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Plant Cell Rep (2004) 22:967–973
DOI 10.1007/s00299-004-0787-x
BIOTIC AND ABIOTIC STRESS
H.-H. Woo · A. M. Hirsch · M. C. Hawes
Altered susceptibility to infection by Sinorhizobium meliloti
and Nectria haematococca in alfalfa roots with altered cell cycle
Received: 20 December 2003 / Accepted: 12 February 2004 / Published online: 23 March 2004
Springer-Verlag 2004
Abstract Most infections of plant roots are initiated in
the region of elongation; the mechanism for this tissuespecific localization pattern is unknown. In alfalfa expressing PsUGT1 antisense mRNA under the control of
the cauliflower mosaic virus (CaMV) 35S promoter, the
cell cycle in roots is completed in 48 h instead of 24 h,
and border cell number is decreased by more than 99%.
These plants were found to exhibit increased root-tip
infection by a fungal pathogen and reduced nodule
formation by a bacterial symbiont. Thus, the frequency
of infection in the region of elongation by Nectria
haematocca was unaffected, but infection of the root tip
was increased by more than 90%; early stages of
Sinorhizobium meliloti infection and nodule morphology
were normal, but the frequency of nodulation was
fourfold lower than in wild-type roots.
Introduction
Root systems anchor plants within soil, provide access to
water and nutrients, and thereby influence virtually every
aspect of higher plant development and function (Aiken
Communicated by H.S. Judelson
H.-H. Woo · A. M. Hirsch
Department of Molecular, Cell, and Developmental Biology,
University of California,
Los Angeles, CA 90095, USA
H.-H. Woo · M. C. Hawes ())
Division of Plant Pathology and Microbiology,
Department of Plant Sciences,
University of Arizona,
Tucson, AZ 85721, USA
e-mail: [email protected]
Tel.: +1-520-6215490
Fax: +1-520-6219290
A. M. Hirsch
Molecular Biology Institute,
University of California,
Los Angeles, CA 90095, USA
and Smucker 1996). As roots penetrate soil, exudates
released from the root stimulate the development of a
complex microbial community in the rhizosphere, the
region immediately surrounding the root (Curl and
Truelove 1986). Organisms in the rhizosphere include
beneficial and parasitic species whose behaviors range
from solubilizing nutrients to causing necrotic lesions that
destroy root function and spread systemically throughout
the plant. The overall health of the plant, therefore, is
defined by the patterns of microbial colonization and
infection that are established as root systems ramify
through the soil environment.
In the absence of external wounding or stress, the
invasion of root tissue by beneficial and pathogenic
organisms occurs according to specific patterns of localized infection. Virtually all organisms that infect roots
initiate relationships in the region a few millimeters
behind the root tip in the region of elongation, where
newly synthesized cells are in the early stages of
differentiation; this pattern is common to infection in
tap roots, adventitious roots, and lateral roots (BaluÐka et
al. 1996; Bauer 1981; Brundrett et al. 1985; Curl and
Truelove 1986; Horan et al. 1988). The root tip—the
apical 1–2 mm of tissue including the root cap and root
apical meristem—generally remains uninfected and free
of surface-colonizing microorganisms (Foster et al. 1983;
Rovira and Campbell 1975; Royle and Hickman 1963).
Since root-infecting organisms include a diverse array of
bacteria and fungi as well as nematodes and higher plants,
it is reasonable to hypothesize that this distinctive pattern
of localized root infection is defined largely by the
properties of the host root rather than by those of the
invading organism (Curl and Truelove 1986).
The mechanism by which plants may control patterns
of root infection is unknown. Some investigators have
speculated that the region of elongation is a predominant
target for infection because an abundance of root exudates
is released there (Pearson and Parkinson 1961; Zentmyer
1961). However, the primary source of exudates in most
species is not the region of elongation but the root tip
itself (Griffin et al. 1976; McDougall and Rovira 1970;
968
Schroth and Snyder 1961). In addition, the release of
specific products—such as flavonoids, that influence
infection by symbiotic bacteria and fungi—also predominates at the root tip rather than at the site of infection
behind the root tip (Peters and Long 1988; Zhu et al.
1997). If exudation patterns do play a role in localized
root infection, then altering root exudation could be
predicted to result in a change in infection patterns. An
alternative hypothesis is that localized infection behind
the root tip is a product of factors controlling root growth
(Wilcox 1996). If this model is correct, then roots with
altered root growth would be predicted to exhibit altered
patterns of root infection by bacteria and fungi.
In previous studies, alfalfa with an altered cell cycle was
developed by inhibiting the expression of a homolog of
a gene encoding PsUGT1, a glycosyltransferase-encoding
gene from Pisum sativum. PsUGT1 activity is closely
correlated with mitosis in the root-cap meristem, and
reducing this activity by PsUGT1 antisense mRNA expression under the control of its own promoter is lethal
in pea and alfalfa (Woo et al. 1999). However, a less
deleterious phenotype was achieved in alfalfa by expressing PsUGT1 antisense mRNA under the control of the
cauliflower mosaic virus (CaMV) 35S promoter. Roots of
clonal alfalfa expressing CaMV 35S::PsUGT1 antisense
mRNA require 48 h instead of 24 h to complete a cell
cycle, and the release of root-border cells from the root tip
during a 24-h period is reduced by more than 99%. If root
growth and/or root exudation patterns underlie root infection patterns, these plants would be predicted to exhibit
changes in localization of infection. Conversely, if these
roots exhibited normal patterns of localized infection, the
results would suggest that factors other than growth and/or
root exudation are key determinants of localized root
infection. Preliminary observations suggested that root tips
of plants expressing CaMV 35S::PsUGT1 antisense
mRNA were more susceptible to fungal infection than
wild-type plants. Therefore, experiments were designed to
examine the possibility that CaMV 35S::PsUGT1 antisense
mRNA expression, which delays growth and the release of
border cells and their associated root exudates (Hawes et
al. 1998), also results in altered root infection localization
patterns.
Materials and methods
Plasmid and bacterial strains
The Escherichia coli uidA gene encoding b-glucuronidase (GUS)
was removed from the pBI121 vector using SstI and BamHI. The
cDNA (+730 to +1,510) from PsUGT1 was ligated into the SstI and
BamHI sites of pBI121 without the uidA gene, which resulted in the
insertion of the PsUGT1 sequence into pBI121, in the opposite
orientation (Woo et al. 1999). The resulting construct expressing
the CaMV 35S::PsUGT1 antisense mRNA was transformed into E.
coli HB101 and then into Agrobacterium tumefaciens LBA4404 by
triparental conjugation.
Plant transformation, selection, and analysis of transgenic plants
Alfalfa leaf disks were transformed as described by Woo et al.
(1999). In brief, leaf disks from Medicago sativa cv. Regen were
transformed with A. tumefaciens strain LBA4404 using the alfalfa
transformation and regeneration procedure described by Fang and
Hirsch (1998). For each experiment, 50 leaf disks were inoculated,
and embryos were selected and regenerated on appropriate media
with kanamycin, as described (Fang and Hirsch 1998). Five
independent transformation experiments were carried out, with
hundreds of kanamycin-resistant embryos obtained for each experiment using the five CaMV 35S::PsUGT1 antisense mRNA
constructs. Among these, clonal plants generated from 23 kanamycin-resistant embryos were selected for further analysis. As a
negative control, transgenic plants transformed with the pBI121
vector only were also generated and used for comparison.
For the selection of transgenic plants, young primary transformed
plants were analyzed for the presence of the transgene by PCR using
Taq polymerase. Template DNA was obtained from leaves by
standard procedures (Sambrook et al. 1989). The following specific
primer set was used for detection of the transgene: CaMV-1 (50 GTTAGAGAGGCTTACGCAGCAGG-TC-30 ) with PsUGT-1 (50 CGGAGGCGTTAATAAGAATTGTGAA-30 ). This will amplify
1,580-bp 35S CaMV-PsUGT1 antisense DNA which is present only
in transgenic plants (not shown).
Since CaMV 35S::PsUGT1 antisense mRNA plants are sterile
(Woo et al. 1999), the transgenic plants were propagated vegetatively. Healthy stems were cut and rooted for 14 days in a 1:1
perlite and vermiculite mixture. The rooted plants then were
transferred into soil. Plants expressing the transgene were propagated for at least ten cycles of subculture before phenotypic
analyses (morphology, rate of root growth, appearance, infection
assays, etc.) were carried out.
For Northern blot analysis, total RNA was isolated from roots of
wild-type and transgenic plants. Total RNA was fractionated on a
1% agarose formaldehyde gel and blotted onto nylon membrane.
[32P]-Labeled probes were generated by random priming using
PsUGT1 cDNA and a Pisum sativum root-cap-expressed gene
encoding ubiquitin conjugating enzyme (PsUBC4) cDNA (Woo et
al. 1994) as a positive control. Northern blot hybridization was
carried out by standard procedures (Sambrook et al. 1989).
Nectria haematococca inoculation and infection assays
The test organism for fungal infection assays was N. haematococca
(Berkeley and Broome) MPVI isolate 3418. Cultures were obtained
from H.D. VanEtten (Department of Plant Pathology, University of
Arizona, Tucson, Arizona, USA) and were maintained on V8 juice
agar (Stevens 1974). Subcultures were incubated at room temperature for 48 h before conidia were harvested by rubbing from the
agar surface; conidial suspensions were washed in water (3,500 g
for 1 min) and adjusted to 105 spores per milliliter. Eighteen-dayold stem cuttings with adventitious roots 3–5 cm in length were
pulled from the propagation mixture (vermiculite:perlite, 1:1) to
avoid wounding; the root-vermiculite mixture was pulled from pots
as a mass, which was gently manipulated by hand until the potting
mixture fell away, leaving the roots free. The roots then were
dipped into water to remove existing border cells and to induce the
production of a new set of cells (Brigham et al. 1998), then returned
to the propagation mixture (vermiculite:perlite, 1:1). Twenty-four
hours later stem cuttings with adventitious roots were again
removed, as described. Each root was inoculated with 200 ml of the
spore suspension distributed uniformly over the entire root, then
inserted into cellophane growth pouches pre-wetted with 10 ml
water, which is sufficient to wet the paper wick without leaving a
pool of free water at the bottom (Gunawardena and Hawes 2002).
Following incubation in a growth chamber [16/8-h (light/dark)photoperiod, 23C] for 2 days, the root tips were briefly rinsed with
water to remove the border cells and unattached hypha just before
observation. The pattern of infection was evaluated based on the
development of necrotic lesions (including all visible lesions, from
969
tan to dark brown to black in color) at the root tip (0–1.5 mm from
the root apex) and the region of elongation (approx. 1.5–10 mm
behind the root apex) by direct observation under magnification(3), as described by Gunawardena and Hawes (2002). The
experiment was carried out twice.
Sinorhizobium meliloti inoculation and infection assays
Eighteen-day-old stem cuttings with adventitious roots 3–7 cm in
length were removed from the propagation mixture (vermiculite:perlite, 1:1) as described above and inoculated by dipping
into a suspension of S. meliloti 1021, a reporter strain constitutively
expressing b-galactosidase (Yelton et al. 1987). Bacteria were
harvested after overnight growth to the logarithmic phase and were
washed twice in water to remove culture medium before being used
to inoculate roots. Inoculated stem cuttings with adventitious roots
were grown in the propagation mixture (vermiculite:perlite, 1:1)
without a nitrogen source in a controlled environment chamber [16/
8-h (light/dark) photoperiod, 23C). Stem cuttings were collected
every 2 days for up to 22 days. Nodulation was measured by
directly counting the number of nodules per root following staining
of the roots with X-gal using standard procedures.
Roots and nodules were fixed in FAA and embedded in paraffin
according to standard procedures (Berlyn and Miksche 1976).
Embedded tissue was sectioned to 10-mm thicknesses. Slides were
stained with toluidine blue O according to Berlyn and Miksche
(1976). Morphology of the root surface, root hairs, and nodules
from both wild-type and antisense roots was compared by a direct
evaluation of intact roots or cross-sectioned tissue using a dissecting microscope at 10 magnification.
Results and discussion
Generation and analysis of transgenic alfalfa expressing
CaMV 35S::PsUGT1 antisense mRNA
1999), flowers from transgenic alfalfa expressing CaMV
35S::PsUGT1 antisense mRNA are sterile and do not
produce seeds. Stem cuttings, which induce adventitious
roots, were used for clonal plant propagation. After ten
cycles of propagation with stem cuttings, the stable
maintenance of CaMV 35S::PsUGT1 antisense mRNA
in transgenic lines was confirmed by PCR analysis (not
shown). Northern blot analysis under both low-stringent
and high-stringent conditions showed that expression of a
single alfalfa transcript corresponding to PsUGT1 was not
detectable in transgenic plants expressing antisense
mRNA (not shown).
Altered root growth in plants expressing
PsUGT1 antisense mRNA
After 8–11 days in perlite:vermiculite, stem cuttings
developed adventitious root initials. After 23 days, wildtype and vector-only transformed transgenic stems
formed 15–16 adventitious roots, with an average length
of 18.6€2.5 cm (not shown). In the same time period,
stems from clones expressing PsUGT1 antisense mRNA
formed six to seven adventitious roots with an average
length of 8.25€1.73 cm (not shown).
Growth rate in wild-type and vector-only transformed
transgenic roots was 2.5-fold faster than in CaMV
35S::PsUGT1 antisense mRNA transgenic roots. Over a
1-week period between 17 days and 23 days, mean root
growth in adventitious roots was 1.9 cm/day in wild-type
and vector-only transformed transgenic clones and 0.77 cm/
day in clones expressing PsUGT1 antisense mRNA.
PsUGT1 antisense mRNA expressed under the control of
the CaMV 35S promoter was transformed into alfalfa
(Woo et al. 1999). As described previously (Woo et al.
Fig. 1a–d Delayed production of root border cells in roots of
transgenic alfalfa plants expressing CaMV 35S::PsUGT1 antisense
mRNA. a Wild-type and vector-only transformed transgenic roots:
after border cells are removed by immersion of the root tip into
water for 60 s with gentle agitation, the root-cap periphery is
smooth and free of cells. b Wild-type and vector-only transformed
transgenic roots: after 24 h, a new set of 1,800€300 cells has been
synthesized by the root cap and is visible at the root-cap periphery
following immersion in water for 5–10 s. c Roots from plants
expressing CaMV 35S::PsUGT1 antisense mRNA: after border
cells are removed, the cap periphery is smooth and free of cells and
indistinguishable in appearance from that of wild-type and vectoronly transformed transgenic root tips. d After 24 h, virtually no
border cells are present at the cap periphery of plants expressing
CaMV 35S::PsUGT1 antisense mRNA; total numbers are reduced
to 15€5 cells per root
970
Fig. 2a–d Increased root-tip
infection by Nectria haematocca in transgenic alfalfa plants
expressing CaMV 35S::
PsUGT1 antisense mRNA. a
Infection in the region of elongation: wild-type alfalfa, vector-only control plants, or plants
expressing CaMV 35S::
PsUGT1 antisense mRNA.
Roots of seedlings (root length
20–25 mm) (left panel) were
inoculated uniformly with
spores. Two days later, more
than 90% of the inoculated
roots had developed light-tan
lesions (right panel) at the site
that had been the region of
elongation, just behind the root
tip, at the time of inoculation
(arrow). Similar results were
obtained with all three plant
lines. b Infection at the root tip:
wild-type and vector-only
transformed transgenic root
tips. In more than 90% of the
inoculated plants, root tips are
white and free of infection. c,d
Infection at the root tip: plants
expressing CaMV 35S::
PsUGT1 antisense mRNA. Necrotic lesions developed at the
root tip of most roots within
2 days of inoculation
Reduced border-cell production in roots
of plants expressing PsUGT1 antisense mRNA
Border cells were removed from wild-type, vector-only
transformed transgenic roots (Fig. 1a), and CaMV
35S::PsUGT1 antisense mRNA transgenic (Fig. 1c) roots
by immersing the tips in water and agitating gently
(Brigham et al. 1998). After incubation for 24 h in sealed
growth pouches with 10 ml water, wild-type and vectoronly transformed transgenic root caps had regenerated the
1,800€300 cells normally present on alfalfa root tips
(Fig. 1b). The morphology of the wild-type and vector-only
transformed transgenic root caps (Fig. 1a,b) was indistinguishable in appearance from that of root caps from plants
expressing PsUGT1 antisense mRNA (Fig. 1c,d). However, during the 24-h period after removal of the border cells
from the transgenic root caps fewer than 20 border cells per
root were produced (Fig. 1d).
Increased lesion development in root tips in response
to fungal inoculation in plants expressing
CaMV 35S::PsUGT1 antisense mRNA
In wild-type and vector-only transformed transgenic
alfalfa, the inoculation of roots with N. haematococca
resulted in the development of a light-tan to brown lesion
localized in the region of elongation (Fig. 2a) in more
than 90% of the inoculated seedlings; when this tissue
was surface-sterilized and plated onto culture medium,
hyphal growth was observed within 24 h (not shown). No
fungal hyphae emerged from tissue without visible lesion
971
Fig. 3a–g Normal binding, hair
curling, and nodule morphology
(a–e) but reduced frequency of
nodulation (f,g) in alfalfa roots
of transgenic plants expressing
CaMV 35S::PsUGT1 antisense
mRNA. a Attachment of
Sinorhizobium meliloti to elongation zone of wild-type or
vector-only transgenic roots visualized by expression of a GFP
(green fluorescent protein) reporter strain. b Attachment to
elongation zone of roots of
transgenic plants expressing
CaMV 35S::PsUGT1 antisense
mRNA. c Root-hair curling in
CaMV 35S::PsUGT1 antisense
transgenic plants is normal, as
in wild-type and vector-only
transgenic lines. Arrow indicates normal infection thread
formation. d Cross-section of
nodules from wild-type or vector-only transgenic roots. e
Cross-section of nodules in
transgenic plants expressing
CaMV 35S::PsUGT1 antisense
mRNA. f Nodule development
in wild-type or vector-only
transgenic roots, with nodules
visualized by histochemical
staining of an S. meliloti LacZ
reporter strain. g Nodule development in plants expressing
CaMV 35S::PsUGT1 antisense
mRNA. Bars (f,g): 2 cm
development (not shown). However, most root tips did
not develop lesions, with more than 90% (50/51 in two
experiments) of the root tips remaining white and free of
visible lesions (Fig. 2b) and producing no fungal growth
when surface-sterilized and plated onto culture medium
(not shown). A similar pattern of localized infection
occurs in pea seedlings inoculated with N. haematococca
(Gunawardena and Hawes 2002).
In roots of clones expressing CaMV 35S::PsUGT1
antisense mRNA, there was no change compared to wild
type in the frequency of lesion development in the region
behind the root tip (as in Fig. 2a). Most seedlings
developed localized necrotic lesions in the region of
elongation just behind the root tip, and there was no
increase in frequency or severity of infection. Thus, the
normal regulation of infection in the region of elongation
appeared to remain intact despite a 24-h slower rate of
root growth and development. Bruehl (1986) proposed
that localization of infection in the region of elongation is
defined based on tissue maturation; thus, infection occurs
only until secondary thickening of cell walls has ensued.
If so, then genes expressed specifically within the region
972
of elongation may be worthwhile targets for examining
mechanisms that dictate susceptibility to pathogenic
invasion (BaluÐka et al. 1996; Beemster and Baskin
2000; Fowler et al. 1999; Kiegle et al. 2000; Lee et al.
2003; Verslues and Sharp 1999; Vissenberg et al. 2000).
Infection in the region of elongation in lines expressing
antisense mRNA was unaffected compared with that of
the wild type, but there was a marked change in lesion
formation at the root tip (Fig. 2). Thus, virtually all
seedlings (46/48 in two independent experiments) developed visible necrosis at the root tip within 24–48 h after
inoculation (Fig. 2c,d). When tips with visible lesions
were surface sterilized and plated onto culture medium,
fungal hyphae emerged from all samples (not shown). No
hyphae emerged from root tips without necrotic lesions.
Reduced nodulation on roots of plants expressing
CaMV 35S::PsUGT1 antisense mRNA
Susceptibility of roots to infection by symbiotic nitrogenfixing bacteria, which also initiate infection predominantly in the region just behind the root tip, was investigated.
The attachment of S. meliloti to root surfaces of the
elongation zone of wild-type and vector-only transformed
transgenic plants (Fig. 3a) was indistinguishable from that
to CaMV35S::PsUGT1 antisense mRNA transgenic roots
(Fig. 3b).
Root-hair curling in CaMV 35S::PsUGT1 antisense
transgenic plants (Fig. 3c) occurred in response to inoculation with S. meliloti, as in wild-type and vector-only
transformed transgenic plants (not shown). Infection
thread formation, cell shape and organization, bacteroid
development, and overall morphology of the nodules that
formed on roots of the wild-type and vector-only transformed transgenic plants (Fig. 3d) were indistinguishable
from those that formed on plants expressing CaMV
35S::PsUGT1 antisense mRNA (Fig. 3e). There were no
distinct differences in the number of infection threads in
the wild type, vector-only transformed transgenic plants
and the CaMV35S::PsUGT1 antisense transgenic plants
(Fig. 3c).
In contrast, the frequency of nodulation on CaMV
35S::PsUGT1 antisense transgenic lines at 12 days postinoculation (dpi) was fourfold lower than that of the wildtype and vector-only transformed transgenic plants. The
number of nodules in wild-type and vector-only transformed transgenic plants ranged from 10 to 15 per root
(mean 12€4) (Fig. 3f). In contrast, in the roots of plants
expressing CaMV 35S::PsUGT1 antisense mRNA, the
number of nodules at 12 dpi ranged from zero to six
nodules per root (mean 3€3) (Fig. 3g). The differences
were statistically significant at P<0.0001.
The nodulation of plant roots is a tightly controlled
process that occurs in a specific tissue during a specific,
narrow window of plant development (Bauer 1981; Fang
and Hirsch 1998; Foucher and Kondorosi 2000; Hirsch
1992; Hirsch and Fang 1994; Hirsch et al. 2003; Penmetsa
et al. 2003; Thimann 1936). Initial recognition involves
plant receptors that are required for early stages of contact
(Catoira et al. 2000; Endre et al. 2002; Stracke et al. 2002).
In the current study, there were no obvious differences in
binding, hair curling, and infection thread formation,
suggesting that early stages of recognition are unaffected
in plants expressing CaMV 35S::PsUGT1 antisense
mRNA. Moreover, the few nodules that formed on
antisense plants were indistinguishable in all aspects from
those of wild-type roots, indicating that processes leading
to nodule formation and function remain intact but that
efficiency of nodulation is severely impacted. The loss of
efficiency could result directly from alterations in the cell
cycle (Foucher and Kondorosi 2000), reduced numbers of
border cells, other root-tip exudates which are a significant
source of nodulation gene-inducing chemicals (Peters and
Long 1988; Zhu et al. 1997), or other indirect effects of an
altered cell-cycle duration.
The results of the current study reveal that altering the
expression of a single plant gene can fundamentally alter
the infection of roots by a pathogenic fungus and a
symbiotic bacterium. Further studies to characterize the
physiological bases for these changes may suggest novel
approaches for controlling beneficial and pathogenic rootmicrobe associations. In light of the current results,
previously hypothesized roles for root growth (Wilcox
1996) and/or release of exudates from the root tip (Bowen
and Rovira 2000; Hawes et al. 1998) in defining the
patterns of localized root infection cannot be ruled out.
Acknowledgments This work was supported in part by grants to
M.C.H. from the United States Department of Agriculture; to
M.C.H. and H.-H.W. from the National Science Foundation and
from the Department of Energy, Division of Energy Biosciences;
by the College of Agriculture and Life Sciences Agricultural
Experiment Station, University of Arizona; and by NIH/NCCAMSPSO AT00151 to the Center for Dietary Supplement Research
Botanicals (CDSRB) at UCLA (A.M.H., Director, Agricultural
Botany Core and H.-H.W., Junior Investigator).
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