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S-RNase and SLF Determine S-Haplotype–Specific Pollen Recognition and Rejection

Department of Biochemistry, University of Missouri, Columbia, MO 65211

mcclureb{at}missouri.edu

Self-incompatibility (SI) is a genetically determined system for recognition and rejection of self-pollen and pollen from closely related plants. Recognition specificity is controlled by the S-locus. This locus is complex in the sense that it contains multiple genes: one controls specificity on the pistil side (pistil S), another on the pollen side (pollen S). It is also highly polymorphic; the S-specificity genes in different haplotypes have highly divergent sequences, giving rise to a set of proteins having a similar function but distinctive recognition characteristics.

S-RNase–based SI occurs in the Solanaceae, Rosaceae, and Scrophulariaceae. S-RNase itself controls specificity on the pistil side. S-RNases were discovered in the mid-1980s (Bredemeijer and Blaas, 1981; Anderson et al., 1986), but it took eight more years to provide direct evidence that they are the genes that determine S-specificity in the pistil (Lee et al., 1994; Murfett et al., 1994). It has recently been determined that SLF (S-locus F-box) genes control specificity on the pollen side. SLF genes were first reported in the Scrophulariaceae (Lai et al., 2002). At first, SLF was not thought to be sufficiently polymorphic to be pollen S, the determinant of S-specificity in pollen. However, highly polymorphic SLF genes were described in Rosaceae in 2003 and early 2004 (Entani et al., 2003; Ushijima et al., 2003; Ikeda et al., 2004). Recent months have seen publication of transformation experiments definitively showing that SLF determines specificity on the pollen side in Petunia inflata (Sijacic et al., 2004). These results are supported by transformation experiments showing that the Antirrhinum SLF₂ gene is implicated in SI, although it was not possible to directly address S-specificity in this system (Qiao et al., 2004b). The compressed time scale between identification of SLF and definitive proof that it is pollen S is a testament to the hard work and skill of the laboratories involved in research in all three S-RNase–based SI systems.

The identification of the S-specificity determining genes on both the pollen and pistil sides brings closure to the first phase of research into the molecular basis of S-RNase–based SI. This is an excellent time to evaluate our understanding of the system, as research moves toward a focus on the biochemistry and physiology of pollen recognition and rejection. This essay describes only the most recent results related to identification of pollen S and highlights select facts that will shape research in the near future. For a more comprehensive review, please see Kao and Tsukamoto (2004).

THE FUNDAMENTALS

S-RNase–based SI is widely distributed among the angiosperms (Igic and Kohn, 2001; Kao and Tsukamoto, 2004). Fundamentally, it is a mechanism for controlling pollination. It can be described as genetic interaction between the sporophyte and the gametophyte that prevents inbreeding. It is a typical single-locus gametophytic SI system—compatibility is determined by the S-haplotype of pollen, the haploid male gametophyte. Pollen is rejected when its S-haplotype is the same as either of the S-haplotypes in the diploid pistil. Conversely, pollen with any S-haplotype not present in the pistil is compatible (de Nettancourt, 2001). This is the origin of the cumbersome but necessary term S-specific pollen rejection (or S-allele–specific pollen rejection). S-RNase is also implicated in interspecific pollen rejection, but the mechanisms are distinct from SI (Murfett et al., 1996). Thus, S-RNase–based systems are the most widespread and diverse pollen rejection systems known.

The genetics of SI are simple. The challenge is to understand how the genetic interaction between the pollen and the pistil is manifested at the biochemical level. Necessarily, the first priority was to identify the biochemical determinants of S-specificity. The approaches used had to be tied to the requirements imposed by the biology and genetics of SI. Putative specificity determinants must meet three criteria: linkage to the S-locus, polymorphism between different S-haplotypes, and expression in the pollen or pistil.

S-RNASE DETERMINES S-SPECIFICITY IN THE PISTIL

S-RNases, the determinants of S-specificity in the pistil, were identified by searching for proteins that met these three requirements. The principle was simple: perform controlled crosses to determine the S-specific pollen rejection phenotypes of a family of plants and analyze for cosegregating proteins. In practice, however, this was difficult. It took years to identify plant materials with protein bands that clearly cosegregated with S-specific pollen rejection (Bredemeijer and Blaas, 1981). Anderson et al. (1986) ultimately obtained N-terminal sequence from a glycoprotein expressed in the Nicotiana alata style that cosegregated with the ability to reject S₂-pollen. Numerous similar clones soon were obtained from other S-haplotypes, species, and families. Sequence analysis led to the discovery that these polymorphic proteins have ribonuclease activity; thus, they were labeled S-RNases (McClure et al., 1989).

It took eight additional years to show definitively that S-RNase genes met the requirements for the determinants of S-specificity in the pistil. Linkage to the S-locus was determined by genetic analyses showing no recombination between S-RNase genes and the pollen rejection phenotype (Murfett et al., 1994). S-RNase gene sequences, and the surrounding genomic regions, proved to be highly polymorphic between different S-haplotypes (Anderson et al., 1989; Ioerger et al., 1990, 1991). S-RNase expression was restricted to the pistil, and the proteins were secreted into the extracellular matrix forming the path from the stigma to the ovary (Cornish et al., 1987; Anderson et al., 1989). Although these data provided strong correlative evidence that S-RNase was the determinant of S-specificity in the pistil, they were not definitive. Articles published in 1994 provided direct evidence that S-RNase is the product of the pistil S gene. They showed that expression of the N. alata S_A2-RNase gene or the P. inflata S₃-RNase gene caused transgenic plants to gain the ability to reject S_A2- or S₃-pollen, respectively (Lee et al., 1994; Murfett et al., 1994).

The basic mechanism of S-RNase–based SI was outlined in the early 1990s. Gray et al. (1991) showed that S-RNase can gain access to the pollen cytoplasm and act as a potent translational inhibitor. A tracer experiment using ³²P-labeled pollen showed that pollen RNA is degraded after incompatible but not compatible pollinations (McClure et al., 1990). Finally, Huang et al. (1994) showed that ribonuclease activity is required for pollen rejection. The basic model that emerged is that each S-haplotype encodes a unique S-RNase protein that is secreted into the transmitting tract extracellular matrix. An interaction between S-RNase and the pollen-side specificity factor, pollen S protein, determines whether the pollination is compatible or incompatible. In incompatible pollinations, S-RNase gains access to the pollen tube cytoplasm where its ribonuclease activity causes a cytotoxic effect; in a compatible pollination, this cytotoxic effect is evaded.

F-BOX PROTEINS DETERMINE S-SPECIFICITY IN POLLEN

The search for the pollen S gene began shortly after the S-RNase gene was cloned. It quickly became obvious that the pollen S protein is not as abundant as S-RNase and different approaches would be needed. Ultimately, pollen S gene candidates were successfully identified by large-scale genomic DNA sequencing in the vicinity of S-RNase genes. This approach succeeded because it was tied directly to the three strict requirements mentioned earlier: candidates must be linked to the S-RNase gene, highly polymorphic, and expressed in pollen. However, proving that a candidate gene is pollen S was not a trivial matter because the only known phenotypes associated with it are pollen tube growth inhibition in SI and a subtle genetic interaction between S-haplotypes that occurs in heteroallelic pollen.

The tight genetic linkage between pollen S and S-RNase provided an obvious route to identifying candidates. However, SI species are not always well suited for genetic experiments, and various SI systems have different strengths and weaknesses. Essential contributions were made by researchers working in Antirrhinum, Nicotiana, Petunia, and Prunus (reviewed in Kao and Tsukamoto, 2004). In Nicotiana and Petunia, historically the best systems for S-RNase studies, a great deal of effort was required to accumulate a collection of pollen-expressed markers linked to S-RNase genes (Li et al., 2000; McCubbin et al., 2000a, 2000b; Wang et al., 2003). In spite of the difficulties, Wang et al. (2003) mapped 13 markers and S-RNase in a large P. inflata population segregating for S₁- and S₂-haplotypes. Nine markers and the S-RNase gene occurred in a 0.25-centimorgan region and could not be separated by recombination. BAC clones were isolated to allow construction of a physical map. Although contigs amounting to 4.4 Mb were constructed from the S₂-haplotype, only one contig contained more than a single marker, revealing that the S-locus is physically very large in Petunia. Ultimately, the pollen expressed genes closest to the P. inflata S₂-RNase gene were identified in an 881-kb contig by cDNA selection and direct sequencing of 328 kb surrounding the S₂-RNase gene (Wang et al., 2004). In other species, the S-locus proved to be more compact. Markers defining the boundaries of the S-locus in P. dulcis turned out to be only 72 kb apart (Ushijima et al., 2001). In Antirrhinum and Prunus mume, genomic contigs spanning only 65 kb proved to contain pollen S candidates (Lai et al., 2002; Entani et al., 2003). Genes that are physically close to the S-RNase gene all fulfill the first requirement for a pollen S candidate; they are linked to the S-locus. Applying the additional requirements of polymorphism and expression in pollen allowed the best candidates to emerge.

Results from genomic sequencing studies showed that the S-locus region contains multiple F-box protein genes. Unfortunately, expression in pollen is common; therefore, this is not a discriminating test for pollen S candidates. Sequence polymorphism proved to be a better way to distinguish among candidates. The polymorphic F-box protein genes so identified (i.e., pollen S) are referred to as SLF_x (S-locus F-box from haplotype X) genes in Antirrhinum, Petunia, and Prunus mume by some authors (Lai et al., 2002; Qiao et al., 2004b, 2004a; Sijacic et al., 2004), others refer to them as SFB genes (Ushijima et al., 2003, 2004; Ikeda et al., 2004).

The Antirrhinum AhSLF₂ gene was the first SLF gene reported (Lai et al., 2002). It was shown to be expressed specifically in pollen, but the polymorphism was not as great as was expected for pollen S. AhSLF₂ probes revealed restiction fragment length polymorphisms in different S-haplotypes but that the degree of cross-hybridization was high, suggesting less sequence polymorphism than S-RNase genes (Lai et al., 2002).

Studies in Prunus strongly pointed to SLF genes as the best pollen S candidates because their sequence polymorphism is comparable to S-RNase. This set of Prunus pollen S candidates (referred to as SLF in P. mume and SFB in P. dulcis) showed S-haplotype–specific hybridization patterns similar to those obtained with S-RNase probes (Entani et al., 2003; Ushijima et al., 2003). Moreover, sequence analyses of 13 of these genes showed a pattern of conserved and variable domains consistent with a role in recognition as well as evidence of positive selection in so-called hypervariable regions (Ikeda et al., 2004). Ushijima et al. (2004) also analyzed Prunus pollen-part mutants having mutations that only affect S-haplotype specificity in pollen while leaving pistil functions intact. The mutant P. avium S₄'-haplotype expressed a truncated SFB₄' gene missing hypervariable regions present in the wild-type SFB₄ gene. In P. mume, the mutant S_f-haplotype contained an SFB_f gene with a large insertion such that the predicted polypeptide would be truncated as well. Evidence for positive selection and the presence of mutated versions of SFB genes in pollen-part mutants strongly supports a connection between SLF and pollen S. Together, the data from Antirrhinum and Prunus provided strong correlative evidence (i.e., close linkage to S-RNase, expression in pollen, and polymorphism between S-haplotypes) that SLF (or SFB) and pollen S were the same gene. What remained lacking was a functional test.

The definitive functional test is a transformation experiment allowing an SLF gene to be directly tested for an S-haplotype–specific effect on pollination. Functional studies in P. inflata have now provided definitive proof that one of the F-box protein genes is indeed pollen S (Sijacic et al., 2004), and a recent study in Antirrhinum also directly implicated an F-box protein gene in SI but did not formally address haplotype specificity (Qiao et al., 2004b). These functional studies of pollen S candidates are much more subtle than those routinely applied to plant genes. A thorough grasp of how pollen behaves in S-RNase–based systems and how S-specificity is expressed in pollen is needed to understand the transformation strategies and, in particular, the separate tests to implicate a gene in SI and to show that it determines S-specificity.

Notwithstanding the fact that S-haplotype–specific pollen rejection is the defining feature of SI, it is essential to grasp the fact that pollen's resistance to the cytotoxic effect of S-RNase is also fundamental in S-RNase–based SI. For example, although S₁-pollen is susceptible to the cytotoxic effect of S₁-RNase, it is also resistant to all other S-RNases. Thus, although a pollen S-haplotype is rejected by one self-S-RNase, it is resistant to all nonself-S-RNases. The popular models to explain this behavior invoke some type of S-RNase inhibitor, and the transformation strategies to test pollen S candidates were strongly influenced by these models (Kao and Tsukamoto, 2004). The simple inhibitor model combines the S-specific recognition function (i.e., the genetic definition of pollen S) and the S-RNase inhibitor function into a single molecule. A modified inhibitor model places these two functions in separate molecules; pollen S determines S-specificity, and a separate inhibitor prevents the cytotoxic activity of S-RNases (Luu et al., 2001).

The inhibitory function of pollen S is supported by mutational studies that suggest that loss of the pollen S gene is lethal to the pollen. Golz et al. (1999) (2001) examined radiation-induced pollen-part mutants in N. alata. All the mutants could be explained by duplication of all or part of the S-locus such that pollen effectively became heterozygous. The absence of mutants with loss of pollen S in the Golz et al. (1999) (2001) studies is easily explained if it is an essential gene. However, the Ushijima et al. (2004) article discussed earlier adds a significant new twist. Their study of Prunus pollen-part mutants shows that at least one class of pollen S mutation is viable. Significantly, the pollen-part mutants do not appear to have lost pollen S entirely. The mutant forms of both SFB₄' and SFB_f retain the N-terminal (i.e., F-box containing) portion of the protein, whereas the C-terminal variable portions were removed. So far as is known, these pollen-part mutants behave as universal pollinators; they retain the ability to pollinate other S-haplotypes but they no longer show rejection by their cognate S-RNase. It is possible to speculate that the N-terminal part of the protein functions in resistance to S-RNase (i.e., compatibility) and the C-terminal portion functions in S-haplotype specific rejection. This interpretation is consistent with the simple inhibitor model, but the modified model is not excluded because it is still possible that a separate inhibitor exists. The Prunus results (Ushijima et al., 2004) open the door for a detailed dissection of pollen S function in Petunia or Solanum where in vitro mutagensis and transformation can be performed. However, such dissection is only meaningful if the two activities (recognition of self-RNase and inhibition of non-self-RNases) definitively have been shown to reside in the same molecule.

Although the details of how pollen resists the effects of nonself-S-RNase are not resolved, and the two inhibitor models make different predictions, it's clear that well-designed pollen S transformation experiments had to account for the possibility that pollen S is an essential gene (i.e., it may function to prevent the cytotoxic action of S-RNase). In particular, antisense or RNA interference approaches were not viable. Thus, Sijacic et al. (2004) and Qiao et al. (2004b) introduced pollen S candidates into pollen already expressing a functional pollen S gene. This approach, though successful, is complicated by the manner in which pollen S genes interact when two different S-haplotypes are expressed in the normally haploid pollen tube.

Heteroallelic pollen may arise from duplications of the S-locus, as described earlier (Golz et al., 1999, 2001), or in tetraploids. When an SI S₁S₂ plant is converted to an S₁S₁S₂S₂ tetraploid, SI is lost (de Nettancourt, 1977). The defect only affects pollen; the tetraploid pistil continues to reject S₁- and S₂-pollen. Moreover, breakdown only occurs in S₁S₂ diploid (i.e., heteroallelic) pollen; S₁S₁ and S₂S₂ pollen is rejected normally on both the diploid S₁S₂ or tetraploid S₁S₁S₂S₂ pistil (Table 1). Thus, the S₁- and S₂-haplotypes interact in such a way that S-specific pollen rejection is abolished.

In P. inflata, the PiSLF₂ gene was transformed into an S₁S₁ background (Sijacic et al., 2004). The resulting phenotype, SI breakdown on the pollen side, was exactly as predicted. Only pollen-part function was disrupted, and significantly, breakdown only occurred in S₁-pollen expressing the PiSLF₂ transgene. Qiao et al. (2004b) performed similar experiments with the Antirrhinum AhSLF₂ gene. Two AhSLF₂ gene constructs were transformed into SI P. hybrida S₃S₃. The Antirrhinum gene behaved as expected, and SI breakdown on the pollen side was observed. Thus, in two systems, SLF transgenes expressed in conjunction with a different SLF gene (i.e., the SLF gene already present at the native S-locus) caused a breakdown in SI on the pollen side. This is completely consistent with predictions based on the behavior of the N. alata pollen-part mutants and the heteroallelic pollen effect (Table 1). However, as noted earlier, the heteroallelic pollen effect is inherently nonspecific; any two different pollen S genes cause SI to breakdown. Moreover, breakdown of SI would be expected after disrupting any gene (i.e., other than pollen S) implicated in pollen SI function.

S-specificity can best be demonstrated by the absence of SI breakdown in the one case where the SLF transgene is from the same S-haplotype as the native SLF gene. Therefore, Sijacic et al. also transformed the PiSLF₂ gene into an S₂S₃ background. Breakdown of pollen-part function was observed again. However, all the self-progeny were either S₂S₃ or S₃S₃ and carried the transgene. This is explained as just described; SI breaks down when the PiSLF₂ transgene is expressed in pollen with the S₃-haplotype. The absence of S₂S₂ progeny shows that the effect of the PiSLF₂ transgene is S-haplotype specific: it only interferes with other S-haplotypes (i.e., S₁ or S₃) and has no effect on the S₂-haplotype (Table 1). As noted, Qiao et al. (2004b) transformed the AhSLF₂ gene into P. hybrida S₃S₃ to test its function in SI. Because it is not possible to examine the effect (or lack of effect) of the transgene on the native Antirrhinum S₂-haplotype, this approach precludes testing for S-specificity. Nonetheless, the S-haplotype–specific effect of PiSLF₂ is definitive. SLF is pollen S.

S-RNASE–BASED SI IS LIKELY TO BE LINKED TO UBIQUITINATION

The most important players in S-RNase–based SI are now known; the field is at a turning point. Research will now shift toward understanding the biochemistry and physiology of S-specific pollen rejection. Until now, only the end points were known. At the pistil end, S-RNase is secreted into the transmitting tract extracellular matrix; at the other end, pollen RNA is degraded or protected, resulting in incompatibility or compatibility, respectively. Identification of pollen S as an F-box protein suggests that the specificity step, the interaction between pollen S (SLF) and pistil S (S-RNase), is linked to ubiquitination.

F-box proteins comprise the F in the SCF (Skp1 Cullin F-box) E3 ubiquitin ligase complex that tags proteins for subsequent degradation (Sullivan et al., 2003). The SCF complex catalyzes the final step in synthesis of a polyubiquitin chain on specific target proteins that become substrates for the 26S proteasome. F-box proteins can act as adaptors, conferring specificity to the E3 complex by binding specific target proteins (although additional proteins also may be required). Complexes with distinct F-box proteins are designated with a superscript, such as SCF^PiSLF2. Plants have an unusually large repertoire of F-box protein genes (Gagne et al., 2002; Smalle and Vierstra, 2004), and ubiquitination has been linked to a wide variety of processes, including sporophytic SI (Stone et al., 2003).

Some have proposed that SCF^SLF functions as an inhibitor of nonself-S-RNase by tagging it for degradation (Qiao et al., 2004a, 2004b; Sijacic et al., 2004; Ushijima et al., 2004). This simple model is attractive and may well be correct, but other models are possible. F-box proteins play roles apart from protein degradation. For example, the yeast F-box protein Rcy1p localizes to areas of polar growth and is required for recycling membrane proteins (Galan et al., 2001). Ubiquitination has been likened to phosphorylation as a signaling mechanism; it has roles in many cellular functions, including membrane traffic, transcriptional regulation, translation, and DNA repair (Weisman, 2001; Smalle and Vierstra, 2004). If protein degradation is the critical process in S-RNase–based SI, it must be determined if the simple nonself-S-RNase degradation model is adequate or whether a more complex model is needed.

Experiments to test aspects of this model have been initiated. The S-specificity step must, by definition, involve an interaction between SLF and S-RNase. Qiao et al. (2004a) reported important pull-down and immunoprecipitation results that show a direct interaction between A. hispanicum AhSLF₂ and S-RNase and presented evidence that AhSLF₂ forms a complex with Skp1- and cullin-like proteins. Thus, SLF interacts with S-RNase and probably participates in an E3 ubiquitin ligase complex. Importantly, there is no biochemical evidence for an S-specific interaction; AhSLF₂ bound both self- and nonself-S-RNases as well as S₃-RNase from P. hybrida (Qiao et al., 2004a, 2004b). By definition, the interaction between S-RNase and SLF determines S-specificity (compatibility). Thus, either the interactions between SLF and self- versus nonself-S-RNases have different outcomes, perhaps, differential activation of an SCF^SLF complex, or the experimental conditions for S-specific interaction have not been found. Only further research will resolve these questions.

Although these experiments represent a considerable advance, some of the conclusions reached by Qiao et al. (2004a) need to be confirmed in other systems before they are generally accepted. For example, a straightforward prediction of the simple model is that levels of S-RNase should decrease after compatible but not incompatible pollination. Qiao et al. tested this by comparing the amount of immunostained S-RNase in whole style extracts after self- and cross-pollination and concluded that S-RNase levels decrease after compatible pollination. However, this has not been observed in other studies. Immunolocalization studies showed large amounts of S-RNase inside both compatible and incompatible Solanum chacoense pollen tubes (Luu et al., 2000). Furthermore, Gray et al. (1991) found no evidence for S-RNase degradation using ³H-labeled S₂-RNase and in vitro–grown N. alata pollen tubes. The hypotheses (Qiao et al., 2004a) that S-RNase is the substrate of the SCF^SLF complex and that it is subsequently degraded by the 26S proteasome are of central importance, but it is also important to be cautious and test them in other systems. An SCF^SLF complex may not be the only E3 ubiquitin ligase that binds to S-RNase. Sims and Ordanic (2001) described a putative RING E3 protein from P. hybrida that binds S-RNase. Unlike SLF, the RING E3 is expressed in nonsexual tissues and, thus far, only has been shown to bind to S-RNase fragments in the yeast two hybrid system. It is not yet known whether the RING E3 is directly involved in SI or whether it interacts with SCF^SLF. The cautions aside, it should be emphasized that Qiao et al. (2004a) initiated important experiments investigating the biochemistry and physiology underlying pollen rejection. Such studies are badly needed, and it is very important to extend their experiments to other systems.

We now know the identity of pistil S and pollen S and we can begin to move on to studies of the biochemistry and physiology of S-RNase–based SI. Sijacic et al. (2004) showed that SLF is the pollen S gene, and Qiao et al. (2004a) (2004b) showed that SLF binds S-RNase. The door is open for investigating the specificity of this interaction using standard domain-swap and mutagenesis techniques. Such studies may clarify coevolution of the S-RNase and SLF genes. The possible roles of SLF-like genes located at the S-locus should be tested. Identification of the substrate(s) of SCF^SLF will help determine whether the simple model involving degradation of nonself-S-RNase needs to be modified. Possible candidates include S-RNase, SLF, and non-S-RNase factors required for pollen rejection, as well as factors that are as yet unknown. Studies of potential SCF^SLF substrates and the origin of specificity are obvious research goals for the immediate future. However, they alone will not provide a comprehensive model of S-RNase–based SI.

ISSUES ARISING

There are essential aspects of S-RNase–based SI of which we are still totally ignorant. There are also established facts that are typically left out of popular illustrations of SI. It is worthwhile to bring some of these to the forefront so that they can contribute to a more comprehensive model.

The mechanism of S-RNase uptake is an area of glaring ignorance. Transmitting tract cells secrete S-RNase into the extracellular matrix. SLF is very likely to be a cytoplasmic protein; the S-specificity step, where SLF and S-RNase interact, therefore probably occurs in the pollen tube cytoplasm. This is also the final site of S-RNase action because incompatible pollen tube RNA is degraded there. Thus, it is important to define the route from the extracellular matrix to the cytoplasm. The only report of S-RNase uptake describes nonspecific uptake of both self- and nonself-S-RNase into pollen tubes (Luu et al., 2000). S-RNase was reported to be in the pollen tube cytoplasm. However, its exact location is difficult to assess because sections were prepared to preserve antigenicity rather than membrane integrity, and established markers for cellular compartments were not reported. Although pollen tube growth is associated with massive fusion of vesicles at the tip, there is also a return flow (Taylor and Hepler, 1997). Therefore, pollen tubes are probably active in endocytosis; perhaps, this is the initial route of S-RNase entry. It is noteworthy that endocytosis and intracellular trafficking are mediated by ubiquitination (Hicke and Dunn, 2003). Membrane proteins may be monoubiquitinated or polyubiquitinated as a signal for incorporation into endocytic vesicles and subsequent sorting. There is precedent for involvement of an F-box protein in yeast membrane recycling (Galan et al., 2001). Clearly, if S-RNase is initially taken up into a membrane-bound compartment, then it would not have immediate access to either SLF or RNA in the cytoplasm. Thus, the question of whether S-RNase is initially taken up into a membrane-bound compartment or whether it somehow gains access directly to the cytoplasm is an area that needs to be addressed.

It is also unclear how the specificity inherent in SI is manifested at the biochemical level. S-RNase mutagenesis experiments are informative but they are seldom discussed in relation to models of SI. Zurek et al. (1997) reported domain-swap experiments in which portions of S_A2- and S_C10-RNase from N. alata were exchanged. Nine chimeric S-RNases were tested; although all were active ribonucleases, none were capable of causing S-specific pollen rejection. Thus, the chimeric S-RNases were capable of acting as cytotoxins but could not be properly recognized. Kao and McCubbin (1996) reported similar results in P. inflata. Because one of the N. alata chimeras was later shown to be active in an interspecific pollen rejection system (Beecher and McClure, 2001) and the evidence suggests that S-RNase uptake is nonspecific, it is unlikely that the chimeras fail to cause pollen rejection because they are not taken up by pollen tubes. At a minimum, the domain-swap experiments show that the S-specificity function of S-RNase is more labile than its enzymatic function. Perhaps, they also suggest that S-specificity and cytotoxic activity are separable functions on the pollen side. In the absence of self-S-RNase recognition, pollen may follow a default pathway that leads to compatibility. Conversely, recognition of self-S-RNase by SLF could either activate a pathway leading to incompatibility or deactivate a compatibility pathway.

Finally, although it is known that factors other than the specificity determinants are required for SI, it is not known how many such factors exist or how they function. If SCF^SLF-mediated ubiquitination and subsequent protein degradation are key elements of S-RNase–based SI, then any factor whose continued presence is required for pollen rejection is a possible target. HT-B is the only factor other than S-RNase and SLF that has been identified with certainty (McClure et al., 1999). It is a small Asn-rich protein secreted into the transmitting tract matrix in Nicotiana, Lycopersicon, and Solanum (McClure et al., 1999; Kondo et al., 2002a, 2002b; O'Brien et al., 2002). Antisense and RNA interference experiments show that HT-B is required for pollen rejection, but its function is unknown (McClure et al., 1999; O'Brien et al., 2002). Other factors acting on both the pollen and pistil side of the SI reaction probably exist. For example, substrates for SCF E3 ubiquitin ligases usually require prior activation. A protein kinase that phosphorylates S-RNase has been identified, but it is not known whether it has a specific function in SI (Kunz et al., 1995). Without better knowledge of the factors required for S-RNase–based pollen rejection, it is not possible to propose a comprehensive model.

Identification of the pollen S gene is the most exciting development in S-RNase–based SI in recent memory. We have a solid foundation from which to proceed, but there are more questions than answers. What is the true mode of SLF-mediated inhibition of nonself-S-RNase: stability, access to the cytoplasm, or something else? What are the roles of non-S-RNase factors, such as HT-B? What determines the specificity of the S-RNase–SLF interaction? What is the connection between S-RNase–SLF interaction and pollen tube growth? What are the constraints on coevolution of S-RNase and SLF genes? Some of these questions can be addressed easily and others cannot. Good tools for cell biological studies relating to S-RNase uptake and the physiology of growth inhibition are needed. A faithful in vitro pollen rejection system would be a boon. One thing, however, is clear: we are much closer to understanding S-RNase–based SI than ever.

Acknowledgments

I thank the members of my laboratory for helpful discussions. I thank Melody Kroll for help in preparing the manuscript. Research in my laboratory is supported by National Science Foundation Grant IBN 0315647.

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