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.     

Identification and characterization of components of a putative petunia S-locus F-box-containing E3 ligase complex involved in S-RNase-based self-incompatibility

Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF₂. We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S₁-, S₂-, and S₃-RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele–specific manner. Native glycosylated S₃-RNase is not degraded to any significant extent; however, deglycosylated S₃-RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF–containing E3 complex is unknown.     

Convergent Evolution at the Gametophytic Self-Incompatibility System in Malus and Prunus

S-RNase-based gametophytic self-incompatibility (GSI) has evolved once before the split of the Asteridae and Rosidae. This conclusion is based on the phylogenetic history of the S-RNase that determines pistil specificity. In Rosaceae, molecular characterizations of Prunus species, and species from the tribe Pyreae (i.e., Malus, Pyrus, Sorbus) revealed different numbers of genes determining S-pollen specificity. In Prunus only one pistil and pollen gene determine GSI, while in Pyreae there is one pistil but multiple pollen genes, implying different specificity recognition mechanisms. It is thus conceivable that within Rosaceae the genes involved in GSI in the two lineages are not orthologous but possibly paralogous. To address this hypothesis we characterised the S-RNase lineage and S-pollen lineage genes present in the genomes of five Rosaceae species from three genera: M. × domestica (apple, self-incompatible (SI); tribe Pyreae), P. persica (peach, self-compatible (SC); Amygdaleae), P. mume (mei, SI; Amygdaleae), Fragaria vesca (strawberry, SC; Potentilleae), and F. nipponica (mori-ichigo, SI; Potentilleae). Phylogenetic analyses revealed that the Malus and Prunus S-RNase and S-pollen genes belong to distinct gene lineages, and that only Prunus S-RNase and SFB-lineage genes are present in Fragaria. Thus, S-RNase based GSI system of Malus evolved independently from the ancestral system of Rosaceae. Using expression patterns based on RNA-seq data, the ancestral S-RNase lineage gene is inferred to be expressed in pistils only, while the ancestral S-pollen lineage gene is inferred to be expressed in tissues other than pollen.     

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.     

On the origin of the widespread self-compatible allotetraploid Capsella bursa-pastoris (Brassicaceae)

Polyploidy, or whole-genome duplication, is a common speciation mechanism in plants. An important barrier to polyploid establishment is a lack of compatible mates. Because self-compatibility alleviates this problem, it has long been hypothesized that there should be an association between polyploidy and self-compatibility (SC), but empirical support for this prediction is mixed. Here, we investigate whether the molecular makeup of the Brassicaceae self-incompatibility (SI) system, and specifically dominance relationships among S-haplotypes mediated by small RNAs, could facilitate loss of SI in allopolyploid crucifers. We focus on the allotetraploid species Capsella bursa-pastoris, which formed ~300 kya by hybridization and whole-genome duplication involving progenitors from the lineages of Capsella orientalis and Capsella grandiflora. We conduct targeted long-read sequencing to assemble and analyze eight full-length S-locus haplotypes, representing both homeologous subgenomes of C. bursa-pastoris. We further analyze small RNA (sRNA) sequencing data from flower buds to identify candidate dominance modifiers. We find that C. orientalis-derived S-haplotypes of C. bursa-pastoris harbor truncated versions of the male SI specificity gene SCR and express a conserved sRNA-based candidate dominance modifier with a target in the C. grandiflora-derived S-haplotype. These results suggest that pollen-level dominance may have facilitated loss of SI in C. bursa-pastoris. Finally, we demonstrate that spontaneous somatic tetraploidization after a wide cross between C. orientalis and C. grandiflora can result in production of self-compatible tetraploid offspring. We discuss the implications of this finding on the mode of formation of this widespread weed.Subject terms: Evolution, Polyploidy in plants, Plant evolution, Haplotypes     

Independent S-Locus Mutations Caused Self-Fertility in Arabidopsis thaliana

A common yet poorly understood evolutionary transition among flowering plants is a switch from outbreeding to an inbreeding mode of mating. The model plant Arabidopsis thaliana evolved to an inbreeding state through the loss of self-incompatibility, a pollen-rejection system in which pollen recognition by the stigma is determined by tightly linked and co-evolving alleles of the S-locus receptor kinase (SRK) and its S-locus cysteine-rich ligand (SCR). Transformation of A. thaliana, with a functional AlSRKb-SCRb gene pair from its outcrossing relative A. lyrata, demonstrated that A. thaliana accessions harbor different sets of cryptic self-fertility–promoting mutations, not only in S-locus genes, but also in other loci required for self-incompatibility. However, it is still not known how many times and in what manner the switch to self-fertility occurred in the A. thaliana lineage. Here, we report on our identification of four accessions that are reverted to full self-incompatibility by transformation with AlSRKb-SCRb, bringing to five the number of accessions in which self-fertility is due to, and was likely caused by, S-locus inactivation. Analysis of S-haplotype organization reveals that inter-haplotypic recombination events, rearrangements, and deletions have restructured the S locus and its genes in these accessions. We also perform a Quantitative Trait Loci (QTL) analysis to identify modifier loci associated with self-fertility in the Col-0 reference accession, which cannot be reverted to full self-incompatibility. Our results indicate that the transition to inbreeding occurred by at least two, and possibly more, independent S-locus mutations, and identify a novel unstable modifier locus that contributes to self-fertility in Col-0.     

Identification of a canonical SCFSLF complex involved in S-RNase-based self-incompatibility of Pyrus (Rosaceae)

S-RNase-based self-incompatibility (SI) is an intraspecific reproductive barrier to prevent self-fertilization found in many species of the Solanaceae, Plantaginaceae and Rosaceae. In this system, S-RNase and SLF/SFB (S-locus F-box) genes have been shown to control the pistil and pollen SI specificity, respectively. Recent studies have shown that the SLF functions as a substrate receptor of a SCF (Skp1/Cullin1/F-box)-type E3 ubiquitin ligase complex to target S-RNases in Solanaceae and Plantaginaceae, but its role in Rosaceae remains largely undefined. Here we report the identification of two pollen-specific SLF-interacting Skp1-like (SSK) proteins, PbSSK1 and PbSSK2, in Pyrus bretschneideri from the tribe Pyreae of Rosaceae. Both yeast two-hybrid and pull-down assays demonstrated that they could connect PbSLFs to PbCUL1 to form a putative canonical SCF^SLF (SSK/CUL1/SLF) complex in Pyrus. Furthermore, pull-down assays showed that the SSK proteins could bind SLF and CUL1 in a cross-species manner between Pyrus and Petunia. Additionally, phylogenetic analysis revealed that the SSK-like proteins from Solanaceae, Plantaginaceae and Rosaceae form a monoclade group, hinting their shared evolutionary origin. Taken together, with the recent identification of a canonical SCF^SFB complex in Prunus of the tribe Amygdaleae of Rosaceae, our results show that a conserved canonical SCF^SLF/SFB complex is present in Solanaceae, Plantaginaceae and Rosaceae, implying that S-RNase-based self-incompatibility shares a similar molecular and biochemical mechanism.     

Electrostatic potentials of the S‐locus F‐box proteins contribute to the pollen S specificity in self‐incompatibility in Petunia hybrida

Self‐incompatibility (SI) is a self/non‐self discrimination system found widely in angiosperms and, in many species, is controlled by a single polymorphic S‐locus. In the Solanaceae, Rosaceae and Plantaginaceae, the S‐locus encodes a single S‐RNase and a cluster of S‐locus F‐box (SLF) proteins to control the pistil and pollen expression of SI, respectively. Previous studies have shown that their cytosolic interactions determine their recognition specificity, but the physical force between their interactions remains unclear. In this study, we show that the electrostatic potentials of SLF contribute to the pollen S specificity through a physical mechanism of ‘like charges repel and unlike charges attract’ between SLFs and S‐RNases in Petunia hybrida. Strikingly, the alteration of a single C‐terminal amino acid of SLF reversed its surface electrostatic potentials and subsequently the pollen S specificity. Collectively, our results reveal that the electrostatic potentials act as a major physical force between cytosolic SLFs and S‐RNases, providing a mechanistic insight into the self/non‐self discrimination between cytosolic proteins in angiosperms.     

S-RNases and sexual incompatibility: structure, functions, and evolutionary perspectives

S-RNase-based gametophytic self-incompatibility appears to be the most phylogenetically widespread form of self-incompatibility found in the angiosperms, having been reported in the Solanaceae, Scrophulariaceae, and Rosaceae. This intraspecific breeding barrier is controlled by a single genetic locus termed S. Rejection of self-pollen has been shown to be mediated in the pistil by a highly polymorphic series of ribonucleases, but as yet the pollen component of this recognition system has not been identified. Here we review our present knowledge concerning the structure, functions, and evolution of S-RNases and the S-loci in which they reside. In addition we present two new phylogenetic analyses of S-RNases which suggest that (1). sequence variability between S-alleles is spread across the whole gene and is not as clustered as is generally believed and (2). there is evidence of recombination and/or diversifying selection in two distinct regions of S-RNases. The implications of these findings are discussed.     

Recognition of S-RNases by an S locus F-box like protein and an S haplotype-specific F-box like protein in the Prunus-specific self-incompatibility system

Key message

S-RNase was demonstrated to be predominantly recognized by an S locus F-box-like protein and an S haplotype-specific F-box-like protein in compatible pollen tubes of sweet cherry.

Abstract

Self-incompatibility (SI) is a reproductive barrier that rejects self-pollen and inhibits self-fertilization to promote outcrossing. In Solanaceae and Rosaceae, S-RNase-based gametophytic SI (GSI) comprises S-RNase and F-box protein(s) as the pistil and pollen S determinants, respectively. Compatible pollen tubes are assumed to detoxify the internalized cytotoxic S-RNases to maintain growth. S-RNase detoxification is conducted by the Skp1-cullin1-F-box protein complex (SCF) formed by pollen S determinants, S locus F-box proteins (SLFs), in Solanaceae. In Prunus, the general inhibitor (GI), but not pollen S determinant S haplotype-specific F-box protein (SFB), is hypothesized to detoxify S-RNases. Recently, SLF-like proteins 1–3 (SLFL1–3) were suggested as GI candidates, although it is still possible that other proteins function predominantly in GI. To identify the other GI candidates, we isolated four other pollen-expressed SLFL and SFB-like (SFBL) proteins PavSLFL6, PavSLFL7A, PavSFBL1, and PavSFBL2 in sweet cherry. Binding assays with four PavS-RNases indicated that PavSFBL2 bound to PavS^{1, 6}-RNase while the others bound to nothing. PavSFBL2 was confirmed to form an SCF complex in vitro. A co-immunoprecipitation assay using the recombinant PavS⁶-RNase as bait against pollen extracts and a mass spectrometry analysis identified the SCF complex components of PavSLFLs and PavSFBL2, M-locus-encoded glutathione S-transferase (MGST), DnaJ-like protein, and other minor proteins. These results suggest that SLFLs and SFBLs could act as predominant GIs in Prunus-specific S-RNase-based GSI.

     

Expression and trans-specific polymorphism of self-incompatibility RNases in coffea (Rubiaceae)

Self-incompatibility (SI) is widespread in the angiosperms, but identifying the biochemical components of SI mechanisms has proven to be difficult in most lineages. Coffea (coffee; Rubiaceae) is a genus of old-world tropical understory trees in which the vast majority of diploid species utilize a mechanism of gametophytic self-incompatibility (GSI). The S-RNase GSI system was one of the first SI mechanisms to be biochemically characterized, and likely represents the ancestral Eudicot condition as evidenced by its functional characterization in both asterid (Solanaceae, Plantaginaceae) and rosid (Rosaceae) lineages. The S-RNase GSI mechanism employs the activity of class III RNase T2 proteins to terminate the growth of "self" pollen tubes. Here, we investigate the mechanism of Coffea GSI and specifically examine the potential for homology to S-RNase GSI by sequencing class III RNase T2 genes in populations of 14 African and Madagascan Coffea species and the closely related self-compatible species Psilanthus ebracteolatus. Phylogenetic analyses of these sequences aligned to a diverse sample of plant RNase T2 genes show that the Coffea genome contains at least three class III RNase T2 genes. Patterns of tissue-specific gene expression identify one of these RNase T2 genes as the putative Coffea S-RNase gene. We show that populations of SI Coffea are remarkably polymorphic for putative S-RNase alleles, and exhibit a persistent pattern of trans-specific polymorphism characteristic of all S-RNase genes previously isolated from GSI Eudicot lineages. We thus conclude that Coffea GSI is most likely homologous to the classic Eudicot S-RNase system, which was retained since the divergence of the Rubiaceae lineage from an ancient SI Eudicot ancestor, nearly 90 million years ago.     

Inbreeding depression in self-incompatible North-American Arabidopsis lyrata: disentangling genomic and S-locus-specific genetic load

Newly formed selfing lineages may express recessive genetic load and suffer inbreeding depression. This can have a genome-wide genetic basis, or be due to loci linked to genes under balancing selection. Understanding the genetic architecture of inbreeding depression is important in the context of the maintenance of self-incompatibility and understanding the evolutionary dynamics of S-alleles. We addressed this using North-American subspecies of Arabidopsis lyrata. This species is normally self-incompatible and outcrossing, but some populations have undergone a transition to selfing. The goals of this study were to: (1) quantify the strength of inbreeding depression in North-American populations of A. lyrata; and (2) disentangle the relative contribution of S-linked genetic load compared with overall inbreeding depression. We enforced selfing in self-incompatible plants with known S-locus genotype by treatment with CO₂, and compared the performance of selfed vs outcrossed progeny. We found significant inbreeding depression for germination rate (δ=0.33), survival rate to 4 weeks (δ=0.45) and early growth (δ=0.07), but not for flowering rate. For two out of four S-alleles in our design, we detected significant S-linked load reflected by an under-representation of S-locus homozygotes in selfed progeny. The presence or absence of S-linked load could not be explained by the dominance level of S-alleles. Instead, the random nature of the mutation process may explain differences in the recessive deleterious load among lineages.     

Ancient Gene Duplications,Rather Than Polyploidization,Facilitate Diversification of Petal Pigmentation Patterns in Clarkia gracilis (Onagraceae)

It has been suggested that gene duplication and polyploidization create opportunities for the evolution of novel characters. However, the connections between the effects of polyploidization and morphological novelties have rarely been examined. In this study, we investigated whether petal pigmentation patterning in an allotetraploid Clarkia gracilis has evolved as a result of polyploidization. Clarkia gracilis is thought to be derived through a recent polyploidization event with two diploid species, C. amoena huntiana and an extinct species that is closely related to C. lassenensis. We reconstructed phylogenetic relationships of the R2R3-MYBs (the regulators of petal pigmentation) from two subspecies of C. gracilis and the two purported progenitors, C. a. huntiana and C. lassenensis. The gene tree reveals that these R2R3-MYB genes have arisen through duplications that occurred before the divergence of the two progenitor species, that is, before polyploidization. After polyploidization and subsequent gene loss, only one of the two orthologous copies inherited from the progenitors was retained in the polyploid, turning it to diploid inheritance. We examined evolutionary changes in these R2R3-MYBs and in their expression, which reveals that the changes affecting patterning (including expression domain contraction, loss-of-function mutation, cis-regulatory mutation) occurred after polyploidization within the C. gracilis lineages. Our results thus suggest that polyploidization itself is not necessary in producing novel petal color patterns. By contrast, duplications of R2R3-MYB genes in the common ancestor of the two progenitors have apparently facilitated diversification of petal pigmentation patterns.     

Self-incompatibility systems: barriers to self-fertilization in flowering plants

Flowering plants (angiosperms) are the most prevalent and evolutionarily advanced group of plants. Success of these plants is owed to several unique evolutionary adaptations that aid in reproduction: the flower, the closed carpel, double fertilization, and the ultimate products of fertilization, seeds enclosed in the fruit. Angiosperms exhibit a vast array of reproductive strategies, including both asexual and sexual, the latter of which includes both self-fertilization and cross-fertilization. Asexual reproduction and self-fertilization are important reproductive strategies in a variety of situations, such as when mates are scarce or when the environment remains relatively stable. However, reproductive strategies promoting cross-fertilization are critical to angiosperm success, since they contribute to the creation of genetically diverse populations, which increase the probability that at least one individual in a population will survive given changing environmental conditions. The evolution of several physical and genetic barriers to self-fertilization or fertilization among closely related individuals is thus widespread in angiosperms. A major genetic barrier to self-fertilization is self-incompatibility (SI), which allows female reproductive cells to discriminate between "self" and "non-self" pollen, and specifically reject self pollen. Evidence for the importance of SI in angiosperm evolution lies in the highly diverse set of mechanisms used by various angiosperm families for recognition of self pollen tube development and preventing self-fertilization.     

Ubiquitin–proteasome‐mediated degradation of S‐RNase in a solanaceous cross‐compatibility reaction

Many plants have a self‐incompatibility (SI) system in which the rejection of self‐pollen is determined by multiple haplotypes at a single locus, termed S. In the Solanaceae, each haplotype encodes a single ribonuclease (S‐RNase) and multiple S‐locus F‐box proteins (SLFs), which function as the pistil and pollen SI determinants, respectively. S‐RNase is cytotoxic to self‐pollen, whereas SLFs are thought to collaboratively recognize non‐self S‐RNases in cross‐pollen and detoxify them via the ubiquitination pathway. However, the actual mechanism of detoxification remains unknown. Here we isolate the components of a SCF^SLF (SCF = SKP1‐CUL1‐F‐box‐RBX1) from Petunia pollen. The SCF^SLF polyubiquitinates a subset of non‐self S‐RNases in vitro. The polyubiquitinated S‐RNases are degraded in the pollen extract, which is attenuated by a proteasome inhibitor. Our findings suggest that multiple SCF^SLF complexes in cross‐pollen polyubiquitinate non‐self S‐RNases, resulting in their degradation by the proteasome.     

Comparison of Petunia inflata S-Locus F-box protein (Pi SLF) with Pi SLF like proteins reveals its unique function in S-RNase based self-incompatibility

Petunia inflata possesses S-RNase-based self-incompatibility (SI), which prevents inbreeding and promotes outcrossing. Two polymorphic genes at the S-locus, S-RNase and P. inflata S-locus F-box (Pi SLF), determine the pistil and pollen specificity, respectively. To understand how the interactions between Pi SLF and S-RNase result in SI responses, we identified four Pi SLF-like (Pi SLFL) genes and used them, along with two previously identified Pi SLFLs, for comparative studies with Pi SLF(2). We examined the in vivo functions of three of these Pi SLFLs and found that none functions in SI. These three Pi SLFLs and two other Pi SLFs either failed to interact with S(3)-RNase (a non-self S-RNase for all of them) or interacted much more weakly than did Pi SLF(2) in vitro. We divided Pi SLF(2) into FD1 (for Functional Domain1), FD2, and FD3, each containing one of the Pi SLF-specific regions, and used truncated Pi SLF(2), chimeric proteins between Pi SLF(2) and one of the Pi SLFLs that did not interact with S(3)-RNase, and chimeric proteins between Pi SLF(1) and Pi SLF(2) to address the biochemical roles of these three domains. The results suggest that FD2, conserved among three allelic variants of Pi SLF, plays a major role in the strong interaction with S-RNase; additionally, FD1 and FD3 (each containing one of the two variable regions of Pi SLF) together negatively modulate this interaction, with a greater effect on interactions with self S-RNase than with non-self S-RNases. A model for how an allelic product of Pi SLF determines the fate of its self and non-self S-RNases in the pollen tube is presented.     

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.