Uncovering an ancestral green ménage à trois: Contributions of Chlamydomonas to the discovery of a broadly conserved triad of plant fertilization proteins

Abstract

During sexual reproduction in the unicellular green alga Chlamydomonas, gametes undergo the conserved cellular events that define fertilization across the tree of life. After initial ciliary adhesion, plus and minus gametes attach to each other at plasma membrane sites specialized for fusion, their bilayers merge, and cell coalescence into a quadri-ciliated cell signals for nuclear fusion. Recent findings show that these conserved cellular events are driven by 3 conserved protein families, FUS1/GEX2, HAP2/GCS1, and KAR5/GEX1. New results also show that species-specific recognition in Chlamydomonas activates the ancestral, viral-like fusogen HAP2 to drive fusion; that the conserved nuclear envelope fusion protein KAR5/GEX1 is also essential for nuclear fusion in Arabidopsis; and that heterodimerization of BELL-KNOX proteins signals for nuclear fusion in Chlamydomonas through early diverging land plants. This review outlines how Chlamydomonas’s Janus-like position in evolution along with the ease of working with its gametes have revealed broadly conserved mechanisms.

INTRODUCTION

FUS1/GEX2, HAP2, and KAR5/GEX1: A Plant “Fertilization Triad”

Recent advances in the genetics, cell biology, biochemistry, imaging, and structural biology of plant and algal fertilization have led to an understanding of the cell and molecular mechanisms of fertilization in these organisms that is deeper than in any animal system. As is described below, three protein families are now identified that are responsible for the core cellular events that define fertilization across algae and plants: FUS1/GEX2 (Gamete Expressed 2) for gamete adhesion, HAP2 (Hapless 2)/GCS1 (Generative Cell Specific 1) for gamete fusion, and KAR5 (Karyogamy Protein 5)/GEX1 for nuclear fusion. Remarkably, two of these families - - HAP2 and KAR5/GEX1 - - are used exclusively for fertilization and are also present in organisms spanning protists to metazoans. Although members of the triad had been compellingly implicated at some step in fertilization in plants, studies in Chlamydomonas reinhardtii (hereafter, Chlamydomonas) were important in identifying the cell or molecular step in fertilization at which they functioned, or for recognizing that they indeed were members of large, cross-taxa families with similar functions. Thus, much like the Roman god, Janus, who wielded the key to life transitions, Chlamydomonas’s position in the tree of life has made it an important model organism for unlocking new understandings of the haploid to diploid transition in plant systems. Here, I will describe our current understanding of the molecules, mechanisms, and signaling events that underlie fertilization in Chlamydomonas and highlight their relationships with those of plants.

Fertilization in Chlamydomonas

Chlamydomonas has been widely used for studies on the assembly, length control, motility, and signaling of cilia (formerly known as flagella); on photosynthesis; and on fertilization [1,2]. Mating type (in microorganisms, the equivalent of plant and animal sexes) is determined by the presence of either the plus or minus mating type locus [3]. The minus mating type locus uniquely harbors the gene for the MID (Minus Dominance) transcription factor that turns on minus-specific genes and turns off plus-specific ones. The FUS1 gene, which encodes the plus-specific membrane adhesion protein, is unique to the plus locus. With only a few exceptions, the several hundred other gamete-specifically expressed genes are distributed throughout the rest of the genome [3,4]. Gametogenesis is induced by transferring haploid plus or minus vegetatively growing cells into medium lacking a nitrogen source (The Chlamydomonas life cycle is illustrated in Figure 1). The random ciliary collisions that occur when plus and minus gametes are mixed and the consequent binding between the ciliary adhesion proteins (agglutinins), SAG1 (Sexual Agglutination 1) on plus gametes and SAD1 (Sexual Adhesion 1) on minus [5] bring the cells together and at the same time induce protein kinase- and adenylyl-cyclase dependent signaling pathways that lead to a 15-20-fold increase in the second messenger cAMP [5–7]. The increased cAMP (whose effects can be mimicked by addition of a cell-permeable cAMP analogue to gametes of a single mating type) brings about key responses in the cell bodies that prepare them for fusion.

Figure 1: Chlamydomonas life cycle.

Gametogenesis is induced by transferring plus or minus vegetatively growing cells, which are haploid, into medium lacking a nitrogen source (N-free medium). Within seconds after mixing plus and minus gametes, random collisions between the highly motile cells lead to their tethering by their cilia through binding between the ciliary adhesion receptor SAG1 on plus gametes and SAD1 on minus gametes. In addition to tethering the cilia, SAG1-SAD1 binding also induces nearly 20-fold increases in cellular concentrations of the second messenger cAMP. The increase in cAMP brings about multiple cellular responses that prepare the gametes for fusion. Gametes release their cell wall, a non-cellulose-containing extracellular matrix composed of hydroxyproline-rich glycoproteins [67,68]; they recruit and activate a store of inactive ciliary adhesion molecules [69] from the cell plasma membrane to the ciliary membrane [8,70]; and they erect mating type-specific membrane protuberances (the plus and minus mating structures) between their two cilia that will be the sites of cell-cell fusion and that bear a second set of gamete-specific membrane receptors, the adhesion protein FUS1 on plus gametes and it binding partner MAR1 on minus gametes.

Of particular importance, the minus mating structure also harbors the conserved fusion protein HAP2. FUS1-MAR1 engagement at the tips of the two mating structures triggers a fusogenic reconfiguration of the latent, prefusion form of HAP2 into homotrimers. The complete cell coalescence that follows bilayer merger generates yetother signals, including blocks to polygamy in which FUS1, HAP2, and MAR1 aredegraded [17,71]; initiation of the zygote developmental pathway through the action the newly formed GSP1-GSM1 heterodimer; and nuclear fusion through the action of the nuclear envelope protein, GEX1. After an obligate time in the dark, incubation in culture medium in the light directly induces germination and meiosis, with no intervening mitoses, to produce the 4 haploid products of meiosis, two plus and two minus vegetative cells. FUS1/GEX2, HAP2, and KAR5/GEX1 are in dark green font to indicate that members of this triad are used from algae through higher land plants. The GSP1-GSM1 heterodimer is in lighter green to indicate its use in algae through early diverging land plants.

This Chlamydomonas cilium-based signaling system has been a particularly important model for investigating conserved mechanisms of ciliary signaling and regulated, bidirectional movement of proteins between the cell body and the cilium [8,9]. Moreover, recent publications have reported evidence for another gamete-specific, cilium-dependent signaling process in Chlamydomonas, gamete chemotaxis. The King-Eipper group showed that Chlamydomonas possesses the enzymes for producing amidated peptides similar to those produced in the nervous systems of animals and that gametes release one amidated peptide that is a chemoattract for minus gametes and a repellent for plus gametes [10]. In future studies, it will be interesting to determine whether the underlying mechanisms depend on glutamate receptor-like ion channels, which function in pollen tube guidance [11,12], and in chemotaxis by the flagellated sperm of the moss Physcomitrium patens [13].

In addition to releasing their cell walls and mobilizing stored agglutinins to the cilia [8], the activated gametes erect membrane protuberances, the plus and minus mating structures, whose tips are the sites of the membrane attachment and bilayer merger that underlie gamete fusion. The plus mating structure, an actin-filled microvillous-like organelle ~100 nm in diameter and 3-5 μm long, harbors the single pass, transmembrane adhesion protein FUS1 [14,15]. The shorter minus mating structure lacks actin filaments and displays the minus gamete-specifically expressed, single-pass transmembrane adhesion protein MAR1 (Minus Adhesion Receptor 1) [16,17] and the membrane fusogen, HAP2 [18]. Attachment of the tips of the organelles through FUS1-MAR1 binding is followed rapidly by HAP2-driven bilayer merger (see middle panel, Figure 1) [18]. The resulting zygotes form an impervious cell wall and enter a dormant state that allows them to survive the harsh environmental conditions in the wild (e.g., nutrient deprivation, freezing, desiccation) that trigger gametogenesis

Trimer formation by HAP2, a homolog of class II viral fusion proteins, drives gamete fusion.

In spite of the major challenges of studying the gamete membrane fusion reaction in plants, innovative experimental strategies had placed HAP2 at the scene of plant gamete fusion - - on the membranes of the 2 pollen tube-delivered sperm within the burst synergid cell near the egg cell and central cell [19–22]. Because of the complexities of the reproductive tissues of flowers, however, along with the consequent reality that the cell biology of fertilization in plants has to be examined 4 gametes at a time, the studies in plants had failed to determine whether HAP2 was required for adhesion or for bilayer merger [19,22]. After its initial discovery in Arabidopsis thaliana (hereafter, Arabidopsis) [20], HAP2 was independently discovered in Lily (where it was called GCS1) [22] and in Chlamydomonas [18].

In Chlamydomonas, it is possible to quantitatively assess discrete steps in fertilization in mixtures composed of millions of pure plus and minus gametes synchronously undergoing the multiple steps of fertilization. Exploiting this system solved the quandary left by the plant studies and showed unequivocally that HAP2 was not required for gamete attachment but was essential for bilayer merger [18] (Figure 2, middle panels), a conclusion that was confirmed in studies with the malaria organism Plasmodium berghei [18,23], and since then in multiple organisms [21,24]; reviewed in [25]. Although HAP2 has not yet been detected in fungi or chordates, the distribution of HAP2 family members across animals (e. g., arthropods, mollusks, and hemichordates), protists, algae, and plants indicates that it is likely the ancestral gamete fusogen [18,24,26–28].

Figure 2: Structures of Arabidopsis thaliana and Chlamydomonas reinhardtii members of the FUS1/GEX2, HAP2, and KAR5/GEX1 families along with the phenotypes of the corresponding Chlamydomonas mutants.

The structures for Chlamydomonas (Cr) and Arabidopsis (At) FUS1/GEX2 family members (ectodomains; upper left panels) were predicted by contact-based methods [72,73]. The data comparing mating structure adhesion by WT and fus1 mutant plus gametes with WT minus gametes (% attachment to minus gametes; upper right panel) are from Figure 4B l· in [14]. The HAP2 structures (middle left panels) are atomic models of postfusion trimers of Chlamydomonas HAP2 (pdb file 6DBS) and Arabidopsis HAP2 (pdb file 5OW3). The HAP2 fusion loops are depicted in red. The upper two subpanels of the middle right panel show low (left) and high (right) magnification TEM images of WT plus and minus gametes just after mating structure fusion. The low and high magnification TEM images in the lower two subpanels of the middle right panel show that the mating structures of a hap2 mutant minus gamete and a WT plus gamete had undergone attachment but had failed to fuse (Images are from [18]). The structures for Chlamydomonas (Cr) and Arabidopsis (At) KAR5/GEX1 family members (ectodomains; upper left panels) were predicted by contact-based methods [72,73]. The TEM images in the right lower panels show images of nuclei that have fused after fusion of WT plus and minus gametes (upper subpanel) and nuclei that have attached to each other but have failed to fuse in a zygote formed by fusion of gexl plus and minus gametes (lower subpanel). (Images are from [4].

A recent breakthrough in understanding gamete fusion came with the discovery that HAP2 is a eukaryotic homolog of the class II fusion proteins of alpha-, flavi-, and bunyaviruses (e. g., dengue and Zika) [27]. The first atomic model of HAP2, a Chlamydomonas HAP2 homotrimer, revealed an unambiguous structural homology to class II fusion proteins, with the β sheet-rich domains I, II, and III, arranged in the folded-back (hairpin) conformation typical for the viral proteins in the post-fusion trimer [17]. In their prefusion form on virus particles, class II proteins exist as homodimers or heterodimers in the viral envelope, with their single, C-terminal transmembrane domain (TMD) embedded in the virus membrane. During target cell entry, the acidic environment of the endosome triggers key conformational changes that release the proteins from their pre-fusion state. Hydrophobic residues displayed on the “fusion loop” in domain II (DII) at the distal tip of each monomer insert into the lipid bilayer of the endosome. Monomers align side by side to form trimers that bridge the two membranes. Further, extensive folding brings the TMDs and the embedded fusion loops towards each other, forcing the two bilayers together and leading to bilayer merger and formation of a fusion pore that expands to bring about complete fusion [31,32].

Recently, several international research groups using an array of in vitro and in vivo strategies to study HAP2 family members in multiple species have made exciting progress towards a fuller understanding of the structure and function of this intriguing fusogen family [33–35]. Their conclusions are the following: The fusion loop of HAP2 indeed is essential for membrane insertion during fusion. HAP2 has evolved multiple modes of fusion loop-directed membrane insertion that are in keeping with the multiple membrane environments it has encountered during eukaryotic evolution. Species-specific motifs in regions critical for the fusogenic conformational change of viral class II proteins, including a cystine ladder-like module and a mucin-like region, are potential interaction sites with other proteins that could regulate HAP2 function before and during gamete interactions. And, also serving as a reminder that studying plant fertilization can catalyze research that leads to globally important consequences not related only to nutrition, multiple studies, including field studies in Africa with antibodies that bind the fusion loop of Plasmodium falciparum HAP2, have established the protein as a promising target for a transmission-blocking malaria vaccine [18,36–38].

More recent experiments on endogenous Chlamydomonas HAP2 using size-exclusion chromatography, velocity sedimentation, and semi-native SDS-PAGE confirmed that endogenous Chlamydomonas HAP2 indeed forms homotrimers during fusion and only during fusion, and that trimer formation is essential for fusion [39]. Mutation of residues designed to disrupt the homotrimer interface blocked trimer formation of recombinant HAP2 in vitro, blocked formation of endogenous HAP2 trimers in vivo, and blocked gamete fusion. Thus, the molecular mechanism of HAP2 function is similar to that of viral class II fusion proteins. These quantitative biochemical findings argue against a mixed or heterotrimer model that has been gaining traction in the literature [29,40–42]. Based on microscopic observations of fusion in animal cells overexpressing recombinant Arabidopsis HAP2 and a member of the only other known eukaryotic class II fusion protein family, EFF-1, the model proposed that HAP2 formed trimers composed of HAP2 from one gamete and HAP2 or an unidentified protein on the other gamete. In light of these new results and the cautionary tales from the field of animal fertilization [43], in future experiments testing the mixed trimer model, it will be important to use biochemical methods to document that trimers form and that they are composed of monomers from the apposing membranes of two cells.

FUS1 and plant sperm GEX2 belong to a conserved family of gamete adhesion proteins. In Chlamydomonas FUS1-dependent gamete adhesion activates HAP2 for fusion.

Zhang et al. [39] also addressed another fundamental question about HAP2-driven gamete fusion. What is the mechanism that triggers this powerful fusogen into action? They found that unlike with viral class II fusion proteins, low pH failed to drive trimer formation. Rather, FUS1-dependent membrane attachment between the plus and minus mating structures initiated trimer formation and bilayer merger. They further showed that less than 10% of the cellular complement of HAP2 was required for fusion. Experiments with Chlamydomonas gametes expressing HAP2 lacking hydrophobic residues in one of the 3 helices in the large, bipartite DII fusion loop showed that fusion failed, but nearly the entire population of HAP2 formed trimers. The interpretation of these results, which were consistent with related in vitro findings from the Rey group [34], was that the two remaining fusion helices were sufficient to promote interaction with the membrane of the plus mating structure and initiate trimerization, but insufficient to support the forces required to pull the two lipid bilayers together for fusion. In the absence of fusion, FUS1-dependent adhesion continued to trigger (futile) trimer formation. Taken together, these results finally provided a molecular function for the long-studied cellular phenomenon of species-specific gamete adhesion.

Of particular relevance to plants, Pinello et al. [17] discovered that FUS1 is the founding member of a broadly conserved family of proteins that includes the more recently discovered plant sperm adhesion protein, GEX2 [44–47], which is present in organisms across plant taxa. Echoing evidence for similarities of portions of FUS1 and GEX2 in the earlier studies, BLAST analyses and contact-based structural modeling showed that the ectodomains of Chlamydomonas FUS1 and GEX2 proteins from Arabidopsis and multiple other plants are similar over their entire lengths and are composed solely of closely linked Ig-like domains, suggesting a common origin [17] (Figure 2). As the authors noted, not only is this domain architecture similar to that of bacterial intimins and adhesins, but it underlies the extensive protein binding variability of immunoglobulins in the adaptive immune system of vertebrates. Similar variations of Ig-like domains in FUS1/GEX2 proteins during evolution could permit interaction with new binding partners that might emerge on the other gamete, thereby potentially contributing to speciation.

Newly identified adhesion receptor MAR1 on minus gametes is bifunctional: MAR1 is associated with HAP2 on minus gametes, and it binds directly to FUS1 on plus gametes.

Pinello et al. [17] also uncovered yet a third actor and a potential HAP2 regulator in fertilization in Chlamydomonas, this one, though, not conserved. They identified the protein MAR1 on the minus mating structure as the binding partner for FUS1 on plus gametes. MAR1 is a species-specific, single-pass transmembrane protein expressed only by minus gametes and required for mating structure adhesion and for gamete fusion. Biochemical studies with endogenous and recombinant proteins showed that MAR1 binds directly to FUS1. Thus, FUS1 and MAR1 join mouse sperm Izumo1 [48] [17] and mouse egg Juno in being the only receptor pairs in eukaryotes known to be essential for gamete adhesion during fertilization [49]. A vertebrate fusogen remains unidentified.

A potentially more far-reaching discovery also emerged from these findings. Pinello et al. [17] determined that MAR1 was biochemically and functionally associated with HAP2 on the minus mating structure. Consistent with the biochemical association, HAP2 expression and localization were impaired in marl gametes lacking a functional MAR1 protein. Thus, MAR1 is at the nexus of species-specific gamete membrane adhesion and the fusogenic reconfiguration of HAP2. A parsimonious model for these findings is that in naive minus gametes, MAR1 interacts with and restrains HAP2, and that binding of FUS1 to MAR1 releases the restraint, thereby triggering the fusogenic reconfiguration of HAP2 [17].

A much more speculative prediction posits a direct, central role for FUS1 in regulating HAP2 (Figure 3, left panels). In this idea, FUS1 binding to MAR1 is followed by a direct interaction of FUS1 with HAP2, thereby triggering HAP2 reconfiguration. The predicted more general notion (Figure 3, right panels) would apply across species and proposes that it is the membrane adhesion-dependent interaction of HAP2 with the FUS1/GEX2 family member that triggers HAP2. This concept accounts for two important features of these protein families. First, in Chlamydomonas, the FUS1/GEX2 family member is on the non-HAP2-expressing (plus) gamete, whereas in plants, the FUS1/GEX2 family member is on the HAP2-expressing gamete, the sperm [44,46,47]. Second, their broad distributions indicate that members of the FUS1/GEX2 and HAP2 families have functioned simultaneously in the gamete membrane fusion reaction throughout the evolution of green algae and plants. A direct, functional link between the two proteins might provide a selective advantage that could underlie their evolutionary link. In this idea, the putative FUS1-HAP2 interaction In Chlamydomonas would be in trans and facilitated by the FUS1-MAR1 interaction. In plants, the GEX2-HAP2 interaction would be in cis, and facilitated by GEX2 binding to its partner on the egg (and central cell). Notably, the finding that seed formation is strongly reduced but not completely blocked in Arabidopsis GEX2 mutants, indicates that other unidentified proteins could act redundantly with or in addition to GEX2 [46]. In keeping with Okabe’s admonition [43], it will be critical in futures studies to test for direct biochemical interactions between members of the two families and to assess possible co-evolving regions of FUS1/GEX2 and HAP2 family members.

Figure 3: Speculative proposal that binding of a FUS1/GEX2 family member with its partner on the other gamete facilitates a subsequent direct interaction between the FUS1/GEX2 protein and HAP2 that that triggers HAP2 for fusion.

The panels on the left depict Chlamydomonas HAP2 and MAR1 in the membrane of the minus gamete and FUS1 in the membrane of the plus gamete. The panels on the right depict HAP2 and GEX2 in the sperm membrane and a putative, unidentified GEX2-binding partner in the egg membrane of Arabidopsis. The upper subpanels in each panel show the sets of membranes before membrane interactions, with the MAR1-HAP2 interaction represented in yellow to reflect the uncertainty of a direct interaction. The middle sets of panels depict the documented interaction (in black) between FUS1 and MAR1 and the predicted interaction (in yellow) of Arabidopsis GEX2 on the sperm with its putative binding partner on a female gamete. The red set of lines in both panels illustrate the proposed interaction of the two proteins with their respective HAP2s. The lower panels represent HAP2 in its triggered form with its fusion loop inserted into the membrane of the opposite gamete. Note that the proposed FUS1/GEX2 interactions with HAP2 illustrated here lack any experimental support and are purely speculative.

Potentially adding yet another layer of complexity to models for the membrane fusion reaction, Chlamydomonas gametes express a protein (encoded by Cre07.g312850) [4] with sequence similarity to Arabidopsis sperm membrane proteins, DMP8 and DMP9. In Arabidopsis, this pair of multi-span transmembrane proteins participates in fertilization [50], with fusion reduced by ~50% in double mutants [51,52]. Although their functions are still unclear, they are required after sperm adhesion, and could influence membrane properties that support HAP2 action. Or they could be important in the trafficking of Arabidopsis HAP2 from intracellular sites to the plasma membrane that was shown to be induced by EC1 peptides released from the egg after it senses sperm are in the vicinity [53].

The nuclear envelope protein GEX1 is essential for nuclear fusion in protists, fungi, vertebrates, green algae, and plants. In Chlamydomonas and Marchantia polymorpha zygotes nuclear fusion also depends on a signaling pathway activated by formation of a heterodimer of gamete-specific TALE domain transcription factors.

In studies following up on their landmark discoveries of GEX1 and GEX2 in Zea mays, the McCormick laboratory had compellingly implicated GEX1 in zygote development in Arabidopsis [47,54]. Using low stringency BLAST methods along with structure-aided search algorithms, Ning et al. [4] subsequently determined that a protein identified in their gamete transcriptome studies of Chlamydomonas was a homolog of GEX1. Ning et al. further showed that the protein was a nuclear envelope protein in gametes of Chlamydomonas and Plasmodium berghei, that it was essential for timely nuclear fusion in Chlamydomonas zygotes, that it was essential for production of viable zygotic progeny in Chlamydomonas and P. berghei, and that, like HAP2, P. berghei GEX1 was essential for mosquito transmission of malaria. Furthermore, they found that the overall domain architectures of Chlamydomonas, apicomplexan, and plant GEX1s were similar to the nuclear envelope fusion proteins Brambleberry in zebrafish [55] and (the much earlier discovered) KAR5 in yeast [28,56,57], and therefore compose the KAR5/GEX1 family of nuclear fusion proteins. A new study in Arabidopsis has elegantly confirmed the nuclear location and function in nuclear fusion of GEX1 [58]. In addition to a requirement for GEX1 in fusion of the sperm nuclei with the nuclei of the egg and central cell, GEX1 was also found to be essential for fusion of the 2 polar nuclei in the central cell that occurs before pollination. The contact-based structural predictions of Chlamydomonas and Arabidopsis GEX1 shown in Figure 2 compelling document the conservation of overall domain architecture of the two proteins, confirming and extending the similarities shown by Ning et al, [4] and Nishikawa et al. [58].

Finally, exciting, recent findings on the roles of members of the TALE (three-amino-acid-loop-extension) family of homeodomain transcription factors in fertilization in Chlamydomonas and in the liverwort Marchantia polymorpha have refined our understanding of regulation of nuclear fusion and the haploid to diploid transition. Studies in Saccharomyces cerevisiae first showed that a TALE and a non-TALE transcription factor expressed in cells of opposite mating types formed a heterodimer in the diploid required for the haploid to diploid transition [59]. Subsequent studies showed that Chlamydomonas used the identical strategy, with the exception that each gamete expressed a cytosol-localized member of a TALE homeodomain protein subfamily, KNOX (GSP1 [Gamete-Specific Plus [mating type] molecule 1] in plus) [60] and BELL (GSM1 [Gamete-Specific minus 1] in minus) [61,62]. A recent and compellingly thorough study in Chlamydomonas showed that the GSP1-GSM1 heterodimer was also essential in a pathway for signaling for nuclear fusion in the zygote as well as for chloroplast and mitochondria fusion [63]. Family members of these proteins also function widely in plant reproduction. A pair of recent papers on Marchantia polymorpha, found that this ancestral mechanism for regulating nuclear fusion and diploid gene expression, although later lost, was retained in early diverging land plants [64,65]. Intriguingly, expression of a BELL-like protein essential for zygote development in the moss Physcomitrium patens depends on the action of the glutamate receptor-like channels that regulate sperm chemotaxis in the organism [13].

Gamete-specific minus1

CONCLUSIONS

Recent research on fertilization in algae and land plants has led to the recognition, that in a large portion of the earth’s eukaryotic “endless forms most beautiful” [66], the creation of each new individual depends on members of 3 protein families that function exclusively in gametes, FUS1/GEX2, HAP2 and KAR5/GEX1. Remarkably, not one member of this fertilization triad is an enzyme and not one fits into the conventional definition of a signaling protein, yet all 3 catalyze cellular reactions that result in essential downstream responses. Furthermore, each functions in just a single cell type and just a single time in the life of each organism. The tremendous sequence divergence within each of these families, which hindered initial recognition of their existence, belies the conservation of the overall domain architectures within each family and is a strong reminder that natural selection acts on structure. Several questions about members of the fertilization triad now emerge. What are the molecular mechanisms that restrain HAP2 in its prefusion state? Will species-specific membrane attachment be a conserved mechanism for HAP2 triggering? Do FUS1/GEX2 family members directly bind to HAP2? Have regions within the two proteins co-evolved? Are the GEX2 binding partners on female plant gametes also members of a conserved family? What mechanisms regulate KAR5/GEX1 function during nuclear fusion? Do KAR5/GEX1 family members undergo a HAP2-like molecular reconfiguration? Chlamydomonas is likely to continue to be an important contributor to addressing these and other fundamental questions in fertilization in the joint quest with plant biologists to understand conserved mechanisms of sexual reproduction in green organisms.