Physiological and Molecular Plant Pathology (1999) 55, 341–348

Article No. pmpp.1999.0239, available online at http:}}www.idealibrary.com on

Identification of genes whose transcripts accumulate rapidly in tomato

after root-knot nematode infection
K. N. L A M B E R T, B. J. F E R R I E*, G. N O M B E L A†, E. D. B R E N N E R‡  V. M. W I L L I A M S O N§
Department of Nematology, UniŠersity of California, DaŠis, CA 95616, U.S.A.

(Accepted for publication September 1999)

Root-knot nematodes (Meloidogyne spp.) penetrate tomato (Lycopersicon esculentum) and other hosts near root
tips and migrate intercellularly to the developing vascular tissue where they induce formation of feeding
cells or, in the case of resistant tomato, trigger a localized necrosis. To investigate early events in the host
response to nematode infection, a seedling inoculation procedure was designed that produced hundreds
of root tips synchronously infected with Meloidogyne jaŠanica. RNA extracted from these tips was used to
produce a cDNA library. Differential screening of a subset of this cDNA library identified eight cDNA
clones representing genes that are reproducibly increased in level after nematode infection. Sequence
analysis revealed that two clones correspond to previously isolated genes encoding ascorbate free radical
reductase and an extensin. A third appears to encode a peroxidase. A fourth encodes a product similar to
a tumor-induced tobacco gene and belongs to the Kunitz trypsin inhibitor family of proteins. Another
encodes a protein highly similar to a tobacco LEA5-like protein. Transcripts of all eight genes are present
at higher levels after nematode infection in both susceptible and resistant tomato cultivars.
# 1999 Academic Press

Keywords : Meloidogyne jaŠanica ; Lycopersicon esculentum ; Mi ; differential screening ; host resistance.

INTRODUCTION root and migrates to the developing vascular tissue.

Root-knot nematodes (Meloidogyne spp.) are obligate,
sedentary endoparasites of many cultivated plants and
cause significant economic losses worldwide [33 ]. The
motile, second-stage juvenile (J2) is attracted to root tips
where it penetrates in the zone of elongation and migrates
intercellularly to the developing vascular tissue, causing
minimal damage to cells [8, 36 ]. In the vascular cylinder,
the nematode initiates a feeding site consisting of large,
multinucleate, metabolically active giant-cells in the host
[35 ]. Concurrently with giant cell formation, nearby cells
of the pericycle, cortex and vascular parenchyma enlarge
and divide forming the root-knot or gall that is charac-
teristic of infection by this group.
Some tomato lines are resistant to root-knot nematodes
due to the presence of a single, dominant gene, Mi [11 ].
In resistant, as in susceptible plants, the J2 penetrates the
*Present address : Celera AgGen, 1756 Picasso Ave., Davis,
CA 95616, U.S.A.
†Present address : Departmento de Proteccio! n Vegetal,
Centro de Ciencias Medioambientales (CSIC), Serrano 115
Dpdo, 28006 Madrid, Spain.
‡Present address : Department of Biology, 1009 Main Build-
ing, Washington Square, New York University, New York, NY
10003, U.S.A.
§To whom all correspondence should be addressed. E-mail :
vmwilliamson!ucdavis.edu
0885-5765}99}120341­08 $30.00}0
However, galls do not develop in resistant tomato. Instead,
a hypersensitive response (HR), consisting of a localized
necrosis, occurs in plant cells near the anterior end of the
nematode. The earliest visible symptoms of the resistance
response occur about 12 h after nematode penetration of
the root [27 ]. Mi has recently been cloned and was shown
to encode a plant gene of the nucleotide binding
site}leucine rich repeat class of genes that includes many
resistance genes [24 ].
Little is known about the changes in gene expression
that occur in the early stages of nematode invasion of
tomato roots. Altered gene expression is likely to be
critical for the establishment of the resistant or susceptible
state or may reflect early attempts of the plant at defense
against invasion in both susceptible and resistant hosts.
Molecular analysis of the early host response has been
difficult because of the highly localized interaction site.
Not only is the affected tissue found in the soil, but the
response may be limited to the few cells along the infection
path and proximal to the nematode’s attempted feeding
site. Producing sufficient numbers of synchronously
infected root tips is difficult and is the limiting step in the
analysis of the interaction. Here we present a seedling
infection assay and demonstrate its utility for the
identification of genes whose transripts are present at
increased levels early after infection by nematodes.
# 1999 Academic Press
342 K. N. Lambert et al.

MATERIALS AND METHODS (a)

Plants
Root-knot nematode-resistant tomato (Lycopersicon
esculentum Mill. cv VFN8) seed was obtained from Petoseed
(Woodland, CA, U.S.A.). Susceptible tomato (UC82b)
seed was obtained from Sunseeds Genetics, Inc. (Hollister,
CA, U.S.A.).

Preparation of nematodes
One day old Meloidogyne jaŠanica (Treub) Chitwood
juveniles (strain VW4) were collected from a hydroponic
culture [20], poured through a 245 µm pore sieve and
collected on a 20 µm pore sieve. The J2s were washed into
a 300 ml filtration apparatus (Millipore, Bedford, MA,
U.S.A.) lined with a 10 µm polycarbonate membrane
(Poretics, Livermore, CA, U.S.A.) and concentrated
using gentle vacuum. For the nematode cleaning, a
500 ml beaker with a metal screen fitting on top and
paper tissues (Kimwipes, Kimberly-Clark, Roswell, GA,
U.S.A.) were autoclaved. The beaker with the screen was
filled with sterile water, paper tissues were placed on top
so that they were wetted and the concentrated J2s were
added. After 6–12 h, J2s that had migrated through the
tissues were concentrated in an autoclaved filtration
apparatus containing a polycarbonate membrane as
described. The nematodes were allowed to migrate
through tissues a second time, then collected on a
polycarbonate membrane and resuspended in 2 ml of
sterile water. The suspension was adjusted to the desired
concentration for inoculation. All manipulations were
carried out aseptically in a laminar flow hood. In
experiments where antibiotic treatment is indicated,
nematodes were exposed to an antibiotic mix (0±05 %
each of Penicillin G and streptomycin sulfate) for the
6–12 h period during which they migrated through the
paper tissues.
Seedling growth and inoculation
Tomato seeds were sterilized and germinated as previously
described [14 ]. Germination of VFN8 seeds begun 1 day
prior to UC82b seeds to synchronize growth. When
seedlings reached 2–3 cm they were transferred to an
inoculation box consisting of a 23¬12¬8±3 cm poly-
propylene container (Fisher Scientific, Pittsburgh, PA,
U.S.A.) with a Teflon sheet (0±125 thick) (Small Parts
Inc., Miami Lakes, FL, U.S.A.) cut to form racks with
slots for holding glass plates (8 cm¬20 cm) [Fig. 1(a)].
The racks were taped inside each box and the bottom of
each container was lined with filter paper. Glass plates
with filter paper cut to the size of the plate were placed in
the racks. Each box was enclosed in a Sun Transparent
Bag (Sigma, St. Louis, MO, U.S.A.) and autoclaved. The
(b)
F. 1. Seedling growth and inoculation. (a) Inoculation box
showing notched teflon sheet and glass plates. (b) Inoculation of
seedling root tips with nematodes.
filter papers in the inoculation boxes were moistened with
50 ml of sterile water. Germinated seedlings were aligned
at least 0±5 cm apart on the filter paper lined glass plates
[Fig. 1(b)] and a sterile nylon mesh (44 µm pore size,
Small Parts Inc.) was laid over the seedlings.
The nematodes were suspended in an equal volume of
sterile 1 % carboxymethyl cellulose in water. For each
seedling, 50 µl of inoculum containing from 15–200
nematodes was pipetted 2 mm below the root tip on top
of the nylon mesh [Fig. 1(b)]. When the inoculum had
soaked into the filter paper, each plate was returned to the
box, the box was resealed in its plastic bag and placed
back into the incubator. For experimental controls, a
mock-inoculation using a sterile solution of 0±5 % carboxy-
methyl cellulose was performed.
After the specified incubation time, the seedlings were
removed from the boxes and about five were set aside.
The remaining seedlings were placed on water-saturated
filter paper with the tips of the roots aligned, and 3–5 mm
of root tip was excized and quickly frozen in liquid
nitrogen. The seedlings previously set aside were pressed
between two glass slides and the number of nematodes per
root counted under a dissecting microscope.
Construction of cDNA library
Following the procedure of Rochester et al. [29 ], total
RNA was extracted from 50 VFN8 tomato root tips which
had each been inoculated with 200 J2 and incubated for
 Nematode-induced genes
12 h. A cDNA library was constructed from poly (A)+
RNA using paramagnetic beads and PCR as previously
described [21 ]. A plasmid library of 1±2¬10& recom-
binants with inserts of about 300–1500 bp was constructed
in pBluescript (Stratagene, La Jolla, CA, U.S.A.).
Preparation of blots for differential screening
For colony blots, the cDNA library was plated and
screened as described by Conkling et al. [5 ]. For cDNA
blot preparation, bacterial colonies from the cDNA library
were transferred individually to a master plate and grown
overnight. A swipe of cells from each colony was
transferred to 0±5 ml microfuge tubes containing 10 µl of
sterile water. Forty µl of PCR mix was added to each tube
to give a final concentration of 8 m tetramethyl-
ammonium chloride, 20 m Tris-HCl (pH 8.3), 2±5 m
MgCl , 25 m KCl, 0±05 % Tween 20, 100 µg ml−"
# #
gelatin, 0±2 m dNTPs, 10 pmol each of M13 forward and
reverse primers and 2±5 U Taq polymerase (Promega,
Madison, WI, U.S.A.). The tubes were placed in a 95°C
pre-heated thermocycler for 2 min and subjected to 30
cycles of 95°C for 1 min, 50°C for 1 min, 72°C for 3 min.
The products were run on a 2 % agarose gel and stained
with ethidium bromide. For those clones containing
inserts, 2 µl of PCR products were spotted in a grid
pattern onto nylon membranes which had been pre-
conditioned in 10¬SSC and air dried. The membranes
were allowed to air dry and the DNA was denatured for
5 min on a filter saturated with 0±5  NaOH, 1±5  NaCl.
The membranes were neutralized and u.v. cross-linked.
All dot-blots were carried out in duplicate.
cDNA probe production and hybridization
Total RNA used to produce the cDNA probes was
extracted from approx. 150 tomato root tips each
inoculated with 200 J2 nematodes, and from tomato root
tips that had been mock-inoculated. The RNA pellet was
resuspended in 4 µl of DEPC-treated water. To produce
each cDNA probe, 1 µl of RNA solution was added to a
20 µl final volume reverse transcription mix containing
50 ng oligo (dT) – , 0±01  DTT, 0±2 m each dTTP,
"# ")
dATP, dGTP, 0±02 m dCTP, 10 U placental ribo-
nuclease inhibitor, 90 µCi [α-$#P]dCTP, 200 U MMLV
reverse transcriptase (Gibco BRL, Gaithersburg, MD,
U.S.A.), in the supplied reaction buffer. The reaction was
carried out at 42°C for 1 h followed by the addition of
0±5 µl of 25 m dNTPs and 100 U of reverse transcriptase
and a further 30 min incubation. The reaction was
stopped by the addition of 2 µl of 0±5  EDTA, diluted to
50 µl with water and passed through a CHROMA SPIN-
100 gel filtration column (Clontech, Palo Alto, CA,
U.S.A.).
The membranes containing the cDNAs were pre-
hybridized at 42°C in 5¬SSC, 5¬Denhardt’s solution,
343
0±5 % SDS, 200 µg ml−" ssDNA, 50 % formamide for at
least 2 h. The two first-strand cDNA probes were boiled
in a solution identical to the prehybridization solution
and hybridized with duplicate membranes at 42°C
overnight. The membranes were washed with increasing
stringency to 0±1¬SSC, 0±1 % SDS at 65°C before
autoradiography.
DNA and RNA blot analysis
Approximately 10 µg of tomato and 5 µg of nematode
genomic DNA [14, 31 ] were cleaved with EcoRI and
fractionated on a 1 % agarose gel. The DNA was
denatured and transferred to a nylon membrane [31 ].
Total RNA was isolated from root tips that had been
either mock-inoculated or nematode-inoculated for 11–
12±5 h as described previously, resuspended in DEPC-
treated H O, denatured using glyoxal}DMSO, electro-
phoresed on a 2 % agarose gel in phosphate buffer and
transferred to a nylon membrane [31 ]. The blots were
prehybridized and hybridized as described above. [α-$#P]-
labelled DNA probes were prepared using 25 ng of
purified insert from the chosen cDNA clones using the
Multiprime DNA labelling system (Amersham Life
Science, U.K.). Cyclophilin, an abundantly expressed
gene in tomato that is thought to be constitutively
expressed [10 ], was to normalize transcript levels. The
blots were washed as above prior to autoradiography. In
some experiments total RNA in blots was normalized by
comparison of ethidium bromide stained rRNA. In these
experiments, relative levels of cyclophilin compared to
rRNA were comparable or slightly lower after nematode
infection.
Nucleic acid sequencing
Nucleic acid sequences of the cDNA inserts were
determined by the dideoxynucleotide chain termination
method using the Sequenase kit (United States Bio-
chemical, Cleveland, OH, U.S.A.) or by automated
sequencing carried out by the Iowa State University DNA
Sequencing and Synthesis Facility (Ames, Iowa, U.S.A.).
Sequence similarities were identified using the BLAST
program of the National Center for Biotechnology
Information [12 ].
RESULTS AND DISCUSSION
Tomato seedling infection
The seedling infection system used here is a modification
of one developed by McClure and Robertson [23 ]. This
infection procedure is capable of generating hundreds of
synchronously infected root tips. At 12 h after inoculation,
most nematodes inside the root were found within 0±5 cm
of the apex. The percentage of nematodes that had
penetrated the roots ranged from 20–92 % with an
344 K. N. Lambert et al.
T    1. Properties of cDNAs

Clone cDNA Transcript Corresponding or related protein %

name size (bp) size (nt) sequences ident. References

23a 508 800 LeMir 100 [4 ]

Tobacco TID91 81 [9 ]
F3 500 1700 Tomato ascorbate free radical 100 [13 ]
reductase
D7 450 1500 Extensin (Lemmi8) 100 [34 ]
A9 651 1500 Azuki bean peroxidase, cationic 67 [15 ]
84 453 770 Tobacco LEA-5 like protein 66 GenBank g2981167
Tomato green fruit gene 57 GenBank g170507
Potato tuber induced gene 56 [16 ]
37 501 780 Hypothetical protein from 40 GenBank gZ99708
Arabidopsis
H8 342 530 Hypothetical protein from 52 GenBank gZ47668
Arabidopsis
94 378 615 no match

(a) R RN
(b) D7 A9 H8 94 84
23a R RN R RN R RN R RN R RN

F3

37
CyP

CyP

F. 2. Induction of tomato genes by nematodes. RNA gel blots contain total RNA from mock-inoculated nematode resistant tomato
(VFN8) root tips (R lanes) or from root-knot nematode infected root tips (RN lanes). Roots were inoculated with about 200
nematodes per tip. Names of clones used as probes (see Table 1) are noted to the left of or above the autoradiographs. (a) The blot
was hybridized and stripped sequentially with probes 23a, F3 and 37, and cyclophilin (CyP). (b) Separate blots were probed with
D7, A9, H8, 94 or 84. All blots were reprobed with cyclophilin (CyP).

average of about 50 %. All roots examined contained
nematodes, including those with inoculation levels of 15
per tip.
Isolation of cDNAs of transcripts that accumulate after
nematode infection
A PCR-based cDNA plasmid library was constructed
with RNA isolated from VFN8 tomato root tips inoculated
with nematodes for 12 h. For differential screening, RNA
samples extracted from control and nematode-inoculated
roots were used as template to generate first strand
radiolabelled cDNA probes.
Two methods of differential screening were used. For
the first 200 clones, the colony blot method was used.
DNA from lysed bacterial colonies was fixed to two
replicate membranes and screened with cDNA probes
made from nematode-inoculated and mock-inoculated
root tips. Five clones were isolated that produced stronger
hybridization signals with cDNA probes from nematode-
infected tissue. However, upon retesting, only one clone,
23a, showed increased hybridization with RNA from
nematode-infected tissue. The second method of screening
used inserts from individual cDNA colonies amplified by
PCR. Equal amounts of the PCR products were spotted
onto duplicate nylon membranes and hybridized to the
cDNA probes from infected or uninfected root tips. Of 225
cDNA clones screened using this second method, seven
cDNAs were identified that represent genes that appear to
be more highly expressed after nematode infection (Table
1). All seven produced stronger hybridization with RNA
from nematode-infected root tips upon retesting.
 Nematode-induced genes 345
1 2 3 4 5 6

F3

23a

CyP

F. 3. Transcript levels in susceptible and resistant tomato root tips. RNA blot contains total RNA from susceptible (UC82), lanes
1–3, or resistant (VFN8), lanes 4–6, tomato root tips hybridized with cDNA clones F3 and 23a. Lanes 3 and 6 contain RNA from
uninfected root tips ; lanes 1 and 4, RNA from root-tips infected with M. jaŠanica strain VW4 ; lanes 2 and 5, RNA from root-tips
infected with M. jaŠanica strain VW5. Roots were inoculated with about 200 nematodes per tip. M. jaŠanica strain VW4 cannot
reproduce on tomato with the nematode resistance gene Mi ; M. jaŠanica strain VW5 is able to reproduce on tomato with Mi.

lines developed at UC Davis, they are not isolines. To

DNA and RNA blot analysis
To determine whether the clones that we had identified
represented tomato genes or contaminating nematode
DNA, each was hybridized to blots containing EcoRI-
digested genomic tomato and nematode DNA. All
hybridized to the tomato DNA under highly stringent
conditions indicating they were of tomato origin, and
each produced one or a few bands (not shown) indicating
that they correspond to single copy genes or small gene
families. None hybridized to the nematode DNA.
Total RNA from nematode infected and uninfected
tomato (VFN8) root tips was isolated and subjected to
RNA blot analysis. The transcript size for each clone is
recorded in Table 1. The blots confirmed the increased
transcript level in nematode infected roots for each of the
eight genes (Fig. 2). To determine if there were differences
between the changes in transcript patterns in resistant
and susceptible tomato, RNA was extracted from
nematode-infected root tips of VFN8 (resistant, Mi}Mi)
and UC82 (susceptible, mi}mi). RNA blots showed that
F3 and 23a transcript accumulation was similar in
susceptible and resistant tomato after nematode infection
(Fig. 3). Increased levels of transcript after nematode
infection for each of the other six clones was measured in
at least two independent experiments (not shown). Among
experiments the amount of increase was variable, but all
of the genes tested were increased in amount in both
resistant and susceptible root tips.
Although the susceptible and resistant tomato lines
used, UC82 and VFN8, are both processing-type tomato
control for the possibility that induction patterns are due
to differences in the plant genetic background in addition
to Mi, we infected each tomato line with each of the
closely related M. jaŠanica strains, VW4 and VW5, that
differ in their ability to reproduce on plants with Mi
(Williamson, unpublished). VW4 produces a resistant
(incompatible) response on tomato lines with Mi, whereas
VW5 infection results in a susceptible (compatible)
response on the same lines. No clear difference in the
levels of 23a and F3 mRNA was seen between the
compatible and incompatible response on tomato line
VFN8 (Fig. 3).
The relative transcript levels of the eight genes in
uninfected or infected resistant and susceptible plants
varied among experiments. There are several features of
our protocol that could account for this variability. For
example, the expression levels of the genes could change
during the early stages of seedling development. Ad-
ditionally, the number of nematodes that penetrate the
root and the distribution of nematodes within the root can
differ from experiment to experiment resulting in
differences in the exact stage of the infection. Since
defense genes are often transiently expressed [6 ], this
could result in large differences in expression levels.
Defense genes transcripts are often increased in both
resistant and susceptible responses of the host, with faster
induction in the resistant plant [1, 2 ]. Time course
experiments will be required to determine if there is any
difference in the timing and extent of induction between
nematode resistant and susceptible plants.
346 K. N. Lambert et al.
T    2. Changes in RNA leŠels in response to wounding or low leŠels of nematodes

Susceptible roots Resistant roots

probe controla inoculated controla inoculated woundedb
30c 10c 30c 10c

A9 ­ ­­ ­­ ­ ­­ ­­ ­­
F3 ­ ­­ ­­ ­ ­­ ­­ ­­
23a ­ ­­ ­­ ­ ­­ ­­ ­­

aMock inoculated. bRNA was collected from seedling roots 2 h after wounding as previously described [4 ].
cAverage number of nematodes counted per root tip. Nematodes were incubated with antibiotic mix before application.
­ indicates transcript is detected. ­­ indicates that transcript in increased at least two-fold compared to mock inoculated control.

Nucleotide sequence analyses of identified cDNAs
DNA sequence of each cDNA was determined. Novel
sequences were submitted to Genbank (accession numbers
AF116866, AF116867, AF116868, AF116869 and
AF116870 for sequences of 37, A9, H8, 94 and 84,
respectively). Derived protein sequences of the clones
were compared with those in GenBank and known genes
with highest similarities are listed in Table 1.
The deduced amino acid sequence of clone 23a was
most similar to that encoded by TID91, a gene expressed
in genetic tumors of tobacco [9, 17 ]. Due to the similarity
in sequence to miraculin, a protein isolated from the
berries of Richadella dulcifica [9, 22], we have called the 23a
gene LeMir (L. esculentum miraculin) [4 ]. The encoded
protein is present at higher levels after wounding and is
secreted from the plant into the rhizosphere.
Clone F3 corresponds to the 3« region of a previously
characterized tomato gene encoding ascorbate free radical
(AFR) reductase [13 ]. Ascorbic acid is a sink for reactive
oxygen species (ROS) produced by many cellular
processes. Ascorbate is oxidized by the ROS to AFR.
AFR reductase reduces AFR to ascorbate, replenishing
the ascorbic acid pool [32 ]. Ascorbate is an essential
reducing agent for hydroxylation of proline residues in
hydroxyproline-rich glycoproteins such as extensin [19 ].
Crosslinking of extensin via hydroxyproline residues may
function to strengthen cell walls in defense responses and
after wounding. AFR reductase has been shown to be
induced by wounding in tomato [13 ].
The DNA sequence of D7 corresponds to that of
Lemmi8, a previously reported tomato extensin gene
identified as being expressed at elevated levels in 2–5
week old root-knot nematode-induced galls of tomato
[34 ]. A closely related extensin was also shown, using a
reporter gene, to be increased 2 days after root-knot
nematode infection in tobacco [26 ], suggesting that
expression is associated with feeding site initiation. At this
time, expression was localized to the center of the initiating
gall and was not found along the migration path of the
nematode.
Clone A9 had an open reading frame with similarity to
a wide variety of peroxidases. Peroxidase levels are often
environmentally regulated, and these enzymes have been
shown to be increased after wounding, fungal infection
and increase in plant hormones [18 ].
The most similar sequence to clone 84 in the public
database is a LEA5-like protein from tobacco. Clone 84
also shows similarity to a cDNA expressed in green
tomato fruit and to a protein whose level increases in
leaves during tuberization in potato [16 ]. These genes
carry signal sequences suggesting that they are trans-
located into the ER. A homologue of another LEA-like
gene, Lemmi9, was shown to be highly expressed in root-
knot nematode giant cells and galls and was postulated to
be an osmoprotectant for the giant cell [34 ]. In contrast
to most LEA genes, the Lea5-like genes from various
plants have been shown to be induced by salt, drought
stress or heat [25 ].
Nematode or wounding response ?
Transcripts of several of the genes that we identified or
related genes have been found to increase after wounding.
We have confirmed that F3, 23a, and A9 are increased in
expression after wounding in tomato seedling roots (Table
2). Potenza et al. [28 ] also found some similarities between
in Šitro translation products of wounded and root-knot
nematode infected alfalfa roots. Although an individual
root-knot nematode infecting a root causes little damage
as it migrates between the cells, large numbers of
nematodes may produce tissue damage. Wounding
produced by the large numbers of nematodes used in our
experiments could be the cause of some of the changes in
gene expression in our system. To minimize the con-
tribution of tissue damage, we isolated RNA from root
tips with low numbers of nematodes (30 and 10 per tip).
We also treated the nematodes with antibiotics to reduce
the possibility that gene expression was altered by
contaminating microbes rather than a specific response to
nematodes. Under these conditions, 23a, F3 and A9
transcripts were increased to the same level as in our
previous experiments (Table 2).
 Nematode-induced genes
Transcripts of many genes have been shown to increase
after both wounding and pathogen infection. This is not
surprizing as wounding and pathogens both trigger a
defense response [3, 7 ]. In addition, some genes may have
different roles in each process. For example, extensin
transcripts have been reported to increase after wounding
as well as after infection by pathogens [30, 37 ], but the
pattern of expression after nematode infection (present at
the site of gall initiation and absent from the nematode
infection path) indicates that in this case the increased
transcript is not caused exclusively by wounding [26 ].
Conclusions
We hypothesized that some genes with increased ex-
pression after nematode infection of resistant tomato
would be involved with nematode resistance mediated by
the Mi gene, while others would be part of a more general
root defense system, present in both resistant and
susceptible tomato. We felt that a cDNA library that was
made from RNA of tissue highly enriched in cells
responding to nematode infection would contain a high
proportion of responsive genes. The fact that we isolated
eight such genes from 425 clones indicates our strategy
was successful. Most of these genes encode or are related
to previously characterized genes associated with defense
or stress responses. No clones specific to the resistance
response were identified. Nonetheless, the inoculation
procedure that we describe here is a useful system for
generating RNA for a more extensive screen for genes
with altered regulation in the early hours after nematode
infection.
The authors thank F. F. Del Campo for helpful comments
on the manuscript. The cyclophilin cDNA was kindly
provided by C. Gasser, University of California, Davis.
This work was supported by grants from the US
Department of Agriculture – National Research Initiative
Competitive Grants Program (nos. 94–37302–0569 and
97–35302–4570). G. Nombela was financially supported
by a FPI fellowship ‘‘ Subprograma de Perfeccionamiento
para Doctores y Techno! logos en el Extranjero ’’ from the
Spanish Ministry of Education and Science.
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