Download Resistance of Cultivated Tomato to Cell Content

Resistance of Cultivated Tomato to Cell
Content-Feeding Herbivores Is Regulated by the
Octadecanoid-Signaling Pathway1
Chuanyou Li2, Mark M. Williams2, Ying-Tsu Loh, Gyu In Lee, and Gregg A. Howe*
Department of Energy-Plant Research Laboratory (C.L., M.M.W., Y.-T.L., G.I.L., G.A.H.), and Department of
Biochemistry and Molecular Biology (G.A.H.), Michigan State University, East Lansing, Michigan 48824
The octadecanoid signaling pathway has been shown to play an important role in plant defense against various chewing
insects and some pathogenic fungi. Here, we examined the interaction of a cell-content feeding arachnid herbivore, the
two-spotted spider mite (Tetranychus urticae Koch), with cultivated tomato (Lycopersicon esculentum) and an isogenic mutant
line (defenseless-1 [def-1]) that is deficient in the biosynthesis of the octadecanoid pathway-derived signal, jasmonic acid (JA).
Spider mite feeding and fecundity on def-1 plants was significantly greater than on wild-type plants. Decreased resistance
of def-1 plants was correlated with reduced JA accumulation and expression of defensive proteinase inhibitor (PI) genes,
which were induced in mite-damaged wild-type leaves. Treatment of def-1 plants with methyl-JA restored resistance to
spider mite feeding and reduced the fecundity of female mites. Plants expressing a 35S::prosystemin transgene that
constitutively activates the octadecanoid pathway in a Def-1-dependent manner were highly resistant to attack by spider
mites and western flower thrips (Frankliniella occidentalis), another cell-content feeder of economic importance. These
findings indicate that activation of the octadecanoid signaling pathway promotes resistance of tomato to a broad spectrum
of herbivores. The techniques of amplified fragment length polymorphism (AFLP) and bulk segregant analysis were used
to map the Def-1 gene to a region on the long arm of chromosome 3 that is genetically separable from the map position of
known JA biosynthetic genes. Tight linkage of Def-1 to a T-DNA insertion harboring the maize (Zea mays) Dissociation
transposable element suggests a strategy for directed transposon tagging of the gene.
Plant resistance to arthropod herbivores is often
mediated by phytochemicals that negatively affect
the feeding, growth, or reproduction of the attacking
pest (Karban and Baldwin, 1997; Walling, 2000). Although many defensive compounds have been identified from diverse plant species, relatively little is
known about the underlying genetic mechanisms
that control their biosynthesis in response to developmental and environmental cues. Lycopersicon spp.
provide an attractive model system to address this
question. Cultivated tomato (Lycopersicon esculentum)
is a natural host to over 100 arthropod herbivores
that feed on roots, leaves, or fruit (Lange and Bronson, 1981). Included among the major pests of tomato
are adult and larval stages of Coleoptera (beetles),
Lepidoptera (moths), Diptera (flies), Thysanoptera
(thrips), Heteroptera (true bugs), Homoptera (aphids
and whiteflies), and Acari (spider mites).
Natural resistance of tomato to many herbivores is
attributed to both constitutive and inducible defen1
This research was supported by the National Institutes of
Health (grant no. GM57795 to G.A.H.), by the U.S. Department of
Energy (grant no. DE–FG02–91ER20021 to G.A.H.), and by the
Michigan Life Science Corridor (grant no. 085P1000466 to G.A.H.).
2
These authors contributed equally to the paper.
* Corresponding author; e-mail [email protected]; fax 517–353–
9168.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.005314.
494
sive phytochemicals (Farrar and Kennedy, 1992).
Among the most thoroughly studied inducible defenses in tomato are proteinase inhibitor (PI) proteins
that inhibit digestive enzymes in the gut of some
insect herbivores (Green and Ryan, 1972; Broadway
and Duffey, 1986). Wound-induced expression of PI
genes is controlled by the jasmonate family of signaling molecules that includes jasmonic acid (JA), its
methyl ester (MeJA), and their metabolic C18 precursor, 12-oxo-phytodienoic acid (Farmer and Ryan,
1992; Ryan, 2000; Walling, 2000; Stintzi et al., 2001).
Jasmonates are synthesized from linolenic acid via
the octadecanoid pathway (Vick and Zimmerman,
1984; Schaller, 2001). In tomato leaves, jasmonate
biosynthesis is positively regulated by wounding
and by leaf-derived wound signals such as systemin
(Ryan, 2000; McGurl et al., 1992). Genetic analysis
indicates that systemin and its precursor protein,
prosystemin, are upstream components of a signaling
cascade that involves both the synthesis and perception of jasmonates (Howe and Ryan, 1999; Li et al.,
2001, 2002). A tomato mutant (defenseless-1 [def-1])
that is deficient in wound- and systemin-induced JA
accumulation and expression of downstream target
genes was shown to be more susceptible to attack by
Manduca sexta larvae, indicating that the octadecanoid pathway is essential for defense against chewing insects (Lightner et al., 1993; Howe et al., 1996).
Theses findings have been extended to field studies
showing that exogenous jasmonate promotes resis-
Plant Physiology, September
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Vol.on
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Octadecanoid-Mediated Resistance to Herbivores
tance of tomato plants to insects in agricultural settings (Thaler et al., 1996; Thaler, 1999).
In contrast to the firmly established role of octadecanoid signaling in plant defense against chewing
insects, much less is known about how this pathway
affects the interaction of plants with herbivores that
use a piercing/sucking mode of feeding (Walling,
2000). The two-spotted spider mite (Tetranychus urticae Koch) represents one such economically important pest of a wide range of plants including many
fruit, vegetable, grain, and ornamental crops, and it is
perhaps the most serious pest in greenhouses around
the world (Lange and Bronson, 1981). The twospotted spider mite uses specialized stylets to puncture epidermal cells of the host tissue; subsequent
withdrawal of cellular contents leads to collapse of
the underlying mesophyll tissue and formation of a
chlorotic lesion at the site of feeding. Severe infestations usually result in complete desiccation and
death of the affected tissue. Resistance of some wild
tomato species to two-spotted spider mite has been
shown to involve trichome-based physical (i.e. entrapment) and chemical (i.e. toxicity) mechanisms
(e.g. Farrar and Kennedy, 1992). However, the role
of octadecanoid signaling in shaping the interaction
between tomato and two-spotted spider mite, or
other cell-content feeders, has not been thoroughly
explored. To address this question, we studied the
performance of two-spotted spider mite on nearisogenic lines of tomato in which this signaling pathway is either attenuated by def-1, or genetically enhanced by overexpression of prosystemin. Our
findings, together the results of previous studies, indicate that octadecanoid signaling plays a critical
role in regulating defense responses of tomato to a
broad spectrum of herbivore pests. As a step toward
understanding the molecular function of Def-1 in octadecanoid defense signaling, we mapped the Def-1
locus to the distal end of the long arm of chromosome 3.
RESULTS
Octadecanoid-Mediated Resistance of Tomato to CellContent Feeding Herbivores
We used the JA-deficient def-1 mutant to investigate the role of the octadecanoid pathway in resistance to the two-spotted spider mite. Two-leaf-stage
(15-d-old) wild-type (WT) and def-1 plants were infested with adult female mites that had been reared
on bean plants, a preferred host of the two-spotted
spider mite. Mites initiated feeding on both host
genotypes within 1 d of challenge as evidenced by
the appearance of small (approximately 0.25 mm2)
chlorotic lesions at the feeding site. Estimation of leaf
damage during a time course of infestation indicated
that def-1 plants were significantly more susceptible
than WT to mite feeding (Fig. 1A). This effect was
accompanied by a significant increase in the number
Plant Physiol. Vol. 130, 2002
Figure 1. Performance of two-spotted spider mite on wild-type and
def-1 plants. Five different sets of 15-d-old wild-type (black bar) and
def-1 (white bar) plants were challenged with adult female mites (10
mites per plant). Leaf damage (A) and egg counts (B) were determined
at various times thereafter, using one set of plants to evaluate each
time point. Ten plants of each genotype were used for each time
point except the 10-d point, where eight plants per genotype were
used. Values represent the mean and SD. Two-way ANOVA was used
to evaluate the statistical significance of differences in leaf damage
and egg count at each time point. Single asterisks denote a significant
difference at P ⬍ 0.05. Double asterisks denote a significant difference at P ⬍ 0.0001.
of mite eggs found on def-1 leaves compared with WT
leaves (Fig. 1B). These results indicate that the Def-1
gene plays an important role in reducing the quality
of tomato leaves as a food source and oviposition
substrate for two-spotted spider mite.
The differential performance of two-spotted spider
mite on WT and def-1 plants suggested that the octadecanoid pathway regulates the production of defensive compounds in leaves of WT plants. To test this
hypothesis, the level of Ser PI-II, a well-characterized
marker of octadecanoid signaling in tomato (Farmer
and Ryan, 1992), was measured in untreated and
mite-infested plants (Fig. 2A). WT plants subjected to
mite feeding for 10 d accumulated high levels of PI-II
relative to untreated control plants. Within this population of WT plants, a positive correlation (r2 ⫽ 0.49)
was observed between the level of leaf damage and
PI-II accumulation. PI-II levels in mite-infested def-1
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495
Li et al.
mato leaves and that this response involves activation of the octadecanoid pathway.
RNA-blot hybridization was used to determine
whether spider mite-induced accumulation of PI-II
protein results from activation of the PI-II gene. In
accordance, plants were infested with spider mites
and RNA was prepared from leaf tissue 1, 2, or 3 d
thereafter (Fig. 3). PI-II mRNA levels in WT plants
were strongly up-regulated (⬎50-fold relative to untreated controls) 1 d after challenge, and remained
high at the 2- and 3-d time points. The expression
pattern of two other PI genes, PI-I and cathepsin D
inhibitor (CDI), was essentially identical to that of
PI-II. Consistent with the data on PI-II protein accumulation (Fig. 2), PI transcript levels in def-1 plants
were less than 10% of that in WT. These findings
indicate that two-spotted spider mite feeding activates the octadecanoid pathway leading to the coordinate expression of several defense-related genes,
and that Def-1 plays an essential role in this induced
response. Feeding of spider mites on WT plants resulted in a modest and gradual increase in the accumulation of LoxD and AOS1, two wound-inducible
transcripts that encode the octadecanoid pathway
Figure 2. The octadecanoid signaling pathway regulates the synthesis of PI-II in response to spider mite feeding. A, Fifteen-day-old
wild-type (WT) and def-1 plants were either not treated (black bars)
or were infested (white bars) with 20 adult female mites per plant (10
mites per leaf). PI-II levels were measured in both leaves of individual
plants 10 d after infestation. Values indicate the mean and SD of each
treatment group (n ⫽ 14). Different lowercase letters denote a significant difference at P ⬍ 0.01 (Student’s t test). B, Sixteen-day-old
WT and def-1 plants were infested with spider mites as described in
“Materials and Methods.” Two days after challenge, leaflets showing
visible signs of damage were harvested for analysis of JA levels (white
bars). JA was also quantified in leaflets of noninfested plants (black
bars). Values indicate the mean and SD of three independent experiments. Different lowercase letters denote a significant difference at
P ⬍ 0.05 (Student’s t test).
plants were only slightly greater than the detection
limit of the assay (approximately 15 ␮g mL⫺1), even
though mutant plants received approximately 3.9fold greater damage than WT. To further test the
hypothesis that the octadecanoid pathway regulates
induced defense responses to spider mites, levels of
endogenous JA were measured in control and infested leaves of WT and def-1 plants. Mite feeding
resulted in a 2.6-fold increase (P ⬍ 0.05) in JA accumulation in WT plants, whereas JA levels of def-1
plants were unaffected by herbivory (Fig. 2B). These
findings demonstrate that two-spotted spider mite
feeding strongly induces PI-II accumulation in to496
Figure 3. Accumulation of defense-related mRNAs in response to
spider mite feeding. Wild-type and def-1 plants (15-d-old) were
challenged with 20 adult female mites as described in the legend to
Figure 2. At various times (1, 2, or 3 d) thereafter, leaf tissue from 10
plants was harvested from control (0 d) and mite-infested leaves for
RNA isolation. RNA-blot hybridization was performed using 32Plabeled cDNAs for Ser PI-I and PI-II, cathepsin D inhibitor (CDI),
lipoxygenaseD (LoxD), and allene oxide synthase (AOS1). Blots were
also hybridized to a probe for translation initiation factor eIF4A as a
loading control.
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Plant Physiol. Vol. 130, 2002
Octadecanoid-Mediated Resistance to Herbivores
enzymes lipoxygenase and allene oxide synthase, respectively (Fig. 3; Heitz et al., 1997; Sivasankar et al.,
2000). Interestingly, the mite-induced expression pattern of LoxD and AOS1 in def-1 plants was nearly
identical to that observed in WT. This finding is
consistent with previous studies suggesting that tomato uses genetically distinct signaling pathways for
the regulation of different classes of woundresponsive genes (Howe et al., 2000).
To test the hypothesis that increased performance
of two-spotted spider mite on def-1 plants results
from a deficiency in JA-induced defense responses,
experiments were conducted to determine whether
exogenous MeJA could restore resistance to the mutant. The results showed that spider mites caused
significantly less damage on MeJA-treated def-1
plants compared with control def-1 plants (Fig. 4A).
Moreover, the fecundity of female mites on MeJAtreated def-1 plants was significantly reduced relative
to controls (Fig. 4B). These findings indicate that
jasmonate is necessary and sufficient to restore defense of the mutant against two-spotted spider mite.
MeJA treatment also reduced spider mite feeding
and fecundity on WT plants, as recently reported by
Thaler et al. (2002). This observation is consistent
with the notion that applied MeJA induces the synthesis of defensive compounds that are normally produced in response to herbivory. Control experiments
demonstrated that MeJA vapor, at concentrations 50fold higher than those used for experiments with
tomato, had no significant effect on the mortality or
fecundity of mites reared on excised bean leaves
(data not shown). Thus, direct toxicity of MeJA vapor
to two-spotted spider mite is not likely responsible
for the observed effects.
To provide additional evidence for a role of the
octadecanoid pathway in resistance to two-spotted
spider mite, we examined the performance of mites
on a transgenic line of tomato that overexpresses
prosystemin from the cauliflower mosaic virus 35S
promoter. Previous studies showed that plants expressing this transgene (called 35S::prosys) constitutively express PI and other defensive genes in the
absence of wounding (McGurl et al., 1994; Constabel
et al., 1995) and that 35S::prosys-mediated signaling
requires octadecanoid biosynthesis and perception
(Howe et al., 1996; Howe and Ryan, 1999; Li et al.,
2001, 2002). We observed that 35S::prosys plants were
much more resistant to mite damage than either def-1
or WT plants (Fig. 5A). A substantial reduction in
mite fecundity on the transgenic line was also evident (Fig. 5B). Given the significance of these effects,
it was of interest to examine the interaction of various
tomato genotoypes with another cell-content feeding
herbivore, western flower thrips (Frankliniella occidentalis). As was the case for spider mites, thrips feeding
resulted in the accumulation of high levels of PI-II in
WT but not def-1 plants (Fig. 6A). The high constitutive levels of PI-II in undamaged 35S::prosys plants
were further increased in response to thrips damage,
similar to the previously reported effects of mechanical wounding on these plants (McGurl et al., 1994).
Thrips larvae inflicted a comparable amount of damage to WT and def-1 plants during the feeding trial
(Fig. 6B). By contrast, 35S::prosys plants were highly
resistant to damage. These findings indicate that
thrips feeding induces octadecanoid-mediated host
responses and that constitutive activation of the signaling pathway by overexpression of prosystemin
enhances resistance to multiple cell-content feeding
herbivores.
Mapping of the Def-1 Gene
Figure 4. Exogenous MeJA protects def-1 plants from spider mite
attack. Wild-type and def-1 plants were treated for 24 h in a closed
container in which MeJA was applied to cotton wicks (white bars). As
a control (black bars), an equivalent volume of ethanol was applied
to wicks in a box containing a separate set of plants. Treated plants
were incubated an additional 24 h in the absence of MeJA before
mite challenge. All plants were challenged with 20 adult female
mites (10 mites per leaf). Ten days after challenge, leaf damage (A)
and egg counts (B) were determined for each of the four treatment
groups. Values indicate the mean and SD of each treatment group
(n ⫽ 8). PI-II measurements showed that the MeJA treatment was
equally effective in both genotypes (data not shown). Asterisks denote a significant difference (P ⬍ 0.05, Student’s t test) in leaf damage
or egg count in comparisons between control and MeJA-treated
plants of the same genotype.
Plant Physiol. Vol. 130, 2002
The essential role of Def-1 in induced responses to
herbivory prompted us to initiate mapping of this
gene as a first step toward understanding its molecular function. The wound response phenotype of
def-1 homozygotes can be scored reliably using an
immunodiffusion assay to measure wound-induced
accumulation of PI-II in two-leaf-stage plants (Lightner et al., 1993; Howe et al., 1996). Phenotypic analysis of an F2 population (168 plants) produced from
self-pollination of a Def-1/def-1 heterozygote showed
that the proportion of wound-responsive (W⫹) to
wound-nonresponsive (W⫺) progeny was 123:45, in
good agreement with the ratio predicted for a single
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497
Li et al.
DNA from 10 W⫹ and 10 W⫺ BC1 plants was pooled
to construct a W⫹ bulk (B⫹) and W⫺ bulk (B⫺),
respectively. Among 64 primer combinations used to
screen the bulks for AFLPs, two combinations (EACA/M-CTG and E-AGC/M-CTC) generated a
polymorphic band that was present in both the W⫹
parent (Def-1/def-1) and the B⫹, but absent in the W⫺
parent (def-1/def-1) and the B⫺ (Fig. 7A). DNA bands
corresponding to the two polymorphisms, designated EM-1 and EM-2, were cloned into a plasmid
vector. Genomic DNA hybridization experiments
showed that EM-1 and EM-2 probes detected singleor low-copy sequences in the genome and easily
scorable RFLPs in genomic DNA digested with
HindIII and HaeIII, respectively (Fig. 7B, lanes 1–3).
Linkage of EM-1 and EM-2 to Def-1 was confirmed
using the 10 W⫹ and 10 W⫺ BC1 individuals that
Figure 5. Activation of the octadecanoid pathway by 35S::prosystemin
confers enhanced resistance to spider mites. Fifteen-day-old wild-type
(WT), def-1, and 35S::prosys (Ps) plants were challenged with 10 adult
female mites on the terminal leaflet of each of two expanded leaves.
Eight days after challenge, leaf damage (A) and egg counts (B) were
determined for each treatment group. Data represent the mean and SD of
12 plants for each genotype. Different lowercase letters denote a significant difference at P ⬍ 0.01.
recessive mutation (␹2 ⫽ 0.26; P ⬎ 0.5). To generate
an interspecific mapping population, L. esculentum
(def-1/def-1) was crossed as a pistillate parent to the
wild tomato species Lycopersicon pennellii (Def-1/Def1). All resulting F1 hybrids (def-1/Def-1; n ⫽ 12) were
W⫹, indicating that def-1 is recessive in the L. pennellii
background. A segregating backcross (BC1) population was generated from a second cross between a F1
plant (staminate parent) and a def-1/def-1 homozygote. Analysis of 509 BC1 progeny showed that 144
plants were W⫺, whereas the remaining plants were
W⫹. This ratio deviated significantly from the expected value of 1:1 (␹2 ⫽ 96; P ⬍ 0.001) and likely
reflects reduced transmission of the def-1 allele
through the pollen (C. Li and G.A. Howe, unpublished data).
Bulk segregant analysis (Michelmore et al., 1991)
was used in combination with AFLP (Vos et al., 1995)
to identify markers that are linked to Def-1. Genomic
498
Figure 6. Octadecanoid signaling mediates defense responses of
tomato to thrips. Fifteen-day-old wild-type (WT), def-1, and
35S::prosys (Ps) plants were challenged with thrips larvae on the
terminal leaflet of each of two expanded leaves (five larvae per leaf).
A, Five days after challenge, PI-II protein levels were measured in
leaves of infested (white bars) and control untreated (black bars)
plants. B, Leaf damage to each host genotype was determined 5 d
after challenge. Different lowercase letters denote a significant difference at P ⬍ 0.005. Data represent the mean and SD of 14 plants for
each genotype.
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Plant Physiol. Vol. 130, 2002
Octadecanoid-Mediated Resistance to Herbivores
Figure 7. Identification and chromosomal localization of AFLP
markers linked to the Def-1 gene. AFLP analysis for Def-1-linked
markers was performed on bulked segregants from a BC1 segregating
population. Four genomic template DNAs were used for each primer
combination: P⫺, W⫺ parent (L. esculentum, def-1/def-1); P⫹, W⫹
parent (L. pennellii, Def-1/Def-1); B⫺ and B⫹, bulks composed of 10
W⫺ and 10 W⫹ progeny, respectively, from a BC1 mapping population (see text for details). A, Arrows indicate the position of the EM-1
(top) and EM-2 (bottom) AFLP markers identified using two different
primer combinations. Only the portion of the autoradiographed AFLP
gel is shown. B, Conversion of EM-1 and -2 to RFLP markers and
mapping of the markers to IL3-5. Cloned AFLP markers were labeled
with 32P and hybridized to genomic DNA digested with HindIII
(EM-1, top) or HaeIII (EM-2, bottom). Genomic DNA was obtained
from plants with the following genotypes: lane 1, F1 hybrid (Def-1/
def-1) between L. esculentum (def-1/def-1) and L. pennellii (LA716);
lane 2, L. esculentum (def-1/def-1) parent; lane 3, L. pennellii (Def1/Def-1) parent; and lane 4, IL (LA3490) harboring the IL3-5 segment
of L. pennellii DNA.
composed the two bulks. This experiment showed
that all W⫹ plants were heterozygous for both markers, whereas all W⫺ plants were homozygous for the
Plant Physiol. Vol. 130, 2002
L. esculentum RFLP pattern (data not shown). The
absence of recombinants in this population of 20 BC1
plants demonstrates that EM-1 and EM-2 are linked
to the Def-1 locus.
The chromosomal location of EM-1 and EM-2 was
determined using a set of 50 introgression lines (ILs)
harboring defined segments of L. pennellii DNA in an
otherwise L. esculentum background (Eshed and
Zamir, 1994). DNA from each IL was screened for the
presence of the EM-1 and EM-2 RFLPs. One line,
LA3490, displayed the L. pennellii RFLP pattern for
both markers (Fig. 7B, lane 4). The introgressed region of DNA contained in LA3490 is located on the
end of the long arm of chromosome 3 and is referred
to here as IL3-5. This region of chromosome 3 includes the RFLP marker TG152 and all other markers
(e.g. TG214 and TG244) distal to the centromere
(Tanksley et al., 1992). Southern hybridization experiments using IL3-5-specific RFLP markers confirmed
the identity of LA3490 (data not shown). To confirm
and refine the position of Def-1 on IL3-5, RFLP markers TG152, TG214, and TG244 were tested for linkage
to Def-1 using 305 plants (144 W⫺ and 161 W⫹) from
the above-mentioned BC1 mapping population. The
results showed that Def-1 is linked to TG152, TG214,
and TG244 at distances corresponding to approximately 17, 9, and 6 centimorgans, respectively (Fig.
8). These findings are consistent with the established
genetic map for chromosome 3 (Tanksley et al., 1992;
Van der Biezen et al., 1994) and position Def-1 distal
to TG244.
The tomato line ET570 carries a T-DNA insertion
approximately 3 centimorgans distal to TG244 (Van
der Biezen et al., 1994). The T-DNA in ET570 harbors
the maize (Zea mays) Dissociation (Ds) transposon,
which was introduced by Agrobacterium tumefaciensmediated transformation for the purpose of
transposon-tagging experiments (Knapp et al., 1994).
We performed a test cross to determine the relative
distance between Def-1 and the T-DNA insertion.
ET570 (Def-1/Def-1 Ds/Ds) was crossed to a def-1/
def-1 homozygote to produce an F1 plant (Def-1/def-1
⫹/Ds) that was subsequently backcrossed to a def-1/
def-1 homozygote. The resulting progeny were analyzed for their wound response phenotype and the
presence of Ds-containing T-DNA. Among 146 plants
tested, three recombinants that were either W⫹ Ds⫺
or W⫺ Ds⫹ were recovered. This finding confirms the
location of Def-1 on the distal end of chromosome 3
and positions the gene approximately 2 centimorgans
from the Ds-containing T-DNA harbored by ET570
(Fig. 8).
Previous studies indicated that the def-1 lesion affects a step in JA biosynthesis (Howe et al., 1996; Fig.
2B). JA biosynthetic enzymes encoded by genes that
map to IL3-5 would thus represent candidates for
Def-1. Among the JA biosynthetic genes identified in
tomato are those encoding two plastidic lipoxygenases (LoxC and LoxD; Heitz et al., 1997), two plastidic
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Li et al.
Figure 8. Genetic map of the long arm of tomato chromosome 3. The
map is oriented with the centromere and telomere at the top and
bottom of the figure, respectively. Recombination distances between
markers and Def-1 are based on segregation analysis of a BC1
population of 305 individuals. Molecular markers are indicated on
the right. Map distances (in centimorgans) indicated on the left were
calculated as the proportion of individuals in the mapping population having a recombination event between the indicated markers.
Markers EM-1 and LoxD co-segregated in all BC1 plants. The T-DNA
insertion present in line ET570 is located approximately 2 centimorgans from Def-1, but its position relative to other markers was not
unambiguously determined.
allene oxide synthases (AOS1; Sivasankar et al., 2000;
AOS2; Howe et al., 2000), plastidic allene oxide cyclase (AOC; Ziegler et al., 2000), and 12-oxophytodienoic acid reductase (OPR3; Strassner et al.,
2002). The location of AOC on chromosome 2 (Ziegler
et al., 2000) indicates that this gene is not a candidate
for Def-1. To test the hypothesis that LoxC, LoxD,
AOS1, AOS2, or OPR3 corresponds to Def-1, these
cDNAs were converted to RFLP markers and
mapped using the ILs described above. The results
showed that LoxD maps to IL3-5, whereas LoxC,
AOS1, AOS2, and OPR3 map to different chromosomes (data not shown). To further test the genetic
relationship between LoxD and Def-1, the position of
LoxD was refined using the aforementioned BC1
mapping population. Among 305 meiotic events analyzed, 15 recombinants between Def-1 and LoxD
were detected. Thus, the LoxD and Def-1 loci appear
to be genetically distinct.
DISCUSSION
In this study, we examined the role of the octadecanoid pathway in resistance of cultivated tomato to
the arachnid herbivore two-spotted spider mite. The
rapid life cycle, ease of rearing, broad host range, and
economic importance of two-spotted spider mite
500
make it well suited for the study of plant-herbivore
interactions. The two-spotted spider mite has proven
particularly valuable for studying herbivore-induced
plant volatiles and their role in influencing tritrophic
interactions (Takabayashi and Dicke, 1996; Arimura
et al., 2000). Increasing evidence indicates that JA and
related signaling molecules play an important role in
regulating volatile-mediated plant defenses against
two-spotted spider mite (Dicke et al., 1990, 1999;
Arimura et al., 2000; Ozawa et al., 2000). By contrast,
relatively little is known about the role of jasmonates
in regulating the synthesis of phytochemicals that
have a direct effect on the two-spotted spider mite.
The availability of isogenic lines of tomato that are
either down-regulated (i.e. def-1) or up-regulated (i.e.
35S::prosys) in the octadecanoid pathway provide
valuable tools to address this question.
Several lines of evidence indicate that induced defense of tomato against two-spotted spider mite is
regulated by the octadecanoid pathway. First, infestation of WT plants with spider mites induced the
expression of several JA-responsive, defense-related
genes (i.e. PI-I, PI-II, CDI). Second, def-1 plants were
deficient in PI expression in response to two-spotted
spider mite feeding. This phenotype was tightly correlated with both increased susceptibility of the mutant to mite damage and increased mite fecundity.
Third, spider mite feeding was accompanied by increased JA accumulation in WT but not def-1 plants.
The increase in JA levels in mite-infested WT plants
was notably less than that observed in mechanically
wounded tomato leaves (e.g. Conconi et al., 1996; Li
et al., 2002). This may reflect differences in the type of
damage caused by mite feeding (i.e. piercing/sucking) and mechanical wounding (i.e. leaf crushing).
Fourth, pretreatment of def-1 plants with exogenous
MeJA before challenge resulted in a significant decrease in two-spotted spider mite performance. This
finding is consistent with recent reports that exogenous jasmonate promotes host plant resistance to
spider mites (Omer et al., 2000; Thaler et al., 2002).
We also observed that activation of the octadecanoid signaling pathway by overexpression of prosystemin significantly reduced the performance of
both spider mites and thrips. To our knowledge, this
finding represents the first report of genetically engineered resistance to cell-content feeding herbivores. Transgene-mediated activation of octadecanoid signaling may have important implications
for the generation of broad-spectrum pest resistance
in agricultural crop plants. It will be interesting to
determine whether 35S::prosys plants are resistant to
other classes of herbivores, such as phloem-feeding
insects that induce both JA-dependent and -independent defense responses (van de Ven et al., 2000;
Moran and Thompson, 2001).
Our results support the idea that the octadecanoid
pathway regulates the synthesis of one or more foliar
compounds that have a negative effect on cell-
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Plant Physiol. Vol. 130, 2002
Octadecanoid-Mediated Resistance to Herbivores
content feeders. Additional studies are needed to
identify these compounds and to determine whether
the mechanism of resistance involves antibiosis (i.e.
toxicity), antixenosis (non-preference), or other factors. As recently noted by Thaler et al. (2002), reduced egg production by spider mites grown on
JA-treated tomato plants suggests a nutritional rather
than toxic mechanism of resistance. It is possible that
JA-regulated proteins such as PIs and polyphenol
oxidases implicated as anti-feedants against Lepidopteran insects (Broadway and Duffey, 1986; Constabel et al., 1995) are also effective against the twospotted spider mite. As an alternative, the observed
effects may be attributed to secondary metabolites
whose biosynthesis is regulated by JA (Keinanen et
al., 2001; Memelink et al., 2001). Resistance of wild
tomato species to two-spotted spider mite is associated with defensive phytochemicals (e.g. methyl ketones, sesquiterpenenes, and acyl sugars) that kill,
repel, or entrap the herbivore (Williams et al., 1980;
Farrar and Kennedy, 1992). However, these compounds are typically found in secretions of glandular
trichomes and generally do not accumulate to high
levels in cultivated tomato.
We determined the chromosomal location of Def-1
as a first step toward molecular characterization of
the gene. The mapping studies relied on the ability to
score wound-inducible PI-II accumulation in a segregating population generated from an interspecific
cross between def-1 and L. pennellii and involved
three basic steps. First, bulk segregant analysis was
used to identify AFLP markers linked to Def-1. Second, ILs harboring defined segments of L. pennellii
DNA were used to map AFLP markers to a specific
region on chromosome 3. Finally, the map position
was refined using RFLP markers on the tomato linkage map. The location of Def-1 was confirmed in
experiments showing linkage of the gene to a T-DNA
insertion previously mapped to the distal end of
chromosome 3 (Van der Biezen et al., 1994). To our
knowledge, genes affecting resistance to herbivores
have not been mapped previously to this region of
the tomato genome.
The inability of def-1 plants to accumulate normal
levels of JA in response to herbivory (Fig. 2B), mechanical wounding, and systemin (Howe et al., 1996)
suggests that Def-1 may encode an enzyme involved
in JA biosynthesis. We tested this hypothesis by determining whether genes encoding known or putative JA biosynthetic enzymes co-map with Def-1. The
finding that LoxC, LoxD, AOS1, AOS2, and OPR3 do
not co-map with Def-1 would appear to exclude them
as candidate genes. Mapping of a tomato AOCencoding cDNA to chromosome 2 (Ziegler et al.,
2000) likewise excludes it as a candidate gene. Given
the existence of several isoforms for JA biosynthetic
enzymes in tomato, it should be emphasized that
these findings do not rule out the possibility that
Def-1 corresponds to a JA biosynthetic gene that has
Plant Physiol. Vol. 130, 2002
not yet been cloned. It is also possible that Def-1
functions indirectly to regulate the activity of a JA
biosynthetic enzyme or the transport or stability of
an octadecanoid intermediate, but virtually nothing
is known about these processes in tomato or other
plants. Map-based cloning methods have been successfully used to isolate tomato genes whose biochemical function was not known (Tanksley et al.,
1995). However, we have found that the telomeric
location of Def-1 hinders the identification of tightly
linked markers that flank the target gene (C. Li and
G.A. Howe, unpublished data), indicating that this
may not be the optimal approach for isolation of
Def-1.
Transposon tagging may provide a useful alternative. The maize Ac/Ds transposon system has been
introduced into the tomato genome by Agrobacterium
tumefaciens-mediated transformation, and numerous
transgenic lines have been developed in which Ds
elements are integrated at defined locations throughout the genome (Emmanuel and Levy, 2002). The
tendency of Ds to transpose from its donor site to
linked acceptor sites has facilitated targeted tagging
of genes whose position is known (e.g. Jones et al.,
1994). The Ds element in line ET570 was previously
used for the nontargeted tagging and isolation of the
feebly gene, which is located approximately 9 centimorgans from the Ds insertion site (Van der Biezen et
al., 1996). The close proximity of Def-1 to this same Ds
element suggests that a targeted transposon tagging
experiment may be useful for identification of Def-1.
MATERIALS AND METHODS
Plant Material and Herbivore Rearing
Tomato (Lycopersicon esculentum Mill cv Castlemart) seedlings were
grown in Jiffy peat pots (Hummert International, Earth City, MO) in a
growth chamber maintained under 17 h of light (200 ␮E m⫺2 s⫺1) at 28°C
and 7 h of dark at 18°C. Seed for def-1 was collected from a def-1/def-1
homozygous line that was backcrossed four times using tomato cv Castlemart as the recurrent parent. Seed for the 35S::prosystemin transgenic plants
was collected from a 35S::prosys/35S::prosys homozygous line (Howe and
Ryan, 1999) that was backcrossed five times using tomato cv Castlemart as
the recurrent parent. Seed for L. pennellii (LA716) and the set of ILs was
obtained from the Tomato Genetics Resource Center (University of California at Davis).
Two-spotted spider mite (Tetranychus urticae Koch) was obtained from a
colony maintained in the Pesticide Research Center greenhouses at Michigan State University. Mites were reared on lima bean (Phaseolus lunatus cv
Fordhook) plants grown in course vermiculite and maintained under 18 h of
light per day. Bean plants were typically 2 to 5 weeks of age, and contained
an average of 50 adult spider mites per leaf. Western flower thrips (Frankliniella occidentalis) were reared on marigolds (cv Golden Boy) in the Pesticide Research Center greenhouses at Michigan State University.
Plant Treatments
Adult female spider mites were transferred, using a small soft-bristled
paintbrush, to the adaxial surface of the terminal leaflets of 14- to 15-d-old
tomato plants. Plants of this age contained two fully expanded leaves and an
emerging third leaf. Care was taken to avoid wounding of plants during the
transfer procedure. Feeding by adult female mites at a single site resulted in
the appearance of a chlorotic lesion, the average size of which was estimated
to be 0.25 mm2. The extent of leaf damage resulting from feeding was
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501
Li et al.
estimated by counting the number of lesions with the aid of a dissecting
microscope. In cases where the area of a single site of damage exceeded 0.25
mm2, the total area of the damaged site was estimated as a multiple of 0.25
mm2. A dissecting microscope was used to count eggs on both the upper
and lower leaf surface. Adult and larval stages of thrips were obtained by
gently shaking infested marigold flowers onto white paper. Larvae were
used for all experiments because, unlike adults, they are fairly immobile.
Larvae were transferred individually to tomato leaves as described above.
Infested plants were confined individually to Magenta boxes covered with
thrips-proof gauze. Leaf damage was estimated by creating a pictorial grid
representation of each leaf. During examination of damaged leaves under a
dissecting microscope, areas representing damage were marked on the
pictorial grid.
For experiments involving MeJA treatment, plants were transferred to a
lucite container (8 L) and exposed to MeJA composed of approximately 20%
(v/v) (⫾)-7-iso-MeJA (product no. 399E, Bedoukian Research, Danbury,
CT). All experiments were performed using 1 ␮L of MeJA, diluted into 100
␮L of ethanol, applied to several cotton wicks distributed evenly throughout
the box. Twenty-four hours after MeJA treatment, the cotton wicks were
removed, and plants were acclimated to ambient humidity for an additional
24 h before mite infestation. Control plants were incubated in a separate
container in which ethanol was applied to cotton wicks.
Measurement of JA
Spider mite-infested lima bean leaves were cut into slices and placed
(abaxial side-down) onto the upper surface of leaves of 16-d-old WT and
def-1 plants. Two days later, tomato leaflets (5 g fresh weight) showing
comparable levels of damage were harvested and frozen in liquid nitrogen.
JA was extracted and quantified using gas chromatography-mass spectrometry as previously described (Li et al., 2002).
the autoradiogram. Excised DNA fragments were eluted in 400 ␮L of
high-salt buffer (20% [v/v] ethanol, 1 m LiCl, and 10 mm Tris-HCl, pH 7.5)
for 2 h at 65°C and then precipitated with ethanol. One quarter of the
resuspended DNA was re-amplified with the same primer combination that
was used for the selective amplification. Re-amplified PCR products were
cloned into the pGEM-T easy vector (Promega, Madison, WI) according to
the manufacturer’s instructions. Cloned DNA fragments corresponding to
EM-1 and EM-2 were 183 and 249 bp in length, respectively. To convert
these markers to RFLP markers, survey blots containing restriction enzymedigested (DraI, EcoRI, EcoRV, HaeIII, HindIII, and XbaI) genomic DNA from
parental lines was probed with the radiolabeled AFLP fragments. The
relative position of EM-1 and EM-2 on chromosome 3 was determined by
mapping the markers using the subset of BC1 plants containing recombination events between TG244 and Def-1. A probe for detection of the Dscontaining T-DNA in line ET570 was obtained by PCR amplification with
primers act5b and 35s, as previously described (Van der Biezen et al., 1996).
ACKNOWLEDGMENTS
We thank Dr. David Smitley for his expert advice on experiments involving spider mites and thrips, and Sarah Norris and Liyan Liu for technical
assistance with mapping experiments. We also thank Dr. Steve Tanksley for
providing the RFLP markers used in this study and Dr. Klaus Theres for
providing tomato line ET570. Tomato EST clones cLED1D24 and cLEC9C14
were obtained from the Clemson University Genomics Institute. Seed for
LA716 and the ILs was provided by the Tomato Genetics Resource Center at
University of California (Davis).
Received March 12, 2002; returned for revision May 20, 2002; accepted May
29, 2002.
LITERATURE CITED
Nucleic Acid Gel-Blot Analysis
Total RNA was isolated and analyzed by blot hybridization as previously
described (Howe et al., 2000), except that Hybond-N Plus membranes
(Amersham Biosciences, Sunnyvale, CA) were used in place of nitrocellulose. Gels were run in duplicate, with one set stained with ethidium bromide
to check for equal loading of the samples and intactness of the RNA. DNA
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