Molecular Plant Advance Access originally published online on June 26, 2008
Molecular Plant 2008 1(4):575-585; doi:10.1093/mp/ssn032
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Biochemical Models for S-RNase-Based Self-Incompatibility
a Intercollege Graduate Degree Program in Plant Biology, The Pennsylvania State University, University Park, PA 16802, USA
b Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
c Present address: Department of Genetics, University of Wisconsin, Madison, WI 53706, USA
1 To whom correspondence should be addressed. E-mail txk3{at}psu.edu, fax (814) 863-7024, tel. (814) 863-1042.
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Abstract |
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 TOP Abstract INTRODUCTIONEARLIER MODELSRECENT MODELSNEW MODELCONCLUSIONS AND FUTURE...FUNDING |
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S-RNase-based self-incompatibility (SI) is a genetically determined self/non-self-recognition process employed by many flowering plant species to prevent inbreeding and promote outcrosses. For the Plantaginaceae, Rosaceae and Solanaceae, it is now known that S-RNase and S-locus F-box (two multiple allelic genes at the S-locus) determine the female and male specificity, respectively, during SI interactions. However, how allelic products of these two genes interact inside pollen tubes to result in specific growth inhibition of self-pollen tubes remains to be investigated. Here, we review all the previously proposed biochemical models and discuss whether their predictions are consistent with all SI phenomena, including competitive interaction where SI breaks down in pollen that carries two different pollen S-alleles. We also discuss these models in light of the recent findings of compartmentalization of S-RNases in both incompatible and compatible pollen tubes. Lastly, we summarize the results from our recent biochemical studies of PiSLF (Petunia inflata SLF) and S-RNase, and present a new model for the biochemical mechanism of SI in the Solanaceae. The tenet of this model is that a PiSLF preferentially interacts with its non-self S-RNases in the cytoplasm of a pollen tube to result in the assembly of an E3-like complex, which then mediates ubiquitination and degradation of non-self S-RNases through the ubiquitin–26S proteasome pathway. This model can explain all SI phenomena and, at the same time, has raised new questions for further study.
Received for publication February 29, 2008. Accepted for publication April 8, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONEARLIER MODELSRECENT MODELSNEW MODELCONCLUSIONS AND FUTURE...FUNDING |
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Most flowering plants (angiosperms) produce bisexual flowers, in which both the female (pistil) and male (anther) reproductive organs are in close proximity. This creates a strong tendency for self-pollination, which results in inbreeding. Inbreeding often leads to reduced fitness in the progeny (e.g. increased susceptibility to disease and stress). During the evolution of flowering plants, had they not adopted various mechanisms to prevent self-pollination and promote out-crossing, the highly diverse plant kingdom, which is more than 80% dominated by angiosperms, would not exist (Barnes-Svarney and Svarney, 1999).
One such mechanism is S-RNase-based self-incompatibility (SI), which employs a highly polymorphic genetic locus, named S-locus, to control pollination (Kao and Tsukamoto, 2004; Takayama and Isogai, 2005; Sims, 2007). Variants of the S-locus are referred to as ‘haplotypes’. If the haplotype of pollen matches one of the two haplotypes of the diploid pistil, the pollen is recognized as self-pollen and its tube growth in the style is inhibited. Pollen that carries a haplotype different from the haplotypes of the pistil is recognized as non-self pollen and its tube is allowed to grow through the style to effect fertilization. To date, S-RNase-based SI has been identified in the Solanaceae, Rosaceae and Plantaginaceae (formerly known as Scrophulariaceae) families. Molecular studies carried out since the early 1980s have attempted to address two fundamental questions: (1) How does a pistil distinguish between self-pollen and non-self pollen? (2) How does self-recognition result in growth inhibition of pollen tubes? Through the efforts of a number of research groups worldwide, it is now known that two tightly linked polymorphic S-genes, S-RNase and SLF (S-locus F-box) or SFB (S-locus F-box), located at the S-locus, control the pistil and pollen SI specificity, respectively (Lee et al., 1994; Murfett et al., 1994; Lai et al., 2002; Entani et al., 2003; Ushijima et al., 2003; Qiao et al., 2004b; Sijacic et al., 2004; Sonneveld et al., 2005; Tsukamoto et al., 2006; Hua et al., 2007). For simplicity, the name SLF will be used hereafter to designate the pollen S-gene.
In this review, we first discuss the models that have been proposed for the biochemical mechanism of S-RNase-based SI, based largely on the data obtained in the Solanaceae, and then present a new model based on the recent results from our lab. For comprehensive reviews of S-RNase-based SI, see Kao and Tsukamoto (2004), Takayama and Isogai (2005), and Sims (2007).
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TOPAbstractINTRODUCTIONEARLIER MODELSRECENT MODELSNEW MODELCONCLUSIONS AND FUTURE...FUNDING |
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Receptor Model
The findings that the RNase activity of S-RNase is essential for its function in rejecting the growth of self-pollen tubes (Huang et al., 1994) and that degradation of RNAs in pollen tubes was correlated with incompatible pollination (McClure et al., 1990) suggest that S-RNase must pass through the plasma membrane and the cell wall of the transmitting cell, where it is synthesized, into the transmitting tract of the style, and then enter the cytoplasm of self-pollen tubes to exert its cytotoxic function. Since S-RNase does not affect the growth of non-self pollen tubes, an earlier model, proposed prior to the identification of the pollen S-gene, hypothesized that uptake of S-RNase by pollen tubes was allele-specific (Thompson and Kirch, 1992; Kao and McCubbin, 1996). This model predicted that the product of the pollen S-gene is a receptor located in the plasma membrane or cell wall of the pollen tube, and each allelic variant interacts specifically with its self S-RNase, thereby selectively taking up self S-RNase into the pollen tube. If the pollen S-gene encoded a receptor, one would expect that deletion of the gene would result in the pollen being compatible with pistils of any S-genotype, as no S-RNase would be taken up by the mutant pollen tube. However, Golz et al. (1999, 2001) found that none of the pollen-part self-compatible mutants of Nicotiana alata (Solanaceae) they generated from
-ray radiation had the pollen S-gene deleted. Thus, they concluded that the product of the pollen S-gene is unlikely to be a receptor. The recent finding that the pollen S-gene product, SLF, is localized in the cytoplasm of the pollen tube (Wang and Xue, 2005) supports this conclusion. The most definitive evidence against the receptor model was obtained by Luu et al. (2000) and Goldraij et al. (2006). They used immunolocalization experiments to show that S-RNase is localized in both self and non-self pollen tubes, suggesting that the uptake of S-RNases by pollen tubes is not S-allele-specific.
Inhibitor Model
Since S-RNase is taken up by both self and non-self pollen tubes, and since only self-pollen tubes are inhibited, one likely scenario is that S-RNase is rendered inactive in non-self pollen tubes, but remains active in self pollen tubes. This is the tenet of the inhibitor model, proposed contemporaneously with the receptor model. The original version of the inhibitor model hypothesized that the product of the pollen S-gene is located in the cytoplasm of the pollen tube, and that each allelic variant specifically inhibits the RNase activity of its non-self S-RNases, thereby only allowing its self S-RNase to exert cytotoxic activity (Thompson and Kirch, 1992). Kao and McCubbin (1996) further expanded this model and proposed that (1) the pollen S-gene product possessed both the S-specificity and inhibitor functions; (2) the interaction between the S-allele-specific domain of a pollen S-allele product and the matching S-allele-specific domain of its self S-RNase would preclude the interaction between the inhibitor domain of the pollen S-allele product and the RNase activity domain of the self S-RNase; and (3) the interaction between the matching S-allele-specific domains of a pollen S-allele product and its self S-RNase was thermodynamically favored over that between the inhibitor domain of a pollen S-allele product and the RNase activity domain of its non-self S-RNases (for review, see Kao and Tsukamoto, 2004).
It would seem reasonable that self-interactions are favored over non-self interactions, as evolution of the SI mechanism may have selected for matching allelic products of the male and female S-genes to recognize and interact with each other. However, this model cannot explain a well documented phenomenon in the Solanaceae, termed competitive interaction, which refers to the following scenario. When a pollen grain carries two different pollen S-alleles, this heteroallelic pollen grain fails to function in SI, and when a pollen grain carries two copies of the same pollen S-allele, this homoallelic pollen grain retains the normal SI function. For example, if a diploid self-incompatible plant of S1S2 genotype is converted to a tetraploid S1S1S2S2 plant, three different S-genotypes, S1S1, S1S2 and S2S2, of pollen will be produced, and S1S2 pollen, but not S1S1 or S2S2 pollen, loses the SI function and cannot be rejected by the S1S1S2S2 pistil or the diploid S1S2 pistil (Figure 1). According to the inhibitor model, in the S1S2 heteroallelic pollen, the pollen S1-allele product would preferentially interact with S1-RNase and the pollen S2-allele product would preferentially interact with S2-RNase. As a result, neither S1-RNase nor S2-RNase would be inactivated, and the S1S2 pollen would be rejected by the S1S2 and S1S1S2S2 pistils—an outcome precisely the opposite of what is observed.
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A modified version of the inhibitor model was subsequently proposed (Luu et al., 2001), partly to make the prediction of the SI behavior of heteroallelic pollen consistent with competitive interaction. One major difference between the original and modified versions is that the latter hypothesized that the pollen S-gene product only possesses the S-specificity function, and a separate protein, a general RNase inhibitor, is involved in the inhibition of S-RNase. According to the modified inhibitor model, the general RNase inhibitor would interact with all S-RNases and inhibit their RNase activity unless the S-allele-specificity domain of an S-RNase is bound to the matching S-allele-specificity domain of its cognate pollen S-allele product. To address competitive interaction, this model also predicted that the active form of a pollen S-allele product is a homotetramer. In the case of heteroallelic pollen, the model predicted that the heterotetramer formed between two different pollen S-allele products would not interact with either of their respective cognate S-RNase or any other S-RNases, and, as a result, the activity of all S-RNases would be inhibited by the general RNase inhibitor. This is an interesting model; however, based on some of the recent results from the authors’ lab (discussed below), it does not seem likely that the functional form of a pollen S-allele product is a homotetramer.
Taking advantage of the competitive interaction phenomenon, Sijacic et al. (2004) introduced the S2-allele of PiSLF (Petunia inflata SLF), PiSLF2, into P. inflata plants of S1S1, S1S2 and S2S3 genotypes and showed that expression of PiSLF2 caused the breakdown of SI function in S1 and S3 pollen (both being heteroallelic pollen), but not in S2 pollen (homoallelic pollen), produced by the transgenic plants (Figure 2). This finding established that PiSLF is indeed the pollen S-gene. Subsequently, Hua et al. (2007) used the LAT52 promoter of tomato (Twell et al., 1990)—a much stronger pollen-specific promoter than the PiSLF2 promoter—to express a PiSLF2:GFP transgene in S2S3 plants to produce higher levels of PiSLF2:GFP than the endogenous PiSLF2 and PiSLF3 in S2 and S3 pollen, respectively, of the transgenic plants. Again, expression of PiSLF2:GFP caused the breakdown of the SI function in S3 pollen (heteroallelic pollen), but not in S2 pollen (homoallelic pollen), of the transgenic plants. If a pollen S-allele product forms a homotetramer with itself and heterotetramers with products of other alleles, S3 pollen that produces a high level of PiSLF2:GFP will produce both heterotetramers, formed between PiSLF3 and PiSLF2:GFP, and homotetramers formed by PiSLF2:GFP. Since the amount of PiSLF2:GFP produced in the S3 transgenic pollen is much higher than that of the endogenous PiSLF3, the amount of the homotetramer should be much higher than that of the heterotetramer. Based on the modified inhibitor model, the homotetramer of PiSLF2:GFP would protect S2-RNase from the inhibition by a general inhibitor, and thus S2-RNase would inhibit the growth of the S3 pollen that carries the PiSLF2:GFP transgene. However, this S3 transgenic pollen was fully compatible with S2S3 pistils (Hua et al., 2007).
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TOPAbstractINTRODUCTIONEARLIER MODELSRECENT MODELSNEW MODELCONCLUSIONS AND FUTURE...FUNDING |
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Protein Degradation Model
The recent identification of the pollen S-gene, SLF, whose allelic products contain an F-box domain at the N-terminus, has led to further modification of the inhibitor model. Most of the F-box proteins whose functions have been characterized so far are components of a type of multi-subunit E3 ubiquitin ligase complex, named SCF (Skp1-Cullin–F-box), for it is composed of Skp1, Cullin-1, an F-box protein and Rbx1. This complex, along with E1 ubiquitin activating enzyme and E2 ubiquitin conjugating enzyme, is involved in ubiquitin-mediated protein degradation by the 26S proteasome (for reviews, see Cardozo and Pagano, 2004; Moon et al., 2004; Smalle and Vierstra, 2004). Thus, SLF may be a component of an SCF complex, which functions in ubiquitin–26S proteasome-mediated protein degradation (Qiao et al., 2004a; Sijacic et al., 2004). Results supporting this hypothesis have been obtained. Huang et al. (2006) used yeast two-hybrid screens to identify a Skp1-like protein, named AhSSK1, that interacts with the F-box domain of AhSLF2 (S2-allele product of A. hispanicum SLF) of Antirrhinum (Plantaginaceae), suggesting that AhSLF may be a component of the putative SCFAhSLF complex. PiSLF has been shown by yeast two-hybrid and in-vitro binding assays to interact with a RING-finger protein, named PiSBP1 (P. inflata S-RNase-Binding Protein), which also interacts with a cullin-1, named PiCUL1-G, and an E2-like protein, PhUBC1 (P. hybrida UBC1). To date, no Skp1-like proteins have yet been found to interact with the F-box domain of PiSLF, suggesting that the putative PiSLF-containing complex may be a novel E3-like complex (Hua and Kao, 2006). However, as pointed out by Sims (2007), it remains possible that this complex also contains an as yet unidentified Skp-1-like protein.
PiSBP1 is a homologue of PhSBP1 (P. hybrida SBP1) identified by Sims and Ordanic (2001) as an S-RNase-interacting protein. Subsequently, a homologue in Solanum chacoense, named ScSBP1, was identified by O'Brien et al. (2004) and shown to also interact with S-RNase. In addition to interacting with PiSLF, PiSBP1, like PhSBP1 and ScSBP1, also interacts with S-RNase (Hua and Kao, 2006). Since SBP1 contains a RING–HC domain at its C-terminus and since the SBP1 gene does not show any sequence polymorphism in different S-genotypes, Sims and Ordanic (2001) proposed that it could serve as a general inhibitor to inactivate S-RNase non-specifically by targeting S-RNase for ubiquitination and degradation. An in-vitro reconstitution experiment has shown that PiSBP1, in conjunction with E1 and E2, can act as an E3 ligase to ubiquitinate S-RNase (Hua and Kao, 2008). However, no in-vivo evidence of this proposed function of PiSBP1 has been obtained as of yet.
The protein degradation model proposes that each allelic variant of SLF specifically mediates ubiquitination and degradation, rather than inhibition of the RNase activity, of its non-self S-RNases, thereby only allowing self S-RNase to function inside a pollen tube (Qiao et al., 2004a; Sijacic et al., 2004). One way this could be accomplished is as follows. An SLF interacts with its self S-RNase through the matching S-allele-specific domains, and the interaction would mask the ubiquitination site(s) of S-RNase, thereby allowing the self S-RNase to evade degradation. In the absence of the matching S-allele-specific domains, an SLF would interact with its non-self S-RNases through a domain common to all SLFs and a domain common to all S-RNases to result in ubiquitination and ultimate degradation of all non-self S-RNases. This model also predicts that the interaction between SLF and S-RNase through the matching S-allele-specific domains is stronger than that through the common domains, and thus self-interactions are thermodynamically favored over non-self interactions. For example, when S1-RNase is taken up by an S1 pollen tube, SLF1 would preferentially interact with S1-RNase through the matching S-allele-specific domains and this interaction would not result in ubiquitination/degradation of S1-RNase, allowing the inhibition of growth of the S1 pollen tube by S1-RNase. However, when S2-RNase is taken up by an S1 pollen tube, SLF1 would interact with S2-RNase through the common domains due to the absence of the matching S-allele-specific domains, and, as a result, S2-RNase would be ubiquitinated and degraded, allowing the pollen tube to grow through the style. If both S1-RNase and S2-RNase are taken up by an S1 pollen tube growing in an S1S2 style, SLF1 would preferentially interact with S1-RNase, and this interaction would not result in ubiquitination/degradation of S1-RNase, thereby allowing S1-RNase to inhibit the growth of the S1 pollen tube.
However, the protein degradation model suffers the same problem as the inhibitor model in that it cannot explain competitive interaction (Takayama and Isogai, 2005). For example, if S1-RNase and S2-RNase are taken up by an S1S2 heteroallelic pollen tube, SLF1 would preferentially interact with S1-RNase and SLF2 would preferentially interact with S2-RNase. As a result, neither S1-RNase nor S2-RNase would be ubiquitinated/degraded and thus they would inhibit the growth of this heteroallelic pollen tube. This predicted outcome is precisely the opposite of what is observed.
Compartmentalization Model
Goldraij et al. (2006) recently proposed another model to explain why S-RNase has a specific cytotoxic effect on self pollen tubes, but not on non-self pollen tubes. This model is based on the following findings. Using immunolocalization, Goldraij et al. (2006) observed that most, if not all, S-RNase molecules were initially sequestered in a vacuolar compartment of the pollen tube after both compatible (cross) and incompatible (self) pollinations. At a later time, when morphological changes of incompatible pollen tubes were observed, the S-RNase molecules were found in the cytoplasm of the incompatible pollen tubes, presumably as a result of the disruption of the compartment, whereas the S-RNase molecules remained sequestered in the vacuolar compartment of the compatible pollen tubes. Goldraij et al. (2006) further observed that a non-S-specific protein, named HT-B, appeared to be preferentially degraded in the compatible pollen tubes. This finding, coupled with the findings that expression of HT-B in the pistil is required for SI (McClure et al., 1999) and that the S-RNase-containing compartment is maintained in the pollen tubes of transgenic plants expressing an antisense HT-B gene (Goldraij et al., 2006), suggests that HT-B is required for the disruption of the S-RNase-containing compartments in the incompatible pollen tubes.
The compartmentalization model predicts that the sequestration of all S-RNase molecules (both self and non-self) in a compatible (non-self) pollen tube, rather than the degradation of non-self S-RNases, allows the pollen tube to grow through the style for pollination, and that the disruption of the S-RNase-containing compartment in an incompatible (self) pollen tube releases both self and non-self S-RNases into the cytoplasm to result in growth inhibition of the self pollen tube (Goldraij et al., 2006; McClure, 2006). Goldraij et al. (2006) further invokes a hypothetical pollen protein (PP, possibly a protease) responsible for the degradation of HT-B.
The compartmentalization model as initially proposed by Goldraij et al. (2006) cannot explain competitive interaction either. The model predicts that there is a small amount of S-RNase in the cytoplasm and the interaction between SLF and its self S-RNase in the cytoplasm would prevent the degradation of HT-B by the hypothetical protease, allowing HT-B to cause the disruption of the compartment and release of the initially sequestered S-RNase molecules into the cytoplasm to inhibit the growth of self pollen tubes. If heteroallelic pollen carrying S1 and S2 alleles is used to pollinate an S1S2 pistil, SLF1 would interact with S1-RNase and SLF2 would interact with S2-RNase, and these interactions in the cytoplasm would stabilize HT-B, leading to the disruption of the compartment to release both S1-RNase and S2-RNase into the cytoplasm. Thus, according to this model, the S1S2 heteroallelic pollen tube would be inhibited by the S1S2 pistil (Figure 3A); however, this is precisely the opposite of what is observed, because, as discussed above, competitive interaction between two different pollen S-alleles in the same pollen grain renders heteroallelic pollen compatible with pistils of any S-genotype.
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McClure (2006) subsequently modified the model of Goldraij et al. (2006) to make its prediction of the SI behavior of heteroallelic pollen consistent with the competitive interaction phenomenon. The revised model proposes that an SLF interacts with its non-self S-RNases, rather than with its self S-RNase as originally proposed, to result in the degradation of HT-B. In this case, if heteroallelic pollen carrying S1-haplotype and S2-haplotype is used to pollinate an S1S2 pistil, the interaction between SLF1 and S2-RNase in the cytoplasm would result in degradation of HT-B, as would the interaction between SLF2 and S1-RNase. Thus, both S1-RNase and S2-RNase would remain sequestered in the compartment (Figure 3B), as predicted by the original model (Goldraij et al., 2006), and would not inhibit the growth of the S1S2 pollen tube, the precise outcome based on the competitive interaction phenomenon. However, the revised model cannot explain the ‘normal’ case of SI. For example, when S1 pollen is used to pollinate an S1S2 pistil, both S1-RNase and S2-RNase are taken into the S1 pollen tube non-specifically and would be sequestered in the same compartment (Goldraij et al., 2006). The revised model predicts that SLF1 would interact with its non-self S-RNase, S2-RNase, in the cytoplasm to result in the degradation of HT-B and consequent stabilization of the compartment containing both S1-RNase and S2-RNase. Thus, according to the revised model (McClure, 2006), S1 pollen would be accepted by an S1S2 pistil, because neither S1-RNase nor S2-RNase would be released to inhibit the growth of the S1 pollen tube (Figure 3C). This is not the case.
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NEW MODEL |
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TOPAbstractINTRODUCTIONEARLIER MODELSRECENT MODELSNEW MODELCONCLUSIONS AND FUTURE...FUNDING |
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Modified Protein-Degradation Model
It would seem that the most straightforward approach to ascertain the validity of the protein-degradation model is to examine whether the amount of S-RNase in pollen tubes after compatible pollination is lower than that after incompatible pollination. However, there are several potential problems and complications for the use of this approach. First, if the bulk of S-RNase molecules, including both self and non-self S-RNases, are sequestered in a vacuolar compartment inside a pollen tube as observed by Goldraij et al. (2006), any change in the small amount of cytoplasmically localized S-RNase molecules at different time points post pollination would be difficult to detect. Second, even if some S-RNase molecules are degraded within a pollen tube during compatible pollination, the steady-state level of S-RNase molecules inside the pollen tube may remain largely unchanged, as the pistil continues to synthesize new S-RNase molecules and delivers them to the pollen tube. Third, S-RNase levels have been found to vary over a wide range in pistils from different flowers of even the same plant; for example, up to 20-fold differences were observed in Solanum chacoense (Qin et al., 2006). Indeed, the S-RNase levels determined either from pollinated styles (Figure 10C of Qiao et al., 2004a) or from pollen tubes dissected from pollinated styles (Figure S7 of Goldraij et al., 2006) showed very high degrees of variations among different repeats, making the interpretation of the results difficult. Thus, the failure to observe significant differences in S-RNase levels within pollen tubes between compatible and incompatible pollinations (Goldraij et al., 2006) cannot be used as evidence against the role of protein degradation in the S-RNase-based SI mechanism.
We have developed a cell-free system, based on similar systems commonly used to study the fate of proteins targeted by the ubiquitin–26S proteasome pathway in yeast and mammalian cells (Verma et al., 1997; Jiang et al., 2005), and used it to demonstrate that S-RNases, including E. coli-expressed recombinant S-RNases and native, deglycosylated S-RNases, are degraded through the ubiquitin–26S proteasome pathway in pollen tube extracts, albeit not in an S-allele-specific manner (Hua and Kao, 2006, 2008). We have further shown that the E. coli-expressed GST:S-RNases used for the degradation and ubiquitination assays have RNase activity and are thus functional. In addition, via a site-directed mutagenesis assay, we have found that the C-terminal lysine residues play a major role in targeting the ubiquitination of S-RNase in pollen tube extracts. These results suggest that S-RNases, used for the degradation and ubiquitination assays in pollen tube extracts, are folded normally and that ubiquitin–26S proteasome-mediated S-RNase degradation is likely an integral part of the SI mechanism (Hua and Kao, 2008).
The results from our in-vitro binding assays between PiSLFs and S-RNases showed, unexpectedly, that a PiSLF interacts with its non-self S-RNases more strongly than with its self S-RNase, and that an S-RNase interacts with its non-self PiSLFs more strongly than with its self PiSLF (Hua and Kao, 2006). This finding provides intriguing biochemical support for the prediction made by the protein-degradation model that the PiSLF-containing complex specifically targets non-self S-RNase for ubiquitination and subsequent degradation by the 26S proteasome, and leaves self S-RNase active to perform its cytotoxic function in vivo (Hua and Kao, 2006, 2008).
The finding that a PiSLF preferentially interacts with its non-self S-RNases over its self S-RNase has led us to further examine the biochemical basis for the differential interactions. Since multiple SLF-like genes, sharing some similar properties with SLF, have been found to be tightly linked to SLF and S-RNase in all the S-loci that have been examined so far (McCubbin et al., 2000; Entani et al., 2003; Ushijima et al., 2003; Wang et al., 2003; Zhou et al., 2003; Sassa et al., 2007; Hua et al., 2007), we first studied whether there were any SLF-like genes in P. inflata that function in SI and/or interact with S-RNases. We found that none of the three PiSLF-like genes examined had SI function in vivo, and that all five PiSLF-like proteins (products of five PiSLF-like genes) examined either failed to interact with S-RNase, or interacted with S-RNase much more weakly than did PiSLF2 in vitro (Hua et al., 2007). These results strongly suggest that PiSLF is unique in its function in determining the pollen SI behavior. To determine the sequence features that confer on PiSLF its unique function in SI, we compared the amino acid sequences of PiSLF1, PiSLF2 and PiSLF3 with those of 10 PiSLF-like proteins (deduced from one to three alleles of six PiSLF-like genes), and identified three PiSLF-specific regions (Hua et al., 2007). We then divided PiSLF into three functional domains—FD1, FD2 and FD3—each containing one of the PiSLF-specific regions, and examined the S-RNase-binding property of each domain by an in-vitro binding assay. It should be noted that FD1 and FD3 each contains one of the two variable regions of PiSLF (Hua et al., 2007).
These experiments included testing truncated versions of PiSLF2, containing one or two of the three domains, for their interactions with S3-RNase, a non-self S-RNase for PiSLF2 (Hua et al., 2007). FD2 of PiSLF2 alone interacts with S3-RNase more strongly than does the full-sized protein, and inclusion of FD1 or FD3 reduces the interaction of FD2 with S3-RNase, but these interactions are still stronger than the interaction between the full-sized protein and S3-RNase. These results suggest that FD2 is the primary region for the interaction between PiSLF and S-RNase, and that FD1 and FD3 each negatively regulates the strong interaction between FD2 and S-RNase.
Additional in-vitro binding experiments were carried out using chimeric proteins with one or two of the three domains swapped between PiSLF2 and PiSLFLb-S2 (a PiSLF-like protein that does not interact with S-RNase), and between PiSLF1 and PiSLF2 (Hua et al., 2007). The results are summarized below, and those pertaining to the chimeric proteins between PiSLF1 and PiSLF2 are graphically shown in Figure 4. First, when FD2 of PiSLF2 is replaced with the corresponding domain of PiSLFLb-S2, the resulting chimeric protein interacts with S3-RNase much more weakly than does PiSLF2. Conversely, when FD2 of PiSLFLb-S2 is replaced with FD2 of PiSLF2, the chimeric protein acquires the ability to interact with S3-RNase. These results support the notion that FD2 of PiSLF is the primary region for interactions with S-RNase. Second, when only FD1 of PiSLF2 is replaced with that of PiSLF1, the chimeric protein interacts with S2-RNase as strongly as does PiSLF1 and much more strongly than does PiSLF2, suggesting that FD3 alone is not sufficient to account for the weak interaction between a PiSLF and its self S-RNase. Third, when only FD1 of PiSLF1 is replaced with that of PiSLF2, the chimeric protein interacts with S2-RNase as strongly as does PiSLF1 and much more strongly than does PiSLF2. However, when both FD1 and FD3 of PiSLF1 are replaced with the corresponding regions of PiSLF2, the chimeric protein behaves like PiSLF2, the protein that contributes FD1 and FD3, because the chimeric protein interacts with S2-RNase as weakly as does PiSLF2. These results suggest that FD1 alone cannot account for the binding difference between a PiSLF and its self and non-self S-RNases, and that FD1 and FD3 together determine the binding difference. Finally, consistent with this suggested role of FD1 and FD3, when FD1 and FD3 of PiSLF2 are replaced with the corresponding regions of PiSLF1, the chimeric protein behaves like PiSLF1, the protein that contributes FD1 and FD3, because the chimeric protein interacts with S2-RNase to a similar extent as the non-self interaction between PiSLF1 and S2-RNase.
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Based on the in-vitro binding results discussed above, we propose that (1) FD2 of PiSLF functions as the S-RNase-binding domain (SBD), which, by itself, interacts strongly with a domain common to all S-RNases, and (2) FD1 and FD3 together determine S-allele-specificity of PiSLF, and they function as the S-RNase-binding-regulating domains (SBRD) to significantly weaken the strong interaction between FD2 of PiSLF and S-RNase during self interactions. It might seem counter-intuitive that the otherwise strong interaction between PiSLF and S-RNase would be weakened when the S-allele-specificity domains of the male and female determinants are products of the same S-allele. However, one could envisage a number of possible mechanisms for this negative regulation. For example, there might be steric hindrance or unfavorable interactions between the SBRD of PiSLF and the S-allele-specificity domain of its self S-RNase. In other words, the S-allele-specificity domains of PiSLF and S-RNase might have evolved in such a way as to favor interactions between PiSLF and all S-RNases but self S-RNase.
We put forward a modified protein-degradation model for the biochemical mechanism of S-RNase-based SI (Figure 5), with all the interactions depicted occurring in the cytoplasm of the pollen tube.
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In the case of incompatible pollination, as illustrated in Figure 5A, the interaction between the SBD of PiSLF1 and its self S-RNase, S1-RNase, in an S1 pollen tube would be weakened due to the unfavorable interaction between the SBRD of PiSLF1 and the S-allele-specific domain of S1-RNase. Thus, the PiSLF1-S1-RNase complex would not be stable. Consequently, most of the S1-RNase molecules would exist in the free form and degrade pollen RNA to result in the growth inhibition of the S1 pollen tube.
In the case of compatible pollination, as shown in Figure 5B, PiSLF1 would interact strongly with S2-RNase, a non-self S-RNase, in an S1 pollen tube, because the SBRD of PiSLF1 and the S-allele-specificity domain of S2-RNase would not affect the strong interaction through the SBD of PiSLF1 and a domain common to all S-RNases. This strong interaction would result in the formation of a stable PiSLF1-S2-RNase complex, and allow the assembly of the PiSLF-containing E3-like complex, which would ubiquitinate S2-RNase to target it for degradation. Thus, the growth of the S1 pollen tube is not inhibited.
In the case of competitive interaction, such as pollination of an S1S2 pistil by S1S2 heteroallelic pollen as depicted in Figure 5C, two different PiSLFs, PiSLF1 and PiSLF2, and their respective self-S-RNases, S1-RNase and S2-RNase, are present in the cytoplasm of the same pollen tube.
PiSLF1 and PiSLF2 would preferentially interact with their respective non-self S-RNases through their SBDs to form stable PiSLF1-S2-RNase and PiSLF2-S1-RNase complexes, as in the case of non-self pollination (Figure 5B). PiSLF1 or PiSLF2 might also interact with their respective self S-RNases; however, as discussed in the case of incompatible pollination (Figure 5A), these interactions would not result in stable complexes. The dissociated S1-RNase and S2-RNase would again preferentially interact with their respective non-self PiSLFs due to stronger interactions between the SBD of a PiSLF and its non-self S-RNases. Ultimately, all the S1-RNase and S2-RNase would be in stable complexes with PiSLF2 and PiSLF1, respectively. Thus, as in the case of compatible pollination, all S1-RNase and S2-RNase would be ubiquitinated and degraded.
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CONCLUSIONS AND FUTURE PERSPECTIVES |
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TOPAbstractINTRODUCTIONEARLIER MODELSRECENT MODELSNEW MODELCONCLUSIONS AND FUTURE...FUNDING |
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The phenomenon of competitive interaction has been well documented in self-incompatible species in the Solanaceae (de Nettancourt, 2001), and it has been used as a robust in-vivo functional assay for the pollen S-gene (Sijacic et al., 2004; Qiao et al., 2004b; Hua et al., 2007). An allele of the pollen S-gene, SLF, when introduced into plants of appropriate S-genotypes, causes the breakdown of pollen SI function in heteroallelic transgenic pollen that carries an S-allele different from that of the transgene, but not in homoallelic transgenic pollen that carries the same S-allele as that of the transgene. In contrast, SLF-like genes, though sharing several similar properties with SLF, do not cause competitive interaction in heteroallelic transgenic pollen (Hua et al., 2007). Thus, any model for the biochemical mechanism of S-RNase-based SI possessed by the solanaceous species must be able to account for the breakdown of pollen SI function by competitive interaction. Among the models reviewed in this article, the modified protein-degradation model meets this stringent criterion. However, since this model is largely based on results of in-vitro assays (Hua and Kao, 2006, 2008; Hua et al., 2007), its validity must still be examined by in-vivo approaches. Some of the questions that remain to be addressed are discussed below.
First, what is the nature of the PiSLF-containing complex? Is it a novel E3 ligase complex that contains a cullin-1, PiSLF, and RING-finger protein, PiSBP1, with the dual role of Skp1 and Rbx1 as we proposed (Hua and Kao, 2006), or is it an E3 ligase that, in addition to containing the components mentioned above, also contains a non-canonical SKP1-like protein similar to AhSSK1, as suggested by Sims (2007)?
Second, does the PiSLF-containing complex mediate specific ubiquitination and degradation of non-self S-RNases? The results from the cell-free system we have developed for assaying these activities do not show any S-haplotype specificity, namely both self and non-self S-RNases are ubiquitinated and degraded in pollen tube extracts. Once all the components of the PiSLF-containing complex are identified, one could reconstitute the complex and test whether the reconstituted complex shows the S-haplotype specificity predicted by the modified protein-degradation model.
Third, what effect does down-regulation of the expression of PiSLF have on the SI behavior of pollen? If PiSLF is indeed required for the specific ubiquitination and degradation of non-self S-RNases, down-regulation of its expression in pollen would result in the inability of the pollen to cause the degradation of any S-RNase, and thus the pollen would be rejected by pistils of any S-genotype.
Fourth, how does S-RNase enter the cytoplasm of a pollen tube? McClure (2006) adopted the retrograde transport pathway of Ricin A to explain the entrance of S-RNase into the pollen tube cytoplasm. During its expression, Ricin A re-translocates from the Endoplasmic Reticulum (ER) to the cytosol via the ER Associated Protein Degradation (ERAD) pathway (Di Cola et al., 2001). However, this mechanism may not be responsible for the uptake of S-RNase into the cytoplasm of a pollen tube. First, S-RNase is expressed in the transmitting cell of a pistil, secreted into the intercellular space in the transmitting tract, and enters the cytoplasm of a pollen tube, whereas cytoplasmic localization of Ricin A results from its retrograde movement from the ER during its maturation in the same cell as it is expressed (Di Cola et al., 2001). Second, most Ricin A-like cytotoxins contain very few lysine residues; this presumably allows them to evade degradation through the ubiquitin–26S proteasome-mediated ERAD pathway so that they can exert their cytotoxic function in the cytoplasm. For example, only two of the 267 amino acid residues of Ricin A are lysines (Hazes and Read, 1997; Di Cola et al., 2005). In contrast, S-RNases contain much higher percentages of lysine residues. For example, the lysine residues in S3-RNase of P. inflata comprise 10% of the total amino acid residues (20 lysine residues out of the 200 total amino acid residues). Lastly, ubiquitin–26S-mediated S-RNase degradation does not seem to be related to ERAD (Hua and Kao, 2008), further suggesting that a different mechanism from retrograde transportation is responsible for the uptake of S-RNase into the cytoplasm of pollen tubes.
Fifth, are the in-vivo functions of FD1, FD2 and FD3 of PiSLF indeed as proposed based on the biochemical properties of the chimeric proteins examined in vitro? For example, if the chimeric protein containing FD2 of PiSLF2, and FD1 and FD3 of PiSLF1, behaves as PiSLF1 (Figure 4; Hua et al., 2007), one would expect that this chimeric protein would cause the breakdown of SI function in S2 pollen, but not in S1 pollen, of transgenic plants. If the roles of these domains are confirmed in vivo, it would be of interest to investigate the biochemical basis for the negative effect of FD1 and FD3 on the interactions between FD2 of a PiSLF and its self S-RNase.
Although the modified version of the compartmentalization model as proposed by McClure (2006) cannot explain the normal SI scenario, the observed sequestering of both self and non-self S-RNases in a vacuolar compartment of pollen tubes is intriguing, and it would be of interest to investigate how the disruption of this S-RNase-containing compartment is controlled. Moreover, since HT-B is required for the disruption of the compartment, it would be interesting to study what role HT-B plays in the disruption of the compartment in incompatible pollen tubes, and why HT-B is specifically degraded in compatible pollen tubes. Since SLF and S-RNase are the only two known S-allele-specificity determinants in S-RNase-based SI, it is reasonable to presume that they are involved in this process. Down-regulation of SLF in pollen tubes, as discussed above, may be a way to address this possibility. It is also tantalizing to speculate that the disruption of the S-RNase-containing compartment in incompatible pollen tubes may serve to reinforce the outcome of the SI response, as this would significantly increase the levels of both self and non-self S-RNases in the cytosol.
Finally, it is worth noting that even though the Rosaceae also employs S-RNase and SLF as the female and male determinants, respectively, the role of SLF in SI may be different from that in the Solanaceae and Plantaginaceae based on the following observations. First, deletion of SLF in sweet cherry (Prunus avium) results in breakdown of SI (Sonneveld et al., 2005), whereas deletion of SLF in the Solanaceae is thought to result in rejection of pollen by pistils of any S-genotype (Golz et al., 2001). Second, unlike in the Solanaceae, SI in the Rosaceae does not break down in heteroallelic pollen (Hauck et al., 2006). It would be of interest to investigate how SLF of the Rosaceae controls the fate of S-RNase in a pollen tube, if it indeed functions differently from SLF of the Solanaceae.
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FUNDING |
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TOPAbstractINTRODUCTIONEARLIER MODELSRECENT MODELSNEW MODELCONCLUSIONS AND FUTURE...FUNDING |
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The work carried out in the authors’ lab was support by grants from the US National Science Foundation (most recently IOB 0543201 to T.-.h.K.).
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Acknowledgements |
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We thank Baoyu Chen, Peter Dowd, Hong Ma, Xiaoying Meng, Tracy Nixon, Paja Sijacic, Song Tan and Ning Wang for comments on the manuscript.
No conflict of interest declared.
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