Biotic Host–Pathogen Interactions As Major Drivers of Plastid Endosymbiosis

TRPLSC 1511 No. of Pages 13

Opinion

Biotic Host–Pathogen Interactions [248_TD$IF]As Major Drivers of Plastid Endosymbiosis Ugo Cenci,1 Debashish Bhattacharya,2 Andreas P.M. Weber,3 Christophe Colleoni,1 Agathe Subtil,4 and Steven G. Ball1,* The plastid originated 1.5 billion years ago through a primary endosymbiosis involving a heterotrophic eukaryote and an ancient cyanobacterium. Phylogenetic and biochemical evidence suggests that the incipient endosymbiont interacted with an obligate intracellular chlamydial pathogen that housed it in an inclusion. This aspect of the ménage-à-trois hypothesis (MATH) posits that [249_TD$IF]Chlamydiales provided critical novel transporters and enzymes secreted by the pathogens in the host cytosol. This initiated the efflux of photosynthate to both the inclusion lumen and host cytosol. Here we review the experimental evidence supporting the MATH and focus on chlamydial genes that replaced existing cyanobacterial functions. The picture emerging from these studies underlines the importance of chlamydial host-pathogen interactions in the metabolic integration of the primary plastid.

Trends Phylogenetic analysis has detected over 50 lateral gene transfers shared between Chlamydiales and the ancestor of all photosynthetic eukaryotes. The ‘ménage-à-trois hypothesis’ proposes that Chlamydiales secreted enzymes required for assimilation of photosynthetic carbon in the cytosol of the eukaryote host of plastid endosymbiosis. Recent evidence suggests that the chlamydial genes involved in photosynthate export from the plastid were donated by conjugation in a common inclusion vacuole.

The Origin and Survival of the Plastid 1.5 billion years ago, an ancestor of extant cyanobacteria was internalized by a heterotrophic eukaryote and stabilized as a photosynthate-exporting symbiont that later evolved into a novel eukaryotic organelle: the plastid [1,2]. The prokaryotic cyanobionts (see [250_TD$IF]Glossary) moved with their initial hosts, the Archaeplastida (red algae, glaucophytes, green algae, and plants) [3,4], to over one-half of basal eukaryotic lineages through subsequent secondary endosymbioses (for review see [1]). All of these photosynthetic lineages radiated into diverse environments such as rain forest canopies, acidic water ponds and rocks, freshwater rivers and lakes, polar ice caps, coral reefs, and the surface and depths of the oceans. These plastid endosymbioses permanently changed the geochemistry of the biosphere by increasing atmospheric oxygen and contributing to primary production, which set the stage for the radiation of animal life. A noteworthy feature of plastid endosymbiosis is that unlike the Rickettsiales-like pathogen ancestors of mitochondria, cyanobacteria are free-living organisms; that is, these phototrophs lack the toolkit to survive the plethora of antibacterial defenses available to eukaryotic phagotrophs [5]. So how did the plastid endosymbiont survive in the cytosol of the Archaeplastida host cell? To address this issue, a recent hypothesis (MATH: ménage-à-trois hypothesis) proposes that the nascent plastids were protected from host antibacterial immunity by their localization within a Chlamydiales-controlled membrane vesicle known as the inclusion (for review see [6]). The inclusion, which is derived from the host plasma membrane, is generated through entry of the chlamydial infectious forms: the elementary bodies (EBs). This inclusion is actively modified by the initial elementary body (for reviews see [7,8]) that differentiates rapidly into the bacterial replicative forms: the reticulate bodies (RBs, Figure 1). Depending on the host status and its existing antibacterial defenses, Chlamydiales may go into active cycles of replication followed by differentiation back to EBs, thereby killing their hosts (for reviews

Trends in Plant Science, Month Year, Vol. xx, No. yy

Glycogen metabolism of extant Chlamydiaceae provides evidence that ancient Chlamydiales were preadapted to generate the carbohydrate flux detailed in the MATH. Analysis of tryptophan metabolism genes in plants suggests that the tryptophan metabolism operon of Chlamydiales was donated through conjugation to the evolving plastid genome to ensure tryptophan export to the inclusion.

1

Université des Sciences et Technologies de Lille, Unité de Glycobiologie Structurale et Fonctionnelle, UMR8576 CNRS-USTL, Cité Scientifique, 59655 Villeneuve d’Ascq Cedex, France 2 Department of Ecology, Evolution, and Natural Resources, Rutgers University, New Brunswick, NJ 08540, USA

http://dx.doi.org/10.1016/j.tplants.2016.12.007 © 2016 Elsevier Ltd. All rights reserved.

1

TRPLSC 1511 No. of Pages 13

3

Institute for Plant Biochemistry, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-HeineUniversity, D-40225 Düsseldorf, Germany 4 Institut Pasteur, Unité de Biologie Cellulaire de l’Infection Microbienne, 25 Rue du Dr Roux, 75015 Paris, France

EB

*

N

*

*$$

*

*

*

EB

1

***

* *** *** **** *** *** ** ** **** *** *** *** * ** ** *** ***

*

*

*

* * *

*

* *

*

*

*

*

*

*

*

*

** *

*

4

*

2

* *

*

*

*

*

* * *

EB RB

***

*

N

*

*

*

*

*

*Correspondence: [email protected] (S.G. Ball).

* *

N

***

*

*

*

*

* *

2.2 3

*

*

*

*

*

*

*

* *

*

N

*

*

*

*

*

* AB

*

* * *

* *

*

*

N

*

* * * * *** * * * * * * * ** ** ** *** ** ***** **** * *** *** ***** * * * * *** ** *** ** * ** * * ** ** * * * * * ** * ** * * * *

AB

*

*

*

*

**

*

Figure 1. The Chlamydial Developmental Cycle. The infectious chlamydia form called elementary body (EB) is displayed in grey. The replicative form called reticulate body (RB) is presented in pink. Glycogen particles are depicted as black asterisks. They are shown to accumulate in EBs, in the inclusion lumen and in the host cytosol. Host glycogen synthesis is induced by chlamydial infection. The inclusion membrane is depicted in blue and the host nucleus (N) in grey. The chlamydial developmental cycle proceeds through four steps: (1) The elementary body (EB) binds to the cell surface and triggers its internalization in the ‘inclusion’, a membrane-bounded compartment derived from the plasma membrane. Pre-synthesized T3SS effectors are secreted, to allow entry and modification of the inclusion membrane. (2) The EB converts into the bacterial replicative form: the reticulate body (RB), which starts dividing. The inclusion escapes digestion by lysosomes. During this stage, the bacteria translocate a large variety of proteins into the inclusion membrane, the inclusion lumen and the host cytosol. (2.2) Depending on the nutritional status of the host, reticulate bodies can enter a quiescent state called ‘persistence’: larger in size, these ‘aberrant bodies’ (AB) remain viable, but do not multiply actively. This path is reversible and the bacteria can re-enter the productive cycle. (3) RBs convert to EBs in an asynchronous manner. (4) The last step of the cycle consists of cell exit, either by cell lysis, or by detachment. The cycle detailed here is overall similar for the animal pathogen Chlamydiaceae bacteria and for those of other Chlamydiales infecting diverse protists. Amoeba-infecting Chlamydiales are thought to be more related to the ancient pathogens that are inferred to have triggered plastid endosymbiosis. These ancient pathogens genomes are thought to be more similar to the comparatively larger genomes of Simkanicaeae or Parachlamydiaceae than to those of the smaller genomes of the animal pathogen Chlamydiaceae. However all Chlamydiales including the animal pathogens are characterized by the same obligate intracellular life cycle.

see [9,10]). Alternatively, they may go into a quiescent stage known as chlamydial persistence. Whereas production of novel EBs is either through host lysis or through budding off of novel infectious bacteria, the quiescent persistence stage is often associated with the presence of abnormal yet viable reticulate bodies (aberrant bodies) in the inclusion [11]. These alternatives are reminiscent of the transition of the lysogenic to lytic stages of temperate phage replication. As with temperate phages, transition between the two stages is triggered through intracellular environmental cues. These are, among others, defined essentially by nutrient starvation [9]. Among the nutrients critical to this phase, iron and tryptophan play central roles [12,13]. In this opinion article, we will recapitulate those findings that laid the foundation for the MATH and

2

Trends in Plant Science, Month Year, Vol. xx, No. yy

TRPLSC 1511 No. of Pages 13

present a modified model. We focus on recent developments regarding chlamydial effector enzymes and transporters responsible for initiating export and assimilation of photosynthetic carbon that were previously absent from both the cyanobiont and host. We will also address, using one example, the nature of other genes that replaced pre-existing cyanobacterial functions. We propose that in this case, host-pathogen biotic interactions also prompted selection of particular chlamydial genes. Finally, we will discuss the possibility that the ancestral pathogen behaved as an intracellular Agrobacterium-like pathogen by interacting not only through secreted protein effectors but also through direct gene transfers between Chlamydiales and the cyanobiont.

The Chlamydial Footprint in Archaeplastida Nuclear Genomes The idea that Chlamydiales might have played a key role in the early stages of plastid endosymbiosis was based on an analysis of the genome of the human pathogen Chlamydia trachomatis. This species encodes 35 (out of 894) genes of apparently eukaryotic provenance. These were initially believed to have derived via lateral gene transfer (LGT) from their hosts. Surprisingly, these foreign genes were most closely related to homologs in plants (i.e., the available data from photosynthetic eukaryotes) rather than to the metazoan hosts of Chlamydiae [14]. This surprising result was explained, not as plant-specific LGTs but rather as ancient transfers from chlamydial sources to the phagotrophic ancestor of the green algae and plants (Viridiplantae). Genome data from other Chlamydiales that infect or have symbiotic relationships with amoebae or animals later validated the initial hypothesis of plant LGT [15–18]. Protochlamydia amoebophila contains over 100 genes related to plants [15], with later analyses showing these LGT-derived sequences to share a closer affinity with amoeba-associated symbionts than with animal-infecting [251_TD$IF]Chlamydiaceae [15,19–21]. This finding was put into a broader perspective when the first red algal genome was determined (from Cyanidioschyzon merolae) and the chlamydial LGTs were found in at least two of the three algal clades in the Archaeplastida [19–21]. These results suggested that many of the LGTs likely occurred in the Archaeplastida common ancestor over a billion years ago [22]. This ancestor is believed to have been a phagotroph feeding on cyanobacteria, and as a consequence must have had accessible plasma membranes, which appears to be a prerequisite for chlamydia penetration. To explain the observation of a substantially larger number of candidate lateral gene transfers in Archaeplastida by comparison to animals or fungi, it was proposed that the pathogens were directly involved in plastid endosymbiosis and were therefore persistent. It should be noted that the number and relevance of chlamydial LGTs and their role in primary endosymbiosis has been challenged and remains contentious among some groups (e.g., [23–25]). The reader is referred to recent reviews and commentaries [5,6,26–28] for an in-depth discussion and rebuttal, using biochemistry-based arguments, of the issues raised about controversial phylogenies.

Foundation of the MATH The reconstruction of ancient metabolic pathways in algae started in earnest with the availability of completed genomes from red and green algae. In 2008, reconstruction of storage polysaccharide metabolism in the Archaeplastida ancestor led to an understanding of the ancient metabolic flux that extracted photosynthate to the host cytosol from the cyanobacterial endosymbiont [25_TD$IF][29–31]. From this reconstruction, it was suggested that photosynthetic carbon was initially exported in the form of the bacterium-specific nucleotide sugar ADP-Glucose (ADP-Glc), through an ancient nucleotide sugar translocator (NST), into the host cytosol (Figure 2A). There it was polymerized into the host glycogen pool by the action of ADP-Glc utilizing glycogen synthase present in green algae and plants. Such enzymes are distinctively found in prokaryotes while eukaryotic glycogen synthases use UDP-Glc as substrate and define enzymes of distinct phylogenetic origins. The host would then be able to use this osmotically inert form of photosynthate through its own glycogen metabolism regulatory

Glossary Archaeplastida: the descendants of an ancient and unique primary plastid endosymbiosis that occurred 1.5 billion years ago. The Archaeplastida consist of the Glaucophyta (glaucophytes), Rhodophyceae (red algae) and Chloroplastida (synonymous with Viridiplantae and Chlorobionta: green algae and land-plants). Chlamydiales: order of obligate intracellular bacteria of the PVC super-group (Planctomyces Verrucomicrobia Chlamydia) consisting of the families Simkaniaceae, Waddliaceae, Parachlamydiaceae, Criblamydiaceae, Rhabdochlamydiaceae and Chlamydiaceae. These bacterial pathogens or symbionts infect animal cells or protists. Chlamydiaceae define animal specific pathogens with smaller genomes than most of the other Chlamydiales. Cyanobiont: A cyanobacterial symbiont that has not yet achieved the status of a true ‘organelle’. Effector: in plant and animal pathology, protein effectors or ‘effectors’ are proteins secreted by the pathogens inside their hosts from within or outside of the target animal or plant cells. The term effector is also used in biochemistry, immunology, and neurobiology with different specific meanings. Lateral gene transfer: the movement of genetic material between organisms other than parent to offspring transmission. LGT is synonymous with horizontal gene transfer (HGT). Merozygote: during bacterial conjugation, homologous bacterial genes of the donor can be functionally redundant with genes present in the recipient bacterium. Because bacterial conjugation can involve distantly related bacteria, the donor and recipient genes do not always qualify as true distinct alleles of the same gene which otherwise defines the heterozygote status in eukaryotes. The term ‘merozygote’ is used in such cases and covers a variety of situations where both versions encoded by the donor and recipient are maintained at least transiently and possibly both expressed. Primary endosymbiosis: consists of internalization and stabilization of a

Trends in Plant Science, Month Year, Vol. xx, No. yy

3

TRPLSC 1511 No. of Pages 13

(A)

(B) *

*

*

*

*

* *

*

*

*

*

*

*

* *

*

*

*

*

*

*

***

*

* *

* ** *** ** ** G-6-P * * * * * * * * * * * UhpC* * ***** *

*

*

*

*

*

*

*

*

*

*

*

*

*

* *

*

Escape O2

CO2 *

*

* * *

* * * *

*

ADP-G

*

*

*

*

*

*

*

*

*

****

*

*

* * * ** G-6-P *

*

*

*

*

** *

* G-6-P * * * ADP-G **** * *** * * ** * * * * NSTADP-G * *

*

*

*

*

**

*

*

*

*

G-6-P

*

*

**

*** ** ***

* * ** * *** * ** UhpC * *** * *

*

*

*

*

*

*

NST

ADP-G *

*

*

* * * ** *

*

*

*

O2

CO2

*

* *

*

*

**

*

*

*

* *

*

*

*

*

ADP-G *

G-6-P

UhpC

*

*

Escape

O2

CO2

* * *

*

*

*

ADP-G

* *

*

NST * * *

*

*

* * ** *

*

G-6-P

*

*

*

*

*

* *

*

*

*

*

*

**

*

Figure 2. Two Representations of the [24_TD$IF]Ménage-à-Trois Hypothesis (MATH). In both cases, eukaryotes were initially repeatedly subjected to Chlamydiales infections. The first model (A) developed by Ball et al. [34] hypothesizes that the Chlamydia-related bacterium (the infectious elementary body depicted in grey) and the cyanobacterium (in green) entered the eukaryotic host separately. In this model, the host-encoded nucleotide sugar transporter (NST, displayed in brown at the surface of the cyanobiont) was by chance targeted to the cyanobacterial inner membrane allowing the export of ADP-glucose from the cyanobacteria to the host cytosol, where it fueled glycogen synthesis by chlamydial cytosolic effectors (see text). Model A requires immediate escape of the cyanobacterium from the phagocytosis vacuole. To account for the presence of UhpC (displayed in orange) on the inner membrane of glaucophytes, this model hypothesizes that the gene encoding this transporter was transferred later from the Chlamydia to the nuclear genome, and that the product was targeted to the cyanobacterium inner membrane. In model (B) presented in [42], both the cyanobacterium and the Chlamydia-related bacterium entered simultaneously in a common Chlamydia-controlled vacuole (the inclusion). The gene encoding UhpC and other chlamydial transporters were transmitted by conjugation to the cyanobacterium, and only later to the nuclear genome. In this model, the host NST recruited by the pathogen on the host-derived inclusion membrane also gave the opportunity to export ADP-glucose to the host cytosol where the chlamydial effectors incorporated it into the host glycogen stores. Escape of the cyanobacterium from the inclusion occurred at a later stage of metabolic integration. Abbreviations: Cy, cyanobacteria; EB, chlamydial elementary body; G6P, glucose-6-phosphate; Mt, Mitochondria; N, Nucleus; NST, nucleotide sugar transporter; RB, chlamydial reticulate body; Pl, Plastid.

4

Trends in Plant Science, Month Year, Vol. xx, No. yy

prokaryotic cell by another free-living organism usually a eukaryote. Rickettsiales: order of obligate intracellular a-proteobacteria consisting of ‘the protomitochondrion’ (the putative ancestor of mitochondria), the Anaplasmataceae (including the insect endosymbiont Wolbachia), the Midichloriaceae and the Rickettsiaceae (including Rickettsia prowazekii, the etiologic agent of epidemic typhus). Most of these bacteria replicate in animals and are transmitted through arthropods. An increasing number of early-diverging, protist-infecting Rickettsiales with larger genomes are currently being described. Secondary endosymbiosis: happens when a product of primary endosymbiosis is internalized and retained by another free-living organism. Type three and four secretion systems (T3SS/T4SS): highly conserved heteromultimeric protein appendages that span all bacterial envelopes (T3SS and T4SS) and secrete proteins within a target prokaryotic or eukaryotic cell through one to several additional membranes. The T4SS transfers both proteins (protein effector secretion) and DNA (bacterial conjugation) while the T3SS secretes only protein effectors.

TRPLSC 1511 No. of Pages 13

networks (Figure 2). Consistent with this hypothesis is the finding of a complete set of eukaryote-derived glycogen metabolism genes in the extant starch metabolism networks of Archaeplastida. This reconstructed flux had the major advantage of obviating the problems of unsynchronized supply and demand of carbon resulting from the merger of two disconnected biochemical networks. Two key unexpected components were required to trigger the appearance of this symbiotic flux. The first was the NST in the inner membrane of the endosymbiont and the second was the prokaryotic ADP-Glc utilizing glycogen synthase. Convincing phylogenetic evidence was produced showing a host provenance of most inner membrane plastid carbon transporters in red and green algae (plus plants) [32]. Furthermore, these host proteins defined NSTs from the host endomembrane system. Biochemical data then showed that a widely distributed host NST could efficiently translocate ADP-Glc despite the fact that this metabolite is absent in eukaryotes [33]. Whereas the prokaryotic type of ADP-Glc utilizing glycogen synthase was initially thought to be of cyanobacterial origin, recent phylogenies support an affiliation with [253_TD$IF]Parachlamydiaceae (e.g., [26,34]). Hence, the enzyme that may have established the initial flux of photosynthates in the host cell appears to be of chlamydial origin [21,25,35]. The presence of ADP-Glc-utilizing glycogen synthase in the host cytosol at the time of plastid endosymbiosis merits explanation. Such an enzyme was not expected to be coded by the host, which most probably used, as do extant eukaryotes, very different enzymes to polymerize glucose from UDP-glc into glycogen. Could this enzyme have originated as a protein, called an effector, that is secreted by the type three secretion system (T3SS) of chlamydial pathogens into the host cytosol to manipulate host polysaccharide stores? This hypothesis was supported by both semi-in vitro assays [34] and thereafter, in vivo studies using human cells infected by Chlamydiaceae [36]. The detailed mode of action of Parachlamydiaceae effectors in manipulating host metabolism has been presented in [34]. The glycogen synthase effector was able to connect the three genomes in a tripartite symbiosis (i.e., the MATH hypothesis). Furthermore, of all chlamydial glycogen metabolism effectors, only two gene products would have been required to establish symbiosis: the glycogen synthase required for photosynthate assimilation, and GlgX, the glycogen debranching enzyme required to feed the chlamydial symbiont which encodes the genes establishing symbiosis (for a more detailed view see [34]). Indeed, GlgX generates small malto-oligosaccharides (MOS) as product of glycogen debranching. Such MOS are not recognized by host enzymes and can thus be imported and used only by the pathogens. All other effectors were likely to have been toxic once symbiosis was established and were therefore selected against. This hypothesis explains many features of plastid endosymbiosis including, of course, the presence of high number of genes of chlamydial phylogenetic origin in the Archaeplastida genomes [34]. This initial MATH model presented in Figure 2A was refined to take into account observations made upon analysis of the first glaucophyte (Cyanophora paradoxa) genome sequence [37]. The C. paradoxa genome data not only confirmed the presence in the host cytosol of the chlamydial glycogen synthase but also demonstrated that glaucophytes export carbon using a Glc-6-P/Pi transporter (UhpC) of likely chlamydial origin [38–40]. A modification of the initial hypothesis was made therefore to account for the presence of all chlamydial and host components (Figure 2B). These include UhpC, now proposed to have been the primordial plastid carbon exporter; nucleotide transporter (NTT), a chlamydial ATP import protein that likely compensates during darkness for loss of plastid energy stores [41] that directly results from carbon export; and both the host NST and the chlamydial glycogen synthase, which are proposed to have helped establish the symbiosis in the host cytosol [42]. The modified model places the cyanobiont within an inclusion vesicle where it exports Glc-6-P into the vesicle (Figure 2B). Within this vesicle, chlamydial-directed glycogen synthesis has been demonstrated to occur [43], whereas host symbiosis is triggered through ADP-glucose export from the inclusion to the host cytosol as previously described. The presence of the NST in the

Trends in Plant Science, Month Year, Vol. xx, No. yy

5

TRPLSC 1511 No. of Pages 13

inclusion membrane instead of the cyanobiont inner envelope is also required in this modified version of the MATH (Figure 2B). The modified model not only accommodates all conflicting biochemical observations but also offers three additional insights. The first is a tenable explanation for the presence of both the chlamydial cell and the cyanobiont, vis-à-vis a unique phagocytosis/infection event. Second, it offers an explanation for the escape of the cyanobiont from lysosomal digestion and/or hostmediated autophagy; that is, Chlamydiae-modified inclusion vesicles are tailored to achieve this end. Third, the colocalization of the cyanobiont and the chlamydial pathogen opened up the door for conjugative transfer of chlamydial genes to the cyanobiont. This is because Simkaniaceae and Parachlamydiaceae, which among Chlamydiales are the closest related donors of the Archaeplastida homologs, harbor a complete and functional conjugation machinery: the Type Four Secretion System (T4SS), thereby facilitating such LGTs. In addition, Simkania negevensis is known to harbor a megaplasmid possessing these conjugative functions as well as ‘cargo’ metabolic functions (including a second glycogen phosphorylase gene) [18]. Furthermore, some evidence for such genetic exchanges between Simkaniaceae, cyanobacteria, green alga chloroplasts and amoeba mitochondria has been reported [44,45]. These gene movements would have allowed the early expression of chlamydial hydrophobic transporters in the inner membrane of the incipient endosymbiont. The modified MATH hypothesis offers therefore an attractive and well-supported scenario for early events in plastid establishment. All chlamydial components of this revised hypothesis are either only found in such pathogens or in other bacteria. Most importantly, they define functions for which no eukaryotic (host) counterpart exists. Some of them, such as GlgX (glycogen debranching enzyme) define major differences in the way glycogen metabolism operates in bacteria and eukaryotes [46].

Extant Glycogen Metabolism Studies of the Human Pathogen Chlamydia trachomatis Support the Modified MATH Hypothesis The modified MATH was proposed with the knowledge that the human pathogen Chlamydia trachomatis accumulates large amounts of glycogen in the inclusion [42]. A recently-published study of glycogen metabolism in C. trachomatis shows that two pathways for glycogen synthesis and mobilization operate in the inclusion [43] (Figure 3). The first, minor pathway consists of glycogen particle import from the cytosol, possibly through internal budding of the inclusion membrane. The second operates through the secretion into the inclusion lumen of the same T3SS effectors that also functioned in the host cytosol. In contrast to other Chlamydiales, ADP-glucose pyrophosphorylase does not appear to be an effector enzyme in C. trachomatis and the enzyme is likely only expressed in the bacteria themselves. Bypassing the need for an ADP-glucose pyrophosphorylase in the inclusion lumen or in the cytoplasm, C. trachomatis glycogen synthase is able to use both UDP-Glc and ADP-Glc as substrate [43]. This is unlike all other ADP-Glc specific prokaryotic enzymes (for review see [47]), with the noticeable exception of the gut bacterium Prevotella bryantii [48] which uses exclusively UDP-Glc. However, Prevotella carries a glycogen synthase gene of likely eukaryotic origin and thus, this enzyme may define a LGT from eukaryotes to gut-bacteria in a fashion similar to that described for amylomaltase [49]. Hence, we suspect that C. trachomatis is synthesizing glycogen from ADPGlc in the bacterium, while it drives glycogen synthesis both in the inclusion and in the cytosol of its host from UDP-Glc. Furthermore, Gehre et al. [43] provided evidence that host UDP-Glc reaches the inclusion lumen via the relocation of the host SLC35D2 UDP-Glc NST, which was observed at the inclusion membrane by immunolabeling. Silencing the expression of this transporter led to a large decrease of glycogen accumulation in the inclusion, strongly supporting the hypothesis that this NST is responsible for UDP-Glc import in the inclusion [43]. Knowing that NSTs operate reversibly, depending on nucleotide–sugar versus nucleotide monophosphate concentrations [50], these results strongly suggest that an ancestral Chlamydiales, using ADP-glucose pyrophosphorylase as an effector, and an inclusion-membrane-localized GDP-

6

Trends in Plant Science, Month Year, Vol. xx, No. yy

TRPLSC 1511 No. of Pages 13

***

* * *

*

*

*

*

*

Glycogen parcles *

*

RB

*

*

* *

EB

N

***

*

* *

* * * *** ** * * * * * * * *** * * * *

*

*

*

* *

GlgX UDP DP-G G UDP-G GlgA

G-1-P GlgP Host GS-GT3

GlgB Glg gB

GlgB SLC35D2

G-6-P GG -6 6--P Pii P

UDP-G

G-1-P + UTP Host UGPase GlgA

Figure 3. Extant Glycogen Metabolism in the Human Pathogen Chlamydia trachomatis. C. trachomatis reticulate bodies (RB) are depicted in pink. These actively replicating bacteria secrete, as effector proteins, enzymes of bacterial glycogen metabolism in both the inclusion and cytosol through the T3SS (depicted in pink on the bacterial envelopes facing the inclusion (in white) or the cytosol (in beige). The enzymes of glycogen synthesis consist of GlgA (glycogen synthase), which elongates glucose chains through a-1,4 linkages from activated sugar–nucleotides (UDP-Glc or ADP-Glc), and GlgB (branching enzyme) which introduces the a-1,6 branches into glycogen. GlgC (ADP-glucose pyrophosphorylase), the enzyme responsible for the synthesis of the bacterial-specific substrate ADP-Glc, is not secreted by the pathogens and remains within the bacteria. Glycogen synthesis in the inclusion occurs by two pathways. One minor pathway consists of the import through vesicle budding of host glycogen from the cytosol (depicted as black particles with bound host glycogen synthase (GS-GT3) depicted as blue circles). The major pathway of glycogen synthesis in the inclusion depends on the chlamydial effectors. The chlamydial glycogen synthase is able to use UDP-Glc that is imported in the inclusion by SLC35D2, a human UDP-Glc transporter recruited to the inclusion membrane. In the cytosol, glycogen metabolism involves both the host and chlamydial effector enzymes. The cytosolic host UDP-glucose pyrophosphorylase defines the sole enzyme responsible for UDP-Glc synthesis used in both the inclusion and the cytosol. The bacterial GlgC (ADP-glucose pyrophosphorylase) is thought to direct synthesis of some glycogen in the elementary bodies (EBs, depicted in grey) as the RBs differentiate back into EBs. Chlamydial effectors of glycogen degradation (GlgP (glycogen phosphorylase) and GlgX (glycogen debranching enzyme)) are also secreted in both the cytosol and the inclusion. GlgP degrades the outer chains of the glycogen granules while GlgX debranches the remaining particle to allow further (Figure legend continued on the bottom of the next page.)

Trends in Plant Science, Month Year, Vol. xx, No. yy

7

TRPLSC 1511 No. of Pages 13

mannose NST (Figure 4), is highly likely to have driven inclusion lumen glycogen synthesis. Such a pathogen would have been preadapted for reversible ADP-Glc export, as hypothesized in the modified MATH.

Biotic Interactions May Explain Many Chlamydial Gene Replacements Evidenced in the Archaeplastida Genomes Given the existence of chlamydial-specific LGTs that played a central role in metabolic symbiosis, we turn now to instances where chlamydial LGTs replaced pre-existing cyanobacterial functions. Were these ‘chance’ replacements given the putative coexistence of the chlamydial and cyanobacterial symbionts in the inclusion, or were they driven by selective forces that inform us about specific host-pathogen biotic interactions? Among the many phylogenomic studies that have been done to date, several report the LGT from Chlamydiales of one or two enzymes involved in tryptophan biosynthesis [18–21,34,51]. Our updated study [52] provides evidence for three LGTs from Chlamydiales to Archaeplastida out of a total of seven protein subunits that catalyze the 5-step enzymatic conversion of chorismate to tryptophan. Three other subunits appear to be of cyanobacterial provenance [51,52]. The catalytic subunit of the rate-controlling first step of tryptophan synthesis regulates the partitioning of chorismate into many different end-products. This enzyme in Archaeplastida has been suggested to have originated through LGT from [254_TD$IF]Planctomycete bacteria [51,52]. That the gene encoding this enzyme has experienced very few exchanges in cyanobacteria is suggested by the finding that unlike other enzymes of tryptophan metabolism, all available cyanobacterial sequences form a monophyletic group that largely mirrors the 16S RNA phylogeny with no evidence of LGTs from other bacteria. This can be explained by the fact that the cyanobacterial enzyme has evolved to integrate metabolic signals specific to the photosynthetic physiology of cyanobacteria, resulting in strong conservation of enzyme regulation. The replacement of this enzyme in Archaeplastida by a [254_TD$IF]Planctomycete homolog therefore supports the intriguing scenario that the cyanobiont lost control of anthranilate synthesis early on in the endosymbiotic relationship. Tryptophan is by far the most energy-costly amino acid to synthesize [53]. Availability of tryptophan has recently been shown to be a major determinant impacting amino-acid protein composition in all Chlamydiales (Figure 5) [12,13]. A convincing case has been made that the proteins involved in rapid pathogen replication (UhpC, NTT, TyrP . . . ) are under high tryptophan content selection [12], whereas those involved in the state of persistence are under low tryptophan content selection (Figure 5). [25_TD$IF]Host and pathogens have evolved a series of biotic interactions based on tryptophan starvation and starvation evasion (for review see [13][256_TD$IF]). Although the tryptophan starvation mechanisms described for Chlamydiaceae are likely to reflect very recent innovations in mammals, a variety of mechanisms tending to the same end have likely evolved in distinct lineages[257_TD$IF]. Given these observations, it has been suggested that Chlamydiales donated the entire chlamydial tryptophan operon by conjugative transfer to the cyanobiont, together with the aforementioned UhpC and NTT transporter genes [52]. This would increase the flux to tryptophan which had previously been under cyanobacterial control. The operon existed in a merozygote status that increased flux to this amino acid without initially losing the cyanobacterial enzymes that may have been involved in other essential interactions. Why would the symbiont/pathogen increase this flux while it was in a persistent phase of replication? One possible explanation is the abundance of tryptophan-rich carbohydrate and ATP transporters (Figure 5) required on the cyanobiont inner membrane which could have necessitated large tryptophan pool sizes. Alternatively, there may have been a need to extract tryptophan from the cyanobiont to obviate exacerbated amino-acid starvation induced by host antibacterial defenses. The aromatic amino-acid transporter of b and g proteobacteria, TyrP, is also considered the major tryptophan transporter responsible for digestion by GlgP. In this process short malto-oligosaccharides (depicted as small black lines) are produced which can only be used by the pathogens. At variance with most prokaryotic glycogen synthases, the C. trachomatis GlgA enzyme was proven to use both UDP-Glc and ADP-Glc efficiently.

8

Trends in Plant Science, Month Year, Vol. xx, No. yy

TRPLSC 1511 No. of Pages 13

***

* * *

*

*

*

*

EB RB

*

N

***

*

Glycogen parcles

*

* *

*

* *

*

*

* * * ** * * * * * * * *** * * *

*** *

*

*

* * *

GlgX

ADP-G DP-G GlgA g

G-1-P GlgP

GlgB Glg gB

GlgB

NST ADP-G

G-6-P GG -66-P Pii P

GlgC G-1-P + ATP GlgA

Figure 4. R [24_TD$IF] econstituted Glycogen Metabolism in the Hypothetical Chlamydiales-Related Pathogens Involved in the MATH. Chlamydiales genes that are the most closely related to homologs in Archaeplastida are most often present in Simkaniaceae and Parachlamydiaceae rather than in the animal pathogens (including C. trachomatis depicted in Figure 3). Unlike C. trachomatis, Parachlamydiaceae have been reported to use ADP-glucose pyrophosphorylase (GlgC) as an effector enzyme suggesting that ADP-Glc rather than UDP-Glc may be used as substrate for glycogen synthesis in the inclusion and in the bacteria. If we interpret the results summarized in Figure 3, which displays the situation in extant Chlamydiaceae (Chlamydia trachomatis), and extrapolate this to what could have happened in a hypothetical ancestral pathogen that used ADP-Glc as sole substrate, we obtain [245_TD$IF]Figure 4. This reconstruction is likely to be identical to what could be happening today in Parachlamydiaceae but this still needs to be demonstrated. In this latter figure, the reticulate bodies secrete all enzymes of glycogen metabolism (synthesis and degradation) in both the inclusion lumen and the host cytosol. ADP-Glc synthesis occurs predominantly in the cytosol where the substrates of GlgC (ATP and glucose-1P) are abundant. ADP-Glc enters the inclusion through a host membrane associated purine–sugar NST (possibly a GDP–mannose translocator able to transport ADPGlc efficiently) that supports inclusion glycogen synthesis. As in Figure 3, RBs are depicted in pink and EBs in grey. The host cytosol is shown in beige and the inclusion in white, whereas the inclusion membrane is depicted in blue. [246_TD$IF]Figure 4 thus displays the hypothetical function of glycogen metabolism in those chlamydiales that would later trigger the ménage à trois. If a cyanobacterium enters together with such a pathogen in the same inclusion it becomes easy to envision how MATH could have been established [42]. The increase of ADP-Glc in the inclusion triggered by the cyanobacterium would have reversed the flux through the inclusion membrane NST, thereby feeding cytosolic glycogen and establishing symbiosis as detailed in [42].

Trends in Plant Science, Month Year, Vol. xx, No. yy

9

Ch l gal amyd lina i a cea

Proto c naeg hlamydia leriop hila

Chlamy dia muridaru m

ia myd Chla suis a ydi lam Ch i s a ibi d hil op yd iae l a m on C h eum pn

Ch pe lam co yd ru ia m

Chlamydia trachomas

(A)

Protoch la amoeb mydia ophila

TRPLSC 1511 No. of Pages 13

di a e my eba a l o ch ra am Pa anth ac

Chlamydia avium

Rub

i la

Wa ch d d l i on a dr o ph il

Chlamydop hila abortus

Chlam y psia dia ci

Verrucomicrobia /Planctomycetes

C h la felis mydop h

ila

ph

ia yd s a m ensi ibl C r uan q se an i a Simk vensis nege

o yd lam e h C via ca

Neochlamydia sp.

idu

sm

ass

il i e n

sis

a

(B) trpE

trpG

trpD

trpC

trpF

trpB

trpA

aroH

trpR

Figure 5. Chlamydiales Phylogeny and Tryptophan Metabolism. (A) Schematic phylogenetic tree of the order Chlamydiales, adapted from [247_TD$IF][62], based on a concatenation of 31 markers genes. The animal pathogens (Chlamydiaceae) characterized by their restrictive host spectra and small genomes (often < 1000 genes) are highlighted by a blue line while the so called ‘environmental Chlamydiales’ with their wider host infection spectra and larger genomes (between 1000 to 2000 genes) are highlighted by a green line. All Chlamydiales are obligate intracellular pathogens with a life cycle analogous to that described in Figure 1. (B) Organization of Simkania negevensis tryptophan operon. The gene nomenclature followed is that of E. coli with trpE trpG trpD trpC trpF trpB and trpA encoding the first [anthranilate synthase subunits a and b (respectively TrpE and TrpG)], second [anthranilate phosphoribosyltransferase (TrpD)], fourth [indoleglycerol phosphate synthase (TrpC)], third [N-phosphoribosylanthranilate isomerase (TrpF)] and fifth [tryptophan synthase a and b subunits (respectively TrpA and TrpB)] steps of the pathway. TrpE is often called the catalytic subunit of anthranilate synthase and carries the sequences required for allosteric regulation of the enzyme activity. Anthranilate synthase is rate-controlling for tryptophan synthesis from chorismate. Tryptophan is considered as the most energy-costly amino acid to synthesize [53]. Hence, many Chlamydiales rely entirely on their host for tryptophan supply and have lost the tryptophan operon. Chlamydiaceae however most often lack the first step of the pathway (anthranilate synthase) while a complete tryptophan operon is presently only reported for Simkania negevensis. Studies on tryptophan content of Chlamydiales proteomes have highlighted that those genes that are required for active multiplication are selectively enriched in tryptophan residues by comparison to those genes required for maintenance of the quiescent stage (persistence). Hence the intracellular pool of tryptophan is a cue for the pathogens to trigger either persistence or the lytic cycle. Among the functions relevant to this opinion that are subjected to TRP-up selection prior to diversification of the Chlamydiales, are the TyrP tryptophan transporters and ATP and other related nucleotide triphosphate transporters (NTT) [13]. UhpC underwent TRP-up selection during Chlamydiaceae diversification [13]. Recently evolved mechanisms of tryptophan starvation implemented by animal cells have been described. It is plausible that a diversity of mechanisms tailored to achieve the same end have evolved repeatedly in other eukaryotes.

amino-acid import in Chlamydiales [13]. TyrP had been found as a candidate LGT from Chlamydiales to Archaeplastida [20,52]. Hence plastid endosymbiosis was selected, as expected, through photosynthate export but may also have involved the supply of costly amino acids, putatively coupled to nitrate reduction and ammonium assimilation. It may have also supplied other very specific compounds required by the pathogens. The energy constraints placed on the cyanobiont were therefore significant, leading to deregulated

10

Trends in Plant Science, Month Year, Vol. xx, No. yy

TRPLSC 1511 No. of Pages 13

photosynthesis and possibly photooxidative stress. It is remarkable that ATP starvation at night, which resulted from photosynthate export and the consequent depletion of carbon and energy stores, was resolved by the LGT of yet another transporter of chlamydial origin: the ATP import protein [54]. We expect that other Chlamydiales LGTs will yield analogous stories of host– pathogen biotic interactions that play key roles in metabolic integration of the nascent plastid and thereby Archaeplastida genome evolution.

Concluding Remarks: Was There a Single Path toward Primary Plastid Origin? Based on the evidence and ideas presented above, we propose that the genome of the captured cyanobacterial endosymbiont became highly chimeric and enriched at the merozygote stage with operons and individual genes of chlamydial origin. However, this is not the case for the only other known case of primary plastid endosymbiosis in the thecate amoeba Paulinella chromatophora [55–57]. In this instance, it has been demonstrated using single cell genomics with wild-caught cells of Paulinella ovalis (a phagotrophic sister lineage of P. chromatophora) that it feeds on members of the same bacterial lineage (a-cyanobacteria) that gave rise to the chromatophore (plastid) [58,59]. These data connect phagotrophy of specific prey to primary plastid origin in closely-related taxa. The current evidence suggests that chromatophore establishment in Paulinella, which occurred ca. 90–140 million years ago, is explained by cessation of the well-established phagocytic process in these amoebae, and not a chlamydial-type interaction [59–61]. Other contrasting traits relative to Archaeplastida are that the chromatophore is present in a lineage with a restricted environmental distribution and low abundance, and the organelle has not spread to other taxa through secondary endosymbiosis. These observations may be explained by the relatively lower fitness of the photosynthetic amoebae when compared to other algae and plants due to its high light sensitivity and less developed metabolic integration into the host cell [56,60]. This latter point is highlighted by the chromatophore gene inventory, which at 867 protein-coding genes is over five-fold greater than in Archaeplastida plastids; that is, most critical chromatophore functions are primarily organelle encoded. In addition, phylogenetic analysis of chromatophore genes provides no support for extensive [258_TD$IF]LGT (a single well-supported case is found in P. chromatophora for a YGGT family membrane protein) from noncyanobacterial sources [259_TD$IF][56,61]. Hence, Paulinella primary plastid origin likely did not rely on pre-adaptations encoded on the endosymbiont genome. It is expected that terminated host–prey interactions are common in nature, and were also occurring at or before the time of the primary endosymbiosis that led to the Archaeplastida. Yet, despite the unlimited potential that exists for generating the Archaeplastida type of primary plastid, this organelle is the putative single ‘winner’ that has spread throughout the tree of life. It is possible therefore, that the intracellular Agrobacterium-like biotic interactions discussed here define a significant level of pre-adaptation that facilitated the success, vis-àvis plastid metabolic integration, in Archaeplastida. Significantly, the only other energetic organelle of primary endosymbiotic origin, the mitochondrion, is also derived from a highly pre-adapted ancestor. It is now widely accepted that the mitochondrion was derived from a Rickettsiales-like intracellular bacterium that had co-evolved with the nascent eukaryote host [5]. Given these diverse sources of data, we propose that some aspects of organellogenesis are shared across different origins (e.g., well-established means of entry into the host cell, metabolic integration, and genome reduction post-endosymbiosis), whereas others will diverge depending on the nature of the particular biotic interaction, leading to different outcomes (see Outstanding Questions).

Outstanding Questions How long did the tripartite symbiotic relationship between host, cyanobiont, and a [249_TD$IF]Chlamydiales pathogen persist before the pathogen was eliminated? Are all extant Archaeplastida free from chlamydial infections or can the pathogens be found in algal lineages with exposed membranes? Can we explain the divergence of the three Archaeplastida lineages by a temporal series of independent losses of the [249_TD$IF]Chlamydiales partner (glaucophytes first, then red algae, then the green lineage)? Does the increasing number of [249_TD$IF]Chlamydiales gene transfer events from glaucophytes to the green lineage support this scenario? How many additional chlamydial contributions to plastid origin can be identified by deeper sampling of chlamydial genomes? What are the functions of currently unknown gene products of chlamydial provenance and how did these contribute to the establishment of endosymbiosis? Are there other examples of multipartite intracellular symbioses and how do they inform our understanding of the formation of plastid endosymbiosis? Will it become possible to recapitulate experimentally the MATH scenario by applying the growing synthetic biology toolkit?

Acknowledgments U[261_TD$IF].C., S.B., C.C., and A.S. were supported by the CNRS, the Université de Lille CNRS, the Institut Pasteur and the ANR grants ‘Expendo’ and ‘Ménage à Trois’. D[265_TD$IF].B. was supported by NSF grants MGSP 0625440 and MCB 0946528, and A[26_TD$IF].W. was supported by German Research Foundation grants CRC-TR1, CRC 1208, and EXC 1028.

Trends in Plant Science, Month Year, Vol. xx, No. yy

11

TRPLSC 1511 No. of Pages 13

References 1. McFadden, G.I. (2014) Origin and evolution of plastids and photosynthesis in eukaryotes. Cold Spring Harb. Perspect. Biol. 6, a016105 2. Rockwell, N.C. et al. (2014) Primary endosymbiosis and the evolution of light and oxygen sensing in photosynthetic eukaryotes. Front. Ecol. Evol. Published online February 27, 2015. http://dx.doi.org/10.3389/fevo.2014.00066 3. Rodriguez-Ezpeleta, N. (2005) Monophyly of primary photosynthetic eukaryotes: green plants, red algae, and glaucophytes. Curr. Biol. 15, 1325–1330 4. Chan, C.X. et al. (2011) Red and green algal monophyly and extensive gene sharing found in a rich repertoire of red algal genes. Curr. Biol. 21, 328–333 5. Ball, S.G. et al. (2016) Pathogen to powerhouse. Science 351, 659–660 6. Ball, S.G. et al. (2015) Toward an understanding of the function of Chlamydiales in plastid endosymbiosis. Biochim. Biophys. Acta 1847, 495–504 7. Cossé, M.M. et al. (2016) One face of Chlamydia trachomatis: the infectious elementary body. Curr. Top. Microbiol. Immunol. 12, 1– 24 8. Subtil, A. et al. (2014) Tracing the primordial Chlamydiae: extinct parasites of plants? Trends Plant Sci. 19, 36–43 9. Abdelrahman, Y.M. and Belland, R.J. (2005) The chlamydial developmental cycle. FEMS Microbiol. Rev. 29, 949–959 10. Omsland, A. et al. (2014) Chlamydial metabolism revisited: interspecies metabolic variability and developmental stage-specific physiologic activities. FEMS Microbiol. Rev. 38, 779–801 11. Kebbi-Beghdadi, C. et al. (2011) Permissivity of Vero cells, human pneumocytes and human endometrial cells to Waddlia chondrophila. Microbes Infect. 13, 566–574 12. Lo, C.C. et al. (2012) The alternative translational profile that underlies the immune-evasive state of persistence in Chlamydiaceae exploits differential tryptophan contents of the protein repertoire. Microbiol. Mol. Biol. Rev. 76, 405–443

26. Qiu, H. et al. (2013) Assessing the bacterial contribution to the plastid proteome. Trends Plant Sci. 18, 680–687 27. Ball, S.G. and Greub, G. (2015) Blurred pictures from the crime scene: the growing case for a function of Chlamydiales in plastid endosymbiosis. Microbes Infect. 17, 723–726 28. Ball, S.G. et al. (2016) Commentary: plastid establishment did not require a chlamydial partner. Front. Cell Infect. Microbiol. 6, 43 29. Deschamps, P. et al. (2008) Metabolic symbiosis and the birth of the plant kingdom. Mol. Biol. Evol. 25, 536–548 30. Deschamps, P. et al. (2008) The relocation of starch metabolism to chloroplasts: when, why and how. Trends Plant Sci. 13, 574–582 31. Ball, S. et al. (2011) The evolution of glycogen and starch metabolism in eukaryotes gives molecular clues to understand the establishment of plastid endosymbiosis. J. Exp. Bot. 62, 1775–1801 32. Weber, A.P. et al. (2006) Single, ancient origin of a plastid metabolite translocator family in Plantae from an endomembranederived ancestor. Eukaryot. Cell 5, 609–612 33. Colleoni, C. et al. (2010) Phylogenetic and biochemical evidence supports the recruitment of an ADP-glucose translocator for the export of photosynthate during plastid endosymbiosis. Mol. Biol. Evol. 27, 2691–2701 34. Ball, S.G. et al. (2013) Metabolic effectors secreted by bacterial pathogens: essential facilitators of plastid endosymbiosis? Plant Cell 25, 7–21 35. Brinkman, F.S. et al. (2002) Evidence that plant-like genes in Chlamydia species reflect an ancestral relationship between Chlamydiaceae, cyanobacteria, and the chloroplast. Genome Res. 12, 1159–1167 36. Lu, C. et al. (2013) Chlamydia trachomatis GlgA is secreted into host cell cytoplasm. PLoS One 8, e68764 37. Price, D.C. et al. (2012) Cyanophora paradoxa genome elucidates origin of photosynthesis in algae and plants. Science 335, 843–847

13. Bonner, C.A. et al. (2014) Chlamydia exploit the mammalian tryptophan-depletion defense strategy as a counter-defensive cue to trigger a survival state of persistence. Front. Cell. Infect. Microbiol. 4, 17

38. Schlichting, R. and Bothe, H. (1993) The cyanelles (organelles of a low evolutionary scale) possess a phosphate-translocator and a glucose-carrier in Cyanophora paradoxa. Botanica Acta 106, 428–434

14. Stephens, R.S. et al. (1998) Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282, 754–759

39. Schwoppe, C. et al. (2002) Properties of the glucose-6-phosphate transporter from Chlamydia pneumoniae (HPTcp) and the glucose-6-phosphate sensor from Escherichia coli (UhpC). J. Bacteriol. 184, 2108–2115

15. Horn, M. et al. (2004) Illuminating the evolutionary history of chlamydiae. Science 304, 728–730 16. Bertelli, C. et al. (2015) Sequencing and characterizing the genome of Estrella lausannensis as an undergraduate project: training students and biological insights. Front. Microbiol. 6, 101 17. Bertelli, C. et al. (2010) The Waddlia genome: a window into chlamydial biology. PLoS One 5, e10890 18. Collingro, A. et al. (2011) Unity in variety – the pan-genome of the Chlamydiae. Mol. Biol. Evol. 28, 3253–3270 19. Huang, J. and Gogarten, J.P. (2007) Did an ancient chlamydial endosymbiosis facilitate the establishment of primary plastids? Genome Biol. 8, R99

40. Facchinelli, F. et al. (2013) Proteomic analysis of the Cyanophora paradoxa muroplast provides clues on early events in plastid endosymbiosis. Planta 237, 637–651 41. Tyra, H.M. et al. (2007) Host origin of plastid solute transporters in the first photosynthetic eukaryotes. Genome Biol. 8, R212 42. Facchinelli, F. et al. (2013) Chlamydia, cyanobiont, or host: who was on top in the ménage à trois? Trends Plant Sci. 18, 673–679 43. Gehre, L. et al. (2016) Sequestration of host metabolism by an intracellular pathogen. Elife 16, 5

20. Becker, B. et al. (2008) Chlamydial genes shed light on the evolution of photoautotrophic eukaryotes. BMC Evol. Biol. 8, 203

44. Everett, K.D. et al. (1999) An unspliced group I intron in 23S rRNA links Chlamydiales, chloroplasts, and mitochondria. J. Bacteriol. 181, 4734–4740

21. Moustafa, A. et al. (2008) Chlamydiae has contributed at least 55 genes to Plantae with predominantly plastid functions. PLoS One 3, e2205

45. Haugen, P. et al. (2007) Cyanobacterial ribosomal RNA genes with multiple, endonuclease-encoding group I introns. BMC Evol. Biol. 7, 159

22. Yoon, H.S. et al. (2004) A molecular timeline for the origin of photosynthetic eukaryotes. Mol. Biol. Evol. 21, 809–818

46. Cenci, U. et al. (2014) Transition from glycogen to starch metabolism in Archaeplastida. Trends Plant Sci. 19, 18–28

23. Dagan, T. et al. (2013) Genomes of Stigonematalean cyanobacteria (subsection V) and the evolution of oxygenic photosynthesis from prokaryotes to plastids. Genome Biol. Evol. 5, 31–44

47. Roach, P.J. et al. (2012) Glycogen and its metabolism: some new developments and old themes. Biochem. J. 441, 763–787

24. Deschamps, P. (2014) Primary endosymbiosis: have cyanobacteria and Chlamydiae ever been roomates? Acta Soc. Bot. Pol. 83, 291–302 25. Domman, D. et al. (2015) Plastid establishment did not require a chlamydial partner. Nat. Commun. 6, 6421

12

Trends in Plant Science, Month Year, Vol. xx, No. yy

48. Lou, J. et al. (1997) Glycogen biosynthesis via UDP-glucose in the ruminal bacterium Prevotella bryantii B1(4). Appl. Environ. Microbiol. 63, 4355–4359 49. Arias, M.C. et al. (2012) Eukaryote to gut bacteria transfer of a glycoside hydrolase gene essential for starch breakdown in plants. Mob. Genet. Elements 2, 81–87

TRPLSC 1511 No. of Pages 13

50. Martinez-Duncker, I. et al. (2003) The nucleotide-sugar transporter family: a phylogenetic approach. Biochimie 85, 245–260 51. Reyes-Prieto, A. and Moustafa, A. (2012) Plastid-localized amino acid biosynthetic pathways of Plantae are predominantly composed of non-cyanobacterial enzymes. Sci. Rep. 2, 955 52. Cenci, U. et al. (2016) Was the Chlamydial adaptative strategy to tryptophan starvation an early determinant of plastid endosymbiosis? Front. Cell. Infect. Microbiol. 6, 67 53. Akashi, H. and Gojobori, T. (2002) Metabolic efficiency and amino acid composition in the proteomes of Escherichia coli and Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 99, 3695–3700 54. Linka, N. et al. (2003) Phylogenetic relationships of non-mitochondrial nucleotide transport proteins in bacteria and eukaryotes. Gene 306, 27–35 55. Marin, B. et al. (2005) A plastid in the making: evidence for a second primary endosymbiosis. Protist 156, 425–432 56. Nowack, E.C. et al. (2008) Chromatophore genome sequence of Paulinella sheds light on acquisition of photosynthesis by eukaryotes. Curr. Biol. 18, 410–418

57. Bhattacharya, D. et al. (2012) Single cell genome analysis supports a link between phagotrophy and primary plastid endosymbiosis. Sci. Rep. 2, 356 58. Reyes-Prieto, A. et al. (2010) Differential gene retention in plastids of common recent origin. Mol. Biol. Evol. 27, 1530–1537 59. Nowack, E.C. et al. (2016) Gene transfers from diverse bacteria compensate for reductive genome evolution in the chromatophore of Paulinella chromatophora. Proc. Natl. Acad. Sci. U. S. A. 113, 12214–12219 60. Nowack, E.C. and Grossman, A.R. (2012) Trafficking of protein into the recently established photosynthetic organelles of Paulinella chromatophora. Proc. Natl. Acad. Sci. U. S. A. 109, 5340– 5345 61. Delaye, L. et al. (2016) How really ancient is Paulinella chromatophora? PLoS Curr. Published online March 15, 2016. http://dx. doi.org/10.1371/currents.tol. e68a099364bb1a1e129a17b4e06b0c6b 62. Domman, D. and Horn, M. (2015) Following the footsteps of chlamydial gene regulation. Mol. Biol. Evol. 32, 3035–3046

Trends in Plant Science, Month Year, Vol. xx, No. yy

13