622 Biochemical Society Transactions (2010) Volume 38, part 2

Protein interactions and subcellular localization inS-RNase-based self-incompatibility

Thomas L. Sims1, Avani Patel and Pratima ShresthaPlant Molecular Biology Center and Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, U.S.A.

AbstractThe recent identification of several proteins playing key roles in S-RNase-based gametophytic self-incompatibility has led both to a greater understanding of the molecular biology of this response, aswell as to questions regarding the precise mechanism by which compatible pollen tubes are recognizedand accepted. A proposed variant SCFSLF (where SCF is SSK1/cullin/F-box and SLF is S-locus F-box) ubiquitinligase complex is thought to play a central role in recognizing and inhibiting non-self S-RNases, but theexact role of ubiquitination remains unclear. How the possible sequestration of non-self S-RNases in a pollenvacuolar compartment can be reconciled with the need for protein interaction between S-RNase and theSCFSLF complex needs to be determined. Current work to answer these questions focuses on more preciselydefining quantitative protein interactions and subcellular localization of proteins involved in S-RNase-basedgametophytic self-incompatibility.

S-RNase-based GSI (gametophytic self-incompatibility)Self-incompatibility is the ability of an otherwise fertilehermaphroditic plant to recognize and reject its own (‘self’)pollen, thus preventing fertilization, while accepting crossor ‘non-self’ pollen from a different individual in the samespecies. This behaviour was first recognized and described byDarwin, in his book The Effects of Cross and Self Fertilisationin the Vegetable Kingdom [1]. Later, Mather [2], Linskens[3] and de Nettancourt [4], among others, recognized thatself-incompatibility recognition, and pollen rejection, wascontrolled by a single genetic locus with multiple alleles,termed the S-locus. In subsequent years, different self-incompatibility systems have been studied and characterized[4–13]. These include S-RNase-based GSI (the subject ofthe present paper), GSI in Papaver, SSI (sporophytic self-incompatibility), self-incompatibility in heterostylous plantsand self-incompatibility in grasses. Of these systems, theones that have been best characterized at the molecularlevel are S-RNase-based GSI in Solanaceae, Plantaginaceaeand Rosaceae [5–9], the GSI system in Papaver rhoeas [10]and SSI in Brassicaceae [11–13]. Although all three of thesesystems have the same end effect (i.e. the rejection of self-pollen), the genetics, timing and specific genes and proteinsinvolved differ among the three systems. In the two GSIsystems, recognition specificity is based on the haploid

Key words: gametophyte, protein interaction, self-incompatibility, S-locus F-box protein,

S-RNase, yeast two-hybrid assay.

Abbreviations used: GSI, gametophytic self-incompatibility; RBX1, RING box 1; SBP1,

S-RNase-binding protein 1; SCF, Skp1/cullin/F-box; SLF/SFB, S-locus F-box; SSI, sporophytic self-

incompatibility; SSK1, SLF-interacting Skp1-like1; AhSSK1, Antirrhinum hispanicum SSK1; PhSSK1,

Petunia hybrida SSK1.1To whom correspondence should be addressed (email tsims@niu.edu).

genotype of individual pollen grains. In such systems, so-called ‘half-compatible’ pollinations are possible with self-pollen being rejected, and non-self pollen accepted withinthe same pistil [9]. In SSI, pollen recognition specificity isdetermined by the diploid genotype of the pollen parent,because recognition proteins synthesized by the diploidparent are deposited in pollen cell walls during pollenmaturation. Advances in our understanding of the molecularbasis of GSI in Papaver rhoeas and of SSI are discussed byother authors in this issue of Biochemical Society Transactions.The remainder of the present paper therefore concentrateson our current understanding of the molecular biology ofGSI in the S-RNase-based system, with the emphasis on ourunderstanding of this response in Petunia hybrida.

Genes and proteins involved in S-RNase-based GSIOver the last several years, a number of different genes havebeen identified and shown to play critical or presumed rolesin GSI. Two of these genes, S-RNase and SLF/SFB (S-locusF-box), have been identified as pistil-S and pollen-S (i.e. thegenes controlling recognition specificity in pistil and pollenrespectively). Three other genes, HT-B, 120K and SSK1 (SLF-interacting Skp1-like1), have been shown to be essential forpollen rejection or acceptance. A sixth gene, SBP1 (S-RNase-binding protein 1), is known to interact with S-RNase andSLF and (along with SLF and SSK1) may be a componentof a proposed SCFSLF (where SCF is SSK1/cullin/F-box)ubiquitin ligase complex.

S-RNase is the pistil component of GSIThe ability to selectively inhibit the growth of self pollen isdetermined in the style by an S-locus-encoded ribonuclease

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known as the S-RNase. All S-RNase proteins characterized todate contain five conserved and two hypervariable domains.Conserved domains C3 and C4 contain the active site of theribonuclease, and ribonuclease activity is required for the abi-lity to reject pollen [14]. The two hypervariable domainshave been demonstrated to control allele specificity [15], andthe protein crystal structure has been determined for theSF11 S-RNase of Nicotiana alata [16]. Immunolocalizationexperiments have demonstrated that both incompatible andcompatible S-RNases are imported into pollen tubes [17,18].The extensive knowledge of S-RNase structure, expressionand function has enabled researchers to design differentgenetic and biochemical experiments (e.g. protein interactionassays) to dissect further the molecular basis of GSI.

SLF/SFB is the pollen-recognition componentof GSIThe pollen-recognition component of GSI, SLF (SFB inRosaceae) was initially cloned via chromosome walking andsequencing to identify pollen-expressed genes linked to theS-locus [19,20]. Confirmation that SLF was pollen-S camefrom experiments that phenocopied the well-documentedphenomenon of competitive interaction, where expressionof two different pollen-S alleles in pollen results in thebreakdown of GSI [21,22]. SLF has an N-terminal F-boxdomain characteristic of the recognition components of SCFE3 ubiquitin ligase complexes [23].

SBP1 interacts with S-RNase and SLF and is an E3ubiquitin ligaseTo attempt to identify pollen-expressed proteins that interactwith the S-RNase, Sims and Ordanic [24] screened a yeasttwo-hybrid library from mature pollen of P. hybrida witha bait construct for the N-terminal half of the P. hybridaS1-RNase. This screen identified a gene, named PhSBP1(P. hybrida SBP1) that bound to N-terminal, but not C-terminal, regions of the S-RNase. Sequence characterizationof SBP1 (as PhSBP1 will be referred to henceforth) indicatedthat it contained a C-terminal RING-HC (or C3HC4) proteindomain. Such domains have been shown to be characteristicof E3 ubiquitin ligases, the components of the ubiquitin–proteasome system that interact with specific substratestargeted for ubiquitination and protein turnover [25].Subsequently, SBP1 was cloned from Solanum tuberosum,Petunia inflata and Nicotiana alata [26–28].

HT-B and 120K proteins are required for pollenrejectionTwo style-expressed proteins, HT-B and 120K, are also im-ported into growing pollen tubes, and transgenic experimentshave shown that both are required for the ability to rejectincompatible pollen [29,30] Recently, Puerta et al. [31] clonedHT-B from P. hybrida. Down-regulation of this gene byRNAi (RNA interference) caused a partial breakdown ofincompatibility, with only highly suppressed plants showingthis partial breakdown.

SSK1 interacts with SLF and cullin and may be anessential component of a SCFSLF complexHuang et al. [32] identified a Skp1-like protein, AhSSK1(Antirrhinum hispanicum SSK1), in yeast two-hybrid screenswith AhSLF1 (A. hispanicum SLF1) as bait. AhSSK1 inter-acted with another scaffold component of SCF complexes,CUL1. Zhao et al. [33] have cloned the orthologue of AhSSK1from P. hybrida (PhSSK1), and confirmed that it interacts withPhSLF-S1L and PhSLF-S3L. Furthermore, down-regulationof PhSSK1 in transgenic P. hybrida, although not affectingthe ability to reject incompatible pollen, severely reduced thefertility of compatible pollen.

Models for pollen recognition and rejectionin GSIAn understanding of the biochemical properties of the pro-teins described above, along with the genetic characteristicsof GSI have allowed the development of models for pollenrejection (incompatible pollination) and pollen acceptance(compatible pollination) for S-RNase-based GSI. A generallyaccepted model for pollen rejection proposes a cytotoxicmechanism resulting in incompatibility. According to thismodel, in an incompatible pollination, S-RNase importedinto pollen tubes acts to degrade pollen-tube RNA, leading toinhibition of protein synthesis and decreased growth. What isalso generally accepted is that compatible pollination resultsfrom the inhibition of S-RNase activity inside compatiblepollen tubes [5–9]. At present, however, two different models(which are not necessarily mutually exclusive) have beenproposed to explain the mechanism of S-RNase inhibitionin self-compatible pollinations. One model proposes theinvolvement of ubiquitination of S-RNase by an SCFSLF

E3 ubiquitin ligase complex, the other model proposes thatcompatible S-RNases remain sequestered in a pollen vacuoleduring compatible pollinations.

The ubiquitination modelMuch of the evidence for this model comes from proteininteraction assays, along with the known characteristics ofthe interacting proteins. Pollen-S (SLF) is an F-box protein,and F-box proteins are the recognition components of SCFubiquitin ligase complexes [22]. SBP1, which interacts withS-RNase, SLF, PhUBC (P. hybrida ubiquitin-conjugatingenzyme, an E2 conjugation protein), and Cullin1 [24,27,34]is a RING-HC protein, which are also E3 ubiquitin ligases[25], and SBP1 has E3 ubiquitin ligase activity in vitro [27].AhSSK1 [32] and PhSSK1 [33] are SKP1-like proteins thatinteract with SLF and Cullin. Pollen extracts have beenshown to ubiquitinate S-RNase proteins, albeit in an allele-independent manner [27]. Together, these results have led tothe proposal that a SCFSLF-like complex acts to recognizeand ubiquitinate S-RNases, leading to the inhibition ofS-RNase activity in compatible pollen tubes. This complexis proposed to differ from a canonical SCF complex, becauseneither SKP1 orthologues [7,27,33] nor RBX1 (RING box 1)

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624 Biochemical Society Transactions (2010) Volume 38, part 2

Figure 1 Proposed SCFSLF ubiquitin ligase complex

Ub, ubiquitin.

[27] interact with SLF or cullin. Instead either (or both)SBP1 and SSK1 have been proposed to replace RBX1 and/orSKP1 in this complex [5–7,33]. According to the simplestversion of this model, recognition of non-self S-RNases bythe SCFSLF complex would lead to polyubiquitination anddegradation of S-RNase by the 26S proteasome complex[5–7]. To date, however, there is little evidence for degradationof S-RNase in compatible pollinations [18]. An alternativepossibility is that mono-ubiquitination of S-RNase couldresult in directing this protein to an endosomal compartment[33,35], although, again, there is little evidence for this. Oneprediction of the SCFSLF ubiquitin ligase complex model isthat down-regulation of SLF, SBP1 or SSK1 should render allpollen tubes incompatible, regardless of genotype. To date,results of experiments to down-regulate SLF or SBP1 havenot been published, but experiments where SSK1 was down-regulated convert compatible pollinations into incompatiblepollinations. Conversely, mutants in the Rosaceae that lackeither the 3′ end of SFB or appear to have deleted this genealtogether, are self-compatible [36]. This result has led tothe suggestion that details of pollen rejection may differ inSolanaceae and Rosaceae [9]. Figure 1 summarizes the knownprotein interactions and components of the proposed SCFSLF

E3 ubiquitin ligase complex.

The sequestration modelGoldraij et al. [18], working with Nicotiana, hybridized anti-S-RNase antibodies along with anti-callose, anti-aleurain(a marker for vacuolar lumen) and anti-vPPase (a markerfor vacuolar membrane) to fixed paraffin-embedded sections,then visualized fluorescence using confocal microscopy.These authors concluded that S-RNase was initiallysequestered in a vacuolar compartment in pollen in bothcompatible and early-stage (16 h) incompatible pollen tubes,but that this compartment broke down at later stages (36 h) of

incompatible pollinations, releasing S-RNase into the pollen-tube cytoplasm. The 120K protein appeared to localize to theperiphery of this presumed vacuolar compartment, whereasHT-B, which could be detected in incompatible pollen tubes,was not detected in compatible pollen tubes. This modelsuggests possible roles for the requirement of HT-B and 120Kexpression for rejection of incompatible pollen tubes, but isdifficult to reconcile with the need for SLF and S-RNase tointeract (since, presumably the two proteins would be onopposite sides of the vacuolar membrane).

Current experiments and future prospectsThe above models suggest certain predictions for both proteininteraction strength and subcellular localization that we arecurrently attempting to test. For example, the ubiquitinationmodel predicts that protein interactions between SCFSLF

complex proteins should be stronger with non-self S-RNasecompared with self-S-RNase. This model also predictsthat SLF, S-RNase and other SCFSLF complex componentsshould co-localize in the same subcellular compartment. Forthe sequestration model, it will be important to confirm,using approaches giving the highest degree of resolutionand tissue preservation, the precise subcellular location ofdifferent proteins at different times following compatible andincompatible pollinations.

Quantitative protein interaction assaysAs a first step to determine the relative strength of interactionof non-self S-RNase–SCFSLF interactions compared withself-interactions, we have used a quantitative yeast two-hybrid assay to measure the relative degree of lacZ reportergene expression between different subdomains of S1- and S3-RNase alleles with SLF-S1 from P. hybrida. These assaysused a fluorescent substrate for β-galactosidase activity

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Cell–Cell Communication in Plant Reproduction 625

Figure 2 Quantitative yeast two-hybrid assays

Self-incompatible (SI) or self-compatible (SC) combinations of SLF-S1 and different bait constructs of S1- (SI) or S1- (SC)

RNases were used in quantitative yeast two-hybrid assays. NT, N-terminal half of S-RNase; CHVC, C2-HVa-HVb-C3; HV,

HVa-HVb domains of the S-RNase.

that is substantially more sensitive than previous substrates[37]. When subdomains of the S1-RNase were used intwo-hybrid assays with the SLF-S1 pollen-S allele fromP. hybrida, the level of β-galactosidase activity detected wassignificantly less than the level seen when the equivalentdomains of the S3-RNase were used in interaction assayswith SLF-S1 (Figure 2). These data are consistent withthe predictions of the ubiquitination model (and possiblywith the sequestration model as well). These experimentsare currently being refined and extended using a variety ofbait and prey constructs, by expressing potential competingprotein constructs from the pTFT1 vector [38], and by usingBiFC (bimolecular fluorescence complementation) [39] toassay protein interactions in planta.

Future prospectsThe isolation of the pollen-S protein, SLF/SFB, along withputative SCFSLF complex proteins SBP1 and SSK1, and thefurther defining of the roles of HT-B and 120K proteinshas given us a better understanding of self-incompatibilityinteractions. These advances have also led to alternative,possibly competing, models for the detailed mechanism ofhow S-RNases are recognized and either inhibited or releasedin an apparent cytotoxic response. In the future, it will be

critical to determine more precisely the exact subcellularlocation of the different proteins involved in S-RNase-basedGSI, at different times following compatible or incompatiblepollinations. The use of high-resolution immunoelectronmicroscopy as well as in planta protein interaction assaysshould help to resolve these questions.

Funding

This work was supported by the Plant Molecular Biology Center of

Northern Illinois University.

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Received 14 September 2009doi:10.1042/BST0380622

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