Legumes have evolved the capacity to maintain a mutualistic association with endosymbiotic nitrogen-fixing rhizobia, hosting them in specialized lateral root organs, the nodules, where bacteria convert atmospheric nitrogen (N₂) to ammonia (NH₄⁺) and release it to the plant in exchange for carbohydrates. By granting them access to an additional source of nitrogen, the RNS allows plants to overcome nitrogen limitations occurring in terrestrial ecosystems and agriculturally used sites. Thus, the transfer of this ability to cereals has been identified as one possible strategy to reduce the use of environmentally harmful and expensive industrial fertilizers (Bloch et al., 2020; Mus et al., 2016; Oldroyd and Dixon, 2014; Pankievicz et al., 2019; Rogers and Oldroyd, 2014). Such an engineering approach, however, requires an in-depth understanding of the molecular machinery allowing rhizobial infections of legume roots.

In most cases, this host-controlled process requires a molecular dialogue between the partners, which involves the perception of bacterial lipo-chitooligosaccharide (LCO) molecules called Nod factors (NFs) by plasma membrane-resident receptor complexes including the M. truncatula LysM receptor-like kinase NFP and its Lotus japonicus ortholog NFR5 (Limpens et al., 2003; Radutoiu et al., 2003). Symbiont recognition triggers the onset of a signaling cascade involving the generation and decoding of nuclear-associated calcium oscillations, which mediates symbiosis-related gene expression (Singh et al., 2014). Interestingly, the same pathway is activated by the perception of diffusible signals produced by arbuscular mycorrhizal (AM) fungi, including both LCOs and short-chain chitooligosaccharides (COs), although these are perceived by a different combination of receptors (Feng et al., 2019; Sun et al., 2015). This common symbiosis signaling pathway shares a number of genetic components including the machinery to decode the specific calcium signature (Kistner et al., 2005) and a few downstream factors such as VPY, a Major Sperm Domain- and ankyrin-repeat-containing protein, that is required for both symbiotic interactions (Murray et al., 2011; Pumplin et al., 2010). Although some structural features are shared between the AM symbiosis and RNS during intracellular colonization, others are remarkably different. A whole set of those RNS-specific responses is controlled by the master regulator NIN, a transcription factor orchestrating the transcriptional reprogramming of cells required to sustain bacterial accommodation and concomitant cell proliferation to induce the formation of a nodule primordium (Schauser et al., 1999; Liu et al., 2019a; Vernié et al., 2015). This spatial and temporal coordination of infection and nodule organogenesis represents a hallmark of RNS (Oldroyd and Downie, 2008).

Intracellular invasion of rhizobia is uniquely enabled by a membrane-confined transcellular tunnel called the IT. In many legumes such as M. truncatula and L. japonicus, IT formation is initiated from an infection chamber (IC) (Fournier et al., 2015) that forms upon curling of a growing root hair and subsequent entrapping of rhizobia (Callaham and Torrey, 1981). Repolarization of targeted secretion towards this compartment enables local cell wall modifications (e.g. by secretion of the NPL to the IC Liu et al., 2019a) leading to its radial expansion (Fournier et al., 2015) and a highly confined local invagination of the plasma membrane, which marks the onset of IT initiation (Gage, 2004). The IT then extends through the trichoblast and subsequently progresses transcellular through the root cortex (Libbenga and Harkes, 1973). IT maintenance and progression require a highly coordinated interplay between the migrating nucleus, a cytosolic column ahead of the IT, actin, and dense microtubule arrays (Fournier et al., 2015; Perrine-Walker et al., 2014; Qiu et al., 2015; Timmers et al., 1999; Yokota et al., 2009). IT propagation in underlying cortical tissues is supported by local cell wall modifications (Su et al., 2023a) and repolarization of these cells, forming pre-infection thread (PIT) structures (van Brussel et al., 1992). This ensures guidance of the symbionts towards the newly divided cortical cells constituting the core of the nodule primordium. There, rhizobia are released from the ITs into the host cell, where they differentiate into nitrogen-fixing bacteroids (Vasse et al., 1990).

Over the last decades, reverse and forward genetics have identified several genes required for bacterial infection (Roy et al., 2020; Tsyganova et al., 2021). Among these is RPG, which is specifically expressed upon rhizobial inoculation and encodes a protein of unknown function (Arrighi et al., 2008). A loss of RPG in M. truncatula results in the development of aberrant ITs and poorly colonized nodules (Arrighi et al., 2008). Nuclear localization of RPG when heterologously over-expressed in Nicotiana benthamiana leaf epidermal cells suggested a putative role as a transcription factor (Arrighi et al., 2008), but its cellular and molecular function in M. truncatula has not been understood in detail. The protein, however, recently re-gained great attention, when two independent comparative phylogenomic studies identified NFP, NIN, and RPG as the only genes that have been consistently lost in non-nodulating species belonging to the Fagales/Fabales/Cucurbitales/Rosales (FaFaCuRo) clade (Griesmann et al., 2018; van Velzen et al., 2018). This evolutionary pattern linking these genes and the ability to form RNS makes them prime candidates for engineering symbiotic nitrogen fixation in cereals.

Here, we demonstrate that RPG is an essential component of the infectosome complex (Liu et al., 2019b; Roy et al., 2020). This multi-protein assembly associates with the very tip of growing ITs and with the nuclear periphery. A loss-of-function mutation in RPG entirely blocks the recruitment of VPY to infectosome foci. Furthermore, a highly confined membrane polarity domain at the IT tip, cytoskeleton connectivity between this site, and the nucleus and targeted secretion to the IT are aberrant in a rpg mutant. Thus, we propose that RPG provides specificity to a conserved cellular machinery that enables rhizobial accommodation.

The capacity to intracellularly accommodate nitrogen-fixing bacteria is a hallmark of the RNS. Bacterial internalization requires profound restructuring of the host cell to form an infection thread compartment via polarized secretion of plasma membrane and cell wall material to a single focal point. Spatially and temporally coordinated polar progression of IT structures determines the establishment of a functional nitrogen-fixing association.

Here, we demonstrate that RPG is a crucial modulator of the infectosome machinery sustaining the polar growth of intracellular ITs. RPG coalesces with VPY and LIN in IT tip- and perinuclear-associated puncta (infectosome foci, Figure 3D–G, Figure 3—figure supplement 6A–C) and is indispensable for VPY recruitment into these cytoplasmic structures during IT progression (Figure 4A–B and Figure 4—figure supplement 1A–B). A cooperative function of RPG with the infectosome complex in regulating IT polar growth is consistent with the phenotypes of rpg, vpy, and lin mutants, exhibiting strong defects in the establishment and/or the maintenance of IT progression but not being affected in nodule organogenesis (Kiss et al., 2009; Murray et al., 2011; Liu et al., 2019b; Li et al., 2023, Figure 1B–D and Figure 1—figure supplement 2A–B). Moreover, the peculiar enlargement of ITs developed by rpg mutants, indicative of a loss of unidirectional polarized growth, is well explained by the failure of rpg-1 to maintain the typical VPY punctate localization observed in WT root hairs (Figure 4A–B and Figure 4—figure supplement 1A–B). Interestingly, infectosome-like and VPY-positive compartments have also been observed during the AM symbiosis (Pumplin et al., 2010; Zhang et al., 2015) and vpy mutants exhibit strong defects in this association (Feddermann et al., 2010; Pumplin et al., 2010). Functionally, the accumulation of VPY in such compartments seems more important for full arbuscule development rather than for controlling initial fungal infections (Lindsay et al., 2022). In contrast, the correlation between loss of VPY-recruitment into infectosome foci in the rpg mutant and its strong infection defects (Figure 1B–C and Figure 4A–B) argues for a higher relevance, and perhaps extended function, of these cytoplasmic structures during bacterial accommodation. In line with this, we hypothesize that RPG serves as a specificity determinant for infectosome-related endo- and exocytotic events during RNS (Figure 7A). This is further supported by the transcriptional dependency of RPG from NIN (Liu et al., 2019a; Soyano and Hayashi, 2014) and is in line with RPG being one of the few essential factors that have been lost in non-nodulating species of the FaFaCuRo clade (Griesmann et al., 2018; van Velzen et al., 2018). Upon recruitment of VPY into the infectosome complex, it is further stabilized by LIN (Liu et al., 2019b). This can also be assumed for CERBERUS, the ortholog of LIN in L. japonicus, which has been shown to stabilize VPY protein levels (Liu et al., 2021) and to interact with RPG in this closely related legume (Li et al., 2023). Different from RPG, the genes encoding for VPY and LIN are retained in angiosperm species forming intracellular symbioses, together with those encoding for the central components of the common symbiosis signaling pathway CCaMK and CYCLOPS, which are essential for both AM and RNS (Radhakrishnan et al., 2020). Intriguingly, VPY was recently proposed to coordinate signaling and cellular accommodation during AM by negatively regulating the nuclear-localized symbiosis signaling kinase CCaMK via direct interaction (Lindsay et al., 2022). If such regulatory function would be conserved during rhizobial infection, the ability of RPG to sequester VPY to cytoplasmic foci might additionally represent a way to modulate the input/output of a conserved endosymbiotic signaling circuit to adequately mediate bacterial infection (Figure 7A).

Figure 7

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While the existence of infectosomes is supported by this and other studies (Liu et al., 2019b; Zhang et al., 2015), the full nature of this presumably membrane-less compartment remains to be investigated further. It is, however, tempting to draw parallels to the so-called ‘polarisome,’ a macromolecular complex coordinating the polarized growth of yeasts and fungal hyphae (Xie and Miao, 2021). Polarisomes are dense spot-like structures localized at the site of polarized growth and are formed by a number of core components and many cooperative interactors (Xie and Miao, 2021). The polarisome is also required to maintain the Spitzenkörper (Crampin et al., 2005), a vesicle supply center located at the tip during hyphal growth, whose positioning is correlated with growth directionality (Riquelme and Sánchez-León, 2014). Interestingly, a myosin protein has recently been shown to mediate focal packing of the polarisome (Dünkler et al., 2021). The structural similarity of the RPG C-terminal extension with myosins, as supported by structural predictions and as previously suggested (Arrighi et al., 2008), and the RPG-dependent recruitment of VPY into discrete cytoplasmic foci (Figure 4A–F), further supports the hypothesis that RPG functions as an organizational hub protein for infectosome assembly and polarity maintenance during intracellular rhizobial infections (Figure 7B). It should, however, be considered that a vesicle-associated fraction of RPG, which is compatible with the predicted lipid binding capability of its NT-C2 domain (Zhang and Aravind, 2010), as well as of the other infectosome components outside the puncta might contribute to their functionalities. Although the diffuse cytoplasmic signal exhibited by RPG, VPY, LIN, and EXO70H4 (Figure 3—figure supplement 3C–D; Liu et al., 2019b) might represent a such a fraction, it cannot be reliably investigated at present due to the vesicle size and/or fluorescence intensity. Within the RPG protein, the core functional scaffolding element is the coiled-coil domain as it is sufficient to mediate the punctate localization of RPG and to complement IT progression in root hairs (Figure 2A–C and Figure 3—figure supplement 3A-D). The latter is unlikely to be determined by transcript stability as all deletion variants were consistently detected as fluorescent proteins when performing live-cell imaging (Figure 3—figure supplement 3A-D).

Rendering the infectosome complex dysfunctional in rpg mutants results in the loss of the highly polarized membrane enrichment of PI(4,5)P₂ at the IT tip (Figure 5A–C) and unconfined secretion of key factors such as NPL (Figure 6D–F) required for IT progression to the growing IT tip (Liu et al., 2019a; Xie et al., 2012). As in many eukaryotic polarized systems, where localized exocytosis targets PI(4,5)P₂-rich membrane domains, accumulation of this phosphoinositide was also shown to occur on perimicrobial membranes during pathogenic infections (Qin et al., 2020) and during AM symbiosis, where PI(4,5)P₂ specifically accumulated at the tips of intracellular linear hyphae (Ivanov and Harrison, 2019). In eukaryotes, the generation of PI(4,5)P₂ involves the phosphorylation of position 5 of PI4P catalyzed by phosphatidylinositol-4-phosphate 5-kinases (PIP5K) localized at the plasma membrane and in the nucleus (Colin and Jaillais, 2020). At least ten genes encoding for isoforms of this enzyme are contained in the Medicago genome and, interestingly, two of them are induced during infection thread development within root hairs (Medtr3g073100, Medtr7g089950; Liu et al., 2019a), one of which (Medtr7g089950) in a NIN-dependent manner (Liu et al., 2019a). This further supports our data and suggests that increased PI(4,5)P₂ accumulation during infection might occur, at least in part, via transcriptional upregulation of its biosynthetic enzymes. However, the trafficking/activity of these and/or other PI(4,5)P₂-metabolizing enzymes are also likely to contribute to the observed membrane enrichment, which is possibly a result of complex feedback loops occurring between those enzymes and their protein effectors such as, for instance, small GTPases, as reported for other systems (Di Paolo and De Camilli, 2006). In line with this, a ROP (Rho of a plant) GTPase and its GEF effector were shown to be required for infection thread growth in L. japonicus (Liu et al., 2020), and expression of at least one gene encoding for a RopGEF is induced during IC and IT formation in Medicago (Breakspear et al., 2014). Ultimately, the generation of a stably polarized cell arises from the functional interplay between membrane-associated polarity circuits and consequent downstream cellular reorganization (Ramalho et al., 2022), with localized exocytosis being central to the process of tip growth, as shown for pollen tube growth guidance in Arabidopsis (Luo et al., 2017). It is noteworthy that maintenance of the IC and secretion of NPL into the symbiotic apoplastic compartment is unaltered in rpg mutants, but remains unconfined (Figure 6D–F). The capacity of RPG to interact with EXO70H4 (Figure 3—figure supplement 7) residing within infectosome foci (Liu et al., 2019b) is compatible with a model where RPG-mediated recruitment of this exocyst subunit could contribute to restrict the tethering of exocytotic vesicles at a single focal site to enable rapid tip growth and IT extension. In the absence of functional RPG, the slow growth caused by unrestricted secretion might negatively feedback on membrane polarization, as shown in yeast and fungi, where reduced growth rates have been linked to the destabilization of polar domains (Haupt et al., 2018). Such depolarization might, in turn, alter the cytoskeleton organization with consequent loss of active polar tip growth. Alongside with an alteration of the microtubule-based connectivity between the IT and the nucleus (Figure 6A–C), it is likely that actin rearrangements might as well be affected. While the organization of the actin cytoskeleton during infection thread progression will have to be investigated, several genes encoding for actin remodeling proteins were reported to be important for IT growth (Liang et al., 2021; Hossain et al., 2012; Yokota et al., 2009; Qiu et al., 2015; Miyahara et al., 2010) and actin dynamics were recently shown to be reduced upon inducible depletion of PI(4,5)P₂ in Arabidopsis root cells (Doumane et al., 2021).

Taken together, our data strongly support the hypothesis that IT polarity requires few specific factors like RPG but predominantly relies on a highly conserved mechanism as suggested earlier (Gage, 2004; Yamazaki and Hayashi, 2015). While the secretion domain in curled root hairs comprises the entire IC (Fournier et al., 2015), the loss of highly restricted focal polarization results in uncoordinated expansion of such compartment and failure to maintain IT elongation. Indeed, while thin and elongated ITs are formed in root hairs of WT over the time period of our live-cell imaging analysis (4–8 dpi), the growth of infection structures developed within root hairs of the rpg-1 mutant appears retarded and their shape is highly variable and irregular (Figure 4A, Figure 5A–B, Figure 5—figure supplement 3A, Figure 6A–D). Continuous multiplication of bacteria within these structures then likely leads to their slow and unguided progression across root hairs (Figure 1B–D), as previously suggested (Arrighi et al., 2008) and further supported by the progressive disintegration of the EMT array and the increase distancing between the nucleus and the IT tip (Figure 6A–C), indicative of aborted active growth. Although polarization of the basal membrane domain in the respective trichoblasts and formation of PITs in the outer cortical cells are unaltered (Figure 4A, Arrighi et al., 2008), effective infection of nodules is impaired (Figure 1—figure supplement 1B), likely because a strictly defined time window that allows transcellular IT growth through recently divided cells is missed by these slowly growing ITs in the rpg mutant. While the molecular nature of this time window remains unknown, this temporal confinement of cellular infectability coupled with the highly controlled and paced growth of ITs represents an essential layer of host control over intracellularly progressing microbes.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Beatrice Lace

   University of Freiburg, Faculty of Biology, Freiburg, Germany

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Writing - original draft, Project administration, Writing – review and editing

   For correspondence

   beatrice.lace@biologie.uni-freiburg.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4732-573X

2. Chao Su

   University of Freiburg, Faculty of Biology, Freiburg, Germany

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

3. Daniel Invernot Perez

   University of Freiburg, Faculty of Biology, Freiburg, Germany

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

4. Marta Rodriguez-Franco

   University of Freiburg, Faculty of Biology, Freiburg, Germany

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1183-2075

5. Tatiana Vernié

   LRSV, Université de Toulouse, CNRS, UPS, INP Toulouse, Castanet-Tolosan, France

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

6. Morgane Batzenschlager

   University of Freiburg, Faculty of Biology, Freiburg, Germany

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

7. Sabrina Egli

   University of Freiburg, Faculty of Biology, Freiburg, Germany

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

8. Cheng-Wu Liu

   School of Life Sciences, Division of Life Sciences and Medicine, MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics, University of Science and Technology of China, Hefei, China

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6650-6245

9. Thomas Ott

   1. University of Freiburg, Faculty of Biology, Freiburg, Germany
   2. CIBSS – Centre of Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing – review and editing

   For correspondence

   Thomas.Ott@biologie.uni-freiburg.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4494-9811

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