# Hereditary Leaf Symbiosis in Tropical Plants: Evolution and Function of Vertically Transmitted Bacterial Symbionts

Léa Ninzatti, Marie-Françoise Jardinaud, Aurélien Carlier

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Léa Ninzatti, Marie-Françoise Jardinaud, Aurélien Carlier. Hereditary Leaf Symbiosis in Tropical Plants: Evolution and Function of Vertically Transmitted Bacterial Symbionts. Phytobiomes Journal, 2025, 9 (3), pp.343 - 353. 10.1094/pbiomes-11-24-0111-rvw. hal-05514735

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A Transdisciplinary Journal of Sustainable Plant Productivity

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# Hereditary Leaf Symbiosis in Tropical Plants: Evolution and Function of Vertically Transmitted Bacterial Symbionts

Léa Ninzatti,<fnref n="1" /> Marie-Françoise Jardinaud,<fnref n="1" /> and Aurélien Carlier<fnref n="1" />,<fnref n="2" />,†

Accepted for publication 14 February 2025.

## Abstract

The presence of bacteria within leaf galls of some tropical plant species of the Rubiaceae and Primulaceae families was first described in the early 1900s and later recognized as signs of permanent associations, with the presence and morphology of the bacteria-hosting structures even serving as taxonomic markers. Until recently, the inability to culture the bacterial symbionts and the difficulty of obtaining bacteria-free plants reliably have limited the understanding of the functions of these symbioses and the mechanisms enabling transmission across generations. The development of culture-free methods has

made it possible to identify bacterial endosymbionts and to realize that these symbioses are highly specific, with each host plant interacting with only one bacterial species. Here, we review the current knowledge of these highly specific leaf symbiotic associations, focusing on recent experimental evidence. We explore hypotheses regarding the mechanisms enabling these highly specific interactions.

**Keywords:** bacteria, specialized metabolism, symbiosis, vertical transmission

All plant surfaces, including roots, shoots, seeds, or propagules engage with microorganisms, collectively called the plant microbiome. These include bacteria, fungi, archaea, protists, and viruses. Most of these interactions are neutral, but some have a beneficial outcome, improving access to nutrients or providing protection against pathogens (Fitzpatrick et al. 2020). This is, for example, the case for root nodule or arbuscular mycorrhiza symbiosis, where potential partners establish sophisticated molecular dialogs (Martin et al. 2017). In addition to these well-documented root symbioses, some Primulaceae, Rubiaceae, and Dioscoreaceae species also form highly specific associations with bacteria in their leaves. In these species, bacteria are often seed-transmitted and grow to very high densities in the leaves.

**Funding:** L. Ninzatti and A. Carlier acknowledge support by the French Laboratories of Excellence Project "TULIP" from the French National Research Agency (ANR-10-LABX-41; ANR-11-IDEX-0002-02). This study is set within the framework of the "École Universitaire de Recherche (EUR)" TULIP-GS (ANR-18-EURE-0019). A. Carlier also acknowledges funding from the French National Research Agency under grant agreements ANR-22-CE92-0042 and ANR-23-CE11-0015 and the Région Occitanie through the "RAMSY" project.

The author(s) declare no conflict of interest.

## Taxonomic Diversity and Leaf Symbiotic Structures

The main feature of leaf symbiosis is the consistent presence of a single endosymbiont species in large numbers within the leaves of host plants. These particular associations have been referred to historically as leaf nodule symbiosis, owing to the common presence of structures on the leaves hosting the bacteria (Lersten and Horner 1976).

### Bacterial leaf nodule symbiosis

Leaves of some species of the *Ardisia* genus (Primulaceae), and from the genera *Psychotria*, *Pavetta*, or *Sericanthe* (Rubiaceae), harbor obvious round or oblong galls that encase dense colonies of symbiotic bacteria (Lemaire et al. 2011a, 2012c). These structures, sometimes called leaf nodules, are morphologically variable. For example, leaf nodules of *Ardisia crenata* and other *Ardisia* species are located at the leaf margin, in structures reminiscent of hydathodes (Fig. 1A and B) (Lersten and Horner 1976). In *Psychotria* species, including *P. kirkii* and *P. umbellata*, small nodules are scattered on the abaxial side of the leaf blade (Fig. 1C and D), whereas in *Pavetta* species, the nodules tend to protrude on the adaxial side (Von Faber 1912; Zimmermann 1902). In most leaf nodulating Rubiaceae species, the number and spatial distribution of nodules can vary widely even within a single plant, indicating that the environment may influence nodule development

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<footnote n="1">¹ LIPME, Université de Toulouse, INRAE, CNRS, 31320 Castanet-Tolosan, France</footnote>

<footnote n="2">² Laboratory of Microbiology, Ghent University, 9000 Ghent, Belgium</footnote>

<footnote>†Corresponding author: A. Carlier; aurelien.carlier@inrae.fr</footnote>
(Lersten and Horner 1976). In contrast, leaves of the monocot *Dioscorea sansibarensis* display two longitudinal glands located at the leaf tip (Fig. 1E and F) (Orr 1923). Leaf nodules or glands typically contain large numbers of bacteria. Unfortunately, because nodule bacteria of Rubiaceae and Primulaceae have not been successfully cultured yet, precise estimates of the number of bacteria

in leaf nodules are not available. Light and electron microscopy images of nodule sections of *Psychotria kirkii* and *Ardisia crenata* show densely packed masses of bacteria (Lemaire et al. 2011b, 2012b). In both of these species, nodules are roughly spherical, with a diameter of at least 1 mm. With dozens to hundreds of nodules per leaf, the numbers of bacteria hosted by the plant are likely large. The bacterial symbiont of *Dioscorea sansibarensis* is culturable, allowing for estimates of $3.4 \times 10^8$ CFU/gland (equivalent to about $4.0 \times 10^9$ CFU/g) (Carlier et al. 2017).

<figure><img src="image_11.png" /><figcaption>**Fig. 1.** Diversity of leaf symbiotic structures. A, Top-down view of an *Ardisia crenata* seedling. Nodules are located in crenations at the margin of the leaf. B, Detail of a mature leaf of *A. crenata*, showing a leaf nodule and its vascularization. Leaves were cleared in Hoyer's solution and observed under a dissecting microscope. C, The abaxial leaf surface of *Psychotria kirkii* is dotted with protruding dark nodules. D, Cross-section of a leaf nodule (n) of *P. punctata* stained with toluidine blue, showing elongated parenchyma cells (pa), surrounded by spongy mesophyll cells (sp). The symbiotic bacteria are contained in between three layers of compressed mesophyll cells. E, *Dioscorea sansibarensis* is a perennial vine with heart-shaped leaves with an elongated acumen (red arrow). The leaf acumen presents two glands containing symbiotic bacteria. F, Cross-section of a leaf acumen of *D. sansibarensis* stained with auramine O and calcofluor showing leaf tissue (visible as red autofluorescence of the chloroplasts) and trichomes (blue) surrounded by a thick cuticle (yellow) that closes up at the adaxial side into a seam and forms a physical barrier to the symbiont. Sources: microscopic picture of *P. punctata* leaf nodules (D) reproduced from Sinnesael et al. (2018) (under the terms of a Creative Commons CC BY license). Images of *A. crenata*: M.-F. Jardinaud. Images of *D. sansibarensis*: A. Carlier (E) and Olivier Coen (F).</figcaption></figure>

## Non-nodulated bacterial leaf symbiosis

Not all leaf symbionts are hosted within immediately visible structures (van Wyk et al. 1990). Some so-called non-nodulating species have been described in the Rubiaceae family, for example, *Fadogia homblei*, as well as several species of *Psychotria*, *Vangueria*, *Tricalysia*, and *Empogona* (Lemaire et al. 2012b; Verstraete et al. 2013, 2023). In these species, endophytic bacteria are not confined to specific structures but grow between the mesophyll cells within a mucus-like substrate (Stanton et al. 2013; van Wyk et al. 1990). What sets these associations apart from typical endophytic colonization is the sheer quantities of bacteria present in the leaves, which otherwise show no outward symptoms of infection. Although the exact cellular proportions are difficult to estimate, Danneels and colleagues reported proportions varying from 5 to 57% of sequencing reads assigned to specific bacterial taxa in DNA extracts of whole leaves of 16 non-nodulating Rubiaceae species belonging to the genera *Psychotria*, *Fadogia*, and *Vangueria* (Danneels et al. 2023). Similarly, sequencing reads assigned to bacteria made up from 14 to 37% of the total from DNA extracts of whole leaves of the *Trycalysia* species *T. semidecidua*, *T. lasiodelphys*, *T. hensii*, and *Empogona congesta* (Verstraete et al. 2023). Although these proportions are affected by numerous factors, such as biases in DNA extraction procedures, relative genome sizes, or contamination with plastids, they are indicative of massive colonization levels, given that bacterial genomes are typically 2 to 3 orders of magnitude smaller than plant genomes.

Interestingly, leaf symbiosis is not a monophyletic trait, even within a single plant genus (Fig. 2A). For example, leaf symbiotic nodulated and non-nodulated *Psychotria* species belong to phylogenetically distinct clades (Lemaire et al. 2012b). Furthermore, leaf symbiotic associations seem to have evolved independently in several taxa. In the Rubiaceae, phylogenetic analysis indicates separate origins for leaf symbiosis in the genera *Psychotria*, *Pavetta*, and *Sericanthe* (Lemaire et al. 2011c). In addition, leaf symbiosis seems to have evolved at least 13 Mya in *D. sansibarensis*, the only known leaf symbiosis in the genus *Dioscorea* (Danneels et al. 2021). Similarly, nodulated species in the *Ardisia* genus form a monophyletic clade that diverged from nonsymbiotic species at least 5 Mya (Lemaire et al. 2011c). Together, these observations indicate that leaf symbiosis has evolved independently multiple times in several plant lineages.

## Suspected leaf bacterial symbiosis

Aside from the relatively well-documented examples above, other plant species have been reported to host leaf bacterial symbionts, but little is yet known about those symbioses. These include *Amblyanthus* spp. and *Amblyanthopsis* spp. (Primulaceae) that were reclassified as a sister clade to *Ardisia crispa* (Zhou et al. 2021). Two Vangueriae species of the *Keetia* genus: *K. nodulosa* and *K. gueinzii* were also reported to host bacterial symbionts in nodules and in the plant intercellular space, respectively, although the taxonomy of the bacteria has not yet been determined (Cheek and Onana 2024; Tilney et al. 2009). Finally, *Styrax camporum* has been reported to engage in leaf symbiosis based on microscopic observation of what
seems to be bacteria within specialized leaf trichomes (Machado et al. 2014). However, no other *Styrax* species were reported to be symbiotic, and the identity of the bacteria has not been determined.

## Taxonomy and Origins of the Symbiotic Bacteria

In contrast to the high diversity of rhizobia species, bacterial leaf endosymbionts are restricted to three genera of the Burkholderiales. All the symbiotic bacteria of Rubiaceae and Primulaceae belong to the related genera *Caballeronia* or *Paraburkholderia*, which are part of the group *Burkholderia sensu lato* (*Burkholderia s.l.*).

### Leaf symbionts of the *Caballeronia* and *Paraburkholderia* genera

Most leaf symbiotic taxa do not have cultured representatives and are known from DNA extracted directly from leaves or nodules (Danneels et al. 2018, 2021; Lemaire et al. 2012c; Pinto-Carbó et al. 2018; Van Oevelen et al. 2001). The genomes of *Caballeronia* and *Paraburkholderia* leaf symbionts reconstructed from metagenome assemblies are considerably smaller than those of free-living related species: from 2.4 to 6.1 Mb versus an average of 8 Mb in free-living *Burkholderia s.l.* Coding capacity is also low because of numerous pseudogenes and insertion sequence elements, in the range of 40% for ‘*Candidatus* Caballeronia ardisicola’ to 80% for the *Paraburkholderia caledonica* symbionts (Danneels et al. 2023). Smaller genomes with a high proportion of pseudogenes are commonly found in obligate symbionts or pathogens that undergo frequent transmission bottlenecks. The effects of genetic drift, combined with a mutational bias toward deletions in prokaryotes, are thought to be responsible for these features (Danneels et al. 2018; Murray et al. 2021). Sequencing of taxonomic markers and

phylogenetic analyses have placed a vast majority of leaf symbiotic bacteria in novel *Candidatus* taxa, that is, taxa for which no cultured representative exists. The only exceptions so far are the symbiotic bacteria of the *Vangueria* species, *V. infausta*, *V. escu-lenta*, *V. madagascariensis*, and *V. randii*, which are uncultured but fall within the species boundaries of *Paraburkholderia phenolirup-trix* (Danneels et al. 2023). Similarly, symbionts of *V. pygmaea* are also uncultured but belong to the *Paraburkholderia caledonica* species. Interestingly, at least one Rubiaceae species harbors culturable *P. caledonica* symbionts: *Fadogia homblei*, a shrub widely distributed in grasslands of sub-Saharan Africa (Danneels et al. 2023).

### Multiple evolutionary origins of *Burkholderia s.l.* leaf symbionts

Leaf symbiosis in the *Burkholderia s.l.* is polyphyletic, with symbionts of *Sericanthe*, *Psychotria*, *Pavetta*, *Tricalysia*, *Empogona*, and *Ardisia* belonging to the *Caballeronia* genus and the symbionts of the *Vangueria* and *Fadogia* plant lineages in the *Paraburkholderia* genus (Lemaire et al. 2011a, b, 2012b, c; Verstraete et al. 2023). The picture is equally complex within each bacterial lineage. The *Ardisia* symbionts form a monophyletic clade within *Caballeronia*, perhaps indicative of a strict mode of vertical transmission and co-speciation. However, the Rubiaceae endosymbionts belong to distinct clades (Fig. 2B). For example, the *Caballeronia* symbionts of *Sericanthe* and *Pavetta* species are nested within a clade of *Psychotria* endophytes (Lemaire et al. 2011a, b, 2012c), as are the symbionts of *Tricalysia* and *Empogona* (Verstraete et al. 2023). So far, *Paraburkholderia* symbionts have only been detected in non-nodulated species but form at least three distinct lineages (Danneels et al. 2023).

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**Fig. 2.** Phylogenetic relationships among plants engaging in leaf symbiosis with *Burkholderia sensu lato* (*Burkholderia s.l.*). **A**, Cladogram of a selection of representative plant species. Black star symbols indicate species involved in a form of leaf symbiosis. Gray stars denote species with uncertain symbiotic status. Colored ranges highlight specific genera. When available, common names are shown in parentheses. Whole chloroplastic sequences or sequencing reads sets of representative Asterids species were downloaded using the NCBI datasets tool. The maximum likelihood phylogeny was inferred using REALPHY (Bertels et al. 2014) and FastTree2 (Price et al. 2010). **B**, Phylogeny of *Caballeronia* and *Paraburkholderia* species, including leaf endophytes. Genome sequences of representative *Caballeronia* and *Paraburkholderia* species were downloaded from the NCBI RefSeq database. Sets of single-copy core genes were identified with OrthoFinder (Emms and Kelly 2019) and aligned using MAFFT v.7.4751 (Katoh and Standley 2013). A maximum likelihood phylogeny was constructed with FastTree2 (Price et al. 2010). Black circles denote branch support values (Shimodaira-Hasegawa test) < 75%. Colored sections indicate the genera of the host plants for the symbiotic species or strains (green, *Ardisia*; blue, *Psychotria*; red, *Fadogia*; yellow, *Vangueria*; purple, *Pavetta*; pink, *Tricalysia*; orange, *Empogona*; white, free-living).
To add further confusion, most leaf-symbiotic *Caballeronia* and *Paraburkholderia* species are nested within broader clades that include highly diverse free-living bacteria (Fig. 2B). For example, free-living strains of *P. caledonica* are frequently isolated from soil and the rhizosphere (Coenye et al. 2001; Verstraete et al. 2014). Similarly, *Caballeronia* strains related to leaf-symbiotic species have been isolated from soil, fungi, or as insect symbionts but relatively rarely as endophytes (Eberl and Vandamme 2016).

## Common features of leaf symbiotic bacteria

Comparative genomics analyses of leaf-symbiotic *Paraburkholderia* and *Caballeronia* failed to reveal clear sets of genes that were shared exclusively among the leaf endophytes and absent from the genomes of other free-living *Burkholderia* *s.l.* (Danneels et al. 2023). This indicates that the genetic prerequisites for leaf symbiosis may be relatively common in the *Burkholderiaceae*, allowing leaf symbiotic associations to emerge from independent lineages. One of the features of *Burkholderia* *s.l.* is the ability to withstand stress, such as low pH (Stopnisek et al. 2014), and a propensity for host association (Eberl and Vandamme 2016). These features may enable a lifestyle in the endophytic niche, which is characterized by oxidative stress, UV radiation, and the low pH of the apoplast (Vorholt 2012).

## Leaf symbiosis in other taxa

The ability to withstand environmental stress is not exclusive to the *Burkholderia* *s.l.*, and neither is leaf symbiosis. In fact, leaf symbiosis in the monocot *D. sansibarensis* involves another species of the order Burkholderiales: *Orrella dioscoreae* (*Alcaligenaceae*). So far, strains of *O. dioscoreae* have been isolated only from leaf glands of *D. sansibarensis*, and strains of related species have been isolated from aquatic environments (Carlier et al. 2017; Jiang et al. 2022; Sheu et al. 2020). Similar to leaf symbiosis in Rubiaceae and Primulaceae species, the *D. sansibarensis/O. dioscoreae* association is specific: leaf glands of plants collected from tropical and subtropical Africa as well as Madagascar all contained *O. dioscoreae* (Danneels et al. 2021). Finally, *Paraburkholderia dioscoreae* Mtsb3T was isolated from the leaf surface of *Dioscorea bulbifera*, a relative of *D. sansibarensis* (Herpell et al. 2020). It is unknown how widespread the association is, or whether it is vertically transmitted. The *P. dioscoreae* Mtsb3T strain is closely related to *P. dioscoreae* Ch1-1 isolated from soil (Herpell et al. 2020, 2021; Vacca et al. 2005), and we have isolated several strains with an identical or nearly identical 16S rRNA sequence to *P. dioscoreae* Mtsb3T from leaf washes of various, unrelated plant species over the years (A. Carlier and M.-F. Jardinaud, *unpublished data*). Whether *P. dioscoreae* Mtsb3T is in the early stages of leaf symbiosis with *D. bulbifera* cannot be known with certainty, but the findings again highlight the propensity of bacteria of the *Burkholderia* *s.l.* to form stable associations with host plants.

## Transmission of Leaf-Symbiotic Bacteria

Early authors who studied leaf symbiosis in *Ardisia crispa* and *Psychotria bacteriophila* (likely a synonym of *Psychotria punctata* var. *punctata*) suggested that leaf-symbiotic bacteria were transmitted through the seeds (Lersten and Horner 1976; Miehe 1911; Von Faber 1912). This was based on microscopic observations, which showed the presence of bacteria at all life stages of the plant, as well as the fact that seeds germinated in sterilized soil produced nodulated seedlings.

## Seeds and bulbils contain the symbiotic bacteria

Using 16S rRNA gene sequencing and fluorescence in situ hybridization, Sinnesael et al. were the first to show unequivocal evi-

dence of bacterial endophytes within the seeds of *P. punctata*, near the shoot apical meristem of the embryo (Sinnesael et al. 2018). The *Caballeronia* symbiont of the closely related species *Psychotria kirkii* var. *hirtella* (probably again a heterotypic synonym of *P. punctata* var. *punctata*) was also detected in dissected seeds using PCR (Lemaire et al. 2012a). These results led to the speculation that the symbiont is vertically transmitted in all other *Psychotria* species (Sinnesael et al. 2019). To our knowledge, the most direct evidence for vertical transmission in leaf symbiosis concerns *D. sansibarensis* and its symbiont *O. dioscoreae*. Fluorescently labeled *O. dioscoreae* introduced at the apical bud of bacteria-free plants was found in all leaf glands and vegetative tissue that developed postinoculation. Bulbils, which are vegetative propagules that form the dominant mode of reproduction of the plant, also contained the tagged bacteria. This engineered association with labeled bacteria was stable for at least two generations in the greenhouse, indicating that inoculation at the shoot tip is sufficient to ensure stable colonization and transmission to the progeny through bulbils (Acar et al. 2022). Interestingly, leaf glands that had formed prior to the inoculation with labeled bacteria remained bacteria-free throughout the experiment, suggesting that inoculated bacteria do not spread to mature tissue. Spread of the bacteria to leaves and aerial reproductive organs in *D. sansibarensis* is therefore likely based on colonization of emerging organs at the shoot tip (Fig. 3). The hypothesis of a passive mode of dissemination, or at least the idea that the plant actively controls colonization by the symbiotic bacteria, is supported by the fact that motility-impaired *O. dioscoreae* strains colonize plants normally when applied at the shoot tip and are even passed on via bulbils (Acar et al. 2022). This central role of the shoot tip in leaf symbiosis is most likely conserved in other taxa as well, although evidence is less direct.

<figure><img src="image_14.png" /><figcaption>**Fig. 3.** Life cycles of *Orrella dioscoreae* and *Dioscorea sansibarensis*. *O. dioscoreae* cells are present in mucilage at the apical meristem that acts as a reservoir to ensure the colonization of the newly formed leaf glands and bulbils. The allocation of bacteria into the bulbils ensures the vertical transmission to the next generation.</figcaption></figure>
Earlier authors had recognized the presence of the endosymbiont in the apical bud of *Ardisia* and *Psychotria* nodulated species as most likely the key feature that enables vertical transmission via the seed (Acar et al. 2022; Lersten and Horner 1967; Miller and Donnelly 1987; Sinnesael et al. 2018). Based on morphological and microscopic observations, Lersten and Horner proposed that secretory trichomes surrounding the shoot apices of *Psychotria bacteriophila* provide a growing environment to the bacteria (Lersten and Horner 1967). Miller et al. later proposed that mucilage, possibly produced by these trichomes, carries the bacteria to the leaves and flowers as they emerge from axillary buds (Miller et al. 1983). However, whether mucilage plays an active role in disseminating the endophyte remains untested. Genome analysis of ‘*Ca*. C. kirkii’ and ‘*Ca*. C. crenata’ revealed that the organisms both lack a flagellum, suggesting that transport of the bacteria to newly formed organs does not depend on motility, similar to what has been demonstrated in the *D. sansibarensis*/*O. dioscoreae* symbiosis (Carlier and Eberl 2012; Carlier et al. 2016).

## Vertical transmission and host-switching

The hypothesis that vertical transmission is a common feature of leaf symbiosis is further supported by phylogenetic analysis, although with a few caveats. In *Ardisia* for example, general phylogenetic congruence between host and symbiont gene markers is consistent with exclusive vertical transmission of the endosymbiont and a single infection event (Lemaire et al. 2011b). However, later analysis using a larger dataset indicated that some horizontal transmission or host-switching likely occurred in some clades (Yang and Hu 2022). Horizontal transfer or host-switching may even occur within a single clade or species in *Psychotria*. For example, a distinct population of *P. leptophylla* harbored specific bacterial lineages consistent with the vertical transmission of the symbiont, although symbionts of one *P. leptophylla* population belonged to a clearly incongruent lineage (Lemaire et al. 2012a). Furthermore, molecular phylogenetic analysis from endosymbiont 16S rRNA, *recA*, and *gyrB* marker sequences indicated the absence of strict host symbiont co-speciation in Rubiaceae species of *Pavetta*, *Psychotria*, and *Sericanthe* (Lemaire et al. 2011c). This is also the case in *D. sansibarensis*, where co-phylogenetic analysis of several populations suggested a mixed mode of transmission (Danneels et al. 2021). However, rejection of the co-speciation hypothesis does not equate a horizontal mode of transmission. Indeed, co-phylogenetic analyses are easily confused by horizontal transmission events, with even rare host-switching events sufficient to reject a strict co-speciation hypothesis (de Vienne et al. 2013). Finally, little is known about transmission of the recently discovered symbiotic bacteria in non-nodulating species of Rubiaceae. However, the associations seem highly specific, and *Fadogia homblei* grown from seeds collected in the wild were colonized by its natural endosymbiont *P. caledonica*, suggesting the vertical transmission of the bacteria through the seeds (Verstraete et al. 2011). RAMPANT genome erosion of *Paraburkholderia* leaf symbionts (although notably not those of *Fadogia homblei*), a well-documented consequence of repeated transmission bottlenecks, is also consistent with vertical transmission (Danneels et al. 2023).

Overall, evidence from these diverse leaf-symbiotic associations is highly suggestive of a dominant vertical mode of transmission and occasional host-switching or horizontal transmission. Interestingly, *O. dioscoreae* mutant strains impaired in flagellar motility were outcompeted by wild-type strains in co-infection experiments. Host-switching might therefore be possible in the early stages of the evolution of an association, and genome erosion and subsequent loss of a flagellar apparatus might effectively “lock” the bacteria inside a host. This transient capacity for horizon-

tal transmission may perhaps explain the complex phylogenetic patterns observed in some clades, such as *Ardisia* spp., where co-phylogenetic patterns are consistent with co-speciation within crown groups but not between deeper rooting clades (Yang and Hu 2022).

## Specificity of Leaf Symbiosis

Specificity of bacterial leaf symbiosis at the species and population levels has been demonstrated by multiple studies (Danneels et al. 2021; Lemaire et al. 2011b, c; Verstraete et al. 2011). The mode of transmission is likely a major factor explaining the stability and high specificity of these associations across evolutionary time, as well as additional constraints imposed by the host of the symbiotic bacteria themselves. Because vertical transmission ensures that a pool of functional symbionts is transmitted to the next generation, associations may be stabilized by partner fidelity feedback, that is, the idea that cooperation is favored when species stay together in stable associations that align their fitness interests (Sachs et al. 2004). To be effective, partner fidelity feedback must rely on an exclusive transmission route or strict environmental filtering (Kaltenpoth et al. 2014). The fact that host-switching is known to occur in all leaf-symbiotic associations studied thus far indicates that it is possible for new microbes to hijack established transmission routes and that additional mechanisms promoting specificity must exist.

## Vulnerability of the transmission route to infection

The shoot apex is central to dissemination of symbiotic bacteria to leaves and reproductive organs, but it is a particularly vulnerable transmission route. In contrast to animals where heritable symbiont transmission may occur only once per life cycle, aboveground plant organs are formed during post-embryonic development initiated at the shoot apical meristem, and contamination of the shoot apex at any time during the growth season may have consequences for the following generations. Although the shoot apical meristem itself is relatively immune to bacterial infection in multiple plant species, leaf-symbiotic bacteria are typically enclosed at the surface of the tissue between newly formed pairs of leaves (Naseem et al. 2020). For example, in *Psychotria punctata*, this “leaf-enclosed chamber” is partially open to the outside and to potential contamination (Sinnesael et al. 2019). Indeed, this semi-open system explains the ease with which aposymbiotic *D. sansibarensis* may be inoculated by dropping a suspension of bacteria directly on the shoot tip (Acar et al. 2024). This means that leaf-symbiotic plants must continuously ensure that the proper bacteria gain access to symbiotic structures. In this context, how host-symbiont specificity is maintained remains largely unknown.

## Adaptation to the leaf endophytic compartment as a potential driver of specificity

Environmental filtering may play a role, either through the presence of specific substrates or highly selective growth conditions inside the plant. Although it is difficult to test the metabolic niche of most leaf-symbiotic bacteria due to a lack of laboratory cultures, clues can be drawn from largely intact pathways in otherwise heavily eroded genomes. First, none of the sequenced endosymbiont genomes contains genes coding for the synthesis of Nod factors or phytohormones, ruling out a potential role of these classes of signaling molecule in the symbiosis (Carlier and Eberl 2012; Carlier et al. 2016, 2017; Danneels et al. 2023; Pinto-Carbó et al. 2016). The genomes of ‘*Ca*. C. kirkii’ and ‘*Ca*. C. crenata’ have been investigated in some detail, and the presence of putative dicarboxylic acid transporters, as well as the partial or complete inactivation of
carbohydrate utilization pathways, indicates that the bacteria rely on acids of the tricarboxylic acid cycle, such as malate or citrate, for growth inside the plant (Carlier et al. 2013, 2016; Schindler et al. 2021). Similarly, various *O. dioscoreae* strains were shown to grow preferentially on carboxylic acids, with few strains able to utilize sugars (Carlier et al. 2017). The genomes of ‘*Ca*. C. kirkii’ and ‘*Ca*. C. crenata’ lack pathways for amino acid utilization and, conversely, retain pathways for their biosynthesis (Carlier et al. 2013, 2016). Exclusive metabolic niches are therefore unlikely to explain the specificity of leaf symbiosis. Metabolomics analysis of *A. crenata* and *P. punctata* leaf nodules revealed significant differences from leaf lamina at every life stage of the plants, which may indicate that the nodules offer a drastically different environment than the leaf lamina (Schindler et al. 2021). This would favor selection of the symbiont through adaptation to compounds available rather than selection through molecular recognition. In addition, physicochemical properties may provide a level of selectivity. Reactive oxygen species are known to be involved in plant defenses against pathogens, and the presence of reactive oxygen species might constrain growth inside the leaf nodule. Indeed, functions linked to resistance to oxidative stress are enriched in the core genomes of leaf nodule symbionts (Carlier et al. 2016). In addition, *A. crenata* and *P. punctata* nodules are enriched in putrescine and spermidine (Schindler et al. 2021), which could also contribute to resistance to oxidative stress if produced by the bacteria (Tkachenko et al. 2012). Trehalose has been reported to be an important component of the osmoregulation pathway of bacteria (Ruhl et al. 2013). A trehalose synthase found in the ‘*Ca*. C. kirkii’ proteome may be involved in osmoregulation through the conversion of maltose to trehalose (Carlier et al. 2013), and trehalose was found to be enriched in leaf nodules of *A. crenata* and *P. punctata* (Schindler et al. 2021). Similarly, *O. dioscoreae* genes involved in the response to oxidative stress are overexpressed in planta compared with laboratorial cultures (De Meyer et al. 2019).

## Antagonistic interactions and specificity

Alternatively, the host may provide some level of filtering through antimicrobial compounds. For instance, the flavonols catechin, epi-gallocatechin, and a newly described epicatechin 3'-O-3-hydroxy-2-methyl-propanoate were found to be enriched in leaf nodules of *A. crenata* compared with noncolonized tissue (Schindler et al. 2021). Flavonoids may play a role as antioxidants, perhaps sheltering leaf nodule bacteria from reactive oxygen species (Ullah et al. 2020). Flavonoids may also display antimicrobial properties, perhaps allowing for selective growth of symbiotic bacteria in the leaf nodule (Taylor et al. 2005). Finally, the symbionts themselves may act directly to ensure that other microorganisms do not invade their niche. In support of this hypothesis, the genome of *O. dioscoreae* LMG 2930<sup>T</sup> harbors two type VI secretion system (T6SS) gene clusters, both of which were highly expressed in planta (De Meyer et al. 2019). T6SSs are mostly known for their antimicrobial activity through the delivery of antimicrobial effectors directly into target cells (Hood et al. 2010). The presence of T6SS genes clusters in the genomes of *P. caledonica* strains isolated from *F. homblei* may indicate an important role of bacterial competition for the specificity, in particular when strict codependence has not yet evolved.

## Ecological Functions of Leaf Symbionts

Few studies so far have focused on the ecological function of leaf symbiosis. However, specialized metabolism or the production of allelopathic compounds appears to be central to these associations.

### C<sub>7</sub>-cyclitol production is widespread along leaf endosymbionts

Proteins linked to bacterial specialized metabolism made up more than 9% of all detected peptide spectral matches in proteomics studies of leaf nodules of *P. kirkii* (Carlier et al. 2013). This suggests that leaf symbiont ‘*Ca*. C. kirkii’ seems to invest considerable resources into the biosynthesis of metabolites, including molecules of the C<sub>7</sub>-cyclitol family. C<sub>7</sub>-cyclitols are carbohydrate analogs formed from the cyclization of sugar phosphates by enzymes of the 2-epi-5-epi-valiolone synthase family (EEVS) (Osborn et al. 2017). At least three distinct C<sub>7</sub>-cyclitols have been isolated from leaves of *P. kirkii*: (+)-streptol, its glycolsylated derivative (+)-streptol glucoside, and the C<sub>7</sub>N-cyclitol kirkamide (Fig. 4A to C) (Hsiao et al. 2019; Sieber et al. 2015). Kirkamide is a weak inhibitor of human N-acetyl glucosaminidase, indicating that carbohydrate-active enzymes are a target (Sieber et al. 2020). However, a high IC50 (in the 240 µM range) indicates that N-acetyl glucosaminidase may not be a primary target. Alternatively, kirkamide might be metabolized in living cells to UDP-kirkamide, potentially acting as an inhibitor of O-GlcNAc transferases, which are critical for chitin biosynthesis. Unfortunately, assays using a per-acetylated derivative of kirkamide failed to support this hypothesis, without ruling it out entirely. The high mortality of beetles (*Meligethes aeneus*) fed a diet tainted with kirkamide and the sub-micromolar LC50 against larvae of *Artemia salina* rather indicate that arthropods are particularly sensitive to kirkamide (Sieber et al. 2015). This suggests that kirkamide might protect plants against feeding by insect herbivores.

In contrast, (+)-streptol and (+)-streptol glucoside isolated from leaves of *P. kirkii* display strong herbicidal activities in lettuce germination assays (Hsiao et al. 2019). Consistent with a role in allelopathic interactions, water extracts of *Psychotria* shoots, flowers, fruits, and roots are effective against a range of land plants, targeting the development of the root system (Georgiou et al. 2021). In particular, streptol and streptol glucoside appear systematically distributed, with inhibitory concentrations of streptol detected in root exudates of symbiotic, but not aposymbiotic, plants. The precise mechanism of action of streptol and streptol glucoside remains unknown. However, leaf extracts of *P. kirkii* appear to disturb growth anisotropy at the onset of root elongation, without affecting root meristem cell proliferation or patterning (Georgiou et al. 2021). This phenotype is most likely due to streptol alone, because as little as 5 µM (+)-streptol is sufficient to mimic the effects of *Psychotria* extracts, whereas up to 100 µM kirkamide or (−)-streptol shows no activity. Moreover, results from in vitro seed germination assays indicate that streptol glucoside might be a prodrug, because plants grown in presence of streptol glucoside accumulate significant amounts of streptol (Georgiou et al. 2021). Streptol does not inhibit the growth of single-celled or filamentous Chlorophyta, mosses, or ferns but inhibits a range of Gymno- and Angiosperms. Remarkably, *Psychotria kirkii* was the only plant insensitive to streptol in an assay including 27 species of the Spermatophyta (Georgiou et al. 2021).

The biosynthesis of C<sub>7</sub>-cyclitols is widespread in leaf-symbiotic bacteria. In fact, of the 26 leaf-symbiotic *Caballeronia* and *Paraburkholderia* genomes surveyed by Danneels and coworkers, all but two contained gene clusters linked to C<sub>7</sub>-cyclitol biosynthesis (Danneels et al. 2023). Genes linked to the biosynthesis of streptol glucoside appeared in 15 genomes of *Caballeronia* and *Paraburkholderia* symbionts of *Ardisia*, *Psychotria*, *Pavetta*, and *Vangueria* species. Evidence of streptol or streptol glucoside could moreover be detected by mass spectrometry in leaf extracts of *Psychotria kirkii*, *Vangueria dryadum*, and *Vangueria pygmaea*, which host ‘*Ca*. C. kirkii’, ‘*Ca*. Paraburkholderia dryadicola’, and *Paraburkholderia caledonica*, respectively. In contrast, complete
gene clusters linked to kirkamide biosynthesis were only detected in *Caballeronia* leaf symbiont genomes, specifically within a clade comprising leaf symbionts of *Psychotria* and *Pavetta*. Our own unpublished analysis of *Tricalysia* and *Empogona* leaf metagenomes also revealed that metagenome-assembled genomes of the *Caballeronia* symbionts of *Empogona congesta*, *Tricalysia hensii*, and *Tricalysia semidecidua* contained complete or partial kirkamide gene clusters (Verstraete et al. 2023). Additional partial or unidentified gene clusters containing EEVS-coding sequences were detected in some genomes of *P. caledonica*, ‘*Ca*. C. ardisicola’, and ‘*Ca*. P. dryadicola’. Importantly, gene clusters linked to C~7~-cyclitol biosynthesis are otherwise exceedingly rare in free-living *Burkholderia s.l*. (Danneels et al. 2023).

## Leaf nodule symbionts of *Ardisia* produce nonribosomal peptides

There is one notable exception to the near-omnipresence of C~7~-cyclitols in leaf symbionts: ‘*Ca*. C. crenata’. ‘*Ca*. C. crenata’ forms associations with two related species of *Ardisia* (*A. crenata* and *A. virens*), and genomes of the species do not encode the EEVS enzyme necessary for C~7~-cyclitol synthesis. However, both *Ardisia* species are a source of the cyclic depsipeptide FR900359 (FR), which was initially isolated in a screen for inhibitors of platelet aggregation and vasodilators (Fig. 4D) (Fujioka et al. 1988). A biosynthetic gene cluster encoded on a plasmid of ‘*Ca*. C. crenata’ was later shown to be responsible for the synthesis of the nonribosomal peptide FR (Carlier et al. 2016; Crüsemann et al. 2018). FR and the related YM-254890, also known as chromodepsins, are potent inhibitors of G protein-coupled receptor signal transduction, specifically targeting the Gq protein family (Hermes et al. 2021). Gq proteins are heterotrimers composed of the three subunits α, β, and λ and are highly conserved in the metazoan (Milligan and Kostenis 2006). In silico structural studies indicate that FR targets virtually all members of the Gα~q~ family (Hanke et al. 2023). Heterologous expression of *Caenorhabditis elegans* and *Heterodera schachtii* Gα~q~ in HEK293 cells genetically edited to lack the endogenous Gq and G11 proteins and overexpressing RIC-8 and M3 showed that FR interferes with Gq signaling even at low micromolar concentrations.

This shows that FR targets the Gq pathway from nematodes to mammals, highlighting its value as a broad-spectrum bioactive metabolite. This is confirmed by the fact that exposure of *C. elegans* to FR impairs movement and egg-laying, similar to mutants impaired in the Gq signaling pathway, and inhibits hatching of the plant-parasitic nematode *H. schachtii* from cysts (Hanke et al. 2023). FR has also been demonstrated to bind to insect Gq proteins and is toxic to nymphs of the pest *Riptortus pedestris* (Crüsemann et al. 2018). It is thus tempting to conclude that production of FR by an *A. crenata* symbiont plays a role in protecting the plant from various pests. Interestingly, the genomes of symbionts of other *Ardisia* species, such as *A. cornudentata* and *A. mamillata*, do not encode the *frs* gene cluster responsible for the synthesis of FR (Danneels et al. 2023). Because homologs of the *frs* gene cluster have been detected in distantly related, free-living *Chromobacterium* species (Hermes et al. 2021), the most likely scenario is that acquisition of the *frs* gene cluster by horizontal gene transfer effectively replaced C~7~-cyclitol biosynthetic pathways in ‘*Ca*. C. crenata’. This exchange of functions by horizontal gene transfer, possibly combined with incomplete lineage sorting, seems to be common in leaf symbionts. This is illustrated by the fact that the phylogenies of EEVS are highly incongruent with core genome phylogenies in leaf symbionts (Danneels et al. 2023).

## Unidentified secondary metabolism of leaf symbionts

The genomes of leaf symbionts encode more unidentified biosynthetic gene clusters in addition to C~7~-cyclitol and FR biosynthetic pathways. For example, the genome of *O. dioscoreae* encodes two novel biosynthetic gene clusters with similarities to nonribosomal peptide and polyketide synthesis that are highly expressed in the leaf gland but whose function remains to be determined. Several authors have also speculated that pavettamine, an unusual toxic polyamine found in leaves of several nodulated Rubiaceae species, is actually synthesized by leaf-symbiotic *Caballeronia* and *Paraburkholderia* (Schindler et al. 2021; Van Elst et al. 2013; van Wyk et al. 1990; Verstraete et al. 2011). However, the link between leaf bacteria and pavettamine production remains to be proven, and the biosynthetic genes are unknown.

<figure><img src="image_15.png" /><figcaption>**Fig. 4.** Chemical structures of bioactive compounds produced by bacterial leaf endophytes. Structures of C~7~-cyclitols isolated from *Psychotria kirkii*. **A**, (+)-streptol; **B**, kirkamide; and **C**, (+)-streptol glucoside. All three contain a common valiolone group formed by the cyclization of sugar phosphates by enzymes of the 2-epi-5-epi-valiolone synthase family, highly conserved in the leaf endophyte genomes. **D**, The cyclic depsipeptide FR900359, produced by the *Ardisia crenata* endophyte ‘*Candidatus* Caballeronia crenata’.</figcaption></figure>
## Codependence: Consequence or Accident?

One features of leaf symbiosis that has attracted attention as early as the beginning of the twentieth century is that some nodulated *Ardisia* and *Psychotria* seeds spontaneously give rise to symbiont-free seedlings (Miehe 1911; Von Faber 1912). The loss of the symbiont is most likely due to imperfect transmission but can be exaggerated by treating the seeds with heat or antibiotics (Yamada 1955). Invariably, these seedlings develop into small and malformed plants, and *Psychotria* seedlings do not present the typical nodules at the leaf surface (Fig. 5) (Miller 1990; Van Oevelen et al. 2003). Production of aposymbiotic plants has been attempted on plant models including *Ardisia crispa*, *A. punctata* (syn. *A. cymosa* var. *cymosa*), *P. umbellata*, and *P. punctata*, but healthy aposymbiotic plants could never be obtained (Fig. 5) (Govaerts et al. 2021; LaMotte and Lersten 1972; Sinnesael et al. 2019; Yamada 1955).

### Attempts to explain codependence

Several hypotheses were put forward to explain the apparent reliance of nodulated *Psychotria* or *Ardisia* on their symbionts, including synthesis of phytohormones by the bacteria or the produc-

tion of yet unidentified growth-promoting factors (Becking 1971; de Jongh 1938; Edwards and Lamotte 1975; Miehe 1917; Miller and Donnelly 1987). Although hormonal imbalance may explain the striking growth phenotypes of aposymbiotic plants, direct evidence of cytokinin or auxin synthesis or degradation by the endophytes is lacking. Because aposymbiotic seedlings germinate normally and the first few leaves usually do not present defects, it is likely that the effect of symbiont loss is felt locally, possibly at the level of the shoot meristem. Further, *A. crenata* and *A. virens* plants from which leaf nodules were clipped immediately after emergence developed normally, indicating that the main role of bacteria in the leaf nodules is unlikely to be the production of a systemic growth factor (Nakahashi et al. 2005). Indeed, the authors conclude the study by writing “the symbiosis might never even have provided an ultimate benefit, but rather the plant may have evolved dependence on the bacteria, and is now effectively held as a slave.” Considering that we now know that the symbiont contributes insecticidal peptides to the association, it becomes clearer that codependence may have evolved as a side effect, rather than a feature, of leaf symbiosis.

Codependence could be, for example, the result of partial loss of redundant metabolic pathways from the host and its symbiont. The presence of amino acids and polyamines in lower quantity in lamina tissue compared with nodules of *A. crenata* and *P. punctata* might suggest that the endosymbiont uses those compounds as nitrogen and carbon sources, perhaps participating in metabolite turnover (Schindler et al. 2021). However, several surveys of the genomes of symbiotic bacteria failed to uncover genes or bacterial pathways involved in plant hormone metabolism or phenylpropanoid synthesis, indicating that the detection of these metabolites in nodules may instead be a reaction of the plant tissue to bacteria (Carlier and Eberl 2012; Danneels et al. 2023; Pinto-Carbó et al. 2016). The many assumptions and contradictory results highlight the complexity of the interaction but do not give actionable clues about the obligate nature of those symbiosis nor its involvement in plant development. The main reasons are the lack of rigorous comparison between aposymbiotic and symbiotic plants due to developmental defects of the former and the absence of genetic information on the plant hosts. It is therefore unclear what roles leaf symbionts may play in addition to the production of specialized metabolites and whether codependence came as a consequence of integrating bacterial metabolism in host pathways or evolved as a result of drift across millions of years of exclusive and permanent association. Disentangling these two possibilities will require future efforts to concentrate on understanding the plant side of the interaction through the development of tractable experimental systems, including methods to reliably remove and inoculate symbiotic bacteria.

### Codependence is not a universal trait of leaf symbiosis

In this regard, we may be able to gain insights from associations that are not obligate and hence more easily manipulated in the lab. The *D. sansibarensis* and *O. dioscoreae* association is an example of such a facultative symbiosis. In this case, axenic plants can be reliably obtained in the laboratory by treating explants (node cuttings) with antibiotics, and symbiotic bacteria are culturable on standard microbiological media. Moreover, under controlled conditions, aposymbiotic *D. sansibarensis* are morphologically and physiologically indistinguishable from symbiotic plants regenerated from node cuttings (Acar et al. 2024). Interestingly, aposymbiotic plants develop fully formed leaf glands, which only differ from symbiotic glands at the microscopic level, with smaller trichomes that present fewer vesicles and cisternae. The absence of codependence in this association may be linked with a greater physical separation of the symbiotic partners than with other leaf symbioses. Indeed, bacteria are separated from plant tissue by a thick

<figure><img src="image_16.png" /><figcaption>**Fig. 5.** Phenotypes of aposymbiotic *Ardisia crenata* and *Psychotria kirkii*. **A**, Development of aposymbiotic seedlings of *A. crenata* generally stops after the emergence of the second or third pair of leaves. The apical meristem then becomes dormant. **B**, After some weeks, protuberances or knobs develop on the stem. **C**, Deformed leaves emerging from an *A. crenata* knob. **D**, Unstained cross-section of a knob showing the presence of numerous vascular vessels and photosynthetic tissue. **E**, Leaves of aposymbiotic *P. kirkii* plants do not present nodules. **F**, The apical bud of aposymbiotic *P. kirkii* stops producing new leaves soon after germination.</figcaption></figure>
cuticle layer, except at the tip of the trichomes lining the inside of the gland (Acar et al. 2024; Miller and Reporter 1987). This is in stark contrast to the close-fitting parenchyma cells layers observed in *Pavetta* spp. or the leaf nodules of *Psychotria* in which plant cells form a mesh inside the bacterial mass (Zimmermann 1902). Similarly, although genomes of *O. dioscoreae* show signs of undergoing reductive evolution, this is to a much lesser degree than the majority of leaf symbionts of Rubiaceae or Primulaceae (Danneels et al. 2021). The absence of codependence, together with the presence of physical barriers between plant and bacterial cells in the gland and the lack of extensive genome erosion in the symbiont, could perhaps be seen as evidence of a younger association. However, phylogenetic dating analysis places the origins of the *D. sansibarensis* and *O. dioscoreae* association to between 3 and 13 Mya (Danneels et al. 2021). This is of course to be taken cautiously, as the analysis rests on a limited number of samples, notably of related nonsymbiotic *Dioscorea* species. Still, other leaf symbionts that are co-dependent are not significantly older (3 to 12 Mya). Different population sizes, modes of reproduction, and the frequency of horizontal acquisition (or host-switching) could all have an effect on the speed of evolution and the rise of codependence in different lineages. The actual function of the *Dioscorea/Orrella* leaf symbiosis remains to be established, but these results show that codependence is not an inevitable consequence of establishing hereditary, highly specific interactions in the phyllosphere.

## Conclusions and Perspectives

Leaf symbioses present an intriguing paradox: although they exhibit high specificity, they also demonstrate considerable taxonomic diversity and variability in hosting structures. Despite this diversity, certain common features begin to emerge, such as the production of secondary metabolites that may play a role in the symbiotic relationship. Much of the structural and functional diversity of these metabolites remains to be characterized and is, in our opinion, key to understanding the evolution and ecology of leaf symbiosis. Testing whether the production of metabolites is essential for partner recognition, such as by interacting with specific receptors at the plant cell level and/or suppressing immune responses, will also be important to understand how plants manage beneficial bacteria in the phyllosphere. Identifying the adaptations enabling the vertical transmission of endophytic bacteria through seeds or propagules can have broad impacts for agriculture, perhaps through the selection of crops permissive to stable inoculation of biofertilizers or biocontrol agents.

## Acknowledgments

We thank Olivier Coen for providing microscopy images.

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