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1.  The amino terminal F-box domain of Petunia inflata S-locus F-box protein is involved in the S-RNase-based self-incompatibility mechanism 
AoB Plants  2011;2011:plr016.
We showed that the F-box domain of PiSLF, Petunia inflata S-locus F-box protein, is essential for SI function and that a P. inflata ortholog of Antirrhinum SSK1 does not interact with PiSLF, consistent with our previous finding that PiSLF might be in a novel E3 complex.
Background and aims
Pistils of flowering plants possessing self-incompatibility (SI) can distinguish between self and non-self pollen, and only allow non-self pollen to effect fertilization. For Petunia inflata, the S-RNase gene encodes pistil specificity and multiple S-locus F-box (SLF) genes encode pollen specificity. Each SLF produced in pollen interacts with a subset of non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome.
Rationale
S-locus F-box has been proposed to function as a component of the conventional SCF (SKP1-CULLIN-F-box protein) complex, based on the finding that two SKP1-like proteins, AhSSK1 (Antirrhinum hispanicum SLF-interacting SKP1-like1) and PhSSK1 (Petunia hybrida SSK1), interact with the F-box domain of some allelic variants of SLF. However, we previously showed that PiSLF (P. inflata SLF) did not interact with any SKP1 of P. inflata or Arabidopsis thaliana, but instead interacted with a RING-finger protein, PiSBP1 (P. inflata S-RNase-Binding Protein1), which may also play the role of SKP1. Thus, the biochemical nature of the SLF-containing complex is as yet unclear.
Principal results
To examine whether the F-box domain of PiSLF is required for SI function, we expressed a truncated PiSLF2 (S2 allelic variant) without this domain in S2S3 plants and showed that, unlike the full-length PiSLF2, it did not cause breakdown of SI in S3 pollen. We identified PiSSK1 (P. inflata SSK1) and found that it did not interact with PiSLF1, PiSLF2 or PiSLF3.
Conclusions
The finding that the truncated PiSLF2 did not cause breakdown of SI in S3 transgenic pollen suggests that the F-box domain of PiSLF2 is required for mediating degradation of S3-RNase, a non-self S-RNase, in S3 pollen, and thus is required for SI function. The finding that PiSSK1 did not interact with three allelic variants of PiSLF is consistent with our previous finding that PiSLF might not be in a conventional SCF complex.
doi:10.1093/aobpla/plr016
PMCID: PMC3158857  PMID: 22476486
2.  Identification of SFBB-Containing Canonical and Noncanonical SCF Complexes in Pollen of Apple (Malus × domestica) 
PLoS ONE  2014;9(5):e97642.
Gametophytic self-incompatibility (GSI) of Rosaceae, Solanaceae and Plantaginaceae is controlled by a single polymorphic S locus. The S locus contains at least two genes, S-RNase and F-box protein encoding gene SLF/SFB/SFBB that control pistil and pollen specificity, respectively. Generally, the F-box protein forms an E3 ligase complex, SCF complex with Skp1, Cullin1 (CUL1) and Rbx1, however, in Petunia inflata, SBP1 (S-RNase binding protein1) was reported to play the role of Skp1 and Rbx1, and form an SCFSLF-like complex for ubiquitination of non-self S-RNases. On the other hand, in Petunia hybrida and Petunia inflata of Solanaceae, Prunus avium and Pyrus bretschneideri of Rosaceae, SSK1 (SLF-interacting Skp1-like protein1) is considered to form the SCFSLF/SFB complex. Here, we isolated pollen-expressed apple homologs of SSK1 and CUL1, and named MdSSK1, MdCUL1A and MdCUL1B. MdSSK1 was preferentially expressed in pollen, but weakly in other organs analyzed, while, MdCUL1A and MdCUL1B were almost equally expressed in all the organs analyzed. MdSSK1 transcript abundance was significantly (>100 times) higher than that of MdSBP1. In vitro binding assays showed that MdSSK1 and MdSBP1 interacted with MdSFBB1-S9 and MdCUL1, and MdSFBB1-S9 interacted more strongly with MdSSK1 than with MdSBP1. The results suggest that both MdSSK1-containing SCFSFBB1 and MdSBP1-containing SCFSFBB1-like complexes function in pollen of apple, and the former plays a major role.
doi:10.1371/journal.pone.0097642
PMCID: PMC4029751  PMID: 24847858
3.  SCFSLF-mediated cytosolic degradation of S-RNase is required for cross-pollen compatibility in S-RNase-based self-incompatibility in Petunia hybrida 
Frontiers in Genetics  2014;5:228.
Many flowering plants adopt self-incompatibility (SI) to maintain their genetic diversity. In species of Solanaceae, Plantaginaceae, and Rosaceae, SI is genetically controlled by a single S-locus with multiple haplotypes. The S-locus has been shown to encode S-RNases expressed in pistil and multiple SLF (S-locus F-box) proteins in pollen controlling the female and male specificity of SI, respectively. S-RNases appear to function as a cytotoxin to reject self-pollen. In addition, SLFs have been shown to form SCF (SKP1/Cullin1/F-box) complexes to serve as putative E3 ubiquitin ligase to interact with S-RNases. Previously, two different mechanisms, the S-RNase degradation and the S-RNase compartmentalization, have been proposed as the restriction mechanisms of S-RNase cytotoxicity allowing compatible pollination. In this study, we have provided several lines of evidence in support of the S-RNase degradation mechanism by a combination of cellular, biochemical and molecular biology approaches. First, both immunogold labeling and subcellular fractionation assays showed that two key pollen SI factors, PhS3L-SLF1 and PhSSK1 (SLF-interacting SKP1-like1) from Petunia hybrida, a Solanaceous species, are co-localized in cytosols of both pollen grains and tubes. Second, PhS3L-RNases are mainly detected in the cytosols of both self and non-self-pollen tubes after pollination. Third, we found that PhS-RNases selectively interact with PhSLFs by yeast two-hybrid and co-immunoprecipitation assays. Fourth, S-RNases are specifically degraded in compatible pollen tubes by non-self SLF action. Taken together, our results demonstrate that SCFSLF-mediated non-self S-RNase degradation occurs in the cytosol of pollen tube through the ubiquitin/26S proteasome system serving as the major mechanism to neutralize S-RNase cytotoxicity during compatible pollination in P. hybrida.
doi:10.3389/fgene.2014.00228
PMCID: PMC4106197  PMID: 25101113
self-incompatibility; self-pollen incompatibility; cross-pollen compatibility; S-RNase localization; SCFSLF; ubiquitin/26S proteasome system
4.  Insight into S-RNase-based self-incompatibility in Petunia: recent findings and future directions 
S-RNase-based self-incompatibility in Petunia is a self/non-self recognition system that allows the pistil to reject self-pollen to prevent inbreeding and to accept non-self pollen for outcrossing. Cloning of S-RNase in 1986 marked the beginning of nearly three decades of intensive research into the mechanism of this complex system. S-RNase was shown to be the sole female determinant in 1994, and the first male determinant, S-locus F-box protein1 (SLF1), was identified in 2004. It was discovered in 2010 that additional SLF proteins are involved in pollen specificity, and recently two S-haplotypes of Petunia inflata were found to possess 17 SLF genes based on pollen transcriptome analysis, further increasing the complexity of the system. Here, we first summarize the current understanding of how the interplay between SLF proteins and S-RNase in the pollen tube allows cross-compatible pollination, but results in self-incompatible pollination. We then discuss some of the aspects that are not yet elucidated, including uptake of S-RNase into the pollen tube, nature, and assembly of SLF-containing complexes, the biochemical basis for differential interactions between SLF proteins and S-RNase, and fate of non-self S-RNases in the pollen tube.
doi:10.3389/fpls.2015.00041
PMCID: PMC4318427
Petunia; SCFSLF complex; self-incompatibility; S-locus F-box protein; Solanaceae; S-RNase
5.  Identification and functional analysis of pistil self-incompatibility factor HT-B of Petunia 
Journal of Experimental Botany  2009;60(4):1309-1318.
Gametophytic self-incompatibility (GSI) in Solanaceae, Rosaceae, and Plantaginaceae is controlled by a multiallelic S-locus. The specificities of pistil and pollen are controlled by separate S-locus genes, S-RNase and SLF/SFB, respectively. Although the S-specificity is determined by the S-locus genes, factors located outside the S-locus are also required for expression of GSI. HT-B is one of the pistil non-S-factors identified in Nicotiana and Solanum, and encodes a small asparagine/aspartate-rich extracellular protein with unknown biochemical function. Here, HT-B was cloned from Petunia and characterized. The structural features and expression pattern of Petunia HT-B were very similar to those of Nicotiana and Solanum. Unlike other solanaceous species, expression of HT-B was also observed in self-compatible Petunia species. RNA interference (RNAi)-mediated suppression of Petunia HT-B resulted in partial breakdown of GSI. Quantitative analysis of the HT-B mRNA accumulation in the transgenics showed that a 100-fold reduction is not sufficient and a >1000-fold reduction is required to achieve partial breakdown of GSI.
doi:10.1093/jxb/erp005
PMCID: PMC2657546  PMID: 19282427
HT-B; Petunia inflata; pistil; RNAi; self-incompatibility
6.  Compatibility and incompatibility in S-RNase-based systems 
Annals of Botany  2011;108(4):647-658.
Background
S-RNase-based self-incompatibility (SI) occurs in the Solanaceae, Rosaceae and Plantaginaceae. In all three families, compatibility is controlled by a polymorphic S-locus encoding at least two genes. S-RNases determine the specificity of pollen rejection in the pistil, and S-locus F-box proteins fulfill this function in pollen. S-RNases are thought to function as S-specific cytotoxins as well as recognition proteins. Thus, incompatibility results from the cytotoxic activity of S-RNase, while compatible pollen tubes evade S-RNase cytotoxicity.
Scope
The S-specificity determinants are known, but many questions remain. In this review, the genetics of SI are introduced and the characteristics of S-RNases and pollen F-box proteins are briefly described. A variety of modifier genes also required for SI are also reviewed. Mutations affecting compatibility in pollen are especially important for defining models of compatibility and incompatibility. In Solanaceae, pollen-side mutations causing breakdown in SI have been attributed to the heteroallelic pollen effect, but a mutation in Solanum chacoense may be an exception. This has been interpreted to mean that pollen incompatibility is the default condition unless the S-locus F-box protein confers resistance to S-RNase. In Prunus, however, S-locus F-box protein gene mutations clearly cause compatibility.
Conclusions
Two alternative mechanisms have been proposed to explain compatibility and incompatibility: compatibility is explained either as a result of either degradation of non-self S-RNase or by its compartmentalization so that it does not have access to the pollen tube cytoplasm. These models are not necessarily mutually exclusive, but each makes different predictions about whether pollen compatibility or incompatibility is the default. As more factors required for SI are identified and characterized, it will be possible to determine the role each process plays in S-RNase-based SI.
doi:10.1093/aob/mcr179
PMCID: PMC3170157  PMID: 21803740
S-RNase; self-incompatibility; Solanaceae; Rosaceae; Nicotiana; Prunus
7.  Genetic evidence that two independent S-loci control RNase-based self-incompatibility in diploid strawberry 
Journal of Experimental Botany  2009;61(3):755-763.
The self-incompatibility mechanism that reduces inbreeding in many plants of the Rosaceae is attributed to a multi-allelic S locus which, in the Prunoideae and Maloideae subfamilies, comprises two complementary genes, a stylar-expressed S-RNase and a pollen-expressed SFB. To elucidate incompatibility in the subfamily Rosoideae, stylar-specific RNases and self-(in)compatibility status were analysed in various diploid strawberries, especially Fragaria nubicola and F. viridis, both self-incompatible, and F. vesca, self-compatible, and in various progenies derived from them. Unexpectedly, two unlinked RNase loci, S and T, were found, encoding peptides distinct from Prunoideae and Maloideae S-RNases; the presence of a single active allele at either is sufficient to confer self-incompatibility. By contrast, in diploid Maloideae and Prunoideae a single locus encodes S-RNases that share several conserved regions and two active alleles are required for self-incompatibility. Our evidence implicates the S locus in unilateral inter-specific incompatibility and shows that S and T RNases can, remarkably, confer not only allele-specific rejection of cognate pollen but also unspecific rejection of Sn Tn pollen, where n indicates a null allele, consistent with the the presence of the pollen component, SFB, activating the cognitive function of these RNases. Comparison of relevant linkage groups between Fragaria and Prunus suggests that Prunus S-RNases, unique in having two introns, may have resulted from gene conversion in an ancestor of Prunus. In addition, it is shown that there is a non-S locus that is essential for self-incompatibility in diploid Fragaria.
doi:10.1093/jxb/erp340
PMCID: PMC2814107  PMID: 20008462
Fragaria; Rosaceae; Rosoideae; self-incompatibility; S/T RNases; unilateral incompatibility
8.  Related polymorphic F-box protein genes between haplotypes clustering in the BAC contig sequences around the S-RNase of Japanese pear 
Journal of Experimental Botany  2010;62(6):1887-1902.
Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB-α–γ) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649 kb around S4-RNase and 378 kb around S2-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB4-u1–u4, 4-d1–d2 and PpSFBB2-u1–u5, 2-d1–d5, respectively) were found, but PpSFBB4-α–γ and PpSFBB2-γ were absent. The PpSFBB4 genes showed 66.2–93.1% amino acid identity with the PpSFBB2 genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3–94.9% identity, while group II consisted of gene pairs with higher identities (>92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes.
doi:10.1093/jxb/erq381
PMCID: PMC3060677  PMID: 21172811
BAC contig; F-box protein; pollen S gene; Pyrus pyrifolia; self-incompatibility; S-locus; S-RNase
9.  Expression and Trans-Specific Polymorphism of Self-Incompatibility RNases in Coffea (Rubiaceae) 
PLoS ONE  2011;6(6):e21019.
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.
doi:10.1371/journal.pone.0021019
PMCID: PMC3120821  PMID: 21731641
10.  A novel gene, MdSSK1, as a component of the SCF complex rather than MdSBP1 can mediate the ubiquitination of S-RNase in apple 
Journal of Experimental Botany  2014;65(12):3121-3131.
Summary
The related components of the SCF complex in apple were cloned and it was proved that an SCF complex containing MdSSK1 rather than MdSBP1 can mediate the ubiquitination of S-RNase.
As a core factor in S-RNase-based gametophytic self-incompatibility (GSI), the SCF (SKP1–Cullin1–F-box-Rbx1) complex (including pollen determinant SLF, S-locus-F-box) functions as an E3 ubiquitin ligase on non-self S-RNase. The SCF complex is formed by SKP1 bridging between SLF, CUL1, and Rbx1; however, it is not known whether an SCF complex lacking SKP1 can mediate the ubiquitination of S-RNase. Three SKP1-like genes from pollen were cloned based on the structural features of the SLF-interacting-SKP1-like (SSK) gene and the ‘Golden Delicious’ apple genome. These genes have a motif of five amino acids following the standard ‘WAFE’ at the C terminal and, in addition, contain eight sheets and two helices. All three genes were expressed exclusively in pollen. In the yeast two-hybrid and pull-down assays only one was found to interact with MdSFBB and MdCUL1, suggesting it is the SLF-interacting SKP1-like gene in apple which was named MdSSK1. In vitro experiments using MdSSK1, S2-MdSFBB1 (S2-Malus domestica S-locus-F-box brother) and MdCUL1 proteins incubated with S 2-RNase and ubiquitin revealed that the SCF complex ubiquitinylates S-RNase in vitro, while MdSBP1 (Malus domestica S-RNase binding protein 1) could not functionally replace MdSSK1 in the SCF complex in ubiquitinylating S-RNase. According to the above experiments, MdSBP1 is probably the only factor responsible for recognition with S-RNase, while not a component of the SCF complex, and an SCF complex containing MdSSK1 is required for mediating the ubiquitination of S-RNase.
doi:10.1093/jxb/eru164
PMCID: PMC4071834  PMID: 24759884
Apple; SCF complex; self-incompatibility; S-RNase; SSK; ubiquitin.
11.  Interhaplotypic heterogeneity and heterochromatic features may contribute to recombination suppression at the S locus in apple (Malus×domestica) 
Journal of Experimental Botany  2012;63(13):4983-4990.
Gametophytic self-incompatibility (GSI) is controlled by a complex S locus containing the pistil determinant S-RNase and pollen determinant SFB/SLF. Tight linkage of the pistil and pollen determinants is necessary to guarantee the self-incompatibility (SI) function. However, multiple probable pollen determinants of apple and Japanese pear, SFBBs (S locus F-box brothers), exist in each S haplotype, and how these multiple genes maintain the SI function remains unclear. It is shown here by high-resolution fluorescence in situ hybridization (FISH) that SFBB genes of the apple S 9 haplotype are physically linked to the S 9 -RNase gene, and the S locus is located in the subtelomeric region. FISH analyses also determined the relative order of SFBB genes and S-RNase in the S 9 haplotype, and showed that gene order differs between the S 9 and S 3 haplotypes. Furthermore, it is shown that the apple S locus is located in a knob-like large heterochromatin block where DNA is highly methylated. It is proposed that interhaplotypic heterogeneity and the heterochromatic nature of the S locus help to suppress recombination at the S locus in apple.
doi:10.1093/jxb/ers176
PMCID: PMC3428002  PMID: 22760470
Fluorescence in situ hybridization; Malus × domestica, recombination suppression; self-incompatibility; S locus F-box brothers (SFBB); Fluorescence in situ hybridization; Fluorescence in situ hybridization
12.  A new dual-specific incompatibility allele revealed by absence of glycosylation in the conserved C2 site of a Solanum chacoense S-RNase 
Journal of Experimental Botany  2013;64(7):1995-2003.
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.
doi:10.1093/jxb/ert059
PMCID: PMC3638826  PMID: 23530129
Allelic recognition; gametophytic self-incompatibility; glycosylation; site-directed mutagenesis; S-RNase; Solanum chacoense.
13.  Progress towards elucidating the mechanisms of self-incompatibility in the grasses: further insights from studies in Lolium 
Annals of Botany  2011;108(4):677-685.
Background and Scope
Self-incompatibility (SI) in flowering plants ensures the maintenance of genetic diversity by ensuring outbreeding. Different genetic and mechanistic systems of SI among flowering plants suggest either multiple origins of SI or considerable evolutionary diversification. In the grasses, SI is based on two loci, S and Z, which are both polyallelic: an incompatible reaction occurs only if both S and Z alleles are matched in individual pollen with alleles of the pistil on which they alight. Such incompatibility is referred to as gametophytic SI (GSI). The mechanics of grass GSI is poorly understood relative to the well-characterized S-RNase-based single-locus GSI systems (Solanaceae, Rosaceae, Plantaginaceae), or the Papaver recognition system that triggers a calcium-dependent signalling network culminating in programmed cell death. There is every reason to suggest that the grass SI system represents yet another mechanism of SI. S and Z loci have been mapped using isozymes to linkage groups C1 and C2 of the Triticeae consensus maps in Secale, Phalaris and Lolium. Recently, in Lolium perenne, in order to finely map and identify S and Z, more closely spaced markers have been developed based on cDNA and repeat DNA sequences, in part from genomic regions syntenic between the grasses. Several genes tightly linked to the S and Z loci were identified, but so far no convincing candidate has emerged.
Research and Progress
From subtracted Lolium immature stigma cDNA libraries derived from S and Z genotyped individuals enriched for SI potential component genes, kinase enzyme domains, a calmodulin-dependent kinase and a peptide with several calcium (Ca2+) binding domains were identified. Preliminary findings suggest that Ca2+ signalling and phosphorylation may be involved in Lolium GSI. This is supported by the inhibition of Lolium SI by Ca2+ channel blockers lanthanum (La3+) and verapamil, and by findings of increased phosphorylation activity during an SI response.
doi:10.1093/aob/mcr186
PMCID: PMC3170160  PMID: 21798860
Lolium perenne; perennial ryegrass; grasses; Poaceae; self-incompatibility; calcium inhibitors; lanthanum chloride; verapamil
14.  Genetic and molecular characterization of three novel S-haplotypes in sour cherry (Prunus cerasus L.) 
Journal of Experimental Botany  2008;59(11):3169-3185.
Tetraploid sour cherry (Prunus cerasus L.) exhibits gametophytic self-incompatibility (GSI) whereby the specificity of self-pollen rejection is controlled by alleles of the stylar and pollen specificity genes, S-RNase and SFB (S haplotype-specific F-box protein gene), respectively. As sour cherry selections can be either self-compatible (SC) or self-incompatible (SI), polyploidy per se does not result in SC. Instead the genotype-dependent loss of SI in sour cherry is due to the accumulation of non-functional S-haplotypes. The presence of two or more non-functional S-haplotypes within sour cherry 2x pollen renders that pollen SC. Two new S-haplotypes from sour cherry, S33 and S34, that are presumed to be contributed by the P. fruticosa species parent, the complete S-RNase and SFB sequences of a third S-haplotype, S35, plus the presence of two previously identified sweet cherry S-haplotypes, S14 and S16 are described here. Genetic segregation data demonstrated that the S16-, S33-, S34-, and S35-haplotypes present in sour cherry are fully functional. This result is consistent with our previous finding that ‘hetero-allelic’ pollen is incompatible in sour cherry. Phylogenetic analyses of the SFB and S-RNase sequences from available Prunus species reveal that the relationships among S-haplotypes show no correspondence to known organismal relationships at any taxonomic level within Prunus, indicating that polymorphisms at the S-locus have been maintained throughout the evolution of the genus. Furthermore, the phylogenetic relationships among SFB sequences are generally incongruent with those among S-RNase sequences for the same S-haplotypes. Hypotheses compatible with these results are discussed.
doi:10.1093/jxb/ern172
PMCID: PMC2504349  PMID: 18617504
Prunus cerasus; self-incompatibility; SFB; S-RNase
15.  The S-locus and unilateral incompatibility. 
Plants have many ways to regulate the type of pollen that arrives on the stigma surface. Once there, further control mechanisms regulate compatibility. The latter controls are largely based on biochemical interactions that support compatible pollination and prevent incompatible matings. S-RNase-based self-incompatibility (SI) systems are the most phylogenetically widespread mechanisms for controlling pollination. Studies of Nicotiana establish a firm link between SI and unilateral interspecific incompatibility. Although implicated in both inter- and intraspecific compatibility, S-RNase operates through at least three distinct genetic mechanisms that differ in their dependence on non-S-RNase factors. Identification and characterization of these non-S-RNase factors is currently an area of active research. Searching for genetic and biochemical interactions with S-RNase can identify candidate non-S-RNase factors. HT-protein is one factor that is required for S-allele-specific pollen rejection in the Solanaceae. Major style arabinogalactan proteins such as TTS interact biochemically with S-RNase. These glycoproteins are known to interact with compatible pollen tubes and have long been suggested as possible recognition molecules. Their binding to S-RNase implies a link between stylar systems for compatibility and incompatibility. Thus, genetic and biochemical studies suggest a highly networked picture of pollen-pistil interactions.
doi:10.1098/rstb.2003.1284
PMCID: PMC1693195  PMID: 12831479
16.  Penetration of the Stigma and Style Elicits a Novel Transcriptome in Pollen Tubes, Pointing to Genes Critical for Growth in a Pistil 
PLoS Genetics  2009;5(8):e1000621.
Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell–cell interaction.
Author Summary
For successful reproduction in flowering plants, a single-celled pollen tube must rapidly extend through female pistil tissue, locate female gametes, and deliver sperm. Pollen tubes undergo a dramatic transformation while growing in the pistil; they grow faster compared to tubes grown in vitro and become competent to perceive and respond to navigation cues secreted by the pistil. The genes expressed by pollen tubes in response to growth in the pistil have not been characterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. Importantly, we identify a set of genes that are specifically expressed in pollen tubes in response to their growth in the pistil and are not expressed during other stages of pollen or plant development. We analyzed mutants in 33 pollen tube–expressed genes using a sensitive series of pollen function assays and demonstrate that seven of these genes are critical for pollen tube growth; two specifically disrupt growth in the pistil. By identifying pollen tube genes induced by the pistil and describing a mutant analysis scheme to understand their function, we lay the foundation for functional genomic analysis of pollen–pistil interactions.
doi:10.1371/journal.pgen.1000621
PMCID: PMC2726614  PMID: 19714218
17.  An S-Locus Independent Pollen Factor Confers Self-Compatibility in ‘Katy’ Apricot 
PLoS ONE  2013;8(1):e53947.
Loss of pollen-S function in Prunus self-compatible cultivars has been mostly associated with deletions or insertions in the S-haplotype-specific F-box (SFB) genes. However, self-compatible pollen-part mutants defective for non-S-locus factors have also been found, for instance, in the apricot (Prunus armeniaca) cv. ‘Canino’. In the present study, we report the genetic and molecular analysis of another self-compatible apricot cv. termed ‘Katy’. S-genotype of ‘Katy’ was determined as S1S2 and S-RNase PCR-typing of selfing and outcrossing populations from ‘Katy’ showed that pollen gametes bearing either the S1- or the S2-haplotype were able to overcome self-incompatibility (SI) barriers. Sequence analyses showed no SNP or indel affecting the SFB1 and SFB2 alleles from ‘Katy’ and, moreover, no evidence of pollen-S duplication was found. As a whole, the obtained results are compatible with the hypothesis that the loss-of-function of a S-locus unlinked factor gametophytically expressed in pollen (M’-locus) leads to SI breakdown in ‘Katy’. A mapping strategy based on segregation distortion loci mapped the M’-locus within an interval of 9.4 cM at the distal end of chr.3 corresponding to ∼1.29 Mb in the peach (Prunus persica) genome. Interestingly, pollen-part mutations (PPMs) causing self-compatibility (SC) in the apricot cvs. ‘Canino’ and ‘Katy’ are located within an overlapping region of ∼273 Kb in chr.3. No evidence is yet available to discern if they affect the same gene or not, but molecular markers seem to indicate that both cultivars are genetically unrelated suggesting that every PPM may have arisen independently. Further research will be necessary to reveal the precise nature of ‘Katy’ PPM, but fine-mapping already enables SC marker-assisted selection and paves the way for future positional cloning of the underlying gene.
doi:10.1371/journal.pone.0053947
PMCID: PMC3544744  PMID: 23342044
18.  Molecular modeling of S-RNases involved in almond self-incompatibility 
Gametophytic self-incompatibility (GSI) is a mechanism in flowering plants, to prevent inbreeding and promote outcrossing. GSI is under the control of a specific locus, known as the S-locus, which contains at least two genes, the RNase and the SFB. Active S-RNases in the style are essential for rejection of haploid pollen, when the pollen S-allele matches one of two S-alleles of the diploid pistil. However, the nature of their mutual interactions at genetic and biochemical levels remain unclear. Thus, detailed understanding of the protein structure involved in GSI may help in discovering how the proteins involved in GSI may function and how they fulfill their biological roles. To this end, 3D models of the SC (Sf) and two SI (S8 and S23) S-RNases of almond were constructed, using comparative modeling tools. The modeled structures consisted of mixed α and β folds, with six helices and six β-strands. However, the self-compatible (Sf) RNase contained an additional extended loop between the conserved domains RC4 and C5, which may be involved in the manifestation of self-compatibility in almond.
doi:10.3389/fpls.2012.00139
PMCID: PMC3384224  PMID: 22754558
almond; self-(in)compatibility; 3D modeling; RNase T2
19.  Evolutionary patterns at the RNase based gametophytic self - incompatibility system in two divergent Rosaceae groups (Maloideae and Prunus) 
Background
Within Rosaceae, the RNase based gametophytic self-incompatibility (GSI) system has been studied at the molecular level in Maloideae and Prunus species that have been diverging for, at least, 32 million years. In order to understand RNase based GSI evolution within this family, comparative studies must be performed, using similar methodologies.
Result
It is here shown that many features are shared between the two species groups such as levels of recombination at the S-RNase (the S-pistil component) gene, and the rate at which new specificities arise. Nevertheless, important differences are found regarding the number of ancestral lineages and the degree of specificity sharing between closely related species. In Maloideae, about 17% of the amino acid positions at the S-RNase protein are found to be positively selected, and they occupy about 30% of the exposed protein surface. Positively selected amino acid sites are shown to be located on either side of the active site cleft, an observation that is compatible with current models of specificity determination. At positively selected amino acid sites, non-conservative changes are almost as frequent as conservative changes. There is no evidence that at these sites the most drastic amino acid changes may be more strongly selected.
Conclusions
Many similarities are found between the GSI system of Prunus and Maloideae that are compatible with the single origin hypothesis for RNase based GSI. The presence of common features such as the location of positively selected amino acid sites and lysine residues that may be important for ubiquitylation, raise a number of issues that, in principle, can be experimentally addressed in Maloideae. Nevertheless, there are also many important differences between the two Rosaceae GSI systems. How such features changed during evolution remains a puzzling issue.
doi:10.1186/1471-2148-10-200
PMCID: PMC2909234  PMID: 20584298
20.  The Papaver Self-Incompatibility Pollen S-Determinant, PrpS, Functions in Arabidopsis thaliana 
Current Biology  2012;22(2):154-159.
Summary
Many angiosperms use specific interactions between pollen and pistil proteins as “self” recognition and/or rejection mechanisms to prevent self-fertilization. Self-incompatibility (SI) is encoded by a multiallelic S locus, comprising pollen and pistil S-determinants [1, 2]. In Papaver rhoeas, cognate pistil and pollen S-determinants, PrpS, a pollen-expressed transmembrane protein, and PrsS, a pistil-expressed secreted protein [3, 4], interact to trigger a Ca2+-dependent signaling network [5–10], resulting in inhibition of pollen tube growth, cytoskeletal alterations [11–13], and programmed cell death (PCD) [14, 15] in incompatible pollen. We introduced the PrpS gene into Arabidopsis thaliana, a self-compatible model plant. Exposing transgenic A. thaliana pollen to recombinant Papaver PrsS protein triggered remarkably similar responses to those observed in incompatible Papaver pollen: S-specific inhibition and hallmark features of Papaver SI [11–15]. Our findings demonstrate that Papaver PrpS is functional in a species with no SI system that diverged ∼140 million years ago [16]. This suggests that the Papaver SI system uses cellular targets that are, perhaps, common to all eudicots and that endogenous signaling components can be recruited to elicit a response that most likely never operated in this species. This will be of interest to biologists interested in the evolution of signaling networks in higher plants.
Highlights
► PrpS, a Papaver SI determinant, functions in Arabidopsis thaliana pollen ► A “self” interaction with PrsS reveals Papaver SI hallmark features in A. thaliana ► The first evidence for transfamily functionality of an SI system (>140 my apart) ► Evidence of recruitment of signaling components for novel SI function
doi:10.1016/j.cub.2011.12.006
PMCID: PMC3695568  PMID: 22209529
21.  Patterns of evolution at the gametophytic self-incompatibility Sorbus aucuparia (Pyrinae) S pollen genes support the non-self recognition by multiple factors model 
Journal of Experimental Botany  2013;64(8):2423-2434.
S-RNase-based gametophytic self-incompatibility evolved once before the split of the Asteridae and Rosidae. In Prunus (tribe Amygdaloideae of Rosaceae), the self-incompatibility S-pollen is a single F-box gene that presents the expected evolutionary signatures. In Malus and Pyrus (subtribe Pyrinae of Rosaceae), however, clusters of F-box genes (called SFBBs) have been described that are expressed in pollen only and are linked to the S-RNase gene. Although polymorphic, SFBB genes present levels of diversity lower than those of the S-RNase gene. They have been suggested as putative S-pollen genes, in a system of non-self recognition by multiple factors. Subsets of allelic products of the different SFBB genes interact with non-self S-RNases, marking them for degradation, and allowing compatible pollinations. This study performed a detailed characterization of SFBB genes in Sorbus aucuparia (Pyrinae) to address three predictions of the non-self recognition by multiple factors model. As predicted, the number of SFBB genes was large to account for the many S-RNase specificities. Secondly, like the S-RNase gene, the SFBB genes were old. Thirdly, amino acids under positive selection—those that could be involved in specificity determination—were identified when intra-haplotype SFBB genes were analysed using codon models. Overall, the findings reported here support the non-self recognition by multiple factors model.
doi:10.1093/jxb/ert098
PMCID: PMC3654429  PMID: 23606363
gametophytic self-incompatibility; molecular evolution; positively selected amino acid sites; SFBB; Sorbus aucuparia; S-RNase.
22.  Genetic Features of the Spontaneous Self-Compatible Mutant, ‘Jin Zhui’ (Pyrus bretschneideri Rehd.) 
PLoS ONE  2013;8(10):e76509.
‘Jin Zhui’ is a spontaneous self-compatible mutant of ‘Ya Li’ (Pyrus bretschneideri Rehd. S21S34), the latter displaying a typical S-RNase-based gametophytic self-incompatibility (GSI). The pollen-part mutation (PPM) of ‘Jin Zhui’ might be due to a natural mutation in the pollen-S gene (S34 haplotype). However, the molecular mechanisms behind these phenotypic changes are still unclear. In this study, we identified five SLF (S-Locus F-box) genes in ‘Ya Li’, while no nucleotide differences were found in the SLF genes of ‘Jin Zhui’. Further genetic analysis by S-RNase PCR-typing of selfed progeny of ‘Jin Zhui’ and ‘Ya Li’ × ‘Jin Zhui’ progeny showed three progeny classes (S21S21, S21S34 and S34S34) as opposed to the two classes reported previously (S21S34 and S34S34), indicating that the pollen gametes of ‘Jin Zhui’, bearing either the S21- or S34-haplotype, were able to overcome self-incompatibility (SI) barriers. Moreover, no evidence of pollen-S duplication was found. These findings support the hypothesis that loss of function of S-locus unlinked PPM expressed in pollen leads to SI breakdown in ‘Jin Zhui’, rather than natural mutation in the pollen-S gene (S34 haplotype). Furthermore, abnormal meiosis was observed in a number of pollen mother cells (PMCs) in ‘Jin Zhui’, but not in ‘Ya Li’. These and other interesting findings are discussed.
doi:10.1371/journal.pone.0076509
PMCID: PMC3792025  PMID: 24116113
23.  Unilateral Incompatibility in Capsicum (Solanaceae): Occurrence and Taxonomic Distribution 
Annals of Botany  2004;94(2):289-295.
• Background and aims Unilateral incompatibility (UI) occurs when pollinations between species are successful in one direction but not in the other. Self-incompatible (SI) species frequently show UI with genetically related, self-compatible (SC) species, as pollen of SI species is compatible on the SC pistil, but not vice versa. Many examples of unilateral incompatibility, and all those which have been studied most intensively, are found in the Solanaceae, particularly Lycopersicon, Solanum, Nicotiana and Petunia. The genus Capsicum is evolutionarily somewhat distant from Lycopersicon and Solanum and even further removed from Nicotiana and Petunia. Unilateral incompatibility has also been reported in Capsicum; however, this is the first comprehensive study of crosses between all readily available species in the genus.
• Methods All readily available (wild and domesticated) species in the genus are used as plant material, including the three genera from the Capsicum pubescens complex plus eight other species. Pollinations were made on pot-grown plants in a glasshouse. The number of pistils pollinated per cross varied (from five to 40 pistils per plant), depending on the numbers of flowers available. Pistils were collected 24 h after pollination and fixed for 3–24 h. After staining, pistils were mounted in a drop of stain, squashed gently under a cover slip and examined microscopically under ultra-violet light for pollen tube growth.
• Key results Unilateral incompatibility is confirmed in the C. pubescens complex. Its direction conforms to that predominant in the Solanaceae and other families, i.e. pistils of self-incompatible species, or self-compatible taxa closely related to self-incompatible species, inhibit pollen tubes of self-compatible species.
• Conclusions Unilateral incompatibility in Capsicum does not seem to have arisen to prevent introgression of self-compatibility into self-incompatible taxa, but as a by-product of divergence of the C. pubescens complex from the remainder of the genus.
doi:10.1093/aob/mch139
PMCID: PMC4242164  PMID: 15229125
Capsicum pubescens complex; chile pepper; incompatibility; incongruity; interspecific hybridization; pollen tube growth; S gene
24.  Identification of the pollen self-incompatibility determinant in Papaver rhoeas 
Nature  2009;459(7249):992-995.
Higher plants produce seed through pollination, using specific interactions between pollen and pistil. Self-incompatibility (SI) is an important mechanism used in many species to prevent inbreeding, and is controlled by a multi-allelic S locus1,2. “Self” (incompatible) pollen is discriminated from “non-self” (compatible) pollen, by interaction of pollen and pistil S locus components, and is subsequently inhibited. In Papaver rhoeas, the pistil S locus product is a small protein that interacts with incompatible pollen, triggering a Ca2+-dependent signalling network, resulting in pollen inhibition and programmed cell death3-7. Here we have cloned three alleles of a highly polymorphic pollen-expressed gene, PrpS, from Papaver and provide evidence that this encodes the pollen S locus determinant. PrpS is a single copy gene linked to the pistil S gene, PrsS. Sequence analysis indicates that PrsS and PrpS are equally ancient and are likely to have co-evolved. PrpS encodes a novel ~20 kDa protein. Consistent with predictions that it is a transmembrane protein, PrpS is associated with the plasma membrane. We show that a predicted extracellular loop segment of PrpS interacts with PrsS and, using PrpS antisense oligonucleotides, we demonstrate that PrpS is involved in S-specific inhibition of incompatible pollen. Identification of PrpS represents a major advance in our understanding of the Papaver SI system. As a novel cell-cell recognition determinant it contributes to the available information concerning the origins and evolution of cell-cell recognition systems involved in discrimination between “self” and “non-self”, which also include histocompatibility systems in primitive chordates and vertebrates.
doi:10.1038/nature08027
PMCID: PMC2699350  PMID: 19483678
Self-incompatibility; pollen S receptor; Papaver rhoeas; pollen tube inhibition
25.  Pollen–pistil interactions and self-incompatibility in the Asteraceae: new insights from studies of Senecio squalidus (Oxford ragwort) 
Annals of Botany  2011;108(4):687-698.
Background
Pollen–pistil interactions are an essential prelude to fertilization in angiosperms and determine compatibility/incompatibility. Pollen–pistil interactions have been studied at a molecular and cellular level in relatively few families. Self-incompatibility (SI) is the best understood pollen–pistil interaction at a molecular level where three different molecular mechanisms have been identified in just five families. Here we review studies of pollen–pistil interactions and SI in the Asteraceae, an important family that has been relatively understudied in these areas of reproductive biology.
Scope
We begin by describing the historical literature which first identified sporophytic SI (SSI) in species of Asteraceae, the SI system later identified and characterized at a molecular level in the Brassicaceae. Early structural and cytological studies in these two families suggested that pollen–pistil interactions and SSI were similar, if not the same. Recent cellular and molecular studies in Senecio squalidus (Oxford ragwort) have challenged this belief by revealing that despite sharing the same genetic system of SSI, the Brassicaceae and Asteraceae molecular mechanisms are different. Key cellular differences have also been highlighted in pollen–stigma interactions, which may arise as a consequence of the Asteraceae possessing a ‘semi-dry’ stigma, rather than the ‘dry’ stigma typical of the Brassicaceae. The review concludes with a summary of recent transcriptomic analyses aimed at identifying proteins regulating pollen–pistil interactions and SI in S. squalidus, and by implication the Asteraceae. The Senecio pistil transcriptome contains many novel pistil-specific genes, but also pistil-specific genes previously shown to play a role in pollen–pistil interactions in other species.
Conclusions
Studies in S. squalidus have shown that stigma structure and the molecular mechanism of SSI in the Asteraceae and Brassicaceae are different. The availability of a pool of pistil-specific genes for S. squalidus offers an opportunity to elucidate the molecular mechanisms of pollen–pistil interactions and SI in the Asteraceae.
doi:10.1093/aob/mcr147
PMCID: PMC3170154  PMID: 21752792
Asteraceae; Senecio; pistil; stigma; pollen; pollen–pistil interactions; self-incompatibility; transcriptome

Results 1-25 (1027914)