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\DOI{10.5802/crbiol.176}
\datereceived{2025-02-06}
\dateaccepted{2025-03-26}
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\dateposted{2025-06-11}
\begin{document}

\begin{noXML}

\CDRsetmeta{articletype}{review}

\title{Obligate intracellular bacterial pathogens as major players in
the metabolic integration of organelles}

\alttitle{Les bact\'{e}ries pathog\`{e}nes intracellulaires
obligatoires, protagonistes majeurs de l'int\'{e}gration
m\'{e}tabolique des organites}

\author{\firstname{Steven} \lastname{Ball}\CDRorcid{0000-0003-1629-1650}\IsCorresp}
\address{Unit\'{e} de Glycobiologie Structurale et Fonctionnelle 
UMR8576 CNRS/Universit\'{e} de Lille, 
B\^{a}timent C9, Cit\'{e} Scientifique, 
59655 Villeneuve d'Ascq, 
France}
\email[S. G. Ball]{steven.ball@univ-lille.fr}

\author{\firstname{Denis} \lastname{Baurain}\CDRorcid{0000-0003-2388-6185}}
\address{Laboratoire de Phylog\'{e}nomique des Eucaryotes B\^{a}t. B22 
Quartier Vall\'{e}e 1
chemin de la Vall\'{e}e 4, 4000 Li\`{e}ge 1, 
Belgium}

\author{\firstname{Marie} \lastname{Leleu}\CDRorcid{0009-0008-1446-665X}}
\addressSameAs{1}{Unit\'{e} de Glycobiologie Structurale et Fonctionnelle 
UMR8576 CNRS/Universit\'{e} de Lille, 
B\^{a}timent C9, Cit\'{e} Scientifique, 
59655 Villeneuve d'Ascq, 
France}
\addressSameAs{2}{Laboratoire de Phylog\'{e}nomique des Eucaryotes B\^{a}t. B22 
Quartier Vall\'{e}e 1
chemin de la Vall\'{e}e 4, 4000 Li\`{e}ge 1, 
Belgium}

\author{\firstname{Ingrid} \lastname{Lafontaine}\CDRorcid{0000-0003-4544-8055}}
\address{Unit\'{e} de Biologie du chloroplaste et perception de la 
lumi\`{e}re chez les micro-algues 
UMR7141 CNRS/Sorbonne Universit\'{e}, 
Institut de Biologie Physico-Chimique, 
13, rue Pierre et Marie Curie, 75005 Paris, 
France}

\author{\firstname{Ugo} \lastname{Cenci}\CDRorcid{0000-0001-9725-9833}}
\addressSameAs{1}{Unit\'{e} de Glycobiologie Structurale et Fonctionnelle 
UMR8576 CNRS/Universit\'{e} de Lille, 
B\^{a}timent C9, Cit\'{e} Scientifique, 
59655 Villeneuve d'Ascq, 
France}

\author{\firstname{Christophe} \lastname{Colleoni}\CDRorcid{0000-0002-6534-6120}}
\addressSameAs{1}{Unit\'{e} de Glycobiologie Structurale et Fonctionnelle 
UMR8576 CNRS/Universit\'{e} de Lille, 
B\^{a}timent C9, Cit\'{e} Scientifique, 
59655 Villeneuve d'Ascq, 
France}

\author{\firstname{Olivier} \lastname{Vallon}\CDRorcid{0000-0001-8827-4927}}
\addressSameAs{3}{Unit\'{e} de Biologie du chloroplaste et perception de la 
lumi\`{e}re chez les micro-algues 
UMR7141 CNRS/Sorbonne Universit\'{e}, 
Institut de Biologie Physico-Chimique, 
13, rue Pierre et Marie Curie, 75005 Paris, 
France}

\author{\firstname{Gilbert} \lastname{Greub}\CDRorcid{0000-0001-9529-3317}}
\address{Institut de microbiologie de l'Universit\'{e} de Lausanne, 
D\'{e}partement des laboratoires Bureau IMUL 02-222, 
Bugnon 48, CH-1011 Lausanne, 
Switzerland}

\author{\firstname{Andreas} \lastname{Weber}\CDRorcid{0000-0003-0970-4672}}
\address{Institute for Plant Biochemistry, 
Heinrich-Heine-University, 
Geb. 26.14.02, Room 106, 
Universitaetsstrasse 1, D-40225 Duesseldorf, 
Germany}

\author{\firstname{Debashish} \lastname{Bhattacharya}\CDRorcid{0000-0003-0611-1273}}
\address{Department of Biochemistry and Microbiology, Rutgers, The State
University of New Jersey, Foran Hall, Room 102, 59 Dudley Road, New
Brunswick, NJ 08901-8520, USA}

\shortrunauthors

\keywords{\kwd{Endosymbiosis}
\kwd{Mitochondria}
\kwd{Plastids}
\kwd{Chlamydia}
\kwd{Rickettsiales}
\kwd{M\'{e}nage \`{a} trois 
\mbox{hypothesis}}}

\altkeywords{\kwd{Endosymbiose}
\kwd{Mitochondries}
\kwd{Plastes}
\kwd{Chlamydia}
\kwd{Rickettsiales}
\kwd{Hypoth\`{e}se du m\'{e}nage \`{a} trois}}

\thanks{CNRS, Universit\'{e} de Lille CNRS,  ANR grants ``Mathtest''
(ANR-18-CE13-0027) ``expendo'' (ANR-14-CE11-0024) and ``m\'{e}nage
\`{a} trois'' (ANR-12-BSV2-0009), R\'{e}gion Hauts de France and
European project Alibiotech, ANR grant ``Mathtest''
(ANR-18-CE13-0027).}

\thanks{\textbf{Note.} Steven Ball is the recipient of the 2022 George
Morel prize.}

\begin{abstract} 
Endosymbionts are very common in nature, offering multiple occasions to
recapitulate events that have led to the generation of mitochondria and
plastids. However, both these organelles are unique because they are
thought to derive from two individual events that gave rise to all
eukaryotes and the plastids in algae and plants (excluding
\textit{Paulinella chromatophora}), respectively. This review focuses
on the differences and similarities existing between extant
endosymbionts and the two major endosymbiont derived organelles: the
mitochondria and plastids. Emphasis is put on recent developments that
point to the major role of intracellular pathogens in the establishment
of these organelles. We argue that metabolic integration of bacterial
endosymbionts into mitochondria and plastids required an unusually high
degree of preadaptation not shared by most extant endosymbionts. We
propose that this was achieved by either recruiting intracellular
bacterial pathogens as ``helper genomes'' providing needed gene
products, or by selecting endosymbionts destined to become organelles
directly from such obligate intracellular bacteria.
\end{abstract}

\begin{altabstract} 
Les endosymbiontes sont tr\`{e}s r\'{e}pandus dans la nature, offrant
de multiples occasions de reproduire les \'{e}v\'{e}nements qui ont
engendr\'{e} les mitochondries et des plastes. Cependant, ces deux
organites sont uniques, car ils sont suppos\'{e}s d\'{e}river de deux
\'{e}v\'{e}nements individuels qui ont donn\'{e} naissance d'une part
\`{a} tous les eucaryotes et d'autre part aux plastes dans les algues
et les plantes (\`{a} l'exception de \textit{Paulinella
chromatophora}). Cette revue se concentre sur les diff\'{e}rences et
les similitudes existant entre les endosymbiontes actuels et les deux
principaux organites d\'{e}riv\'{e}s des endosymbiontes : la
mitochondrie et les plastes. L'accent est mis sur des d\'{e}couvertes
r\'{e}centes qui soulignent le r\^{o}le majeur des pathog\`{e}nes
intracellulaires dans l'\'{e}tablissement de ces organites. Nous
d\'{e}fendons l'id\'{e}e que l'int\'{e}gration m\'{e}tabolique des
endosymbiontes bact\'{e}riens dans les mitochondries et les plastes a
n\'{e}cessit\'{e} un degr\'{e} exceptionnellement \'{e}lev\'{e} de
pr\'{e}adaptation dont sont d\'{e}pourvus la plupart des endosymbiontes
actuels. Nous proposons que cela se soit r\'{e}alis\'{e} soit en
recrutant des bact\'{e}ries pathog\`{e}nes intracellulaires apportant
les fonctions indispensables au succ\`{e}s de l'endosymbiose, soit en
s\'{e}lectionnant les endosymbiontes destin\'{e}s \`{a} devenir des
organites directement \`{a} partir de ces bact\'{e}ries
intracellulaires obligatoires. 
\end{altabstract}

\maketitle


\end{noXML}

\vspace*{10pt}

\twocolumngrid

\section{Introduction: comparing extant\newline endosymbionts with
endosymbiont-derived organelles}\label{sec1}
Unlike the primary endosymbioses that gave rise to mitochondria and
plastids (excluding Paulinella, see below), which are single events
involving two different bacterial donors, endosymbiosis of bacteria by
eukaryotes is common, particularly in invertebrates and protists. These
diverse endosymbioses rely on mutualism, commensalism, or parasitic
interactions. Such common endosymbionts can be distinguished from
mitochondria and plastids by the apparent absence within the
endosymbiont compartment of proteins that are not encoded by the
residual symbiont genome itself. This correlates with the absence of a
specialized protein targeting machinery that directs host
nuclear-encoded proteins to the ancestral bacterial compartment or
membranes. Some of the associations of extant endosymbionts are
obligate, whereas others are facultative. Endosymbiont-derived
organelles such as mitochondria and plastids are derived from obligate
mutualistic endosymbionts, which have managed to target to the symbiont
compartment proteins now encoded by the host. Obligate endosymbionts
and endosymbiont-derived organelles share many properties. Upon
entering symbiosis with the eukaryotic host, ancestral bacteria were in
all cases isolated from their ``free-living'' progenitors, and unable
to exchange genes with them through recombination. As a result, these
genomes accumulated mutations leading to pseudogenes and, ultimately,
gene losses, a phenomenon known as ``Muller's 
ratchet''~\cite{Muller1964}. The presence of a nutrient-rich cytosol
within the eukaryotic host likely further favoured the loss of all
functions that are not essential for symbiosis and maintenance through
the selection of symbiotic genes~\cite{Moran1996}. Some of the extant
obligate endosymbiont genomes, such as that of \textit{Nasuia
deltocephalinicola}, an amino acid supplying symbiont of hemipteran
insects, have thus undergone reduction. It went from a genome several
Mbp in size in the hypothetical ancestral Burkholderiales
$\upbeta$-proteobacterium to a 112~kbp relic genome with 137
~predicted protein-coding genes, which is comparable to or less than
what is found in endosymbiont-derived
organelles~\cite{BennettMoran2013,MoranBennett2014}. This correlates
with a significant increase in genome AT content and changes in the
genetic code, traits also present in mitochondria. Hence, these
endosymbionts show convergent evolution with respect to genome size and
nucleotide content in mitochondria and plastids. However, unlike
standard endosymbionts, organelle genome size and gene number are not
robust indicators of the organelle proteome complexity. Both
mitochondria and plastid symbioses have relied on the presence of
electron transport systems coupled to specific complex membrane
structures. The number of genes required to optimize the function or to
encode components of these complex machineries vastly exceed the number
of genes involved, for example, in amino acid metabolism, which form
the basis of the symbiosis uniting the aforementioned insect
endosymbionts to their host. To avoid the deleterious effects of
Muller's ratchet-induced accumulation of mutations affecting the
fitness of their genes, the organelle-generating endosymbionts relied
increasingly on the host genetic system. This took the form of either
the host providing host-derived functions or the host genome being the
recipient of lateral gene transfers providing compromised symbiont
functions. In most cases, the foreign gene source is the endosymbiont
itself, thereby generating \textbf{EGTs} (endosymbiotic gene
transfers). However, in a significant number of cases, the source of
the gene is from another bacterial origin not directly related to the
symbiont. LGTs that specifically impact the host genome at the time of
organelle endosymbiosis are hereby termed \textbf{ERGTs}
(Endosymbiosis-Related lateral Gene Transfer). In this context, the
foreign substitute of the original symbiont gene only accounts for part
of the observed ERGT signal. Equally important are ERGTs corresponding
to genes not previously encoded by the symbiont, which bring novel
functions required during the process of organelle-host metabolic
integration. In the case of EGTs and replacement ERGTs, the now
nuclear-encoded organelle-derived genes escaped Muller's ratchet, due
to the population size and the gene recombination and repair systems of
the host. Nevertheless, accumulation of mutations followed by
pseudogenization and gene losses was the hallmark of the early phases
of reductive genome evolution in organelles. In time, and in addition,
both organelle genomes have evolved mechanisms that attenuate the
ratchet, including, among others, an increased genome copy number and
efficient recombination and repair\unskip\break 
systems~\cite{MarechalBrisson2010,NaitoPawlowska2016}.

However, the present evidence gathered from bacterial non-organellar
endosymbionts and their insect host genomes suggests that if any EGT or
ERGT can eventually be associated with such symbioses, these will
likely be less frequent. Nevertheless, some extant endosymbionts not
related to mitochondria or plastids are presently 
suspected~\cite{Spethetal2024} and even recognized to be able to
display either  limited~\cite{Bublitzetal2019,Nakabachietal2014} or
even extensive symbiont targeting of host encoded 
proteins~\cite{Coaleetal2024a,NowackGrossman2012}. This further blurs
the distinction between true organelles and endosymbionts. This review
will focus on the ancient and recently gathered evidence pointing to an
active role of intracellular bacterial pathogens in the
organelle-generating endosymbioses. For a more general outlook at the
process of primary endosymbiosis and alternative hypotheses, the reader
is redirected to more general, excellent and comprehensive
reviews\break \cite{Archibald2015,McFadden2014,Ponce-Toledoetal2019,Stephensetal2021}.

\section{Consequences of Muller's ratchet on our\newline 
understanding of the
initial symbiosis\newline biochemistry}\label{sec2}
Pseudogenization followed by gene loss is very rapid, frequent, and
universal in all past and present endosymbiotic events. This, in turn,
limits the number of ways in which the nature of the symbiotic
relationship between endosymbionts and their host can be altered. For
instance, extant mitochondria and plastids are obviously providing
oxidative phosphorylation and oxygenic photosynthesis, respectively, to
their host; however, it has been and is proposed that other symbiotic
relationships initiated the partnership. For instance, it has been
proposed that the symbiosis uniting the ancestral mitochondrion to its
archaea-derived host was based on the supply of molecular hydrogen to
the host under anoxia~\cite{MartinMuller1998}. The molecular supply of
hydrogen by a symbiont hydrogenase can indeed form the basis of a very
efficient symbiosis under anaerobiosis. We argue, however, that if such
a symbiotic link drove mitochondrial endosymbiosis for any significant
length of time, Muller's ratchet would have made it quickly
irreversible. Indeed, the production of molecular hydrogen most often
acts as an electron valve for recycling NADH during fermentation.
Hydrogen production would thus occur in conditions where oxidative
phosphorylation is prevented. Free-living facultative anaerobic
{$\upalpha$-proteobacteria} do not respire and produce molecular
hydrogen simultaneously, but they do so at distinct times under
different conditions in their changing environments. Upon
endosymbiosis, within a strictly anaerobic archaea-like organism,
unless oxygen consumption has some symbiotic advantage (in which case,
however, symbiosis would not rely solely or even chiefly on hydrogen
production), Muller's ratchet would have quickly and irreversibly
affected the capacity of a hydrogen-producing symbiont to switch to
oxidative phosphorylation in the constant nutrient-rich but anoxic
environment of the eukaryote ancestors' cytosol. Nitrogen fixation has
also been proposed as a starter symbiosis for plastid 
endosymbiosis~\cite{Daganetal2013} on the grounds that nitrogen
fixation is the most frequently found symbiosis involving extant
cyanobacteria and protists. Yet irreversible losses of oxygenic
photosynthesis are also evidenced dramatically in the extant
cyanobacterial endosymbionts that fix
nitrogen~\cite{Kneipetal2008,NakayamaInagaki2017}. These are derived
from photosynthetically competent unicellular diazotrophic
cyanobacteria with large genomes (around 5~Mbp with up to 5000~ORFs).
The extant endosymbionts fix nitrogen for their photosynthetic diatom
or Prymnesiophyceae (haptophyte) hosts. These symbionts, called
spheroid bodies within diatoms, are presently at comparable stages of
genome reduction (from 1.5 to 3~Mbp genomes with approximately 1200 to
1700~deduced ORFs), due to mutations followed by deletions in several
distinct metabolic\unskip\break 
pathways~\cite{FosterZehr2019}.

These gene mutations and losses encompass essential elements of ad
minima PSII  (haptophyte symbionts) or of both PSI and PSII  (spheroid
bodies) and of the Calvin cycle and respiration, among many other
pathways. It is proposed that the bacteria have evolved an essentially
fermentative metabolism and import several metabolites from the
nutrient-rich host cytosol to ensure anoxygenic photosynthesis through
PSI in haptophytes. These recently acquired endosymbionts testify to
the irreversible nature imposed by Muller's ratchet on the evolution of
endosymbionts. Even at the earliest stages of their evolution, these
nitrogen-fixing symbionts had definitively lost the opportunity to
revert to oxygenic photosynthesis and become plastids because of the
loss of essential proteins\unskip\break  
of PSII. 

Interestingly, in the case of \textit{Braarudosphaera bigelowii}, a
Prymnesiophyceae containing such diazotrophic endosymbionts, extensive
targeting of host-encoded proteins into the symbiont seems to occur,
leading to the proposal that the latter would define a true organelle:
the  nitroplast~\cite{Coaleetal2024b}.

We argue that a facultative hydrogen-producing anaerobic
$\upalpha$-proteobacterium would have experienced a similar evolution
of its genome towards anaerobic metabolism through mutations and gene
losses, precluding its evolution into an oxygen-consuming organelle,
such as the mitochondrion, at a very early stage. In other words, if a
bacterium engages in an endosymbiotic relationship, it had better get
it right from the start. Once a particular evolutionary path is
followed, there is no turning back! An important consequence of these
considerations is that both the nature of the initial bacterium and
that of the initial symbiosis biochemistry will irreversibly affect the
outcome of endosymbiosis a fact that is overlooked or underestimated by
many researchers in this field. These considerations do not mean that
the initial ancestral symbiosis that subsequently led to mitochondria
and plastids necessarily entailed the export of ATP through oxidative
phosphorylation and carbohydrates through oxygenic photosynthesis,
respectively. However, they do mean that the initial symbiotic
relationship required biochemical conditions that must have necessarily
relied on respiration and photosynthesis and thus conserved all the
components of oxidative phosphorylation and oxygenic photosynthesis to
prevent their irreversible loss through Muller's ratchet. These could
indeed lead later to the evolution of ATP and photosynthate supply by
mitochondria and plastids, respectively. In this respect, the
respiratory consumption and protection from oxygen by respiring
bacteria in the anoxic host cytosol, or the supply of oxygen to protist
ancestors by photosynthetic symbionts, qualifies, among other examples,
as possible initial symbiotic relationships. On the other hand,
production of hydrogen or nitrogen fixation by
$\upalpha$-proteobacterial or cyanobacterial ancestors does not, as
neither requires the maintenance of oxidative phosphorylation or
oxygenic photosynthesis  (on the contrary, most nitrogenases and
hydrogenases  are highly sensitive to inhibition by oxygen) in the
intracellular conditions that prevailed for the progenitors of the
mitochondrion or plastid.

\section{Endosymbiotic and endosymbiosis-related lateral gene
transfers: the organelle\newline solutions to Muller's ratchet}\label{sec3} 
Both mitochondria and plastids have evolved sophisticated protein
targeting machineries that allowed the transfer of genes present in the
symbiont genome to the host nuclear genome  (for reviews concerning
plastids  see Ballabani et~al.~\cite{Ballabanietal2023}, Flores-P\'erez
and Jarvis~\cite{Flores-PerezJarvis2013}). Interestingly, mitochondria
and plastids use analogous systems based on N-terminal targeting
peptides of 20 to 70 amino acids present on the proteins targeted to
organelles.

These peptides are of similar amino acid composition, except for an
additional segment of approximately 16 residues found on plastidial
targeting peptides. Most importantly, all such organelle-targeting
peptides share the ability to spontaneously form an amphiphilic
$\upalpha$-helix when interacting with their target organelle
membranes. These structural and functional properties are also shared
by a subclass of Helical-Amphiphilic-Ribosomal  antimicrobial (HA-RAMP)
peptides, which are ubiquitous in both prokaryotes and eukaryotes.
HA-RAMPs are an ancient antimicrobial defence system against invasion
by intracellular pathogenic bacteria. The phagotrophic habit of
ancestral eukaryotes would have required the evolution of strong
antibacterial immunity very early on. Membrane permeabilization by
HA-RAMPs is well documented and could explain their antibacterial
activity through the unselective leakage of essential 
metabolites~\cite{Wangetal2016}. In a recent insightful paper, it was
proposed that the acquisition of resistance to eukaryotic HA-RAMPs
through the import of such peptides by intracellular bacteria, and
their proteolytic destruction within the bacteria by a selective
protease, would have formed a possible mechanism explaining the
emergence of a complex and efficient protein import 
machinery~\cite{Wollman2016}. Indeed, eukaryotic or bacterial genes 
(of endosymbiont or foreign bacterial origin) transferred to the
eukaryote nucleus, when fortuitously located downstream of an
eukaryotic HA-RAMP gene directed against such bacteria, would have
resulted in the inadvertent targeting of the hybrid protein to the
evolving symbiont, followed by the selective degradation of the HA-RAMP
through the bacterial protease and the possible folding of the 
protein\unskip\break 
remnant. 

Such antimicrobial peptide resistance mechanisms have been well
documented in  bacteria
(\cite{Groismanetal1992,Guefrachietal2015,Sheltonetal2011}; reviewed
in~\cite{CaspariLafontaine2021}). Furthermore, two out of the four
suspected peptidases active during mitochondrial and plastidial
endosymbiosis display a bacterial phylogenetic affinity in agreement
with this  hypothesis~\cite{Garridoetal2022}.

In any case, whatever the origin of these targeting machineries of
mitochondria and plastids, both targeting systems currently require a
distinct set of chaperones specialized in the translocation of the
unfolded targeting peptide-protein to the outer membrane of the 
organelle~\cite{Ballabanietal2023,Flores-PerezJarvis2013}, followed by
import, peptide cleavage, and subsequent folding. We argue that native
bacterial and host proteins have not evolved under such constraints and
may have, by chance, been quite differently suited to following such a
complex route of unfolding, folding, and interaction with a specific
set of chaperones. This could explain why, whenever a native
endosymbiont protein was poorly suited to follow this route, a foreign
alternative may have been selected faster. Hence, the genes that were
selected for transfer to protect the evolving organelle from the impact
of Muller's ratchet became a mix of original endosymbiont and foreign
genes. The origin of the gene depended on the one hand on functional
constraints and, on the other hand, on the rate of endosymbiont
compared to foreign DNA transfer. At first glance, these conditions
would apparently favour the transfer of endosymbiont genes by
endosymbiotic gene  transfer (EGT). However, the origin of the selected
gene equally depended on the number of mutations that must have
followed the transfer, to facilitate the targeting of the protein. In
many instances other than the photosynthetic machinery per se, or a few
core proteins needed for replication or expression of genes encoded in
the endosymbiont genome, there was little need to reproduce the
original cyanobacterial protein network with all its interactions. A
more integrated alternative, allowing for a significant amount of
intraorganellar proteome chimerism was favoured because this happened
faster and was suitable. Thus, both the EGT of the endosymbiont genes
and the ERGT of foreign bacterial genes, or the redirection of the
product of corresponding host genes to the plastid, allowed for the
loss of the corresponding gene in the symbiont genome and achieved
protection of these organelle functions from mutation, 
inactivation,\unskip\break 
and loss.

\section{Are antimicrobial peptides at the core of non-organellar
endosymbioses?}\label{sec4} 
It has recently been shown that extant nitrogen-fixing endosymbionts of
legumes, such as the $\upalpha$-proteobacteria of the Rhizobiales 
order (reviewed in Mergaert~\cite{Mergaert2018}), or the actinobacteria
of the Frankia  genus~\cite{Carroetal2015} both interact with
cysteine-rich antimicrobial peptides released by the plant host.
Targeting of these symbiotic peptides to symbiont membranes has been
demonstrated to be required for symbiosis. These antimicrobial peptides
are probably derived from the mechanisms of antibacterial host immunity
described above that interfere with a number of bacterial processes,
such as membrane permeability and cell division. During symbioses, at
sublethal doses, the peptides limit uncontrolled cell division and
induce a differentiation process that is compatible with nitrogen
fixation and host cell viability. Sometimes this differentiation is
cytotoxic for the symbiont, and occasionally the bacteria do not
recover from these  interactions (reviewed in
Mergaert~\cite{Mergaert2018}). In the case of the \textit{Frankia}
symbiosis, it has been demonstrated that reduced nitrogen-containing
molecules such as glutamine and glutamic acid are released through
interaction with host-encoded  peptides~\cite{Carroetal2015}. Hence, as
with HA-RAMPs, membrane interaction with these toxic peptides provokes
an increased permeability of the bacterial membranes, possibly leading
to the non-specific leakage of the two amino acids. These anyhow
account for a substantial proportion of the metabolites present in the
Frankia cytosol during symbiosis. Therefore leakage specificity is not
an issue for this symbiosis. It was recently suggested that the
apparent absence or paucity of transporters on bacterial membranes of
insect  endosymbionts~\cite{Charlesetal2011,Fengetal2019} could be
explained if one assumes the existence of antibacterial
peptide-mediated non-specific leakage of molecules and 
metabolites~\cite{Mergaert2018,Mergaertetal2017}. This would happen in
insect endosymbioses in the small volume defined by the lumen of the
host-controlled symbiosome membrane that contains the symbionts within
the bacteriome. Limited by this restricted volume, this leakage would
not be lethal or even detrimental to the bacteria. Hence, here again,
sublethal alterations of membrane permeability followed by non-specific
leakage could be occurring. Interestingly, such a system would obviate
the need for a complex protein targeting machinery to locate crucial
transporters on the bacterial envelopes. We believe that this property
may account for the high frequency and diversity of endosymbionts
observed in both animals and other eukaryotes. We are in agreement with
those who have proposed this function to account for endosymbiosis
onset and function in extant non-organellar endosymbiosis such as the
nitrogen-fixing \textit{Frankia alni}/Alnus 
symbiosis~\cite{Carroetal2015} or even the few organellar-generated
endosymbionts that are not derived from, nor apparently assisted by,
obligate intracellular pathogens such as the Paulinella 
chromatophores~\cite{Mergaert2018,Mergaertetal2017,Stephensetal2021}.
Indeed, despite all efforts, no convincing case for the presence of
metabolite transporters targeted to the chromatophore membranes could
be made in  Paulinella~\cite{Singeretal2017} leaving microbial
peptide-induced membrane permeability as the sole possible mechanism
for the installation of the symbiotic metabolic fluxes.

\section{Are antimicrobial peptides at the core of\newline 
endosymbiosis of the
future organelles?}\label{sec5} 
The host's demonstrated use of antimicrobial peptides to control
nitrogen fixation by bacterial endosymbionts has led to the recent
proposal that such interactions may offer an alternative to the
requirement for transporters to explain the onset of symbiosis, in the
case of the mitochondria or primary 
plastids~\cite{Carroetal2015,Massonetal2016,Mergaertetal2017}. It must
be pointed out, however, that tinkering with membrane permeability to
ensure nonspecific leakage of metabolites is highly unlikely to have
benefited symbionts that heavily relied on membrane integrity for the
normal function of membrane-embedded electron transport chains. These
chains must have been at the core of the symbiotic relationship in the
case of the future mitochondria in order to obviate their loss through
M\"{u}ller's ratchet. We therefore propose that, on the contrary, the
use of antibacterial peptides by a majority of extant endosymbioses not
involving intracellular bacterial pathogens distinguishes  them from
endosymbiont-derived ``true'' organelles. This proposal could explain
the apparent contradiction between the high frequency of endosymbiosis
observed in nature and the extreme rarity of 
\mbox{primary} \mbox{endosymbiosis} of
eukaryotic organelles, as evidenced by the apparent monophyly of these
events. This implies that the bacteria used as the progenitors of both
the mitochondria and plastids must have had the full suite of
transporters required to establish symbiosis at the very onset of the
symbiotic interactions. This also distinguishes the Paulinella
chromatophore from the primary plastid, and limits the frequency of
primary endosymbiosis of bacteria destined to become organelles. The
level of preadaptation required for the progenitors of mitochondrion or
plastid had to be much higher than for many extant endosymbionts. In
this respect, the bacteria that offer the highest level of
preadaptation to living within eukaryotes are by far the obligatory
intracellular bacterial  pathogens~\cite{Balletal2016c}.

\section{The restricted diversity but high\newline 
connectivity and flexibility
of the obligate intracellular pathogens}\label{sec6} 
Bacteria that have lost the ability to multiply out of their eukaryotic
hosts are constantly evolving from facultative intracellular bacteria.
Some of these evolutionary transitions are fairly recent, as
exemplified by the leprosy agent \textit{Mycobacterium leprae}, the
aphid symbiont \textit{Buchnera aphidicolae}, or the Q-fever pathogen
\textit{Coxiella burnettii}, which have evolved through genome
reduction of a facultative intracellular bacterial pathogen of the
order Mycobacteriales, Enterobacteriales or Legionellales,
respectively, in  animals~\cite{vanSchaiketal2013,Seshadrietal2003}.
Some of these are, however, far more ancient and define large and
diverse groups of bacteria in which most members of bacterial orders
are intracellular. These are defined by the $\upalpha$-proteobacteria
Rickettsiales and the PVC supergroup member order 
Chlamydiales~\cite{Collingroetal2011,Munoz-Gomezetal2019}.
Representatives of these two orders can be found multiplying in hosts
from the whole spectrum of the eukaryotic domain and not only within
the animal kingdom, as initially thought. This makes them candidates
for very ancient transitions to intracellular life, possibly as far
back as, or prior to, the time of plastid and mitochondrial
endosymbioses. While reductive genome evolution has been at work at
some point to explain their relatively simple genomes, the most
ancestrally derived members of Rickettsiales or Chlamydiales still
encode over 2000 protein-coding candidate genes, a respectable number
compared to the amino acid or vitamin supplying insect bacterial 
symbionts~\cite{Collingroetal2011,WangWu2015}. The mitochondria have
often been phylogenetically found closely related to the Rickettsiales
order among $\upalpha$-proteobacteria. What has been and still is
controversial is their precise relative position, nested 
within~\cite{WangWu2015} or sisters to 
Rickettsiales~\cite{Rogeretal2017}. This is of importance, since a
nested topology would strongly suggest that mitochondria evolved from
an ancestral bacterium that already had  an obligate intracellular
lifestyle. Apparently, the latest evidence points at mitochondria
being, at best, sisters of Rickettsiales, with one study questioning
the  sisterhood~\cite{Martijnetal2018} and another placing mitochondria
only as sisters of $\upalpha$-proteobacteria as a 
whole~\cite{Munoz-Gomezetal2022}. For the time being, the prospects of
clarifying the origin of mitochondria through phylogenetics alone seem
remote. However, because of the considerations summarized in this
review, we do not view the close phylogenetic placement of mitochondria
relative to the Rickettsiales as 
coincidental. 

At variance with the non-Wolbachia insect endosymbionts with highly
reduced genomes discussed above, obligate intracellular pathogens have 
retained the potential to infect eukaryotes and multiply by horizontal
transmission, often implying additional selection for the retention of
active genes. Furthermore, the life cycles of these pathogens imply
extracellular infection phases and transmission to novel hosts, making
them less isolated and less subjected to the deleterious effects of
Muller's ratchet than obligate non-infectious endosymbionts.
Nevertheless, obligate intracellular pathogens have in common the loss
of several metabolic steps, in particular biosynthetic pathways,
suggesting that they import of a vaster array of metabolites than
facultative, intracellular 
pathogens~\cite{Collingroetal2011,Seshadrietal2003}. The most ancestral
Rickettsiales lineages are now suggested to have a facultative
intracellular or even an ectosymbiotic  lifestyle~\cite{Schonetal2022}.
Their genomes nevertheless testify to the presence of multiple
opportunities for symbiotic or pathogenic biotic interactions with
their hosts. This suggests a higher degree of connectivity to the host
with an additional number of transporters, most of which remain to be
characterized in  Rickettsiales~\cite{Driscolletal2017}. In addition,
their obligate or facultative intracellular lifestyles implies the
presence of active resistance mechanisms against the very aggressive
antibacterial immunity of the host. Moreover, they share with
facultative intracellular pathogens the secretion into the host cytosol
of many and probably more effector proteins through sophisticated
secretion machineries that facilitate their active multiplication or 
maintenance~\cite{Driscolletal2017}.

Finally, and most importantly, as do temperate phages, they often
display both a ``productive'' pathogenic cycle leading to the massive
production of infectious bacteria through lysis and a temperate cycle
where the bacteria coordinate their replication and maintenance with
that of their host to various degrees. This temperate phase can be
accompanied or not by the production of infectious bacteria through
budding. This state, also called ``bacterial persistence'', is
reversible and the pathogen cycle can resume when the environmental
conditions allow  it~\cite{Hoganetal2004}. Prophages can also be
considered to be in a stage of persistence. Prophages are rarely
neutral: in the few cases where this has been studied, they seem to
provide a significant selective advantage to the bacterial clone
hosting them. The best described fitness advantage is the resistance to
infection by both  virulent (productive) phages of the same and
different  types (reviewed in Bondy-Denomy and
Davidson~\cite{Bondy-DenomyDavidson2014}). Curiously, a similar
situation has been recently described for the infection of
\textit{Acanthamoeba castellanii} by facultative intracellular bacteria
such as \textit{Legionella  pneumophila}~\cite{Konigetal2019}.
Pre-infection of the amoeba by persistent forms of the Chlamydiales
obligatory intracellular pathogens \textit{Neochlamydia} or
\textit{Protochlamydia amoebophila} protects the host against
superinfection by pathogenic  (lytic) forms of Legionella. The notions
of pathogen/symbiont persistent or temperate phases are particularly
blurred in these organisms, and a specific bacterium can easily switch
from one status to the other, depending on the host's nature. Temperate
obligate intracellular bacteria have often evolved sophisticated means
by which they can spread both horizontally and vertically in their
hosts. For instance, Wolbachia, a Rickettsiales symbiont with over a
thousand genes, has evolved several means by which they can spread in
the germ line of insect populations through sophisticated manipulations
of their sexual life  cycle (for review see
Landmann~\cite{Landmann2019}). They can induce feminization,
cytoplasmic incompatibility, parthenogenesis, male killing, and various
types of other manipulations, thanks to which they have achieved the
status of the most abundant symbiont on our 
planet~\cite{Landmann2019}. Their presence can lead to both \mbox{parasitic}
or \mbox{facultative} and obligate mutualistic interactions depending on the
benefits for the host of such manipulations. Transition from parasitic
symbiosis to obligatory mutualism is also common and some Wolbachia
strains have been shown to supply essential vitamins to their 
host~\cite{Nikohetal2014}. Conversely, transition from symbiosis to a
full-fledged pathogen is equally observed and can be reproduced 
experimentally~\cite{LeClechetal2017}. At the time of mitochondrial
endosymbiosis, a highly connected parasitic persistent
Rickettsiales-like symbiont would have scored very high on the
preadaptation scale with respect to the yet to be developed
mitochondrial endosymbiosis. 

The list of preadaptations of Rickettsiales to primary endosymbiosis is
particularly long and has been reviewed by Wang and
Wu~\cite{WangWu2014}. Suffice it to say here that they are able to
resist to the host antibacterial immunity and yet replicate in a
persistent fashion, are able to rapidly invade populations of hosts
vertically and horizontally, have a rich suite of transporters although
many remain to be characterized, and are perfectly adapted to the
intracellular environment. The anciently diverged Rickettsiales are
also sufficiently gene rich to offer many distinct routes to reductive
evolution that can lead to the evolution of a true novel organelle.
They are also somewhat generally resistant to Muller's ratchet,
provided they do not go too far in mutualistic interactions and keep on
producing infectious particles. These advantages were also shared with
Chlamydiales, but unlike Rickettsiales the latter do not harbour the
complete set of genes needed for oxidative phosphorylation, which was
quite obviously at the core of mitochondrial endosymbiosis.

\section{The onset of mitochondrial endosymbiosis: lessons from
Legionella and Neorickettsia}\label{sec7}
Eukaryogenesis, the process by which the first eukaryotes emerged from
the archaea, is a highly controversial issue, and multiple conflicting
hypotheses have been proposed to describe the evolution of the first 
eukaryotes~\cite{Donoghueetal2023}. Nevertheless, a consensus is
emerging for a rather late occurrence of mitochondrial endosymbiosis on
the way to  LECA (Last Common Eukaryotic
Ancestor)~\cite{Tobiassonetal2024}.

With such unknowns, proposing the nature of the primordial symbiosis
biochemistry uniting the
\mbox{future} mitochondrial and archaeal relatives,
and deducing the implementation of this pathway during eukaryogenesis,
is a speculative and perilous exercise. We may not have to do this if
we simply hypothesize that the ancestor of the mitochondrion was an
ancestral Rickettsiales symbiont/pathogen that had already managed to
spread in or at the surface of the host ancestor populations, through
both parasitic and facultative mutualistic interactions. We may not
even have to necessarily imagine that this organism was an obligate
intracellular symbiont: it might very well have been a facultative
endosymbiont or a tightly associated ectosymbiont displaying many
critical biotic interactions with the eukaryote ancestors. This is
indeed suggested by the recent study of several ancestrally diverged
Rickettsiales lineages, which suggest the presence of multiple
transitions to the obligate intracellular 
lifestyle~\cite{Castellietal2019,Schonetal2022}. We suggest that a
transition to an obligate intracellular life was selected on ATP
transport relying on  MCFs (Mitochondrial Carrier Family) exchange of
host cytosolic ADP for bacterial endosymbiont generated ATP rather than
the reverse. Indeed, the ATP import protein common to most Chlamydiales
and Rickettsiales catalyses the unidirectional import of ATP from the
host, and would have driven the bacteria into parasitism rather than
symbiosis. If the host was still an oxygen-sensitive anaerobe, one can
propose protection from oxygen by its respiratory symbiotic consumption
as a possible initial symbiotic interaction. The loss of
glycolysis-mediated fermentation by many ancestral Rickettsiales is in
line with such biotic interactions.

As hypothesized by  FA Wollman~\cite{Wollman2016}, the evolution of
resistance to the host antimicrobial  peptides 
\mbox{(HA-RAMPs)} by the
progenitor of mitochondria would have further preadapted this lineage
to primary endosymbiosis. Other bacteria or the host would have evolved
distinct antibacterial peptides or proteins to tame or compete with
this symbiont. Among such proteins, the MCF ADP/ATP  transporter
(hereafter called MCF for simplicity) can be viewed as antibacterial
proteins responsible for ATP leakage of potentially pathogenic
intracellular bacteria. The evolution of such transporters is often
believed to have signalled the onset of mitochondrial endosymbiosis 
(for review, see Haferkamp and
Schmitz-Esser~\cite{HaferkampSchmitz-Esser2012}); they would have
driven or consolidated the ancestor of mitochondria into a symbiotic
relationship. This led to the assumption that MCF was 
\mbox{necessarily} of
host origin, which \mbox{immediately} raised the question of protein
targeting. Novel findings bring this view into question. Quite
recently, \textit{Legionella pneumophila} was demonstrated to harbour a
\textit{bona fide} MCF effector protein secreted in an unfolded fashion
by the type~IV secretion system, affording for its secretion through
three Legionella-controlled  membranes~\cite{Dolezaletal2012}. The
highly hydrophobic protein is thought to interact in the cytosol with
the chaperones responsible for protein targeting to the mitochondrial
inner membrane. Expressed in yeast, the Legionella MCF interfered with
normal mitochondrial ATP export in particular genetic backgrounds
involving mutations of the yeast MCF ATP transport complement. In
addition, the MCF was active for ATP transport on its own. The
rationale for the evolution of such a function in Legionella remains
unknown. Interestingly, two out of the five candidate bacterial MCFs
identified in the Legionella study were reported in Neorikettsia, a
\textit{bona fide}  Rickettsiales~\cite{Dolezaletal2012}. One could
argue that specific ATP export is very unlikely to have originated in
the ancestor of the mitochondria prior to endosymbiosis. Nevertheless,
toxins that induce specific ATP leakage are known, for instance, to be
encoded by the \textit{E.~coli} chromosome and not only on 
plasmids~\cite{Wilmaertsetal2018}. It has been recently demonstrated
that induction of the synthesis of such a toxin occurs under specific
stress conditions, resulting in attenuated growth and multidrug
resistance of pathogens normally sensitive to antibiotics. Evidently,
the toxin is cytotoxic for \textit{E. coli} but allows for the recovery
of viable ``persisters'' in the treated population. This gene, however,
does not encode an MCF and, most importantly, affects respiration.
Hence, these novel findings concerning intracellular bacteria may
suggest that, after all, the mitochondrial carriers could have
originated in bacteria, either in the form of effectors designed to
affect competitors in an intracellular environment, or in the form of a
sophisticated chromosomal toxin--antitoxin system designed to prompt
attenuated growth. In any case, whatever the origin of MCF, permanent,
yet highly specific and controlled leakage of ATP signals, in our view,
the onset of mitochondrial endosymbiosis. This can indeed be viewed as
the biochemical innovation that separated the future organelle from its
infectious relatives. It is indeed hard to envision that such an
ATP-leaking symbiont would remain\unskip\break 
infectious, thereby signalling an
irreversible switch from a parasitic symbiont to an obligate
mutualistic symbiont. Therefore, this switch implies that the former
pathogen had now become completely isolated in the intracellular
environment of the eukaryote ancestors, with little ability to
recombine with its original population, making it thus fully sensitive 
to reductive genome evolution through Muller's ratchet. This evidently
boosted eukaryogenesis to generate LECA and allowed for the evolution
of many free-energy demanding properties.

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Simplified view of the proposed metabolic
reconstruction of the function of starch metabolism in the last
Archaeplastida common ancestor, based on~\cite{Deschampsetal2008a,Deschampsetal2008b}.
Origins of the various enzymes are indicated with different colours,
grey indicates a protein from host origin, while yellow indicates a
protein from bacterial origin. Synthesis of the bacterial specific
nucleotide-sugar ADP-glucose occurs in the cyanobiont. ADP-glucose is
then exported from the plastid to the host cytosol through a host
encoded NST transporter, where it is used by a bacterial glucan
synthase using ADP-glucose. The latter consists of the ancestor of
SSIII/SSIV starch synthase present in green plants and algae and in
glaucophytes. This flux is combined to the cytosolic eukaryotic flux
that relies solely on UDP-glucose through a UDP-glucose specific glucan
synthase of host origin found in both glaucophytes and red algae. The
linear glucans are then branched into preamylopectin. The step
performed by isoamylase (Isa) consists in trimming the soluble
preamylopectin precursor, thereby leading to crystallization and
aggregation of the starch granule. Catabolism is first initiated by the
two dikinases GWD and PWD, which destabilize the crystalline structure
and allow degradation by $\upbeta$-amylase and  glycogen/starch
phosphorylase.\unskip\break Nc:~Nucleus, BE:~Branching enzyme, GS:
Glucan synthase, AGPase: ADP-glucose pyrophosphorylase,
Isa:~Isoamylase, GWD/PWD:  Glucan/Phosphoglucan Water Dikinase, Pho:
Glycogen phosphorylase, $\upbeta$-amy: $\upbeta$-amylase, G1P:
Glucose-1-phosphate, NST: nucleotide sugar transporter, ADP/UDP-glc:
ADP/UDP  \mbox{glucose.}}
\end{figure*}

\section{Obligate intracellular bacteria as helper genomes of plastid
endosymbiosis:\newline
proposal for the carbohydrate metabolism symbiotic
fluxes}\label{sec8}
Cyanobacteria are by no means preadapted to survive in the
intracellular environment of eukaryotic phagotrophs. As is also the
case for present-day plant cells, and by comparison to intracellular
bacteria or free-living heterotrophs, their connectivity, albeit 
significant~\cite{Senguptaetal2022,Stebeggetal2019}, is comparatively
low, while these photoautotrophs most often require only, in addition
to light and water, a few major minerals and trace elements. Yet, there
is no question that an ancient cyanobacterial endosymbiont has achieved
the status of a true eukaryotic organelle. But how did these organisms
escape the deadly antimicrobial response of its phagotrophic host, and
how did they establish the required connectivity while maintaining
their membrane embedded electron transport chains fully functional?
Possible answers to this question came from the finding of an
unexpected phylogenetic signal in Archaeplastida 
genomes~\cite{Balletal2013,Beckeretal2008,Brinkmanetal2002,Collingroetal2011,Everettetal1999,HuangGogarten2008,HuangGogarten2007,Moustafaetal2008,Stephensetal1998}. Archaeplastida comprise the three lineages 
(red algae, green algae and plants, and glaucophytes) that are direct
descendants of the eukaryotic lineage that experienced primary plastid
endosymbiosis. During the process of metabolic integration of the
plastid, genes from the cyanobiont were transferred to the host
nucleus. This protected the prokaryotic genes from Muller's ratchet, as
discussed above, and generated a strong EGT signal in phylogenies
amounting to 600/700 cyanobacterial genes that were transferred to the
host nuclear  genome(s)~\cite{Qiuetal2013}. However, using the same
phylogenomic techniques, another signal was found, uniting
Archaeplastida and obligate intracellular bacterial pathogens in the
order Chlamydiales. In this case, 50 to over 100~genes of chlamydial
origin were identified in Archaeplastida 
genomes~\cite{Collingroetal2011,Stephensetal1998}. Several groups
investigating this phylogenetic signal proposed that these pathogens
played a role in endosymbiosis, possibly by supplying helper functions
during metabolic integration of the nascent 
plastid~\cite{Balletal2013,Beckeretal2008,HuangGogarten2007,Moustafaetal2008}. 
A possible function was proposed by looking at the evolution of storage polysaccharide metabolism in 
Archaeplastida~\cite{Balletal2013,Colleonietal2010,Deschampsetal2008a}. 
By comparing the corresponding extant enzyme
networks, the simplest possible metabolic pathway for starch synthesis
in the last 
\mbox{common} ancestor of Archaeplastida was 
\mbox{proposed~\cite{Deschampsetal2008a}}. This hypothetical pathway displayed
several interesting characteristics  (Figure~\ref{fig1}). First, it
likely defined a major flux of reduced carbon from the plastid to the
eukaryote host cytosol, where it was polymerized into starch or
glycogen. Indeed, starch/glycogen, together with  TAGs (lipid
droplets), can be considered as the major form of carbon storage in the
eukaryotic cytosol. Second, unlike eukaryotic glycogen metabolism,
which relies exclusively on UDP-glucose as a synthesis substrate, this
hypothetical pathway relied on the presence within the host cytosol of
both ADP-glucose, the substrate devoted to bacterial glycogen
synthesis, and  UDP-glucose (reviewed in Ball
et~al.~\cite{Balletal2015,Balletal2011,Deschampsetal2008b}). The
metabolic reconstruction had to account for the presence in the
ancestors of the ADP-glucose-utilizing starch synthase SSIII/IV, found
today in the green algae and in plant chloroplasts 
(Figure~\ref{fig1}). This enzyme was initially thought to be encoded in
the nucleus by an EGT from cyanobacteria, and thus to require
ADP-glucose, at least initially. The proposed metabolic reconstructions
clearly indicated that ADP-glucose pyrophosphorylase, the enzyme of
ADP-glucose synthesis now found in the plastids of green algae and
plants, was the only glycogen metabolism enzyme remaining inside the 
cyanobiont~\cite{Deschampsetal2008b,Deschampsetal2008a}. Interestingly,
a recent phylogenetic analysis demonstrates a likely specific
affiliation of ADP-glucose pyrophosphorylase to the
\textit{Gloemargarita lithophora}  enzyme~\cite{Deschampsetal2023}.
Because Gloeomargarita is presently thought to be the closest extant
relative to the plastid donor, we believe that this affiliation
strongly supports an EGT to the green lineage ancestors, rather than
acquisition of the gene through LGT from other cyanobacteria post
endosymbiosis. This further supports the idea that ADP-glucose
pyrophosphorylase experienced a strong selection for its maintenance in
the common ancestor of all Archaeplastida at the very onset of plastid
endosymbiosis, despite all other enzymes of cyanobacterial glycogen
metabolism having been lost! Indeed, enzymes of glycogen metabolism, in
general, have been noted to be most often lost through the 
ratchet~\cite{Henrissatetal2002} as bacteria become facultative
obligate intracellular pathogens or symbionts. In line with Henrissat's
finding~\cite{Henrissatetal2002}, the carbon-fixing Paulinella
cyanobacterial chromatophore or the nitrogen-fixing cyanobacterial
``spheroid bodies'' mentioned above have had their genomes stripped
clean of glycogen metabolism genes, while these are universally present
in all their free-living relatives. Interestingly, ADP-glucose
pyrophosphorylase is entirely and exclusively devoted to glycogen
synthesis in cyanobacteria, where it defines the major rate-controlling
step of the  pathway (reviewed in Ball et~al.~\cite{Balletal2015}).
This enzyme is finally tuned by photosynthesis through its substrates
ATP and glucose-1-P and through its allosteric activator 3-PGA or
through its Pi inhibitor. As a result, it generates a flux to storage
optimized as a function of the photosynthetic activity and of the
cyanobacterial energy charge, which 
\mbox{justifies} its maintenance within
the plastid stroma. Unlike the ADP-glucose pyrophosphorylase, proposed
to have remained in the plastids in the presence of its substrates and
allosteric effectors, the ADP-glucose utilizing starch/glycogen
synthase was, on the contrary, quite logically proposed to be located
in the host cytosol. There, its glucan primer and polysaccharide
products were selectively present, as is the case today for red algae
and glaucophytes. For the flux to be operating, the ADP-glc synthesized
within the plastid necessarily had to be transported from the
cyanobiont to the cytosol. It was therefore proposed that an ancient
host-derived nucleotide sugar transporter was responsible for this 
function~\cite{Deschampsetal2008b}. At the time of the reconstruction
proposal, solid phylogenetic evidence had been produced showing that
most present plastidial carbohydrate transporters exporting
photosynthate from the plastid to the cytosol in red and green algae or
plants could be traced back to a unique ancestor that belonged to the
nucleotide sugar translocator (NST) family of endomembrane eukaryotic 
transporters~\cite{Tyraetal2007,Weberetal2006}. Of particular interest,
some of the proximal eukaryotic endomembrane NSTs were GDP-mannose
translocators, and such NSTs were subsequently shown to be very
efficient ADP-Glc translocators, as demonstrated by uptake assays using
recombinantly produced transporter protein reconstituted into 
proteoliposomes~\cite{Colleonietal2010}. The reconstructed ancestral
starch metabolism represents an optimal symbiotic flux to initiate a
photosymbiosis based on the export of photosynthetic carbon to the
cytosol of the eukaryotic host. Such a flux was challenging to
establish due to the distinct and disconnected biochemical pathways of
both partners. The cyanobacterium would provide carbon substrates
during the day, independently of the host's demand, potentially
flooding the exquisitely osmotically sensitive eukaryotic cytosol with
unwanted and potentially cytotoxic osmotically active metabolites. On
the other hand, the host may have strong carbon demands at night, at a
time where the cyanobiont is unable to provide it. Linking the two
disconnected biochemical networks through the osmotically inert carbon
stores of the host neatly solves this asynchrony of demand and supply
of carbon at the onset of plastid endosymbiosis, effectively buffering
and synchronizing the biochemical fluxes of symbiosis. The export of
ADP-glucose as 
\mbox{photosynthate} supply molecules that are unrecognized by
eukaryotes 
\mbox{ensured} that 
photosynthetic carbon would not affect the
host's physiology and would be immediately incorporated into the host's
glucan stores through the ADP-Glc specific glycogen/starch synthases.
In addition, having the flux of photosynthate escape the cyanobiont
through ADP-glucose is not expected to affect its central carbon
metabolism in the light, as ADP-glucose synthesis only concerns the
part of the cyanobiont's metabolism that was anyhow devoted to storage.
However, NSTs exchange nucleotide sugars  with AMP; consequently,
export of NDP-sugars results in the net export of phosphate from the
cyanobiont. If not balanced by an inverse flux of Pi, this NDP-sugar
export will deplete the cyanobiont's phosphate pools and thereby
affect, for example, ATP biosynthesis by photophosphorylation. This
suggests the need for a Pi import mechanism coupled with carbon export
in the light. 

Mutants of \textit{Synechocystis} defective for glycogen metabolism
grow well  photoautotrophically~\cite{Grundeletal2012}. However, the
same mutants display severely impaired growth and reduced viability
when exposed to day and night  cycles~\cite{Grundeletal2012}. The
absence of glucan stores in cyanobacteria is indeed logically expected
to lead to severe ATP starvation in darkness in photoautotrophic
cultures. However, when the symbiotic flux of storage polysaccharide
metabolism was  proposed~\cite{Deschampsetal2008a}, this problem was
not addressed. Two key proteins were identified at the time as being
required to operate metabolic symbiosis. These consisted of the NST
transporter and the ADP-Glc utilizing starch/glycogen  synthase (the
ancestor of the green alga/plant  SSIII/IV)~\cite{Deschampsetal2008a}.
This type of glucan synthase is normally never found in the eukaryote
cytosol and displays a distribution restricted to prokaryotes. Its
presence was deduced from the comparisons of red and green algae, due
to its selective presence in the green lineage. The subsequent
unexpected finding of the corresponding sequence in the first
\textit{Cyanophora paradoxa} glaucophyte genome highlighted the ancient
presence of this enzyme for cytosolic starch 
synthesis~\cite{Priceetal2012}. This unexpected finding brought
considerable support to the proposed reconstitution of ancestral starch
metabolism. 

However, while SSIII/IV was originally thought to be of cyanobacterial 
phylogeny~\cite{Deschampsetal2008a}, subsequent analysis rejected a
cyanobacterial ancestry for this enzyme, while displaying a surprising,
likely 
\mbox{chlamydial}
origin~\cite{Balletal2013}. Because the SSIII/IV
enzyme seems to be at the core of the symbiotic connection, it was
proposed that it was present in the cytosol of the host at the onset of
the process. To achieve this, it was suggested that glycogen metabolism
enzymes were, in fact, cytosolic effectors secreted by Chlamydiae in
the cytosol of their hosts, to manipulate the host carbon fluxes at
their advantage. Glycogen metabolism, prior to these studies, had never
been viewed as a provider of effector enzymes. Glucose storage had
mainly been considered as an intra-chlamydial housekeeping function,
and therefore not concerned with highly specialized biotic interactions
of the chlamydial growth cycle. 

One important consequence of this proposal is that, if SSIII/IV was
indeed a chlamydial effector that was targeted to the host cytosol at
the onset of plastid endosymbiosis, it would \textit{ipso facto} be at
the centre of carbohydrate metabolism symbiotic fluxes. This would
immediately tie the chlamydia pathogen/symbiont into the symbiotic
relationship uniting not two but three genomes. This became known as
the  MATH (M\'{e}nage \`{a} Trois Hypothesis) where three rather than
two genomes encoded essential symbiosis functions during plastid
endosymbiosis. However if the gene encoding an essential function was
transferred from the chlamydia to the host genome, ERGTs from this
third genome would thus become the opposing force whereby the host
replaced chlamydia as the genome encoding the chlamydial effectors. As
long as the chlamydial ancestor either initially had, or shaped
thereafter, novel favourable biotic interactions through secretion of
chlamydia encoded effectors, its maintenance would remain necessary to
the tripartite symbiosis because it housed indispensable genes. However
when the supply of novel chlamydial effectors was exhausted and after
the corresponding genes had all been secured by ERGT in the host
nuclear genome, the pathogen/symbiont vanished. Indeed, its cellular
compartment, at variance with the electron transport membranes carrying
cyanobiont, was not hosting any indispensable biochemical reaction. The
ancestor of the SSIII/IV starch synthase was not the only chlamydial
effector transmitted through ERGT to the storage polysaccharide
metabolism network of Archaeplastida. GlgX, encoding a ``direct
debranching  enzyme'' (also called isoamylase), was also 
\mbox{transmitted} to
all three Archaeplastida lineages.  Isoamylase (ISA) is never found in
eukaryotes, save Archaeplastida. Glycogen-accumulating eukaryotes use
another, more complex enzyme named ``indirect debranching enzyme
(iDBE)'' to hydrolyse the $\upalpha$-1,6 branch linkage through
an altogether different mechanism  (reviewed in Ball
et~al.~\cite{Balletal2015,Balletal2011}). Mutants of isoamylase in all
plants tested revert to the accumulation of hydrosoluble glycogen. The
latter was likely the ancestral form of hydrosoluble storage
polysaccharide present in the cytosol of the host  (reviewed in Cenci
et~al.~\cite{Cencietal2014}). It was therefore proposed that
isoamylase was responsible for trimming out the branches that supported
those chains during synthesis; otherwise, these branches would have
prevented the proper alignment of chains required for the
crystallization of glucans into huge solid and insoluble aggregates
known as  starch~\cite{Balletal1996}. That ISA assumes an analogous
function in red algae is suggested by the recent description of mutants
of \textit{Cyanidioschizon merolae} defective for ISA and starch 
synthesis~\cite{Maenoetal2022}. However, we do not think that the
initial function of the chlamydial effector was to generate starch from
glycogen metabolism outright. GlgX ISA enzymes in bacteria have evolved
to ensure glycogen breakdown through the release of small unbranched
malto-oligosaccharides (MOS). Eukaryotes do not produce MOS in the
cytosol and do not have MOS breakdown enzymes in this compartment.
Hence, having GlgX together with other chlamydial effectors in the
cytosol would not only induce glycogen synthesis through chlamydial
effectors using host substrates and energy, but it would also give
privileged and exclusive access to the Chlamydiae of the glycogen
metabolism breakdown products. The function of ISA in starch
crystallization likely evolved later and required the co-evolution of
novel host-encoded glucan-water dikinases, which are indispensable to
the breakdown of glucan crystallites in eukaryotes  (reviewed in detail
in Ball et~al.~\cite{Balletal2015}, Cenci
et~al.~\cite{Cencietal2014}).

\section{Obligate intracellular bacteria as helper genomes of plastid
endosymbiosis:\newline
establishing the nitrogen metabolism\newline symbiotic
fluxes}\label{sec9}
Carbohydrate (starch) metabolism was not the only pathway impacted by
multiple chlamydial ERGTs; tryptophan and isoprenoid metabolism were
equally impacted. Of a total of seven protein subunits \mbox{involved} in the
selective biosynthesis of tryptophan from chorismate, three are
entirely cyanobacterial in all Archaeplastida, two are entirely
chlamydial, one is entirely planctomycetal, and one is partly
chlamydial  (red algae) and partly cyanobacterial (glaucophytes and
green  algae)~\cite{Cencietal2016}. The Planctomycete ERGT of TrpE is
of high significance, since this subunit controls and regulates the
flux to tryptophan through its feedback sensitivity to the final\break
product~\cite{Cencietal2016}.

Of all 20 amino acids, tryptophan displays the most energy-consuming
synthesis pathway and, as such, has led to the evolution of several
documented biotic interactions, whereby chlamydia subverts its host to
supply either tryptophan itself, tryptophan metabolites, or substitutes
such as kynurenine or  indole (reviewed in Bonner
et~al.~\cite{Bonneretal2014}). According to the species and the
strategy of tryptophan scavenging involved, chlamydia would have either
lost or kept most of the genes for tryptophan synthesis. Chlamydiales,
rather than Chlamydiaceae, are closer to the suspected donors of ERGTs
to Archaeplastida. Among them, \textit{Simkania negevensis} contains
the full suite of tryptophan synthesis genes from chorismic acid  (7
genes) on an operon hosting, in addition, a gene encoding
3-Deoxy-D-arabinoheptulosonate 
\mbox{7-phosphate} synthase  (DAHP
synthase)~\cite{Bonneretal2014}. DAHP synthase is a major
flux-controlling enzyme in the shikimate pathway leading to chorismic
acid, the substrate for aromatic amino acid biosynthesis, including
tryptophan, and for the building of many other aromatic compounds. If
such an 8-gene set was introduced into a eukaryote by LGT either to an 
organelle (mitochondrion or plastid) or to another intracellular
pathogenic/symbiotic bacterium, then these compartments could be
induced to massively overproduce tryptophan. However, for such a
conjugative gene-transfer type of biotic interaction to be useful to an
incipient Simkania-like Chlamydiales, the latter would have to also
trigger the release of tryptophan to the cytosol from which it could be
harnessed for subsequent use. It was thus proposed that an ancient
Chlamydiales transferred both the genes of tryptophan synthesis and
tryptophan export to the cyanobiont. The phylogeny of TyrP/Mtr,
previously identified as the major tryptophan transporters of 
Chlamydiae~\cite{Bonneretal2014} was thus 
investigated~\cite{Cencietal2016}. TyrP was thereby proven to define
another well supported yet previously undetected ERGT from Chlamydiae
to all three Archaeplastida lineages, arguing for a very early
implementation of this pathway during 
endosymbiosis~\cite{Cencietal2016}. It is very difficult to consider
that ERGT of TyrP and those of several tryptophan biosynthetic genes
from Chlamydiae to Archaeplastida could be coincidental. This
observation thus lends considerable support to what could have been
otherwise considered as far-fetched speculations. In addition, during
these investigations, evidence was provided for a direct exchange of
genes in the intracellular environment between Chlamydiales and other
obligate or facultative intracellular  bacteria (Legionella, Coxiella,
Piscirickettsia), in both the phylogenies of the tryptophan synthesis
genes and that of the TyrP  transporters~\cite{Cencietal2016}.

It must be emphasized that, before these analyses, some evidence had
been obtained very early on concerning possible conjugative gene
transfers of group~I introns containing single LAGLIDADG Homing
Endonuclease Genes  (HEGs). These transfers involved the Chlamydiales
\textit{Simkania negevensis}, green alga plastids and protist 
mitochondria~\cite{Everettetal1999}. These intron transfers were
reinvestigated a few years later  by Brinkman
et~al.~\cite{Brinkmanetal2002}, and nearly a decade later  by Haugen
et~al.~\cite{Haugenetal2007}, who added cyanobacteria to the
previously reported list of organisms and organelles involved in these
group~ I intron gene transfers. What was not previously noted in these
studies is that the few thermophilic freshwater cyanobacteria reported
to house these introns are phylogenetically close to what is now
considered by many as the likeliest candidate relatives of the plastid 
donor~\cite{Haugenetal2007,Ponce-Toledoetal2017,RockwellLagarias2023}.
However, because of the patchy distribution of the strains concerned by
these exchanges, no firm conclusions can be made as to the source of
these introns. The suspicion thus remains that they could reflect
ancient gene transfers between Chlamydiales and the cyanobacterial
ancestors of plastids.

We do believe, nevertheless, that a strong case exists for an initial
interaction between chlamydia, cyanobiont and host that involved both
the export of photosynthate through the action of chlamydial encoded
cytosolic effector enzymes secreted through the chlamydial T3SS, and
the induction of tryptophan synthesis within the evolving plastids
through direct operon and gene transfers from chlamydial genes or
plasmids. The presence of a robust planctomycete ERGT for  TRPE
(anthranilate synthase subunit~I) in glaucophytes as well as green and
red algae proves anyhow that the cyanobiont had lost very early on the
cyanobacterial control of flux to tryptophan by anthranilate synthase
feedback inhibition. The shikimate pathway, which provides the
chorismate common substrate for aromatic amino acid biosynthesis, is
absent in most eukaryotes except for fungi and Archaeplastida. While
some evidence suggests the presence of multiple cyanobacterial EGTs in
this prokaryotic  pathway~\cite{Richardsetal2006}, the first
rate-controlling step of this  pathway (DAHP synthase) is suggestive of
a non-cyanobacterial ERGT from a non-identified bacterial 
source~\cite{Richardsetal2006}, again arguing for early loss of
cyanobacterial control of these fluxes.

\section{Obligate intracellular bacteria as helper genomes of plastid
endosymbiosis:\newline 
protection from ATP starvation and anoxia}\label{sec10}
Under ATP starvation conditions in darkness, chlorophyll biosynthesis
is impaired because magnesium chelatase cannot assemble correctly. This
results in the accumulation of ROS-generating intermediates of
chlorophyll synthesis during the night, and cell death when light 
returns (reviewed in Deschamps et~al.~\cite{Deschampsetal2008a}). It
has been proposed that the redirection of the starch metabolism pathway
to the plastid was selectively favoured in the rising green algae to
prevent the photooxidative stresses due to the increased demand on
chlorophyll synthesis in this  lineage (reviewed in Deschamps
et~al.~\cite{Deschampsetal2008a}). This increase resulted from the
building of the novel chlorophyll~b-containing light-harvesting
complexes. Starch is thought to ensure ATP homeostasis in darkness in
the stroma, thanks to the in situ availability of storage glucans. A
very insightful  study~\cite{Reinholdetal2007} proved that knockout
mutants of the Arabidopsis ATP transporters (NTT), responsible for the
unidirectional ATP import into plastids at night, experience
photooxidative stresses. Furthermore, it proved that the severity of
the stresses experienced depended chiefly on the amount of starch
remaining in the chloroplasts at the end of the night phase, which is
in line with the aforementioned proposal. It can be concluded that both
plastidial starch accumulation and ATP import from the cytosol through
the NTT transporters protect the plastid from otherwise lethal
photooxidative stresses. In addition, a plastidial starch pool
facilitates the recycling of organically bound phosphate, through the
release of Pi from sugar \mbox{phosphates}
\mbox{during} starch biosynthesis, thereby
increasing the robustness of photosynthetic metabolism in the light.

Cyanobacteria do not have ATP import proteins and thus entirely rely on
their glycogen stores to ensure ATP homeostasis during the night, and
prevent the accumulation of ROS-generating intermediates of chlorophyll
synthesis. The slow growth and viability loss of glycogen metabolism
mutants of \textit{Synechocystis} sp.\ PCC~6803~\cite{Grundeletal2012}
grown under alternating day and night cycles can be, at least partly,
understood in this light. The MAT hypothesis posits that chlamydial
effectors generate a flux of carbohydrates from the plastid to the host
cytosol that is expected to substantially diminish or even wipe out
glycogen accumulation in the cyanobiont. This is, of course, expected
to generate severe ATP starvation during the night. It is therefore
quite remarkable to report that all three Archaeplastida  lineages
(glaucophytes and red and green algae, and plants) have experienced a
clear-cut chlamydial ERGT of a gene encoding the ATP-import protein,
the hallmark of ``energy parasitism'' in Chlamydiales and
Rickettsiales. It was proposed that the gene encoding this transporter
was transfered by conjugation to the cyanobiont, together with other
critical transporters, thereby obviating the need for an as yet
inexistent organelle protein targeting machinery. Hence, not only did
chlamydia initiate the symbiotic fluxes, but it also provided the tools
to survive the ensuing ATP starvation generated from such fluxes.

Two other pathways (namely those of isoprenoid (MEP) and
2-methyl-1,4-naphthoquinone (DHNA) syntheses) have also been targeted
by multiple chlamydial LGTs. Interestingly, the naphtoquinone is
coupled to polyprenyl chains, which are themselves synthesized through
the polymerization of isoprenoids to generate both  menaquinone
(vitamin K2) and phylloquinone (vitamin K1). The prenylated quinone
yields the physiologically active electron carrier menaquinone.
Menaquinones shuttle electrons between different respiratory complexes
in anaerobic respiration, or aerobic respiration of bacteria in a
microaerophilic environment. Reduction of the prenyl side chain leads
to phylloquinone, which, unlike the membrane-diffusing respiratory
menaquinones, is a fixed electron carrier of PSI found in cyanobacteria
and Archaeplastida, or their derivatives. Menaquinone and phylloquinone
share a common pathway, and all Archaeplastida contain such quinones,
which are essential for PSI function and, therefore, for
photosynthesis. Two distinct  pathways (Men and Futalosine) have been
documented for the conversion of chorismate to menaquinone or
phylloquinone in bacteria and  archaea (for review see Zhi
et~al.~\cite{Zhietal2014}). All red and green algae use the so-called
``Men'' pathway involving the production of  OSB (o-succinyl benzoate),
which is transformed into DHNA in the peroxisome. Our present knowledge
of menaquinone/Phylloquinone metabolism in glaucophytes does not allow
the identification of early Men pathway  enzymes~\cite{Cencietal2018},
save for the presence of MenB in \textit{Cyanoptyche gloeocystis}. MenB
uses O-succinyl benzoyl CoA as a substrate. This therefore suggests the
presence of an OSB-generating Men rather than a Futalosine pathway in
glaucophytes. However, we must presume in this case that the
glaucophyte enzymes have diverged beyond recognition. Interestingly,
freshwater basal cyanobacteria, such as Gloeobacter and Gloeomargarita,
seem to lack both the Men and Futalosine pathways. Yet Gloeobacter, at
least, can produce menaquinone under axenic growth conditions and hosts
menaquinone in PSI as do all organisms performing oxygenic
photosynthesis, which host either menaquinone or 
phylloquinone~\cite{Mimuroetal2005}. Because Gloeomargarita-like
cyanobacteria are the closest relatives to the plastid ancestor, it is
thus entirely possible that glaucophytes contain cyanobacterial Men
pathway genes that remain unrecognizable.

Mesophilic red algae and green algae (and plants) contain nuclear
sequences that yield the production of various fusion protein
arrangements in the Men pathway. They are collectively named PHYLLO.
However, unicellular thermophilic red  algae (the Cyanidiales), which
harbour the most gene-rich plastid genomes among Archaeplastida,
contain a seven-gene cluster encoding all the genes required for the
production of the DHNA menaquinone core, in addition to MenA, which is
required for its prenylation. Cyanidiales, unlike other red and green
algae, do not contain nuclear PHYLLO genes. While initial studies
suggested that this gene cluster may have been monophyletic with the
nuclear PHYLLO  proteins~\cite{Grossetal2008}, more recent analyses
question this result while not rejecting  it~\cite{Cencietal2018}. The
relationship between PHYLLO and this cluster remains, unfortunately,
\mbox{unresolved.}

No extant cyanobacteria contain these genes, and the cluster must have
entered the plastome at minimum before Cyanidiales  diversification (if
the PHYLLO fusions and the cluster genes are considered polyphyletic)
and perhaps even before Archaeplastida diversification  (if PHYLLO and
plastidial cluster genes have at least in part a common origin). Single
gene phylogenies of the small Men proteins are particularly
problematic. Few trees of the Men cluster are indeed congruent, which
is due to the intensive gene sharing among bacteria, thereby defeating
phylogenetic signal increases by concatenating the analysis of these
small proteins. Despite this, the largest Men protein (MenD) displays
robust phylogenies and clearly shows a chlamydial 
origin~\cite{Cencietal2018}. Moreover, the smaller MenF gene shows
enough congruence with MenD to allow for concatenation, further proving
a chlamydial origin. Despite the unresolved phylogenies for the other
Men genes of the cluster, all trees show Cyanidiales monophyly,
strongly suggesting that the cluster came in one piece. Hence, we can
safely conclude that the plastome acquired this cluster through LGT
from  Chlamydia~\cite{Cencietal2018}. We propose that the mechanism
involved is identical to that hypothesized for tryptophan metabolism,
and consisted of conjugative transfer. Indeed, those Chlamydiales
suspected of having donated genes to the Archaeplastida ancestors are
fully equipped to handle not only protein effector secretion through
the T3SS in the host cytosol or elsewhere, but also conjugative DNA
transfer through the  T4SS~\cite{Vougaetal2017}. That such organellar
cluster transfers are indeed occurring between obligatory intracellular
pathogens/symbionts and their hosts is exemplified by the relationship
uniting Phycorickettsia and its secondary plastid endosymbiosis host
ochrophyte  lineages~\cite{Sevcikovaetal2019,Yurchenkoetal2018}. In
this case, Phycorickettsia has also donated a 6 ``ebo'' gene cluster of
unknown function to the plastome of several eustigmatophyte algae. The
cluster conditions the biosynthesis of an unknown prenylated compound,
which in other bacteria is likely involved in the transport of small
molecules across  membranes~\cite{Klickietal2018}. Interestingly,
evidence of multiple losses from the plastome of genes encoding the
plastidial  ACP (acyl carrier protein) gene responsible for plastidial
fatty acid biosynthesis have been recently 
reported~\cite{Sevcikovaetal2019}. These losses were allowed by the
prior presence of a nuclear LGT in eustigmatophyte algae. This LGT
consists of a phycorickettsial ACP gene whose product was targeted to
the secondary plastids. This is reminiscent of the chlamydial ERGTs to
the host of primary plastid endosymbiosis. However, there is no present
indication that the LGT is correlated to the secondary plastid
endosymbiosis that generated the ochrophytes. Nevertheless, the timing
of this nuclear LGT seems to coincide with the base of the
eustigmatophyte algae and could still very well define a
eustigmatophyte-specific acquisition resulting from a bacterial ERGTs
that occurred during secondary endosymbiosis of ochrophytes. Unlike
Chlamydiales, which disappeared a long time ago from Archaeplastida,
Phycorickettsia is still actively replicating in eustigmatophyte algae
today. Interestingly, only those eustigmatophyte lineages that have
conserved the naked zoospores life stage in the eustigmatophytes life
cycle seem to be prone to Phycorickettsia 
infection~\cite{Yurchenkoetal2018}. Other lineages devoid of this stage
might have closed the door to infection by building a permanent cell
wall. This, again, could recapitulate what has happened with
Chlamydiales after primary plastid endosymbiosis. As with the
menaquinone chlamydial gene cluster in Cyanidiales, the plastidial ebo
gene cluster is present in strains that are not currently infected by
Phycorickettsia. We can thus presume that it provides an important,
albeit unknown, selection advantage to the extant ochrophyte host,
which results from the exceptionally high levels of expression of these
genes afforded by  plastids~\cite{Oeyetal2009}. The selection advantage
yielded by the final biosynthetic product of ebo gene activity might
very well be one of several reasons for the maintenance of the
Phycorickettsia symbiont in those lineages lacking the ebo plastidial
cluster.

Does the plastidial menaquinone gene cluster yield a comparable
selection advantage in Cyanidiales? We argue that this is indeed the
case. Because warmer waters are notably impoverished in oxygen,
expression of the menaquinone gene cluster to very high levels from
plastidial genes would have increased significantly and flooded the
small Cyanidiales cell volume with menaquinone with no need for
specific transporters, as such molecules readily cross membranes.
Menaquinone supplied in the diet is known to be used in animal
mitochondria, where its substitutes for ubiquinone to ensure optimal
ATP synthesis in a microaerophilic environment. This would selectively
protect extant Cyanidiales from anoxic stress. We argue, nevertheless,
despite the 
\mbox{unresolved} nature of the relationship between PHYLLOs and
the plastidial gene cluster, that this function might also have applied
to the common ancestor of Archaeplastida. Overexpression of the Men
pathway in the cyanobiont would have equally protected the cyanobiont
from anoxic stresses. In addition, if the speculations concerning early
induction of both tryptophan synthesis and the shikimate pathway that
generates the chorismic acid substrate are correct, then the
implementation of massive menaquinone synthesis at endosymbiosis would
have been greatly facilitated because of chorismate overproduction.
Green alga photosymbionts of salamander embryo cells have been recently
documented to express a severe hypoxic stress response as they
penetrate within the animal cell 
cytosol~\cite{BallCenci2017,Burnsetal2017}. It is likely that
free-living cyanobacteria that have been enslaved within eukaryotes in
a chlamydial inclusion would likely experience similar hypoxic
stresses. Free-living cyanobacteria contain high affinity terminal
oxidases to manage such stresses in the extracellular environment.
These would have benefited from an increase in menaquinone supply to
further optimize respiratory ATP synthesis. Under these conditions, a
large increase in menaquinone synthesis afforded by conjugative
transfer of the Men genes would have been mutually beneficial not only
for both Chlamydiae and cyanobacteria, but also for mitochondrial
respiration by the host. Indeed, this would have further protected the
cyanobiont from ATP starvation in darkness, in conjunction with the
expression of the ATP import protein discussed above.

However, for menaquinone to assume this protective function,
overproduction of the core quinone must be in line with the supply of
its C20 prenyl tails. The latter would have depended on the synthesis of
geranylgeranyl diphosphate from the isoprenoids DMAPP and IPP. DMAPP
and IPP are generated from the bacterial MEP pathway, which is
otherwise absent from eukaryotes unrelated to Archaeplastida. The MEP
pathway seems to have been massively impacted by chlamydial 
ERGTs~\cite{Cencietal2018,ZengDehesh2021}. Of a total of seven genes,
two are certainly of chlamydial origin, while a third is possibly of 
chlamydial (or PVC) origin in all three Archaeplastida lineages. In
addition to this, a fourth gene displays a clear-cut chlamydial origin
in green algae, while both glaucophyte and red algae have conserved the
cyanobacterial copy of the  gene~\cite{Cencietal2018,ZengDehesh2021}.
Only two genes 
\mbox{display} a clear cyanobacterial ancestry in all three
Archaeplastida lineages, while the first committed step of isoprenoid 
synthesis (DOXP synthase (Dxs)) that initially gated the flux of the
cyanobacterial pathway is now of alphaproteobacterial origin. This
again, as was the case for tryptophan synthesis, suggests that the
cyanobacteria had lost control of the MEP pathway early on. As for
tryptophan metabolism, and possibly menaquinone biosynthesis, we
propose that the MEP genes were donated by conjugative transfer to the
cyanobiont, resulting in a chimeric merozygote genome. We speculate
that the high levels of synthesis of isoprenoids that possibly resulted
from the biotic interaction between chlamydia and the cyanobiont were
not desirable to maintain while forging a novel cellular organelle,
thereby leading to the preference for another bacterial source for ERGT
of the flux-limiting step. We indeed believe that flux-controlling
enzyme genes of chlamydial origin in general were tailored initially to
favour chlamydia replication. After the plastid protein-targeting
machines evolved, these genes were counterselected when the gene
encoding such enzymes was transferred. This happened to effectively
match the flux required by the host, rather than the pathogen/symbiont.
Likewise, expression of the menaquinone core genes from the host
nuclear ``PHYLLO'' gene fusion instead of the plastidial cluster
significantly decreased the flux to menaquinone.

\section{Obligate intracellular bacteria as helper genomes of plastid
endosymbiosis:\newline 
protection from host immunity and the modified
MATH}\label{sec11}
When the first glaucophyte genome sequence became available, the genome
was screened for the presence of the triose phosphate/phosphate
translocators (TPT) which are members of the plastidic phosphate
translocator (pPTs) family of transporters. TPTs are known to export
reduced carbon from plastids of both red and green algae, as well as
land plants. pPTs have been shown to be evolutionarily derived from a
unique host nucleotide-sugar transporter of the NST3 family. This NST3
family encodes several GDP-mannose transporters of the endomembrane
system that are known to display transport activity for the
bacterial-specific ADP-glucose 
\mbox{substrate,} a structural analogue of
GDP-mannose. As~detailed above, this observation led to the proposal of
the MATH, according to which carbon was originally exported from
plastids as ADP-glucose, which was then polymerized into host glycogen
by an ADP-Glc-specific glycogen synthase that was encoded and secreted
by Chlamydia. The enzyme was secreted by the T3SS as an effector. This
ensured full membership of Chlamydia in an exclusive tripartite
photosymbiosis club.

However, no TPTs or even worse, no pPTs, were found in the Cyanophora
genome sequence. Only six NSTs were found in the genome, none of which
was phylogenetically related to the pPT family of transporters. These
results were confirmed by the purification of the glaucophyte  plastids
(the muroplasts) and an experimental characterization of the muroplast 
proteome~\cite{Facchinellietal2013b}. This analysis identified only 14
transporters, which is at least one order of magnitude lower than the
number found by detailed proteomic analysis in the green lineage, as
well as the number suggested by bioinformatic analysis of red alga
genomes. Surprisingly, 25\% of the transporter complement was found to
be of chlamydial origin, including the major glucose-6-P transporter of
Chlamydiales, UhpC. This bacterial transporter exchanges glucose-6-P
for orthophosphate in a manner reminiscent of pPTs. Although this
finding can be seen as a spectacular confirmation of the importance of
Chlamydiales in early events of plastid endosymbiosis, it challenges
the original version of the MATH, which was based on ADP-glucose rather
than glucose-6-P export from the ancestral cyanobiont. Because the
MATH, as originally proposed, was supported by many other observations,
Facchinelli et~al.~\cite{Facchinellietal2013a} proposed an alternative
MATH model in which the cyanobiont entered together with a Chlamydiales
via phagocytosis  (Figure~\ref{fig2}). The phagocytic vacuole was
transformed into a chlamydial inclusion, preventing its acidification
and thus protecting the cyanobiont from phagocytosis. The cyanobiont
thus temporarily evaded the highly aggressive antibacterial immunity of
the phagotrophic host. The secluded inclusion environment also
facilitated direct gene transfer from chlamydia to the cyanobiont,
equipping it with genes encoding essential transporters, such as the
ATP import protein, TyrP, and most importantly, UhpC. These gene
transfers may also have included deregulated but redundant pathways,
such as those of 
\mbox{tryptophan,} isoprenoid, and menaquinone core
biosynthesis. Chlamydia would also secrete its usual set of glycogen
metabolism enzyme effectors, both into the inclusion lumen, where only
bacterial enzymes including the enzyme of ADP-glucose synthesis were
active, and into the host cytosol, in the presence of host glycogen
metabolism, as described in the original MATH.  Facchinelli
et~al.~\cite{Facchinellietal2013a} therefore proposed that UhpC was the
initial translocator that exported photosynthate into the lumen of the
inclusion, and that the host membrane-derived inclusion contained a
NST3 transporter for ADP-glucose export from the lumen of the inclusion
to the host cytosol. It should be emphasized that in this scheme, the
host only receives the overflow of inclusion glycogen metabolism 
(Figure~\ref{fig2}). Nevertheless, the paradox of the missing pPTs in
the glaucophyte genomes has been neatly solved and reconciled with the
MATH. Furthermore, counter exchange of glucose 6-phosphate with
orthophosphate by UhpC allows for metabolic coupling via phosphate
balancing, and it avoids depletion of the cyanobiont's phosphate pool,
which would be detrimental for photosynthetic carbon assimilation.

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Simplified representation of the  MATH (M\'{e}nage
\`{a} Trois Hypothesis) suggested in \cite{Facchinellietal2013a}. 
Chlamydia and cyanobacteria were engulfed together leading to the
localization of the cyanobacteria inside the chlamydial inclusion. This
allowed two important types of transfers for plastid symbiosis: 
(i)~Protein secretion in the host cytosol through the chlamydial type
III secretion system (T3SS), which allowed to hijack host metabolism. 
(ii)~Direct conjugation through the type 4 secretion system (T4SS)
between chlamydia and cyanobacteria, allowing for direct gene transfer
leading to the programmed enslavement of cyanobacterial metabolism, and
transport of various metabolites inside the inclusion. This led to
direct exchange between host, cyanobacteria, and chlamydia. Nc:
Nucleus, Cy:~Cyanobacteria, Inc V: Inclusion vesicule, Ch: Chlamydial
cell.} 
\end{figure*}

\section{Support for MATH from extant host-{\ubreak}chlamydia
interactions}\label{sec12} 
Can we support such seemingly far-fetched speculations with hard
experimental data? As for the discovery of the chlamydial LGT signal in
plants~\cite{Stephensetal1998} and the demonstration of the effector
nature of glycogen metabolism enzymes both in vitro and in 
vivo~\cite{Balletal2013,Luetal2013}, the answer once again came from
microbiologists and cell biologists studying these pathogens. By far
the most studied models are defined by the animal-specific infecting
Chlamydiaceae pathogens. Among these, certain strains of
\textit{Chlamydia trachomatis}  (an important human pathogen)
accumulate glycogen to such high levels within their inclusions that it
becomes easy to quantify it through cytological staining. It can thus
be easily distinguished from cytosolic host glycogen. Gehre et al., in
2016, first demonstrated, using gene silencing techniques, that the
eukaryotic genes encoding enzymes of host glycogen metabolism had
little or no effect on inclusion glycogen 
accumulation~\cite{Gehreetal2016}. They further demonstrated that
inclusion glycogen accumulation was under the control of chlamydial
enzymes, thereby confirming the results obtained by Nguyen and Valdivia
through the isolation of glycogen-branching enzyme mutants of
\textit{Chlamydia  trachomatis}~\cite{NguyenValdivia2012}. A noticeable
exception reported  by Gehre et~al~\cite{Gehreetal2016} was the host
UDP-glucose pyrophosphorylase, which greatly impacted inclusion
glycogen synthesis when decreased through silencing. However, the
Chlamydiaceae studied differed from the protist-infecting Chlamydiales
that are thought to have affected plastid endosymbiosis by two
criteria. First, ADP-glucose  pyrophosphorylase (the enzyme of ADP-Glc
synthesis) is the only enzyme of glycogen metabolism in
\textit{Chlamydia trachomatis} that has been shown not to be a
T3SS-secreted effector, whereas all glycogen metabolism enzymes of
protist-infecting Chlamydiales have been validated as T3SS effectors,
including ADP-glucose pyrophosphorylase. Consistent with this observed
difference, the recombinant chlamydial glycogen synthase of
\textit{C.~trachomatis} is able to efficiently use both UDP-Glc and
ADP-Glc, a highly unusual property for a glycogen synthase. However,
the glycogen synthases tested so far in protist-infecting Chlamydiales
were selective for ADP-Glc. The finding of a major impact of host
UDP-Glc synthesis by the host UDP-Glc pyrophosphorylase on inclusion
lumen glycogen accumulation in \textit{C. trachomatis} led to the
hypothesis that the pathogen was importing UDP-Glc across the inclusion
membrane to fuel luminal glycogen synthesis; indeed, no ADP-glucose
pyrophosphorylase can be secreted to the lumen of this organism.
SLC35D2, one of the established human UDP-Glc translocators, was shown 
by Gehre et~al.~\cite{Gehreetal2016} to be recruited to the
\textit{Chlamydia trachomatis} inclusion membrane. Silencing of SLC35D2
had the strongest effect on inclusion glycogen accumulation, validating
this NST as the major host transporter active for chlamydial inclusion
glycogen metabolism in \textit{C.~trachomatis}  (Figure~\ref{fig3}).
Thus, the modified MATH is supported by very solid experimental
evidence demonstrating the presence of a critical NST for inclusion
glycogen synthesis in Chlamydiaceae. Following these studies,  Cenci
et~al.~\cite{Cencietal2017} very simply extrapolated the Chlamydiaceae
results to protist-infecting Chlamydiales. The only differences taken
into account were the additional presence of the chlamydial ADP-glucose
pyrophosphorylase in both the host cytosol and in the inclusion lumen,
and a substrate specificity of the chlamydial glycogen synthase
restricted to ADP-glucose. In this context, ADP-Glc synthesis could
occur in the cytosol from the host's ATP and Glucose-1-P suppies in a
classical pathogenetic ``energy parasite'' fashion. The ADP-glc
synthesized can only be used by the chlamydial glycogen synthase to
feed into either cytosolic host or inclusion lumen glycogen pools. For
such a pathogen flux to be functional in the inclusion, we need only to
imagine that a host NST3 capable of translocating ADP-Glc replaces
SLC35D2 on the inclusion membrane  (Figures~\ref{fig3}, \ref{fig4}) If
we further imagine the presence within the lumen of the chlamydial
inclusion of a cyanobacterium that has been reprogrammed by chlamydial
DNA transfers of transporter genes and deregulated pathways, then we
will have massive ADP-Glc and glycogen synthesis in the inclusion lumen
during the day. This will, of course, immediately benefit the
Chlamydia, but the host will also get its share through the reverse
flux of ADP-Glc through the NST3 transporter and the polymerization of
glucose into glycogen stores  (Figure~\ref{fig4}). However, the host
would only get the overflow of photosynthate left over by Chlamydia. We
do not consider this to be day~1 of plastid endosymbiosis, but rather
day~${-}$1, when a cyanobacterium, otherwise rather unlikely to survive in
the phagotroph's intracellular environment and ill-adapted to install
sophisticated symbiotic fluxes, acquired the necessary tools to do so
and, more importantly, to go all the way to become a true novel
cellular organelle at variance with all currently known photosymbionts,
with the noticeable exception of \textit{Paulinella chromatophora}.

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Simplified view of extant glycogen metabolism of
\textit{C. trachomatis} infected human cells, based 
on~\cite{Gehreetal2016}. Chlamydia is found inside its inclusion, from
where it secretes protein effectors in both the host cytosol and the
lumen of the chlamydial inclusion. Chlamydial enzymes were demonstrated
to control inclusion lumen glycogen accumulation provided that the
pathogen are fed with host UDP-Glc substrate. This occurs thanks to the
recruitment of the human UDP-glucose transporter SLC35D2 on the
inclusion membrane. Besides this major pathway of inclusion lumen
glycogen accumulation, some evidence has been provided for the presence
of a minor pathway of cytosolic glycogen import within the inclusion.
Nc: Nucleus, BE: Branching enzyme, GS: Glucan synthase, AGPase:
ADP-glucose pyrophosphorylase, SLC35D2: nucleotide sugar transporter,
UDP-glc: UDP glucose, Inc V: Inclusion vesicule, Ch: Chlamydial 
cell.}
\end{figure*}

\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Proposed glycogen metabolism under the revised
MATH following Facchinelli et~al.~\cite{Facchinellietal2013a} and Cenci
et~al.~\cite{Cencietal2017}. The cyanobiont entered together with
chlamydia in the common ancestor of plastid endosymbiosis. It is seen
inside the chlamydial inclusion where it received the chlamydial UhpC
gene through T4SS-mediated conjugation leading to G6P export. As was
demonstrated for \textit{C.\ trachomis}-infected extant human cells  (see
Figure~\ref{fig3}), the synthesis of glycogen was likely occuring in
the inclusion thanks to the classical chlamydial enzymes effectors
involved in alpha-glucan storage  (AGPase, GS ADP-Glc, BE), and would
have represented the main flux of glycogen accumulation  (bold arrow).
In addition, ADP-glucose, due to a high concentration of this
metabolite in the inclusion, would have been exported to the host
cytosol by an host encoded NST  (nucleotide-sugar transporter). At this
stage, the host would thus get the overflow of ADP-glucose. This
bacterial-specific metabolite was incorporated in host glycogen thanks
to the chlamydial ADP-glucose specific glucan synthase  (GS-ADP-Glc)
effector as detailed in Figure~\ref{fig1}.
Nc: Nucleus, BE: Branching enzyme, GS: Glucan synthase, AGPase:
ADP-glucose pyrophosphorylase, G6P: glucose-6-phosphate, G1P:
Glucose-1-phosphate Inc: Inclusion, Ch:~Chlamydial cell, NST:
nucleotide sugar transporter, ADP/UDP-glc: ADP/UDP glucose.}
\end{figure*}

\section{What was ``Day~1'' of plastid\newline 
endosymbiosis?}\label{sec13}
In our view, the chlamydial biotic interactions described above do not
signal the onset of plastid endosymbiosis. We believe that these
interactions involve specific pathways that are reflected by the
presence of multiple chlamydial ERGTs in Archaeplastida. Bacterial
ERGTs are common and by no means limited to Chlamydiae, but multiple
bacterial ERGTs from the same source in the same pathway are
exceedingly rare and mostly limited to Chlamydiae. We believe that in
such cases the original interaction consisted of overexpression of
genes from whole pathways, which was achieved by donating deregulated
chlamydial copies of these genes to increase flux. This was accompanied
by the transfer of genes encoding future plastidial transporters, to
ensure that whatever useful compound was being synthesized, the latter
would be accessible, predominantly in the inclusion lumen. The host
would get the leftovers. It must be emphasized that ERGTs of
transporter genes are also exceedingly rare among bacterial ERGTs and
mostly limited to Chlamydiae. 

Nevertheless, the host leftovers were substantial and included most
importantly photosynthetic O$_{2}$, which is of no use for
Chlamydiales but extremely desirable for host mitochondrial
respiration, as well as menaquinone for nocturnal 
\mbox{respiration} in a
\mbox{microaerophilic} environment, some carbohydrates into its glycogen
stores, and tryptophan, among other things. We suggest that the demands
made on cyanobacterial metabolism were considerable, and that, despite
the efforts of chlamydia to ensure ATP homeostasis in the enslaved
cyanobionts through gene transfers, the association may have been
transient. In the absence of extant representatives of the responsible
chlamydial symbionts, we can only speculate about the number and type
of genes involved and the modalities of such gene transfers. We propose
that, at minimum, UhpC, the ATP import protein (NTT), TyrP  (the
tryptophan transporter) the whole plastidial menaquinone cluster and
the isoprenoid MEP and tryptophan pathways were donated by a chlamydial
ancestor, 
\mbox{possibly} on a conjugative plasmid in a fashion similar to the
Agrobacterium/plant biotic interaction, albeit in the intracellular
environment of protists. The host was possibly a phagotroph, grazing
mostly on 
\mbox{freshwater} cyanobacteria. The introduction of photosymbiosis
to alleviate the stress of anoxia may have been highly desirable in the
environment, especially if the latter was warm and the available oxygen
was limited. The reprogrammed cyanobacteria were a dead end as far as
survival in the extracellular environment is concerned, because it was
leaking tryptophan, glucose-6-P, quinones, and isoprenoid. We define
Day~1 of plastid endosymbiosis as the first day following the escape
from the chlamydial inclusion to the cytosol of such a
Chlamydia-enslaved cyanobiont. This escape was not trivial because it
possibly entailed a complex fusion of the prokaryotic cyanobacterial
outer membrane with the eukaryotic-derived but chlamydia remodelled
inclusion membrane, leading to novel outer cyanobiont membranes with
both inner-layer prokaryotic and outer-layer mixed
prokaryotic/eukaryotic features. We can only speculate about the
properties displayed by the cyanobiont's novel outer membrane. We do
not know what level of protection from host immunity is provided by
this membrane, nor how much of the ancient chlamydial protein targeting
to the inclusion has remained functional. This  escape resulted in the
presence of transporters not only of cyanobacterial or chlamydial
origin, encoded in the cyanobacterial merozygote genome, but also of
host origin that were recruited by Chlamydiales on their inclusion,
thereby establishing early connectivity of the evolving plastid. Both
ADP-Glc transport via the host NST and glucose-6-P transport via UhpC
generated suitable carbohydrate fluxes cementing the endosymbiotic
relationship. During the evolution of the glaucophyte lineage, the
NST-mediated transport was lost; in contrast, during evolution of green
and red algae, the NST that had evolved into a sugar phosphate
transporter similar to UhpC replaced the latter. If, unlike the
inclusion membrane, the novel rearranged cyanobiont membranes became
sensitive to RAMPs, the cyanobiont may have needed to become resistant
to such peptides. Several possible resistance mechanisms are
documented. Among these, RAMP import into the cyanobacterium, followed
by proteolytic degradation of the peptides, provided a scaffold on
which the TIC--TOC machinery could evolve to ensure the targeting of
proteins containing RAMP-like plastid targeting sequences, as discussed
above, recapitulating the events that had occurred during mitochondrial
endosymbiosis. It is plausible that the escaped cyanobiont received the
critical genes from other intracellular bacteria present in the host,
as suggested by recent  studies~\cite{Garridoetal2022}. At this stage
(day~1) the cyanobiont still contained all the genes in its free-living
relatives. However, it also contained a moderately chimeric genome,
including the genes required to export photosynthate (UhpC) and
tryptophan in an optimized fashion, as well as those required to
obviate the oxidative, anoxic, and energy stresses generated by this
export, and to survive in an intracellular environment. These are
things that the cyanobacterial genomes were not tailored to achieve.
Most importantly, it did all these things using specific protein
transporters, without resorting to antimicrobial peptide tinkering of
membrane permeability to generate symbiotic fluxes. Such tinkering is
observed in the many extant symbioses studied in plants and insects 
(see above), including in Paulinella, which is devoid of
transporters on the chromatophore membranes. It did this by leaving the
photosynthetic membrane function intact. It is useful here to continue
the comparison between Agrobacterium and the hypothetical chlamydia
symbiont that could have primed plastid endosymbiosis to help grasp the
amount of evolution required in these processes. As was the case for
the Agrobacterium/plant mediated interactions, it probably took
millions of years to evolve the chlamydial biotic interactions that
resulted in high frequency conjugation of transporters and deregulated
genes that prompted tryptophan and photosynthate export, as well as
those genes required to resist ATP starvation and ensure prolonged
survival in the chlamydial inclusion. From the cyanobacterium point of
view, it took only hours, days and weeks to be engulfed by a protist
together with chlamydia and to be reprogrammed by conjugation to become
enslaved in exporting photosynthate, amino acids, quinones, or
isoprenoids. This is similar to the growth of a tumor during the
Agrobacterium/plant interactions. Reprogrammed plant tumor cells have
no future on their own in the environment, and reprogrammed and
metabolite leaking cyanobacteria would have met with very limited
success if accidently released in the environment. 

Successful escape from the inclusion and simultaneous acquisition of
resistance to eukaryotic host cell immunity was a complex and rare
event. The cyanobiont was now isolated from its free-living relatives
and experienced Muller's ratchet, yielding 
\mbox{reductive} genome evolution.
The irreversible nature of the multiple possible patterns of gene
losses began distinguishing several lineages that were selected in
different environments and that experienced the forging of different
types and numbers of new host genes. Finally, this process ended with
the emergence of green algae, red algae and glaucophytes. This process
of reductive genome evolution, leading to different outcomes of gene
transfers to the host genome, together with the evolution of both
common and different sets of novel host genes, is collectively referred
to as metabolic integration of the protoplastid. The Chlamydia-primed
plastid endosymbiosis can thus be considered as a highly successful
process. As pointed out in this review, the precise nature of the
symbiosis occurring at the very beginning of the reductive evolutionary
process would have significantly influenced the outcome and success of
the endosymbiosis. Indeed, the chlamydia-primed symbiosis offered an
unparalleled level of preadaptation at the very beginning of the
process, which may explain the rarity of primary plastid endosymbiosis
compared to the plethora of extant diverse photosymbioses. Despite the
relative speed of action of the ratchet, the entire metabolic
integration may have taken up tens to hundreds of millions of years.
Even after the escape of its enslaved endosymbiont, the first phases of
this evolution still required the maintenance of the chlamydial
symbiont, which secreted beneficial effector enzymes into the host
cytosol. Indeed, the presence of chlamydial genes in the evolving
chimeric plastome did not warrant the maintenance of the symbiont,
since the plastid fully took over the production of these proteins;
rather, it was the evolution of new useful cytosolic effectors for the
tripartite symbiosis that have ensured further maintenance. In the end,
the transfer of these useful genes to the nuclear genome by ERGT would
have rendered chlamydia redundant and probably signalled its
disappearance from\unskip\break 
Archaeplastida.

\section{What next? Can we find the culprit of MATH?}\label{sec14}
Because of the apparent disappearance of Chlamydia pathogens from
Archaeplastida, studies leading to the m\'{e}nage \`{a} trois
hypothesis have mainly {relied} on linking the phylogenetic imprint of
\mbox{chlamydial} ERGTs to knowledge of biochemical pathways and their
possible functions in both chlamydial and cyanobacterial physiology.
While interesting and rich in useful predictions  (see above), this
approach alone is not sufficient to prove the MATH, let alone encourage
young scientists to further investigate a topic that has been met with
such strong and often poorly justified 
opposition~\cite{Balletal2016b,Balletal2016a,Dommanetal2015}. Indeed,
when looking at biochemical pathways and gene transfers in
Archaeplastida genomes, we will always be looking at single gene trees
of mostly enzymes. Depending on the nature of organisms investigated,
the inference of evolutionary trajectories in some trees may be
obscured by repeated gene sharing events, which are particularly
difficult to interpret when occurring in deep time. In addition, these
trees target enzymes that respond to local selection in  lineages
(e.g., subtle substrate changes, shifts in compartmental localization)
rather than being highly conserved, slowly evolving functions such as
ribosomal RNAs. As such, the trees will in most cases be poorly
resolved, with either poor or no bootstrap support, as well as
topological rearrangements that are challenging to reconcile. Everyone
in the field of deep evolution is aware of these problems, and people
who perform such analyses are accustomed to dealing with phylogenetic
uncertainty. It is therefore logical to amend the deep-time toolkit
with robust biochemistry-based arguments whenever possible. For
instance, we believe that knowledge of gene distributions should be
actively pursued to resolve questions about the directionalities of
gene  transfer~\cite{Balletal2016b,Balletal2016a}. For us, the
shortcoming of phylogenetic approaches is not the inevitable
uncertainties, but rather that even a well-established ERGT obviously
does not tell us per se what was the underlying mechanism of its
emergence. For instance, is a specific chlamydial ERGT a mere gene
replacement of a cyanobacterial gene because the protein was poorly
suited to the TOC--TIC targeting mechanism and because Chlamydiae were
present in the environment? Or does it reflect a more sophisticated
biotic interaction whereby the gene was first introduced into the
cyanobiont? We cannot rely on phylogenetic trees to resolve these
issues. One possibility would be to make progress by attempting
endosymbiosis experimentally. Such approaches are ongoing, and will
likely inform us on many issues dealing with the establishment of
\mbox{bacteria} or eukaryotes within host phagotrophs and eukaryotes in
general. However, it is unlikely to yield information about the
particular question dealt with in this review, which is addressing
organelle-generating endosymbioses. Another possibility would be to
find Chlamydiales that infect protists grazing chiefly on freshwater
basal cyanobacteria related to those suspected to be the closest
relatives of the plastid progenitor. Finding a chlamydia interacting
with cyanobacteria in such a phagotroph would indeed be a substantial
leap forward. The recent finding of the Phycorickettsia/ochrophyte
interaction encourages us in this respect.

\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Acknowledgements}
SGB, UC, ML, and CC were supported by the CNRS, the
Universit\'{e} de Lille CNRS, the ANR grants ``Mathtest''
(ANR-18-CE13-0027) ``expendo'' (ANR-14-CE11-0024) and ``m\'{e}nage
\`{a} trois'' (ANR-12-BSV2-0009) and the R\'{e}gion Hauts de France
and European project Alibiotech.
UC, SGB, CC acknowledge BILILLE to provide computational resources. 
IL and OV were supported by ANR grant ``Mathtest'' (ANR-18-CE13-0027).

\CDRGrant[ANR]{ANR-18-CE13-0027}
\CDRGrant[ANR]{ANR-14-CE11-0024}
\CDRGrant[ANR]{ANR-12-BSV2-0009}
\CDRGrant[ANR]{ANR-18-CE13-0027}

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