RNaseI from Escherichia coli cannot substitute for S-RNase in rejection of Nicotiana plumbaginifolia pollen

Unilateral incompatibility often occurs between self-incompatible (SI) species and their self-compatible (SC) relatives. For example, SI Nicotiana alata rejects pollen from SC N. plumbaginifolia, but the reciprocal pollination is compatible. This interspecific pollen rejection system closely resembles intraspecific S-allele-specific pollen rejection. However, the two systems differ in degree of specificity. In SI, rejection is S-allele-specific, meaning that only a single S-RNase causes rejection of pollen with a specific S genotype. Rejection of N. plumbaginifolia pollen is less specific, occurring in response to almost any S-RNase. Here, we have tested whether a non-S-RNase can cause rejection of N. plumbaginifolia pollen. The Escherichia coli rna gene encoding RNaseI was engineered for expression in transgenic (N. plumbaginifolia × SC N. alata) hybrids. Expression levels and pollination behavior of hybrids expressing E. coli RNaseI were compared to controls expressing S_A2-RNase from N. alata. Immunoblot analysis and RNase activity assays showed that RNaseI and S_A2-RNase were expressed at comparable levels. However, expression of S_A2-RNase caused rejection of N. plumbaginifolia pollen, whereas expression of RNaseI did not. Thus, in this system, RNase activity alone is not sufficient for rejection of N. plumbaginifolia pollen. The results suggest that S-RNases may be specially adapted to function in pollen rejection.     

Unilateral incompatibility within the brassicaceae: further evidence for the involvement of the self-incompatibility (S)-locus

Unilateral pollen-pistil incompatibility within the Brassicaceae has been re-examined in a series of interspecific and intergeneric crosses using 13 self-compatible (SC, Sc) species and 12 self-incompatible (SI) species from ten tribes. SC x SC crosses were usually compatible, SI x SC crosses showed unilateral incompatibility, while SI x SI crosses were often incompatible or unilaterally incompatible. Unilateral incompatibility (UI) is shown to be overcome by bud pollination or treating stigmas with cycloheximide — features in common with self-incompatibility. Treating stigmas with pronase prevents pollen tubes from penetrating the stigma in normally compatible intra-and interspecific pollinations. The results presented show that the presence of an incompatibility system is important in predicting the outcome of interspecific and intergeneric crosses and, combined with the physiological similarities between UI and SI, would suggest an involvement of the S-locus in UI.     

A farnesyl pyrophosphate synthase gene expressed in pollen functions in S‐RNase‐independent unilateral incompatibility

Multiple independent and overlapping pollen rejection pathways contribute to unilateral interspecific incompatibility (UI). In crosses between tomato species, pollen rejection usually occurs when the female parent is self‐incompatible (SI) and the male parent self‐compatible (SC) (the ‘SI × SC rule’). Additional, as yet unknown, UI mechanisms are independent of self‐incompatibility and contribute to UI between SC species or populations. We identified a major quantitative trait locus on chromosome 10 (ui10.1) which affects pollen‐side UI responses in crosses between cultivated tomato, Solanum lycopersicum, and Solanum pennelliiLA0716, both of which are SC and lack S‐RNase, the pistil determinant of S‐specificity in Solanaceae. Here we show that ui10.1 is a farnesyl pyrophosphate synthase gene (FPS2) expressed in pollen. Expression is about 18‐fold higher in pollen of S. pennellii than in S. lycopersicum. Pollen with the hypomorphic S. lycopersicum allele is selectively eliminated on pistils of the F₁ hybrid, leading to transmission ratio distortion in the F₂ progeny. CRISPR/Cas9‐generated knockout mutants (fps2) in S. pennelliiLA0716 are self‐sterile due to pollen rejection, but mutant pollen is fully functional on pistils of S. lycopersicum. F₂ progeny of S. lycopersicum × S. pennellii (fps2) show reversed transmission ratio distortion due to selective elimination of pollen bearing the knockout allele. Overexpression of FPS2 in S. lycopersicum pollen rescues the pollen elimination phenotype. FPS2‐based pollen selectivity does not involve S‐RNase and has not been previously linked to UI. Our results point to an entirely new mechanism of interspecific pollen rejection in plants.     

HT proteins contribute to S‐RNase‐independent pollen rejection in Solanum

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self‐incompatibility (SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers (IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S‐RNase and HT protein function in a pistil‐side IRB that causes rejection of pollen from self‐compatible (SC) red/orange‐fruited species in the tomato clade. However, S‐RNase‐independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S‐RNase‐independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S‐RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S‐RNase, and then used RNAi to test whether they also function in S‐RNase‐independent pollen rejection. Suppressing HT expression in SC S. pennellii LA0716 allows S. lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S. arcanum LA2157 allows S. lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S‐RNase‐dependent and S‐RNase‐independent pollen rejection. The results support the view that overall compatibility results from multiple pollen–pistil interactions with additive effects.     

UNILATERAL HYBRIDIZATION IN AUREOLARIA RAF. (SCROPHULARIACEAE)

A review of the slowly accumulating data on artificial hybridization in the genus Aureolaria shows a pattern of unilateral hybridization (or unilateral incompatibility) indicative of a type of homomorphic gametophytic self-incompatibility system, involving multiple S alleles at a single locus, which has been found and genetically analyzed in plants of a few genera of both the Solanaceae and Scrophulariaceae. In such systems, if well developed, the pollen of the self-compatible (SC) plants (in this case the annual A. pedicularia) will not grow on the stigma of the self-incompatible (SI) plants (here the three perennial species of Aureolaria) but the reciprocal cross would be effective and the hybridization would thus be unilateral. The fact that some SI stigma x SC pollen crosses were successful indicates a relatively recent evolutionary age for the less strongly developed (Sc) system in A. pedicularia.     

The stylar 120 kDa glycoprotein is required for S-specific pollen rejection in Nicotiana

S-RNase participates in at least three mechanisms of pollen rejection. It functions in S-specific pollen rejection (self-incompatibility) and in at least two distinct interspecific mechanisms of pollen rejection in Nicotiana. S-specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia also require additional stylar proteins. Transmitting-tract-specific (TTS) protein, 120 kDa glycoprotein (120K) and pistil extensin-like protein III (PELP III) are stylar glycoproteins that bind S-RNase in vitro and are also known to interact with pollen. Here we tested whether these glycoproteins have a direct role in pollen rejection. 120K shows the most polymorphism in size between Nicotiana species. Larger 120K-like proteins are often correlated with S-specific pollen rejection. Sequencing results suggest that the polymorphism primarily reflects differences in glycosylation, although indels also occur in the predicted polypeptides. Using RNA interference (RNAi), we suppressed expression of 120K to determine if it is required for S-specific pollen rejection. Transgenic SC N. plumbaginifolia x SI Nicotiana alata (S105S105 or SC10SC10) hybrids with no detectable 120K were unable to perform S-specific pollen rejection. Thus, 120K has a direct role in S-specific pollen rejection. However, suppression of 120K had no effect on rejection of N. plumbaginifolia pollen. In contrast, suppression of HT-B, a factor previously implicated in S-specific pollen rejection, disrupts rejection of N. plumbaginifolia pollen. Thus, S-specific pollen rejection and rejection of N. plumbaginifolia pollen are mechanistically distinct, because they require different non-S-RNase factors.     

Unilateral and bilateral hybridization barriers in inter-series crosses of 4x 2EBN Solanum stoloniferum, S. pinnatisectum, S. cardiophyllum, and 2x 2EBN S. tuberosum haploids and haploid-species hybrids

Wild Mexican potato species are an important untapped source of useful variation for potato improvement. Introgression methods such as 2n gametes, chromosome doubling, and crossing with disomic 4x 2 endosperm balance number (EBN) bridge species have been used to overcome post-zygotic endosperm failure according to the EBN hypothesis. Stylar barriers can prevent zygote formation, bilaterally when zygote formation is blocked in both directions of the cross or unilaterally when zygote formation is blocked in self incompatible (SI) × self compatible (SC) crosses. In several Solanaceae species, the S-locus for SI has been implicated in interspecific incompatibility. The objectives of this research were to determine if: (1) disomic 4x 2EBN Solanum stoloniferum can be used as a bridge species for introgression of the Mexican 2x 1EBN species Solanum cardiophyllum and Solanum pinnatisectum, (2) pre- and/or post-zygotic barriers limit hybridization among EBN compatible Solanum inter-series crosses, and (3) reproductive barriers act unilaterally or bilaterally. Fruit formation and seed set was recorded for inter-pollinations of S. stoloniferum, 4x 2EBN chromosome doubled S. cardiophyllum and S. pinnatisectum, and 2x 2EBN S. tuberosum haploids (HAP) or haploid-species hybrids (H-S). In vivo pollen tube growth was analyzed for each cross combination with fluorescence microscopy. Attempts to create bridge hybrids between S. stoloniferum, and S. cardiophyllum or S. pinnatisectum were not successful. Pre- and post-zygotic barriers prevented seed formation in crosses involving S. cardiophyllum and S. pinnatisectum. Self compatibility in S. stoloniferum and S. pinnatisectum suggests that the S-locus does not contribute to the stylar barriers observed with these species. Alternatively, the presence of functional and nonfunctional (SC) S-alleles may explain interspecific incompatibility in intra- and inter-ploidy crosses. A non-stylar unilateral incongruity was discovered in H-S/HAP × S. stoloniferum crosses, indicating either a post-zygotic barrier, or a pre-zygotic barrier acting at or within the ovary. Furthermore, lack of S. stoloniferum pollen rejection may occur through absence of S. stoloniferum pollen-active genes needed to initiate pollen rejection, or through competitive interaction in S-locus heterozygous S. stoloniferum pollen. Introgression strategies using these species would benefit potato breeding by introducing genetic diversity for several traits simultaneously through co-current introgression.     

Fine Mapping of ui6.1, a Gametophytic Factor Controlling Pollen-Side Unilateral Incompatibility in Interspecific Solanum Hybrids

Unilateral incompatibility (UI) is a prezygotic reproductive barrier in plants that prevents fertilization by foreign (interspecific) pollen through the inhibition of pollen tube growth. Incompatibility occurs in one direction only, most often when the female is a self-incompatible species and the male is self-compatible (the “SI × SC rule”). Pistils of the wild tomato relative Solanum lycopersicoides (SI) reject pollen of cultivated tomato (S. lycopersicum, SC), but accept pollen of S. pennellii (SC accession). Expression of pistil-side UI is weakened in S. lycopersicum × S. lycopersicoides hybrids, as pollen tube rejection occurs lower in the style. Two gametophytic factors are sufficient for pollen compatibility on allotriploid hybrids: ui1.1 on chromosome 1 (near the S locus), and ui6.1 on chromosome 6. We report herein a fine-scale map of the ui6.1 region. Recombination around ui6.1 was suppressed in lines containing a short S. pennellii introgression, but less so in lines containing a longer introgression. More recombinants were obtained from female than male meioses. A high-resolution genetic map of this region delineated the location of ui6.1 to ∼0.128 MU, or 160 kb. Identification of the underlying gene should elucidate the mechanism of interspecific pollen rejection and its relationship to self-incompatibility.FLOWERING plants have evolved several reproductive barriers for preventing illegitimate hybridization with related species. These barriers may be expressed prefertilization and/or postfertilization. Unilateral incompatibility or incongruity (UI) is a prefertilization barrier that occurs when pollen of one species is rejected on pistils of a related species, while no rejection occurs in the reciprocal cross (De Nettancourt 1977). In theory, unilateral incompatibility should reinforce species identity in natural, sympatric populations of related taxa. This barrier also impedes the efforts of plant breeders to transfer traits from wild species into related crop plants. For example, the transfer of cytoplasmic traits from species with maternally inherited chloroplasts and mitochondria may be prevented by unilateral crossing barriers. Nuclear-encoded traits may also be inaccessible if F₁ interspecific hybrids are both male sterile and incompatible as female parents.In the Solanaceae, unilateral incompatibility is observed in crosses between cultivated tomato (Solanum lycopersicum, formerly Lycopersicon esculentum) and some related wild species. In general, pistils of the cultivated tomato act as a “universal acceptor,” in that they fail to recognize and reject pollen of other tomato species. In the reciprocal crosses, pollen of S. lycopersicum is rejected on styles of virtually all of the green-fruited species, but not on styles of other red or orange-fruited species (reviewed by Mutschler and Liedl 1994). This pattern is mostly consistent with the “SI × SC” rule, wherein pollen of self-compatible (SC) species (including cultivated tomato) are rejected on pistils of self-incompatible (SI) species, but not in the reverse direction (Lewis and Crowe 1958). Exceptions to the SI × SC rule in the tomato clade include species or populations that have lost self-incompatibility but retain the ability to reject pollen of tomato. This is the case for the facultative outcrossing species S. chmielewskii, the autogamous S. neorickii (formerly L. parviflorum), as well as marginal SC populations of normally SI species such as S. pennellii and S. habrochaites (formerly L. hirsutum). An SC accession of S. pennellii, LA0716, is exceptional in having lost the ability to reject self pollen, while retaining the ability to serve as pollen parent on styles of SI accessions of this species (and other SI species, including S. peruvianum and S. lycopersicoides) (Hardon 1967; Rick 1979; Quiros et al. 1986). In this regard, S. pennellii LA0716 conforms to the Lewis and Crowe (1958) model in that it behaves like a transitional form lacking SI function in the pistil but not in the pollen.Unilateral incompatibility may also occur in crosses between populations or races of a single species. In S. habrochaites for example, pollen from SC biotypes located at the northern or southern margins of its geographic range is rejected on pistils of the central, SI populations (Martin 1961, 1963). Furthermore, pollen from the northern SC group is rejected by styles of the southern SC populations. Yet pistils of both SC biotypes are able to reject pollen of cultivated tomato. Thus there appear to be at least three distinct unilateral crossing barriers, just within S. habrochaites, possibly indicating different pollen tube recognition and rejection systems. The F₁ N × S hybrid is SC, as expected, but SI plants are recovered in the F₂ generation, suggesting that the loss of SI occurred via independent mutations in the north and the south (Rick and Chetelat 1991).Interspecific F₁ hybrids between SI wild species and SC cultivated tomato are self-incompatible and reject pollen of cultivated tomato, indicating both traits are at least partially dominant (McGuire and Rick 1954; Martin 1963; Hardon 1967). Interestingly, pollen of the F₁ hybrids is incompatible on pistils of the wild species parent (i.e., including other individuals of the same accessions, but with nonmatching S alleles). This observation suggests that there are dominant factors from cultivated tomato that lead to pollen rejection on styles of the wild species, regardless of the pollen genotype. This apparent sporophytic effect contrasts with the purely gametophytic nature of pollen SI specificity in the Solanaceae (De Nettancourt 1977).Early studies of the inheritance of unilateral incompatibility in tomato suggested the involvement of several genes controlling the pistil response; however, the genetics of pollen responses have received little attention. In F₂ S. habrochaites (northern SC accession) × S. habrochaites (central SI accession), the rejection of pollen from the SC parent segregated as if controlled by one to two dominant genes from the SI accession (Martin 1964). In crosses of S. lycopersicum to both SI and SC accessions of S. pennellii, the intra- and interspecific crossing relations were largely consistent with the Lewis and Crowe (1958) model of stepwise mutation at the S locus (Hardon 1967); there was also evidence of a second barrier in the SC S. pennellii accession. In F₁ and BC₁ hybrids of S. lycopersicum × S. habrochaites, the segregation of unilateral and self-incompatibilities was consistent with the action of two major genes, with minor polygenes indicated as well (Martin 1967). More recently, several QTL underlying pistil-side unilateral and self-incompatibilities were mapped in BC₁ S. lycopersicum × S. habrochaites (Bernacchi and Tanksley 1997); the major QTL for both forms of pollen rejection was located at or near the S locus on chromosome 1, which controls SI specificity (Tanksley and Loaiza-Figueroa 1985).There are little data on pollen-side unilateral incompatibility factors in the tomato clade, or any other system. Our previous work identified two to three genetic loci from S. pennellii that are required for pollen to overcome incompatibility on pistils of S. lycopersicum × S. lycopersicoides or S. lycopersicum × S. sitiens hybrids (Chetelat and Deverna 1991; Pertuze et al. 2003). One of these factors mapped to the S locus, the other two were on chromosomes 6 and 10. In this system the female tester stocks were either diploid or allotriploid hybrids, the latter containing one genome of the wild, SI parent, plus two genomes of cultivated tomato; both types of hybrids reject pollen of cultivated tomato. The pollen parents were either F₁ S. lycopersicum × S. pennellii or bridging lines developed by backcrossing the F₁ to cultivated tomato and selecting for the ability to overcome stylar incompatibility. In the progeny, distorted segregation ratios were observed in which the S. pennellii alleles were preferentially transmitted, indicating linkage to gametophytic factors required forcompatibility.This experimental system has several advantages for detecting pollen (gametophytic) unilateral incompatibility genes. First, pollen-expressed factors are readily distinguished from pistil factors because only the former show linkage to S. pennellii specific markers. Second, pollen rejection is by unilateral, not self-incompatibility, since both species contributing to the pollen genotype, S. lycopersicum and S. pennellii, are SC. Finally, as we describe herein, the rejection of tomato pollen by pistils of the interspecific hybrids is weakened by the decreasing dosage of the S. lycopersicoides genome, which reduces the number of pollen factors required for compatibility. Thus, the gametophytic factors on chromosomes 1 and 6 (denoted hereinafter ui1.1 and ui6.1), when present in the same pollen, are sufficient for full compatibility on pistils of allotriploid interspecific hybrids, whereas they confer only partial compatibility on diploid hybrids.Our overall objectives are to identify the genes underlying both the chromosome 1 and chromosome 6 pollen-specific unilateral incompatibility factors from S. pennellii and to determine the nature of their interaction. Toward this goal, we report herein the high-resolution genetic and physical mapping of the ui6.1 region.     

Stylar glycoproteins bind to S-RNase in vitro

S-RNases determine the specificity of S-specific pollen rejection in self-incompatible plants of the Solanaceae, Rosaceae, and Scrophulariaceae. They are also implicated in at least two distinct types of unilateral interspecific incompatibility in Nicotiana. However, S-RNase itself is not sufficient for most types of pollen rejection, and evidence for its direct interaction with pollen tubes is limited. Thus, non-S-RNase factors also are required for pollen rejection. As one approach to identifying such factors, we tested whether SC10-RNase from Nicotiana alata would bind to other stylar proteins in vitro. SC10-RNase was immobilized on Affi-gel, and binding proteins were analyzed by SDS-PAGE and immunoblotting. In addition to SC10-RNase and a small protein similar to lily chemocyanin, the most prominent binding proteins include NaTTS, 120K, and NaPELPIII, these latter three being arabinogalactan proteins previously shown to interact directly with pollen tubes. We also show that SC10-RNase and these glycoproteins migrate as a complex in a native PAGE system. Our hypothesis is that S-RNase forms a complex with these glycoproteins in the stylar ECM, that the glycoproteins interact directly with the pollen tubes and thus that the initial interaction between the pollen tube and S-RNase is indirect.     

S-RNase complexes and pollen rejection

Biochemical interactions between the pollen and the pistil allow plants fine control over fertilization. S-RNase-based pollen rejection is among the most widespread and best understood of these interactions. At least three plant families have S-RNase-based self-incompatibility (SI) systems, and S-RNases have also been implicated in interspecific pollen rejection. Although S-RNases determine the specificity of SI, other genes are required for the pollen rejection system to function. Progress is being made toward identifying these non-S-RNase factors. HT-protein, first identified as a non-S-RNase factor that was required for SI in Nicotiana alata, has now been implicated in other species as well. In addition, several pistil proteins bind to S-RNase in vitro. One hypothesis is that S-RNase forms a complex with these proteins in vivo that is the active form of S-RNase in pollen rejection.     

Expression of 10 S-class SLF-like genes in Nicotiana alata pollen and its implications for understanding the pollen factor of the S locus

The S locus of Nicotiana alata encodes a polymorphic series of ribonucleases (S-RNases) that determine the self-incompatibility (SI) phenotype of the style. The pollen product of the S locus (pollen S) in N. alata is unknown, but in species from the related genus Petunia and in self-incompatible members of the Plantaginaceae and Rosaceae, this function has been assigned to an F-box protein known as SLF or SFB. Here we describe the identification of 10 genes (designated DD1-10) encoding SLF-related proteins that are expressed in N. alata pollen. Because our approach to cloning the DD genes was based on sequences of SLFs from other species, we presume that one of the DD genes encodes the N. alata SLF ortholog. Seven of the DD genes were exclusively expressed in pollen and a low level of sequence variation was found in alleles of each DD gene. Mapping studies confirmed that all 10 DD genes were linked to the S locus and that at least three were located in the same chromosomal segment as pollen S. Finally, the different topologies of the phylogenetic trees produced using available SLF-related sequences and those produced using S-RNase sequences suggests that pollen S and the S-RNase have different evolutionary histories.     

Stylar water potential and unilateral interspecific incompatibility inSolanaceae

Water and osmotic potentials were measured in young and mature styles of selfcompatible (SC) and selfincompatible (SI) species ofSolanaceae. For mature flowers, the stylar water and osmotic potentials were considerably lower in SI speciesNicotiana alata andPetunia hybrida than in SC speciesN. sylvestris andN. tabacum. Stylar water content (in % fresh mass) was also significantly lower in SI species (N. alata, P. hybrida, diploid clones of potato) than in SC species (N. acuminata, N. glauca, N. paniculata, N. sylvestris, N. tabacum). For flower buds younger than 2 d before anthesis, no differences in stylar water potential betweenN. alata (SI) andN. tabacum (SC) were observed. During further flower bud development the stylar water potential inN. alata decreased more expressively than inN. tabacum. Pollen culture in media with various concentrations of sucrose or polyethylene glycol revealed that optimal water potential for pollen tube growth was lower inN. alata than inN. tabacum. The differences were similar as for differences in stylar water potential. The results are considered in relation to possible involvement of stylar water potential in unilateral interspecific incompatibility inSolanaceae.     

烟草种间杂交亲和性

以野生烟草Nicotiana alata、N.rustica、N.repanda、N.stocktonnii与栽培烟草K326、红花大金元、Yun87、Yun97为材料,进行种间正反杂交,研究种间杂交亲和性.田间观察杂交后的坐果情况并统计坐果率,采用显微荧光染色观察授粉后花粉管在雌蕊上的生长情况,并结合杂交后代萌发检测的方法.结果表明:N.rustica、N.repanda、N.stocktonnii与栽培烟草杂交不亲和.N.rustica花粉能够穿过K326花柱,N.repanda和N.stocktonnii花粉在K326柱头上很少萌发生长.N.alata花粉可以穿过K326的花柱,并得到果实,但是萌发实验显示其种子无活力.N.alata作为母本与栽培烟草杂交不亲和.     

烟草种间杂交亲和性

以野生烟草Nicotiana alata、N.rustica、N.repanda、N.stocktonnii与栽培烟草K326、红花大金元、Yun87、Yun97为材料,进行种间正反杂交,研究种间杂交亲和性。田间观察杂交后的坐果情况并统计坐果率,采用显微荧光染色观察授粉后花粉管在雌蕊上的生长情况,并结合杂交后代萌发检测的方法。结果表明：N.rustica、N.repanda、N.stocktonnii与栽培烟草杂交不亲和。N.rustica花粉能够穿过K326花柱,N.repanda和N.stocktonnii花粉在K326柱头上很少萌发生长。N.alata花粉可以穿过K326的花柱,并得到果实,但是萌发实验显示其种子无活力。N.alata作为母本与栽培烟草杂交不亲和。     

INTERSPECIFIC INCOMPATIBILITY IN SOLANUM SPECIES

Pandey , K.K. (Crop Res. Div., D.S. & I.R., Lincoln, Christchurch, New Zealand.) Interspecific incompatibility in Solanum species. Amer. Jour. Bot. 49(8): 874–882. Illus. 1962.—A diallel cross involving 11 self-incompatible and 3 self-compatible species of Solanum was made to study the genetic basis of interspecific incompatibility. Interspecific incompatibility was not limited to crosses in which a self-compatible species was used as the male parent onto a self-incompatible species (unilateral incompatibility). A number of crosses between self-incompatible species were incompatible. In one cross, Q vernei X verrucosum, a self-compatible species was successful as a pollen parent with a self-incompatible species. Unlike other hybrids between self-compatible and self-incompatible species, which are self-incompatible, these F₁ hybrids were self-fertile, and cross-fertile among themselves and with both parents. The self-fertile S. polyadenium was cross-incompatible as a female as well as a male parent with all other species. It is suggested that the unilateral incompatibility is a property of the allele S_C which originated as a consequence of one kind of breakdown of the S_I gene; the S_C allele produces “bare” pollen growth substances which are inactivated in an incompatible style. It is proposed that the failure of the principle of unilateral interspecific incompatibility in solanaceous species may be due to the action of alleles at the second incompatibility locus revealed in certain Mexican species. It is assumed that the South American species are selected intraspecifically only for the action of S alleles but that in certain interspecific crosses and rarely in intraspecific crosses the alleles at the second locus may be expressed, thus interfering with the usual action of S alleles. The F₁ hybrids Q verrucosum (self-fertile) X simplicifolium (self-sterile) were self-incompatible at the tetraploid as well as the diploid level, and their cross-compatibility behavior was consistent with the expected activity of the S_C and S_I alleles of the 2 parents respectively.     

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

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

Transitions between self-compatibility and self-incompatibility and the evolution of reproductive isolation in the large and diverse tropical genus Dendrobium (Orchidaceae)

Background and Aims The evolution of interspecific reproductive barriers is crucial to understanding species evolution. This study examines the contribution of transitions between self-compatibility (SC) and self-incompatibility (SI) and genetic divergence in the evolution of reproductive barriers in Dendrobium, one of the largest orchid genera. Specifically, it investigates the evolution of pre- and postzygotic isolation and the effects of transitions between compatibility states on interspecific reproductive isolation within the genus.Methods The role of SC and SI changes in reproductive compatibility among species was examined using fruit set and seed viability data available in the literature from 86 species and ∼2500 hand pollinations. The evolution of SC and SI in Dendrobium species was investigated within a phylogenetic framework using internal transcribed spacer sequences available in GenBank.Key Results Based on data from crossing experiments, estimations of genetic distance and the results of a literature survey, it was found that changes in SC and SI significantly influenced the compatibility between species in interspecific crosses. The number of fruits produced was significantly higher in crosses in which self-incompatible species acted as pollen donor for self-compatible species, following the SI × SC rule. Maximum likelihood and Bayesian tests did not reject transitions from SI to SC and from SC to SI across the Dendrobium phylogeny. In addition, postzygotic isolation (embryo mortality) was found to evolve gradually with genetic divergence, in agreement with previous results observed for other plant species, including orchids.Conclusions Transitions between SC and SI and the gradual accumulation of genetic incompatibilities affecting postzygotic isolation are important mechanisms preventing gene flow among Dendrobium species, and may constitute important evolutionary processes contributing to the high levels of species diversity in this tropical orchid group.     

Unilateral incongruity in crosses involvingLycopersicon pennellii andL. esculentum is distinct from self-incompatibility in expression,timing and location

Both interspecific and intraspecific mechanisms restrict the exchange of genes between plants. Much research has focused on self incompatibility (SI), an intraspecific barrier, but research on interspecific barriers lags behind. We are using crosses betweenLycopersicon esculentum andL. pennellii as a model with which to study interspecific crossing barriers. The crossL. esculentum×L. pennellii is successful, but the reciprocal cross fails. Since the cross can be successfully made in one direction but not the other, gross genomic imbalance or chromosomal abnormality are precluded as causes. We showed that the lack of seed set observed in the crossL. pennellii×L. esculentum is due to the inability of pollen tubes to grow more than 2–3 mm into the style, whereas S1 crosses show continued slow pollen tube growth but, also, fail to set seed. These results indicate that the unilateral response is a barrier distinct from SI, differing from SI in the timing and location of expression in the style. We therefore suggest that this unilateral response in theL. pennellii×L. esculentum cross is more accurately referred to as unilateral incongruity (UI) rather than interspecific incompatibility. Periclinal chimeras were used to determine the tissues involved in UI. The results of crosses with the available chimeras indicate that the female parent must beL. pennellii at either LI (layer 1) or both LI and LII (layer 2) and the male parent must beL. esculentum at either LII or both LI and LII to observe UI similar to that seen in theL. pennellii×L. esculentum cross. Pollinations with a mixture of pollen fromL. pennellii and from transgenicL. esculentum plants harboring a pollen-specific GUS reporter gene marker were used to ascertain whether the growth of the pollen tubes of either species was modified as a possible means of overcoming UI. We found no evidence of communication between the two types of pollen tubes to either enhance or restrict all pollen tube growth.     

Unilateral incompatibility in Capsicum (Solanaceae): occurrence and taxonomic distribution

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.     

The Role of a Pollen-Expressed Cullin1 Protein in Gametophytic Self-Incompatibility in Solanum

We previously isolated a pollen factor, ui6.1, which encodes a Cullin1 protein (CUL1) that functions in unilateral interspecific incompatibility (UI) in Solanum. Here we show that CUL1 is also required for pollen function in self-incompatibility (SI). We used RNA interference (RNAi) to reduce CUL1 expression in pollen of Solanum arcanum, a wild SI tomato relative. Hemizygous T₀ plants showed little or no transmission of the transfer DNA (T-DNA) through pollen when crossed onto nontransgenic SI plants, indicating that CUL1-deficient pollen are selectively eliminated. When crossed onto a related self-compatible (SC) accession lacking active S-RNase, pollen transmission of the T-DNA followed Mendelian ratios. These results provide further evidence for functional overlap between SI and UI on the pollen side and suggest that CUL1 mutations will reinforce SI-to-SC transitions in natural populations only if preceded by loss of pistil S-RNase expression.