls pistil specificity, and multiple S-locus F-box (SLF) genes that collectively control pollen specificity. Each SLF is a component of an SCF (Skp1/Cullin/F-box) complex that is responsible for mediating degradation of non-self S-RNase(s), with which the SLF interacts, via the ubiquitin-26S proteasome pathway. A complete set of SLFs is required to detoxify all non-self S-RNases to allow cross-compatible pollination. Here, we show that SLF1 of Petunia inflata is itself subject to degradation via the ubiquitin-26S proteasome pathway, and identify an 18 amino acid sequence in the C-terminal region of S2 -SLF1 (SLF1 of S2 haplotype) that contains a degradation motif. Seven of the 18 amino acids are conserved among all 17 SLF proteins of S2 haplotype and S3 haplotype involved in pollen specificity, suggesting that all SLF proteins are probably subject to similar degradation. Deleting the 18 amino acid sequence from S2 -SLF1 stabilized the protein but abolished its function in self-incompatibility, suggesting that dynamic cycling of SLF proteins is an integral part of their function in self-incompatibility.

Transcriptome analysis reveals the same 17 S-locus F-box genes in two haplotypes of the self-incompatibility locus of Petunia inflata.

Petunia possesses self-incompatibility, by which pistils reject self-pollen but accept non-self-pollen for fertilization. Self-/non-self-recognition between pollen and pistil is regulated by the pistil-specific S-RNase gene and by multiple pollen-specific S-locus F-box (SLF) genes. To date, 10 SLF genes have been identified by various methods, and seven have been shown to be involved in pollen specificity. For a given S-haplotype, each SLF interacts with a subset of its non-self S-RNases, and an as yet unknown number of SLFs are thought to collectively mediate ubiquitination and degradation of all non-self S-RNases to allow cross-compatible pollination. To identify a complete suite of SLF genes of P. inflata, we used a de novo RNA-seq approach to analyze the pollen transcriptomes of S2-haplotype and S3-haplotype, as well as the leaf transcriptome of the S3S3 genotype. We searched for genes that fit several criteria established from the properties of the known SLF genes and identified the same seven new SLF genes in S2-haplotype and S3-haplotype, suggesting that a total of 17 SLF genes constitute pollen specificity in each S-haplotype. This finding lays the foundation for understanding how multiple SLF genes evolved and the biochemical basis for differential interactions between SLF proteins and S-RNases.

Identification of the self-incompatibility locus F-box protein-containing complex in Petunia inflata.

The polymorphic S-locus regulating self-incompatibility (SI) in Petunia contains the S-RNase gene and a number of S-locus F-box (SLF) genes. While penetrating the style through the stigma, a pollen tube takes up all S-RNases, but only self S-RNase inhibits pollen tube growth. Recent evidence suggests that SLFs produced by pollen collectively interact with and detoxify non-self S-RNases, but none can interact with self S-RNase. An SLF may be the F-box protein component of an SCF complex (containing Cullin1, Skp1 and Rbx1), which mediates ubiquitination of protein substrates for degradation by the 26S proteasome. However, the precise nature of the complex is unknown. We used pollen extracts of a transgenic plant over-expressing GFP-fused S2-SLF1 (SLF1 of S 2-haplotype) for co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS). We identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (an Rbx1). To validate the results, we raised transgenic plants over-expressing PiSSK1:FLAG:GFP and used pollen extracts for Co-IP-MS. The results confirmed the presence of PiCUL1-P and PiRBX1 in the complex and identified two different SLFs as the F-box protein component. Thus, all but Rbx1 of the complex may have evolved in SI, and all SLFs may be the F-box component of similar complexes.

Self-incompatibility in Petunia inflata: the relationship between a self-incompatibility locus F-box protein and its non-self S-RNases.

The highly polymorphic S (for self-incompatibility) locus regulates self-incompatibility in Petunia inflata; the S-RNase regulates pistil specificity, and multiple S-locus F-box (SLF) genes regulate pollen specificity. The collaborative non-self recognition model predicts that, for any S-haplotype, an unknown number of SLFs collectively recognize all non-self S-RNases to mediate their ubiquitination and degradation. Using a gain-of-function assay, we examined the relationships between S2-SLF1 (for S2-allelic product of Type-1 SLF) and four S-RNases. The results suggest that S2-SLF1 interacts with S7- and S13-RNases, and the previously identified S1- and S3-RNases, but not with S5- or S11-RNase. An artificial microRNA expressed by the S2-SLF1 promoter, but not by the vegetative cell-specific promoter, Late Anther Tomato 52, suppressed expression of S2-SLF1 in S2 pollen, suggesting that SLF1 is specific to the generative cell. The S2 pollen with S2-SLF1 suppressed was compatible with S3-, S5-, S7-, S11-, and S13-carrying pistils, confirming that other SLF proteins are responsible for detoxifying S5- and S11-RNases and suggesting that S2-SLF1 is not the only SLF in S2 pollen that interacts with S3-, S7-, and S13-RNases. Petunia may have evolved at least two types of SLF proteins to detoxify any non-self S-RNase to minimize the deleterious effects of mutation in any SLF.

S-RNase-based self-incompatibility in Petunia inflata.

BACKGROUND: For the Solanaceae-type self-incompatibility, also possessed by Rosaceae and Plantaginaceae, the specificity of self/non-self interactions between pollen and pistil is controlled by two polymorphic genes at the S-locus: the S-locus F-box gene (SLF or SFB) controls pollen specificity and the S-RNase gene controls pistil specificity. SCOPE: This review focuses on the work from the authors' laboratory using Petunia inflata (Solanaceae) as a model. Here, recent results on the identification and functional studies of S-RNase and SLF are summarized and a protein-degradation model is proposed to explain the biochemical mechanism for specific rejection of self-pollen tubes by the pistil. CONCLUSIONS: The protein-degradation model invokes specific degradation of non-self S-RNases in the pollen tube mediated by an SLF, and can explain compatible versus incompatible pollination and the phenomenon of competitive interaction, where SI breaks down in pollen carrying two different S-alleles. In Solanaceae, Plantaginaceae and subfamily Maloideae of Rosaceae, there also exist multiple S-locus-linked SLF/SFB-like genes that potentially function as the pollen S-gene. To date, only three such genes, all in P. inflata, have been examined, and they do not function as the pollen S-gene in the S-genotype backgrounds tested. Interestingly, subfamily Prunoideae of Rosaceae appears to possess only a single SLF/SFB gene, and competitive interaction, observed in Solanaceae, Plantaginaceae and subfamily Maloideae, has not been observed. Thus, although the cytotoxic function of S-RNase is an integral part of SI in Solanaceae, Plantaginaceae and Rosaceae, the function of SLF/SFB may have diverged. This highlights the complexity of the S-RNase-based SI mechanism. The review concludes by discussing some key experiments that will further advance our understanding of this self/non-self discrimination mechanism.

Collaborative non-self recognition system in S-RNase-based self-incompatibility.

Self-incompatibility in flowering plants prevents inbreeding and promotes outcrossing to generate genetic diversity. In Solanaceae, a multiallelic gene, S-locus F-box (SLF), was previously shown to encode the pollen determinant in self-incompatibility. It was postulated that an SLF allelic product specifically detoxifies its non-self S-ribonucleases (S-RNases), allelic products of the pistil determinant, inside pollen tubes via the ubiquitin-26S-proteasome system, thereby allowing compatible pollinations. However, it remained puzzling how SLF, with much lower allelic sequence diversity than S-RNase, might have the capacity to recognize a large repertoire of non-self S-RNases. We used in vivo functional assays and protein interaction assays to show that in Petunia, at least three types of divergent SLF proteins function as the pollen determinant, each recognizing a subset of non-self S-RNases. Our findings reveal a collaborative non-self recognition system in plants.

Ectopic expression of S-RNase of Petunia inflata in pollen results in its sequestration and non-cytotoxic function.

The specificity of S-RNase-based self-incompatibility (SI) is controlled by two S-locus genes, the pistil S-RNase gene and the pollen S-locus-F-box gene. S-RNase is synthesized in the transmitting cell; its signal peptide is cleaved off during secretion into the transmitting tract; and the mature "S-RNase", the subject of this study, is taken up by growing pollen tubes via an as-yet unknown mechanism. Upon uptake, S-RNase is sequestered in a vacuolar compartment in both non-self (compatible) and self (incompatible) pollen tubes, and the subsequent disruption of this compartment in incompatible pollen tubes correlates with the onset of the SI response. How the S-RNase-containing compartment is specifically disrupted in incompatible pollen tubes, however, is unknown. Here, we circumvented the uptake step of S-RNase by directly expressing S(2)-RNase, S(3)-RNase and non-glycosylated S(3)-RNase of Petunia inflata, with green fluorescent protein (GFP) fused at the C-terminus of each protein, in self (incompatible) and non-self (compatible) pollen of transgenic plants. We found that none of these ectopically expressed S-RNases affected the viability or the SI behavior of their self or non-self-pollen/pollen tubes. Based on GFP fluorescence of in vitro-germinated pollen tubes, all were sequestered in both self and non-self-pollen tubes. Moreover, the S-RNase-containing compartment was dynamic in living pollen tubes, with movement dependent on the actin-myosin-based molecular motor system. All these results suggest that glycosylation is not required for sequestration of S-RNase expressed in pollen tubes, and that the cytosol of pollen is the site of the cytotoxic action of S-RNase in SI.

Distribution of self-compatible and self-incompatible populations of Petunia axillaris (Solanaceae) outside Uruguay.

Petunia axillaris occurs in temperate South America and consists of three allopatric subspecies: axillaris, parodii, and subandina. Previous studies have revealed that subsp. axillaris is self-incompatible (SI), subsp. parodii is self-compatible (SC) in Uruguay, and subsp. subandina is SC in Argentina. The SI/SC status over the entire distribution range is not completely understood, however. The objective of this study was to examine the overall SI/SC status of the respective subspecies in comparison with floral morphology. The results confirmed that subsp. parodii and subsp. subandina were SC throughout the distribution range, and that subsp. axillaris was also SC in Brazil and in most of the Argentinean territory. The SI P. axillaris occurs in the natural population only between 34 and 36 degrees S, along the eastern shore of South America. The Brazilian and Uruguayan subsp. axillaris differed in SI/SC status and floral morphology. We discuss the cause of this difference.

Identification and characterization of PiORP1, a Petunia oxysterol-binding-protein related protein involved in receptor-kinase mediated signaling in pollen, and analysis of the ORP gene family in Arabidopsis.

Oxysterol-binding proteins (OSBPs) and oxysterol-binding-protein related proteins (ORPs) are encoded by most eukaryotic genomes examined to date; however, they have not yet been characterized in plants. Here we report the identification and characterization of PiORP1, an ORP of Petunia inflata that interacts with the cytoplasmic kinase domain of a receptor-like kinase, named PRK1, of P. inflata. PiORP1 is phosphorylated by PRK1 in vitro and therefore may be involved in PRK1 signaling during pollen development and growth. RNA gel blot analysis showed that PiORP1 and PRK1 had very similar expression patterns in developing pollen, mature pollen and pollen tubes. GFP fusion proteins of PiORP1 localized in the plasma membrane of pollen tubes at distinct foci and its PH domain alone was sufficient to mediate this localization. The sequence for the oxysterol-binding domain of PiORP1 was used to search the genome of Arabidopsis; 12 ORPs were identified and phylogenetic analysis revealed that they fell into two distinct clades, consistent with the ORPs of other eukaryotes. RT-PCR analysis showed that all 12 Arabidopsis ORPs were expressed; 10 were expressed in most of the tissues examined under normal growth conditions, but only three were expressed in pollen.

Petunia phospholipase c1 is involved in pollen tube growth.

Although pollen tube growth is essential for plant fertilization and reproductive success, the regulators of the actin-related growth machinery and the cytosolic Ca2+ gradient thought to determine how these cells elongate remain poorly defined. Phospholipases, their substrates, and their phospholipid turnover products have been proposed as such regulators; however, the relevant phospholipase(s) have not been characterized. Therefore, we cloned cDNA for a pollen-expressed phosphatidylinositol 4,5-bisphosphate (PtdInsP2)-cleaving phospholipase C (PLC) from Petunia inflata, named Pet PLC1. Expressing a catalytically inactive form of Pet PLC1 in pollen tubes caused expansion of the apical Ca2+ gradient, disruption of the organization of the actin cytoskeleton, and delocalization of growth at the tube tip. These phenotypes were suppressed by depolymerizing actin with low concentrations of latrunculin B, suggesting that a critical site of action of Pet PLC1 is in regulating actin structure at the growing tip. A green fluorescent protein (GFP) fusion to Pet PLC1 caused enrichment in regions of the apical plasma membrane not undergoing rapid expansion, whereas a GFP fusion to the PtdInsP2 binding domain of mammalian PLC delta1 caused enrichment in apical regions depleted in PLC. Thus, Pet PLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell.

Duplication of the S-locus F-box gene is associated with breakdown of pollen function in an S-haplotype identified in a natural population of self-incompatible Petunia axillaris.

We previously identified both self-incompatible and self-compatible plants in a natural population of self-incompatible Petunia axillaris subsp. axillaris, and found that all the self-compatible plants studied carried either SC1- or SC2-haplotype. Genetic crosses showed that SC2 was identical to S17 identified from another natural population of P. axillaris, except that its pollen function was defective, and that the pollen-part mutation in SC2 was tightly linked to the S-locus. Recent identification of the S-locus F-box gene (SLF) as the gene that controls pollen specificity in S-RNase-based self-incompatibility has prompted us to examine the molecular basis of this pollen-part mutation. We cloned and sequenced the S17-allele of SLF of P. axillaris, named PaSLF17, and found that SC2SC2 plants contained extra restriction fragments that hybridized to PaSLF17 in addition to all of those observed in S17S17 plants. Moreover, these additional fragments co-segregated with SC2. We used the SC2-specific restriction fragments as templates to clone an allele of PaSLF by PCR. To determine the identity of this allele, named PaSLFx, primers based on its sequence were used to amplify PaSLF alleles from genomic DNA of 40 S-homozygotes of P. axillaris, S1S1 through S40S40. Sequence comparison revealed that PaSLFx was completely identical with PaSLF19 obtained from S19S19. We conclude that the S-locus of SC2 contained both S17-allele and the duplicated S19-allele of PaSLF. SC2 is the first naturally occurring pollen-part mutation of a solanaceous species that was shown to be associated with duplication of the pollen S. This finding lends support to the proposal, based on studies of irradiation-generated pollen-part mutants of solanaceous species, that duplication, but not deletion, of the pollen S, causes breakdown of pollen function.

Identification of the pollen determinant of S-RNase-mediated self-incompatibility.

Many flowering plants have adopted self-incompatibility mechanisms to prevent inbreeding and promote out-crosses. In the Solanaceae, Rosaceae and Scrophulariaceae, two separate genes at the highly polymorphic S-locus control self-incompatibility interactions: the S-RNase gene encodes the pistil determinant and the previously unidentified S-gene encodes the pollen determinant. S-RNases interact with pollen S-allele products to inhibit the growth of self-pollen tubes in the style. Pollen-expressed F-box genes showing allelic sequence polymorphism have recently been identified near to the S-RNase gene in members of the Rosaceae and Scrophulariaceae; but until now have not been directly shown to encode the pollen determinant. Here we report the identification and characterization of PiSLF, an S-locus F-box gene of Petunia inflata (Solanaceae). We show that transformation of S1S1, S1S2 and S2S3 plants with the S2-allele of PiSLF causes breakdown of their pollen function in self-incompatibility. This breakdown of pollen function is consistent with 'competitive interaction', in which pollen carrying two different pollen S-alleles fails to function in self-incompatibility. We conclude that PiSLF encodes the pollen self-incompatibility determinant.

Breakdown of self-incompatibility in a natural population of Petunia axillaris caused by loss of pollen function.

Although Petunia axillaris subsp. axillaris is described as a self-incompatible taxon, some of the natural populations we have identified in Uruguay are composed of both self-incompatible and self-compatible plants. Here, we studied the self-incompatibility (SI) behavior of 50 plants derived from such a mixed population, designated U83, and examined the cause of the breakdown of SI. Thirteen plants were found to be self-incompatible, and the other 37 were found to be self-compatible. A total of 14 S-haplotypes were represented in these 50 plants, including two that we had previously identified from another mixed population, designated U1. All the 37 self-compatible plants carried either an S(C1)- or an S(C2)-haplotype. S(C1)S(C1) and S(C2)S(C2) homozygotes were generated by self-pollination of two of the self-compatible plants, and they were reciprocally crossed with 40 self-incompatible S-homozygotes (S(1)S(1) through S(40)S(40)) generated from plants identified from three mixed populations, including U83. The S(C1)S(C1) homozygote was reciprocally compatible with all the genotypes examined. The S(C2)S(C2) homozygote accepted pollen from all but the S(17)S(17) homozygote (identified from the U1 population), but the S(17)S(17) homozygote accepted pollen from the S(C2)S(C2) homozygote. cDNAs encoding S(C2)- and S(17)-RNases were cloned and sequenced, and their nucleotide sequences were completely identical. Analysis of bud-selfed progeny of heterozygotes carrying S(C1) or S(C2) showed that the SI behavior of S(C1) and S(C2) was identical to that of S(C1) and S(C2) homozygotes, respectively. All these results taken together suggested that the S(C2)-haplotype was a mutant form of the S(17)-haplotype, with the defect lying in the pollen function. The possible nature of the mutation is discussed.

S-RNase-mediated self-incompatibility.

The Solanaceae, Rosaceae, and Scrophulariaceae families all possess an RNase-mediated self-incompatibility mechanism through which their pistils can recognize and reject self-pollen to prevent inbreeding. The highly polymorphic S-locus controls the self-incompatibility interaction, and the S-locus of the Solanaceae has been shown to be a multi-gene complex in excess of 1.3 Mb. To date, the function of only one of the S-locus genes, the S-RNase gene, has been determined. This article reviews the current status of the search for the pollen S-gene and the current models for how S-haplotype specific inhibition of pollen tubes can be accomplished by S-RNases.