Transposable elements cause the loss of self-incompatibility in citrus.

Self-incompatibility (SI) is a widespread prezygotic mechanism for flowering plants to avoid inbreeding depression and promote genetic diversity. Citrus has an S-RNase-based SI system, which was frequently lost during evolution. We previously identified a single nucleotide mutation in Sm-RNase, which is responsible for the loss of SI in mandarin and its hybrids. However, little is known about other mechanisms responsible for conversion of SI to self-compatibility (SC) and we identify a completely different mechanism widely utilized by citrus. Here, we found a 786-bp miniature inverted-repeat transposable element (MITE) insertion in the promoter region of the FhiS2-RNase in Fortunella hindsii Swingle (a model plant for citrus gene function), which does not contain the Sm-RNase allele but are still SC. We demonstrate that this MITE plays a pivotal role in the loss of SI in citrus, providing evidence that this MITE insertion prevents expression of the S-RNase; moreover, transgenic experiments show that deletion of this 786-bp MITE insertion recovers the expression of FhiS2-RNase and restores SI. This study identifies the first evidence for a role for MITEs at the S-locus affecting the SI phenotype. A family-wide survey of the S-locus revealed that MITE insertions occur frequently adjacent to S-RNase alleles in different citrus genera, but only certain MITEs appear to be responsible for the loss of SI. Our study provides evidence that insertion of MITEs into a promoter region can alter a breeding strategy and suggests that this phenomenon may be broadly responsible for SC in species with the S-RNase system.

Villin controls the formation and enlargement of punctate actin foci in pollen tubes.

Self-incompatibility (SI) in the poppy Papaver rhoeas triggers dramatic alterations in actin within pollen tubes. However, how these actin alterations are mechanistically achieved remains largely unexplored. Here, we used treatment with the Ca2+ ionophore A23187 to mimic the SI-induced elevation in cytosolic Ca2+ and trigger formation of the distinctive F-actin foci. Live-cell imaging revealed that this remodeling involves F-actin fragmentation and depolymerization, accompanied by the rapid formation of punctate actin foci and subsequent increase in their size. We established that actin foci are generated and enlarged from crosslinking of fragmented actin filament structures. Moreover, we show that villins associate with actin structures and are involved in this actin reorganization process. Notably, we demonstrate that Arabidopsis VILLIN5 promotes actin depolymerization and formation of actin foci by fragmenting actin filaments, and controlling the enlargement of actin foci via bundling of actin filaments. Our study thus uncovers important novel insights about the molecular players and mechanisms involved in forming the distinctive actin foci in pollen tubes.

Depletion plays a pivotal role in self-incompatibility, revealing a link between cellular energy status, cytosolic acidification and actin remodelling in pollen tubes.

Self-incompatibility (SI) involves specific interactions during pollination to reject incompatible ('self') pollen, preventing inbreeding in angiosperms. A key event observed in pollen undergoing the Papaver rhoeas SI response is the formation of punctate F-actin foci. Pollen tube growth is heavily energy-dependent, yet ATP levels in pollen tubes have not been directly measured during SI. Here we used transgenic Arabidopsis lines expressing the Papaver pollen S-determinant to investigate a possible link between ATP levels, cytosolic pH ([pH]cyt ) and alterations to the actin cytoskeleton. We identify for the first time that SI triggers a rapid and significant ATP depletion in pollen tubes. Artificial depletion of ATP triggered cytosolic acidification and formation of actin aggregates. We also identify in vivo, evidence for a threshold [pH]cyt of 5.8 for actin foci formation. Imaging revealed that SI stimulates acidic cytosolic patches adjacent to the plasma membrane. In conclusion, this study provides evidence that ATP depletion plays a pivotal role in SI upstream of programmed cell death and reveals a link between the cellular energy status, cytosolic acidification and alterations to the actin cytoskeleton in regulating Papaver SI in pollen tubes.

Self-Incompatibility Triggers Irreversible Oxidative Modification of Proteins in Incompatible Pollen.

Self-incompatibility (SI) is used by many angiosperms to prevent self-fertilization and inbreeding. In common poppy (Papaver rhoeas), interaction of cognate pollen and pistil S-determinants triggers programmed cell death (PCD) of incompatible pollen. We previously identified that reactive oxygen species (ROS) signal to SI-PCD. ROS-induced oxidative posttranslational modifications (oxPTMs) can regulate protein structure and function. Here, we have identified and mapped oxPTMs triggered by SI in incompatible pollen. Notably, SI-induced pollen had numerous irreversible oxidative modifications, while untreated pollen had virtually none. Our data provide a valuable analysis of the protein targets of ROS in the context of SI-induction and comprise a benchmark because currently there are few reports of irreversible oxPTMs in plants. Strikingly, cytoskeletal proteins and enzymes involved in energy metabolism are a prominent target of ROS. Oxidative modifications to a phosphomimic form of a pyrophosphatase result in a reduction of its activity. Therefore, our results demonstrate irreversible oxidation of pollen proteins during SI and provide evidence that this modification can affect protein function. We suggest that this reduction in cellular activity could lead to PCD.

Ectopic Expression of a Self-Incompatibility Module Triggers Growth Arrest and Cell Death in Vegetative Cells.

Self-incompatibility (SI) is used by many angiosperms to reject self-pollen and avoid inbreeding. In field poppy (Papaver rhoeas), SI recognition and rejection of self-pollen is facilitated by a female S-determinant, PrsS, and a male S-determinant, PrpS PrsS belongs to the cysteine-rich peptide family, whose members activate diverse signaling networks involved in plant growth, defense, and reproduction. PrsS and PrpS are tightly regulated and expressed solely in pistil and pollen cells, respectively. Interaction of cognate PrsS and PrpS triggers pollen tube growth inhibition and programmed cell death (PCD) of self-pollen. We previously demonstrated functional intergeneric transfer of PrpS and PrsS to Arabidopsis (Arabidopsis thaliana) pollen and pistil. Here, we show that PrpS and PrsS, when expressed ectopically, act as a bipartite module to trigger a self-recognition:self-destruct response in Arabidopsis independently of its reproductive context in vegetative cells. The addition of recombinant PrsS to seedling roots expressing the cognate PrpS resulted in hallmark features of the P rhoeas SI response, including S-specific growth inhibition and PCD of root cells. Moreover, inducible expression of PrsS in PrpS-expressing seedlings resulted in rapid death of the entire seedling. This demonstrates that, besides specifying SI, the bipartite PrpS-PrsS module can trigger growth arrest and cell death in vegetative cells. Heterologous, ectopic expression of a plant bipartite signaling module in plants has not been shown previously and, by extrapolation, our findings suggest that cysteine-rich peptides diversified for a variety of specialized functions, including the regulation of growth and PCD.

New opportunities and insights into Papaver self-incompatibility by imaging engineered Arabidopsis pollen.

Pollen tube growth is essential for plant reproduction. Their rapid extension using polarized tip growth provides an exciting system for studying this specialized type of growth. Self-incompatibility (SI) is a genetically controlled mechanism to prevent self-fertilization. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy). This utilizes two S-determinants: stigma-expressed PrsS and pollen-expressed PrpS. Interaction of cognate PrpS-PrsS triggers a signalling network, causing rapid growth arrest and programmed cell death (PCD) in incompatible pollen. We previously demonstrated that transgenic Arabidopsis thaliana pollen expressing PrpS-green fluorescent protein (GFP) can respond to Papaver PrsS with remarkably similar responses to those observed in incompatible Papaver pollen. Here we describe recent advances using these transgenic plants combined with genetically encoded fluorescent probes to monitor SI-induced cellular alterations, including cytosolic calcium, pH, the actin cytoskeleton, clathrin-mediated endocytosis (CME), and the vacuole. This approach has allowed us to study the SI response in depth, using multiparameter live-cell imaging approaches that were not possible in Papaver. This lays the foundations for new opportunities to elucidate key mechanisms involved in SI. Here we establish that CME is disrupted in self-incompatible pollen. Moreover, we reveal new detailed information about F-actin remodelling in pollen tubes after SI.

Self-incompatibility in Papaver pollen: programmed cell death in an acidic environment.

Self-incompatibility (SI) is a genetically controlled mechanism that prevents self-fertilization and thus encourages outbreeding and genetic diversity. During pollination, most SI systems utilize cell-cell recognition to reject incompatible pollen. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy), which involves the interaction between the two S-determinants, a stigma-expressed secreted protein (PrsS) and a pollen-expressed plasma membrane-localized protein (PrpS). This interaction is the critical step in determining acceptance of compatible pollen or rejection of incompatible pollen. Cognate PrpS-PrsS interaction triggers a signalling network causing rapid growth arrest and eventually programmed cell death (PCD) in incompatible pollen. In this review, we provide an overview of recent advances in our understanding of the major components involved in the SI-induced PCD (SI-PCD). In particular, we focus on the importance of SI-induced intracellular acidification and consequences for protein function, and the regulation of soluble inorganic pyrophosphatase (Pr-p26.1) activity by post-translational modification. We also discuss attempts to identify protease(s) involved in the SI-PCD process. Finally, we outline future opportunities made possible by the functional transfer of the P. rhoeas SI system to Arabidopsis.

MAP Kinase PrMPK9-1 Contributes to the Self-Incompatibility Response.

Mitogen-activated protein kinases (MAPKs) form important signaling modules for a variety of cellular responses in eukaryotic cells. In plants, MAPKs play key roles in growth and development as well as in immunity/stress responses. Pollen-pistil interactions are critical early events regulating pollination and fertilization and involve many signaling events. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants and also is known to utilize signaling to achieve incompatible pollen rejection. Although several pollen-expressed MAPKs exist, very little is known about their function. We previously identified a pollen-expressed MAPK (p56) from Papaver rhoeas that was rapidly activated during SI; several studies implicated its role in signaling to SI-induced programmed cell death involving a DEVDase. However, to date, the identity of the MAPK involved has been unknown. Here, we have identified and cloned a pollen-expressed P. rhoeas threonine-aspartate-tyrosine (TDY) MAPK, PrMPK9-1 Rather few data relating to the function of TDY MAPKs in plants currently exist. We provide evidence that PrMPK9-1 has a cell type-specific function, with a distinct role from AtMPK9 To our knowledge, this is the first study implicating a function for a TDY MAPK in pollen. We show that PrMPK9-1 corresponds to p56 and demonstrate that it is functionally involved in mediating SI in P. rhoeas pollen: PrMPK9-1 is a key regulator for SI in pollen and acts upstream of programmed cell death involving actin and activation of a DEVDase. Our study provides an important advance in elucidating functional roles for this class of MAPKs.

Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity.

Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important posttranslational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollen-expressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plant Papaver rhoeas were inhibited by phosphorylation. Despite the potential significance, there is a paucity of data on sPPase phosphorylation and regulation. Here, we used liquid chromatographic tandem mass spectrometry to map phosphorylation sites to the otherwise divergent amino-terminal extensions on these pollen sPPases. Despite the absence of reports in the literature on mapping phosphorylation sites on sPPases, a database survey of various proteomes identified a number of examples, suggesting that phosphorylation may be a more widely used mechanism to regulate these enzymes. Phosphomimetic mutants of Pr-p26.1a/b significantly and differentially reduced PPase activities by up to 2.5-fold at pH 6.8 and 52% in the presence of Ca2+ and hydrogen peroxide over unmodified proteins. This indicates that phosphoregulation of key sites can inhibit the catalytic responsiveness of these proteins in concert with key intracellular events. As sPPases are essential for many metabolic pathways in eukaryotic cells, our findings identify the phosphorylation of sPPases as a potential master regulatory mechanism that could be used to attenuate metabolism.

Self-incompatibility-induced programmed cell death in field poppy pollen involves dramatic acidification of the incompatible pollen tube cytosol.

Self-incompatibility (SI) is an important genetically controlled mechanism to prevent inbreeding in higher plants. SI involves highly specific interactions during pollination, resulting in the rejection of incompatible (self) pollen. Programmed cell death (PCD) is an important mechanism for destroying cells in a precisely regulated manner. SI in field poppy (Papaver rhoeas) triggers PCD in incompatible pollen. During SI-induced PCD, we previously observed a major acidification of the pollen cytosol. Here, we present measurements of temporal alterations in cytosolic pH ([pH]cyt); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction and stabilizing by 60 min at pH 5.5. By manipulating the [pH]cyt of the pollen tubes in vivo, we show that [pH]cyt acidification is an integral and essential event for SI-induced PCD. Here, we provide evidence showing the physiological relevance of the cytosolic acidification and identify key targets of this major physiological alteration. A small drop in [pH]cyt inhibits the activity of a soluble inorganic pyrophosphatase required for pollen tube growth. We also show that [pH]cyt acidification is necessary and sufficient for triggering several key hallmark features of the SI PCD signaling pathway, notably activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci. Importantly, the actin binding proteins Cyclase-Associated Protein and Actin-Depolymerizing Factor are identified as key downstream targets. Thus, we have shown the biological relevance of an extreme but physiologically relevant alteration in [pH]cyt and its effect on several components in the context of SI-induced events and PCD.

Identification of the pollen self-incompatibility determinant in Papaver rhoeas.

Higher plants produce seed through pollination, using specific interactions between pollen and pistil. Self-incompatibility is an important mechanism used in many species to prevent inbreeding; it is controlled by a multi-allelic S locus. 'Self' (incompatible) pollen is discriminated from 'non-self' (compatible) pollen by interaction of pollen and pistil S locus components, and is subsequently inhibited. In Papaver rhoeas, the pistil S locus product is a small protein that interacts with incompatible pollen, triggering a Ca(2+)-dependent signalling network, resulting in pollen inhibition and programmed cell death. Here we have cloned three alleles of a highly polymorphic pollen-expressed gene, PrpS (Papaver rhoeas pollen S), from Papaver and provide evidence that this encodes the pollen S locus determinant. PrpS is a single-copy gene linked to the pistil S gene (currently called S, but referred to hereafter as PrsS for Papaver rhoeas stigma S determinant). Sequence analysis indicates that PrsS and PrpS are equally ancient and probably co-evolved. PrpS encodes a novel approximately 20-kDa protein. Consistent with predictions that it is a transmembrane protein, PrpS is associated with the plasma membrane. We show that a predicted extracellular loop segment of PrpS interacts with PrsS and, using PrpS antisense oligonucleotides, we demonstrate that PrpS is involved in S-specific inhibition of incompatible pollen. Identification of PrpS represents a major advance in our understanding of the Papaver self-incompatibility system. As a novel cell-cell recognition determinant it contributes to the available information concerning the origins and evolution of cell-cell recognition systems involved in discrimination between self and non-self, which also include histocompatibility systems in primitive chordates and vertebrates.

Self-incompatibility in Papaver: advances in integrating the signalling network.

Self-fertilization, which results in reduced fitness of offspring, is a common problem in hermaphrodite angiosperms. To prevent this, many plants utilize SI (self-incompatibility), which is determined by the multi-allelic S-locus, that allows discrimination between self (incompatible) and non-self (compatible) pollen by the pistil. In poppy (Papaver rhoeas), the pistil S-determinant (PrsS) is a small secreted protein which interacts with the pollen S-determinant PrpS, a ~20 kDa novel transmembrane protein. Interaction of matching pollen and pistil S-determinants results in self-recognition, initiating a Ca²âº-dependent signalling network in incompatible pollen. This triggers several downstream events, including alterations to the cytoskeleton, phosphorylation of sPPases (soluble inorganic pyrophosphatases) and an MAPK (mitogen-activated protein kinase), increases in ROS (reactive oxygen species) and nitric oxide (NO), and activation of several caspase-like activities. This results in the inhibition of pollen tube growth, prevention of self-fertilization and ultimately PCD (programmed cell death) in incompatible pollen. The present review focuses on our current understanding of the integration of these signals with their targets in the SI/PCD network. We also discuss our recent functional expression of PrpS in Arabidopsis thaliana pollen.

Taking one for the team: self-recognition and cell suicide in pollen.

Self-incompatibility (SI) is an important genetically controlled mechanism used by many angiosperms to prevent self-fertilization and inbreeding. A multiallelic S-locus allows discrimination between 'self' (incompatible) pollen from 'nonself' pollen at the pistil. Interaction of matching pollen and pistil S-determinants allows 'self' recognition and triggers rejection of incompatible pollen. The S-determinants for Papaver rhoeas (poppy) are PrsS and PrpS. PrsS is a small secreted protein that acts as a signalling ligand to interact with its cognate pollen S-determinant PrpS, a small novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates increases in cytosolic free Ca(2+) and involves influx of Ca(2+) and K(+). Data implicate involvement of reactive oxygen species and nitric oxide signalling in the SI response. Downstream targets include the cytoskeleton, a soluble inorganic pyrophosphatase, Pr-p26.1, and a MAP kinase, PrMPK9-1. A major focus for SI-induced signalling is to initiate programmed cell death (PCD). In this review we provide an overview of our understanding of SI, with focus on how the signals and components are integrated, in particular, how reactive oxygen species, nitric oxide, and the actin cytoskeleton feed into a PCD network. We also discuss our recent functional expression of PrpS in Arabidopsis thaliana pollen in the context of understanding how PCD signalling systems may have evolved.

Contrasting self-recognition rejection systems for self-incompatibility in Brassica and Papaver.

Self-incompatibility (SI) plays a pivotal role in whether self-pollen is accepted or rejected. Most SI systems employ two tightly linked loci encoding highly polymorphic pollen (male) and pistil (female) S-determinants that control whether self-pollination is successful or not. In recent years our knowledge of the signalling networks and cellular mechanisms involved has improved considerably, providing an important contribution to our understanding of the diverse mechanisms used by plant cells to recognise each other and elicit responses. Here, we compare and contrast two important SI systems employed in the Brassicaceae and Papaveraceae. Both use 'self-recognition' systems, but their genetic control and S-determinants are quite different. We describe the current knowledge about the receptors and ligands, and the downstream signals and responses utilized to prevent self-seed set. What emerges is a common theme involving the initiation of destructive pathways that block the key processes that are required for compatible pollen-pistil interactions.

Reactive oxygen species and nitric oxide mediate actin reorganization and programmed cell death in the self-incompatibility response of papaver.

Pollen-pistil interactions are critical early events regulating pollination and fertilization. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants. Although data implicate the involvement of reactive oxygen species (ROS) and nitric oxide (NO) in pollen-pistil interactions and the regulation of pollen tube growth, there has been a lack of studies investigating ROS and NO signaling in pollen tubes in response to defined, physiologically relevant stimuli. We have used live-cell imaging to visualize ROS and NO in growing Papaver rhoeas pollen tubes using chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and demonstrate that SI induces relatively rapid and transient increases in ROS and NO, with each showing a distinctive "signature" within incompatible pollen tubes. Investigating how these signals integrate with the SI responses, we show that Ca(2+) increases are upstream of ROS and NO. As ROS/NO scavengers alleviated both the formation of SI-induced actin punctate foci and also the activation of a DEVDase/caspase-3-like activity, this demonstrates that ROS and NO act upstream of these key SI markers and suggests that they signal to these SI events. These data represent, to our knowledge, the first steps in understanding ROS/NO signaling triggered by this receptor-ligand interaction in pollen tubes.

Self-incompatibility in Papaver rhoeas activates nonspecific cation conductance permeable to Ca2+ and K+.

Cellular responses rely on signaling. In plant cells, cytosolic free calcium is a major second messenger, and ion channels play a key role in mediating physiological responses. Self-incompatibility (SI) is an important genetically controlled mechanism to prevent self-fertilization. It uses interaction of matching S-determinants from the pistil and pollen to allow "self" recognition, which triggers rejection of incompatible pollen. In Papaver rhoeas, the S-determinants are PrsS and PrpS. PrsS is a small novel cysteine-rich protein; PrpS is a small novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates S-specific increases in cytosolic free calcium and alterations in the actin cytoskeleton, resulting in programmed cell death in incompatible but not compatible pollen. Here, we have used whole-cell patch clamping of pollen protoplasts to show that PrsS stimulates SI-specific activation of pollen grain plasma membrane conductance in incompatible but not compatible pollen grain protoplasts. The SI-activated conductance does not require voltage activation, but it is voltage sensitive. It is permeable to divalent cations (Ba(2+) ≥ Ca(2+) > Mg(2+)) and the monovalent ions K(+) and NH(4)(+) and is enhanced at voltages negative to -100 mV. The Ca(2+) conductance is blocked by La(3+) but not by verapamil; the K(+) currents are tetraethylammonium chloride insensitive and do not require Ca(2+). We propose that the SI-stimulated conductance may represent a nonspecific cation channel or possibly two conductances, permeable to monovalent and divalent cations. Our data provide insights into signal-response coupling involving a biologically important response. PrsS provides a rare example of a protein triggering alterations in ion channel activity.

Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen.

In higher plants, sexual reproduction involves interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility through rejection of incompatible ('self') pollen. In Papaver rhoeas, S proteins encoded by the stigma interact with incompatible pollen, triggering a Ca2+-dependent signalling network resulting in pollen tube inhibition and programmed cell death. The cytosolic phosphoprotein p26.1, which has been identified in incompatible pollen, shows rapid, self-incompatibility-induced Ca2+-dependent hyperphosphorylation in vivo. Here we show that p26.1 comprises two proteins, Pr-p26.1a and Pr-p26.1b, which are soluble inorganic pyrophosphatases (sPPases). These proteins have classic Mg2+-dependent sPPase activity, which is inhibited by Ca2+, and unexpectedly can be phosphorylated in vitro. We show that phosphorylation inhibits sPPase activity, establishing a previously unknown mechanism for regulating eukaryotic sPPases. Reduced sPPase activity is predicted to result in the inhibition of many biosynthetic pathways, suggesting that there may be additional mechanisms of self-incompatibility-mediated pollen tube inhibition. We provide evidence that sPPases are required for growth and that self-incompatibility results in an increase in inorganic pyrophosphate, implying a functional role for Pr-p26.1.

Self-incompatibility requires GPI anchor remodeling by the poppy PGAP1 ortholog HLD1.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are tethered to the outer leaflet of the plasma membrane where they function as key regulators of a plethora of biological processes in eukaryotes. Self-incompatibility (SI) plays a pivotal role regulating fertilization in higher plants through recognition and rejection of "self" pollen. Here, we used Arabidopsis thaliana lines that were engineered to be self-incompatible by expression of Papaver rhoeas SI determinants for an SI suppressor screen. We identify HLD1/AtPGAP1, an ortholog of the human GPI-inositol deacylase PGAP1, as a critical component required for the SI response. Besides a delay in flowering time, no developmental defects were observed in HLD1/AtPGAP1 knockout plants, but SI was completely abolished. We demonstrate that HLD1/AtPGAP1 functions as a GPI-inositol deacylase and that this GPI-remodeling activity is essential for SI. Using GFP-SKU5 as a representative GPI-AP, we show that the HLD1/AtPGAP1 mutation does not affect GPI-AP production and targeting but affects their cleavage and release from membranes in vivo. Our data not only implicate GPI-APs in SI, providing new directions to investigate SI mechanisms, but also identify a key functional role for GPI-AP remodeling by inositol deacylation in planta.

Actin-binding proteins implicated in the formation of the punctate actin foci stimulated by the self-incompatibility response in Papaver.

The actin cytoskeleton is a key target for signaling networks and plays a central role in translating signals into cellular responses in eukaryotic cells. Self-incompatibility (SI) is an important mechanism responsible for preventing self-fertilization. The SI system of Papaver rhoeas pollen involves a Ca(2+)-dependent signaling network, including massive actin depolymerization as one of the earliest cellular responses, followed by the formation of large actin foci. However, no analysis of these structures, which appear to be aggregates of filamentous (F-)actin based on phalloidin staining, has been carried out to date. Here, we characterize and quantify the formation of F-actin foci in incompatible Papaver pollen tubes over time. The F-actin foci increase in size over time, and we provide evidence that their formation requires actin polymerization. Once formed, these SI-induced structures are unusually stable, being resistant to treatments with latrunculin B. Furthermore, their formation is associated with changes in the intracellular localization of two actin-binding proteins, cyclase-associated protein and actin-depolymerizing factor. Two other regulators of actin dynamics, profilin and fimbrin, do not associate with the F-actin foci. This study provides, to our knowledge, the first insights into the actin-binding proteins and mechanisms involved in the formation of these intriguing structures, which appear to be actively formed during the SI response.