REVIEW Published 27 Jul 2010 | DOI: 10.

1038/ncomms1046

Mechanisms underlying beneficial plant–fungus

interactions in mycorrhizal symbiosis
Paola Bonfante1 & Andrea Genre1

Mycorrhizal fungi are a heterogeneous group of diverse fungal taxa, associated with the
roots of over 90% of all plant species. Recently, state-of-the-art molecular and genetic tools,
coupled to high-throughput sequencing and advanced microscopy, have led to the genome
and transcriptome analysis of several symbionts. Signalling pathways between plants and
fungi have now been described and the identification of several novel nutrient transporters has
revealed some of the cellular processes that underlie symbiosis. Thus, the contributions of each
partner in a mycorrhizal association are starting to be unravelled. This new knowledge is now
available for use in agricultural practices.

O
wing to their filamentous organization, fungi exploit very diverse substrates on the basis of
their nutritional strategy. Saprobes thrive in soil, water and on decaying animal and plant tis-
sues. A smaller group of fungi, the parasitic and mutualistic symbionts, feed on living organ-
isms1. Such a classification cannot easily be applied to mycorrhizal fungi, a heterogeneous group of
species spread over diverse fungal taxa. Although they can spend part of their life cycle as free-living
organisms, mycorrhizal fungi always associate with the roots of higher plants, indeed over 90% of
plant species, including forest trees, wild grasses and many crops. Both partners benefit from the rela-
tionship: mycorrhizal fungi improve the nutrient status of their host plants, influencing mineral nutri-
tion, water absorption, growth and disease resistance, whereas in exchange, the host plant is necessary
for fungal growth and reproduction2.
Mycorrhizal fungi colonize environments such as alpine and boreal zones, tropical forests, grass-
lands and croplands. They have a major role in nutrient cycling through the specific activity of their
mycelium in absorbing soil nutrients and supplying them to the plant, although their role in carbon
flux is less well defined3.
The term mycorrhiza is derived from the Greek words for ‘fungus’ and ‘root’. Mycorrhizal fungi
develop an extensive hyphal network in the soil, the aptly named wood-wide web4, which can con-
nect whole plant communities offering efficient horizontal transfer of nutrients. Mycorrhizas develop
specialized areas, called symbiotic interfaces, to interact with the host plant5–7. Mycorrhizal fungi can
be divided into two major groups: aseptate endophytes such as Glomeromycota, or septate Asco- and
Basidiomycota (see Box 1 Glossary)2. More commonly, mycorrhiza classifications reflect anatomical
aspects and identify two broad categories2, referred to as ectomycorrhizas (EMs) and endomycor-
rhizas, depending on whether the fungus colonizes the root intercellular spaces or develops inside
cells (Fig. 1). Endomycorrhizas are further divided into orchid, ericoid and arbuscular mycorrhizas
(AMs).
A descriptive approach has dominated the investigation of mycorrhizas for at least 50 years until
the advent of molecular biology, and the ‘omics’ era provided insight into their mechanisms. High-
throughput technology, genome sequencing of fungi, plants and associated microbes, transcriptomic

1
Department of Plant Biology, University of Torino and IPP-CNR, Viale P.A. Mattioli 25, Torino 10125, Italy. Correspondence and requests for materials
should be addressed to P.B. (email: [email protected]).

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BOX 1 GLOSSARY

Arbuscule: Highly branched structure produced by arbuscular mycorrhizal fungi inside the cell lumen of their host. Arbuscules are
considered to be the key element of the symbiotic nutrient exchanges between the plant and the fungus.
Arbuscular mycorrhiza: Widespread type of endomycorrhizal interactions involving fungi of the phylum Glomeromycota, the hyphae of
which reach the root inner cortex and develop highly branched exchange structures called arbuscules.
Ascomycetes: Phylum of the kingdom Fungi characterized by a microscopic sexual reproduction structure called ascus, a single cell
containing non-motile spores. Ascomycetes include macroscopic fungi that live in symbiosis with forest trees.
Aseptate endophytes: Plant-colonizing fungi with syncytial hyphae that lack transversal walls (septa).
Basidiomycetes: Phylum of fungi the sexual reproduction of which is accomplished in club-shaped cells called basidia that bear external
spores. Several basidiomycetes develop ectomycorrhizas with trees.
Ectomycorrhiza: Symbiosis between higher plants and fungi belonging to Asco- and Basidiomycetes, in which fungal hyphae surround the
root tips and develop between epidermal cells but never enter the cell lumen.
Endomycorrhiza: Group of mycorrhizal symbioses involving fungal penetration inside living cells of the root epidermis and cortex.
Extraradical mycelium: Hyphal network that develops in the rhizosphere, in which it absorbs inorganic nutrients that are transferred to
the host plant through intraradical hyphae.
Hyphopodium: Specialized hypha of an arbuscular mycorrhizal fungus, often branched and swollen, which adheres to the root epidermis
before intracellular fungal penetration.
Intraradical hyphae: Network of hyphae from mycorrhizal fungi that colonizes the host root tissues.
Metagenomics: Analysis of genetic material obtained from environmental samples.
Nodulation (Nod) factors: Signalling molecules produced by nitrogen-fixing bacteria (rhizobia) and eliciting nodule initiation and bacterial
uptake in the roots of legumes. They are lipochitooligosaccharides consisting of an acylated chitin oligomeric backbone carrying various
functional group substitutions at the terminal or non-terminal residues.
Pathogen: Organism that receives its nutrients from a host causing a disease.
Perifungal interface: Thin apoplastic compartment that surrounds each intracellular fungal structure inside plant tissues. The interface
consists of plant cell wall components and is bordered by an invagination of the plant plasma membrane.
Presymbiotic phase: Phase in the life cycle of mycorrhizal fungi that precedes contact with the host plant. During this phase, fungal
development is not supported by the nutrients of plant origin.
Saprotroph: Organism that obtains nutrients from dead organic matter.

analyses, availability of mutant collections, RNA interference lines have a dual lifestyle: they live as symbionts with plant roots and
and plants transformed with fluorescent tags have all led to new per- as facultative saprotrophs in soil12. However, data on their in situ
spectives on plant–microbe interactions, and on EMs and AMs in activity (metatranscriptomics) are not yet available and the eco-
particular. In this review we focus on the mechanisms that govern logical functions accomplished by EM fungi in the soil remain at
the growth of EM and AM fungi and their interactions with plants. the moment unexplored.
We report how genomics have unwrapped the genomes of at least two
EM fungi, revealing their peculiarities, whereas similar approaches Deciphering the genome of EM fungi
have not fully deciphered any AM fungal genome so far. By contrast, Genome sequencing of two EM fungi, the Basidiomycete Laccaria
recent discoveries have shed light on the plant mechanisms that regu- bicolor13 and the Ascomycete Tuber melanosporum (black truffle)14,
late AM interactions, mostly because of plant-based genetics, ‘omics’ has shed some light on EM biology, highlighting similarities and
and advanced microscopy technologies. differences between the two fungal symbionts and identifying the
primary factors that regulate mycorrhiza development and func-
EM fungi dominate the world forests tion. Although L. bicolor has a 65-Mb genome and an estimated
Trees in the families Pinaceae, Fagaceae, Dipterocarpaceae and 19,102 protein-encoding genes, T. melanosporum, with its 125-Mb
Caesalpinoidaceae found in many forests all interact with hun- genome and an estimated 7500 protein-encoding genes, is in the
dreds of EM species of Basidiomycetes and Ascomycetes, so that odd condition of being the largest sequenced fungal genome,
these fungi can be said to have shaped the present forests2. EM with a relatively small expected proteome compared with other
fungi colonize the lateral roots of these trees with sheathing mycor- filamentous fungi14. This argues that the symbiotic status can be
rhizas, in which a fungal mantle covers the root tip, and a so-called achieved even with a reduced number of protein-coding genes,
Hartig net of intercellular hyphae surround epidermal and outer very few of which (1.5%) are differentially regulated during mycor-
cortical cells (Figs 1 and 2). EM fungi may also live independently rhizal formation14. Another contrasting feature is that the genome
of plant roots, as demonstrated by their growth capabilities in Petri of L. bicolor has undergone extensive expansion of a few gene
dishes2. families with predicted roles in protein–protein interactions and
Molecular techniques have identified EM fungi in the field8, signal-transduction mechanisms13, whereas this trait has not been
and owing to metagenomics, that is, the deep sequencing of observed in T. melanosporum, in which multigene families are rare.
organisms living in an environment9, a multitude of sequences The genome size of both EM fungi is not ascribed to large-scale
from soil10 are catalogued, also highlighting the rhizosphere duplication events, but rather to a high number of transposable
around EMs as a special biome. Use of the Roche 454 Titanium elements that represent more than 20 and 58% of the genome in
genome sequencer has revealed unexpected levels of fungal bio- L. bicolor13 and T. melanosporum14, respectively. In the truffle, the
diversity when applied to the soil of a French forest, in which insertion of transposable elements has been dated to 2–5 million
Agaricomycetes were found to be the dominant class including a years ago, and is considered to have contributed to genome evolu-
large number of EM species11. These data confirm that EM fungi tion and plasticity.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1046 REVIEW
Ectomycorrhiza Arbuscular mycorrhiza

Hyphopodium
Mycelium

Mantle
Arbuscules

Hartig
net Spore

Figure 1 | Illustration of root colonization structures in ectomycorrhizal

(blue) and arbuscular mycorrhizal (pink) interactions. The Figure 2 | The transition from the free-living status of an EM fungus
ectomycorrhizal fungus surrounds the root tip with a thick mantle of to the symbiotic phase. A growing hypha from the mycelium of
closely appressed hyphae, whereas the Hartig net develops around T. melanosporum as observed in fluorescence microscopy is shown
epidermal cells (green). In the case of arbuscular mycorrhizas, the root in a (courtesy: R. Balestrini). Hyphal morphology changes in the
tip is usually not colonized. Hyphae develop from a spore and produce a mantle, in which the repeated branching, characterized by incomplete
hyphopodium on the root epidermis. Intraradical colonization proceeds transversal walls (arrowheads), originates a pseudoparenchymatous
both intra and intercellularly and culminates with the formation of structure, as evident in the electron micrograph shown in b. In both images,
arbuscules, little fungal trees, inside inner cortical cells (brown). the fungal wall is falsely coloured in blue and nuclei in red. The transverse
section of a mycorrhizal root tip stained with Trypan blue is presented
in c, showing the organization of hyphae in the mycelium, mantle and
In filamentous Ascomycetes and Basidiomycetes, sexual fertiliza- Hartig net. Bars, 10 μm.
tion and karyogamy are limited by a system of mating types, whereby
only different and compatible strains can mate. T. melanosporum
possesses the sex-related components identified in other Ascomyc- Malassezia globosa14, a yeast commonly found on the skin of many
etes14, and the presence of HGM and MATa loci in the sequenced animals, including humans.
strain and in environmental samples shows that truffle is an obli-
gate outcrossing species15. This is leading to the development of new Nutrient and signal exchange in EMs. Mycorrhizal fungi, similar
strategies for truffle cultivation. to other plant-interacting fungi, base their life cycle on the uptake of
Comparison of L. bicolor and truffle genomes with those of sapro- organic carbon from a living plant2 (Fig. 3). Fifteen genes encoding
trophic and pathogenic fungi reveals some components of the gene putative hexose transporters have been annotated in the L. bicolor
set required for a symbiotic lifestyle12. Both EM fungi have a relatively genome and many of them are upregulated during symbiosis. How-
small number of enzymes that target plant cell wall components, for ever, the genome lacks genes encoding invertases, the enzymes
example, pectinases and pectin lyases. Remarkably, genes encod- that hydrolyse sucrose (the most abundant sugar in plant tissues)
ing some of these enzymes are upregulated during the symbiotic to glucose and fructose, suggesting that L. bicolor depends on the
steps, when the fungus develops inside plant tissues. These data glucose released by its green host. In contrast, T. melanosporum pos-
strongly suggest that the colonization process is based on limitation sesses one invertase gene, indicating its potential capacity to access
of host damage, avoiding the stimulation of defence reactions. the plant sucrose pool, whereas other EM fungi, Amanita muscaria
The enzyme repertoire of both EM fungi also sheds light on their and Hebeloma cylindrosporum, also metabolize fructose. Interest-
saprotrophic capacities. L. bicolor possesses substrate-degrading ingly, Ustilago maydis, a biotrophic pathogen causing smut disease
enzymes, including glycosyl hydrolases involved in the hydrolysis of on maize, possesses a sucrose transporter localized on the plasma
bacterial and microfaunal polysaccharides, as well as many secreted membrane16 that allows the fungus to directly acquire sucrose with
proteases, chitinases and glucanases, likely involved in the degra- no need for secreted invertases. Such a range of mechanisms to
dation of organic compounds in the litter. The absence of lignino- access plant sugars thus seems to mirror some of the differences in
lytic capacities points to a weak saprotrophic habit for L. bicolor. By nutritional strategies between symbiotic and pathogenic fungi.
contrast, these degradative functions are more limited in the truffle, Mutualistic interactions are based on the exchange of nutrients;
making it intriguingly closer to animal-interacting fungi such as for this reason, genes coding for transporter proteins are expected to

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for communicating with their hosts or whether such functions are

carried out by other small secreted proteins.
The wealth of information derived from the sequencing of
L. bicolor, T. melanosporum and poplar19 provides an opportunity
to dissect the molecular basis of EM and unravel the regulatory
networks that allow uptake, movement and exchange of nutrients
between partners. However, plant transcriptomic responses are still
limited, as only the poplar genes that respond to L. bicolor have been
investigated20. Among the 39,303 genes on the poplar microarray,
2,945 were differentially expressed, including several transcripts
encoding proteins involved in auxin metabolism and root morpho-
genesis. This finding has led to a model in which fungus-induced
auxin accumulation at the root tip stimulates lateral root formation
through auxin redistribution and auxin-based signalling.
Sequencing the genomes of L. bicolor and T. melanosporum has
therefore been a landmark in the study of fungal biology: the data
illustrate similarities and differences among saprotrophic, sym-
Figure 3 | Scheme summarizing the main nutrient exchange processes
biotic and pathogenic fungi and offer the first insights into the
in EM and AM symbiosis. Emphasis is placed on the translocation of
mechanisms behind fungal nutritional strategies. Genomic tools
phosphorus (P), nitrogen (N) and carbon (C) at the soil–fungus and
have transformed our understanding of the molecular mechanisms
fungus–plant interface. Inorganic P and mineral or organic forms of N,
that regulate EMs, which may now become new models in plant–
such as NH4 + , NO3 − and amino acids (AA), are taken up by specialized
microbe interactions. Furthermore, they provide an appreciation
transporters located on the fungal membrane in the extraradical mycelium.
of what makes a fungus a symbiont or a pathogen. Development
NH3/NH4 + and Pi (the latter originated in AM fungi from the hydrolysis of
of transformation protocols for EM fungi will permit the identi-
polyphosphate) are imported from the symbiotic interface to the plant cells
fication of molecular determinants that allow the transition from
through selective transporters. Hexose transporters import plant-derived
pathogenicity to saprotrophism or symbiosis, similar to that carried
carbon to the fungus, whereas transporter proteins involved in the export
out for bacterial21 and fungal species22 in which few genes have been
of nutrients from either the plant or fungus have not been identified yet.
demonstrated to control the symbiotic status.
This questions whether such processes indeed result from active, protein-
mediated transport or involve passive export mechanisms.
The key to success for AM fungi
AM fungi are the most widespread fungal symbionts of plants,
be well represented in EM fungal genomes. In spite of this prevision, being associated with more than 80% of current land plants2 (Fig. 1).
the number of transporters annotated in L. bicolor is comparable to They all belong to the phylum Glomeromycota, a monophyletic
that of other saprotrophic and pathogenic fungi (491 genes), and group that diverged from the same common ancestor as Ascomy-
they are even less abundant in T. melanosporum (381 sequences). cota and Basidiomycota23.
However, many of these genes are upregulated in the symbiotic con- Although AM host plants can survive if deprived of their fungal
dition, implying very intense traffic of amino acids, oligopeptides symbionts, this condition is virtually unknown in natural ecosystems,
and polyamines through the symbiotic interface12. in which AM fungi function as true helper microorganisms, improv-
Analysis of EM genomes highlights the capacity of the free- ing overall plant fitness. Plants grown in artificial non-symbiotic
living mycelium to import organic and inorganic N sources, includ- conditions have shown that AM fungi significantly contribute to the
ing nitrate, ammonium and peptides from the soil, through proteins uptake of soil nutrients, increase plant biomass and confer on the
such as the ammonium transporter or the urea permease (Fig. 3). plant improved resistance to stress and pathogens2.
These functions are probably common to many EM fungi, as they In contrast, AM fungi have so far proved to be unculturable in
were first described in A. muscaria (AmAMT2) and Paxillus involutus the absence of a host. Being unable to absorb carbohydrates except
(PiDur3)12. From the mycelium, N compounds reach the mantle from inside a plant cell, these fungi strictly depend on their green
and the Hartig net, from where they can be exported to the hosts for growth and reproduction, which gives them the status of
plant; even if this mechanism is poorly understood, glutamine obligate biotrophs. From an evolutionary point of view, the ecologi-
and ammonium are the best candidate molecules to cross the cal success of AM fungi demonstrates that the advantages of such a
symbiotic interface12. This implies ammonium assimilation strict association with plants have overcome the risks arising from
into amino acids and their translocation to the plant, or, alter- the loss of saprotrophic capabilities.
natively, the direct release of ammonium ions from the Hartig net
hyphae. A possible confirmation of the second alternative comes The unique biological features of AM fungi. AM fungi are con-
from the upregulation of ammonium importers in EM poplar sidered to be asexual, although the hyphae of genetically distinct
roots17. strains can anastomose and exchange genetic material24,25. The very
Pathogenic fungi possess genes for a large array of secreted/ concepts of species and individual are poorly defined in this group
effector proteins18. The discovery that L. bicolor possesses about of organisms, reflecting a high degree of genetic and functional
300 small cystein-rich secreted proteins that are upregulated during variability. Even the definition of a cell as a hyphal compartment
interaction with poplar and Douglas-fir opens new scenarios. For is inconsistent with the syncytial mycelia of these fungi, in which
example, these proteins could function as effectors during the early nuclei are constantly translocated by rapid cytoplasmic streaming26.
phase of the plant–fungus interaction or could be involved in the Finally, their large asexual spores contain thousands of nuclei,
construction of the symbiotic interface, as suggested by the immuno- making classical genetic approaches unsuitable7.
detection of one of these proteins (MISSP7) in the hyphal mantle13. A further level of complexity arises as several Glomeromycota
These data point to the interface area as a site of recognition host unculturable endobacteria. The meaning of this bacterial/fungal
between the partners; however, comparable effector-like proteins symbiosis has only recently begun to be investigated27,28, but the
have not been detected in T. melanosporum, leading to the question inability to culture both types of endobacteria, their vertical trans-
of whether EM Ascomycetes have developed alternative pathways mission and the small genome size of Candidatus Glomeribacter27

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strongly suggest that these endobacteria are ancient partners was found to be similar to the Gin-N protein from G. intraradices43.
of AM fungi and may have been of evolutionary importance for These findings suggest that some molecular mechanisms underlying
fungal speciation29. the invasion of plant tissues are shared by pathogenic and symbiotic
All these unorthodox traits have so far limited the possibility fungi, in spite of their distant phylogenetic relationships and diverse
of applying standard approaches such as genetic transformation, trophic habits.
mutant generation and characterization, as well as large-scale tran-
scriptomic and genomic analyses, to AM fungi. Although sequenc- Finding the host root
ing methods are evolving fast, the project to sequence the genome The dissection of plant responses illustrates how the mechanisms
of the AM fungus Glomus intraradices has met a bumpy road30. operating to accommodate the AM fungus inside the plant cell
Assembly difficulties for this relatively small genome (estimated lumen are shared by diverse plants and have been conserved dur-
to be around 14–16 Mb) are probably due to a high level of hetero- ing evolution. Root colonization is vital to AM fungi (Fig. 4). Their
zygosity and/or to a larger genome than expected. At the same time, spores feed germinating hyphae through the catabolism of storage
the lack of a uninucleate cell stage in the life cycle of AM fungi is a lipids for just a few days2. During this period, hyphae explore the
major obstacle in obtaining stable transgenic strains. Nevertheless, soil in search of a host but if they never meet one, they arrest their
the transient expression of an exogenous gene in G. intraradices has growth and retract their cytoplasm back into the spore, which may
been achieved through biolistic transformation31. Because of these again become dormant and restart the germination process over
peculiar traits, transcriptomic32 and proteomic33 analyses have so far and over. This situation is not likely to be very frequent in nature
proven to be more successful in investigating the molecular basis of because of the wide host range of these fungi, but is a hallmark
AM fungal growth and function. of their obligate biotrophy, as well as their endurance. This strict
In contrast to AM fungi, genetic tools and genomic information biotrophy has long frustrated attempts to obtain enough material
have helped in investigating several aspects of AM interactions such to perform large-scale nucleic acid extractions or produce sterile
as root colonization and communication between symbionts7,27,34. inoculum. The latter was made possible by root organ cultures that
Nevertheless, this one-way approach has very likely provided us serve as a living substrate to grow several species of AM fungi44.
with a biased picture, in which the plant seems to be the main factor
and determining the contribution of each partner to the establish- Strigolactones as a new class of bioactive molecules. Perception
ment and functioning of the association is still difficult. of the host plant by the presymbiotic mycelium is mediated by root
exudates. The responsible compounds that are released by the root
Understanding the functions of AM fungi. The demonstration and diffuse a short distance, before being washed away or degraded,
that AM fungi possess high-affinity inorganic phosphate (Pi) trans- have been identified as strigolactones45,46. They stimulate AM fungal
porters provided a breakthrough in the understanding of fungal
function. After the identification of a complementary DNA that
encoded a transmembrane Pi transporter from G. versiforme, the
function of the protein was confirmed by complementation of a
yeast mutant affected in Pi transport35. Furthermore, the expression
of this Pi transporter was localized to the extraradical hyphae of
G. versiforme, the site of phosphate uptake from the soil (Fig. 3).
Accumulated as polyphosphate, Pi is then rapidly translocated
along the aseptate mycelium to the host plant36. Nitrogen is another
important element taken up by AM fungi, and genes involved in the
transport of ammonium37 and amino acids38 have been identified,
whereas arginine is probably the preferred molecule for long-
distance transport to the host plant39. A glutamine synthase gene
of G. intraradices is in fact preferentially expressed in extraradical
hyphae, whereas a gene associated with arginine breakdown is
expressed more in the intraradical mycelium.
Although carbon transfer from plants to AM fungi was demon-
strated in the 1960s2, its molecular mechanisms are still unclear.
Surprisingly, with the exception of one gene from the non-AM Figure 4 | Schematic summary of the root colonization process by AM
Geosiphon pyriforme40, which hosts intracellular cyanobacteria, no fungi. The germination of a resting spore is followed by the production of
hexose transporters responsible for C uptake from host cells have so a short explorative mycelium. The perception of plant exudates, released
far been characterized in Glomeromycota. by the host root, induces recursive hyphal branching, increasing the
The availability of expressed sequence tag libraries from different probability of a direct contact between the symbionts. In the meantime,
steps of the life cycle of G. intraradices will offer a more complete fungal exudates are perceived by the root, where they trigger calcium
picture of fungal activity. A first glimpse has been caught by Seddas spiking through the activation of the common SYM pathway (Box 1).
et al.41, who monitored G. intraradices genes involved in transcrip- Signal transduction leads to the activation of cellular and transcriptional
tion, protein synthesis and metabolism in quiescent and germinating responses (green cells and nuclei). The contact between the plant and
spores, as well as during interactions with wild-type and AM-defective fungus is followed by the adhesion of a hyphopodium to the root surface.
(Myc − ) mutants of Medicago truncatula. The expression of only a This triggers the assembly of a broad aggregation of cytoplasm (yellow),
few fungal genes is strongly affected by the plant genotype41. named the prepenetration apparatus (PPA) in the contacted epidermal
Although there are an increasing number of studies on specific cell and underlying outer cortical cell. Subsequent intracellular fungal
fungal genes, the genetic traits that discriminate an AM fungus from colonization strictly follows the route of PPAs from the epidermis to the
an EM fungus or a pathogen are currently unknown. Interestingly, inner cortex. Here, intercellular hyphae can develop along the root axis.
a G. intraradices gene, STE12-like, complements a non-invasive The PPA mechanism is then replicated in the contacted inner cortical cells,
mutant of the pathogenic Colletotrichum lindemuthianum, restoring both before fungal entry and—on a smaller scale—branching. Eventually,
its infectivity42. Similarly, a gene encoding an Era-like GTPase in a highly branched arbuscule occupies most of the cell volume, forming an
the rice pathogen Magnaporthe oryzae and required for virulence extensive surface for nutrient exchange.

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metabolism and branching, a change in mycelial growth pattern Calcium spiking as a Rosetta stone for nodulation and AM.
that is thought to increase the chances of an encounter with the The common SYM pathway seems to be primarily involved in
host. Strigolactones also have a hormonal role in inhibiting lateral controlling early events in AM establishment, and particularly the
branching of shoots47,48. This direct effect on plant development is precontact responses and first steps of root colonization. Mutation
considered to be the main function for this class of molecules, which of one of these genes blocks fungal colonization at the epidermal or
are in fact produced by many plant taxa45,49, including those not asso- subepidermal cell layer. The central factor of this signalling path-
ciated with mycorrhizal fungi. The leakage of strigolactones from way is the most widespread second messenger in eukaryotic cells:
roots into the soil and their rapid hydrolysis have in any case made the calcium ion. In addition to rapid calcium influxes, which are
them ideal for signalling root proximity in the rhizosphere7,49. The induced by both Nod factors54 and AM fungal exudates52, the re-
seeds of parasitic weeds are also stimulated to germinate by strigo- peated oscillation of calcium concentration in the nuclear region
lactones50. These multiple functions recall the pleiotropic effects of of root hairs (Ca2 + spiking) has long been observed in response to
auxins, and promote consideration of strigolactones as hormones. rhizobial signals54 and used to position the activity of gene products
along the SYM pathway. In recent years, specific calcium signatures
The still uncharacterized Myc factors. AM fungi are not inactive have also been described for AMs. The proximity of branched hy-
during their presymbiotic growth. A molecular dialogue precedes phae was reported to induce Ca2 + oscillations in the perinuclear
root colonization, keeping the partners informed about their recip- cytoplasm of M. truncatula root hairs expressing the cytoplasmic
rocal proximity34. Although the responsible molecules have not yet calcium sensor Cameleon55. Although such cells are not usually in-
been identified, AM fungal bioactive molecules were shown to be fected by AM fungi, this observation represents the first evidence
smaller than 3 kDa, partially lipophilic51 and suggested to possess of an AM-induced Ca2 + spiking response. The pattern of such Ca2 +
a chitin backbone34. These diffusible signals, often referred to as oscillations show several distinctive traits compared with those in-
‘Myc factor’52, are known to be perceived by the plant also in the ab- duced by nitrogen-fixing bacteria. The regular peak frequency that
sence of a physical contact with the fungus, inducing, for example, characterizes Nod factor-induced Ca2 + spiking and the much more
a membrane-steroid-binding protein, which is critical for mycor- irregular pattern observed in AM responses have been modelled
rhization53. Plant responses to Myc factors range from the molecular mathematically to suggest their chaotic nature55,56. These differences
to the organ level and are part of a reprogramming under the con- could be the key to understanding one of the central questions in
trol of the so-called common symbiosis (SYM) pathway (Box 2), the the study of legume symbiosis: as the common pathway is shared
signal-transduction pathway that prepares the plant for successful by both AM and nodulation, how does the plant determine which
association with both AM fungi and nitrogen-fixing rhizobia. microorganism is trying to colonize its tissues? This is anyway not

BOX 2 THE COMMON SYMBIOSIS PATHWAY

The establishment of AM interactions and, in particular,

fungus recognition by the host plant are mediated by a partially
characterized signalling pathway partly shared with Rhizobium–
legume symbiosis7,54. At least seven genes (SYM genes) are
known to be required for both symbioses to develop correctly.
Some genes (for example, LjNFR1/LjNFR5) are nodulation specific,
whereas no elements of this pathway have been identified that
are only related to AM7. The hypothetical Myc factor is perceived
by the host plant by a still-to-be-identified Myc factor receptor
(MFR). Myc factor perception has been reported to trigger a
rapid transient elevation of cytosolic calcium54. In Lotus japonicus,
a second membrane protein, LjSYMRK (MtDMI2 in Medicago
truncatula) codes for a receptor-like kinase that has the potential
to directly or indirectly perceive rhizobial and AM fungal signals94,
and transduces the signal to the cytoplasm by phosphorylating
an unknown substrate through its kinase domain. The nuclear
localization of all the downstream elements of the SYM pathway
suggests that the signal is rapidly transduced to the nucleus.
A role for the nuclear pore complex is strongly suggested by the
involvement of two nucleoporins (LjNUP85 and LjNUP133) in this
pathway. LjCASTOR/LjPOLLUX proteins (MtDMI1 in M. truncatula) epidermis colonization by rhizobia95,96. LjCCaMK is known to form
encode a potential cation channel located in the nuclear envelope7 a complex with and phosphorylate the product of the last SYM
and are required for calcium spiking. Both MtDMI1 and MtDMI2 are gene, LjCYCLOPS97, another nuclear protein of unknown function,
required for the onset of a repeated oscillation of Ca2 + concentration orthologue of MtIPD3, eventually leading to gene regulation and
in the nucleus and perinuclear cytoplasm through the alternate colonization (see figure). Besides the several gaps that are still
activity of Ca2 + channels and transporters54,55. LjCCaMK, a calcium- present along the common SYM pathway, increasing data support
calmodulin-dependent protein kinase (orthologue of MtDMI3), the existence of alternative signalling pathways that control other
localizes in the nucleoplasm and is probably responsible for decoding early responses to AM fungi and are largely unknown. Examples
calcium oscillations7. Gain-of-function mutations of LjCCaMK come from the demonstration of a SYM-independent regulation of
have recently been demonstrated to restore root colonization AM-induced gene expression in rice98 and M. truncatula53. AM fungi
by AM fungi in mutants for the SYM genes located upstream of are therefore likely to generate a number of distinct biologically
Ca2 + spiking, and used to uncouple nodule organogenesis and active molecules, activating different signalling pathways.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1046 REVIEW

the case for the majority of AM host plants, which have no obvious
relationship with rhizobia. The search for Ca2 + spiking responses to
AM fungi in non-legumes therefore seems to be a promising field
for providing insight into such mechanisms.

On the root surface

Once a chemical acquaintance has been made between the fungus
and the plant, and roots and hyphae have proliferated and branched
in a small volume of the rhizosphere, the presymbiotic phase of the
AM interaction culminates in a physical encounter between symbi-
onts, when a hyphal tip touches the surface of a root (Figs 4 and 5).
AM fungi carefully select the location for starting root penetration.
Hyphae can wander for several centimetres along the root surface,
forming long, straight or gently curving hyphae; then, unpredict-
ably, they swell, flatten on the cell wall of a few epidermal cells and
branch repeatedly to develop what is known as a hyphopodium57. In
the absence of direct evidence, several clues may shed light on this
process. When the plant and fungus are separated by a permeable
membrane, AM hyphal branching concentrates in the vicinity of
young lateral roots52, which are also commonly acknowledged as the
primary site of AM colonization, at least in laboratory conditions58.
Hyphopodia tightly adhere to the root epidermis and their wall
protrudes with many digitations into the outer layers of the plant
cell wall (Fig. 5). The molecular and developmental mechanisms
mediating the switch from linear apical growth to the swollen and
branched form of the hyphopodium are still completely obscure. By
contrast, hyphopodium development is known to be followed by a
pause in fungal growth that can last for 4–6 h before new tip growth
is initiated to develop the penetration hypha57. During this time,
plant cells and tissues prepare for colonization.
The symbiotic pathway-dependent expression of ENOD11
(a gene coding for a putative secreted protein) is reported in the
hyphopodium area58. The expression of several other plant genes
also changes, many of which are already regulated during the pre-
symbiotic phase59. New genes also become active on hyphopodium
development, including those involved in cell wall remodelling and
defence60, just at the time when epidermal cells reorganize their
cytoplasm to produce an AM-specific structure that is indispen-
sable for successful fungal penetration: the PPA (prepenetration
apparatus)57.
Contacted epidermal cells start to assemble the secretory
machinery that builds the interface compartment where the fungus
will be hosted. Cytoplasm becomes aggregated at the contact site, Figure 5 | Hyphopodium adhesion to the root epidermis in arbuscular
then develops into a thick column that predicts the subsequent mycorrhizas. A top view of the carrot root epidermis is shown in a, in
route of the hypha across the cell57. All elements of the secretory which a branched hyphopodium of Gigaspora gigantea stained with acidic
pathway are concentrated in the PPA: abundant endoplasmic retic- fuchsin contacts the cell surface (bar, 10 μm). The transmission electron
ulum, and many Golgi bodies and secretory vesicles61. But the main micrograph in b shows that the hyphopodium strict adhesion to the root
factor in this display is the nucleus; at an early stage, its movements is achieved through the formation of several protrusions of the fungal
to and from the contact site anticipate PPA development57. Eventu- cell wall into the wall of the epidermal cell (arrows). A prepenetration
ally, the PPA is completed and the fungus starts growing again, with apparatus (PPA), assembled in response to fungal contact, is visible
a hyphal tip heading through the epidermal cell wall and along the underneath the hyphopodium as a broad column of cytoplasm traversing
track of the PPA. At this time, the perifungal membrane is thought the vacuole (bar, 2 μm).
to be assembled, as PPA secretory vesicles are expected to fuse to
produce an invagination of the plant plasma membrane. Perifun- common symbiotic pathway, as, under high inoculum pressure, a
gal membrane development marks the appearance of the symbiotic few SYM mutants can be forced into AM colonization63. In most
interface, the narrow intracellular compartment that allows AM of these cases, the development of arbuscules proceeds normally,
fungi to grow inside the plant cell without breaking its integrity5 although their overall distribution is sometimes reported to be more
(Figs 4 and 6). limited and patchy7. Some genes that affect arbuscule development
have been recently identified by reverse genetics. RNA interference
The arbuscule is at the heart of symbiosis knockdown of the Vapyrin gene, coding for a cytoplasmic protein
All AM fungi are characterized by—and named after—arbuscules with unknown function, induces a marked decrease in epidermal
(from the Latin arbusculum, small tree, Fig. 6). These structures penetration and a total block of arbuscule formation64. In this case,
are formed in the inner root cortex by repeated branching of an only intercellular hyphae develop between cortical cells. This form
intracellular hypha, and considered the site of nutrient exchange62. of hyphal growth also spreads AM infection along the root in wild-
The mechanisms controlling arbuscule development are largely type plants and is likely to be less strictly controlled by the plant, as it
unknown. The colonization step is not strictly dependent on the takes place outside the cells. Similarly, silencing of either a subtilisin

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REVIEW NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1046

b the fungus, interconnected by their stromules69. The nucleus and

nucleolus increase in size, whereas chromatin is decondensed61,
a likely mark of enhanced transcriptional activity. When first the
PPA and later the arbuscule occupy a considerable part of the cell
volume, the vacuole also has to be reorganized. Although a detailed
in vivo analysis of tonoplast dynamics (and the mechanisms
involved) is not yet available, it is clear from confocal microscopy
Perifungal that the vacuole is markedly reduced in size68, closely following
membrane
hyphal branches (Fig. 6).
Fungus
Interface Arbuscules are ephemeral structures with an estimated lifespan
Vacuole of 4–5 days. At the end of this cycle, the fungal walls start to collapse
Fungal in the arbuscule fine branches, as the cytoplasm retracts. Eventually,
wall
this process of senescence extends to the trunk hypha until the whole
arbuscule is shrunk to a compact mass of hyphal cell walls. During
this process, the periarbuscular membrane rearranges once more to
a
adapt to its changing content. The fungus eventually disappears and
Nucleus the host cell regains its previous organization with a large central
vacuole70 and can undergo new colonization. In fact, root infection
Fungus by AM fungi is not a linear or synchronous event. As the fungus
Vacuole
repeatedly attempts cell (and root) colonization, arbuscules of very
different ages coexist in neigbouring cells, and early structures, such
as young hyphopodia, can develop on already colonized roots.

How arbusculated cells operate. The significant cell reorganization

during root colonization is associated with important changes in the
Figure 6 | The organization of an arbusculated cortical cell. The scheme transcriptomic profile of AM roots. Several model plants have been
in a shows how intracellular fungal hyphae are enveloped by the perifungal investigated, including M. truncatula71–73, rice74 and L. japonicus75,
membrane, an extension of the host cell plasma membrane. This outlines in which more than 500 protein-coding genes were found to be
the interface compartment, an apoplastic space that surrounds the differentially regulated during the arbuscular phase. In all cases,
fungus and mediates nutrient exchange. The central vacuole is resized expression level was significantly altered for genes, including those
into a smaller volume as the fungus develops to occupy most of the cell for nutrient transporters, transcription factors and proteins involved
lumen. The nucleus enlarges and is positioned in the middle of the hyphal in cellular dynamics and cell wall synthesis. A mycorrhiza-specific
branches. The transmission electron image in b shows the details of the plant phosphate transporter, which is localized on the periarbuscu-
fungal accommodation process inside an arbusculated cell of carrot. The lar membrane6 (Fig. 3), is essential for active symbiosis66. Among
interface compartment (uncoloured) is clearly visible all around the fungus the regulated L. japonicus genes, 47 putative transporters were
(pink), whereas plant organelles are observed all around the perifungal identified; 28 of them may be important for nutrient acquisition
membrane. In particular, the tonoplast occasionally seems to be in direct and are considered as novel markers for AM roots. The strongest
connection (arrows) with the perifungal membrane (bar, 2 μm). upregulated gene of the array is a putative ammonium transporter76,
the transcripts of which have been quantified and specifically local-
ized in arbusculated cells using laser microdissection.
protease65, a phosphate transporter66 or two ABC transporters67 The availability of microarrays and novel molecular tools has
results in arbuscular morphogenetical defects. Taken together, these stimulated questions on the systemic effects of AM fungi77 in
findings suggest that genes essential for arbuscule development may plants of agronomic interest as well. Significant gene modulation
be independent of the SYM signalling pathway. was reported in shoots of mycorrhizal tomato78. The knockdown of
sucrose synthase affects arbuscule development, and also reduces
The fungal accommodation process. Although the regulatory plant height, shoot weight and seed yield79. All these data support
mechanisms leading to fungal branches filling most of the host cell the idea that, on colonization, plants activate an organism-wide
lumen are poorly understood, the process of arbuscule accommo- reprogramming of their major regulatory networks and argue that
dation, which requires a substantial remodelling of the cortical cell, mobile factors of fungal or plant origin are involved in a generalized
has long been described5–7. Similar to any intracellular hypha, all metabolic change; in this context, hormones80,81 and microRNA82,83
the thin arbuscule branches are enveloped by the perifungal (now may be good candidates. At present, experimental data—for exam-
periarbuscular) membrane. This invagination of the host plasma- ple, for jasmonic acid—are still highly controversial80,81. Analysis of
lemma does not simply surround the arbuscule as a whole, but ethylene, abscisic acid, salicylic acid and jasmonate-related com-
closely follows the surface of each branch, moulded to the arbuscule pounds coupled to transcriptional profiling have documented both
itself. The arbusculated cell is where the interface acquires its maxi- common and divergent responses of tomato roots to G. mosseae and
mum spatial complexity, with a composition very similar to that of G. intraradices84. The emerging picture looks complicated: symbio-
plant primary cell wall5. The assembly of this extensive amount of sis changes the levels of several hormones, in parallel with changes
membrane and cell wall material requires the recursive involvement in the expression of their biosynthetic enzymes, also highlighting a
of PPA-like structures, organized both in advance of fungal penetra- role for oxylipins in AM84. Oxylipin regulation shows parallels with
tion through the cell wall and along the ‘trunk’ hypha, as smaller plant–pathogen interactions; as resistance to Phytophthora para-
aggregates anticipating the formation of fungal branches61. sitica is known to depend partly on oxylipin synthesis in tobacco85,
The periarbuscular interface assembly also involves the prolif- oxylipin regulation might shed light on the enhanced resistance of
eration of endoplasmic reticulum, Golgi apparatus, trans-Golgi net- AM plants to pathogens86.
work and secretory vesicles61,68, as well as AM-induced subtilases In conclusion, the plant processes that permit the accommoda-
secreted at the symbiotic interface65. This is not the only restructuring tion of fungal structures are active in epidermal, outer and inner
that the cortical cell undergoes. Plastids multiply and deploy around cortical cells. They require orchestration among signal-transduction

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© 2010 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1046 REVIEW

pathways, transcriptional and proteomic changes and major cell

reorganization. Recent data clearly show that the long described
‘growth effect’ observed in AM plants depends on systemic con-
sequences of the symbiosis, which go beyond the root system and
affect the physiology of the whole plant.

An evo-devo approach to mycorrhizas

The idea that AM fungi have coevolved with plants since the
past 400 million years, and that their association has helped the
conquest of dry terrestrial ecosystems by Embryophyta87, is very
popular. As lichens clearly show, combining an organism that can
exploit light and atmospheric CO2 with one that can efficiently
exploit the substrate represents a fruitful adaptation to habitats
at the soil–atmosphere interface. However, experimental data
supporting the antiquity of AM associations have until recently
been limited to fossils and the observation that extant AM fungi
colonize basal plant lineages, including hepatics, hornworts and
lycopods. The study of AM interactions in these plants can shed
light on the origins of symbiosis, as they spend most of their life
cycle haploid and do not possess roots87. AM fungi are known to
colonize haploid gametophytic tissues with different degrees of
success: whereas mosses such as Physcomitrella patens, a model
basal plant, do not seem to undergo AM colonization, many
hepatics and hornworts are successfully colonized both in the
field88 and in vitro89. Figure 7 | The distribution of SYM genes in plant taxa. Phylogenetic
The molecular biology of rootless plants and their interaction and molecular evolutionary analyses91 have demonstrated that the SYM
with AM fungi are poorly studied. However, recent findings90 may genes controlling symbiosis establishment in legumes7 and monocots90,91
represent a breakthrough: three SYM genes controlling symbiosis are distributed throughout Embryophyta, including both gametophytic
establishment in legumes7 and monocots91 have also been reported and sporophytic phases of liverworts and other basal plants. By contrast,
in basal plants (Fig. 7), suggesting that AM colonization is a homo- SYM homologues have not been detected in green algae (Chlorophyta),
logous process throughout Embryophyta, including both gameto- apparently relating the occurrence of SYM genes to terrestrial habitats.
phytes and sporophytes. Furthermore, CCaMK orthologues from AM colonization can therefore be envisaged as a homologous process
liverworts and hornworts can complement M. truncatula dmi3 non- that was established by a common ancestor of all extant plant clades
mycorrhizal mutants. These results suggest that SYM genes were (pink node). Red colour highlights plant taxa that possess SYM gene
already present in ancestral plants and have been conserved during homologues but do not establish AM.
evolution. Finally, the finding of SYM genes in taxa that do not asso-
ciate with AM fungi, such as Physcomitrella and Arabidopsis, could
indicate that the corresponding proteins have likely been coopted to this information is available, novel agricultural and forestal treat-
other functions, unrelated to AM symbiosis. ments can be envisaged, whereas the quest for the corresponding
receptors in plants will begin. Another promising field is the study of
Future directions microbes associated with mycorrhizal fungi and their role as a third
When considering mycorrhizal symbioses in the context of current component of symbiosis. Once these basic biological questions have
global challenges (environmental change, ecosystem conservation, answers, we will be ready to exploit the mycorrhizal fungal commu-
sustainable agriculture, development of plants for future needs and nity hidden in the soil but quietly benefiting plants and humans.
food safety), we acknowledge that mycorrhizal fungi may be crucial
in many of these fields: mycorrhizal fungi mobilize P and N, and References
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Acknowledgments
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Contributions to this review were partly funded by the FISR-Soil Sink project, by
colonized by an arbuscular mycorrhizal fungus. New Phytol. 184, 975–987 (2009).
Compagnia di San Paolo, Converging technologies (CIPE-BIOBIT) and MIUR PRIN
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2008. We thank Raffaella Balestrini for generously providing an unpublished picture,
Arbuscule Maturation and Maintenance in Mycorrhizal Roots of Medicago
Maria Teresa Della Beffa for helping to prepare the reference list, Robert Milne for his
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critical reading and Francis Martin for comments. We apologize to all those colleagues
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81. Hause, B. & Schaarschmidt, S. The role of jasmonates in mutualistic symbiosis Additional information
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82. Gua, M. et al. Expression analysis suggests potential roles of microRNAs for Reprints and permissions information is available online at http://npg.nature.com/
phosphate and arbuscular mycorrhizal signaling in Solanum lycopersicum. reprintsandpermissions.
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83. Brancheid, A. et al. Expression pattern suggests a role of MiR399 in the regula- How to cite this article: Bonfante, P. & Genre, A. Mechanisms underlying beneficial
tion of the cellular response to local Pi increase during arbuscular mycorrhizal plant–fungus interactions in mycorrhizal symbiosis. Nat. Commun. 1:48 doi: 10.1038/
symbiosis. MPMI 23, 915–926 (2010). ncomms1046 (2010).

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