What is the endosymbiotic theory explain 3 pieces of evidence that support the endosymbiotic theory why is the endosymbiotic theory significant?

1 Introduction

Endosymbiosis is a primary force in eukaryotic cell evolution. Recent studies of algal evolution have shown that endosymbiosis has occurred several times and has yielded a variety of eukaryotic cells. Despite the importance of this phenomenon, however, molecular mechanisms for the induction of endosymbiosis between different protists are not well known.

Paramecium bursaria cells harbor several hundred symbiotic Chlorella spp. in their cytoplasm (Fig. 2.1A). In P. bursaria, each symbiotic alga is enclosed in a perialgal vacuole (PV) membrane derived from the host digestive vacuole (DV) membrane, which provides protection from lysosomal fusion (Figs. 2.2B and 2.3B) (Gu et al., 2002; Karakashian and Rudzinska, 1981). Timing of cell divisions of both algae and the host cells is well coordinated (Kadono et al., 2004; Takahashi et al., 2007). For that reason, symbiotic algae can be inherited to the daughter cells. Irrespective of the mutual relations (see Section 5) between P. bursaria and symbiotic algae, the algae-free cells and symbiotic algae retain the ability to grow without a partner. Algae-free P. bursaria can be produced easily from algae-bearing cells using one of the following methods: rapid fission (Jennings, 1938); cultivation in darkness (Karakashian, 1963; Pado, 1965; Weis, 1969); X-ray irradiation (Wichterman, 1948); treatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a blocker of electron flow in photosystem II (Reisser, 1976); treatment with the herbicide paraquat (Hosoya et al., 1995; Tanaka et al., 2002); or treatment with a protein synthesis inhibitor, cycloheximide (Kodama and Fujishima, 2008a; Kodama et al., 2007; Weis, 1984). On the other hand, symbiotic algae can be isolated easily by sonication, homogenization, or treatment with detergents. Furthermore, endosymbiosis between algae-free P. bursaria cells and symbiotic algae isolated from algae-bearing P. bursaria cells is easily reestablished by mixing them (Karakashian, 1975; Siegel and Karakashian, 1959). In fact, P. bursaria can be cultivated easily, producing a mass culture. The reinfection process can be observed easily under a light microscope. An algae-free mutant strain of P. bursaria has been collected (Tonooka and Watanabe, 2002, 2007). For these reasons, the symbiotic associations between P. bursaria and Chlorella spp. are considered an excellent model for studying cell-to-cell interaction and the evolution of eukaryotic cells through secondary endosymbiosis (Gerashchenko et al., 2000; Stoebe and Maier, 2002). However, the mechanisms and timings used by the algae to escape from the host DV and to protect themselves from host lysosomal fusion have long remained unknown. Therefore, in this review, we first introduce the algal reinfection process, as revealed using the pulse label and chase method. Then we review recently reported studies of mutual benefit between P. bursaria and symbiotic Chlorella spp.

Primary Endosymbiosis

Endosymbiosis has had a profound impact on the evolution and diversification of eukaryotes. Mitochondria and plastids, the energy-generating organelles of modern-day eukaryotes, evolved from free-living prokaryotes that were taken up by eukaryotic hosts and transformed into permanent subcellular compartments. In ‘modern’ cells, these organelles now function as the sites of respiration and oxygenic photosynthesis, respectively. Unlike the endosymbiotic origin of mitochondria, which appears to have occurred in the common ancestor of all known eukaryotes, the endosymbiosis that gave rise to plastids occurred after the deepest divergences in eukaryotic evolution had taken place. This pivotal event paved the way for the evolution of a diverse array of algal lineages and for the spread of plastids between unrelated groups of eukaryotes by ‘secondary’ (i.e., eukaryote–eukaryote) endosymbiosis.

Photosynthesis is widespread in bacteria, but oxygenic photosynthesis occurs only in the cyanobacteria, a lineage of unicellular and colony-forming prokaryotes once referred to as blue-green algae. Among bacteria, only cyanobacteria possess both photosystems I and II. This allows them to use light energy to split water and produce ATP, NADPH, and glucose, liberating oxygen in the process. These products, together with carbon dioxide, are then converted to carbohydrate via the Calvin cycle. The fundamental similarities of cyanobacterial photosynthesis and that of plastids, which also possess photosystems I and II, argue strongly that eukaryotes derived their plastids from a harnessed cyanobacterium, in what is generally referred to as a ‘primary’ endosymbiotic event (Figure 1(a)).

The evidence supporting a cyanobacterial ancestry for plastids is overwhelmingly strong and is based on a large amount of biochemical, ultrastructural, and molecular phylogenetic data. As discussed below, three eukaryotic lineages – the green algae (and land plants), red algae, and glaucophyte algae – possess primary plastids (Table 1), indicating that they arose directly from this landmark event.

Table 1. Distribution of primary and secondary plastids and their basic characteristics

Abbreviations: Chl, chlorophyll; RER, rough endoplasmic reticulum.

One of the hallmark features of plastids is their membranes. Most bacteria have two membranes, with a layer of peptidoglycan in between. Based on what is understood about the process of phagocytosis in eukaryotes, one would expect plastids to be surrounded by three membranes, two from the cyanobacterial endosymbiont and a third, outermost membrane derived from the phagosomal membrane of the heterotrophic eukaryote (Figure 1(a)). However, primary plastids possess only two membranes, both of which appear to be cyanobacterial in origin (the plastids of glaucophyte algae also possess a peptidoglycan layer, suggesting that they might represent an early divergence from the main line of plastid decent) (Figure 1(b)). One possible explanation is that the engulfed cyanobacterium ‘escaped’ the confines of the host cell phagocytic vacuole and took up residence in its cytoplasm. Regardless, it is reasonable to assume that the photosynthetic abilities of the endosymbiont were of great benefit to the eukaryotic host and provided a strong selective advantage to those cells that happened to retain their endosymbionts for progressively longer periods of time. The two cells became increasingly dependent on one another and, eventually, the cyanobacterial endosymbiont evolved into a fully integrated component of the host.

Complex Plastids, Complex History

Secondary endosymbiosis then is a mechanism by which unrelated groups of eukaryotes can end up sharing the same basic trait of photosynthesis. Furthermore it is clear that secondary endosymbiosis has happened multiple times (Figure 2(b)). For example, there are two main groups of secondary algae – chlorarachniophytes and phototrophic euglenids – that have chlorophylls a and b, like green algae and land plants (see below); however, phylogenies of plastid genome sequences show that chlorarachniophyte and euglenid plastids are related to the plastids of different subgroups of green algae (Rogers et al., 2007; Turmel et al., 2009). This demonstrates that chlorarachniophytes and euglenids acquired their plastids through separate events of secondary endosymbiosis.

The situation in other groups of algae with complex plastids is more complicated. Four important groups of algae – cryptophytes, haptophytes, photosynthetic dinoflagellates, and photosynthetic stramenopiles (also known as ochrophytes, or heterokont algae) – typically have complex plastids that contain a distinctive chlorophyll, chlorophyll c, in addition to chlorophyll a (see Andersen, 2004; Graham et al., 2009). Phylogenies based on sequence data from the plastid genome show that these chlorophyll c-containing organelles are related to red algal plastids (Bachvaroff et al., 2005; Janouskovec et al., 2010; Yoon et al., 2002), demonstrating that they have a separate endosymbiotic ancestry from the chloroplastid-derived complex plastids of chlorarachniophytes and euglenids discussed above. Furthermore, these plastid gene phylogenies also indicate that the plastids of chlorophyll c-containing groups stem ultimately from a single event of secondary endosymbiosis (Bachvaroff et al., 2005; Yoon et al., 2002). However, attempts to estimate the evolutionary trees of the algae themselves, using genes from their nuclear genomes, do not demonstrate that these organisms as closely related. For example, as discussed below, dinoflagellates are more closely related to ciliates (which never have their own plastids), than they are to stramenopiles, haptophytes, or cryptophytes (e.g., Burki et al., 2012; Van de Peer and De Wachter, 1997).

There are two main scenarios that would resolve this apparent contradiction. One possibility is that all of these algae indeed stem directly from a single event of secondary endosymbiosis in a common ancestor, but this photosynthetic ability (and in most cases, the entire plastid) was subsequently lost by ancestors of several major groups of heterotrophs, at a minimum ciliates, Rhizaria, several groups of heterotrophic stramenopiles, and some obscure taxa related to plastid-bearing cryptophytes. The second possibility is that there was a single event of secondary endosymbiosis that gave rise to the chlorophyll c-containing plastid, but this plastid was subsequently acquired by one or more unrelated groups through additional symbiotic events (Bodyl et al., 2009; Cavalier-Smith, 1999; Gould et al., 2008; Keeling, 2013). These additional events could have involved a heterotrophic eukaryote acquiring a secondary alga as a symbiont, and over time, this symbiont becoming reduced to just a plastid, in other words ‘tertiary endosymbiosis’ (Archibald, 2009; Keeling 2013). Tertiary endosymbiosis is actually an independently documented phenomenon: There are some dinoflagellates that have plastids that seem to be derived relatively recently from different secondary algae. In the clearest case, the donor of the plastid was a haptophyte, as evident from both plastid pigments, and phylogenetic analysis of plastid genes (Tengs et al., 2000).

Unfortunately, it is not straightforward to distinguish between secondary and tertiary endosymbioses that happened a long time in the past, and at present it is not clearly resolved which of the two scenarios outlined above best corresponds to the actual evolutionary history (see Archibald, 2015; Keeling, 2013; Gould et al., 2015; Petersen et al., 2014; Figure 2(b)). What is not disputed is that resolving the histories of red-alga-derived complex plastids, and of their host algae, remain amongst the most important questions in the study of the evolution of extant eukaryotes.

Host Controls Over Microbial Infections

Endosymbioses persist, meaning that the population of microorganisms is retained within the host for extended periods, potentially for the full lifespan of the host and, in vertically transmitted associations, through multiple host generations. Furthermore, the density and proliferation rate of the microorganisms are tightly regulated such that the microbial population increases in parallel with the host, neither overgrowing nor being diluted out by host growth. Generally, this requires suppression of microbial growth rates. For example, the doubling time of the dinoflagellate alga Symbiodinium is <24 h in culture and 50–60 days in symbiosis with corals; the symbiotic bacteria Buchnera in aphids with a population doubling time of c. 3 days are allied to enteric bacteria with a capacity to divide every 20 min.

The endosymbiotic microorganisms may be controlled by suppression of growth rates, expulsion from the host, and by lysis, and the relative importance of these different processes varies among associations. Most algal associations in hydra, corals, and related aquatic invertebrates are regulated primarily by controls over algal proliferation, although up to 5% of the algal population in some coral hosts may be expelled from the association per day. The algal density in these symbioses is increased in media with high concentrations of ammonia or other inorganic nutrients, suggesting that these symbionts may be nutrient-limited and that the host control over nutrient supply to the symbionts may be overwhelmed by high levels of exogenous nutrients. Expulsion plays a central role in the regulation of the bioluminescent bacteria Vibrio fischeri in the bobtail squid Euprymna scolopes, with up to 90% of the bacterial population in the squid light organ expelled daily followed by a rapid proliferation of the remaining bacterial population. Lysis of endosymbionts is developmentally controlled in many symbioses. For example, cells bearing the bacteria Buchnera in aphids lyse in mid-reproductive insects, releasing the bacterial cells into the hemolymph (blood), where they are destroyed.

The abundance of microbial symbionts is also influenced strongly by the scale of the benefit they confer on the host. This has been demonstrated experimentally for the symbiosis of Bradyrhizobium with soybean plants. When the bacteria are prevented from fixing nitrogen by replacing air with the N2-free atmosphere of argon and oxygen, the numbers of rhizobia are markedly reduced; this effect is obtained whether the experiment was conducted at the scale of the whole root, part of the root system, or even the individual root nodule (Figure 5(a)). Monitoring of the oxygen relations revealed reduced oxygen tensions in the central infected zone of the nodule, where the rhizobia are located, and depressed oxygen permeability of the outer nodule tissues (Figure 5(b)). These results suggest that legume plants respond to rhizobia that fail to fix nitrogen by decreasing the oxygen supply to the rhizobia.

The host can also control the life history traits of their symbionts, generally suppressing motile or sexual forms. For example, the fungal symbionts of leaf-cutting ants are maintained in a permanently asexual condition, presumably by secretions from the ants; sexual fruiting bodies are produced only in nests abandoned by the ants. The persistence of the obligately anaerobic protists in wood-eating cockroaches Cryptocercus is linked to the molt cycle of the insect. The protists are restricted to the anoxic hindgut of their insect host, where they degrade ingested cellulose. However, they are expelled from the insect at each insect molt. In the hours prior to expulsion, and in response to elevated titers of the insect ecdysteroid (molting) hormones, the symbionts develop into oxygen-resistant cysts, enabling them to survive in the external environment until such time as they are ingested by the insect and return to a metabolically active state in the insect gut.

Endosymbionts are generally restricted to specific locations in their host, such as particular organs or cell types that, in some associations, have the sole function to house and maintain the microorganisms. Examples include the root nodules of leguminous plants, the light organs of various fish and squid housing luminescent bacteria, and the bacteriocytes of diverse insects. A particularly vivid example of spatial control is provided by stratified lichens, that is, lichens in which the photosynthetic symbionts are restricted to a specific zone of the thallus (body) of the lichen. Within this layer, light capture by the symbionts is optimized by minimizing shading of one symbiont cell by another. For example, the algal symbiont Trebouxia in the lichen Parmelia borreri is maintained in regular rows, controlled by hyphae of the fungi. Each Trebouxia is contacted by a single fungal haustorium. When the algal cell divides to produce four daughter cells, the fungal haustorium branches fourfold and lengthens, thereby separating the four daughter cells and maintaining regular spacing between the algal cells.

3.7 Mitochondrial dynamics

The endosymbiosis theory claims that mitochondria were once independent entities that at one point in time became associated with eukaryotic cells. So, it should come as no big surprise that mitochondria do not exist in fixed positions in the cell. In fact, mitochondria have been observed to be highly dynamic organelles with a high degree of motility, constantly transitioning between fusional and fissional states and moving to sites of high-energy demand, such as to synaptic zones. Given this, it is the alteration of mitochondrial dynamics that is now believed to be at the core of many neurodegenerative disorders, including AD. For instance, proteins such as optic atrophy 1 (OPA1), mitofusin 1 (MFN1), and mitofusin 2 (MFN2) have been implicated in regulating mitochondrial morphology (i.e., balancing fusion and fission events) and, when mutated, result in disease (Burte et al., 2015). Moreover, data suggest that tau and Aβ can influence the regulation of mitochondrial dynamics through proteins such as dynamin-related protein 1 (DRP1), which is thought to alter processes of mitochondrial fission in AD. In particular, studies have focused on interactions of DRP1 with Aβ (Manczak, Calkins, & Reddy, 2011). In a study by Manczak et al. (2011), it was found that Aβ interacts with DRP1 in the frontal cortex of AD subjects and in the cerebral cortex of AβPP/PS1 transgenic mice. It was also found that the interaction between DRP1 and Aβ increased as AD progressed.

In addition, Drp1 knockdown has been shown to lead to mitochondrial elongation and apoptosis (DuBoff, Feany, & Gotz, 2013). Altering the downregulation of DRP1 also has an effect on reducing mitophagy, the clean-up mechanism for the cell. Other studies have suggested that Aβ and phosphorylated tau interact with DRP1 causing excessive fragmentation of mitochondria (Kandimalla & Reddy, 2016).

3.3 Intracellular symbiont transmission from the soma to the germline involves cell-to-cell transfer

Obligate endosymbiosis is at a very high frequency among hemipteran insect taxa, such as aphids, mealybugs, whiteflies, and planthoppers, due to an exclusive diet of nutrient-poor plant fluids (Hansen & Moran, 2014). In these associations, symbionts provide metabolites such as amino acids to supplement the host's nitrogen-poor diet (Douglas, 2016). The majority of these taxa host one primary symbiont, which is always present and co-speciates with the host, and one or more secondary symbionts, which are more facultative and have their own evolutionary histories independent of the host. Both primary and secondary symbionts are vertically transmitted in these associations (Douglas, 2016), but they may have different cellular routes of inheritance (Koga et al., 2012). In many of these associations, intracellular symbionts pass from adult bacteriocyte cells to gametes or embryos through cell-to-cell transfer mechanisms. Below, we describe what is known about the best studied of these associations.

In pea aphids, Acyrthosiphon pisum, females reproduce by either sexual or asexual reproduction depending on the season. Over the summer months, many asexual generations are produced via telescoping viviparous parthenogenesis (in which adult aphids contain embryonic aphids, containing embryonic aphids), and sexual eggs are produced for overwintering. In aphid sexual reproduction, the gammaproteobacterial endosymbiont Candidatus Buchnera aphidicola is delivered directly to the posterior pole of the oocyte in the final stages of oogenesis via cell-to-cell transfer from the follicle cells (Miura et al., 2003) (Fig. 4). During parthenogenetic embryogenesis, Buchnera are transported in host bacteriocytes to the posterior pole of the blastula (Miura et al., 2003). There, Buchnera are exocytosed from the bacteriocytes and endocytosed by the blastula membrane, and incorporated into the syncytial cytoplasm (Koga et al., 2012). These bacteria localize near host nuclei in the mesodermal syncytium and become enclosed in individual cells during cellularization, producing a new generation of bacteriocytes (Fig. 4). Interestingly, the secondary symbiont Serratia is taken up by the blastula from the hemolymph, and sorts separately from Buchnera during cellularization (Koga et al., 2012). During later stages of embryogenesis, bacteriocytes cluster together, forming a paired bacteriome organ that remains in close proximity to the germ cells throughout development, maintaining this position in the adult (Koga et al., 2012; Miura et al., 2003). As a limited number of Buchnera are transferred in either reproduction mode, the vertical transmission process imposes a fairly harsh bottleneck on within-host symbiont population sizes (Mira & Moran, 2002).

Appropriation of symbionts to the germline or embryo from somatic tissues late in development is a common strategy for vertical transmission, and routes between tissues can be complex. For example, the Arsenophonus-like bacterial endosymbiont of human lice, Candidatus Riesia pediculicola (Allen, Reed, Perotti, & Braig, 2007), completes a complex pattern of migration across host cells and tissues during development, crossing extracellularly from germline in embryogenesis to the soma during the nymphal stages, and back to the ovary in the adult. There, symbionts pass into fully developed eggs through hydropyle structures in the shell (Perotti, Allen, Reed, & Braig, 2007). Similarly, during embryogenesis the fat-body bacteroidetes symbionts of cockroaches and the related basal termites (genus Mastotermes) migrate extracellularly from the embryonic bacteriome to pre-bacteriocyte cells in the fat body (Bandi et al., 1995; Lambiase, Grigolo, Laudani, Sacchi, & Baccetti, 1997), and then migrate from there to the ovary in the second nymphal instar (Sacchi et al., 1988). During the third and fourth instars, symbionts exit bacteriocytes and migrate extracellularly across the ovariole sheath, between the follicle cells, and to the plasma membrane of the oocyte. There they are surrounded by microvilli until after vitellogenesis when these bacteria are taken up by oocytes via pseudopod-like extensions (Sacchi et al., 1988). In Camponotus floridanus carpenter ants, endosymbionts also have a dynamic pattern of migration during development, ending up in the midgut prior to metamorphosis. They are thought to migrate from this tissue to the ovary (Stoll, Feldhaar, Fraunholz, & Gross, 2010), colonizing oocytes shortly after division from the stem cell (Kupper, Stigloher, Feldhaar, & Gross, 2016). Lastly, in one of the more bizarre localization patterns reported, the gammaproteobacterial symbionts of mealybugs reside within a second betaproteobacterial endosymbiont, and are transported to oocytes in this configuration. The nested symbiont cells are transported within bacteriocytes from the symbiont-housing organ (bacteriome) in the abdomen to the ovary. There, they are released from host cells, cluster around the connection between the oocyte and supporting cells (similar to scale insects, Michalik et al., 2018), and are taken into the germline at this point (von Dohlen, Kohler, Alsop, & McManus, 2001).

Interestingly, several associations demonstrate the phenomenon of the “symbiont ball,” where symbionts cluster together in a clumped, ball-shaped structure during transmission and embryogenesis. While some symbiont balls are bound by the oocyte membrane (Michalik et al., 2018), the examples below are not. In brown planthoppers, yeast-like symbionts appear to migrate from the adult fat body to the ovary, passing between follicle cells to enter the posterior oocyte cytoplasm in late oogenesis. There, they form a ball of symbionts that migrates, and ultimately colonizes the fat body in the embryonic abdomen (Nan et al., 2016). Similarly, the Carsonella and Profftella symbionts of the Asian citrus psyllid, Diaphorina citri, migrate extracellularly from the abdomen to the oocyte where they pass between the follicle cells and are incorporated in the oocyte as a ball (Dan, Ikeda, Fujikami, & Nakabachi, 2017). In the stink bugs Nysius ericae, Nysius plebius, and Nithecus jacobaeae, bacteriocytes containing gammaproteobacterial symbionts exist within membranes adjoining those of previtellogenic oocytes, and transfer symbionts across the membranes. As oocytes mature, symbionts form a ball at the oocyte anterior (Matsuura et al., 2012; Swiatoniowska, Ogorzalek, Golas, Michalik, & Szklarzewicz, 2013). Although it is not its normal distribution, a symbiont ball can also be seen in wMel-infected Drosophila melanogaster when symbiont transport via host microtubules is increased (Russell, Lemseffer, & Sullivan, 2018).

Passage of symbionts between follicle cells for direct uptake by vitellogenic host oocytes is another common theme in ovarial symbiont transmission strategies, as described above for scale insects, termites/cockroaches, planthoppers, and psyllids, and has been shown for the adelgid aphid Adelges viridis and its betaproteobacterial symbionts (Michalik, Gołas, Kot, Wieczorek, & Szklarzewicz, 2013). Fortunately, this is also how the Spiroplasma symbionts of the model organism Drosophila melanogaster are transmitted, enabling experiments to determine the underlying molecular and cellular mechanisms. Work by Herren et al. has shown that in infected D. melanogaster, Spiroplasma colonize the oocyte following extracellular transport from the hemolymph. After passing between the follicle cells of vitellogenic oocytes (stages 8–10), symbionts are endocytosed with yolk granules and use the Yolkless receptor involved in normal yolk uptake from follicle cells (Herren, Paredes, Schupfer, & Lemaitre, 2013). Given the diversity of symbionts that enter the oocyte through the peri-follicular space (Dan et al., 2017; Miura et al., 2003; Nan et al., 2016), the high yolk content of insect embryos (Izumi, Yano, Yamamoto, & Takahashi, 1994), and that intracellular pathogens have also been found to co-opt the yolk machinery for ovarial transmission (Herren et al., 2013), this is a potential mechanism for other endosymbionts.

Infection of oocytes in pre-vitellogenic stages of oogenesis is observed in other associations in addition to those in stink bugs. In the bulrush bug, Chilacis typhae, symbionts are housed in bacteriocyte-like cells in the midgut epithelium as well as in the ovary germaria. Symbionts enter oocytes from the surrounding cells near the posterior of the germarium (Kuechler, Dettner, & Kehl, 2011). Other examples of transfer to pre-vitellogenic oocytes may exist, however, resolving the position of symbionts in the germarium's dense tissue structure may limit the detection of this transmission route.

The full and precise details about how symbionts colonize host tissues during host development are not known for many associations, but much can be inferred from their localization patterns in adults. For example, Rhipicephalus spp. ticks host Coxiella sp. symbionts, which are present in the malpighian tubules of both males and females, as well as in the female gonad. While it is unclear when in development the Coxiella symbionts migrate to the ovary, Lalzar et al. showed that they are at high concentration in the oviduct and interstitial ovary cells, and associate with host oocytes beginning in mid-oogenesis. Interestingly, Coxiella concentrate at opposite poles during mid-oogenesis (stage 3) and become restricted to one pole by late oogenesis (stages 4–5) (Lalzar et al., 2014). Based on studies in Drosophila, this suggests that these symbionts may rely on the host actin and microtubule cytoskeleton and microtubule motor proteins (Ferree et al., 2005; Russell, Lemseffer, & Sullivan, 2018; Serbus & Sullivan, 2007).

B Rhizoxin

An interesting example of endo-symbiosis between a fungus and a bacterium has been discovered in the case of rice seedling blight where the toxic metabolite, rhizoxin (Figure 5.4), originally isolated from the contaminating Rhizopus fungus, was initially thought to be produced by a symbiotic Burkholderia bacterial species [109,110]. This unexpected finding revealed a complex symbiotic-pathogenic relationship, extending the fungal–plant interaction to a third, key bacterial player, thereby offering potentially new avenues for pest control. This observation was consistent with the discovery of four Rhizopus species that would produce rhizoxin on fermentation and two others in the same genus that did not. Later work using combinations of Rhizopus strains that either produced rhizoxin or stopped at the monoepoxy compound WF-1360F (Figure 5.4), which was originally thought to be a by-product of biosynthesis, demonstrated that, by mixing and matching fungal hosts (strains that either produced rhizoxin or only WF-1360F) and the endosymbiotic bacterium from each, the Burkholderia bacterium produced the monoepoxy precursor, and then the fungus produced rhizoxin via a second fungal-specific interaction. These results led to a revison of the bipartite system to a novel tripartite system, and aptly demonstrates what can be done at this moment in time to “interrogate” biochemical processes [111].

7.2 Primary and Secondary Endosymbiosis

A symbiotic relationship where one organism lives inside the other is known as endosymbiosis. Primary endosymbiosis refers to the original internalization of prokaryotes by an ancestral eukaryotic cell, resulting in the formation of the mitochondria and chloroplasts. Two membranes surround mitochondria and chloroplasts. The inner one is derived from the bacterial ancestor and the outer “mitochondrial” or “chloroplast” membrane is actually derived from the host-cell membrane. However, several lineages of protozoans appear to have engulfed other single-celled eukaryotes, in particular algae. Several groups of algae therefore have chloroplasts acquired at second-hand by what is termed secondary endosymbiosis.

In contrast to the typical two membranes of primary organelles, four membranes surround chloroplasts obtained by secondary endosymbiosis. In most cases, the nucleus of the engulfed eukaryotic alga has disappeared without trace. Occasionally, the remains of this nucleus are still to be found lying between the two pairs of membranes (Fig. 26.23). This structure is termed a nucleomorph and can be seen in cryptomonad algae where it represents the remains of the nucleus of a red alga that was swallowed by an amoeba-like ancestor. The nucleomorph contains three vestigial linear chromosomes totaling 550 kb of DNA. These carry genes for rRNA that is incorporated into a few eukaryotic type ribosomes that are also located in the space between the two pairs of membranes.

Cells resulting from secondary endosymbiosis are composites of four or five original genomes. These include the primary ancestral eukaryote nucleus and its mitochondrion, plus the nucleus, mitochondrion, and chloroplast from the secondary endosymbiont. Many genes from the subordinate genomes have been lost during evolution and no trace has ever been found of the secondary mitochondrion. Some genes from the secondary endosymbiont nucleus have been transferred to the primary eukaryotic nucleus. The protein products of about 30 of these are made on ribosomes belonging to the primary nucleus and shipped from the primary eukaryotic cytoplasm back into the nucleomorph compartment. In turn, the nucleomorph contains genes for proteins that are made on the 80 S ribosomes in the nucleomorph compartment and transported across the inner two membranes into the chloroplast. Finally, there are proteins now encoded by the primary nucleus that must be translocated across both sets of double membranes from the primary cytoplasm into the chloroplast!

Box 26.1

Is Malaria Really a Plant?

Malaria is a disease that affects many millions of people world wide and is responsible for two or three million deaths each year, mostly in Africa. Malaria is caused by the single-celled eukaryote Plasmodium. The malaria parasite and other related single-celled eukaryotes are members of the phylum Apicomplexa. Although these parasites live inside humans and mosquitoes, far from the sunlight, they possess plastids as well as mitochondria. These plastids are degenerate, non-photosynthetic chloroplasts with a circular genome. In Plasmodium the plastid DNA is 35 kb and encodes rRNA, tRNA, and a few proteins, mostly involved in translation (Fig. 26.24).

The malarial plastid or “apicoplast” is thought to derive from secondary endosymbiosis. The ancestor of the Apicomplexa appears to have swallowed a single-celled eukaryotic alga that possessed a chloroplast. The algal nucleus has been completely lost, but the plastid was kept and is surrounded by four membranes. Sequence comparisons suggest the malarial apicoplast is most closely related to the chloroplast of red algae.

Although it does not convert light into energy, the apicoplast is essential for the survival of Plasmodium. The apicoplast plays a vital role in lipid metabolism. Several enzymes of fatty-acid synthesis are encoded in the nucleus but translocated into the apicoplast where fatty-acid synthesis occurs. As a result, certain herbicides that prevent fatty-acid synthesis in the chloroplasts of green plants are effective against Plasmodium and other pathogenic apicomplexans such as Toxoplasma and Cryptosporidium. For example, clodinafop targets the acetyl-CoA carboxylase and triclosan inhibits the enoyl ACP reductase of plants and bacteria. These herbicides have no effect on fatty-acid synthesis in animals or fungi. In addition, the herbicide fosmidomycin inhibits the isoprenoid pathway of plants and bacteria, which differs from that of animals. Fosmidomycin inhibits growth of Plasmodium and cures malaria-infected mice. Plasmodium and its relatives are also inhibited by chloramphenicol, rifamycin, macrolides, and quinolones, all of which are antibacterial antibiotics. These are also thought to act via the apicoplast.

Trichoderma + Bacteria = ?

Close associations with bacteria including endosymbiosis have been recently detected in many fungi (Bianciotto et al., 2003; Compant et al., 2008; Coenye et al., 2001; Lim et al., 2003; Partida-Martinez and Hertweck, 2005). Recent advances in molecular ecology and genomics indicate that the interactions of Trichoderma spp. with other organisms such as animals and plants may have evolved as a result of saprotrophy on fungal biomass and various forms of parasitism on other fungi (mycoparasitism), combined with broad environmental opportunism (Druzhinina et al., 2011; see above). Up to date most of Trichoderma–bacteria associations are known to either have a beneficial effect on plant disease protection as combined biocontrol agents in agriculture or Trichoderma secondary metabolites were studied as effectors of bacterial growth. A study showed that application of Trichoderma sp. and several strains of Pseudomonas spp. mixture provided greater suppression of the Gaeumannomyces graminis var. tritici on wheat (Duffy et al., 1996). Further it has been shown that chitinolytic enzymes of Trichoderma sp. enhanced the growth of Enterobacter cloacae in the presence of chitinous substrates and increased the ability of bacterial cells to bind to hyphae of the fungal pathogens (Lorito et al., 1993). Trichoderma spp. were also able to beneficially modify the response of plants to infection by bacteria e.g. Xanthomonas spp., Pseudomonas syringae, etc. (Woo et al., 2006). Recently, Davidson et al. (2009) performed an experimental study on microbial diversity of artificial and natural nests of Camponotus (Colobopsis) cylindricus (COCY) ants which dominate the lowland dipterocarp rain forest on Borneo. They suggested that the filamentous mycoparasitic fungus Trichoderma and bacteria from the nitrogen fixing genus Burkholderia are likely associated with nests of these ants. However, no other study has so far reported about such interactions for Trichoderma.

Endosymbiotic Gene Transfer

One of the important aspects of endosymbiosis is that it can, and does, lead to gene transfer from organelles to the nucleus (Martin et al., 1998; Martin and Herrmann, 1998; Timmis et al., 2004). Ninety years ago, even Wallin sensed that somehow the process of endosymbiosis should be connected to a transfer of genetic material from the organelle to the host. He wrote: “It appears logical, however, that under certain circumstances, [...] bacterial organisms may develop an absolute symbiosis with a higher organism and in some way or another impress a new character on the factors of heredity. The simplest and most readily conceivable mechanism by which the alteration takes place would be the addition of new genes to the chromosomes from the bacterial symbiont” (Wallin, 1925; p. 144). That is a fairly modern formulation of a process that is now called endosymbiotic gene transfer (Martin et al., 1993). About 15–18% of the genes in a higher plant's nuclear genome come from the cyanobacterial antecedent of plastids (Martin et al., 2002; Deusch et al., 2008), and in eukaryotes that lack plastids, such as yeast, the vast majority of genes having prokaryotic homologues come from bacteria, not archaea (Esser et al., 2004; Cotton and McInerney, 2010; Thiergart et al., 2012). The simplest interpretation is that these bacterial genes in nonphotosynthetic eukaryotic lineages come from the mitochondrial ancestor (Pisani et al., 2007; McInerney et al., 2014).

The process of endosymbiotic gene transfer entails the integration of bulk chunks of organellar chromosomes, or in some cases even a whole organelle genome spanning more than 100 kb (Huang et al., 2005). The evidence that this has happened can be seen at the computer by comparing organelle genomes to nuclear genomes (Hazkani-Covo and Covo, 2008) and in laboratory experiments where organelles are transformed with constructs that only become active in the nucleus (Huang et al., 2003, 2004). The mechanism of DNA insertion entails nonhomologous end joining and most eukaryotic genomes are replete with such recent organelle insertions (Hazkani-Covo and Covo, 2008). One might wonder how organelle DNA gets to the nucleus in the first place so that it can recombine. The most likely mechanism is simply stress induced organelle lysis, and there is some evidence for this in plants (Lane, 2011; Wang et al., 2012). Importantly, organelle lysis means that there has to be more than one organelle copy in the cell, one to lyse and one for progeny, and this is the crux of the ‘limited window’ hypothesis (Barbrook et al., 2006).

There is another important aspect to gene transfer to the nucleus. Both at the origin of mitochondria and at the origin of plastids, host, and symbiont possessed a large number of genes for homologous functions. Such genes would include ribosome biogenesis, amino acid biosynthesis, nucleotide biosynthesis, core carbon and energy metabolism, cofactor biosynthesis, and the like. Chloroplasts and mitochondria have both retained their own ribosomes, for example, and divergent members of homologous gene families for ribosomal proteins as one example, but other examples have been well studied, including core carbohydrate metabolism. This phenomenon is called ‘functional redundancy through endosymbiosis’ (Martin and Schnarrenberger, 1997). It generates highly divergent copies of genes homologous to prokaryotes even though they reside on eukaryotic chromosomes.