Analyses of Cullin1 homologs reveal functional redundancy in S-RNase-based self-incompatibility and evolutionary relationships in eudicots.

In Petunia (Solanaceae family), self-incompatibility (SI) is regulated by the polymorphic S-locus, which contains the pistil-specific S-RNase and multiple pollen-specific S-Locus F-box (SLF) genes. SLFs assemble into E3 ubiquitin ligase complexes known as Skp1-Cullin1-F-box complexes (SCFSLF). In pollen tubes, these complexes collectively mediate ubiquitination and degradation of all nonself S-RNases, but not self S-RNase, resulting in cross-compatible, but self-incompatible, pollination. Using Petunia inflata, we show that two pollen-expressed Cullin1 (CUL1) proteins, PiCUL1-P and PiCUL1-B, function redundantly in SI. This redundancy is lost in Petunia hybrida, not because of the inability of PhCUL1-B to interact with SSK1, but due to a reduction in the PhCUL1-B transcript level. This is possibly caused by the presence of a DNA transposon in the PhCUL1-B promoter region, which was inherited from Petunia axillaris, one of the parental species of Pe. hybrida. Phylogenetic and syntenic analyses of Cullin genes in various eudicots show that three Solanaceae-specific CUL1 genes share a common origin, with CUL1-P dedicated to S-RNase-related reproductive processes. However, CUL1-B is a dispersed duplicate of CUL1-P present only in Petunia, and not in the other species of the Solanaceae family examined. We suggest that the CUL1s involved (or potentially involved) in the SI response in eudicots share a common origin.

Sequence analysis of the Petunia inflata S-locus region containing 17 S-Locus F-Box genes and the S-RNase gene involved in self-incompatibility.

Self-incompatibility in Petunia is controlled by the polymorphic S-locus, which contains S-RNase encoding the pistil determinant and 16-20 S-locus F-box (SLF) genes collectively encoding the pollen determinant. Here we sequenced and assembled approximately 3.1 Mb of the S2 -haplotype of the S-locus in Petunia inflata using bacterial artificial chromosome clones collectively containing all 17 SLF genes, SLFLike1, and S-RNase. Two SLF pseudogenes and 28 potential protein-coding genes were identified, 20 of which were also found at the S-loci of both the S6a -haplotype of P. inflata and the SN -haplotype of self-compatible Petunia axillaris, but not in the S-locus remnants of self-compatible potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Comparative analyses of S-locus sequences of these three S-haplotypes revealed potential genetic exchange in the flanking regions of SLF genes, resulting in highly similar flanking regions between different types of SLF and between alleles of the same type of SLF of different S-haplotypes. The high degree of sequence similarity in the flanking regions could often be explained by the presence of similar long terminal repeat retroelements, which were enriched at the S-loci of all three S-haplotypes and in the flanking regions of all S-locus genes examined. We also found evidence of the association of transposable elements with SLF pseudogenes. Based on the hypothesis that SLF genes were derived by retrotransposition, we identified 10 F-box genes as putative SLF parent genes. Our results shed light on the importance of non-coding sequences in the evolution of the S-locus, and on possible evolutionary mechanisms of generation, proliferation, and deletion of SLF genes.

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.

AcsA-AcsB: The core of the cellulose synthase complex from Gluconacetobacter hansenii ATCC23769.

The gram-negative bacterium, Gluconacetobacter hansenii, produces cellulose of exceptionally high crystallinity in comparison to the cellulose of higher plants. This bacterial cellulose is synthesized and extruded into the extracellular medium by the cellulose synthase complex (CSC). The catalytic component of this complex is encoded by the gene AcsAB. However, several other genes are known to encode proteins critical to cellulose synthesis and are likely components of the bacterial CSC. We have purified an active heterodimer AcsA-AcsB from G. hansenii ATCC23769 to homogeneity by two different methods. With the purified protein, we have determined how it is post-translationally processed, forming the active heterodimer AcsA-AcsB. Additionally, we have performed steady-state kinetic studies on the AcsA-AcsB complex. Finally through mutagenesis studies, we have explored the roles of the postulated CSC proteins AcsC, AcsD, and CcpAx.

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.

Isolation and characterization of two cellulose morphology mutants of Gluconacetobacter hansenii ATCC23769 producing cellulose with lower crystallinity.

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of ß-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.

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.

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.

Identification and characterization of non-cellulose-producing mutants of Gluconacetobacter hansenii generated by Tn5 transposon mutagenesis.

The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel(-)) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript "Ax" indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel(-) mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel(-) mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding ß-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis.

Identification and characterization of a cellulose binding heptapeptide revealed by phage display.

Cellulose nanowhiskers (CNWs) were used in conjunction with phage display technology to identify polypeptides which bind the crystalline region of cellulose. A consensus peptide WHWTYYW was identified to efficiently bind the CNWs. The binding affinities of specific phage particles were assessed using biopanning assays and enzyme-linked immunosorbent assay (ELISA). The WHWTYYW peptide was synthesized and isothermal titration calorimetry (ITC) analysis showed that the peptide exhibited a binding constant of ∼10(5) M(-1) toward the crystalline CNWs. In order to understand how the affinity of this peptide differs for noncrystalline cellulose, binding properties were characterized using cello-oligosaccharides as substrates. Binding analysis was performed using UV spectroscopy and fluorescence quenching experiments. The specific molecular interactions of the WHWTYYW peptide with cellohexaose were examined using nuclear magnetic resonance (NMR). Interactions of this peptide with crystalline cellulose were also investigated using classical molecular modeling and quantum mechanical calculations of (13)C NMR chemical shifts. The NMR experiments and calculations indicate that the WHWTYYW peptide exhibits a bent structure when bound, allowing the Y5 amino acid to form a CH/π stacking interaction and H-bond with the glucose ring of cellulose.

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.

Processing of cellulose synthase (AcsAB) from Gluconacetobacter hansenii 23769.

The cellulose synthase protein (AcsAB) is encoded by a single gene in Gluconacetobacter hansenii ATCC 23769. We have examined the processing pattern of this enzyme and the localization of the cleavage products by heterologously expressing the truncated portions of the AcsAB protein and using specific antibodies generated against these regions. We found that the AcsAB protein is processed into three polypeptide subunits of molecular masses 46kDa, 34kDa and 95kDa. The 46kDa polypeptide (AcsA(cat)) harbors the conserved glycosyltransferase domain and hence contains the catalytic subunit of the enzyme. This polypeptide is localized in the cytoplasmic membrane. The 34kDa polypeptide (AcsA(reg)) is the regulatory subunit with the cyclic diGMP-binding PilZ domain. This polypeptide is largely cytoplasmic. The 95kDa subunit (AcsB) is of unknown function and contains a predicted signal peptide at its N-terminus. This subunit is localized in the outer membrane. In addition to this, we have also localized the AcsC protein in the outer membrane, confirming its predicted localization based on the OM-signal sequence at its N-terminus.

Self-incompatibility in Petunia: a self/nonself-recognition mechanism employing S-locus F-box proteins and S-RNase to prevent inbreeding.

Many flowering plants producing bisexual flowers have adopted self-incompatibility (SI), a reproductive strategy which allows pistils to distinguish between self and nonself pollen, and to only permit nonself pollen to effect fertilization. To date, three different SI mechanisms have been identified, and this article focuses on the S-RNase-based mechanism using Petunia (Solanaceae) as a model. The genetic basis of this type of SI was established nearly a century ago; the polymorphic S-locus specifies the genetic identity of pollen and the pistil. Molecular genetic studies carried out since the late 1980s have led to the identification of the polymorphic genes at the S-locus that control self/nonself-recognition between pollen and the pistil. The S-RNase gene, which controls pistil specificity, was identified first, and subsequent sequencing of the S-locus region containing S-RNase led to the identification of the S-locus F-box (SLF) gene (now named SLF1). A transgenic approach was used to show that S2-SLF1 (SLF1 of S2-halotype) of Petunia inflata controls pollen specificity. The S-locus contains additional pollen-expressed F-box genes that show sequence similarity with SLF1, and initially they were thought not to be involved in pollen specificity. However, further studies of SLF1 suggested that it is not the only pollen specificity gene. Indeed, it has recently been shown that two previously identified SLF-like genes in P. inflata (now named SLF2 and SLF3) and a yet unknown number of additional SLF-like genes (named SLF4, SLF5, etc.) collaboratively function to control pollen specificity. The significance and implications of this new finding are discussed.

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.

The amino terminal F-box domain of Petunia inflata S-locus F-box protein is involved in the S-RNase-based self-incompatibility mechanism.

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 PiSLF(2) (S(2) allelic variant) without this domain in S(2)S(3) plants and showed that, unlike the full-length PiSLF(2), it did not cause breakdown of SI in S(3) pollen. We identified PiSSK1 (P. inflata SSK1) and found that it did not interact with PiSLF(1), PiSLF(2) or PiSLF(3). CONCLUSIONS: The finding that the truncated PiSLF(2) did not cause breakdown of SI in S(3) transgenic pollen suggests that the F-box domain of PiSLF(2) is required for mediating degradation of S(3)-RNase, a non-self S-RNase, in S(3) 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.