Background

Plants never live alone. Arbuscular mycorrhizae (AM) offer one of the most powerful examples of the interaction capacity of plants to associate with the phylogenetically distant kingdom of fungi. Mycorrhizal symbioses have been known since the end of the 19th century [1], but the understanding of the AM success in space, being present in more than 72% of current land plants [2], and in time, dating back to 400 MYAs ago, is more recent. Physiological, genetic, molecular biology and -omics approaches have demonstrated some mechanisms which are at the basis of the AM success, such as the exchange of nutrients between the partners. The reduced carbon by the plant flows towards the fungus, which in turn improves the mineral nutrition of its green host [3], leading to a root-shoot axis which often has systemic positive effects [4]. The molecular dialogue and the mutual regulation between the two partners have revealed a surprising intimate interaction [5, 6], where the fungal role remains more enigmatic, even if the genomes of many AM fungi have been sequenced [7].

The benefits of the AM symbiosis are so important that international agencies consider them in the context of safeguarding the planet’s health, preserving soil fertility and pushing towards more sustainable agriculture. However, the steps towards an application still have many obstacles: the systems that are studied in laboratories are very few in comparison to the huge biodiversity found in nature, or even just in agroecosystems. Furthermore, advanced biology that integrates genetic approaches and -omics techniques usually operates on the root system of AM plants. On the contrary, we know that recognition processes occur within the rhizosphere, involving strigolactones (SL) and short-chain chitin oligomers (COs) exuded by the root and fungus respectively [8, 9]. The fungus uptakes mineral elements thanks to its extraradical hyphae, but it is also capable of penetrating the root by the hyphopodium to reach the cortical cells, where throughout hyphal branching, arbuscules are formed. Even if the arbusculated hyphae are found in close proximity with the cytoplasm of the host cells, they remain physically separated by the peri-arbuscular membrane, in continuity with the plant cell plasma membrane, and establishing an extensive surface area where nutrient exchanges occur [10, 11] (Fig. 1). Altogether, it is clear that symbiotic functions are compartmentalised with a specific role for each cell type. These concepts already emerged from transmission electron microscopy studies in the 80–90s [1] which showed the different reactivity of root cells to the advancing fungus as well as the progressive changes of the latter when crossing the root tissues. Ultrastructural observations coupled with in situ labelling techniques allowed to describe changes in the fungal wall mirroring structural chitin modifications, when moving from the extraradical hyphae to the arbuscules [12, 13]. Looking at the plant side, conspicuous molecular changes were detectable in the plant material laid down at the partner interface by using a range of monoclonal antibodies and depending on the root cell typology [14] as well as in the fine nuclear organization [15]. Lastly, the discovery of endobacteria living inside AM hyphae [16] has opened the way to the novel concept of the fungal microbiota [17].

With the current advances in molecular biology, molecular and cellular knowledge therefore necessitates a deeper integration also in the field of plant-microbe interactions. The first aim of this review is to highlight how some techniques already developed in the past (laser microdissection, confocal studies by using plant lines transformed with reporter genes, etc.) have given relevant contributions by highlighting the characteristics of colonized cells versus non-colonized cells within the same AM root. The second objective is to illustrate how new technologies such as single-cell RNA sequencing (scRNA-seq), that recently started to be applied in the study of some plant-microbe interactions, can open new horizons and new questions to better dissect and appreciate the potentiality of the AM symbiosis process.

How technologies have shaped the cellular resolution of arbuscular mycorrhizal association

Arbuscular mycorrhizas, as other plant-microbe systems, illustrate how the development of molecular technologies, beginning with the PCR, paved the way for crucial discoveries. Experimental limitations given by the obligate symbiotic nature of AM fungi were overcome only at the end of the 90s, opening a new era based on molecular and genetic analyses.

Starting from that period, the establishment of new techniques led to crucial discoveries of AM symbiosis functioning coupled with a cellular resolution gain. It has been known for a long time that plants activate a specific phosphate mycorrhizal pathway [18], but only through testing promoter activity with histochemical GUS approaches [19] and protein localization with confocal microscopy it was possible to describe the location of plant phosphate transporters. Many research groups [20,21,22,23] demonstrated the dominant expression of AM-induced phosphate transporters in the root cortical cells containing arbuscules, and more precisely on the peri-fungal membrane which always surrounds the intracellular fungus [11] (Fig. 1).

Similarly, our capacity to follow in vivo single-cell development and rearrangements of plant cellular compartments during the different steps of AM formation has shaped our understanding of this process. Live cell imaging of intraradical hyphal growth using fluorescent markers of the cellular endomembrane system has brought to the identification of a novel plant structure, the plant pre-penetration apparatus [24, 25], and allowed to follow the intricate vesicle trafficking orchestrating the substantial reshaping of the cellular environment [26].

Later on, being able to observe live plant cells hosting fungal arbuscules in greater detail allowed researchers to identify two distinct domains within the peri-arbuscular membrane surrounding the arbuscule-hosting cells, harbouring different sets of proteins and mediating different processes [13]: the trunk domain, which represents the base of the arbuscule and accumulates similar proteins to the ones found in the peripheral plasma membrane, and the branch domain, that encases the fungal hyphal branches and contains specific proteins primarily needed for plant-fungus signalling and nutrient exchange [27] (Fig. 1).

Because mycorrhizal colonization occurs asynchronously, a bulk characterization of whole roots provides an average representation of all developmental stages and cell-types present in a single root (Fig. 2). Hence, numerous studies have employed laser microdissection methodologies [28,29,30,31,32,33,34,35,36,37,38,39] to gain more comprehensive insights into gene expression associated with various stages and cell-types during this interaction. This approach was introduced back in 1976 by pathologists to cut single cells from freeze-dried tissue [40], and applied for the first time in plant tissues 16 years later [41]. Combined with RT-qPCR or microarray to have a genome-wide picture of gene regulation, the laser microdissection technology has surely contributed to the identification of a core set of genes that are regulated by AM interaction and preferentially active in arbusculated cells: among them, most of the so-far identified plant nutrient transporters, such as the iconic phosphate transporter, an ammonium and a sulphate transporter, as well as a fungal inorganic phosphate (Pi) transporter, previously hypothesized to be present exclusively in the extraradical hyphae and fungal-enriched RNA [42]. However, laser microdissection as a method is limited to the selected cells and does not easily differentiate among the diverse coexistent arbuscular stages.

From whole-root to single-cell RNA sequencing: opportunities and challenges

Among the new technologies that enriched plant research in the past few years, transcriptomics stands out, expanding into multiple fields in biology and being applied to numerous organisms. Particularly, when studying plant-microbe interactions, both symbiotic partners create a multi-factorial transcriptomic and proteomic environment [43]. In this context, traditional methods such as bulk RNA-seq and microarrays served as primary techniques for profiling the transcriptome but merely depicted the collective transcriptional patterns averaged across a population of thousands of cells. Single-cell RNA sequencing, as the name states, embraces different techniques used for sequencing the RNA present in individual cells, which means the sequencing of a single transcriptome and gene expression analysis in each cell in an independent way, overcoming the obstacles of bulk RNA-seq (Fig. 2).

The first step to carry out a single-cell study is the dissociation of the cells from the plant tissue. One key attribute of plant cells that has to be considered within this step is the presence of the structural carbohydrate-rich cell wall surrounding the plasma membrane. To isolate plant cells, the cell wall must be removed through enzymatic digestion, a procedure known as protoplasting, which yields single cells without cell walls (protoplasts) [44]. However, this process is often associated with technical challenges, like incomplete cell isolations as a result of the wide range of cell sizes and cell wall compositions, or the position of the layers through the tissue [45], resulting in limited access to certain cellular sub-populations and, in consequence, to a detection bias toward easier-to-protoplast cell-types [46, 47], which could represent an obstacle if the objective is to perform cell annotation [48, 49]. In addition, protoplasting can be difficult or impractical to apply in certain plant tissues or species with diverse cell wall components that need a particular optimization [50, 51]; still, the main problem associated with the protoplasting methods in transcriptomics research is its impact on gene expression due to the introduction of enzymes that can develop plant stress responses or cause mRNA leaking [50, 52]. To overcome these challenges associated with cell isolation, single-nucleus RNA-sequencing (snRNA-seq), which requires nuclei isolation, has lately become a promising alternative to use in plant research as a protoplasting-free method.

Even in other fields that do not involve plants or cellulose cell walls, like mammalian research, the implementation of snRNA-seq has largely increased over the last years due to the advantages that this technique shows for certain aims, like getting the expected cell-type frequencies and the limitations found in scRNA-seq [53]. Moreover, protein-coding genes and long non-coding RNAs are highly detected with snRNA-seq, as well as genes with a large intronic burden, likely because these require more time within the nucleus for an accurate splicing. However, it has to be considered that the amount of data obtained per nucleus is from 3 to 10 times lower than the one obtained per cell through scRNA-seq [53,54,55]. In addition, if the study aims to understand plastid or mitochondrial gene expression, scRNA-seq would represent a better choice.

Alongside the decision between cells or nuclei isolation, there are several strategies for its capture that can be used, intending to physically separate them from the others. Currently, the technology most commonly utilized for single-cell capture in plants is based on droplet microfluidics, which revolutionized single-cell studies by significantly increasing the number of cells that can be profiled in comparison to classic plate-based assays or laser microdissection [56]; in fact, all the plant-microbe interaction studies published until now and reviewed here (Table 1) employ the droplet microfluidics-based 10X Genomics Chromium system, which consists in encapsulating cells/nuclei in single gel beads containing barcoded oligonucleotides that label the RNA molecules through reverse transcription and produce a library for sequencing through next-generation sequencing (NGS) platforms [57, 58]. Nevertheless, new technologies based on combinatorial barcoding that, unlike droplet microfluidic-based methods, do not require complex partitioning equipment or a combination of both approaches, have been described in the past years [59] and are nowadays available as commercial kits, which could promote the accessibility to single-cell resolution research.

Typically, the first step in single-cell data analysis is to cluster and annotate the cells based on their similarities in gene expression profiles. To this aim, researchers focus on well-known specific cell-type marker genes, which exhibit high expression within a particular cell-type, and low or not expression in the others. However, the requirement of cells or nuclei isolation to perform sc/snRNAseq entails a loss of spatial perspective regarding cell location, and this challenge is just partially solved after bioinformatics processing for clustering. Tissues are composed of a multitude of cells with a precise spatial organization, a critical feature for executing their functional roles. Clustering methods neglect cells’ spatial coordinates, not being able to differentiate spatially diverse cells that show similar transcriptomes. As a matter of fact, Pereira and colleagues [60] analyzed M. truncatula early nodule development and found a subset of cells in the nodule meristem cluster that was also present in non-infected plants. This suggested that those were likely cells from the lateral root meristem. In addition, when a plant-microbe interaction is being studied, the cell isolation means the displacement of the physical interaction between the partners.

Therefore, the need to understand cellular gene expression while investigating its spatial environment within a complex organ or tissue has prompted the development of a group of techniques known as spatial transcriptomics (ST) [61, 62]. These technologies are already extensively applied in animal research, but have only recently started to be adopted in the plant research field. Depending on how spatial information is acquired, ST methods can be divided into three groups: (i) laser capture microscopy-based methods, as LCM-seq, where individual or group of cells are harvested directly from the tissue’s region of interest and then gene expression analyses are performed, (ii) imaging-based methods, that consist in in situ hybridization followed by the identification of the target RNAs through high resolution microscopy, like MERFISH (Multiplexed Error Robust Fluorescence In Situ Hybridization), a highly multiplexed single-molecule imaging approach that can detect thousands of RNA species at a single-cell resolution [63], and (iii) in situ capture sequencing-based methods, in which the location of the transcripts of interest is obtained via high-throughput sequencing, e.g. 10x Visium [64], with the last one representing a powerful tool to investigate a plethora of plant models and biological questions, as it combines spatial information with transcriptomic data; recently, You and collaborators [65] compared 11 sequencing-based spatial transcriptomics methods using animal reference tissues and explain how resolution is affected by molecular diffusion.. The progression of technology has facilitated the application of these techniques in studying the development of plant organs [66] or in spatially describing metabolic pathways [67] and, subsequently, researchers are delving into investigating plant-microbe interactions.

In summary, the single-cell resolution is transforming the quality and depth of the results obtained in the plant-microbe interaction field, especially when combined with ST approaches (Table 1). However, the conventional bulk RNA-seq methodology is indeed significantly more affordable, the data analysis is less complex and it presents a high sensitivity and little genome amplification required, reducing noise and biases. So, in the end, the choice of approach would depend on the particular objective of the study and the practical considerations, like the budget available or the characteristics of the samples [48, 68].

Single-cell level insights into plant-microbe interaction

In early 2022, Bai and colleagues [69] pioneered the first single-cell scale research of a plant-microbe interaction, delving into the infection of woodland strawberry leaves by the fungus that causes grey mold disease, a significant threat in agriculture. They generated a high-resolution transcriptome atlas through scRNA-seq. Subsequently, they examined the individual responses of each cell-type to the infection, identifying differentially expressed genes, particularly those associated with stress response and secondary metabolism. Notably, the hydathode, upper epidermal and mesophyll cells showed the most robust initial responses to the infection. Altogether, each cell-type showed a specific expression pattern in the transcriptional regulation process, forming a sophisticated regulatory network to respond to fungal infections.

Nearly one year later, two groups generated the first single-cell resolution studies of the nodulation process, using the model legume M. truncatula [70, 71]. Both studies illuminated the impact of nodulation on the different cell-types, whether in the entire root [71] or focusing exclusively on nodule tissue cells [70], providing crucial resources and insights for advancing rhizobial nodulation research. Cervantes-Pérez and colleagues [71] studied the early response to the bacterial inoculation of the 25 different cell-types they found, including epidermal, endodermal, cortical and stem cells, and so forth. Interestingly, they identified an upregulation of genes involved in cell wall modifications and vesicle transport in root hairs, likely linked to the formation of curling root hairs associated with rhizobial infections. On the other hand, Ye et al. [70] focused their research only on mature nodule cells, identifying 13 spatially and functionally interconnected cell clusters that included apical, vascular and parenchyma cells, but also discovered functional clusters with diverse roles in the symbiotic process, including pre-infected, infected or nitrogen fixation clusters, among others. The authors revealed a diverse distribution of synthesis, transportation and metabolism of nutrients and substances across the different nodule cell-types; describing for example how symbiotic cells primarily perform nitrogen fixation, while un-symbiotic cells maintain homeostasis and provide energy for symbiotic cells, while surrounding them. Curiously, two unknown clusters were reported, because of their lack of specific marker genes. Later, other publications about single-cell resolution research on nodule development were released, employing diverse model legumes [60, 72,73,74]. They generated new cell atlases classifying major cell-types in both root and nodules and revealed a complex spatial compartmentalization of biochemical reactions during nitrogen fixation; particularly, Liu and colleagues analyzed two different nodule stages, and included GUS staining and RNA in situ hybridization assays to validate their observations, identifying diverse expression patterns and cell-type frequencies according to the nodule stage [75].

Serrano and collaborators [76] presented the first single-cell resolution study of the AM symbiosis, using M. truncatula and R. irregularis interaction as model. Through the integration of ST and snRNA-seq, they generated a map showing simultaneous plant and fungal transcriptomes during the symbiosis, in which they unravel a coordinated gene regulation between them. They analyzed the expression of AM-responsive genes reported in literature, inherent to each stage of colonization, through the different obtained clusters. They report a symbiosis-specific upregulation of several genes in both plant and fungus, identifying previously known and unknown transcripts and cell-type specific responses to the symbiosis. Among those, they observed the localized expression of five phosphate, three ammonium and two sugar transporters in a symbiosis-responsive cell cluster; which correlates with the continuous exchange of nutrients that occurs between the partners. The authors report as a major limitation of the study the low resolution of ST techniques, which do not reach the cell size, leading to a less accurate clustering [61].

On the other hand, three cell-resolution atlases of Arabidopsis leaves infected by Pseudomonas syringae have been released until now [77,78,79], revealing the complexity and heterogeneity of the response to bacterial infection among the different cell-types, showing coexistent cell populations that exhibit opposite states: immune and susceptible. Nobori et al. [79] introduced snMultiome in plant-pathogen research, a novel approach combining snRNA-seq and snATAC-seq (single-nucleus Assay for Transposase Accessible Chromatin sequencing), which allowed them to study single-cell transcriptome and epigenome in leaf cells responding to bacteria. To complement their results, they performed MERFISH, which allowed them to model the spatial heterogeneity of the plant immune response associated with pathogen distribution. Furthermore, Saarenpää et al. [80] introduced another innovative spatial transcriptomics technology to study P. syringae infection: Spatial metatranscriptomics (SmT), a sequencing-based approach that simultaneously considers the heterogeneity of not only the plant cells but the bacterial and fungal communities that interact with them, characterizing the microbiome signals from the tissues with a microscopic resolution.

Several publications studying the same plant-microbe interaction have yielded different or even opposite results regarding clustering of plant cell-types and how they respond to the symbiotic interaction. These differences could be attributed to the scarcity of published literature and the complexity of the data obtained. Additionally, the diversity on cell/nuclei isolation protocols, library preparation techniques and sequencing platforms available, among other factors, may have introduced biases into their findings. In summary, there is a low consensus and correlation among experiments conducted by the different laboratories, as a result of the early stages of employing single-cell methodologies in the plant field. Indeed, the majority of the results obtained in the publications that are described here (Table 1) comprise high-resolution gene atlases, which give a spatial sense of the different cell-type clusters within a plant-microbe interaction but, above all, are valuable and powerful resources that will empower researchers to answer, or at least ask, new biological questions. As a common goal to coordinate efforts in generating plant single-cell atlases, The Plant Cell Atlas aims to serve as a community asset providing an exhaustive portrayal of diverse plant cell types. It will merge detailed location data on nucleic acids, proteins, and metabolites within plant cells at a high resolution, with guidelines for common practices and for sharing results and raw data [55, 81] and comprehensive datasets for multiple plant species [82].

Leveraging scRNA-seq to shed new light on AM development and regulation

In summary, plant single-cell NGS offers a comprehensive, but complex, analysis of thousands of individual cells, organizing them based on their statistical proximity. However, there still remains a diffused degree of scepticism regarding the validity of newly formed insights or hypotheses from this data that could go beyond the mere application of a “new” technique and the creation of an extra gene expression atlas [61]. Nevertheless, understanding the molecular bases of a highly complex tissue demands a high-resolution technique: this is particularly important when two distinct organisms interact and modulate their transcriptome depending on specific time and space. In the case of AM symbiosis, several long-lasting and new biological questions could arise and, eventually, be answered through these new approaches; here we discuss some of them: from the regulation of arbuscular development and degeneration, to the control of nutrient sensing in arbusculated cells and the identification of key allelic variants shaping plant responsiveness to AM fungi.

One of the biggest open questions concerns the molecular underpinnings and genetic regulation governing arbuscule turnover and degeneration. This phenomenon seems to be tightly regulated by plants, and suppressive genetic screenings pinpointed a MYB-like transcription factor that orchestrates this process [83, 84]. Nevertheless, the transcriptomic blueprint governing the short lifespan of arbuscules remains elusive, and the potential existence of feedback-loop control mechanisms remains unexplored. The ability to sequence the complete transcriptomes of plant cells harbouring arbuscules at various developmental stages, and link it to the specific fungal counterpart, or the access to mutant lines that are affected in different steps of arbuscule formation [85,86,87], will be indispensable for elucidating the molecular checkpoints that govern this intricate process.

The same approach could help us disentangling the molecular mechanisms governing the mutual recognition between AM fungi and plants during the development of arbuscules. Current knowledge has partially revealed the complexity of plant-fungus communication in the pre-symbiotic phase, where fungal short-chain chitin oligomers (COs) trigger plant nuclear and cytosolic calcium spiking and initiate a specific symbiosis [9, 88], but much less is known regarding the arbuscular phase. Recent studies have identified several kinases implicated in this process across various plant species that, together with fungal effectors [89] and miRNAs [90, 91] that are delivered into plant cells, could play a role during this step of the interaction [92,93,94,95,96]. Moreover, cell wall-associated kinases (WAKs), triggered by pectin disassembling, seem to play a pivotal role in tuning plant defence responses upon fungal growth [92, 93]. It appears clear that AM fungal intraradical growth represents a process at the boundary between plant defence suppression and precise activation of defence-related pathways, eventually leading and mediating the arbuscule degeneration. Currently, we know at least three different levels of modulation of plant defence mechanisms during the colonization by AM fungi: at local root level both a suppression of cellular immunity is necessary for hyphal growth, counterbalanced by specific activation of plant defence genes, such as chitinases and pathogenesis-related genes. At the systemic level, AM symbiosis often leads to priming effects [94, 95] and increasing our resolution on cells will indeed be instrumental in deciphering this conundrum.

Another open question that has been partially addressed regards the mechanisms by which plants can perceive cellular phosphate concentration and, consequently, induce or repress arbuscular mycorrhizal. Indeed PHOSPHATE STARVATION RESPONSE 1 (PHR1) is a master transcription factor regulating plant phosphate starvation responses and is necessary for the transcription of several AM-inducible genes [96]. This has been demonstrated in different plant species, but the key metabolites perceived by plants allowing to sense Pi concentration and modulate AM development are still not known. Hints come from Arabidopsis thaliana and yeast, where inositol phosphate (IP7/IP8) mirrors phosphate levels and mediates PHR1 sequestration by proteins containing the SPX domain [97,98,99]. It is foreseeable that plant single-cell metabolomics [100] and proteomics [101, 102] could reveal key metabolites and proteins differentially accumulating in plant cells hosting arbuscules at different stages and mediating plant cell nutrient perception both at local and systemic levels. This would increase our knowledge of the links between plant-fungal recognition and plant nutrient sensing where, so far, only a few actors, such as NODULATION SIGNALING PATHWAY 1 and 2 (NSP1/2), have been described [5].

Finally, single-cell atlases generated from mycorrhizal roots will be instrumental in selecting candidate genes from Genome Wide Association Studies, similarly to what has been done for maize genomics, where a detailed transcriptome atlas of the developing maize ear inflorescence allowed the identification of potential loci linked to ear yield traits [103]. Several recent studies have attempted to unveil the genetic base of variation in plant growth responsiveness to AM fungi. This is a challenging goal, being plant responsiveness to AM fungi highly influenced by the environment. Therefore, only through the integration of multiple approaches and datasets, we will be able to discover genes and alleles underlying colonization levels or growth effects upon mycorrhization, beyond the ones that have already been identified [104,105,106].

Moving to more specific aspects of colonized cells, transmission electron microscopy has revealed interesting changes in both nuclear structure (chromatin moves to a very loose organization) and plastid morphology (stromules abundance) depending on the colonization phase [15, 107, 108]. Single-cell ATAC combined with RNA-seq could help to detect potential epigenetic events controlling nuclear features, as already identified in nematode-infected cells [109]. Another still open question concerns the signal(s) from the root meristem which block the AM growth. It is known that shoot apical meristem activates a sort of resistance to viruses [110], suggesting the potential presence of a common mechanism which makes stem cells resistant to exogenous microorganisms. On the other hand, the presence of AM fungi stimulates the production of new root meristems, increasing the branching, but again the specific activation site is unknown.

Altogether, due to the available genetics and molecular tools, most of our current knowledge on the AM symbiosis is plant-centric. In addition to the many questions that we have identified, single-cell level sequencing should surely help to better discover the mechanisms which underlie the morphogenesis of the symbiotic fungus, starting from the hyphopodium and the penetrating intracellular not-branched hyphae till the arbuscules or the vesicles. In the meantime, it could provide information on the saprotrophic potentialities of the fungus and on its use of mineral nutrients for its own metabolism. New knowledge could therefore lead to a more balanced information on the partners, allowing researchers to look at the interaction also from the fungal part. This could eventually lead to changing our ideas on the biology of the association, challenging the concept of mutualism for a more complex opportunistic relationship.

Conclusions

Decades of research on AM symbiosis and the efforts of an always growing scientific community have allowed us to disentangle many aspects of the intricate plant-fungal interactions. AM symbiosis is unique for the intimate contact of their partners and for its features which are surprisingly constant notwithstanding the huge biodiversity of the organisms involved [111]. This stability probably mirrors the antiquity of this alliance, suggesting that plant speciation occurred after AM establishment. The process of AM fungi accompanying most plants during evolution might have caused a strong link between organ development, especially of roots, and AM fungal presence.

It is evident that our understanding across multiple fields in biology, including plant research, has deeply advanced since we have gained access to single-cell resolution. Particularly, the ever expanding sequencing technologies could shed some light on the profound cellular heterogeneity inherent in this intimate plant-fungal interaction, offering new insights into the dynamic complexities of symbiotic relationships across evolution and within different ecosystems. The recent publication of the first AM symbiosis study using snRNAseq and ST, in which the authors generated an atlas showing plant and fungal transcriptomes simultaneously throughout their symbiotic relationship (72), is just the starting point in disentangling the molecular bases of this complex process with single-cell resolution. Certainly, these new technologies combined with the emerging ones in the coming years may address the questions that still lack answers and, in turn, promote the generation of new hypotheses.

Fig. 1
figure 1

Root colonization by AM fungi. Plants recognize fungal molecules, such as short-chain chitin oligomers (COs) or Myc factors, found in AM fungi exudates; in turn, plant strigolactones (SLs) induce spore germination and hyphal branching. Upon initial contact with the root epidermis, tip-growing hyphae differentiate, forming a structure known as a hyphopodium. Subsequently, the fungus penetrates the root guided by a plant structure called the pre-penetration apparatus (PPA). Once inside the root, the hypha traverses intercellular spaces of the inner cortex root cells and, following PPA formation, enters the cell and initiates the trunk formation. Then, young arbuscules are produced, which expand within the cortical cell through continuous branching until mature, occupying the entire space and engaging in mutualistic nutrient exchanges. These mature functional arbuscules persist for several days before undergoing senescence. Created with BioRender.com

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Fig. 2
figure 2

Bulk versus sc/sn RNA sequencing in mycorrhizal roots. Bulk RNA sequencing offers an average gene expression profile of all the root and fungal cells. On the other hand, single-cell and single-nucleus RNA sequencing offer a distinct transcriptome profile for each individual plant and fungal cell, enabling the grouping of the different cell populations into physiological or functional clusters. Created with BioRender.com

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Table 1 An overview of single-cell research studies in plant-microbe interactions ordered by publication time. scRNA-seq = single-cell RNA sequencing, snRNA-seq = single nucleus RNA sequencing, snATAC-seq = assay for transposase-accessible chromatin using single nucleus sequencing, MERFISH = multiplexed error-robust fluorescence in situ hybridization
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