How flowering plants discriminate between self and non-self pollen to prevent inbreeding.

Flowering plants have evolved various genetic mechanisms to circumvent the tendency for self-fertilization created by the close proximity of male and female reproductive organs in a bisexual flower. One such mechanism is gametophytic self-incompatibility, which allows the female reproductive organ, the pistil, to distinguish between self pollen and non-self pollen; self pollen is rejected, whereas non-self pollen is accepted for fertilization. The Solanaceae family has been used as a model to study the molecular and biochemical basis of self/non-self-recognition and self-rejection. Discrimination of self and non-self pollen by the pistil is controlled by a single polymorphic locus, the S locus. The protein products of S alleles in the pistil, S proteins, were initially identified based on their cosegregation with S alleles. S proteins have recently been shown to indeed control the ability of the pistil to recognize and reject self pollen. S proteins are also RNases, and the RNase activity has been shown to be essential for rejection of self pollen, suggesting that the biochemical mechanism of self-rejection involves the cytotoxic action of the RNase activity. S proteins contain various numbers of N-linked glycans, but the carbohydrate moiety has been shown not to be required for the function of S proteins, suggesting that the S allele specificity determinant of S proteins lies in the amino acid sequence. The male component in self-incompatibility interactions, the pollen S gene, has not yet been identified. The possible nature of the pollen S gene product and the possible mechanism by which allele-specific rejection of pollen is accomplished are discussed.     

The pollen determinant of self-incompatibility in Brassica campestris

Many flowering plants possess self-incompatibility (SI) systems that prevent inbreeding. In Brassica, SI is controlled by a single polymorphic locus, the S locus. Two highly polymorphic S locus genes, SLG (S locus glycoprotein) and SRK (S receptor kinase), have been identified, both of which are expressed predominantly in the stigmatic papillar cell. We have shown recently that SRK is the determinant of the S haplotype specificity of the stigma. SRK is thought to serve as a receptor for a pollen ligand, which presumably is encoded by another polymorphic gene at the S locus. We previously have identified an S locus gene, SP11 (S locus protein 11), of the S(9) haplotype of Brassica campestris and proposed that it potentially encodes the pollen ligand. SP11 is a novel member of the PCP (pollen coat protein) family of proteins, some members of which have been shown to interact with SLG. In this work, we identified the SP11 gene from three additional S haplotypes and further characterized the gene. We found that (i) SP11 showed an S haplotype-specific sequence polymorphism; (ii) SP11 was located in the immediate flanking region of the SRK gene of the four S haplotypes examined; (iii) SP11 was expressed in the tapetum of the anther, a site consistent with sporophytic control of Brassica SI; and (iv) recombinant SP11 of the S(9) haplotype applied to papillar cells of S(9) stigmas, but not of S(8) stigmas, elicited SI response, resulting in inhibition of hydration of cross-pollen. All these results taken together strongly suggest that SP11 is the pollen S determinant in SI.     

Genetic analysis of Nicotiana pollen-part mutants is consistent with the presence of an S-ribonuclease inhibitor at the S locus

Self-incompatibility (SI) is a genetic mechanism that restricts inbreeding in flowering plants. In the nightshade family (Solanaceae) SI is controlled by a single multiallelic S locus. Pollen rejection in this system requires the interaction of two S locus products: a stylar (S)-RNase and its pollen counterpart (pollen S). pollen S has not yet been cloned. Our understanding of how this gene functions comes from studies of plants with mutations that affect the pollen but not the stylar SI response (pollen-part mutations). These mutations are frequently associated with duplicated S alleles, but the absence of an obvious additional allele in some plants suggests pollen S can also be deleted. We studied Nicotiana alata plants with an additional S allele and show that duplication causes a pollen-part mutation in several different genetic backgrounds. Inheritance of the duplication was consistent with a competitive interaction model in which any two nonmatching S alleles cause a breakdown of SI when present in the same pollen grain. We also examined plants with presumed deletions of pollen S and found that they instead have duplications that included pollen S but not the S-RNase gene. This finding is consistent with a bipartite structure for the S locus. The absence of pollen S deletions in this study and perhaps other studies suggests that pollen S might be required for pollen viability, possibly because its product acts as an S-RNase inhibitor.     

Functional test of Brassica self-incompatibility modifiers in Arabidopsis thaliana

The self-incompatibility (SI) system of the Brassicaceae is based on allele-specific interactions among haplotypes of the S locus. In all tested self-incompatible Brassicaceae, the S haplotype encompasses two linked genes, one encoding the S-locus receptor kinase (SRK), a transmembrane kinase displayed at the surface of stigma epidermal cells, and the other encoding its ligand, the S-locus cysteine-rich (SCR) protein, which is localized in the pollen coat. Transfer of the two genes to self-fertile Arabidopsis thaliana allowed the establishment of robust SI in several accessions, indicating that the signaling cascade triggered by this receptor-ligand interaction and the resulting inhibition of "self" pollen by the stigma have been maintained in extant A. thaliana. Based on studies in Brassica species, the membrane-tethered kinase MLPK, the ARM repeat-containing U-box protein ARC1, and the exocyst subunit Exo70A1 have been proposed to function as components of an SI signaling cascade. Here we tested the role of these molecules in the SI response of A. thaliana SRK-SCR plants. We show that the A. thaliana ARC1 ortholog is a highly decayed pseudogene. We also show that, unlike reports in Brassica, inactivation of the MLPK ortholog AtAPK1b and overexpression of Exo70A1 neither abolish nor weaken SI in A. thaliana SRK-SCR plants. These results do not support a role for these molecules in the SI response of A. thaliana.     

Specificity determinants and diversification of the Brassica self-incompatibility pollen ligand

Self-incompatibility in crucifers is effected by allele-specific interactions between the highly polymorphic stigmatic S locus receptor kinase (SRK) and its pollen ligand, the S locus cysteine-rich protein (SCR). Here we show that specificity in SCR function is determined by four contiguous amino acids in one variant, indicating that the minimum sequence requirement for gaining a new specificity can be low. We also provide evidence for an extraordinarily high degree of evolutionary flexibility in SCR, whereby SCR can tolerate extensive amino acid changes within the limits of maintaining the same predicted overall structure. This remarkable adaptability suggests a hypothesis for generation of new self-incompatibility specificities by gradual modification of SRK-SCR affinities and, more generally, for functional specialization within families of homologous ligands and receptors.     

Non-cell-autonomous regulation of crucifer self-incompatibility by Auxin Response Factor ARF3

In many angiosperms, outcrossing is enforced by genetic self-incompatibility (SI), which allows cells of the pistil to recognize and specifically inhibit “self” pollen. SI is often associated with increased stigma-anther separation, a morphological trait that promotes cross-pollen deposition on the stigma. However, the gene networks responsible for coordinate evolution of these complex outbreeding devices are not known. In self-incompatible members of the Brassicaceae (crucifers), the inhibition of “self”-pollen is triggered within the stigma epidermal cell by allele-specific interaction between two highly polymorphic proteins, the stigma-expressed S-locus receptor kinase (SRK) and its pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein. Using Arabidopsis thaliana plants that express SI as a result of transformation with a functional SRK–SCR gene pair, we identify Auxin Response Factor 3 (ARF3) as a mediator of cross-talk between SI signaling and pistil development. We show that ARF3, a regulator of pistil development that is expressed in the vascular tissue of the style, acts non-cell-autonomously to enhance the SI response and simultaneously down-regulate auxin responses in stigma epidermal cells, likely by regulating a mobile signal derived from the stylar vasculature. The inverse correlation we observed in stigma epidermal cells between the strength of SI and the levels of auxin inferred from activity of the auxin-responsive reporter DR5::GUS suggests that the dampening of auxin responses in the stigma epidermis promotes inhibition of “self” pollen in crucifer SI.     

Loss of a histidine residue at the active site of S-locus ribonuclease is associated with self-compatibility in Lycopersicon peruvianum.

Gametophytic self-incompatibility in the Solanaceae is controlled by a single, multiallelic locus, the S locus. We have recently described an allele of the S locus of Lycopersicon peruvianum that caused this normally self-incompatible plant to become self-compatible. We have now characterized two glycoproteins present in the styles of self-compatible and self-incompatible accessions of L. peruvianum: one is a ribonuclease that cosegregates with a functional self-incompatibility allele (S6 allele); the other cosegregates with the self-compatible allele (Sc allele) but has no ribonuclease activity. The derived amino acid sequences of the cDNAs encoding the S6 and Sc glycoproteins resemble sequences of other ribonucleases encoded by the S locus. The derived sequence for the Sc glycoprotein differs from the others by lacking one of the histidine residues found in all other S-locus ribonucleases. These findings demonstrate the essential role of ribonuclease activity in self-incompatibility and lend further weight to evidence that this histidine residue is involved in the catalytic site of the enzyme.     

Early steps of angiosperm pollinator coevolution

The hypothesis that early flowering plants were insect-pollinated could be tested by an examination of the pollination biology of basal angiosperms and the pollination modes of fossil angiosperms. We provide data to show that early fossil angiosperms were insect-pollinated. Eighty-six percent of 29 extant basal angiosperm families have species that are zoophilous (of which 34% are specialized) and 17% of the families have species that are wind-pollinated, whereas basal eudicot families and basal monocot families more commonly have wind and specialized pollination modes (up to 78%). Character reconstruction based on recent molecular trees of angiosperms suggests that the most parsimonious result is that zoophily is the ancestral state. Combining pollen ornamentation, size, and aperture characteristics and the abundance of single-species pollen clumps of Cenomanian angiosperm-dispersed pollen species from the Dakota Formation demonstrates a dominance of zoophilous pollination (76% versus 24% wind pollination). The zoophilous pollen species have adaptations for pollination by generalist insects (39%), specialized pollen-collecting insects (27%), and other specialized pollinators (10%). These data quantify the presences of more specialized pollination modes during the mid-Cretaceous angiosperm diversification.     

Cloning and expression of a distinctive class of self-incompatibility (S) gene from Papaver rhoeas L.

We present the identification, cloning, and characterization of a self-incompatibility (S) gene from Papaver rhoeas that has no significant homology to any previously reported gene sequences, including S genes from other species. This result suggests that a different self-incompatibility mechanism may be operating in this species and has important implications for the evolutionary relationships between the S genes. The S1 cDNA was cloned by using an oligonucleotide based upon N-terminal amino acid sequence data from stigmatic proteins that show complete linkage with the S1 gene. The single-copy gene has been expressed in Escherichia coli to test biological activity. Although the recombinant S1 protein (S1e) is not processed in the same way as the protein produced in the plant, it exhibits, in vitro, the specific pollen inhibitory activity expected of an S gene product; pollen carrying the S1 allele is inhibited, whereas pollen not carrying S1 is not inhibited. These results provide definitive demonstration that the product of a cloned S gene has S-specific pollen inhibitory activity.     

The S locus glycoprotein and the S receptor kinase are sufficient for self-pollen rejection in Brassica

Self-incompatibility (SI) is one of several mechanisms that have evolved to prevent inbreeding in plants. SI in Brassica is controlled by the polymorphic S locus complex. Two S locus-encoded proteins are coordinately expressed in the stigma epidermis: the cell wall-localized S locus glycoprotein (SLG) and the plasma membrane-anchored S receptor kinase (SRK). These proteins are thought to recognize a pollen factor that leads to the rejection of self-pollen. Evidence has accumulated that indicates that both proteins are necessary for the ability of the stigma to inhibit self-pollen. However, it has not been possible to prove this necessity definitively or to demonstrate that these genes are sufficient for this phenotype, because previous attempts to transfer this phenotype via transformation have not been successful. In this study, two overlapping S locus genomic clones, which cover approximately 55 kilobases of DNA and contain the SLG, SRK, and an anther-expressed gene in the region common to the two, were introduced into a self-compatible Brassica napus line. The resulting transgenic plants were shown to carry the female part of the SI phenotype, rejecting pollen in a haplotype-specific manner. However, the pollen SI phenotype was not found in any of the transgenic plants. These data show that the SLG and SRK are sufficient for the female side but not the male side of the SI phenotype in Brassica and that there must be an independent pollen S factor encoded outside the cloned region.     

From the Cover: Evolutionary relationships among self-incompatibility RNases

T2-type RNases are responsible for self-pollen recognition and rejection in three distantly related families of flowering plants-the Solanaceae, Scrophulariaceae, and Rosaceae. We used phylogenetic analyses of 67 T2-type RNases together with information on intron number and position to determine whether the use of RNases for self-incompatibility in these families is homologous or convergent. All methods of phylogenetic reconstruction as well as patterns of variation in intron structure find that all self-incompatibility RNases along with non-S genes from only two taxa form a monophyletic clade. Several lines of evidence suggest that the best interpretation of this pattern is homology of self-incompatibility RNases from the Scrophulariaceae, Solanaceae, and Rosaceae. Because the most recent common ancestor of these three families is the ancestor of approximately 75% of dicot families, our results indicate that RNase-based self-incompatibility was the ancestral state in the majority of dicots.     

Organ-specific and Agamous-regulated expression and glycosylation of a pollen tube growth-promoting protein.

Transmitting tissue-specific (TTS) protein is a pollen tube growth-promoting and attracting glycoprotein located in the stylar transmitting tissue extracellular matrix of the pistil of tobacco. The TTS protein backbones have a deduced molecular mass of about 28 kDa, whereas the glycosylated stylar TTS proteins have apparent molecular masses ranging between 50 and 100 kDa. TTS mRNAs and proteins are ectopically produced in transgenic tobacco plants that express either a cauliflower mosaic virus (CaMV) 35S promoter-TTS2 transgene or a CaMV 35S-promoter-NAG1 (NAG1 = Nicotiana tabacum Agamous gene) transgene. However, the patterns of TTS mRNA and protein accumulation and the quality of the TTS proteins produced are different in these two types of transgenic plants. In 35S-TTS transgenic plants, TTS mRNAs and proteins accumulate constitutively in vegetative and floral tissues. However, the ectopically expressed TTS proteins in these transgenic plants accumulate as underglycosylated protein species with apparent molecular masses between 30 and 50 kDa. This indicates that the capacity to produce highly glycosylated TTS proteins is restricted to the stylar transmitting tissue. In 35S-NAG transgenic plants, NAG1 mRNAs accumulate constitutively in vegetative and floral tissues, and TTS mRNAs are induced in the sepals of these plants. Moreover, highly glycosylated TTS proteins in the 50- to 100-kDa molecular mass range accumulate in the sepals of these transgenic, 35S-NAG plants. These results show that the tobacco NAGI gene, together with other yet unidentified regulatory factors, control the expression of TTS genes and the cellular capacity to glycosylate TTS proteins, which are normally expressed very late in the pistil developmental pathway and function in the final stage of floral development. The sepals in the transgenic 35S-NAG plants also support efficient pollen germination and tube growth, similar to what normally occurs in the pistil, and this ability correlates with the accumulation of the highest levels of the 50- to 100-kDa glycosylated TTS proteins.     

Gametophytic self-incompatibility is controlled by a single major locus on chromosome 1 in Lycopersicon peruvianum

By using a number of previously mapped enzyme-coding genes as genetic markers, it has been possible to scan the genome of Lycopersicon peruvianum for gene(s) controlling the gametophytic self-incompatibility reaction. Regardless of genetic background or level of inbreeding, only a single locus (S), mapping to chromosome 1, was found to control the self-incompatibility reaction. Despite the widespread occurrence of this form of self-incompatibility in higher plants, to the best of our knowledge, the locus underlying the response has not been confirmed previously through genetic mapping, and the results cast doubts on hypotheses requiring multifactoral or dynamic control of gametophytic self-incompatibility.     

Chemocyanin, a small basic protein from the lily stigma, induces pollen tube chemotropism

In plant reproduction, pollination is an essential process that delivers the sperm through specialized extracellular matrices (ECM) of the pistil to the ovule. Although specific mechanisms of guidance for pollen tubes through the pistil are not known, the female tissues play a critical role in this event. Many studies have documented the existence of diffusible chemotropic factors in the lily stigma that can induce pollen tube chemotropism in vitro, but no molecules have been isolated to date. In this study, we identified a chemotropic compound from the stigma by use of biochemical methods. We purified a lily stigma protein that is active in an in vitro chemotropism assay by using cation exchange, gel filtration, and HPLC. Tryptic digestion of the protein yielded peptides that identified the protein as a plantacyanin (basic blue protein), and this was confirmed by cloning the cDNA from the lily stigma. Plantacyanins are small cell wall proteins of unknown function. The measured molecular mass by electrospray ionization ion source MS is 9898 Da, and the molecular mass of the mature protein (calculated from the cDNA) is 9900.2 Da. Activity of the lily plantacyanin (named chemocyanin) is enhanced in the presence of stigma/stylar cysteine-rich adhesin, previously identified as a pollen tube adhesin in the lily style.     

Pex1, a pollen-specific gene with an extensin-like domain.

We report here the identification of a pollen-specific gene from Zea mays that contains multiple Ser-(Pro)n repeats, the motif found in the cell wall-associated extensins. Sequence analysis reveals that the encoded protein has a putative globular domain at the N terminus and an extensin-like domain at the C terminus. The Pex1 (pollen extensin-like) gene is expressed exclusively in pollen, not in vegetative or female tissues, and is not induced in leaves upon wounding. We propose that the encoded protein may have a role in reproduction, either as a structural element deposited in the pollen tube wall during its rapid growth or as a sexual recognition molecule that interacts with partner molecules in the pistil.     

Isolation and characterization of pollen coat proteins of Brassica campestris that interact with S locus-related glycoprotein 1 involved in pollen-stigma adhesion

Adhesion of pollen grains to the stigmatic surface is a critical step during sexual reproduction in plants. In Brassica, S locus-related glycoprotein 1 (SLR1), a stigma-specific protein belonging to the S gene family of proteins, has been shown to be involved in this step. However, the identity of the interacting counterpart in pollen and the molecular mechanism of this interaction have not been determined. Using an optical biosensor immobilized with S gene family proteins, we detected strong SLR1-binding activity in pollen coat extracts of Brassica campestris. Two SLR1-binding proteins, named SLR1-BP1 and SLR1-BP2, were identified and purified by the combination of SLR1 affinity column chromatography and reverse-phase HPLC. Sequence analyses revealed that these two proteins (i) differ only in that a proline residue near the N terminus is hydroxylated in SLR1-BP1 but not in SLR1-BP2, and (ii) are members of the class A pollen coat protein (PCP) family, which includes PCP-A1, an SLG (S locus glycoprotein)-binding protein isolated from Brassica oleracea. Kinetic analysis showed that SLR1-BP1 and SLR1-BP2 specifically bound SLR1 with high affinity (K(d) = 5.6 and 4.4 nM, respectively). The SLR1-BP gene was specifically expressed in pollen at late stages of development, and its sequence is highly conserved in Brassica species with the A genome.     

Polymorphism at the self-incompatibility locus in Solanaceae predates speciation.

Sequences of 11 alleles of the gametophytic self-incompatibility locus (S locus) from three species of the Solanaceae family have recently been determined. Pairwise comparisons of these alleles reveal two unexpected observations: (i) amino acid sequence similarity can be as low as 40% within species and (ii) some interspecific similarities are higher than intraspecific similarities. The gene genealogy clearly illustrates this unusual pattern of relationships. The data suggest that some of the polymorphism at the S locus existed prior to the divergence of these species and has been maintained to the present. In support of this hypothesis, the number of shared polymorphic sites was found to exceed the number found in simulations with independent accumulation of mutations. Strictly neutral evolution is exceedingly unlikely to maintain the polymorphism for such a long time. The allele multiplicity and extreme age of the alleles is consistent with Wright's classic one-locus population genetic model of gametophytic self-incompatibility. Similarities between the plant S locus and the mammalian major histocompatibility complex are discussed.     

Unilateral incompatibility gene ui1.1 encodes an S-locus F-box protein expressed in pollen of Solanum species

Unilateral interspecific incompatibility (UI) is a postpollination, prezygotic reproductive barrier that prevents hybridization between related species when the female parent is self-incompatible (SI) and the male parent is self-compatible (SC). In tomato and related Solanum species, two genes, ui1.1 and ui6.1, are required for pollen compatibility on pistils of SI species or hybrids. We previously showed that ui6.1 encodes a Cullin1 (CUL1) protein. Here we report that ui1.1 encodes an S-locus F-box (SLF) protein. The ui1.1 gene was mapped to a 0.43-cM, 43.2-Mbp interval at the S-locus on chromosome 1, but positional cloning was hampered by low recombination frequency. We hypothesized that ui1.1 encodes an SLF protein(s) that interacts with CUL1 and Skp1 proteins to form an SCF-type (Skp1, Cullin1, F-box) ubiquitin E3 ligase complex. We identified 23 SLF genes in the S. pennellii genome, of which 19 were also represented in cultivated tomato (S. lycopersicum). Data from recombination events, expression analysis, and sequence annotation highlighted 11 S. pennellii genes as candidates. Genetic transformations demonstrated that one of these, SpSLF-23, is sufficient for ui1.1 function. A survey of cultivated and wild tomato species identified SLF-23 orthologs in each of the SI species, but not in the SC species S. lycopersicum, S. cheesmaniae, and S. galapagense, pollen of which lacks ui1.1 function. These results demonstrate that pollen compatibility in UI is mediated by protein degradation through the ubiquitin–proteasome pathway, a mechanism related to that which controls pollen recognition in SI.Self-incompatibility (SI) in Solanum and other Solanaceae is the S-RNase–based, gametophytic type, in which S-specificity is determined by S-RNases in the pistil (1) and S-locus F-box proteins (SLFs) in pollen (2). F-box proteins, together with Skp1 and Cullin1 proteins, are components of SCF-type (Skp1, Cullin1, F-box) ubiquitin E3 ligases that mark target proteins for degradation by the 26S proteasome (3, 4). The ubiquitin–proteasome pathway controls the pollen compatibility phenotype in SI (5). In the “collaborative non–self-recognition” model (6), the S-locus encodes multiple SLF proteins that together recognize different sets of S-RNases. In a compatible pollination, the SLF/S-RNase interaction leads to protection of pollen tubes against cytotoxic S-RNase, whereas in incompatible pollinations, a failure to recognize self–S-RNase results in pollen tube arrest. In addition, modifier genes, such as those encoding HT-B, NaStEP, and 120-kDa proteins in the pistil, and CUL1 and SSK1 proteins in pollen, are required for SI function but do not confer S-specificity (7–11).Unilateral incompatibility (UI) is a reproductive barrier related to SI in which pollen from one species or population is rejected on pistils of a related species or population, whereas in the reciprocal crosses, no pollen rejection occurs. Pollen of SC species or populations is almost always rejected on pistils of related SI species or populations, whereas in the reciprocal crosses (SC pollinated by SI), pollen rejection rarely occurs. This unidirectional pattern of pollen rejection is referred to as the “SI × SC rule” (12). Although the mechanisms of pollen recognition and rejection by UI are complex (13), several SI factors, including S-RNase, CUL1, and HT, also function in UI (8, 14, 15).Cultivated and wild tomatoes provide a powerful model system to study the mechanisms of reproductive barriers in the Solanaceae (16). They display wide variation in mating systems, both between and within species (17). Cultivated tomato, S. lycopersicum, is SC and accepts pollen of its SI or SC wild relatives; conversely, pollen of S. lycopersicum is rejected by pistils of the SI species. Three other red- or orange-fruited species, S. cheesmaniae, S. galapagense, and S. pimpinellifolium, are bilaterally cross-compatible with each other and with S. lycopersicum. There are notable exceptions to SI × SC rule in the tomato clade (18). One is that pollen of all of the red/orange-fruited species (SC) are rejected on pistils of the SC green-fruited species. Another exception is that pollen of some SC biotypes of SI species are compatible on pistils of conspecific SI populations. Therefore, pollen rejection is complex and likely involves more than one mechanism (13). The ability to reject tomato pollen is dominant in interspecific F₁ hybrids between cultivated tomato and related wild SI species (i.e., SC × SI hybrids), although pollen tube arrest occurs lower in the style of hybrids, suggesting that expression of the pistil side barrier is weakened (19). Allotriploid hybrids comprised of two genomes from S. lycopersicum (SC) and one genome from S. lycopersicoides (SI) reject tomato pollen tubes lower in the style than corresponding diploid hybrids (19).We previously reported that two pollen factors from S. pennellii, ui1.1 and ui6.1, are required and sufficient to overcome the UI barrier on allotriploid S. lycopersicum × S. lycopersicoides hybrids (19, 20). These two factors are not sufficient for compatibility on diploid S. lycopersicum × S. lycopersicoides hybrids (19). The ui6.1 locus encodes a pollen specific Cullin1 (CUL1) protein (21) that functions in pollen recognition by UI and SI (8). Pollen lacking ui6.1 are incompatible on pistils expressing active S-RNases, but compatible on pistils expressing a mutant S-RNase lacking RNase activity (8). This observation suggested that ui6.1—and by extension, ui1.1—is required for pollen resistance to S-RNase–based rejection in the pistil. Interestingly, neither ui6.1 nor ui1.1 is required for resistance to S-RNase itself, because tomato pollen is fully compatible on pistils expressing active S-RNase in the absence of a functional HT protein (15, 22). Thus, both SI and UI require multiple pollen and pistil factors. The ui1.1 locus is located at or near the S locus region on the short arm of chromosome 1, suggesting it might encode one or more SLF proteins. The goal of the present research was to isolate ui1.1 from S. pennellii to elucidate the nature of pollen rejection by UI and its relationship to SI.