Download Signal Transduction Pathways in Mycorrhizal Associations

Fungal Genetics and Biology 23, 205–212 (1998)
Article No. FG981046
REVIEW
Signal Transduction Pathways in Mycorrhizal
Associations: Comparisons with the
Rhizobium–Legume Symbiosis
Ann M. Hirsch*,1 and Yoram Kapulnik†
*Department of Molecular, Cell and Developmental Biology and Molecular Biology Institute, University of California,
Los Angeles, Los Angeles, California 90095-1606; and †Institute of Field and Garden Crops, Agricultural Research
Organization, The Volcani Center, Bet Dagan 50-250, Israel
Accepted for publication March 26, 1998
Hirsch, A. M., and Kapulnik, Y. 1998. Signal transduction pathways in mycorrhizal associations: Comparisons with the Rhizobium–legume symbiosis. Fungal
Genetics and Biology 24, 205–212. A number of
genera of soil fungi interact with plant roots to establish symbiotic associations whereby phosphate acquired by the fungus is exchanged for fixed carbon from
the plant. Recent progress in investigating these associations, designated as mycorrhizae (sing., mycorrhiza), has led to the identification of specific steps in
the establishment of the symbiosis in which the fungus
and the plant interact in response to various molecular
signals. Some of these signals are conserved with
those of the Rhizobium–legume nitrogen-fixing symbiosis, suggesting that the two plant–microbe interactions
share a common signal transduction pathway. Nevertheless, only legume hosts nodulate in response to
Rhizobium, whereas the vast majority of flowering
plants establish mycorrhizal associations. The key
questions for the future are: what are the signal
molecules produced by mycorrhizal fungi and how are
they perceived by the plant? r 1998 Academic Press
1 To whom correspondence should be addressed. Department of
Molecular, Cell and Developmental Biology, University of California, Los
Angeles, 405 Hilgard Avenue, Los Angeles, CA 90095-1606. Fax: (310)
206-5413. E-mail: [email protected]. URL: www.lifesci.ucla.edu/mcdbio/
Faculty/Hirsch/index.html.
1087-1845/98 $25.00
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Index Descriptors: endomycorrhiza; VAM fungi; appressoria; flavonoids; phytohormones; Myc2 and Nod2
phenotypes.
Mycorrhizal associations are found in more than 80% of
all terrestrial plants. Nevertheless, very little is known at
the molecular level about this important plant–fungal
symbiosis although much is known about the anatomy,
physiology, and ecology of the association. Mycorrhizal
fungi are involved in procuring and transporting phosphate
and other nutrients from the soil to plant roots; in turn, the
plant provides fixed carbon to the fungal partner. In
addition, mycorrhizal associations are said to increase the
resistance of plants to environmental stresses and plant
pathogens. The goal of this review is to summarize what is
currently known about signal transduction pathways in
these important symbiotic associations.
Mycorrhizal associations can be broadly divided into
endo- and ectomycorrhizal interactions (Barker et al.,
1998; Smith and Read, 1997). Endomycorrhizal associations are interactions between woody or herbaceous angiosperms and six genera of fungi in the order Glomales of the
Zygomycetes (Morton and Benny, 1990). Roots are colonized both extraradically and interradically; for the latter,
the fungi grow both intercellularly and intracellularly.
Ectomycorrhizal interactions are formed between fungal
partners that are basdiomycetes, ascomycetes, or zygomy-
205
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Hirsch and Kapulnik
FIG. 1. Colonization of sweet clover (Melilotus alba Desr.) roots by Glomus intraradices, stained with acid fuchsin. (A) Branches of the appressorium
(arrow) have begun to form intercellular hyphae. Bar, 50 µm. (B) Occasionally, fungal hyphae penetrate a root hair (arrow). Bar, 25 µm. (C) Intercellular
hyphae (IH) penetrate cortical cells and differentiate to form arbuscules (arrows). Bar, 25 µm.
cetes and hosts that can be either gymnosperms or woody
angiosperms. The hyphae are wholly intercellular and form
a dense sheath around lateral roots that cease to elongate
once colonized. For a review of recent research on gene
expression in ectomycorrhizae, see Martin et al. (1997).
Other types of mycorrhizal associations are also present in
nature, and these are described in Smith and Read (1997).
THE AM/VAM SYMBIOSIS
Because of their ancient lineage (Remy et al., 1994) and
their apparent similarity to the Rhizobium–legume interac-
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tion (see below), this review concentrates on endomycorrhizae, especially those symbioses referred to as arbuscular
mycorrhizal (AM)2 or vesicular arbuscular mycorrhizal
(VAM). Arbuscules are highly branched, tree-like elaborations of fungal hyphae (arrow, Fig. 1C) that have a high
capacity for absorption of mineral nutrients, whereas
vesicles, if present, are oval-shaped structures filled with
lipid bodies and are proposed to have a storage or
propagule function (Fig. 2A). For recent reviews, see
Gianinazzi-Pearson (1996) and Harrison (1997).
VAM or AM fungi are obligate symbionts which in the
2
Abbreviations used: AM, arbuscular mycorrhizal; VAM, vesicular
arbuscular mycorrhizal.
Signaling in the Mycorrhizal Symbiosis
207
FIG. 2. Diagrammatic illustration of mycorrhiza and nodule development. (A) Mycorrhiza formation. Flavonoids released from the host stimulate spore
germination (Germ), preinfection growth (Pif), and hyphal branching (Pab). Later, appressoria (Apr) form and hyphal branches colonize the root. Some
hyphae penetrate cortical cells (Pen) and may form coils or arbuscules (Arb). Phenotypic codes after Smith and Reid (1997). (B) Early stages of nodule
development. Flavonoids from the root induce rhizobial nod genes. Nod factor is synthesized and stimulates root hair deformation (Had). Firm attachment
of compatible rhizobia leads to root hair curling (Hac) and infection thread formation (Inf). Cellular divisions in the cortex (Ccd) trigger nodule initiation
(Noi). Phenotypic codes from Caetano-Anollés and Gresshoff (1991).
absence of a plant are present in the soil as multinucleate
spores surrounded by thick cell walls or as hyphal fragments in dead or dying roots. Spore germination (Germ)
and preinfection hyphal growth (Pif) and branching (Pab)
are stimulated by signal molecules synthesized and secreted by the root. CO2 also has a positive effect on fungal
growth (Bécard et al., 1992). The root-produced molecules
are often flavonoids (Siqueria et al., 1991; Tsai and Phillips,
1991), several of which serve as signals in the Rhizobium–
legume symbiosis—as chemoattractants and as inducers of
Rhizobium nod (nodulation) genes, which are responsible
for the synthesis of Nod factor (Fig. 2B), a primary
morphogen that by itself can deform root hairs (Had) and,
in some legumes, trigger cortical cell divisions (Ccd) or
nodule initiation (Noi). Nevertheless, rhizobia are required
for the formation of shepherd’s crooks or 360° hair curling
(Hac) and infection thread formation (Inf) (Fig. 2B).
However, Bécard et al. (1995) found that Agrobacterium
rhizogenes-transformed roots of carrot and maize chalcone
synthase mutants underwent normal VAM fungal coloniza-
tion even though flavonoids were not detected in either
plant system. Previously, Bel Rhlid et al. (1993) reported
that flavonoids were present in transformed carrot root
extracts, but only caffeic acid derivatives were found in
exudates (Bécard et al., 1995).
As further evidence for a plant-generated signaling
molecule, Giovannetti et al. (1993a, b) used a membrane to
separate a host plant from its fungal partner and showed
that even though the fungus was physically isolated from its
host, hyphal differentiation, as evidenced by extensive
branching, took place. This indicates that: (1) a diffusible
signal emanates from the host (nonhosts did not elicit the
same response) and (2) cell–cell contact is not required for
the branching response. The compound(s) responsible for
the branching response has not been fully characterized,
but appears to have a molecular mass of less than 500 Da
(Giovannetti et al., 1996). Flavonoids or phenolics are
smaller than this, so it is possible that the branching signal
is a member of either of these classes of compounds.
The above-described experiments indicate that cell–cell
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208
contact is required for the differentiation of the appressorium (a specialized structure formed upon adhesion and
prior to penetration of host tissues) (arrow, Fig. 1A; Apr,
Fig. 2A) because true appressoria did not develop under
these experimental conditions. Supporting this idea are the
findings of Nagahashi and Douds (1997), who determined
that appressoria developed on isolated cell wall fragments
from host plants but not from nonhosts. These results
suggest that the signal for appressorium formation is
independent of host exudates or cytoplasmic contents.
From the appressorium, which adheres to the root
surface, branches of the fungal body either directly penetrate root hair or epidermal cells (Fig. 1B) forming
intracellular coils (‘‘Paris’’-type) or invade by way of
intercellular spaces, degrading the middle lamella between
host cells as the hyphae elongate (‘‘Arum’’-type) (Fig. 2A)
(see Smith and Reid, 1997). Eventually, the hyphae
penetrate cortical cells via infection pegs (Pen; Fig. 2A);
VAM fungi have been shown to produce hydrolytic enzymes (Garcia-Romera et al., 1991; Garcia-Garrido et al.,
1992). The internalized hypha then branches repeatedly to
form an arbuscule (Arb; Fig. 2A), which is surrounded by a
peri-arbuscular membrane derived from the host plasma
membrane. Phosphate and fixed carbon molecules are
exchanged at this interface. A phosphate-transporter gene
has been cloned from Glomus versiforme and localized to
the external hyphae (Harrison and van Buuren, 1995). A
hexose transporter from the plant has also been identified
(Harrison, 1996). In addition, Burleigh and Harrison
(1997) have identified genes that are down-regulated by
the interaction between fungus and plant, and these also
appear to be phosphate-regulated. Thus, phosphate status
also serves as a signal for mycorrhizal formation (Menge et
al., 1978; Graham et al., 1981).
LACK OF A HOST-DEFENSE
RESPONSE
The possibility that VAM fungi do not induce typical
defense responses in host plants was suggested previously
(Codignola et al., 1989), but nonetheless, the normal
pathogen-response proteins, such as chitinase and b-1,3glucanase (Spanu et al., 1989; Lambais and Mehdy, 1993),
peroxidase (Spanu and Bonfante-Fasolo, 1988), PR-1a
(Gianinazzi-Pearson et al., 1996), and others, have been
detected in host roots during the early stages of colonization. In alfalfa, genes for enzymes utilized early in the
phenylpropanoid pathway, i.e., phenylalanine ammonia
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Hirsch and Kapulnik
lyase, chalcone synthase, and chalcone isomerase, were
expressed at low levels and transiently, whereas expression
of the gene encoding isoflavone reductase, an enzyme
required later in the pathway for medicarpin biosynthesis,
was completely suppressed (Harrison and Dixon, 1993;
Volpin et al., 1995). This contrasts with the strong and
sustained response that plants exhibit following a pathogen
attack. These findings suggest that mycorrhizal fungi either
do not trigger or are able to suppress certain host-defense
reactions (see Gianinazzi-Pearson et al., 1996; Kapulnik et
al., 1996; Harrison, 1997).
To test this hypothesis, tobacco plants were either left
uninoculated or inoculated with VAM fungi and treated
with chemical activators of host-defense proteins. Expression of genes encoding pathogenesis-related proteins such
as basic chitinase and PR-1a was reduced following Glomus intraradices colonization of the host roots and treatment with the chemical activators 2,6-dichloroisonicotinic
acid and (1,2,3)-thiadiazole-7-carbothioic acid S-methyl
ester compared to uninoculated controls (Ginzberg et al.,
1998; R. David, unpublished results). The suppression of
PR-1a and basic chitinase coincided with a significant
increase in the level of zeatin riboside-like cytokinin
compounds in mycorrhizal roots compared to controls. It is
tempting to speculate that the elevated level of cytokinins
found in mycorrhizal roots (see below) was involved in
suppressing the induction of some PR protein genes.
Supporting this speculation is the observation that direct
application of cytokinins to tobacco leaves resulted in a
reduced accumulation of chitinase and PR-1a proteins
(Ginzberg et al., 1998; O. Shaul-Keinan, Y. Elad, and Y.
Kapulnik, personal communication). Thus, VAM fungi may
be inducing changes in the phytohormone balance of roots,
thereby effecting a down-regulation of defense-related
gene expression (see references in Gianinazzi-Pearson et
al., 1996). Alternatively, the mycorrhizal fungi may be
producing nonhormonal suppressors of the plant-defense
responses.
COMPARISON BETWEEN THE
RHIZOBIUM–LEGUME SYMBIOSIS
AND THE MYCORRHIZAL
ASSOCIATION
A comparison between nitrogen-fixing nodules and
phosphate-acquiring mycorrhizae is presented in Table 1.
Because of many striking similarities between these two
plant–microbe symbioses, including a suppression of host-
209
Signaling in the Mycorrhizal Symbiosis
TABLE 1
Comparison between Nodular and VA/A-Mycorrhizal Symbioses
Nodule
Function
Nitrogen fixation
Specificity
Variable, low
(NGR234) to high
(R. meliloti, R. leguminosarum, etc.);
restricted to
legumes (exception:
Parasponia)
Appears universal;
induction of nod
genes, chemotaxis,
and other functions
Response to flavonoids
Cytological responses
of host
Root hair deformation; cell divisions
in cortical cells to
generate a nodule
Cytological responses
of symbiont
Together with host,
formation of infection thread; differentiation into bacteroids
Yes, for both host and
symbiont
Expression of symbiosis-related genes
VAM/AM
Mainly phosphate
acquisition
Very low; ca. 80% of
angiosperms
(exceptions: Chenopodiaceae, Brassicaceae, Juncaceae,
Cyperaceae, etc.)
May not be universal,
but flavonoids
involved in germination and hyphal
growth
Proliferation of
plasma membrane
around arbuscule;
no cell division or
elongation
Appressorium formation; hyphal proliferation; differentiation into arbuscules
(and vesicles)
Yes, for both host and
symbiont
defense responses in rhizobia–legume interactions (see
McKhann and Hirsch, 1994), LaRue and Weeden (1994)
proposed that nodulation by Rhizobium evolved from the
more ancient VAM symbiosis. The most compelling evidence for this hypothesis is the fact that Nod2 (nonnodulating) legumes are frequently Myc2 (nonmycorrhizal) (Duc
et al., 1989; Bradbury et al., 1991, 1993; Sagan et al., 1995;
T. A. LaRue, J. H. Norris, and A. M. Hirsch, unpublished
results) [there are exceptions, the most notable are certain
2
Nod mutants of soybean and Phaseolus bean, which
establish functional mycorrhizae (Wyss et al., 1990; Shirtliffe and Vessey, 1996)]. Moreover, Nod factor, the rhizobial signal molecule which is a substituted oligomer of
N-acetyl-D-glucosamine residues (see Long, 1996), is related to a class of molecules that are more typical of fungal
cell walls (Bonfante-Fasolo et al., 1990) than of gramnegative bacterial surfaces. Other points of similarity are
that: (1) rhizobial Nod factors stimulated mycorrhizal
formation in both nodulating and nonnodulating soybeans
(Xie et al., 1994) and (2) the early nodulin genes ENOD2
and ENOD40 were expressed in alfalfa mycorrhizae (van
Rhijn et al., 1997). The latter fact strongly suggests that the
signal transduction pathways between the Rhizobium–
legume symbiosis and the mycorrhizal association are
conserved. Indeed, in well-established legume mycorrhizae, genes similar to those encoding late nodulins, such as
leghemoglobin (Frühling et al., 1996) and nodulin 26
(Wyss et al., 1990; Roussel et al., 1997), are expressed.
However, using PCR and Southern hybridization analysis, van Rhijn et al. (1997) could not detect, in DNA
isolated from mycorrhizal fungi, any genes homologous to
nodC, a Rhizobium nodulation gene that exhibits sequence
similarity to a yeast chitin synthase gene (Atkinson and
Long, 1992; Debellé et al., 1992). This finding, and the fact
that numerous nonlegume plants colonized by mycorrhizal
fungi are Myc1 but always Nod2, argues that the interaction between host and fungus may be more complex than
suggested by the simple correlations in gene expression
and appearance described earlier for mycorrhizal interactions and nodulation.
Could the conservation in signal pathways be not at the
ligand–receptor end of the pathway but rather downstream
of signal perception? Perhaps the two different signal
transduction pathways converge on one key intracellular
molecular switch, such as a switch involving the plant
hormones. Plant hormones are involved in various aspects
of the mycorrhizal interaction (see Beyrle, 1995). van Rhijn
et al. (1997) determined that levels of cytokinin, a plant
hormone which induces ENOD2 and ENOD40 gene
expression in legumes (Dehio and deBruijn, 1992; Fang
and Hirsch, 1998), were elevated in mycorrhizal alfalfa
roots. However, as both the fungus and the plant could be
the source of this hormone (see Hirsch et al., 1997), it is
difficult to determine whether a change in cytokinin levels
in mycorrhizal root is a direct or an indirect consequence
of fungal colonization.
CONCLUDING REMARKS
Research thus far suggests that some of the components
of the signaling pathways used in establishing nitrogenfixing nodules or phosphate-acquiring mycorrhizae are
conserved. (1) Although not yet shown to be obligatory,
flavonoids (or other phenolics) are important for the
earliest stages of the interaction between the plant and its
symbiont. (2) Nod factor is known to be a potent signal
molecule for inducing a distinctive developmental program in legumes, and chitin from pathogenic fungi serves
as an elicitor of the hypersensitive response. Do chitinaceous fragments from the walls of VAM fungi serve as
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210
signals for mycorrhizal formation? (3) The product of a
single gene, at least as indicated by legume mutants that
are both Nod2 and Myc2, appears to encode a protein that
perceives both rhizobia (Nod factor) and VAM fungi. Is
this protein a receptor for a chitin-based signal molecule?
Is this potential receptor conserved between the two
symbioses? (4) Both symbiotic interactions are characterized by a weak and transient host-defense response as well
as by an increase in endogenous cytokinin concentrations.
Which downstream components are conserved between
the two symbioses?
Despite these similarities, the vast majority of flowering
plants are Myc1 but yet Nod2—incapable of establishing a
symbiotic association with rhizobia. Why is it that only
legumes nodulate as opposed to all plants colonized by
VAM fungi? Indeed, are nonlegumes truly unresponsive to
Nod factor? Staehelin et al. (1994) have shown that tomato
cells respond not only to chitin but also to Nod factors.
Bono et al. (1995) identified a nonselective Nod factorbinding site (NFBS1) in both Medicago and tomato roots.
Perhaps Myc1 nonlegumes perceive Nod factor molecules
via a generic chitin-binding protein; this hypothesis is
testable.
So far, the two distinct Myc2 phenotypes described,
Myc21 and Myc22, refer to a lack of colonization of the
plant (Myc21) or of arbuscule formation (Myc22) (see
Gianinazzi-Pearson, 1996; Harrison, 1997). Of the two, the
Myc21 phenotype is the more commonly described. Are
there other Myc phenotypes or are these the only two early
points where mycorrhizal formation can be interrupted?
The identification of genes and gene products involved in
the establishment of mycorrhizae will yield definitive
information about signaling in this very important symbiosis and in addition may lead to a greater understanding of
the Rhizobium–legume symbiosis.
ACKNOWLEDGMENTS
We thank Drs. Charles West and Stefan Kirchanski and members of
our laboratories for their helpful comments on the manuscript. Thanks
also to various researchers studying mycorrhizal symbiosis for answering
e-mail requests and sending preprints and reprints. Rakefet David and
Ying Li are gratefully acknowledged for preparing the roots illustrated in
Fig. 1. We also thank Margaret Kowalczyk for preparing the final figures.
This research was supported in part by NSF 96-30842 and 97-23982 to
A.M.H. and by German Israel Foundation Grant G388-199.12/94 to Y.K.
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Hirsch and Kapulnik
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