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Novel infection process in the indeterminate root nodule
symbiosis between Chamaecytisus proliferus (tagasaste)
and Bradyrhizobium sp.
Blackwell Science Ltd
M. C. Vega-Hernández1, R. Pérez-Galdona1, F. B. Dazzo2, A. Jarabo-Lorenzo1, M. C. Alfayate1 and
M. León-Barrios1
1
Departamento de Microbiología y Biología Celular. Facultad de Farmacia. Universidad de La Laguna. 38071 La Laguna. Tenerife. Canary Islands. Spain;
2
Department of Microbiology and Center for Microbial Ecology. Michigan State University, East Lansing. MI 48824, USA
Summary
Author for correspondence:
M. León-Barrios
Tel: +34 922318481
Fax: +34 922630095
Email: [email protected]
Received: 17 October 2000
Accepted: 21 December 2000
• The main characteristics of the symbiosis of tagasaste (Chamaecytisus proliferus
ssp. proliferus var. palmensis), a woody legume forming N2-fixing indeterminate
nodules in response to infection by strains of Bradyrhizobium sp. (Chamaecytisus),
are reported here.
• The infection process in this legume was examined by bright field, phase contrast
and transmission electron microscopy, and was found to be unlike any other previously described.
• First steps in the infection process involve initiation of infection threads within
deformed root hairs and induction of foci of host-cell divisions in the inner root
cortex. However, infection of root hairs aborts early, and instead, the bacteria use
the crack-entry mode of host infection, whereby they penetrate the periphery of the
emerging nodule through an intercellular route, eventually infecting host nodule
cells directly through altered cell walls. No successful infection threads were detected
at any stage of primary-host infection or nodule invasion. Indeterminate nodules
were mainly formed on unbranched areas of lateral roots.
• This is the first description of such a combination of events in an infection process
in the Rhizobium–legume root-nodule symbiosis.
Key words: tagasaste (Chamaecytisus proliferus), Rhizobium-legume symbiosis,
Bradyrhizobium sp., crack entry, infection, nodulation.
© New Phytologist (2001) 150: 707–721
Introduction
The rhizobia (Rhizobium, Bradyrhizobium, Azorhizobium,
Sinorhizobium, Mesorhizobium and Allorhizobium) are a group
of Gram-negative soil bacteria that can infect legumes and
establish a symbiotic relationship. As a result of this association,
a new organ, the root-derived nodule, is formed. Within the
nodule, rhizobia differentiate into endosymbiotic bacteroids
that can fix atmospheric N2.
Two main modes of infection have been described in the
root nodule symbiosis. The mode most thoroughly studied
involves primary entry of bacteria into deformed root hairs
through infection threads, which progress to the root cortex
and eventually reach the emerging nodule primordia (Napoli
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& Hubbell, 1976; Callaham & Torrey, 1981; Newcomb, 1981;
Hirsch, 1992). The other infection mode is called crack entry,
in which the rhizobia invade the root interior through natural
wounds on the epidermis (Napoli et al., 1975; Boogerd &
van Rossum, 1997). In addition, a mode of direct infection
through undamaged epidermis has been described for the
hairless tree legume, Mimosa scabrella (de Faria et al., 1988).
This has been proposed as a third mode of infection for woody
legumes that neither produce root hairs regularly nor produce
nodules associated with lateral roots (Sprent, 1989).
The best-studied mode of primary host infection takes
place through infection thread formation (Inf ), where the
rhizobia induce a marked curling of the root hair tip (Hac),
forming the typical ‘shepherd crook’ deformation. The bacteria
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infect the root through the formation of a tubular infection
thread that grows to the base of the root hair cell and penetrates
into the underlying root cortex, releasing the rhizobia into
newly divided cortical cells. Collectively referred to as the Inf
phenotype, this mode of infection can be subdivided into Iti
(Infection Thread Initiation), Itr (Infection Thread development
in Root hairs), and Itn (Infection Thread development in
Nodules) phenotypes. This commonly yields two different
nodule morphotypes depending on the host. These are the
indeterminate nodules as in clover and alfalfa, and determinate
nodules as in soybean and cowpea (Hirsch, 1992). This is a
well-documented infection process.
The alternative mode of crack entry infection occurs by
intercellular penetration between root cells. After colonizing
the root epidermis, the bacteria invade the root cortex through
natural wounds caused by splitting of the epidermis where
young lateral roots or nodule primordia have been stimulated
to develop and emerge. The crack entry mode of primary host
infection has been described in various (sub)tropical legumes
such as Aeschynomene (Napoli et al., 1975; Alazard & Duhoux,
1990), Arachis (Chandler, 1978; Boogerd & van Rossum, 1997),
Stylosanthes (Chandler et al., 1982), Sesbania rostrata (Dreyfus
et al., 1984; Duhox, 1984; Ndoye et al., 1994; Rana &
Krishnan, 1995), and Neptunia (Subba-Rao et al., 1995). Two
types of crack entry can be distinguished depending on the
mode of bacterial dissemination within the nodule. In the case
of Aeschynomene, Arachis, and Stylosanthes ( Napoli et al., 1975;
Chandler, 1978; Chandler et al., 1982) the intercellular rhizobia
directly invade some root cortical cells and their dissemination within the nodule takes place by division of the infected cells
without involving infection threads. In the case of Sesbania
and Neptunia, dissemination of the microsymbiont involves
an initial intercellular spread, followed later by intracellular
infection involving formation of true tubular infection threads
that penetrate nodule cells and release the endosymbiotic
bacteria at infection droplets that have protruded from localized
eroded areas of the infection thread wall (Ndoye et al., 1994;
Subba-Rao et al., 1995). Crack entry has also been described in
the infection of the nonlegume Parasponia by Bradyrhizobium
in which both mechanisms of dissemination appear to take place
(Trinick, 1979; Lancelle & Torrey, 1984; Bender et al., 1987).
Tagasaste, Chamaecytisus proliferus (L. fil.) Link ssp. proliferus
var. palmensis (Christ) Hansen & Sunding, is a temperate
woody legume in the tribe Genisteae of the Papilionoideae
subfamily endemic to the Canary Islands. This evergreen shrub
can grow to a height of 5 m and is of great agronomic and
ecological value (Pérez de Paz et al., 1986; Francisco-Ortega
et al., 1991). Its high nutritive value and palatability makes
this plant a suitable forage for grazing animals, pigs and poultry
(Borens & Poppi, 1986; Dann & Trimmer, 1986; Pérez de
Paz et al., 1986; Borens & Poppi, 1990; Oldham et al., 1991;
Snook, 1996). For centuries, tagasaste has been used as fodder
in the Canaries (Pérez de Paz et al., 1986). It was introduced
in the last century in other parts of the world, especially
in Australia and New Zealand (Snook, 1996) where it was
misnamed tree-lucerne (European name for alfalfa) alluding
to its comparative forage value with alfalfa. In Australia,
tagasaste has been reported to be nodulated by strains of
rhizobia and bradyrhizobia (Gault et al., 1994), but in the
Canaries only the slow-growing bradyrhizobia isolated from
endemic woody legumes have been found to nodulate tagasaste
effectively (León-Barrios et al., 1991; Santamaría et al., 1997),
typically forming cylindrical, indeterminate nodules. The
legume host range of these Canarian bradyrhizobia also
includes Macroptilium atropurpureum (siratro), where the
mature nodules are spherical rather than elongated. Genomic
studies indicate that some of the Canarian bradyrhizobia
isolated from tagasaste and other endemic woody legumes form
a distinctive group of strains that could constitute a new species
of the genus Bradyrhizobium (Jarabo-Lorenzo et al., 2000).
The aim of this work was to document the developmental
morphology of the infection and nodulation processes in
tagasaste by two strains of Bradyrhizobium sp. (Chamaecytisus)
indigenous to the Canary Islands. Portions of this work were
presented at the 12th International Congress on Nitrogen
Fixation (Vega-Hernández et al., 2000).
Materials and Methods
Bacterial and plant growth conditions
Bradyrhizobium sp. (Chamaecytisus) strain BTA-1 was isolated
from root nodules of tagasaste, Chamaecytisus proliferus (L. fil.)
Link ssp. proliferus var. palmensis (Christ) Hansen & Sunding
(Acebes-Ginovés et al., 1991), and strain BGA-1 from nodules
of Teline stenopetala (Webb & Berth) Webb & Berth. var.
stenopetala (León-Barrios et al., 1991). Inocula were prepared
by growing bacterial cultures to late exponential phase in
shaken flasks containing yeast mannitol broth at 28°C.
In order to facilitate germination, tagasaste seeds were
scarified with concentrated sulphuric acid for at least 30 min,
and then sterilized for 15 min in 5% (v/v) aqueous sodium
hypochlorite. Macroptilium atropurpureum seeds were similarly
sterilized for 15 min in 5% (v/v) sodium hypochlorite.
Surface-sterilized seeds were rinsed six times in sterile distilled
water, and germinated on 1% (w/v) agar plates in darkness
at room temperature. Young seedlings with roots approx.
1 cm long were removed from the germination plates and
immersed for 2 h in a suspension of strain BTA-1 or BGA-1.
Uninoculated seedlings were used as control.
The first steps of the infection process were examined using
the Fåhraeus slide culture technique (Fåhraeus, 1957), and
nodule development was examined on inoculated plants
grown in Leonard jars containing a 1 : 1 mixture of sand and
vermiculite. In both cases, nitrogen-free Fåhraeus solution was
added as nutrient (Fåhraeus, 1957). Plants were incubated in a
growth chamber programmed with a 16-h photoperiod, 70%
rh, and a day/night temperature of 25°C and 17°C, respectively.
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Microscopy analyses
Slide cultures were observed daily by brightfield and phase
contrast microscopy to examine the early steps of the infection
process. Foci of cortical meristems were visualized by the
hypochlorite clearing/methylene blue staining procedure of
Truchet et al. (1989). Segments of inoculated plants were cleared
by vacuum-infiltration with commercial sodium hypochlorite
solution diluted to 70% of its original volume with water,
rinsed in water, stained briefly with an aqueous solution of
methylene blue (0.2 mg ml–1) and examined by brightfield
microscopy.
Mature nodules from 6–8-wk-old plants were fixed in
ethanol-acetic acid-formaldehyde (90 : 5 : 5, by volume),
infiltrated in Paraplast, sectioned to 10 µm, stained with
safranine/fast green (Johansen, 1940) and examined by brightfield and phase contrast microscopy.
Mature nodules on 2–4-wk-old plants were fixed by
vacuum infiltration for 2 h with 3% (v/v) glutaraldehyde in
0.1 M phosphate buffer (PB; pH 7.2) at room temperature.
Submerged nodules were washed in PB, postfixed in 1%
(w/v) osmium tetroxide in the same buffer for 2 h at 4°C,
washed again in PB, dehydrated in a graded ethanol series and
embedded in Spurr’s resin (Spurr, 1969). Semi-thin-sections
obtained using glass knives were stained with toluidine blue
and examined by brightfield microscopy. Ultra-thin sections
obtained using a diamond knife were stained with aqueous
uranyl acetate and lead citrate, and examined by TEM using
Jeol 1010 (Tokyo, Japan), and Philips CM-10 electron
microscopes (Hillsboro, Oregon, USA).
Results
Appearance of nodulated tagasaste plants
The typical morphology of an inoculated tagasaste plant after
1 month of growth under the conditions for this study and a
closer view of its nodulated roots are shown in Fig. 1(a),(b),
Fig. 1 Example of a typical nodulated tagasaste plant (Chamaecytisus proliferus) used in this study. (a) The entire plant was place in a Petri dish
and photographed at low magnification. The arrows indicate nodulated lateral roots. Bar, 2 cm (main picture). Bar, 1.2 mm (inset). (b) Higher
magnification stereomicrograph of the same lateral roots. Note the cylindrical shape of the mature indeterminate nodule (arrow) on the dense
hairy root. Bar, 1 mm.
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Fig. 2 Phase contrast micrographs illustrating
various typical alterations in root hairs of
tagasaste (Chamaecytisus proliferus)
inoculated with bradyrhizobia. (a) Markedly
curled (‘shepherd crook’) deformation at the
root hair tip. (b) An aborted infection of a
deformed root hair. Arrows point to the
infection thread that arrested shortly after
formation. (c) Refractile, circular eroded areas
(arrow) at a root hair tip. (d) A lysed root hair
with its cytoplasm expressed through a hole
at the tip. Residual portions of the root hair
protoplast have separated from the inner face
of the cell wall. Bar, 12 µm.
respectively. Nodules developed frequently on lateral roots
but not on the main root or at the lateral root axils. Nodules
were typically cylindrical when mature.
indication of unsuccessful root hair infections and suggest that
these bradyrhizobia probably use an alternate mode of primary
host infection in order to nodulate tagasaste effectively.
Light microscopy of aborted root hair infections
Light microscopy of nodule development and histology
Both bacterial strains showed the same pattern of symbiotic
infection with tagasaste. Bacteria polarly attached to the root
hair tips (figure not shown), and induced various types of
root hair deformation within 24–48 h after inoculation
(Fig. 2a,b). Root hairs with typical markedly curled shepherd’s
crooks were found on inoculated seedlings 48 h and older
(Fig. 2a). At this point, the initial formation of refractile infection threads within deformed root hairs (Iti+) could also be
distinguished, but these initial infections always aborted at or
near the site of inception (Fig. 2b). No infection threads were
found that had grown to the base of the root hair and/or
penetrated to the subepidermal cortex (Itr–). Further examination of 2 and 3 wk-old seedlings revealed no successful
infection threads in the vicinity of emerged root nodules, or
elsewhere within the entire areas of the bulging epidermis
overlying nodule primordia on lateral roots at this time.
Possibly related to the unusual Ini+ Inr– phenotypes of this
symbiosis was the frequent development of localized areas of
wall erosion at the tips of root hairs on inoculated plants only
(Fig. 2c). Occasionally, root hair cytoplasm leaked out from
these localized erosions (Fig. 2d). These results are a further
Brightfield microscopy indicated that the initial foci of host
cell divisions induced by the symbiotic bacteria occurred in
the inner cortex, as is typical of indeterminate nodule formation (Fig. 3a). These foci of cortical cell divisions developed more
frequently in lateral roots than in the main root. The mitotic
activity of these cortical cells gave rise to nodule primordia
(Fig. 3b) that emerged as nodules (Figs 1a, 1b). Longitudinal
sections of mature tagasaste nodules exhibited the typical
histological organization of elongated, indeterminate nodules
with apical meristem, infection and fixation zones, and a
proximal senescence zone (Fig. 4).
Ultrastructural morphology of the tagasaste nodule
Transmission electron microscopy confirmed that the nodule
invasion stage of the infection process did not involve any
infection threads. Instead, primary invasion of young nodules
by the bacteria involved crack entry through intercellular
spaces of the outer apical cell layers (Figs 5a,b, 6a). Deeper
within the nodule, the rhizobia proliferated in enlarged
intercellular spaces that served as foci to facilitate further
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Fig. 3 Brightfield micrographs of cleared/
stained roots exhibiting development of foci of
cortical cell divisions initiated in the inner root
cortex (a, arrows) and a nodule primordium
prior to emergence on a lateral root (b).
Bar, 250 µm.
dissemination of intercellular invasion. Bacteria entered other
cell layers through the middle lamellae within the nodule
(Fig. 6b) resulting in a network of intercellular infection
(Fig. 6c). Accompanying the intercellular dissemination of
the bacteria was the successive collapse of host cells in the
apical layers of the nodule, causing the deformation and convolution of their cell walls (Fig. 7a). This collapse phenomenon
was seen only in uninfected host cells neighbouring intercellular bacteria in the outer cortical or invasion zone of the
nodule. This was clearly observed in nodules aged 4 wk or
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older, probably due to more extensive rhizobial proliferation
(Fig. 8c), whereas 2- or 3-wk-old nodules had flatter, nonconvoluted cells in this region. Furthermore, plant cells
infected with endosymbiotic bacteria showed a normal cell
shape (Fig. 7b,c).
Closer examination of the invaded intercellular spaces
within nodules revealed bacteria surrounded by a matrix
of electron-transparent material that approximately followed
their contour (perhaps capsule) and more electron-dense fibrillar
material loosely associated with, and possibly derived from,
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Fig. 4 Brightfield micrograph of a semi-thin
longitudinal section of an 8-wk-old
indeterminant tagasaste nodule. The apical
meristem (m), the central zone of N2 fixation
(fz) and the proximal senescence zone (sz) are
labelled. Bar, 250 µm.
the host cell wall (Fig. 8a). The extent of alterations in the
nodule host cell walls was such that, sometimes, they appeared
to be pliable and became invaginated, forming pockets
that were slightly larger than individual bacteria (Fig. 8b,c).
However, instead of inducing infection threads, the bacteria
presumably entered nodule host cells directly from intercellular
spaces by continued, localized erosion of their structurally
altered cell walls followed by invagination of their plasmamembrane. Several lines of TEM evidence strongly suggest
that the bacteria transverse the host cell wall. First, intercellular
bacteria were frequently found to occupy the cell wall pockets
that were invaginated into the adjacent host cell (Fig. 8c).
Similarly, Fig. 8(d),(e) show bacteria embedded within altered
structures of the host cell wall in contact with the host
cytoplasmic membrane, and at the same time, adjacent to
other bacteria that had differentiated into bacteroids with
well-defined nucleoids. Sometimes extracellular bacteria with
internal poly-β-hydroxybutyrate (PHB) granules (characteristic
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Fig. 5 Brightfield micrographs of semi-thin
sections at the periphery of an 18-d-old
tagasaste nodule. Arrows indicate opened
areas between epidermal host cells providing
crack portals of entry for the symbiotic
bradyrhizobia. Bar, 12 µm.
of bacteria in the intercellular spaces) were observed in the
process of invaginating the plasmamembrane during invasion
of the plant cell (Fig. 8f ). Moreover, localized areas of the
host cell walls that were thinner and/or altered in electrondensity were often found near the symbiosomes (Fig. 9a–d).
These observations are very suggestive of the mode of invasion
of the host nodule cells. We interpret these changes in
ultrastructure as reflecting localized areas of structurally
altered host cell walls that constitute regions more easily
penetrated by the bacteria during their invasion of the
nodule cell. Division of recently infected meristematic
nodule cells appears to expand the infected area without
the involvement of infection threads (Fig. 10a,b).
The central fixation zone had the typical organization of an
indeterminate nodule characterized by the presence of densely
infected plant cells (Figs 4, 7c). By contrast to intracellular
vegetative bacteria, bacteroids commonly appeared as enlarged,
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elongated (and occasionally pleomorphic) rods that were
singly enclosed within peribacteroid membranes (Figs 7c, 11).
The centrally localized nucleoid region of the endosymbiotic
bacteroids appeared densely compacted and associated with
electron-dense (possibly polyphosphate) inclusions (Fig. 11).
Interestingly, although the bacteria in the intercellular spaces
frequently contained large intracellular inclusions of poly
β-hydroxybutyric acid (Figs 6a,b 8a–c,f ), these electrontransparent granules were rarely, if ever, found in the endosymbiotic bacteroid stage (Figs 7c, 8d,e, 11).
The proximal senescence zone was characterized by the
presence of degenerated bacteroids with associated membranes in a disorganized host cytoplasm (not shown). In no
case were any infection threads found within the senescing
nodule cells.
In striking contrast to the above description of symbiotic
development with tagasaste, these same strains of Canarian
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Fig. 6 Transmission electron microscopy
(TEM) of intercellular invasion of tagasaste
nodules by bradyrhizobia. (a) Bacteria
(arrow) that have entered a peripheral
intercellular space of the outer nodule
cortex. (b) Bradyrhizobia colonizing an
intercellular space deeper within the
nodule. Note the packing of bacteria in
the middle lamella between two separated
host cell walls. (c) Low magnification view
of the intercellular infection network of
disseminating bradyrhizobia within the
tagasaste nodule. Note the convoluted
cell walls of the host cells neighbouring
the intercellular bacteria. Bar, 2 µm.
bradyrhizobia infected siratro roots via formation of infection
threads within deformed root hairs, induction of the initial
foci of cell divisions in the root outer cortex and development
of mature nodules of the spherical determinate type with a
central infected tissue formed by groups of host cells containing true infection threads and symbiosomes surrounded by
uninfected interstitial cells (not shown).
Discussion
Tagasaste formed indeterminate nodules with bradyrhizobia
indigenous to the Canary Islands. The first stages of the infection
process included rhizobial attachment to and deformation of
root hairs, followed by marked curling, localized formation of
bright spots, and initiation of intracellular infection threads.
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Fig. 7 Cytology of tagasaste nodule cells.
(a) Collapse of host cells in the nodule cortex,
where intercellular dissemination of symbiotic
bradyrhizobia is occurring. (b) Brightfield
micrograph showing the intercellular
dissemination of the bacteria and sparsely
infected host cells containing released
vegetative bacteria. (c) Transmission electron
microscopy (TEM) of the fixation zone
showing infected host cells completely filled
with singly enclosed bacteroids. Bar, 5 µm in
(a) and (b), and 1 µm in (c).
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Fig. 8 Transmission electron microscopy (TEM)
of nodule host cell invasion. (a,b,c) Rhizobia
surrounded by an electron-transparent zone
and electron-dense fibrillar material in the
intercellular space. (b) Accumulation of loose
fibrillar material (arrowhead) suggestive of wall
loosening associated with the invading bacteria,
and the presence of rhizobia near pockets of
invaginated host cell wall (double arrowheads).
(c) Extensive rhizobial colonization of an
intercellular space. Double arrowhead points to
an invaginated host cell wall. Line-bordered
insert is an enlargement of the corresponding
area. (d) Bacterium embedded within the wall
forming a host cell wall loop (hcw) associated
with electron-dense material (arrowhead).
(e) Bacterium included in the host cell wall
(hcw), surrounded by loosened cell-wall
material (arrowheads) near a symbiosome
containing a bacteroid with condensed
nucleoid. The interface of the symbiosome
and host cell wall is indicated by a double
arrowhead. (f) Bacterium (asterisk) in the
process of entering the plant cell. Note the
proximity of the intercellular space, an altered
host cell wall, an invaginated host cell
plasmamembrane, and the presence in the
bacterial cytoplasm of poly-β-hydroxybutyrate
(PHB) granules (characteristics of vegetative
state). Bar, 0.5 µm.
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Fig. 9 Transmission electron microscopy (TEM) of infected nodule cells. (a) Infected host cell with the cell wall thinned at several sites
(arrowheads). (b– d) Symbiosomes containing bacteroids near the inner face of the host cellular wall (hcw) where a distinct electron-dense
material has accumulated (arrowhead). Peribacteroid membrane (pbm), peribacteroid space (pbs). Bar, 0.5 µm.
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Fig. 10 (a,b) Phase contrast micrographs of
semi-thin cross-sections at the interface of the
meristem-infection zone of a tagasaste
nodule invaded by bradyrhizobia. Bacteria
are present within recently divided host cells.
Bar, 10 µm.
These infection threads always aborted before they grew to the
base of the root hair. This phenotype has been described in
other symbioses with rhizobial mutants defective in the genes
that specify the synthesis of superficial polysaccharides (Finan
et al., 1985; Carlson et al., 1987; de Maagd et al., 1988; Long,
1989; Hirsch, 1992; Rolfe et al., 1996; van Workum et al.,
1998; Pellock et al., 2000). However, the two wild-type strains
used in this study produce normal surface polysaccharides
(León-Barrios et al., 1992a,b; Santamaría et al., 1997), and are
fully capable of infecting deformed root hairs of Macroptilium
through true infection threads that reached the root cortex
(data not shown).
A thorough search, using combined light and transmission
electron microscopy, confirmed the absence of infection threads
in tagasaste nodules. Instead, both strains of bradyrhizobia
invaded the periphery of nodules by the crack entry mode,
followed by deeper dissemination though intercellular spaces,
infection of host cells without infection thread formation
and extension of infection by division of the recently infected
meristematic cells.
The unique character in development of the tagasaste symbiosis lies in the fact that it shows a hybrid mix of combined
events representing several different types of infection collectively unlike any other previously described. The preinfection
stages of interaction with root hairs are triggered on tagasaste
by strains BTA-1 and BGA-1 in the same way as the Rhizobiumclover or Bradyrhizobium-siratro symbioses. Although deformation of axillary hairs of Aeschynomene, Arachis and Stylosanthes
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Fig. 11 Ultrastructure of an endosymbiotic
bacteroid of Bradyrhizobium sp. strain BTA-1
within a tagasaste nodule cell. Shown are
the peribacteroid membrane (pbm),
peribacteroid space (pbs), and a bacteroid cell
with a centrally localized nucleoid associated
with electron-opaque polyphosphate
inclusions (pp). Note the absence of electrontransparent intracellular granules of poly
β-hydroxybutyrate. Bar, 0.15 µm.
occurs when inoculated with homologous rhizobia, these
other crack-entry types of symbioses do not progress further
in root hair infection (Napoli et al., 1975; Chandler, 1978;
Chandler et al., 1982). By contrast, in the Bradyrhizobiumtagasaste symbiosis, these early interactions progress further
to initiate infection threads within deformed root hairs,
but these intracellular infection structures always abort before
growing to the base of the root hair. The formation of infection threads within root hairs but not within root nodules,
the initiation of meristematic foci in the inner (rather than
the outer) cortex and the nodule morphology, all represent
characteristics that distinguish the Bradyrhizobium-tagasaste
symbiosis from the Rhizobium-lupin symbiosis (Tang et al.,
1993; James et al., 1997).
Another important difference in symbiotic development
between tagasaste and other legumes infected by crack entry
is that the nodules emerge along lateral roots rather than
being restricted to lateral root axils as is usually the case for
Aeschynomene, Arachis, Stylosanthes, Sesbania, and Neptunia
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(Napoli et al., 1975; Chandler, 1978; Chandler et al., 1982;
Ndoye et al., 1994; Rana & Krishnan, 1995; Subba-Rao et al.,
1995). This fact, together with the early development of
nodule primordia in the inner cortex of the tagasaste root,
suggests that the strategy used by these strains to produce
wounds to penetrate the epidermis is similar to the one used
by bradyrhizobia to infect the nonlegume Parasponia. In this
symbiosis, the stimulation of cell divisions in epidermal root
hairs or divisions in the outermost cortical cells by the rhizobia
leads eventually to the separation of epidermal cells and
enlargement of the intercellular spaces, thereby developing
an access point of entry (Lancelle & Torrey, 1984; Bender
et al., 1987). Presumably, strains BTA-1 and BGA-1 stimulate
the development of an expanding nodule primordium that
splits the epidermis as it emerges, thus creating the portal
of crack entry through which these bacteria can access the
nodule surface. Young nodules contain wide enough open
spaces in the epidermis through which the bacteria can enter
their interior.
719
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720 Research
The Bradyrhizobium-tagasaste symbiosis shares additional
characteristics with other symbioses using crack entry as the
mode of infection. For instance, the bacterial dissemination through the nodule cortex and into the infection zone
takes place by separating cortical cells at the middle lamellae
as occurs in Arachis (Dongre et al., 1985). Like tagasaste,
collapse of cells in the outer layers of the nodule also occurs
in Aeschynomene and Stylosanthes (Chandler et al., 1982;
Alazard & Duhoux, 1990).
It must be highlighted that the bacteria in intercellular
spaces were often associated with structurally altered and
weakened host cell walls, and accumulation of broken host
cell wall fragments. This suggests the presence of highly
active enzymes with lytic activity against walls. The holes that
developed on the tips of tagasaste root hairs during the first
stages of the infection process are also suggestive of the activity
of wall-degrading enzymes. Similar holes have been detected
on root hair tips of axenic white clover seedlings incubated
with purified cellulases from Rhizobium leguminosarum bv.
trifolii (Mateos et al., 1992; Mateos et al., 1996; Mateos et al.,
2000). Although low cellulase activities have been detected in
pure cultures of BTA-1 and BGA-1 (data not shown), the in
situ concentrations of these and other wall-degrading enzymes
made by the tagasaste bradyrhizobia in planta is not known.
Other events that could possibly influence this process of
localized wall modification in planta include the induction
of host plant polygalacturonase by rhizobial components
(Muñoz et al., 1998), the inhibition of the expression of
peroxidase genes encoding the enzymes involved in lignification of the plant cell wall (Klotz & Lagrimini, 1996), the
transient suppression of plant wall-bound peroxidase activity
by homologous rhizobial components (Salzwedel & Dazzo,
1993) and localized disruption in crystalline wall architecture
in host cells grown with chitolipooligosaccharide Nod factors
(Dazzo et al., 1996). Some of these activities, documented in
other plant systems, could contribute to the magnitude of the
injuries found in the host cell wall during the infection process
in tagasaste. The direct invasion of tagasaste nodule cells by
bradyrhizobia without infection thread formation is similar to
rhizobial invasion in Arachis nodules (Chandler, 1978), but
distinctly different from rhizobial invasion via infection thread
formation as occurs in nodules of the tropical legumes, Mimosa
(de Faria et al., 1988), Sesbania (Ndoye et al., 1994) and
Neptunia (Subba-Rao et al., 1995).
Another unique feature of the bradyrhizobia-tagasaste
symbiosis is that, in spite of infection by crack entry, it nevertheless forms the developmental gradient of indeterminate
nodules characterized in the mature stage by their cylindrical
shape, within which the apical meristem, host cell infection,
bacteroid N2 fixation and senescence structures are separated
into discrete, distal-to-proximal zones. Indeed, this is the first
reported case of a wild-type rhizobia-legume symbiosis in which
crack entry leads to the development of an indeterminate N2fixing nodule. The necessity of studying other root-nodule
symbioses established between rhizobia and (sub)tropical
legumes (especially woody legumes) must be highlighted to
determine if this crack entry mode of infection evolved
uniquely in the Bradyrhizobium-tagasaste symbiosis, or alternatively, is more common than is assumed among N2-fixing
rhizobia-legume symbioses.
Acknowledgements
We thank Pedro Mateos for measuring the hydrolytic enzymatic activities. This work was supported by a grant PI1998/
016 from the Canary Autonomous Government, and by the
MSU Center for Microbial Ecology supported by National
Science Foundation grant DEB 91–20006.
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