277
Molecular Breeding 11: 277–285, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Overexpression of glutamate decarboxylase in transgenic tobacco plants
confers resistance to the northern root-knot nematode
Michael D. McLean 1,4, Dmytro P. Yevtushenko 1,5, Alice Deschene 1, Owen R. Van
Cauwenberghe 1,6, Amina Makhmoudova 1, John W. Potter 2, Alan W. Bown 3 and Barry J.
Shelp 1,*
1
Department of Plant Agriculture, Biotechnology Division, University of Guelph, Bovey Bldg, Guelph, N1G
2W1, ON, Canada; 2Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research
Centre, Vineland Station, L0R 2E0, ON, Canada; 3Department of Biological Sciences, Brock University, St
Catharines, L2S 3A1, ON, Canada; 4Current address: Department of Environmental Biology, University of
Guelph, N1G 2W1, ON, Canada; 5Department of Biochemistry and Microbiology, University of Victoria,
Victoria, V8W 3P6, BC, Canada; 6Lilly Analytical Research Laboratory, Eli Lilly Canada Inc., 3650 Danforth
Ave., Toronto, M1N 2E8, ON, Canada; *Author for correspondence (e-mail: [email protected]; phone:
519-824-4120 ext 53089; fax: 519-767-0755)
Received 1 July 2002; accepted in revised form 20 December 2002
Key words: Gamma-aminobutyrate, Glutamate decarboxylase, Plant defense, Root-knot nematode, Tobacco,
Transgenic plants
Abstract
Previous research suggests that the endogenous synthesis of gamma-aminobutyrate (GABA), a naturally occurring inhibitory neurotransmitter, serves as a plant defense mechanism against invertebrate pests. Here, we tested
the hypothesis that elevated GABA levels in engineered tobacco confer resistance to the northern root nematode
(Meloidogyne hapla). This nematode species was chosen because of its sedentary nature and economic importance in Canada. We derived nine phenotypically normal, homozygous lines of transgenic tobacco (Nicotiana
tabacum L.), which contain one or two copies of a full-length, chimeric tobacco glutamate decarboxylase (GAD)
cDNA or a mutant version that lacks the autoinhibitory calmodulin-binding domain, under the control of a chimeric octopine synthase/mannopine synthase promoter. Regardless of experimental protocol, uninfected transgenic lines consistently contained higher GABA concentrations than wild-type controls. Growth chamber trials
revealed that 9–12 weeks after inoculation of tobacco transplants with the northern root-knot nematode, mature
plants of five lines possessed significantly fewer egg masses on the root surface when the data were expressed on
both root and root fresh weight bases. Therefore, it can be concluded that constitutive transgenic expression of
GAD conferred resistance against the root-knot nematode in phenotypically normal tobacco plants, probably via
a GABA-based mechanism.
Abbreviations: CaM – calmodulin, CaMBD – calmodulin-binding domain, GABA – gamma-aminobutyrate,
GAD – glutamate decarboxylase, GAD plants – plants expressing the full-length GAD, GAD⌬C plants – plants
expressing a mutant GAD that lacks the CaMBD, WT – wild type
Introduction
Parasitic root nematodes cause estimated worldwide
losses of US$100 billion annually and average yield
losses in excess of 10% to a variety of field, vegeta-
ble, fruit and ornamental crops (Sasser 1980; Sasser
and Freckman 1987; Potter and Olthof 1993; Atkinson et al. 1995). They comprise a large phylum of
animals, with three genera (i.e., the cyst nematodes
Heterodera and Globodera, and the root-knot nema-
278
todes or Meloidogyne) causing the most agricultural
damage (Barker 1998; Ferris and Ferris 1998). Females of the root-knot nematodes exhibit a prolonged
sedentary phase during which they modify root cells
into feeding sites (Niebel et al. 1994).
Nematode management in traditional and subsistence agricultural systems has involved the use of
cultural practices such as crop rotation, fallows, antagonistic plant species, organic soil amendments and
biological control (Bridge 1996). These practices
have been largely replaced in high-input agricultural
systems with nematicides (including soil fumigants)
and resistant cultivars. However, the active ingredients of fumigants are either known human carcinogens or chlorinated hydrocarbons that contribute to
the deterioration of the ozone layer. Legislation in
Canada and the United States has restricted the usage
of some of these materials (Nolling and Becker 1994;
Marcotte and Tibelius 1998). The use of resistant cultivars can be the most useful and economical practice
for controlling nematodes; however, natural crop resistance is often limited by its lack of durability under varying environmental conditions or challenge
from different species, pathotypes or races (Niebel et
al. 1994; Bridge 1996; Atkinson et al. 1998).
In recent years the ability to genetically engineer
plants has revolutionized the possibilities for nematode control (Atkinson et al. 1998; Jung and Wyss
1999; Williamson 1999). Transgenic approaches include the transfer and manipulation of natural resistance genes such as Hs1 pro-1, as well as direct interference with feeding cells or the nematode itself. In
the latter case, strategies have targeted proteinases,
proteinase inhibitors, lectins, antibodies and endotoxins. Transgenic resistance to nematodes will soon be
available; however, new targets and strategies must be
identified to keep pace with resistance-breaking abilities of nematodes (Atkinson et al. 1998).
Gamma-aminobutyrate (GABA) is a naturally occurring inhibitory neurotransmitter that has ready access to the central nervous system of invertebrates
when ingested, but not that of vertebrates such as man
(Kuriyama and Sze 1971; Cooper et al. 1982). The
addition of GABA to the saline solution bathing Ascaris muscle preparations causes hyperpolarization of
muscle cells, suggesting that functioning of the nervous system of invertebrates would be disrupted with
the ingestion of GABA (Del Castillo et al. 1964).
Also, the growth and development of oblique-banded
leafroller larvae are deterred by inclusion of GABA
in an artificial diet (Ramputh and Bown 1996), and
the ambulatory activity of this insect, as well as that
of the tobacco budworm, causes the accumulation of
GABA in leaves (Bown et al. 2002). Together, these
observations suggest that the endogenous synthesis of
GABA serves as a plant defense mechanism.
Typically, GABA levels are low in plants, but increase several times in response to many diverse stimuli such as oxygen or temperature shock (Bown and
Shelp 1997; Shelp et al. 1999; Kinnersley and Turano
2000). This result can be attributed to increases in
cytosolic H + or calcium/calmodulin (CaM) concentrations that directly affect the activity of the enzyme
responsible for the synthesis of GABA, glutamate decarboxylase (GAD; EC 4.1.1.15). In particular, calcium/CaM binds to a carboxyl-terminal domain of the
plant GAD, thereby relieving an autoinhibition of
GAD activity. Transgenic tobacco plants constitutively overexpressing a petunia GAD contain elevated
GABA concentrations; those plants with a mutant
GAD lacking the calmodulin-binding domain
(CaMBD) have the highest GABA concentrations, but
are sterile and exhibit severe morphological abnormalities (Baum et al. 1996).
Here, we tested the hypothesis that elevated
GABA levels in engineered tobacco confer resistance
to the northern root-knot nematode. This nematode
species was chosen as the test organism because of
its sedentary nature and agricultural importance in
Canada. Phenotypically normal, homozygous lines of
transgenic tobacco, which overexpressed either a fulllength native GAD (GAD plants) or a mutant GAD
lacking the CaMBD (GAD⌬C plants), were generated
and found to exhibit consistently higher GABA concentrations and resistance to the northern root-knot
nematode than control plants.
Materials and methods
Plant materials and growth conditions
Tobacco (Nicotiana tabacum L cvs. Delgold and
Samsun NN) plants were grown in a University of
Guelph greenhouse in a soil mix containing Sunshine
Mix 2 (Sun Gro Horticulture Inc., Bellevue, WA) as
described previously (Scott-Taggart et al. 1999), or in
controlled environment chambers (Conviron Ltd,
Winnipeg, Canada) using a combination of inflorescence and incandescent lamps to supply a 16-h photoperiod (a photosynthetic photon flux density of 250
µmol m −2 s −1 at the top of the seedling tray), a day/
279
night temperature of 23/18 °C, and 40–65% relative
humidity. Where appropriate, seeds were also sterilized and grown aseptically in Magenta vessels under
tissue-culture-room conditions (16-h photoperiod
with a day/night temperature of 22/19 °C, inflorescence lamps providing a photosynthetic photon flux
density of 80 µmol m −2 s −1 at the shelf surface), or
grown on filter paper in petri dishes at room temperature inside a dark cupboard.
cDNA library preparation and screening
A cDNA library was constructed with reverse transcribed poly(A) + RNA isolated from mature leaves of
greenhouse-grown ‘Samsun NN’ tobacco and cloned,
using the ZAP-cDNA synthesis kit, into the Uni-ZAP
XR vector (Stratagene, La Jolla, CA). Recombinant
bacteriophage was packaged in vitro using the Gigapack kit (Stratagene). A 1.35-kb BamHI/EcoRI fragment of a petunia GAD cDNA (Baum et al. 1993) was
labeled with [␣- 32P]dCTP using the Prime-it II random primer labeling kit (Stratagene), and used to
probe 1.1 × 10 6 recombinant bacteriophage cDNA
clones which were blotted onto Gene Screen Plus hybridization transfer membranes (NEN Life Science
Products, Boston, MA). Prehybridization and hybridization were performed at 55 °C in an aqueous buffer
containing 10% dextran sulphate (Na salt, MW
500,000) (w/v), 1% SDS (w/v), 1 M NaCl and 100
µg mL −1 denatured and sonicated salmon sperm
DNA. The final wash of the membranes was in 0.2X
SSC and 0.1% SDS at 60 °C for 30 min (Sambrook
et al. 1989). Positive plaques were isolated and subjected to secondary and tertiary screening under the
same conditions. The cDNA inserts in positive
plaques from tertiary screening were excised in vivo
according to the Stratagene protocol, and insert-containing clones of Bluescript phagemids in the E. coli
SOLR strain were analyzed by restriction enzyme digestion and hybridization using the petunia GAD
probe, and by DNA sequencing performed at the
Guelph Molecular Supercenter on a ABI prism 377
DNA sequencer using the dideoxy termination
method (Sanger et al. 1977).
Transgene construction
One cDNA clone appeared to contain a full-length
GAD cDNA sequence; however, a two-nucleotide deletion in codon 18 was detected, causing a frame-shift
mutation with a premature stop codon after amino
acid 29. This was repaired with a DNA fragment produced using PCR with primers 5⬘-GGAGTCCATCATAAGCTTATC-3⬘ and 5⬘-CTTCTAGATCGTACTACCACCACTACGCC-3⬘ and tobacco ‘Samsun
NN’ cDNA as a template. This fragment was cloned
into the 5⬘ end of the GAD cDNA taking advantage
of an EcoRI site between codons 34 to 36.
The repaired GAD cDNA was subcloned downstream of the chimeric octopine synthase/mannopine
synthase ‘superpromoter’ between the XbaI and SacI
restriction endonuclease sites in pE1068 (Ni et al.
1995). The resulting superpromoter/GAD gene cassette was excised using SalI and SacI restriction endonucleases and cloned into pMDM8, a plant binary
transformation vector produced in this laboratory as a
derivative of pBIN19 (Frisch et al. 1995). pMDM8
differs from pBIN19 by the introduction of two yeast
FLP recombination target sequences (Lyznik et al.
1996) at the PmeI and ClaI restriction sites which
flank the nptII resistance gene (nptII; Beck et al.
(1982)), and a 260-bp polyadenylation sequence from
the nos terminator (Depicker et al. 1982) between the
SacI and EcoRI sites. The T-DNA region of the resultant derivative pMDM8 plasmid bearing the superpromoter/GAD was designated pSPGAD.
One CaMBD-deletion plasmid, pSPGAD⌬C40,
lacking the forty C-terminal amino acids, was made
by digesting pSPGAD with NheI, and then preparing
and ligating the 9.0- and 4.7-kb fragments together in
order to delete the 235-bp NheI fragment, which contains the GAD CaMBD. The resulting coding sequence has a serine-tyrosine-cysteine three-aminoacid sequence after amino acid number 456 (alanine),
resulting in a predicted polypeptide of 459 amino acids. The pMDM8 plasmid was used as a ⬙no insert⬙
negative control plasmid for plant transformation.
Transgenic plant production
Primary transgenic ‘Delgold’ tobacco plants (T 0), harboring the T-DNA regions from pSPGAD (GAD
plants) or pSPGAD⌬C40 (GAD⌬C plants) were produced under tissue-culture-room conditions, using
Agrobacterium-mediated leaf disk transformation
(Horsch et al. 1985). Selection on 200 mg L −1 kanamycin sulfate was performed in the presence of 500
mg L −1 cefotaxime on Murashige and Skoog basal
medium with 0.1 mg L −1 1-naphthaleneacetic acid
and 1 mg L −1 6-benzylaminopurine. Healthy regenerated shoots were rooted in hormone-free Murashige
and Skoog basal medium containing 100 mg L −1 kan-
280
amycin, transferred to soil and grown, after one week
under shade cloth, to maturity in the greenhouse.
Wild-type (WT) ‘Delgold’ was also grown under tissue culture conditions, but kanamycin selection was
omitted.
Derivation of homozygous lines, and determination
of GABA levels
WT ‘Delgold’ and all kanamycin-resistant plants were
grown individually in 9-L pots that were placed randomly on the greenhouse benches. Two months after
transplanting, about five T 0 plants with small stature,
possibly due to root damage during transplanting,
were discarded. The tip of a young leaf was removed
from each of the remaining plants (62 GAD and 39
GAD⌬C), frozen in liquid nitrogen, and ground in
five volumes of sulphosalicylic acid (30 mg mL −1).
The homogenate was centrifuged, and the supernatant
removed and adjusted to pH 7 with 4 N NaOH and
stored at −20 °C prior to GABA analysis by highperformance liquid chromatography (Oaks et al.
1986)
Using genomic blot hybridization and standard
protocols, transgene copy number was determined in
the ten primary transformants of both GAD and
GAD⌬C plants with the highest GABA concentrations (Southern 1975; Sambrook et al. 1989). Hybridization was performed with a 789-bp GAD DNA fragment (from a BclI restriction site at codon number
310 to the end of the 3⬘ untranslated region) as a
probe labeled with [␣- 32P]dCTP to a specific activity
of > 10 9 cpm µg −1. Plants with one or two transgene
copies were allowed to set T 1 seed and 10 to 20 progeny from each were grown and allowed to set T 2 seed.
To reduce the requirements for greenhouse space and
save time, DNA was purified from T 1 plants at about
two months of age, and analyzed by BclI digestion
and Southern blot hybridization as described above.
As the inheritance of each transgene band was expected to follow a Mendelian 1:2:1 ratio of inheritance, the relative intensities of bands from a homozygous transgene locus should therefore be twice the
relative intensity of that from a hemizygous transgene
locus. Native GAD gene bands served as internal
loading controls, and a plant was judged to be homozygous for a transgene locus when its band had
double intensity with respect to sibling transgene
bands (Krakowsky et al. 1993). Nine plants, which
were judged to be homozygous, were propagated to
obtain sufficient T 3 or T 4 progeny for further inves-
tigation of GABA concentration and nematode resistance. Homozygosity was verified by the observation
of 100% resistance of seedlings on kanamycin plates.
The progeny derived from each T 0 plant was designated with a whole number, with lines 1–7 representing GAD plants and lines 1–2⌬C representing
GAD⌬C plants. Lines 1–5, 7, and 1–2⌬C contained
one transgene copy, whereas line 6 contained two
transgene copies. Furthermore, lines 1–2, 3, 4–6, 7,
and 1–2⌬C were derived from separate transformation events. Kanamycin-resistant plants created with
the empty pMDM8 vector were also grown to maturity and homozygous T 1 plants selected by genomic
blot hybridization with an nptII specific probe. A homozygous line with a single T-DNA insertion was
chosen for use as a negative control in the nematode
resistance bioassays described below.
Seeds of the WT and homozygous transgenic lines
were grown at room temperature in the dark in petri
dishes containing filter paper soaked with 1 mM calcium sulphate. After two weeks, liquid nitrogen was
poured over the seedlings and allowed to evaporate.
Then the frozen seedlings were scraped from the filter paper into additional liquid nitrogen and prepared
for HPLC determination of GABA concentrations as
described above. Alternatively, seeds were individually germinated in Magenta vessels on hormone-free
Murashige and Skoog agar medium containing 2%
(w/v) sucrose, and grown in a tissue-culture room.
After twelve weeks, each shoot was rapidly removed
with scissors and placed in liquid nitrogen. Then the
roots were slowly removed from the medium using
tweezers and frozen in liquid nitrogen. All samples
were allowed to thaw for 10 min (this was designated
as the freeze-thaw protocol) before the GABA concentrations were determined.
Nematode resistance bioassay
Resistance of the transgenic lines against the northern root-knot nematode (Meloidogyne hapla) was assessed as the number of egg masses present on the
root surface (Omwega et al. 1990). Separate, staggered experiments were conducted for each line in
order to minimize requirements for growth- chamber
space and the length of time spent at the microscope.
The experiments were conducted in a Rubbermaid ™
storage bin (45.7 × 35.6 × 30.5 cm) located in a controlled environment chamber, drilled in the bottom for
drainage, and filled with 29 L of a Fox sandy loam
(pH 6.5), which had been used previously to main-
281
tain a northern root-knot nematode culture on a tomato (Lycopersicon esculentum L. cv. Bonnie Best)
host. Two rootrainers (Model Hillson #170-4, Spencer-Lemarie Rootrainers, Edmonton, AB), each containing four 170-mL cells, were positioned in the soil
about 10 cm from either side of the bin. WT tobacco
and transgenic plants from one line were transplanted
at the three-leaf-stage, from seedling trays filled with
the sandy loam, to alternate cells of the rootrainers.
The roots within the rootrainer were inoculated, using a syringe, with 2000–4000 eggs and/or J2 juveniles that were freshly collected from infested tomato
plants using a sieving method as described elsewhere
(Barker 1985). The plants were supplied with tap water and liquid fertilizer as required (Scott-Taggart et
al. 1999). Two weeks after transplanting, plants were
culled so that four-six healthy plants of each genotype
remained. Six weeks after transplanting, the plants
were detopped to the sixth node. Nine to twelve
weeks after transplanting the fresh weight of roots
within the cell was determined, as well as the number
of egg masses present on the root surface by microscopic examination. Mean (± S.E.) egg mass number
of the WT controls ranged from 15 ± 5 to 133 ± 14
per root in the various experiments.
Statistical analyses
Variability is indicated by standard error in Figures 1,
2 and 3. Statistical analyses of the raw data in Figures 1, 2 and 3 were performed using a Kruskal-Wallis one-way ANOVA and comparison of mean ranks
for transgenic and control plants (Anonymous 2000).
Results
Two types of GAD genes were constructed, one containing a full-length cDNA sequence encoding a predicted 496-amino acid polypeptide (GenBank Accession No. AF506366) with 86 to 99% identity to
known tobacco GADs (GenBank Accession Nos
AF020425,
U54774, AF352732, AF020424,
AF253615), the other identical but lacking the carboxyl-terminal 40 amino acids (i.e., nucleotides 13681488 being deleted). The resulting genes, cloned behind a constitutive promoter (Ni et al. 1995), were
used to transform tobacco. Five independent T 0 plants
with elevated GABA concentration and one or two
copies of the transgene, as indicated by Southern
analysis, were used to derive nine homozygous trans-
genic lines. These lines were subjected to two different experimental protocols to assess the impact of
GAD overexpression on GABA concentrations. In the
first protocol, seedlings grown in the dark for two
weeks possessed GABA concentrations that were
about 150–270% of that in the WT control (Figure 1).
In the second protocol, plants were grown for twelve
weeks in Magenta vessels under light conditions, and
then subjected to a freeze-thaw protocol. Shoot
GABA concentrations in seven of the nine lines were
significantly higher than that in the WT (140–380%
WT, Figure 2A). Also, root GABA concentrations
were higher in seven lines (160–2500% WT, Figure
2B).
When infested with the northern root-knot nematode, mature plants of seven of the nine transgenic
lines possessed significantly fewer egg masses than
WT plants whether the data were expressed on a root
basis (Figure 3A) or a root fresh weigh basis (Figure
3B). Five of the nine lines possessed fewer egg
masses than WT on both root and root fresh weigh
bases. On a root basis, GAD and GAD⌬C lines, respectively, had 0–42% and 3–33% of the egg masses
found on WT controls (Figure 3A). The egg mass
number, as well as the root fresh weight of a homozygous transgenic line expressing the empty vector was
not significantly different from the WT (data not
shown).
Discussion
GABA, a naturally occurring inhibitory neurotransmitter found in the nervous systems of both vertebrates and invertebrates, increases the membrane conductance of neurons to chloride ions, resulting in
hyperpolarization (Del Castillo et al. 1964; Satya
Narayan and Nair 1990). It is not toxic to vertebrates
when ingested or injected because it does not sufficiently penetrate the blood-brain barrier (Kuriyama
and Sze 1971; Cooper et al. 1982). In contrast, the
GABA receptors of the nervous system of invertebrates are exposed to the haemolymph and are not
protected by a blood-brain barrier (Irving et al. 1976,
1979). GABA receptors have been the target of many
synthetic pesticides, including the avermectins, but
their use has diminished or been inappropriate for
nematode control because of persistence, toxicology
or handling difficulties (Stretton et al. 1987; Casida
1993).
282
Figure 1. GABA concentrations of two-week-old seedlings of nine transgenic tobacco lines grown on filter paper in the dark and supplied
with 1 mM calcium sulphate only. Values, which represent the mean ± S.E. of three replicate petri dishes containing approximately 0.1 g of
seed each, can be directly compared to each other. Asterisks indicate that the transgenic line was significantly different from the WT control
(P ⭐ 0.05).
In plants, the function(s) of GABA is uncertain
(Bown and Shelp 1997; Shelp et al. 1999; Kinnersley
and Turano 2000), yet the enzyme responsible for the
synthesis of GABA from glutamate, GAD, is conserved in the plant kingdom. GAD genes from petunia, tomato, tobacco, Arabidopsis and rice have been
identified (Shelp et al. 1999; Akama et al. 2001), and
all but OsGAD2 (Akama et al. 2001) are likely activated via a stress-mediated signal transduction pathway involving the interaction of a calcium/CaM complex with an autoinhibitory domain on the protein.
The CaMBD of GAD is highly variable and generally located at the carboxyl-terminal end of the protein (Arazi et al. 1995; Yuan and Vogel 1998; Shelp
et al. 1999). Thus, deletion of the CaMBD should allow unrestricted synthesis and possibly accumulation
of GABA.
In the present study, homozygous transgenic plants
of tobacco overexpressing one or two copies of a tobacco GAD, under the control of the chimeric octopine synthase/mannopine synthase promoter, had an
enhanced capacity to accumulate GABA in both root
and shoot prior to nematode infection (Figures 1 and
2), as well as physical characteristics substantially
similar to those of the WT plant. Thus, both fulllength and mutant genes encoded proteins with GAD
activity, and GAD overexpression in tobacco was a
stable and heritable character. Recently, recombinant
expression of two native tobacco GAD cDNAs (GenBank Accession Nos. AF352732 and AF353615),
confirmed the existence of a regulatory CaMBD
within the carboxyl-terminal 40 amino acids of the
chimeric GAD used here (Yevtushenko D.P., McLean,
M.D., Peiris S. and Shelp BJ, unpublished data).
Baum et al. (1996) have produced T 0 tobacco
plants expressing, under the control of the constitutive CaMV 35S promoter, either a full length petunia
GAD or a mutant version lacking the carboxyl-terminal 27 amino acids that encompass the CaMBD.
While both GAD and GAD⌬C plants possess elevated
GABA concentrations in the stem or leaf, the GAD⌬C
plants also exhibit an altered phenotype (e.g., stunted
stature and sterility), which may be attributed to severe overexpression from the use of the CaMV 35S
promoter, the presence of multiple transgene copies
(McLean M.D. and Shelp B.J., unpublished data), or
higher shoot GABA concentrations than found here.
Unfortunately, the GABA data were expressed on a
mol% basis making direct comparison with the
present study difficult. Mammalian GAD, unlike plant
GAD, does not possess a CaMBD and expression of
a full-length GAD65 or GAD67 in transgenic tobacco
plants does not cause any phenotypic alterations (Ma
et al. 1997; Porceddu et al. 1999), although GAD67
283
Figure 2. GABA concentrations of shoots (A) and roots (B) of
twelve-week-old plants of nine transgenic tobacco lines grown in
Magenta vessels under light conditions, after exposure to a freezethaw cycle (i.e., abrupt freezing in liquid nitrogen, followed by a
10-min period at room temperature). Values, which represent the
mean ± S.E. of five to thirteen plants, can be directly compared to
each other. ⴱ and ⴱⴱ indicate that the transgenic line was significantly different from the WT control at P ⭐ 0.05 and P ⭐ 0.10,
respectively.
is inactive (McLean M.D. and Shelp B.J., unpublished data).
Unfortunately, determination of GABA concentration in the plant after nematode infection is complicated by the presence of the nematode, and the root
damage that occurs during sampling. This damage results in GAD activation and GABA accumulation
Figure 3. Number of egg masses on the root surface of chambergrown plants of nine transgenic tobacco lines, nine to twelve weeks
after inoculation with the root-knot nematode. For each line, the
value is expressed as a percent of the appropriate WT control, both
on a root basis (A) and a root fresh weight basis (B). Values, which
represent the mean ± S.E. of four to six plants, cannot be compared directly with each other, line 2⌬C was used in two separate
experiments. Statistical analysis was performed on the raw data. ⴱ
and ⴱⴱ indicate that the transgenic line was significantly different
from the WT control at P ⭐ 0.05 and P ⭐ 0.10, respectively.
(Ramputh and Bown 1996; Bown and Shelp 1997),
which complicates interpretation of the relationship
between GABA and nematode resistance.
Microscopic assessment of the root surface indicated that five of the nine transgenic lines possessed
significantly fewer egg masses than WT plants (Figure 3), suggesting that either the number or fecundity
284
of the reproductive females was reduced. Therefore,
it can be concluded that constitutive transgenic expression of GAD conferred resistance against the
root-knot nematode in phenotypically normal tobacco
plants, probably via a GABA-based mechanism. It is
envisaged that the engineered tobacco host would
contain elevated GABA concentrations under resting
conditions, and that GAD activity and GABA synthesis could be stimulated upon nematode infection and
feeding via independent increases in either cytosolic
calcium/calmodulin or H + (Shelp et al. 1999).
While this research provides further evidence to
support the view that plant GABA is a natural defense
against invertebrate pests, the root-knot nematode has
a prolonged sedentary phase in the host root, which
optimizes the impact of higher endogenous GABA
concentrations in engineered tobacco. Many invertebrate pests, unlike the root-knot nematode, are not
associated with the host for long periods, feeding
briefly at one or more sites on a plant and then moving on. To manage these pests using a GABA-based
mechanism, additional strategies such as the manipulation of specific calmodulins (Snedden and Fromm
1998), may be required to engineer higher endogenous concentrations of plant GABA.
Acknowledgements
The authors wish to thank: Dr. Hillel Fromm (Leeds
University) for providing petunia cDNA and tobacco
plants containing petunia GAD; Dr. Jim Brandle (Agriculture & Agri-Food Canada) for providing ‘Delgold’ seed; Dr. Stanton Gelvin (Purdue University)
for providing the superpromoter; and Dr. A.M. Jevnikar (University of Western Ontario) for providing tobacco plants containing mouse GAD67. This research
was supported by grants from the Natural Sciences
and Engineering Research Council (NSERC) of Canada Strategic Grants Program to B.J.S., A.W.B. and
J.W.P., the NSERC Canada Research Grants Program
to B.J.S., and the Ontario Ministry of Agriculture
Food and Rural Affairs to B.J.S.
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