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 of self-incompatibility (S-) locus linked pollen cDNA markers in Petunia inflata.

Solanaceous type self-incompatibility (SI) is controlled by a single polymorphic locus, termed the S-locus. The only gene at the S-locus that has been characterized thus far is the S-RNase gene, which controls pistil function, but not pollen function, in SI interactions between pistil and pollen. One approach to identifying additional genes (including the pollen S-gene, which controls pollen function in SI) at the S-locus and to study the structural organization of the S-locus is chromosome walking from the S-RNase gene. However, the presence of highly repetitive sequences in its flanking regions has made this approach difficult so far. Here, we used RNA differential display to identify pollen cDNAs of Petunia inflata, a self-incompatible solanaceous species, which exhibited restriction fragment length polymorphism (RFLP) for at least one of the three S-haplotypes (S1, S2, and S3) examined. We found that the genes corresponding to 10 groups of pollen cDNAs are genetically tightly linked to the S-RNase gene. These cDNA markers will expedite the mapping and cloning of the chromosomal region of the Solanaceae S-locus by providing multiple starting points.     

Genetic mapping and molecular characterization of the self-incompatibility (S) locus in Petunia inflata

Gametophytic self-incompatibility (SI) possessed by the Solanaceae is controlled by a highly polymorphic locus called the S locus. The S locus contains two linked genes, S-RNase, which determines female specificity, and the as yet unidentified pollen S gene, which determines male specificity in SI interactions. To identify the pollen S gene of Petunia inflata, we had previously used mRNA differential display and subtractive hybridization to identify 13 pollen-expressed genes that showed S -haplotype-specific RFLP. Here, we carried out recombination analysis of 1205 F2 plants to determine the genetic distance between each of these S -linked genes and S-RNase. Recombination was observed between four of the genes (3.16, G211, G212, and G221) and S-RNase, whereas no recombination was observed for the other nine genes (3.2, 3.15, A113, A134, A181, A301, G261, X9, and X11). A genetic map of the S locus was constructed, with 3.16 and G221 delimiting the outer limits. None of the observed crossovers disrupted SI, suggesting that all the genes required for SI are contained in the chromosomal region defined by 3.16 and G221. These results and our preliminary chromosome walking results suggest that the S locus is a huge multi-gene complex. Allelic sequence diversity of G221 and 3.16, as well as of 3.2, 3.15, A113, A134 and G261, was determined by comparing two or three alleles of their cDNA and/or genomic sequences. In contrast to S-RNase, all these genes showed very low degrees of allelic sequence diversity in the coding regions, introns, and flanking regions.     

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.

     

Construction of a binary bacterial artificial chromosome library of Petunia inflata and the isolation of large genomic fragments linked to the self-incompatibility (S-) locus.

The Solanaceae family of flowering plants possesses a type of self-incompatibility mechanism that enables the pistil to reject self pollen but accept non-self pollen for fertilization. The pistil function in this system has been shown to be controlled by a polymorphic gene at the S-locus, termed the S-RNase gene. The pollen function is believed to be controlled by another as yet unidentified polymorphic gene at the S-locus, termed the pollen S-gene. As a first step in using a functional genomic approach to identify the pollen S-gene, a genomic BAC (bacterial artificial chromosome) library of the S2S2 genotype of Petunia inflata, a self-incompatible solanaceous species, was constructed using a Ti-plasmid based BAC vector, BIBAC2. The average insert size was 136.4 kb and the entire library represented a 7.5-fold genome coverage. Screening of the library using cDNAs for the S2-RNase gene and 13 pollen-expressed genes that are linked to the S-locus yielded 51 positive clones, with at least one positive clone for each gene. Collectively, at least 2 Mb of the chromosomal region was spanned by these clones. Together, three clones that contained the S2-RNase gene spanned approximately 263 kb. How this BAC library and the clones identified could be used to identify the pollen S-gene and to study other aspects of self-incompatibility is discussed.     

Pollen-expressed F-box gene family and mechanism of S-RNase-based gametophytic self-incompatibility (GSI) in Rosaceae

Many species of Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI) in which pistil-part specificity is controlled by S locus-encoded ribonuclease (S-RNase). Although recent findings revealed that S locus-encoded F-box protein, SLF/SFB, determines pollen-part specificity, how these pistil- and pollen-part S locus products interact in vivo and elicit the SI reaction is largely unclear. Furthermore, genetic studies suggested that pollen S function can differ among species. In Solanaceae and the rosaceous subfamily Maloideae (e.g., apple and pear), the coexistence of two different pollen S alleles in a pollen breaks down SI of the pollen, a phenomenon known as competitive interaction. However, competitive interaction seems not to occur in the subfamily Prunoideae (e.g., cherry and almond) of Rosaceae. Furthermore, the effect of the deletion of pollen S seems to vary among taxa. This review focuses on the potential differences in pollen-part function between subfamilies of Rosaceae, Maloideae, and Prunoideae, and discusses implications for the mechanistic divergence of the S-RNase-based SI.     

Segregation distortion of T-DNA markers linked to the self-incompatibility (S) locus in Petunia hybrida

In plants with a gametophytic self-incompatibility system the specificity of the pollen is determined by the haploid genotype at the self-incompatibility (S) locus. In certain crosses this can lead to the exclusion of half the gametes from the male parent carrying a particular S-allele. This leads to pronounced segregation distortion for any genetic markers that are linked to the S-locus. We have used this approach to identify T-DNA insertions carrying a maize transposable element that are linked to the S-locus of Petunia hybrida. A total of 83 T-DNA insertions were tested for segregation distortion of the selectable marker used during transformation with Agrobacterium. Segregation distortion was observed for 12 T-DNA insertions and at least 8 of these were shown to be in the same linkage group by intercrossing. This indicates that differential transmission of a single locus (S) is probably responsible for all of these examples of T-DNA segregation distortion. The identification of selectable markers in coupling with a functional S-allele will allow the preselection of recombination events around the S-locus in petunia. Our approach provides a general method for identifying transgenes that are linked to gametophytic self-incompatibility loci and provides an opportunity for transposon tagging of the petunia S-locus.     

Allelic diversity of S-RNase at the self-incompatibility locus in natural flowering cherry populations (Prunus lannesiana var. speciosa)

In the Rosaceae family, which includes Prunus, gametophytic self-incompatibility (GSI) is controlled by a single multiallelic locus (S-locus), and the S-locus product expressed in the pistils is a glycoprotein with ribonuclease activity (S-RNase). Two populations of flowering cherry (Prunus lannesiana var. speciosa), located on Hachijo Island in Japan's Izu Islands, were sampled, and S-allele diversity was surveyed based on the sequence polymorphism of S-RNase. A total of seven S-alleles were cloned and sequenced. The S-RNases of flowering cherry showed high homology to those of Prunus cultivars (P. avium and P. dulcis). In the phylogenetic tree, the S-RNases of flowering cherry and other Prunus cultivars formed a distinct group, but they did not form species-specific subgroups. The nucleotide substitution pattern in S-RNases of flowering cherry showed no excess of nonsynonymous substitutions relative to synonymous substitutions. However, the S-RNases of flowering cherry had a higher Ka/Ks ratio than those of other Prunus cultivars, and a subtle heterogeneity in the nucleotide substitution rates was observed among the Prunus species. The S-genotype of each individual was determined by Southern blotting of restriction enzyme-digested genomic DNA, using cDNA for S-RNase as a probe. A total of 22 S-alleles were identified. All individuals examined were heterozygous, as expected under GSI. The allele frequencies were, contrary to the expectation under GSI, significantly unequal. The two populations studied showed a high degree of overlap, with 18 shared alleles. However, the allele frequencies differed considerably between the two populations.     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S ₂, encoded by the S ₂ allele, with the expected features of the Sp gene was identified. AhSLF-S ₂ is located 9 kb downstream of S ₂ RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S ₂ are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S ₂ is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S2, encoded by the S2 allele, with the expected features of the Sp gene was identified. AhSLF-S2 is located 9 kb downstream of S2 RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S2 are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S2 is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

Style proteins of a wild tomato (Lycopersicon peruvianum) associated with expression of self-incompatibility

The identification, isolation and aminoterminal sequencing of two S-genotype-associated proteins from style extracts of Lycopersicon peruvianum Mill. is reported. There is a high level of homology between these two sequences and with the amino-terminal sequences of other S-allele-associated glycoproteins isolated from Nicotiana alata Link et Otto. These sequences were obtained by a new high-sensitivity method of selected twodimensional gel analysis followed by electroelution and purification of proteins by inverse-gradient high-performance liquid chromatography before sequencing.Abbreviations HPLC high-performance liquid chromatography - Mr relative molecular mass - PTH phenylthiohydrantoin - SDS sodium dodecyl sulphate     

Breakdown of self-incompatibility in a natural population of Petunia axillaris (Solanaceae) in Uruguay containing both self-incompatible and self-compatible plants

 Many members of the Solanaceae display a type of gametophytic self-incompatibility which is controlled by a single multiallelic locus, called the S-locus. From our previous survey of more than 100 natural populations of Petunia axillaris (a solanaceous species) in Uruguay, we had found that the majority of the populations of subspecies axillaris were comprised of virtually all self-incompatible individuals. The rest were ”mixed populations” which contained mostly self-incompatible and some self-compatible individuals. In this study, we examined the self-incompatibility behavior and determined the S-genotypes of 33 plants raised from seeds obtained from one such mixed population, designated U1. We found that 30 of the 33 plants (designated U1–1 through U1–33) were self-incompatible and a total of 18 different S-alleles were represented. To determine the S-genotypes of the three self-compatible plants (U1–2, U1–16, and U1–22) and the possible causes for the breakdown of their self-incompatibility, we carried out reciprocal crosses between each of them and each of the 18 S-homozygotes (S ₁ S ₁ through S ₁₈ S ₁₈ ) obtained from bud-selfed progeny of 14 of the 30 self-incompatible plants. For U1–2 and U1–16, we also carried out additional crosses with U1–25 (with S ₁ S ₁₃ genotype) and an S ₁₃ S ₁₅ plant (obtained from a cross between an S ₁₃ -homozygote and an S ₁₅ -homozygote), respectively. Based on all the pollination results and analysis of the production of S-RNases, products of S-alleles in the pistil, we determined the S-genotypes of U1–2, U1–16, and U1–22, and propose that the breakdown of self-incompatibility in these three plants is caused by suppression of the production of S₁₃-RNase from the S ₁₃ -allele they all carry. We have termed this phenomenon ”stylar-part suppression of an S-allele” or SPS. Received: 25 September 1998 / Revision accepted: 22 December 1998     

High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata

A strategy was developed for the high-throughput localization of unknown expressed proteins in Nicotiana benthamiana. Libraries of random, partial cDNAs fused to the 5' or 3' end of the gene for green fluorescent protein (GFP) were expressed in planta using a vector based on Tobacco mosaic virus. Viral populations were screened en masse on inoculated leaves using a confocal microscope fitted with water-dipping lenses. Each viral infection site expressed a unique cDNA-GFP fusion, allowing several hundred cDNA-GFP fusions to be screened in a single day. More than half of the members of the library carrying cDNA fusions to the 5' end of gfp that expressed fluorescent fusion proteins displayed discrete, noncytosolic, subcellular localizations. Nucleotide sequence determination of recovered cDNA sequences and subsequent sequence searches showed that fusions of GFP to proteins that had a predicted subcellular "address" became localized with high fidelity. In a subsequent screen of >20,000 infection foci, 12 fusion proteins were identified that localized to plasmodesmata, a subcellular structure for which very few protein components have been identified. This virus-based system represents a method for high-throughput functional genomic study of plant cell organelles and allows the identification of unique proteins that associate with specific subcompartments within organelles.     

Identification of major lysine residues of S(3)-RNase of Petunia inflata involved in ubiquitin-26S proteasome-mediated degradation in vitro

S-RNase-based self-incompatibility has been identified in three flowering plant families, including the Solanaceae, and this self/non-self recognition mechanism between pollen and pistil is controlled by two polymorphic genes at the S -locus, S-RNase and S-locus F-box ( SLF ). S-RNase is produced in the pistil and taken up by pollen tubes in a non- S- haplotype-specific manner. How an allelic product of SLF interacts with self and non-self S-RNases to result in growth inhibition of self pollen tubes is not completely understood. One model predicts that SLF targets non-self S-RNases for ubiquitin/26S proteasome-mediated degradation, thereby only allowing self S-RNase to exert cytotoxic activity inside a pollen tube. To test this model, we studied whether any of the 20 lysine residues in S₃-RNase of Petunia inflata might be targets for ubiquitination. We identified six lysines near the C-terminus for which mutation to arginine significantly reduced ubiquitination and degradation of the mutant S₃-RNase, GST:S₃-RNase (K141–164R) in pollen tube extracts. We further showed that GST:S₃-RNase (K141–164R) and GST:S₃-RNase had similar RNase activity, suggesting that their degradation was probably not caused by an ER-associated protein degradation pathway that removes mis-folded proteins. Finally, we showed that PiSBP1 ( P. inflata S-RNase binding protein 1), a potential RING-HC subunit of the PiSLF ( P. inflata SLF)-containing E3-like complex, could target S-RNase for ubiquitination in vitro . All these results suggest that ubiquitin/26S proteasome-dependent degradation of S-RNase may be an integral part of the S-RNase-based self-incompatibility mechanism.     

Ectopic expression of S-RNase of <Emphasis Type="Italic">Petunia inflata</Emphasis> in pollen results in its sequestration and non-cytotoxic function

The specificity of S-RNase-based self-incompatibility (SI) is controlled by two S-locus genes, the pistil S-RNase gene and the pollen S-locus-F-box gene. S-RNase is synthesized in the transmitting cell; its signal peptide is cleaved off during secretion into the transmitting tract; and the mature “S-RNase”, the subject of this study, is taken up by growing pollen tubes via an as-yet unknown mechanism. Upon uptake, S-RNase is sequestered in a vacuolar compartment in both non-self (compatible) and self (incompatible) pollen tubes, and the subsequent disruption of this compartment in incompatible pollen tubes correlates with the onset of the SI response. How the S-RNase-containing compartment is specifically disrupted in incompatible pollen tubes, however, is unknown. Here, we circumvented the uptake step of S-RNase by directly expressing S₂-RNase, S₃-RNase and non-glycosylated S₃-RNase of Petunia inflata, with green fluorescent protein (GFP) fused at the C-terminus of each protein, in self (incompatible) and non-self (compatible) pollen of transgenic plants. We found that none of these ectopically expressed S-RNases affected the viability or the SI behavior of their self or non-self-pollen/pollen tubes. Based on GFP fluorescence of in vitro-germinated pollen tubes, all were sequestered in both self and non-self-pollen tubes. Moreover, the S-RNase-containing compartment was dynamic in living pollen tubes, with movement dependent on the actin–myosin-based molecular motor system. All these results suggest that glycosylation is not required for sequestration of S-RNase expressed in pollen tubes, and that the cytosol of pollen is the site of the cytotoxic action of S-RNase in SI.     

Isobaric tag for relative and absolute quantitation based quantitative proteomics reveals unique urinary protein profiles in patients with preeclampsia

Preeclampsia (PE) is one of the most significant pregnancy‐related hypertensive disorders. Currently, there are no useful markers to predict the onset of the condition in pregnant women. To provide further insights into the pathogenesis of PE and identify biomarkers of the condition, we used isobaric tags for relative and absolute quantitation (iTRAQ) proteomics coupled with 2‐D LC‐MS/MS, to analyze urinary protein profiles from 7 PE patients and 7 normotensive pregnant women. A total of 294 proteins were abnormally expressed in PE patients. Of these, 233 were significantly down‐regulated and 61 proteins were significantly up‐regulated. Bioinformatics analysis using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, found that the most differentially expressed proteins (DEPs) were involved in coagulation and complement pathways, the renin‐angiotensin system and cell adhesion molecules (CAMs) pathways. We further validated three of the DEPs, including serotransferrin (TF) and complement factor B (CFB) by immunoblottingand serum paraoxonase/arylesterase 1 (PON1) by ELISA using 14 pairs of urine samples from PE patients and normal pregnant women. Taken together, our results provide the basis for further understanding the pathogenesis of PE and identifying predictive biomarkers.     

Gametophytic self-incompatibility in Lycium parishii (Solanaceae): allelic diversity, genealogical structure, and patterns of molecular evolution at the S-RNase locus

We characterized allelic diversity at the locus controlling self-incompatibility (SI) for a population of Lycium parishii (Solanaceae) from Organ Pipe National Monument, Arizona. Twenty-four partial sequences of S-RNase alleles were recovered from 25 individuals. Estimates of allelic diversity range from 23 to 27 alleles and, consistent with expectations for SI, individuals are heterozygous. We compare S-RNase diversity, patterns of molecular evolution, and the genealogical structure of alleles from L. parishii to a previously studied population of its congener L. andersonii. Gametophytic SI is well characterized for Solanaceae and although balancing selection is hypothesized to be responsible for high levels of allelic divergence, the pattern of selection varies depending on the portion of the gene considered. Site-specific models investigating patterns of selection for L. parishii and L. andersonii indicate that positive selection occurs in those regions of the S-RNase gene hypothesized as important to the recognition response, whereas positive selection was not detected for any position within regions previously characterized as conserved. A 10-species genealogy including S-RNases from a pair of congeners from each of five genera in Solanaceae reveals extensive transgeneric evolution of L. parishii S-RNases. Further, within Lycium, the Dn/Ds ratios for pairs of closely related alleles for intraspecific versus interspecific comparisons were not significantly different, suggesting that the S-RNase diversity recovered in these two species was present prior to the speciation event separating them. Despite this, two S-RNases from L. parishii are identical to two previously reported alleles for L. andersonii, suggesting gene flow between these species.