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    Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999. 50:36189

    Copyrightc 1999 by Annual Reviews. All rights reserved




    Maria J. HarrisonThe Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73402;

    e-mail: [email protected]

    KEY WORDS: root, fungus, plant-microbe interaction, phosphate transport, Rhizobium-legumesymbiosis


    Arbuscular mycorrhizae are symbiotic associations formed between a wide range

    of plant species including angiosperms, gymnosperms, pteridophytes, and some

    bryophytes, and a limited range of fungi belonging to a single order, the Gloma-

    les. The symbiosis develops in the plant roots where the fungus colonizes the

    apoplast and cells of the cortex to access carbon supplied by the plant. The fungal

    contribution to the symbiosis is complex, but a major aspect includes the transfer

    of mineral nutrients, particularly phosphate from the soil to the plant. Devel-

    opment of this highly compatible association requires the coordinate molecular

    and cellular differentiation of both symbionts to form specialized interfaces over

    which bi-directional nutrient transfer occurs. Recent insights into the molecular

    events underlying these aspects of the symbiosis are discussed.


    INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

    ARBUSCULAR MYCORRHIZAL FUNGI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

    Molecular Analyses of the Fungal Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363


    Early Signaling Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364Appressorium Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365Penetration of the Root. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366Internal Growth and Development of Arbuscules in an Arum-Type Mycorrhiza . . . . . . . . 368

    The External Mycelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371









    362 HARRISON


    MYCORRHIZAL SYMBIOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372

    Expression of Defense Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374Expression of Nodulation Genes in the Mycorrhizal Symbiosis . . . . . . . . . . . . . . . . . . . . . 376


    INTERFACES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376Carbon Transfer from the Plant to the Fungus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377Phosphate Transfer from Soil to the Plant via the Fungus . . . . . . . . . . . . . . . . . . . . . . . . . 378

    CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379


    The symbiotic associations of plant roots and fungi have intrigued many gener-

    ations of biologists, and in the late 1880s these associations were given the name

    mycorrhizaderived from the Greek for fungus-root (68). Recent observations

    of fossil plants from the Devonian era suggest that one type of mycorrhizal as-sociation, the arbuscular mycorrhiza, existed approximately 400 million years

    ago (MYA), indicating that plants have formed associations with arbuscular

    mycorrhizal (AM) fungi since they first colonized land (143, 151). Today, the

    arbuscular mycorrhiza is the most widespread type of mycorrhizal association

    and exists in ecosystems throughout the world where it creates an intimate link

    between plants and the rhizosphere (6, 95, 180, 190). Despite the ubiquitous

    occurrence of this association and its importance in nutrient movement be-

    tween plants and the soil, our understanding of the mechanisms underlying the

    development and functioning of the symbiosis is limited.This review builds on the information from two earlier Annual Reviews of

    the arbuscular mycorrhizal symbiosis, in which the physiology and regulation

    of the symbiosis were discussed (110, 178). In the conclusions to their 1988

    review concerning the physiological interactions between the symbionts (178),

    Smith & Gianinazzi-Pearson noted a need for approaches to provide fundamen-

    tal information at a molecular level about the development and functioning of

    the association. Over the past ten years there has been an explosion of molecu-

    lar, genetic, and biochemical analyses of the AM fungi and the AM symbiosis.

    This review emphasizes insights into aspects of the development of the sym-biosis emerging from these studies. It focuses briefly on the AM fungi and the

    advances in our understanding of these obligate symbionts and then on aspects

    of development of the association, and finally briefly considers the mechanisms

    underlying nutrient transport, which are still almost entirely unknown.


    The arbuscular mycorrhizal fungi are all members of the zygomycota and

    the current classification contains one order, the Glomales, encompassing sixgenera into which 149 species have been classified (27, 129). A major factor









    hampering studies of the AM fungi, including the taxonomy, is their obli-

    gately biotrophic nature; so far, they have not been cultured in the absence of

    a plant host. Despite the lack of axenic culture, it is possible to grow them

    in sterile culture with plant roots, or with so-called hairy roots transformed

    with Agrobacterium rhizogenes (23, 56, 130, 132). Recent adaptations of thesemethods utilize petri plates in which the fungus and root are cultured together

    in one compartment while the external mycelium is permitted to ramify into

    a second compartment separate from the roots (185). Increasing numbers of

    fungal species are being established in this culture system (54, 57, 60), which

    is proving useful for studies of the fungal symbiont (12, 13, 25, 47, 106, 116).

    Such systems also provide access to pure, sterile fungal spores and mycelium

    that are essential for molecular analyses and useful for the taxonomy of these


    Molecular Analyses of the Fungal Genome

    There has been one report of sexual structures in AM fungi, but it remains

    unconfirmed. Recent genetic analyses also suggest that the fungi are asexual

    and reproduce clonally (153). The large resting spores formed by these fungi

    are unusual in that they are multinucleate and, depending on the species, may

    contain thousands of nuclei per spore (24). The nuclei in quiescent spores are ar-

    rested in the G0/G1 phase (29) and although initial studies suggested that DNA

    replication did not take place without a plant host (40), this was later demon-

    strated to be incorrect; both DNA replication and nuclear division occur duringthe initial growth of the hyphal germ tube, regardless of the presence or absence

    of a host plant (24). Using nuclear stains and flow cytometry, the DNA content

    of the nuclei has been estimated to range from 0.13 to 1.0 pg per nucleus for

    12 species tested (30, 103). Analysis of the base composition of nine different

    species of glomalean fungi, containing representatives from four different gen-

    era, indicated that the genomes of these fungi have a relatively low GC content,

    averaging 33%, and in contrast to other fungal taxa, a high level of methylcy-

    tosine (2.234.26%), although not as high as that of plant genomes (102).

    The first AM fungal genes to be sequenced were the small subunit rRNAgenes (SSU) and the internal transcribed spacer (ITS) regions that were targets

    for phylogenetic analyses (119, 149, 169, 170). From the SSU sequence data

    it was possible to estimate a date for the origin of the AM fungi and the time

    at which further divergence within the group occurred. The origin of the AM

    fungi was placed between 462 and 353 MYA and the ancestral fungi were

    probably most like the extant Glomus species. The families Acaulosporaceae

    and Gigasporaceae emerged later and were estimated to have diverged from

    each other 250 MYA (170).

    The SSU and ITS sequence data also allowed the design of specific primersthat, when coupled with PCR amplification, enabled the identification of AM








    364 HARRISON

    fungi from both spores and within plant roots in field situations (49, 55, 119, 149,

    159, 160, 170, 171). Additional DNA-based methods of identification followed

    (120, 195, 209), including random amplification of polymorphic DNA (RAPD)

    analysis. This enabled the development of primer-pairs specific for a number of

    species including Glomus mosseae, Gigaspora margarita, and Scutellosporacastenea (1, 114, 206). These primers have utility in taxonomic studies and in

    competitive PCR assays to quantify the amount of fungus within mycorrhizal

    roots (59, 66).

    A particularly interesting finding that emerged during the molecular analy-

    ses of the ITS regions was the variability in the genetic composition within and

    between spores of a single fungal species. A single spore may contain more

    than one ITS sequence and individual spores have ITS sequences that differ

    from other spores of the same species (119, 159). This variability was further

    confirmed by analyses of other loci (153, 210). Using minisatellite-based mark-ers, it was observed that the first generation of spores arising from single-spore

    cultures displayed a high level of variation, which suggests that the multinucle-

    ate spores are heterokaryotic. It seems likely that reassortment of genetically

    different nuclei provides a mechanism by which these fungi maintain genetic

    diversity (210).

    Genomic libraries prepared from spores and cDNA libraries from spores

    and mycorrhizal roots have been constructed for a limited number of species

    (69, 192, 208), and the first cDNA clones representing genes other than the

    rRNAs have been identified. Glyceroldehyde-3-phosphate dehydrogenase(GAPDH), -tubulin, ATPase, nitrate reductase, and DNA-binding protein se-

    quences were among the first to be reported. Although most of these are con-

    sidered housekeeping genes, they will be useful molecular markers for analysis

    of these fungi during the development of the symbiosis (42, 69, 70a, 107).

    Future dissection of gene function in arbuscular mycorrhizal fungi will be

    difficult without the possibility to transform these fungi genetically. Progress

    toward this goal is being made and transient expression of a reporter gene

    construct in spores ofGigaspora margarita has been achieved. This in itself is

    an important technological achievement and will enhance molecular analysesof the AM fungi (67).


    Early Signaling Events

    Germination of AM fungal spores and the initial growth of hyphal germ tubes

    can occur in the absence of the plant root; however, both root exudates and









    volatiles such as CO2 can stimulate both of these processes (15, 22, 25, 47, 84,

    108, 135, 144, 152). In some cases, root exudates also elicit rapid and extensive

    branching of the hyphae as they enter the vicinity of the root (89, 91), a response

    that has been observed as hyphae approach the roots of host plants but not

    when they encounter non-host roots, which suggests that recognition of thehost occurs. In this case, lack of recognition of the non-host could be due to

    lack of a signal; however, in other instances non-host status is probably due to

    inhibitory compounds (164, 165, 199).

    The range of active components present in root exudates is unknown. How-

    ever, some of the activities are probably due to flavonoid and phenolic com-

    pounds that stimulate the growth of some AM fungal species while inhibiting

    others (22, 62, 144, 172, 191). The particular molecule(s) responsible for elic-

    iting hyphal branching is also unknown but, based on its estimated size, it

    could also be a phenolic or flavonoid derivative (90). Since the flavonoid com-pounds are active at very low concentrations, it is assumed that they do not

    have a nutritional effect but rather that they act as signals to stimulate or inhibit

    growth. Plant flavonoid/isoflavonoids bind to estrogen receptors, and recent ex-

    periments utilizing estrogens and anti-estrogens provide preliminary evidence

    for the presence of an AM fungal receptor capable of binding biochanin A and

    estrogens. Based on the structures of these molecules, it is suggested that the

    A and C rings of the isoflavonoid and the hydroxyl group at position A-7 are

    important features for recognition by the receptor (145). Although flavonoid

    derivatives can influence the initial stages of the fungal life cycle, experimentswith flavonoid-deficient mutants of maize indicate that they are not essential

    for the development of the symbiosis (26). Maybe in natural environments, the

    flavonoid-mediated stimulation of growth and branching in the vicinity of the

    root helps to ensure contact with the root and the establishment of the sym-

    biosis. The differential effects of flavonoids/isoflavonoids on different fungal

    species could be envisaged to influence the fungal populations associated with

    particular plants.

    Appressorium FormationDevelopment of the symbiosis is initiated when a fungal hypha contacts theroot of a host plant where it differentiates to form an appressorium. Although

    components of root exudates are capable of stimulating hyphal growth and

    branching, they are unable to elicit the formation of appressoria, which were

    initially only observed on intact plant roots (88). Recently, it was demonstrated

    that Gigaspora margarita could form appressoria in vitro on purified epidermal

    cell walls isolated from carrot roots, a host for Gigaspora margarita, but not on

    walls isolated from sugar beet, a non-host (134). The fungus also recognized

    specifically the epidermal cell walls and did not form appressoria on cortical or






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    366 HARRISON

    vascular cell walls. These experiments indicate that the signal for appressorium

    formation lies within the epidermal cell wall, a hypothesis suggested earlier by

    Tester et al (188). The experiments also confirm that the branching signal is

    either loosely bound to the wall or exuded from the roots, since the purified

    wall fragments did not elicit the extensive hyphal branching observed in intactroots. These purified walls probably consist of a mixture of polysaccharides,

    including cellulases and polygalacturonans and some proteins. Carbohydrate

    molecules act as signals in a number of other plant/fungal interactions and are

    likely candidates for the induction of appressoria in the AM symbiosis (121).

    Penetration of the Root

    Appressorium formation is followed by the development of a penetration hypha

    and penetration of the root. This can occur in different ways; in some species,

    the hypha enters by forcing its way between two epidermal cells, whereasin other cases, the hypha penetrates an epidermal or root hair cell wall and

    grows through the cell (34). The exact mechanisms involved in penetration are

    unknown; however, by analogy to a number of the biotrophic pathogens (127),

    it has been suggested that specific, localized production of cell walldegrading

    enzymes, in combination with mechanical force, may facilitate entry of the

    hyphae without inducing defense responses (33). AM fungi produce exo- and

    endoglucanases, cellulases, xyloglucanases, and pectolytic enzymes including

    polygalacturonase (7678, 80, 150), all of which would expedite their passage

    through a cell wall.Since the appressoria that developed on the purified epidermal cell wall frag-

    ments failed to form a viable penetration hypha and did not penetrate the wall,

    processes subsequent to appressorium formation likely require an intact cell

    (134). A wide range of plant mutants on which the AM fungi can form ap-

    pressoria but cannot develop further are direct proof that the plant controls

    this developmental step in the association. Mutants blocked at this stage of

    this symbiosis have been described in Pisum sativum (63, 86), Medicago sativa

    (37, 38), Vicia faba (63), Phaseolus vulgaris (168), Medicago truncatula (155),

    Lotus japonicus (203), and Lycopersicon esculentum (21). The phenotypes ofthese mutant associations are fairly similar at the morphological level and fall

    into two broadly defined groups. In association with the P. sativum, L. esculen-

    tum, and Medicago mutants, the fungus forms appressoria that are frequently

    large and deformed and that become septate when the fungus fails to enter

    the root (21, 37, 86). In one of the Medicago sativa mutant lines, the number

    of appressoria formed on the mutant increases, a possible consequence of the

    failure to penetrate (37); however, increased numbers of appressoria were not

    reported for the P. sativum mutants (86). In P. sativum, the non-penetrating

    phenotype is referred to as myc(1)

    and 21 such mutants have been described.






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    They belong to five complementation groups, which indicates that entry into

    the root is under complex genetic control. The traits segregate as single reces-

    sive loci and the condition is determined by the roots (86). Grafting wild-type

    scions onto mutant stocks did not rescue the mutants (86, 200). Hairy roots

    prepared from these genotypes also maintain their nonmycorrhizal status (14).Cytochemical analyses of one of the P. sativum and one of the M. sativa mutant

    interactions indicated that cell wall depositions, including callose and pheno-

    lics, were present in the walls of cells adjacent to the appressoria (93, 142).

    Such depositions were not seen in wild-type interactions, which suggests that

    a defense response has been elicited in these mutants. Based on these data, it is

    possible that a suppressor of defense responses has been mutated such that the

    plant now views the fungus as a pathogen. This situation is reminiscent of the

    barley/Erysiphe graminis interaction where mutation-induced recessive alleles

    of the Mlo locus confer resistance to a wide range of isolates ofErysiphe (71).Resistance is mediated by the formation of appositions on the cell walls below

    the appressoria and wild-type Mlo is a negative regulator of defense responses

    as well as leaf cell death (71). The Mlo gene has been cloned and the encoded

    protein is predicted to be an integral membrane protein; however, the function

    of the protein and mechanism of regulation of defense responses remain to be

    determined (43).

    The P. vulgaris and L. japonicus mutants show a slightly different phenotype

    from the other species and appressorium formation is followed by penetration

    of the first cell layer (203). The association then aborts in the root epidermiswhere swollen and deformed hyphae are visible within these cells (203). In the

    Lotus mutants, the hyphae occasionally manage to overcome the block in the

    epidermis and growth from the deformed hyphae continues, producing normal

    internal mycorrhizal structures. Since these are indistinguishable from wild-

    type, it has been suggested that the mutated genes are not required for the later

    phases of growth (203). All of the legume mycorrhizal mutants are also affected

    in their ability to form a nitrogen-fixing symbiosis with Rhizobium species and

    thus define a set of genes, termed sym genes, essential for both symbioses.

    Similarities between these symbioses are just beginning to emerge, and some ofthe signaling pathways and downstream events occurring during the formation

    of the symbioses clearly are conserved (2, 194).

    A nonpenetrating mutant identified in L. esculentum is the first mutant of this

    type to be identified from a nonleguminous species. This mutant shows a similar

    phenotype to the legume mutants, although slightly different responses were

    noted depending on the fungal symbiont involved. Glomus mosseae was unable

    to penetrate the L. esculentum mutant roots, whereas Gigaspora margarita was

    occasionally able to enter. In contrast to the L. japonicus mutants, this mutation

    appears to affect the internal stages of development of the mycorrhiza, and








    368 HARRISON

    following entry, G. margarita did not develop extensively within the roots and

    was unable to form arbuscules (21). The future cloning of the mutated genes,

    which should be feasible for L. esculentum and also for thelegumes, L. japonicus

    and M. truncatula, due to their small genome size, will provide insight into the

    controlling mechanisms.Internal Growth and Development of Arbuscules

    in an Arum-Type Mycorrhiza

    Following entry into the root, internal development of the fungus is influenced

    by the plant, and a single species of fungus may show significantly different

    morphological growth patterns depending on the plant partner in the association

    (82, 105). The two main patterns are referred to as the Paris and Arum types,

    named after the species in which they were first described (74, 174). Much of

    the laboratory research focuses on crop species that form the Arum type andmolecular investigations of the Paris type have not been undertaken. Thus the

    Arum type is the focus of these discussions.

    In the Arum-type mycorrhiza, penetration of the root is initially followed

    by intercellular hyphal growth, although in some instances the fungus will

    penetrate the exodermis and form hyphal coils in the exodermal cells as it

    passes through (34). On reaching the inner cortex, branches arising from the

    intercellular hyphae penetrate the cortical cell walls and differentiate terminally

    within the cell to form dichotomously branched structures known as arbuscules

    (Figure 1). Although an arbuscule develops within a cell, it remains essentiallyapoplastic as the plant plasma membrane extends to completely surround it.

    The fungal cell wall becomes progressively thinner as the arbuscule develops

    and consequently in these cells, there is an extensive intracellular interface

    in which the two symbionts are in extremely close contact, separated only

    by their membranes and a narrow plant-derived apoplast (35, 36, 178). This

    interface is thought to be the site at which phosphate and possibly carbon are

    transferred between symbionts, although some speculate that the intercellular

    hyphae might be responsible for carbon uptake (173, 176, 181). Despite the

    intensive effort expended by both symbionts to develop the arbuscule and thearbuscular interface, the life span of an arbuscule is only a few days, after

    which it collapses and decays leaving the cell undamaged and capable of hosting

    another arbuscule(4, 5). Both thevariable growth patterns observed in the Arum-

    and Paris-type hosts and a P. sativum myc(2) mutant in which the arbuscules

    barely develop, indicate that the plant also controls this stage of the association

    (83, 86).

    Following formation of arbuscules, some species of AM fungi also form

    lipid-filled vesicles within the roots, which are presumed to act as a storage

    reserve for the fungus (178).






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    Figure 1 Diagram indicating possible sites of plant and fungal phosphate transporters and fungal

    glucose transporters in membranes of an arbuscular mycorrhiza (Arum type). Phosphate transport:

    1, phosphate uptake across membranes in the external hyphae; 2, phosphate efflux across the

    arbuscule membrane; 3, phosphate uptake across the peri-arbuscular membrane. Carbon transport:

    2 and 4, possible sites of glucose uptake by the fungus.


    DEVELOPMENT As the fungal hypha penetrates a cortical cell wall and beginsto differentiate into an arbuscule, the invaded cell responds with fragmentation

    of the vacuole, migration of the nucleus to a central position within the cell, and

    an increase in the number of organelles (17, 33, 46). This response seems to be

    arbuscule specific and it does not occur in the exodermal cells during the de-

    velopment of coils. The plasma membrane extends approximately fourfold to

    form the peri-arbuscular membrane (5) that envelops the arbuscule, and there-

    fore concomitant increases in membrane biosynthesis must be occurring. Al-

    though the peri-arbuscular membrane is connected to the peripheral membrane

    of the plant cell, cytochemical analyses indicate that it has high levels of ATPase






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    370 HARRISON

    activity, whereas the remainder of the plasma membrane shows little staining

    for ATPases (87). Since H+-ATPase activity gives rise to the proton gradient

    required for many secondary active transporter processes, these data support

    the suggestion that active transport of nutrients may be occurring across this

    membrane. Currently, little else is known about activities associated with theperi-arbuscular membrane and the fact that it develops deep within the tissues

    of the root makes it recalcitrant to many of the modern techniques of membrane

    biology. It is assumed to retain the ability to synthesize and deposit cell wall

    components including -1,4 glucans since these have been found in the new

    apoplastic compartment formed between the peri-arbuscular membrane and

    the arbuscule (33). Immunocytochemical analyses have also demonstrated the

    presence of a matrix composed of pectins, xyloglucans, nonesterified polygalac-

    turonans, arabinogalactans, and hydroxyproline-rich glycoproteins (HRGP) in

    this compartment (20, 33, 92, 140a). Consistent with this composition, genesencoding both a putative arabinogalactan protein (AGP) and an HRGP have

    been shown to be induced in mycorrhizal roots ofM. truncatula and maize, re-

    spectively, and the transcripts are localized specifically in the cells containing

    arbuscules (19, 193).

    Although the interface compartment is continuous with the cell wall and the

    molecular content tends to reflect the composition of the cortical cell wall (18),

    the physical structure is considerably different. The components of the inter-

    face matrix do not become cross-linked and most closely resemble primary cell

    walls (33). The lack of cross-linking has been suggested to be the result of lyticenzymes released from the arbuscule, and in support of this hypothesis, en-

    dopolygalacturonase has been immunolocalized to the arbuscule and interface

    compartment (33, 139). In pea mycorrhizae, immunocytochemical analysis of

    the interface matrix indicates the presence of components in common with the

    peribacteroid compartment of pea nodule cells. These epitopes are not present

    in the peripheral cell walls, providing further evidence for the specialization of

    the interface matrices and reiterating similarities between the two symbioses

    (140a, 141). Whether the components of the arbuscular interface matrix have

    any symbiosis-specific functions is unclear; however, AGP-like proteins havebeen reported in two other symbiotic associations. They are induced both dur-

    ing nodule development in the Rhizobium-legume symbiosis (163) and also in

    the Gunnera/Nostoc association, where they are abundant in the Gunnera stem

    gland mucilage, which plays a central role in communication between the sym-

    bionts (147). In plant/biotrophic fungal pathogen associations, it was shown re-

    cently that the fungus produces a proline-rich glycoprotein that is very similar to

    the plant cell wall proline-rich and hydroxyproline-rich glycoproteins. The pro-

    tein is deposited in the extrahaustorial matrix, a component analogous to the ar-

    buscular interface matrix, where it is suggested to mimic plant cell wall proteins






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    and prevent the plant from perceiving the pathogen (140). Components of the

    arbuscular interface matrix might conceivably function in a similar manner.

    The specific changes associated with formation of the arbuscular interface

    clearly require significant reorganization of the cytoskeleton of the cell. Recent

    confocal microscopy studies confirm this hypothesis and demonstrate the pres-ence of new cortical microtubule networks running along the hyphae and also

    between the hyphae and the nucleus of the cortical cell. This latter observation

    is consistent with earlier studies documenting the movement of the nucleus

    of the colonized cortical cell to a central position adjacent to the arbuscule

    (17, 81). A maize -tubulin promoter is also activated specifically in cells in

    which arbuscules are developing, providing further evidence of an increase in

    cytoskeletal components in these cells (32).

    It is predicted that development of the arbuscular interface will be accompa-

    nied by many other alterations in the cortical cells, and documentation of thesechanges is just beginning. In M. truncatula, transcripts encoding enzymes of the

    flavonoid biosynthetic pathway, phenylalanine ammonia lyase (PAL) and chal-

    cone synthase (CHS), but not the defense-specific enzyme isoflavone reductase

    (IFR), are induced specifically in cells containing arbuscules (98). Since sec-

    ondary metabolites from this pathway accumulate in mycorrhizal M. truncatula

    roots, the location of expression of these two genes has led to the speculation that

    they might be synthesized in these cells (97, 98). The potential function of the

    flavonoids/isoflavonoids within these cells is unclear; however, some of them

    stimulate growth of AM fungi both in the presymbiotic phase of their life cycleand during the symbiosis (47, 191, 207). In other cases, flavonoids act as auxin

    transport inhibitors (104) and therefore alter the hormone balance in the roots.

    Hormone levels are known to change in mycorrhizal roots and are probably

    responsible for the induction of expression of some of the mycorrhiza-induced

    genes (194). Transcription of an auxin-inducible gene encoding glutathione

    S-transferase (GST) is also induced in cells containing arbuscules (186). GSTs

    are stress-response enzymes that add glutathione (GSH) to a variety of com-

    pounds including phenylpropanoids/flavonoids to facilitate their transport via a

    specific carrier into the vacuole (53, 124). Sequestration in the vacuole preventsphytotoxic effects on the cell that can occur if high levels of these compounds

    accumulate. Although in this case the flavonoid and GST experiments were

    performed in different plants, it is not surprising that they are co-induced in the

    same cell type.

    The External Mycelium

    Following colonization of the root cortex, fungal hyphae develop extensively

    within the soil. This external mycelium plays a pivotal role in the AM symbiosis

    where its functions include the acquisition of mineral nutrients from the soil and






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    372 HARRISON

    their subsequent translocation to the plant, colonization of additional roots, and,

    in many cases, the production of spores. In addition to its role in the symbiosis,

    the extraradical mycelia contributes to soil stability by the aggregation of soil

    particles, probably mediated in part by glycoproteins produced by the hyphae

    (204, 205).Studies of this phase of the symbiosis have lagged behind that of the internal

    phase, mainly owing to difficulties with observation, collection, and quantifi-

    cation of the mycelium. However, new methods that overcome many of these

    problems are now available (137, 185). Early studies of the external mycelium

    had indicated that it is comprised of different types of hyphae, including large

    runner hyphae and finer absorptive hyphae (72). Recently, these findings have

    been confirmed, and an ultrastructural examination of the fine absorptive hy-

    phae has revealed features consistent with a role in nutrient absorption (10, 11).

    Measurements of both the internal pH of the hyphae and the external pH of themedia surrounding the hyphae are also possible in this system; therefore, the

    physiological state of the hyphae can be monitored. (13, 106). Since appro-

    priate tools are now available, studies of this phase of the symbiosis should be

    emphasized in the future.


    Over the past few years, a search for genes and proteins induced in mycorrhizalroots has been initiated in a number of laboratories. Analysis of protein pro-

    files, differential screening (133, 187, 193), and differential display (125, 126)

    have all been used to provide insight into molecular changes occurring in both

    the plant and fungal symbionts during development of the symbiosis. The ini-

    tial comparisons of the protein profiles of noncolonized and colonized roots

    indicated the presence of new proteins in mycorrhizal roots but did not per-

    mit differentiation between fungal proteins and newly induced plant proteins

    (8, 65, 79, 158, 162). These differences are more readily resolved via molecular

    analyses where comparison of cDNA and genomic sequences can be used todetermine the genome of origin.

    Consistent with the increased synthesis of plasma membrane in the mycor-

    rhizal roots, many of the mycorrhiza-induced plant cDNAs identified so far

    encode membrane proteins. Differential screening of a cDNA library prepared

    from barley mycorrhizal roots resulted in the identification of a barley cDNA

    sharing sequence identity with a H+-ATPase (133). This clone hybridizes to

    transcripts that are induced in mycorrhizal roots and although the cell and mem-

    brane location of the protein are still unknown, it is a potential candidate for the

    gene encoding the ATPase located on the peri-arbuscular membrane. A cDNA






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    encoding a member of the membrane intrinsic protein (MIP) family is induced

    in mycorrhizal parsley roots. These integral membrane proteins facilitate the

    movement of small molecules across membranes and might be predicted to have

    a role in transport at the arbuscular interface (154). Differential display anal-

    yses of mycorrhizal pea roots resulted in the identification of a cDNA psam1,predicted to encode a novel protein sharing some similarities with phospholam-

    ban, a membrane-anchored protein that regulates the activity of Ca2+-ATPase

    (125). The function of the psam1 gene product in mycorrhizae is unknown.

    Since the development of the matrix at the arbuscular interface requires the

    de novo synthesis of cell wall proteins, it might also be predicted that this class

    of proteins would be induced in mycorrhizal roots. Consistent with this hypoth-

    esis, genes encoding an HRGP, a putative AGP, and a member of the xyloglucan

    endo-transglycosylase (XET) family are all induced during development of the

    association (193). XETs are enzymes that cleave and reform xyloglucan bondswithin the cell walls, and it is speculated that this activity might be employed to

    loosen cells walls to permit penetration of the fungal hyphae or, alternatively, to

    function in maintaining the structure of the arbuscular interface matrix (193).

    In addition to the growing numbers of genes up-regulated during the sym-

    biosis, there are clearly groups of genes that are specifically down-regulated in

    the mycorrhiza. In Medicago truncatula, it was observed that four phosphate

    starvation-inducible genes including two phosphate transporter genes, a gene

    of unknown function (Mt4), and an acid phosphatase homolog were all down-

    regulated following colonization of the root by a mycorrhizal fungus (41, 118).Elevated levels of phosphate in the external medium down-regulate the expres-

    sion of these genes, and since the symbiosis generally results in an increased

    level of phosphate in the roots, the fact that these genes are down-regulated in

    the symbiosis is not so surprising (41, 118). However, studies of the Mt4 gene

    indicate that down-regulation of this transcript also occurs in one of the Med-

    icago mutants in which the fungus fails to penetrate the root and grows only

    on the external surface. In this association, it seems unlikely that the fungus

    delivers phosphate to the root. Therefore, this suggests that down-regulation

    of the Mt4 gene can occur also via a signal from the fungus and is consistentwith the presence of two, initially independent pathways for the regulation of

    the Mt4 gene (41).

    Although the identification of genes differentially regulated in mycorrhizal

    roots provides initial information about the changes that occur during develop-

    ment of the mycorrhiza, real insight into mechanisms underlying its develop-

    ment will occur in the future when the roles of these proteins in the symbiosis

    are elucidated. New technologies, such as confocal laser scanning microscopy

    coupled with the use of fluorescent reporter proteins such as green fluorescent

    protein (GFP) permit the in vivo monitoring of gene and protein expression








    374 HARRISON

    within living cells and provide a powerful addition to the current arsenal of

    molecular tools with which to analyze the symbiosis.

    Expression of Defense Responses

    In many plant-fungal pathogen interactions, invasion of plant tissues by the fun-gus results in the induction and sustained expression of a varied battery of plant

    defenses that prevent further pathogen ingress (58). This is not the case, how-

    ever, with some of the biotrophic pathogens that form ostensibly compatible

    but parasitic associations with their hosts and probably avoid eliciting defenses

    by the conservative and local production of hydrolytic wall-degrading enzymes

    and minimal damage to the plant cell (122, 127, 128). The arbuscular mycor-

    rhiza seems to be the most highly attuned plant-fungal association. Data from

    many AM associations indicate that in the AM symbiosis plant defense re-

    sponses generally show small transient increases in the early stages of thesymbiosis, followed by suppression to levels well below those of noncolonized

    plants (85, 109).

    In Allium porrum, chitinase and cell wallbound peroxidase activities showed

    a transient increase in expression during the initial stages of development of

    the mycorrhiza; however, activity in a well-established mycorrhizal association

    was notably lower than in the controls (183, 184). In addition, immunocyto-

    chemical analysis suggested that the chitinase, when present, was located in the

    vacuole and was not in contact with the hyphae (183). Similar observations were

    later made in bean and tobacco roots where colonization by an AM fungus wasaccompanied by a transient increase and then a decrease in both the transcript

    levels and activity of chitinase, -1,3 endoglucanase, and chalcone isomerase,

    an enzyme of isoflavonoid biosythesis (52, 112, 197, 201). These data indi-

    cated that regulation was occurring probably at the level of gene expression,

    a finding that is also supported by studies of the expression of genes encod-

    ing enzymes of the isoflavonoid biosynthetic pathway in Medicago species.

    Medicago sativa and M. truncatula respond to attack by fungal pathogens with

    the rapid induction of genes and enzymes of the isoflavonoid pathway, which

    results in the production of the defense compound, medicarpin. In contrast,colonization by AM fungi results in the down-regulation of the gene encoding

    isoflavone reductase (IFR), the penultimate enzyme of medicarpin biosynthesis,

    and medicarpin does not accumulate (97, 202). In situ hybridization revealed

    that down-regulation of the IFR transcript occurred exclusively in the areas of

    the root in which arbuscules had formed, indicating that this is a specific and lo-

    cal effect (98). Investigations of soybean roots colonized with different strains

    ofG. intraradices suggested that the suppression of endochitinase expression

    was correlated with the infectivity of the different strains, with the most infec-

    tive strains resulting in the most down-regulation (113). This finding might






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    explain the results from parsley and bean roots where defense responses were

    not altered following colonization by mycorrhizal fungi (31, 70).

    Although the suppressionof plant defense responses seems to be a widespread

    occurrence in AM associations, the necessity for this suppression is challenged

    by data indicating that Nicotiana tabacum and Nicotiana sylvestris plants over-expressing various chitinases, glucanases or pathogenesis-related (PR) pro-

    teins were apparently entirely unaffected in their ability to form mycorrhizae

    (196, 198). In contrast, overexpression of these defense proteins inhibits the

    growth of fungal pathogens. Plants overexpressing chitinase were more re-

    sistant to the root pathogen Rhizoctonia solani, whereas those overexpressing

    PR-1a were more resistant to Peronospora tabacina and Phytopthora parasit-

    ica (3, 39, 198). The only gene whose overexpression was observed to inhibit

    colonization of plants by AM fungi was PR-2, a protein with -1,3 glucanase

    activity (196). Although the data from these transgenic plants cast doubts onthe requirement for suppression of these particular defense responses, there are

    other instances in which defense responses seem to be impeding the interac-

    tion. In Salsola kali, which is a non-mycotrophic species, AM fungi colonize

    the roots and initially form coils and arbuscules; however, the root responds very

    quickly with the production of autofluorescent compounds and the colonized

    areas of the root turn brown and die (7). It might be argued that this is a non-host

    response; however, similar responses are seen in alfalfa (a mycorrhizal species)

    in response to colonization by G. margarita. The fungus enters the root, but

    colonized cells show a hypersensitive response and become necrotic. Phenolicand isoflavonoid compounds characteristic of a defense response accumulate

    in these regions of the root (61). Thus, if defense responses are elicited in the

    appropriate cells, they can apparently prevent development of the association.

    These results also indicate that the AM fungi do not simply fail to elicit de-

    fense responses, as has been occasionally suggested, but rather that at some

    level, there is compatibility between the fungus and the plant, and recognition

    of compatibility prevents induction of defense responses. Incompatible com-

    binations clearly exist, and as different genera and species of fungi are utilized

    for laboratory experiments, it might be predicted that more of the incompatibleinteractions will be identified. Additional evidence supporting the hypothesis

    that recognition and suppression of defense responses is required for successful

    colonization is provided by the pea locus a myc(1) mutant in which defenses

    seem to be induced and prevent further development of the appressorium (93).

    Although in general a sustained defense response is not induced during a suc-

    cessful AM symbiosis, there are some exceptions. For example, the defense

    gene, PR-1, is expressed in pea root cells containing arbuscules, and in a number

    of plants new symbiosis-related forms of chitinase are induced in the roots fol-

    lowing colonization by mycorrhizal fungi (51, 64, 146). In these instances, the






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    376 HARRISON

    proteins could have roles other than classical defense. Chitinases released from

    spruce cells do not have deleterious effects on ectomycorrhizal fungi, but actu-

    ally destroy fungal elicitors released from fungal cell walls by cleaving them into

    smaller, inactive units. In this way, the elicitation of defense responses can be

    prevented and development of the symbiosis can proceed (157). The new chiti-nase isoforms induced in arbuscular mycorrhizal roots may have a similar role.

    Expression of Nodulation Genes

    in the Mycorrhizal Symbiosis

    The emerging similarities between the Rhizobium-legume symbiosis and the

    arbuscular mycorrhizal symbiosis have stimulated investigations of the expres-

    sion of nodulation genes during the AM symbiosis. Leghemoglobin transcripts

    were detected in mycorrhizal roots ofVicia faba (73), whereas in Medicago

    sativa, two nodulation genes, MsENOD40 and MsENOD2, were induced inmycorrhizal roots with similar tissue-specific patterns of expression as in roots

    inoculated with Rhizobium (194). Both genes can be induced in roots in the

    absence of a symbiosis via the application of cytokinin, and since cytokinin

    levels are elevated during nodulation and also in mycorrhizal roots (194), it is

    speculated that cytokinin is one component of the signal transduction pathway

    mediating induction of these genes during the symbioses. Further evidence of

    signal transduction pathways common to both symbioses is provided by studies

    of the PsENOD5 and PsENOD12A genes, which are induced in pea roots dur-

    ing interactions with either AM fungi or Rhizobium. In the pea sym8 mutant,which is unable to form either of these symbioses, expression of both genes is

    blocked, suggesting that SYM8 functions in the signal transduction pathway

    for induction of these genes in both symbioses (2). Based on these studies and

    also on the legume symbiosis mutants, it is clear that some mechanisms are

    shared between the two symbioses and this has fuelled speculation that the

    Rhizobium-legume symbiosis arose by exploiting signaling pathways from ar-

    buscular mycorrhizae (194). Since our understanding of the Rhizobium-legume

    symbiosis is more advanced than that of the arbuscular mycorrhizal symbio-

    sis, it will be fruitful to exploit these overlaps to elucidate these aspects of theassociation.


    Nutrient transport between the symbionts is a central aspect of the symbiosis;

    however, the membrane transporters responsible for the movement of carbon or

    phosphate between the symbionts are unknown (Figure 1). In addition, there is

    speculation as to the interfaces involved in carbon transport between the plant









    and the fungus. [For in depth discussions of these issues, readers are referred to

    a number of comprehensive reviews (173, 179, 181).] A brief summary of the

    current opinions on transport in the symbiosis and insights obtained from recent

    data that will facilitate identification of the transport proteins are included here.

    Carbon Transfer from the Plant to the Fungus

    Although it has been known for over 20 years that carbon is transferred from

    the plant to the fungus, evidence as to the form of carbon has been lacking

    (28, 101). Recent in vivo 13C nuclear magnetic resonance spectroscopy data

    strongly suggest that glucose is the form of carbon utilized by these fungi

    (167) and this is further supported by studies of isolated arbuscules that were

    observed recently to use glucose for respiration (182). Although carbon allo-

    cation to the roots increases during mycorrhizal associations, the amounts of

    carbon estimated to leak out of intact root cells into the apoplast are thought tobe insufficient to account for the amount of fungal growth occurring in mycor-

    rhizal roots. Therefore, enhanced efflux, or a decrease in the level of competing

    host uptake systems, has been proposed (138, 166, 173, 181). In M. truncatula,

    expression of a hexose transporter gene is induced in mycorrhizal roots, specif-

    ically in the cortical cells in the vicinity of the fungus, which suggests that in

    this case potentially competing host mechanisms are not suppressed (96). In

    other symbioses, enhanced efflux of nutrients, stimulated by the demand of the

    microsymbiont, has been observed and in some plant/fungal pathogen interac-

    tions, the fungi produce toxins that alter membrane transport processes to favorrelease of metabolites (75, 100, 123). Similar events may well occur during the

    AM symbiosis, and the fungal symbiont may possess the capability to stimulate

    efflux of carbon from the plant. So far, there is no molecular information about

    transport proteins responsible for the efflux of carbon out of plant cells; how-

    ever, this is currently an active area of research since this type of transporter is

    also expected to exist in the mesophyll and vascular tissues where sugar export

    occurs (161).

    Although the arbuscule presents a large area of close contact between the

    symbionts and was traditionally assumed to be the interface over which carbonwould be transferred, the observation that the membrane of the arbuscule lacks

    ATPase activity has led to the suggestion that carbon uptake might occur via

    the intercellular hyphae, whose membranes have been observed to have a high

    ATPase activity and thus are energized for active transport processes (87, 176)

    (Figure 1). It is unclear whether uptake of carbon by the AM fungus requires

    an active transport mechanism similar to those of plant transporters, or whether

    concentrations of carbon at the interfaces could be sufficient to permit uptake

    by facilitated diffusion, as occurs in yeast (111). In the absence of information

    about the concentrations of carbon present in the various apoplastic interfaces, it








    378 HARRISON

    is difficult to speculate on potential mechanisms of transport either out of plant

    cells or into the fungus. Amanita muscaria, an ectomycorrhizal fungus, utilizes

    both fructose and glucose but relies on plant invertases for release of these

    hexoses from sucrose (48, 156). A monosaccharide transporter was cloned

    recently from Amanita muscaria and is probably the transporter responsible forhexose uptake in both the free-living and symbiotic stages of its life (136). A

    similar transporter might be envisaged for AM fungi, although it seems unlikely

    that it will be present in the absence of the plant host. Sequence information

    from the Amanita transporter coupled with those from yeast and Neurospora

    crassa may facilitate the cloning of transporters from arbuscular mycorrhizal


    Phosphate Transfer from Soil to the Plant via the Fungus

    Phosphate movement in the symbiosis involves a number of membrane transportsteps, beginning with uptake across membranes in the external hyphae. This

    is followed by translocation back to the internal fungal structures where it is

    thought to be released from the fungus across the arbuscule membrane and

    then taken up into the plant by transporters on the peri-arbuscular membrane

    (Figure 1) (173, 178, 181). There has been some progress toward understanding

    the mechanisms of uptake of phosphate by the external AM fungal hyphae and

    a high-affinity phosphate transporter has been cloned from G. versiforme (99).

    The transporter has a Km of 18 M as determined by expression in yeast cells, a

    value that is consistent with previous measurements of phosphate uptake by AMfungi (189). The transporter transcripts are present in the external mycelium and

    not in the structures of thefungus internal to theroot and therefore the transporter

    may be responsible for the initial uptake of phosphate into the mycorrhiza

    (99). Unfortunately, the inability to perform gene disruption experiments in

    arbuscular mycorrhizal fungi currently prevents direct proof of its role in the


    Phosphate flux across the symbiotic interfaces in the mycorrhiza has been

    estimated at 13 nmol m2s1, although this value increases if extra phosphate

    is supplied to the mycorrhiza (50, 177). In contrast, the general rate of effluxof phosphate from fungal hyphae growing in culture was measured at 12 pmol

    m2s1 (45). Based on these findings, the AM fungi likely have some type of

    specialized efflux mechanism operating in the arbuscule membrane to permit

    sufficient phosphate efflux to the arbuscular interface. Efflux of phosphate

    from hyphae of an ectomycorrhizal fungus was shown to be stimulated by

    divalent cations, and a similar mechanism, possibly triggered by a component

    of the interface matrix, might be envisaged for the AM fungi (45). Since the

    peri-arbuscular membrane has a high ATPase activity (87), the subsequent

    uptake of phosphate by the plant could then occur by proton-coupled transport









    mechanisms. However, as with the previous discussion of carbon transport, the

    lack of information about physiological conditions at the arbuscular apoplastic

    interface prevents informed speculation as to the type of transport mechanisms.

    Analysis of the conditions in the apoplastic interfaces in mycorrhizal roots is

    extremely challenging, although progress is being made (9).In the past two years, phosphate transporters have been cloned from the roots

    of a number of plant species (115, 117, 118, 131, 175). These transporters are

    expressed during growth in low-phosphate environments and mediate high-

    affinity phosphate transport into the epidermal and cortical cells. In Medicago

    truncatula, the expression of these transporters is down-regulated in mycor-

    rhizal roots (118). This suggests that the plant does not use these transporters

    during the symbiosis and therefore it seems unlikely that these transporters

    operate at the peri-arbuscular membrane. Studies of phosphate uptake in myc-

    orrhizae have revealed that in some associations, phosphate uptake directly viathe root cells was considerably reduced during the symbiosis and the bulk of

    the phosphate uptake occurred via the fungal symbiont (138a), which is con-

    sistent with the down-regulation of the root phosphate transporters during the


    The cloning and functional analysis of the transport proteins operating in

    the symbiosis is just one of the challenges for the future. Part of this process

    might be accelerated by utilizing approaches that have been successful in other

    research fields, such as the plant/biotrophic fungal pathogen associations, where

    there have been recent advances in the identification of transporters operatingin the haustorial membranes. The cloning of an amino acid transporter from a

    rust fungus was achieved from libraries prepared from isolated haustoria (94).

    With the refinements in methods for isolation of arbuscules, similar approaches

    might permit access to the fungal transport proteins operating at the arbuscule



    Over the past few years, the first insights into molecular mechanisms underlyingdevelopment of the symbiosis have been achieved. The initial similarities to,

    and differences from, other plant-microbe interactions have been explored and

    exploited. Despite this progress, there is a still a vast amount to learn, and the

    molecular approaches that have been initiated recently need to be integrated with

    other disciplines to address both development and functioning of the symbiosis.

    Although the current range of mycorrhizal mutants is limited, they have

    played a pivotal role in proving that the plant controls various stages of devel-

    opment of the association. The complexity of this symbiosis renders genetic

    approaches essential, and the identification of additional mutants, particularly








    380 HARRISON

    those that are specific for the mycorrhizal symbiosis, should be emphasized in

    the future.


    I thank Sally Smith (University of Adelaide, Australia) for critical reading ofthe manuscript and both Richard Dixon (Samuel Roberts Noble Foundation,

    USA) and Sally Smith for encouragement and advice. The work in the authors

    lab is funded by The Samuel Roberts Noble Foundation.

    Visit the Annual Reviews home page at

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