A Diffusible Factor from Arbuscular Mycorrhizal Fungi

Induces Symbiosis-Specific MtENOD11 Expression in

Roots of Medicago truncatula1

Sonja Kosuta, Mireille Chabaud, Géraldine Lougnon, Clare Gough, Jean Dénarié, David G. Barker, and
Guillaume Bécard*
Equipe Mycologie Végétale, V7R 5546 Centre National de la Recherche Scientifique (CNRS)/Université
Toulouse III, Pôle de Biotechnologie Végétale, 24 chemin de Borde-Rouge, 31326 Castanet-Tolosan cedex,
France (S.K., G.L., G.B.); and Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes,
Institut National de la Recherche Agronomique-CNRS, Boite Postale 27, 31326 Castanet-Tolosan cedex,
France (M.C., C.G., J.D., D.G.B.)

Using dual cultures of arbuscular mycorrhizal (AM) fungi and Medicago truncatula separated by a physical barrier, we
demonstrate that hyphae from germinating spores produce a diffusible factor that is perceived by roots in the absence of
direct physical contact. This AM factor elicits expression of the Nod factor-inducible gene MtENOD11, visualized using a
pMtENOD11-gusA reporter. Transgene induction occurs primarily in the root cortex, with expression stretching from the
zone of root hair emergence to the region of mature root hairs. All AM fungi tested (Gigaspora rosea, Gigaspora gigantea,
Gigaspora margarita, and Glomus intraradices) elicit a similar response, whereas pathogenic fungi such as Phythophthora
medicaginis, Phoma medicaginis var pinodella and Fusarium solani f.sp. phaseoli do not, suggesting that the observed root
response is specific to AM fungi. Finally, pMtENOD11-gusA induction in response to the diffusible AM fungal factor is also
observed with all three M. truncatula Nod⫺/Myc⫺ mutants (dmi1, dmi2, and dmi3), whereas the same mutants are blocked
in their response to Nod factor. This positive response of the Nod⫺/Myc⫺ mutants to the diffusible AM fungal factor and
the different cellular localization of pMtENOD11-gusA expression in response to Nod factor versus AM factor suggest that
signal transduction occurs via different pathways and that expression of MtENOD11 is differently regulated by the two
diffusible factors.

Arbuscular mycorrhizal (AM) fungi have existed in
symbiosis with plant roots for over 460 million years,
since the appearance of the earliest land plants
(Remy et al., 1994). This group of fungi, recently
renamed Glomeromycota (Schüssler et al., 2001), is
one of the most widely distributed; 95% of present-
day plant species belong to families that are charac-
teristically mycorrhizal (Smith and Read, 1997). AM
fungi are able to transfer rare or poorly soluble nu-
trients such as phosphorous, copper, and zinc from
the soil to the plant, which in turn provides carbo-
hydrates to the fungus. This nutrient exchange may
be of critical importance when soil fertility and water
availability are low, conditions that severely limit
agricultural production in most parts of the world.
Although AM fungi are both agriculturally and eco-
1
This work was supported by the French Ministry of National
Education, Research, and Technology (IFR40 grant “Root Endo-
symbioses” to D.G.B., G.B., and J.D., 2000/2001), by the Region
Midi-Pyrénées (grant no. 990 090 70 to D.G.B., G.B., and J.D.), and
by the Quebec Fonds pour la Formation de Chercheurs et l’Aide à
la Recherche (doctoral scholarship to S.K.).
* Corresponding author; e-mail becard@smcv.ups-tlse.fr; fax
33–5– 62–19 –35– 02.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.011882.
952 Plant Physiology, March 2003, Vol. 131, pp. 952–962, www.plantphysiol.org © 2003 American Society of Plant Biologists
logically important, very little is known about the
cellular and molecular events that occur during es-
tablishment of the association, and in particular
events that play a role in signaling and recognition of
both symbiotic partners.
Before infection, AM fungi recognize and respond
to their potential hosts. Compounds constitutively
secreted by the roots of host plants, but not non-host
plants, stimulate ramifications in hyphae from ger-
minating spores of Gigaspora and Glomus spp. (Mosse
and Hepper, 1995; Giovannetti et al., 1993b; Buée et
al., 2000). These morphological changes increase the
possibility of contact between hyphae and host roots,
but also signal a physiological “switch” to active
presymbiotic fungal growth without which hyphal
attachment and appressorium formation may not oc-
cur (Giovannetti et al., 1994). Upon contact, the topo-
graphical and/or biochemical properties of host root
epidermal cell walls induce the formation of AM
fungal appressoria (Giovannetti et al., 1993a; Naga-
hashi and Douds, 1997). Although rapid stimulation
of spore germination, hyphal growth, and appresso-
rium formation by host-roots has obvious advantages
for the survival of the obligately symbiotic AM fun-
gus, no evidence to date indicates plant recognition
of the fungus before contact, nor the existence of
fungal signals before root penetration.

Gene expression studies indicate an active plant
response to the AM fungus during the earliest stages
of hyphal penetration. Studies using reverse
transcriptase-PCR and northern analyses in pea (Pi-
sum sativum) suggest that induction of PsENOD12A
and Psam5 is concurrent with appressorium forma-
tion and hyphal proliferation in the cortex (Albrecht
et al., 1998; Roussel et al., 2001). Use of gene-
promoter ␤-glucuronidase (GUS) fusions in rice
(Oryza sativa) has revealed that expression of the
lipid transferase protein (Ltp) gene in epidermal cells
is associated with appressorium formation (Blilou et
al., 2000). By a similar approach, Chabaud et al.
(2002) have recently shown that the Medicago trunca-
tula ENOD11 gene is transcriptionally activated in
epidermal and cortical cells containing penetration
hyphae during infection by Gigaspora rosea. This early
nodulin gene is also expressed in M. truncatula epi-
dermal cells in response to purified Nod factors,
during infection of the root by Sinorhizobium meliloti,
and in arbuscule-containing cells in mycorrhizal
roots (Journet et al., 2001). Thus, in AM infection, as
in root infection by Rhizobium sp. bacteria, the host
plant actively and specifically responds to penetra-
tion of host root cells.
Mycorrhization and nodulation are very different
processes, involving unrelated microbial symbionts,
and giving rise to very different physiological struc-
tures in the host plant root. Nonetheless, the estab-
lishment of these two root symbioses appears to in-
volve a number of related plant responses, including
the expression of common plant genes (for refer-
ences, see Gianinazzi-Pearson and Dénarié, 1997;
Hirsch and Kapulnik, 1998; Harrison, 1999). The ge-
netic evidence is the most striking: nodulation-
defective (Nod⫺) mutants that are also non-
mycorrhizal (Myc⫺) have been found in pea (Duc et
al., 1989), alfalfa (Medicago sativa; Bradbury et al.,
1991), M. truncatula (Sagan et al., 1995; Catoira et al.,
2000), bean (Phaseolus vulgaris; Shirtliffe and Vessey,
1996), and Lotus japonicus (Wegel et al., 1998; Bon-
fante et al., 2000). The recent characterization of sev-
eral M. truncatula, L. japonicus, and pea Nod⫺/Myc⫺
mutants blocked for very early steps in Nod factor
signal transduction and also required for the estab-
lishment of arbuscular mycorrhizas, indicate that cer-
tain elements of signal transduction are common to
both symbiotic interactions (Catoira et al., 2000;
Walker et al., 2000; Stracke et al., 2002).
These analogies have led to the suggestion that
so-called “Myc factors” may be produced by AM
fungi, acting as fungal signals recognized by host
roots and necessary for the establishment of a suc-
cessful mycorrhizal association. Although many cel-
lular, molecular, and developmental root responses
to Nod factors have been characterized in several
legume systems, no such responses have been de-
scribed preceding infection of plant roots by AM
fungi. This is probably because of the difficulty in
Plant Physiol. Vol. 131, 2003
Diffusible AM Fungal Factor
studying the early steps of AM fungal symbiosis, and
the non-synchronous nature of AM infection.
Chabaud et al. (2002) recently developed an in vitro
technique for studying the early stages of AM fungal
infection using Agrobacterium rhizogenes-transformed
roots of M. truncatula carrying a chimeric gusA gene
fusion under the control of the MtENOD11 promoter.
The authors observed that during early stages of the
interaction between M. truncatula Ri T-DNA-
transformed roots and G. rosea, strong cortical
pMtENOD11-gusA expression was often observed in
noninfected roots in the vicinity of fungal-root con-
tacts. This suggested that pMtENOD11-gusA gene
expression might be induced in M. truncatula roots by
G. rosea without direct contact. To test this hypothe-
sis, we have developed a coculture system, also ap-
plicable to seedlings, where roots and fungi are sep-
arated by a membrane barrier. In this way, we have
been able to demonstrate unequivocally that germi-
nated spores of AM fungi synthesize a diffusible
factor capable of triggering the expression of an early
nodulin gene in host roots in the absence of fungal-
plant contact.
RESULTS
Membrane-Separated M. truncatula/G. rosea Coculture
To test whether a fungal diffusible factor was re-
sponsible for inducing pMtENOD11-gusA expres-
sion, we inserted a cellophane membrane between
the fungus and the Ri T-DNA-transformed roots
from the start of coculture (Fig. 1a). In this experi-
mental system, the two symbiotic partners are able to
grow in close proximity (⬍1 mm), thus facilitating
the exchange of pre-infection signals. Furthermore,
the transparency of the cellophane membrane used
made it possible to follow fungal growth and mor-
phology, which were essential parameters in defin-
ing the bio-assay conditions. Germination of G. rosea
spores occurred after 4 to 7 d and was followed 2 d
later by the appearance of the hyphal ramifications
characteristically produced by AM fungi in response
to diffusible root factors (Figs. 1c and 2a). Histochem-
ical GUS staining, carried out after 7 to 14 d of cocul-
ture, systematically revealed strong pMtENOD11-
gusA transcriptional activation in numerous root
laterals, with staining initiating just behind the root tip
and extending some distance up the root (Fig. 2, a and
b). This expression pattern was almost exclusively
observed in the immediate proximity of the develop-
ing fungus. In control roots, grown without fungus
but with the cellophane membrane, pMtENOD11-
gusA expression was generally only observed at three
locations in the root (root caps, the base of root
laterals, and occasionally in vascular tissues: Fig. 2, c
and e), corresponding to the non-symbiotic “consti-
tutive” expression of MtENOD11 in roots of intact
plants (Journet et al., 2001).
953
Kosuta et al.
Figure 1. Schematic representation of the membrane-separated coculture of AM fungi and M. truncatula. a, Excised (Ri
T-DNA-transformed) roots; b, seedlings. c, Detail of the corridor containing dense hyphal growth and ramifications, in which
MtENOD11 induction occurs. 1, Spores; 2, location of membrane; 3, primary root; 4, secondary root; 5, tertiary root; 6,
negatively geotropic germ tubes; 7, corridor containing hyphal ramifications.
To verify that the observed reporter gene expres-
sion is also valid for whole plants, we developed an
axenic petri dish experimental system for AM colo-
nization of M. truncatula seedlings. In this whole-
plant system (see “Materials and Methods”), spore
germination occurred after 5 to 7 d of coculture,
when seedling roots passed close to spores. Fungal
hyphae generally made contact with roots 2 to 3 d
later. After 1 month of coculture, daughter spores
were observed, indicating that a viable symbiosis had
formed between the AM fungus and the host M.
truncatula plant. We then applied the membrane-
separated approach to germinating spores and trans-
genic pMtENOD11-gusA seedlings (Fig. 1b). After 10
to 14 d of coculture, GUS expression was observed in
the cortex, epidermis, endodermis, pericycle, and
vascular tissues of secondary roots growing in close
proximity to AM fungal germ tubes and hyphal ram-
ifications (Fig. 2i), whereas in control roots, only
constitutive expression was seen in vascular tissues
(Fig. 2j), sites of root lateral initiation, and root tips
(not shown) as described above.
A Diffusible Fungal Factor Induces
pMtENOD11-gusA Expression
To confirm that the root response we have ob-
served is attributable to a diffusible factor, we veri-
fied by several methods that the fungus had not
traversed the membrane barrier. First, careful and
extensive microscopic examination of all roots
stained with Chlorazol Black E failed to detect fungal
structures either at the root surface or within roots
(data not shown). Second, diagnostic PCR using
fungal-specific primers (see “Materials and Meth-
ods”) revealed no fungal signals in DNA samples
extracted either from control root cultures or roots
recovered 10 d after coculture with membrane-
954
separated fungus (Fig. 3). On the other hand, DNA
extracted from both G. rosea spores and colonized
roots gave a positive PCR signal. Third, replacing
the cellophane membrane with a polycarbonate
membrane (0.6-␮m pore size) resulted in the same
pMtENOD11-gusA expression pattern. Finally, the
addition of a second cellophane membrane, increas-
ing the physical distance between the fungus and the
root to approximately 3 mm, did not prevent the
characteristic reporter gene expression. Although
the appearance of hyphal ramifications was delayed
by 24 h in the double membrane tests, the number of
responding roots and the intensity of GUS staining
was not different from single-membrane experiments
(data not shown). In addition, replacing the cello-
phane membrane by a dialysis membrane (3.5 kD
molecular mass cut-off) also permitted pMtENOD11-
gusA induction. Thus, we conclude that an AM fun-
gal factor, possibly less than 3.5 kD in size and capa-
ble of diffusing across a variety of membrane
barriers, is capable of eliciting early nodulin gene
expression in host roots.
Localization of Diffusible AM Factor-Elicited
MtENOD11 Expression
By staining for GUS activity directly in the petri
dish containing the membrane-separated coculture,
we were able to show that pMtENOD11-gusA induc-
tion occurred mainly in the proximity of the advanc-
ing fungal germ tubes and hyphal ramifications (Fig.
2a; for schematic localization, see Fig. 1a). Within
a “corridor” containing all fungal germ tubes and
hyphal ramifications (Fig. 1c), pMtENOD11-gusA
induction was seen in approximately one-quarter
(25% ⫾ 4%) of the tertiary roots present, whereas in
control roots only 5% ⫾ 2% of the tertiary roots
showed expression of the transgene (Table I).
Plant Physiol. Vol. 131, 2003

MtENOD11 expression was also induced to a lesser
extent in secondary roots (7% ⫾ 2% compared with
0% ⫾ 0% in controls). Primary roots never showed
transgene induction. GUS activity was limited to
roots growing within the culture medium, because
expression of the gusA reporter was never observed
in aerial roots. Microscopic examination of stained
roots revealed that pMtENOD11-gusA activity initi-
ated 0.1 to 0.7 cm behind the root cap and continued
up to 1.5 cm from the root cap (Fig. 2d). This com-
prises the zone of root hair emergence, root hair
development, and part of the region of mature root
hairs. Semithin sections of stained root segments
showed that in roots exhibiting strong GUS activity,
staining was observed in the vast majority of cortical
cells and often in the root epidermis (including root
hairs), the root endodermis, and vascular tissue (Fig. 2,
f and g). In weaker-stained roots, GUS expression was
primarily seen in the cortex and was often limited to
isolated cells or small groups of cells in one or all of
the root tissues mentioned above (data not shown).
Plant Physiol. Vol. 131, 2003
Diffusible AM Fungal Factor
Figure 2. Root pMtENOD11-gusA induction in
membrane-separated coculture. a, b, d, f, and g, Ri
T-DNA-transformed roots membrane-separated
from G. rosea. c, e, and h, Control roots cultured
with membrane separation but without fungus. a,
GUS activity colocalizes with hyphal ramifica-
tions on other side of membrane (white arrow-
heads), bar ⫽ 0.2 cm. b, MtENOD11 induction
occurs mainly in tertiary Ri T-DNA-transformed
roots; c, only constitutive expression (see also e) is
observed in control roots, bars ⫽ 2 cm. d, AM
factor-induced GUS activity stretches from 0.1 cm
behind the root cap as far as the zone of mature
root hair growth, bar ⫽ 0.2 cm; e, in control roots,
only constitutive expression is present in root caps
and at the base of lateral roots, bar ⫽ 0.1 cm. f,
Detail of pMtENOD11-gusA induction in epider-
mal cells including root hairs, bar ⫽ 50 ␮m. g and
h, Seventy-five-micrometer-thick transverse sec-
tions of AM-inoculated (g) and control (h) roots,
bars ⫽ 150 ␮m. i, Membrane-separated coculture
of G. rosea with whole plants; j, control plants. i,
pMtENOD11-gusA is induced in young lateral
roots, bar ⫽ 0.3 cm; j, only background GUS
activity is seen in vascular tissues in control roots,
bar ⫽ 0.3 cm. k and l, pMtENOD11-gusA induc-
tion in membrane-separated coculture with other
fungi. Induction occurs in Ri T-DNA-transformed
roots cocultured with G. margarita (k), but not
with Phy. medicaginis (l), bars ⫽ 0.6 cm. m,
pMtENOD11-gusA induction in membrane-
separated coculture of G. rosea and with Ri
T-DNA-transformed roots derived from the M.
truncatula Myc⫺ mutant dmi2-2, bar ⫽ 0.7 cm.
All AM Fungi Tested Produce a Diffusible Factor
Capable of Inducing MtENOD11 Expression
All of the AM fungi tested (G. rosea, Gigaspora mar-
garita, Gigaspora gigantea and Glomus intraradices; Table
Table I. pMtENOD11-gusA induction
Percentage of Ri T-DNA-transformed roots of M. truncatula ex-
pressing pMtENOD11-gusA after 10 d of membrane-separated co-
culture with G. rosea (⫹AM) and without (⫺AM). Roots were
counted in the “corridor” of dense fungal growth and hyphal rami-
fications. Values listed are means ⫾ SE. Data presented are from a
single representative experiment. Similar results were obtained in at
least five independent experiments. Data in each column followed
by a different letter (a or b) are significantly different according to
Student’s t test (P ⬍ 0.05, n ⫽ 12).
Percentage of MtENOD11-Expressing Roots
Secondary lateral roots Tertiary lateral roots
⫺AM 0 ⫾ 0a 5 ⫾ 2a
⫹AM 7 ⫾ 2b 25 ⫾ 4b
955
Kosuta et al.
Figure 3. PCR analysis of potential fungal contamination. Products
from plant and fungal DNA amplified with universal primers ITS1/4
(odd lanes) and fungal specific primers ITS1F/4 (even lanes). Lanes 1
and 2, DNA of M. truncatula control roots; Lanes 3 and 4, DNA from
roots harvested from a membrane-separated coculture of M. trunca-
tula and G. rosea at 10 dai; Lanes 5 and 6, DNA of M. truncatula
roots colonized by G. rosea; Lanes 7 and 8, DNA from G. rosea
spores; and Lanes 9 and 10, water control. Note that the band (550
bp) amplified by fungal primers in spores (lane 8) and colonized roots
(lane 6; arrowheads), could not be detected in roots separated from
AM fungi by a membrane (lane 4). Two faint non-specific bands (600
and 680 bp) can be seen in lanes 2 and 4 corresponding to root DNA.
II) produced diffusible factors with pMtENOD11-
gusA-inducing activity, although reporter gene ex-
pression varied in intensity between fungi (data not
shown). Under our membrane-separated coculture
conditions, pMtENOD11-gusA expression was stron-
gest and most uniform in response to G. rosea and G.
gigantea. Per equivalent fungal inoculum, G. margarita
elicited weaker and more localized reporter gene
expression (Fig. 2k), and G. intraradices elicited even
weaker gene expression. We therefore conclude that
the capacity to produce this diffusible factor may be
common to all AM fungi tested, irrespective of phy-
logenetic diversity.
The MtENOD11-Activating Factor Is Specific to
AM Fungi
Non-mycorrhizal fungi were also tested to see
whether the response we have observed is specific to
AM fungi or whether it might correspond to a gen-
eral stress response to fungi. The three M. truncatula
root pathogens tested (F. solani f.sp. phaseoli, Phoma
medicaginis var pinodella, and Phythophthora medicagi-
nis; Table I) were first tested for their effect on Ri
T-DNA-transformed roots under our in vitro growth
conditions. Inoculation of root explants with all three
fungi resulted in negative growth effects within 4 d
after inoculation (dai). For F. solani var phaseoli and
Phy. medicaginis, root growth ceased at approxi-
mately 8 dai, whereas Pho. medicaginis var pinodella
was less aggressive, with growth of infected roots
continuing, despite signs of necrosis, until at least 20
dai. We also tested pathogenicity on seedlings, where
Suc was absent from the medium. After inoculation
of M. truncatula seedlings with F. solani var phaseoli
956
and Phy. medicaginis, the first visible signs of patho-
genicity (necrosis of root tissue in the primary root)
were visible 2 to 3 dai, with plant death occurring 10
to 14 dai. Again, Pho. medicaginis var pinodella was
less aggressive, with the plant surviving for at least 3
weeks with only moderate signs of necrosis. To cover
the entire infection period, histochemical staining for
GUS activity was therefore performed at 3, 6, and 10
dai. Our experiments failed to reveal any cortical or
epidermal pMtENOD11-gusA expression in either Ri
T-DNA-transformed root cultures or seedlings inoc-
ulated with any of the three pathogenic fungi.
Finally, we investigated whether reporter gene ex-
pression could be induced in either root organ cul-
tures or seedlings separated from pathogenic fungi
by a cellophane membrane. F. solani var phaseoli and
Phy. medicaginis developed rapidly and digested the
cellophane membrane within 4 dai, leading to infec-
tion of the host roots. Pho. medicaginis var pinodella
had not crossed the membrane barrier by 10 dai but
nonetheless elicited some necrosis in roots. Despite
this, pMtENOD11-gusA expression was never ob-
served in roots confronted with pathogens across a
membrane, stained for GUS activity at 3, 6, and 10 dai
(Fig. 2l). The absence of pMtENOD11-gusA induction
in interactions with three different pathogenic fungi
strongly suggests that the diffusible factor is specific
to AM fungi.
M. truncatula Nodⴚ/Mycⴚ dmi Mutants Also
Respond to the Diffusible AM Factor
Mutations in the three DMI genes of M. truncatula
result in Nod⫺/Myc⫺ phenotypes and correspond-
ing mutants are blocked at early stages of a Nod
factor signal transduction pathway (Catoira et al.,
2000). To evaluate possible mechanistic parallels be-
tween the Rhizobium sp. Nod factor and the diffusible
AM factor, we established pMtENOD11-gusA root
cultures derived from three representative dmi mu-
tants (dmi1-1, dmi2-2, and dmi3-1; Table II). After
several weeks of coculture with G. intraradices, no
new spores were formed, and in all cases no fungal
growth was visible after 14 d (results not shown). In
addition, microscopic observations of Chlorazol
Black-stained roots from 11-d-old cocultures with G.
rosea revealed that the fungus was blocked at the
stage of appressorium formation, and no hyphal pen-
etration had occurred. Despite their defective mycor-
rhization phenotype, these root cultures still produce
the factors that stimulate AM hyphal ramification.
Using the membrane barrier approach described ear-
lier, Ri T-DNA-transformed root cultures of dmi1,
dmi2, and dmi3 mutants were tested for their capacity
to respond to the diffusible AM factor produced by
G. rosea. Expression of pMtENOD11-gusA was in-
duced in dmi1, dmi2, and dmi3 mutant root cultures
with the same histological localization, intensity, and
timing as wild-type M. truncatula roots (Fig. 2m).
Plant Physiol. Vol. 131, 2003
 Diffusible AM Fungal Factor

Table II. Fungal isolates, plants, and Ri T-DNA-transformed roots used in this study
Designation Relevant Characteristics Reference/Source

Fungal isolates
G. gigantea AM fungus R. Koske (University of Rhode Island, RI)
G. rosea (BEG 9) AM fungus Biorhize (Dijon, France)
G. margarita (BEG 34) AM fungus Biorhize
G. intraradices (DAOM 197198) AM fungus Chabot et al. (1992)
Fusarium solani f.sp. phaseoli (CBS 190 –35) Deuteromycota, aggressive pathogen CBSnr (Utrecht, The Netherlands)
Phoma medicaginis var. pinodella (CBS 110 –32) Deuteromycota, non-aggressive pathogen CBSnr
Phytophthora medicaginis (M2019) Oomycota, aggressive pathogen Silo-Suh et al. (1994)
Plants
M. truncatula
Jemalong (A17) Wild type, Nod⫹/Myc⫹ Penmetsa and Cook (1997)
Jemalong (L416) Transgenic wild type with a single-copy Journet et al. (2001)
fusion between the MtENOD11
promoter and thegusA gene
C71 (dmi1-1) ⫻ L416 Transgenic Nod⫺/Myc⫺ mutant Catoira et al. (2000)
TR26 (dmi2-2 ) ⫻ L416 Transgenic Nod⫺/Myc⫺ mutant Vernoud et al. (2000)
TRV25 (dmi3-1) ⫻ L416 Transgenic Nod⫺/Myc⫺ mutant E.-P. Journet (unpublished data)
Ri T-DNA-transformed roots
L416-Ar4 Derived from L416 Chabaud et al. (2002)
C71 ⫻ L416-Ar1 Derived from dmi1/L416 This study
TR26 ⫻ L416-Ar8 Derived from dmi2/L416 Chabaud et al. (2002)
TRV25 ⫻ L416-Ar2 Derived from dmi3/L416 This study

Membrane-separated coculture with G. rosea led to
statistically significant (P ⬍ 0.001) pMtENOD11-gusA
induction compared with control roots (Fig. 4). The
percentage of roots showing GUS activity varied
somewhat between mutants. In particular, dmi3 roots
showed less GUS activity than wild-type and other
dmi mutant roots in response to the diffusible AM
factor, however, differences were not statistically
significant (P ⫽ 0.20). Thus, mutations in the dmi1,
dmi2, and dmi3 genes apparently do not alter the
pMtENOD11-gusA induction response to the AM
factor.
DISCUSSION
Evidence for a Diffusible Factor Produced by
AM Fungi

We demonstrate that pMtENOD11-gusA expres-

Figure 4. pMtENOD11-gusA induction in M. truncatula Nod⫺/Myc⫺
dmi mutants. Percentage of Ri T-DNA-transformed roots of M. trun-
catula wild-type and dmi mutants that show induction of
pMtENOD11-gusA after 10 d of membrane-separated coculture with
G. rosea (⫹AM) and without (⫺AM). Roots were counted in the
“corridor” of dense fungal growth and hyphal ramifications. Data
from three independent experiments were pooled and treated as
subsets (blocks) of a single data set for statistical analysis. Means ⫾
SE labeled with a different letter (a and b) are significantly different
according to analysis of variance followed by Tukey’s test (P ⬍ 0.05,
n ⫽ 7).
Plant Physiol. Vol. 131, 2003
sion is induced in M. truncatula roots by AM fungi in
cocultures separated by a variety of membrane bar-
riers. We have verified that in our experiments, fun-
gal hyphae did not cross the membrane both by
microscopic examination of stained roots and PCR
analysis using fungus-specific primers. Furthermore,
the addition of a second membrane barrier, increas-
ing the distance between the fungus and the roots to
at least 3 mm, did not alter induced pMtENOD11-
gusA reporter gene expression in roots. We therefore
conclude that a diffusible factor produced by AM
fungi induces pMtENOD11-gusA expression in M.
truncatula roots.
In these experiments, we have predominantly
made use of Ri T-DNA-transformed roots expressing
a pMtENOD11-gusA reporter as a convenient exper-
imental tool. Such root cultures have been validated
for studies of plant/AM fungal associations in the
case of carrot (Daucus carota), pea, and M. truncatula
(Bécard and Fortin, 1988; Balagi et al., 1994; Boisson-
Dernier et al., 2001; Chabaud et al., 2002). Further-
more, Ri T-DNA-transformed roots derived from
penetration mutants exhibit the same phenotype as
957
Kosuta et al.
the intact plant for all legume mutants tested so far
(this study; Balagi et al., 1994; Chabaud et al., 2002).
Despite this, we have also verified that induction of
pMtENOD11-gusA expression also occurs in the same
root tissues of whole plants in response to the diffus-
ible AM factor.
All of the AM fungi tested elicited similar spatio-
temporal expression of the pMtENOD11-gusA re-
porter. However, the intensity of GUS expression
varied with fungal species. It is therefore possible
that the quantity of diffusible AM factor differs be-
tween AM fungi or that factors from different fungi
may have different gene-inducing activities or differ-
ent diffusion rates/stabilities. By staining for GUS
activity directly in the petri dish containing the co-
culture, we were able to conclude that pMtENOD11-
gusA-expressing roots were always in the vicinity of
fungal germ tubes and highly ramified hyphal struc-
tures on the other side of the membrane, whereas
reporter gene activity was not observed in roots dis-
tant from the fungus. Our observations are therefore
consistent with the hypothesis of a slowly diffusible
or relatively unstable factor. In addition, our results
with dialysis membranes suggest that the factor may
have a low molecular mass (⬍3.5 kD). Nevertheless,
because the pathogenic fungi efficiently digested the
cellulose membranes, we cannot exclude the possi-
bility that the AM fungi, in contact with the mem-
brane for several days, might alter its cellulose struc-
ture and hence its molecular cut-off.
Significance of MtENOD11 Expression in
Response to the Diffusible AM Factor
Similar results were obtained with all four AM
fungal species tested, including the phylogenetically
distant G. intraradices, implying that diverse AM
fungi produce a diffusible factor that can be per-
ceived by host roots without direct physical contact
between the two organisms. On the other hand,
pMtENOD11-gusA induction was not observed in
interactions with fungal pathogens (aggressive and
nonaggressive) in our study or in interactions with
the biotrophic root fungus Rhizoctonia sp. (Journet et
al., 2001). Because infection with aggressive fungal
pathogens alters root growth and therefore may alter
gene expression, we cannot directly interpret the ab-
sence of pMtENOD11-gusA induction in response to
these fungi. However, our results with a range of
fungal pathogens suggest that the pMtENOD11-gusA
induction we observe is specific to AM fungi. Com-
parison with biotrophic fungi in future may be use-
ful, because these fungi can enter host root tissues
without negatively affecting root growth, and there-
fore may be more appropriate controls for AM fungal
studies.
Diffusible AM factor-dependent induction of
pMtENOD11-gusA occurred mainly in root laterals,
the preferred site for AM colonization both for whole
958
plants and in vitro root cultures (Mosse and Hepper,
1995; Chabaud et al., 2002). However, within the
zone perceiving the AM factor, certain roots re-
sponded more strongly than others, and some roots
not at all. We do not yet understand the reason for
this variability. It is possible that phytohormonal
balance or some other internal regulatory mechanism
may control the susceptibility of a given root to the
AM factor, as has been suggested for ectomycorrhizal
fungal and Rhizobium sp. bacterial infection (Smith
and Read, 1997; Mathesius et al., 2000). In the absence
of a membrane, appressoria were often observed on
roots showing strong cortical pMtENOD11-gusA ex-
pression (data not shown), suggesting a correlation
between the gene expression pattern that we ob-
served and the subsequent passage of the fungus
through these tissues. However, a direct link be-
tween cortical pMtENOD11-gusA expression and
subsequent appressorium formation and AM root
infection remains to be established. Finally, it should
be underlined that the expression of pMtENOD11-
gusA in response to the diffusible AM factor differs
significantly from that elicited at a later stage during
fungal root infection (Chabaud et al., 2002). In the
latter case, expression is strictly limited to individual
epidermal and cortical cells penetrated by hyphae,
and furthermore no expression is observed with
infection-defective Nod⫺/Myc⫺ mutants.
The physiological function of the MtENOD11 gene
product is so far unknown, but based on its nucleo-
tide sequence, MtENOD11 is predicted to encode an
extracellular repetitive Pro-rich protein with low
overall Tyr content. A possible role for MtENOD11 in
the construction of a cell wall with reduced cross-
linking and thus higher plasticity and/or matrix po-
rosity has been suggested (Journet et al., 2001). So far,
no major alterations in cell size or shape have been
observed in pMtENOD11-gusA-expressing cells in
semithin sections, but a more detailed analysis is
clearly required to evaluate whether these cells are
modified in their cellular morphology. Some other
ENOD genes have been found to be induced during
mycorrhizal symbiosis, including Psam5, which is
expressed very early in AM fungal interactions with
both Myc⫹ and Myc⫺ mutants of pea (Roussel et al.,
2001). However, little is known about the products of
these genes or whether they are also expressed in
non-legumes during mycorrhization. Further spatio-
temporal expression studies of known genes and the
identification of novel genes induced by the diffus-
ible AM factor should help toward understanding
the physiological significance of both pMtENOD11-
gusA induction and the activity of the diffusible fac-
tor produced by AM fungi.
Is the Diffusible AM Factor a Symbiotic Signal?
When roots and AM fungus are separated by a
membrane, pMtENOD11-gusA induction correlated
Plant Physiol. Vol. 131, 2003

both spatially and temporally with the appearance of
the hyphal ramifications that indicate that the fungus
has “recognized” its host (Giovannetti et al., 1994).
No transgene expression was observed when hyphal
ramifications were not already present. Thus, a cru-
cial step of fungal-host recognition may be required
for synthesis of the diffusible AM factor, suggesting
that, in the same way that legume root flavonoids
activate nodulation genes in rhizobia, host root com-
pounds may activate mycorrhization genes in AM
fungi. Overall, our results suggest that a molecular
dialogue takes place between germinating AM fungi
and their potential host roots.
Analysis of legume Nod⫺/Myc⫺ mutants blocked
for Nod factor signal transduction has led several
authors to suggest an analogous signaling mecha-
nism in the AM fungal and rhizobial symbiosis, in-
cluding a putative “Myc” factor equivalent to Nod
factor (LaRue and Weeden, 1994; Albrecht et al.,
1998; Catoira et al., 2000; Walker et al., 2000; Stracke
et al., 2002). As the chitin backbone of the Nod factor
molecule is more typical of fungi than bacteria, the
diffusible AM factor could indeed be a Nod factor-
like molecule or simply chitin oligomers. It has been
shown that such chito-oligosaccharides can induce
rapid alkalinization of tomato cell suspension cul-
tures (Felix et al., 1993), transient GmENOD40 induc-
tion in soybean (Glycine max; Minami et al., 1996),
and calcium spiking in pea (Walker et al., 2000).
However, in this case, we would hypothesize that the
chitin oligomers released by AM fungi during sym-
biotic infection are not the same as chitin fragments
derived from plant chitinase-degradation of the fun-
gal pathogens Pho. medicaginis var pinodella and F.
solani.
Evidence for a DMI-Independent Symbiotic
Transduction Pathway
Our study has revealed some important differences
between responses induced by Nod factors and the
diffusible AM fungal factor that must be considered.
MtENOD11 induction by Nod factor and diffusible
AM factor is not identical; pMtENOD11-gusA is ex-
pressed mainly in epidermal cells in response to Nod
factor (nod-ENOD11 response), whereas the AM fac-
tor induces expression of the same gene most
strongly in the cortex of a larger region of the root
(myc-ENOD11 response). This indicates that, al-
though the same gene responds to a diffusible signal
from both microsymbionts, spatial and temporal
gene expression is probably regulated differently in
the two symbioses. In addition, the M. truncatula dmi
mutants are totally blocked for certain Nod factor
responses, in particular for epidermal MtENOD11
induction (Catoira et al., 2000). In our study, dmi1,
dmi2, and dmi3 all responded positively to the diffus-
ible AM factor, indicating that the DMI gene prod-
ucts are not required for the AM factor-induced
Plant Physiol. Vol. 131, 2003
Diffusible AM Fungal Factor
ENOD11 expression that we have revealed. The dif-
fusible AM factor is thus likely to activate this re-
sponse by a signal transduction pathway indepen-
dent of the DMI genes. This pathway may be specific
to the AM factor, i.e. not activated by Nod factor
signaling.
Finally, is our diffusible AM factor the “Myc fac-
tor” proposed to activate the DMI cascade in M.
truncatula (Catoira et al., 2000)? Our diffusible AM
fungal factor may activate only the pathway leading
to myc-ENOD11 induction, whereas a different fac-
tor, the Myc factor, would activate the DMI path-
way. As an alternative, we cannot rule out the hy-
pothesis that the AM factor activates steps in the
DMI pathway that are common to both Nod and
Myc signaling in addition to the pathway leading to
myc-ENOD11 induction. Whether or not our diffus-
ible factor is the Myc factor, the signaling pathway
that we have revealed might temporally precede
DMI-dependent fungal penetration of root tissues.
This would be consistent with the fact the dmi Myc⫺
phenotype is blocked at appressorium formation
and requires physical contact between plant and
fungus, whereas our myc-ENOD11 induction occurs
in the absence of contact. Further studies are clearly
now needed to distinguish between these alterna-
tives and to firmly establish the role of this diffus-
ible factor in the plant/AM fungal symbiotic
dialogue.
CONCLUSIONS
This paper describes a novel system developed for
the study of early signaling in AM fungal-root in-
teractions. Our membrane-separated coculture ap-
proach provides the first evidence that AM fungi
produce a diffusible symbiotic factor. This factor
seems to be specific to AM fungi, and thus poten-
tially important in a molecular dialogue associated
with symbiotic infection. The transgenic root/
reporter gene strategy that we have used to detect
the plant response to the fungal factor, combined
with our novel technique for separating the AM
fungus from its host in axenic culture, should now
provide the essential tools for the isolation and char-
acterization of AM fungal diffusible factors. Recent
breakthroughs in the cloning of receptor-like kinase
genes (MtDMI2 and orthologous genes in alfalfa, L.
japonicus, Melilotus alba, and pea) involved in Nod/
Myc signaling (Endre et al., 2002; Stracke et al.,
2002) now open the way to the molecular analysis of
early recognition and transduction of Nod/Myc fac-
tor signals. In the foreseeable future, we can expect
rapid advances in our understanding of early sig-
naling events in AM infection and the mechanisms
underlying the signaling pathways leading to
mycorrhization.
959
Kosuta et al.
MATERIALS AND METHODS
Plant Material
Mycorrhizal interactions were studied in vitro using the Medicago trun-
catula Gaertn. excised roots (Ri T-DNA-transformed) and whole plants listed
in Table II. Ri T-DNA-transformed roots derived from the Nod⫹/Myc⫹ M.
truncatula transgenic line expressing the pMtENOD11-gusA fusion (L416)
and the Nod⫺/Myc⫺ mutant TR26 (dmi2-2) expressing the same fusion have
been described previously (Chabaud et al., 2002). Analogous transformed
root lines were obtained following Agrobacterium rhizogenes (isolate ARqua1-
A4T) transformation (Boisson-Dernier et al., 2001) of C71 (dmi1-1) and
TRV25 (dmi3-1) Nod⫺/Myc⫺ mutant plants into which the pMtENOD11-
gusA fusion had been introduced by genetic crossing (kindly provided by
E.-P. Journet [IPM, Toulouse, France]). Before use, we confirmed that root
clones possessed a normal non-symbiotic pENOD11-gusA expression pat-
tern by staining for GUS (see below). Their Myc⫺ phenotype (absence of AM
infection and fungal sporulation) was confirmed by staining roots for the
presence of intraradical colonization (see below) 11 d after inoculation with
G. rosea, and by examining fungal growth 6 weeks after inoculation and
coculture with G. intraradices. By that time, dual culture with wild-type M.
truncatula root cultures resulted in numerous mycorrhizal infection sites and
de novo spore production (data not shown). All transformed roots were
cultured vertically, at an angle of 70°, at 24°C in the dark (Fig. 1a; Chabaud
et al., 2002) on M medium (in mg L⫺1: 80 KNO3, 731 MgSO4䡠 7H2O, 65 KCl,
4.8 KH2PO4, 288 Ca(NO3)2䡠4H2O, 6 MnCl2䡠 4H2O, 1.5 H3BO3, 2.65
ZnSO4䡠 7H2O, 0.0024 Na2MoO4䡠 2H2O, 0.13 CuSO4䡠 5H2O, 0.75 KI, 8 NaFe-
EDTA, 3 Gly, 50 myo-inositol, 0.5 nicotinic acid, 0.1 pyridoxine-HCl, 0.1
thiamine-HCl, and 10,000 Suc) (Bécard and Fortin, 1988) containing 0.5%
(w/v) gellan gum (Phytagel, Sigma-Aldrich, St. Louis).
When using seedlings, seeds of M. truncatula transgenic line L416 (Jour-
net et al., 2001) were scarified with a 4-min treatment with concentrated
H2SO4, surface-sterilized with 3% (w/v) calcium hypochlorite for 4 min,
then germinated for 48 h at 15°C in the dark in petri dishes containing M
medium gelled with 0.5% (w/v) gellan gum without Suc. All seedlings were
grown axenically on M medium without Suc, using a culture system
adapted from Wong and Fortin (1989) in which the aerial portion of the
plant develops outside the petri dish, permitting optimal photosynthesis
and gas exchange, while the root system remains in the humid, sterile
environment of the petri dish (Fig. 1b). In brief, a 3-mm-diameter hole was
burned into the top edge of a 90- ⫻ 90-mm square plastic petri dish, through
which the 2-cm-long root of a germinated seed was inserted. After seedling
insertion, petri dishes were sealed with parafilm, covered with aluminum
foil, propped vertically at an angle of 70°, and cultured for 10 to 40 d in a
controlled growth chamber at 24°C/20°C with a 16-h photoperiod and 3,200
cd m⫺2 light intensity.
Fungal Inoculum
Symbiotic and pathogenic fungal inocula used are listed in Table II.
Gigaspora gigantea (Nicol. & Gerd.) Gerd. & Trappe, Gigaspora margarita
Becker & Hall, and Gigaspora rosea Nicol. & Schenck spores were surface-
sterilized and stored at 4°C according to Bécard and Fortin (1988). Sterile
Glomus intraradices Schenck & Smith inoculum was stored in sterile water at
4°C. The phytopathogenic fungi Fusarium solani var phaseoli (Burkholder)
Snyder & Hansen, Phoma medicaginis var pinodella (L.K. Jones) G. Morgan,
Jones & Bunch, and Phytophthora medicaginis Drechs. were cultured on the
above M medium with 1% (w/v) Suc for 2 weeks before use as inoculum in
dual cultures.
Dual Cultures and Membrane Separation
Fast-growing Ri T-DNA-transformed root explants with a “fishbone”
morphology were prepared according to Chabaud et al. (2002) for dual
cultures. Three 5-cm-long explants were transferred to the upper one-third
of 120- ⫻ 120-mm square petri dishes containing 50 mL of M medium, and
fungal inoculum was added at the same time to the center of the petri dish
(Fig. 1a). The AM fungal inoculum consisted of 10 surface-sterilized spores
of Gigaspora spp. or approximately 500 sterile spores of G. intraradices
pressed into the medium. Inocula of non-mycorrhizal fungi consisted of a
0.5-cm-diameter plug taken from 14-d-old cultures grown on M medium
with Suc. Because mycelial growth of these fungi was significantly faster
960
than that of AM fungi under our experimental conditions, the inoculated
plants were harvested at 3, 6, and 10 dai. Dual cultures were incubated
vertically, as for Ri T-DNA-transformed root cultures. After spore germi-
nation, AM fungal growth was observed daily using a dissecting microscope
and marked on the petri dish using colored markers. Roots were harvested
at 10, 20, and 30 dai to determine fungal infection and monitor
pMtENOD11-gusA expression using histochemical GUS staining (see
below).
Germinated seedlings were grown as described above in square petri
dishes (90 ⫻ 90 mm) containing 25 mL of M medium without Suc, one
seedling per petri dish. Dual cultures were incubated vertically, as described
above for plant growth. At the time of seedling insertion, fungal inoculum
was added as described above for root coculture inoculations (Fig. 1b).
To physically separate fungal inoculum from roots, membranes were
inserted as a physical barrier. A cellophane membrane (Couvre-Confitures,
Hutchinson, Chalette sur Loing, France) was generally used, or alternately,
a polycarbonate membrane of 0.6-␮m pore size (catalog no. 7062 4706,
Whatman International, Maidstone, UK) or a dialysis membrane with a
molecular mass cut-off of 3.5 kD (Spectra/Por, Spectrum Laboratories, Inc.,
Rancho Dominguez, CA). All membranes were rinsed with distilled water
for 30 min and autoclaved in distilled water before use. For experiments
with Ri T-DNA-transformed roots, fungal inocula were inserted into the
medium in the center of a 120- ⫻ 120-mm square petri dish containing 50 mL
of M medium (with Suc). The membrane (120 ⫻ 120 cm) was then laid on
top, 25 mL of the same medium with Suc was then added, and finally, three
rapidly growing root explants were transferred to the petri dish, as de-
scribed above (Fig. 1a). This system was modified to increase the distance
between the fungus and the roots, by adding first a cellophane membrane,
followed by 25 mL of M medium with Suc, then a second membrane,
followed by 25 mL of the same medium, and finally three explants.
For experiments with seedlings, fungal inocula were inserted into the me-
dium in the center of a 90- ⫻ 90-mm petri dish containing 25 mL of
M medium without Suc, the membrane was laid on top, 12 mL of the same
M medium without Suc was added, and finally, the single seedling (L416)
was inserted into the petri dish as described above (Fig. 1b). For both plant
and Ri T-DNA-transformed culture systems, fungus and roots were cocul-
tured for 10 to 14 d and then harvested for study. All plant and transformed
root experiments were performed at least three times, with three to five
replicates per experiment. In all experiments, control roots were grown
under identical conditions, including membrane inserted in the medium,
but in the absence of fungal inocula.
Histochemical Localization of GUS Activity
Roots were evaluated for GUS activity after 6 h incubation at 37°C with
the substrate 5-bromo-4-chloro-3-indolyl glucuronide, cyclohexylammo-
nium salt (X-gluc, Biosynth AG, Staad, Switzerland), as described previ-
ously (Journet et al., 1994). To visualize the spatial relationship between
fungal structures and pMtENOD11-gusA expression, whole-root systems
were stained in situ by adding the X-gluc substrate-buffer combination
directly to the petri dish (0.3 mg X-gluc mL⫺1 medium). The number of roots
showing pMtENOD11-gusA induction and the total number of roots were
counted in membrane-separated coculture experiments. Differences be-
tween AM treatments were assessed by pair wise Student’s t Tests (Mi-
crosoft Word 97 SR-1, Microsoft, Redmond, WA), and differences between
dmi mutants were compared by analysis of variance followed by Tukey’s
test (STATISTICA v6, StatSoft, Tulsa, OK) at the P ⫽ 0.05 significance level.
For histological observations, stained root segments were embedded in
3% (w/v) low-gelling temperature agar (type III, Sigma-Aldrich). Semithin
sections (50–75 ␮m) were prepared using a Microcut H1250 vibrotome
(Energy Beam Sciences, Agawam, MA), and observed immediately with
bright-field microscopy (LEICA DM IRB/E, Leica Microsystems, Wetzlar,
Germany).
Observation of Fungal Root Infection
To check for fungal colonization and to verify that the fungus had not
crossed the membrane and contacted the roots in membrane-separated
coculture, whole-root systems were cleared with 10% (w/v) KOH for 1 h at
90°C, thoroughly rinsed with distilled water, and stained with 0.05% (w/v)
Plant Physiol. Vol. 131, 2003

Chlorazol Black for 1 h at 90°C. Roots were destained overnight in 50%
(v/v) glycerol and carefully examined by bright-field microscopy.
PCR Analysis of Potential Fungal Contamination
To further verify that the fungus had not crossed the cellophane barrier,
the presence of fungal DNA in the root compartment was tested by PCR
using fungus-specific and universal primers. Total DNA was extracted from
liquid-nitrogen frozen plant and fungal tissue using the Wizard Genomic
DNA Purification kit (Promega, Madison, WI) and stored at 4°C. DNA
samples were collected from control roots, from roots grown in the presence
of G. rosea but physically separated by a cellophane membrane, from roots
colonized by G. rosea, and from spores of G. rosea. Two independent PCRs
were performed concurrently on each DNA sample, using primers specific
to the internal transcribed spacer (ITS) region in the nuclear ribosomal
repeat unit. The first primer set, a so-called “universal” primer combination
(ITS1 and ITS4) designed to amplify DNA from a broad range of organisms
including fungi, plants, protists, and animals (White et al., 1990), was used
to amplify plant and fungal DNA. The second primer set, ITS1-F coupled
with a universal primer ITS4, was used to amplify fungal DNA more
specifically (Gardes and Bruns, 1993). The following PCR conditions were
used: denaturation at 93°C for 3 min, followed by 35 cycles of denaturation
at 93°C for 30 s, annealing at 62°C for 1 min, and extension at 72°C for 1 min,
with a final extension at 72°C for 10 min. Negative controls (no DNA
template) were included in every experiment to test for contamination of
reagents and reaction mixtures. DNA from three independent experiments
was tested in this way, and similar results were obtained.
ACKNOWLEDGMENTS
We thank A. Jauneau, A. Boisson-Dernier, and B. Olah for technical
advice, E.-P. Journet for the C71 and TRV25 Nod⫺/Myc⫺ mutant plants
carrying the pMtENOD11-gusA fusion, R. Koske for the G. gigantea spores,
Premier Tech for the G. intraradices inoculum, and A. Bottin for the fungal
culture of Phy. medicaginis.
Received July 27, 2002; returned for revision August 24, 2002; accepted
October 19, 2002.
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