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. 2013 Sep 10;163(3):1242–1253. doi: 10.1104/pp.113.222802

Disarming the Jasmonate-Dependent Plant Defense Makes Nonhost Arabidopsis Plants Accessible to the American Serpentine Leafminer1

Hiroshi Abe 1,2,3,4,5,6,7,*, Ken Tateishi 1,2,3,4,5,6,7, Shigemi Seo 1,2,3,4,5,6,7, Soichi Kugimiya 1,2,3,4,5,6,7, Masami Yokota Hirai 1,2,3,4,5,6,7, Yuji Sawada 1,2,3,4,5,6,7, Yoshiyuki Murata 1,2,3,4,5,6,7, Kaori Yara 1,2,3,4,5,6,7, Takeshi Shimoda 1,2,3,4,5,6,7, Masatomo Kobayashi 1,2,3,4,5,6,7
PMCID: PMC3813647  PMID: 24022267

Loss of jasmonate-dependent plant defense converts nonhost plants to host plants that are accessible to leafminers, and demonstrates a role for jasmonate in regulating host plant suitability of herbivores.

Abstract

Here, we analyzed the interaction between Arabidopsis (Arabidopsis thaliana) and the American serpentine leafminer (Liriomyza trifolii), an important and intractable herbivore of many cultivated plants. We examined the role of the immunity-related plant hormone jasmonate (JA) in the plant response and resistance to leafminer feeding to determine whether JA affects host suitability for leafminers. The expression of marker genes for the JA-dependent plant defense was induced by leafminer feeding on Arabidopsis wild-type plants. Analyses of JA-insensitive coi1-1 mutants suggested the importance of JA in the plant response to leafminer feeding. The JA content of wild-type plants significantly increased after leafminer feeding. Moreover, coi1-1 mutants showed lower feeding resistance against leafminer attack than did wild-type plants. The number of feeding scars caused by inoculated adult leafminers in JA-insensitive coi1-1 mutants was higher than that in wild-type plants. In addition, adults of the following generation appeared only from coi1-1 mutants and not from wild-type plants, suggesting that the loss of the JA-dependent plant defense converted nonhost plants to accessible host plants. Interestingly, the glucosinolate-myrosinase defense system may play at most a minor role in this conversion, indicating that this major antiherbivore defense of Brassica species plants probably does not have a major function in plant resistance to leafminer. Application of JA to wild-type plants before leafminer feeding enhanced feeding resistance in Chinese cabbage (Brassica rapa), tomato (Solanum lycopersicum), and garland chrysanthemum (Chrysanthemum coronarium). Our results indicate that JA plays an important role in the plant response and resistance to leafminers and, in so doing, affects host plant suitability for leafminers.

Insect attack is one of the most important factors affecting agricultural production, together with pathogen infections and abiotic stress conditions such as dehydration, high salinity, and heat. Insect attacks retard plant growth and decrease harvest levels (Hendrix, 1988). Plants exhibit many types of defenses to insect attack, which are classified into two major classes: constitutive defense and induced defense (Kessler and Baldwin, 2002; Howe and Jander, 2008; Howe and Schaller, 2008). Plant defenses have been well analyzed at the molecular, metabolic, and physiological levels (Van Poecke, 2007; Howe and Schaller, 2008). However, we currently understand only some aspects of plant defenses to herbivore attacks. Insects constitute the most numerous species on Earth, with approximately 920,000 insect species having been described (Matthews and Matthews, 2010). Among them, more than 400,000 herbivorous insect species exist (Schoonhoven et al., 2005). Insect herbivores have various feeding styles, and those of agriculturally important insect pests have been well studied. The chewing-type feeding by lepidopteran larvae (caterpillars) and the sucking-type feeding by aphids and whiteflies are the best understood feeding mechanisms from the viewpoint of the plant response to herbivore attacks (Rossi et al., 1998; Kahl et al., 2000; Reymond et al., 2000; Winz and Baldwin, 2001; Li et al., 2003; Nombela et al., 2003). Piercing and sucking-type feeding, which are characteristic of thrips and spider mites, respectively, have also been extensively analyzed (Parrella, 1995; Abe et al., 2008, 2009). The other important feeding manner with agricultural impact is mining-type feeding by leafminer larvae. Leafminers are the larvae of various beetles, flies, and moths. The adult lays its eggs on the leaf, and the larvae feed inside the leaf and stem tissues, creating tunnels (Connor and Taverner, 1997; Yamazaki, 2010).

Numerous studies analyzing the plant response to various insect herbivores indicate the importance of the plant hormone jasmonate (JA). JA mediates many processes in plant growth and development and regulates part of the plant’s basal defense system, such as plant responses to insect feeding (Browse and Howe, 2008; Schaller and Stintzi, 2008), pathogen attack, mechanical wounding, UV irradiation, ozone exposure, and osmotic stress (Thomma et al., 1998; Sasaki-Sekimoto et al., 2005). Reymond et al. (2004) reported the importance of JA in plant resistance to cabbage butterfly (Pieris rapae), whereas Ellis et al. (2002) reported that the JA-dependent plant defense is also involved in aphid resistance. JA-dependent plant defense also has a role in the response and resistance to thrips attack (Abe et al., 2008, 2009). Interestingly, several reports indicate that application of JA can reduce the feeding, oviposition, and population growth of herbivores (Thaler et al., 2001; Lu et al., 2004; Rodriguez-Saona and Thaler, 2005; Abe et al., 2009). However, the effect of the JA-dependent plant defense on host suitability for herbivores is unknown.

The American serpentine leafminer (Liriomyza trifolii), a member of the family Agromyzidae, is a highly polyphagous pest insect that causes serious damage to many crops, vegetables, fruits, and flower plants, including members of the Brassicaceae, Solanaceae, Asteraceae, Fabaceae, Cucurbitaceae, and Apiaceae (Parrella, 1987). Leafminer larvae are mining-type feeders. In addition to the American serpentine leafminer, the tomato leafminers (Liriomyza sativae and Liriomyza bryoniae) and pea leafminer (Liriomyza huidobrensis) cause major problems worldwide. Because of the frequent emergence of insecticide resistance, it is difficult to control leafminers with insecticides (Parrella, 1987). Therefore, elucidation of the molecular mechanisms responsible for the plant response and resistance to leafminers is important to contribute to the development of new methods to prevent damage.

Here, we analyzed the role of JA in the plant response to American serpentine leafminer attack and the function of the JA-dependent plant defense in leafminer resistance and host suitability by using Arabidopsis (Arabidopsis thaliana).

RESULTS

Expression of Marker Genes for the JA Response

The larvae of American serpentine leafminers have a unique feeding style in that they make tunnels in plant leaves (mining-type feeding; Fig. 1); the adults of American serpentine leafminers have a piercing-type feeding style, which may also cause serious problems for some agricultural crops. To characterize the plant response to leafminer feeding, we analyzed the JA-, ethylene (ET)-, and salicylic acid (SA)-dependent marker genes for the plant defense response in Arabidopsis. In this analysis, five adult females were allowed to feed on each whole plant in a cylindrical acryl chamber with air ventilation windows covered with a fine mesh. We sampled the aboveground portions of the plants after 3 d to analyze adult feeding, because the larvae did not hatch within these 3 d. We also sampled at 7 d after the start of the assay to analyze larval feeding in addition to adult feeding. Expression of the VEGETATIVE STORAGE PROTEIN2 (VSP2) and LIPOXYGENASE2 (LOX2) genes, markers for the JA-dependent plant defense, was induced in wild-type plants by leafminer feeding after 3 and 7 d (Fig. 2, A and B). Similarly, expression of the JA- and ET-dependent marker genes β-CHITINASE (chiB) and PLANT DEFENSIN1.2 (PDF1.2) was also induced by leafminer feeding after both 3 and 7 d (Fig. 2, C and D). However, expression of the SA-dependent marker genes PATHOGENESIS-RELATED PROTEIN1 (PR1) and β-1,3-GLUCANASE2 (BGL2) was not induced (Fig. 2, E and F). These results indicate the possible involvement of JA in the plant response to leafminer feeding. Therefore, we subsequently used the JA-insensitive coi1-1 mutant (Xie et al., 1998) to analyze the expression of these marker genes in response to leafminer feeding. Induction of all four JA-related marker genes, VSP2, LOX2, PDF1.2, and chiB, by leafminer feeding was notably decreased or abolished in the coi1-1 mutants after 3 and 7 d (Fig. 2, A–D). Expression of SA-dependent PR1 and BGL2 was not affected in the coi1-1 mutant, as was found with the wild-type plants (Fig. 2, E and F).

Figure 1.

Figure 1.

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Life cycle of the American serpentine leafminer. A, An adult female American serpentine leafminer. Bar = 500 μm. B, Diagram of the life cycle of the American serpentine leafminer. Adult females oviposit their eggs into the leaf mesophyll, and the eggs subsequently hatch into first-instar larvae. The first-instar larvae feed inside the mesophyll tissue; the feeding scars resemble mining tunnels. The first-instar larvae mature to second-instar larvae and subsequently to third-instar larvae. The third-instar larvae become pupae, which appear on the leaf surface and develop into adults.

Figure 2.

Figure 2.

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Expression of marker genes for JA, ET, and SA signals induced by leafminer feeding on Arabidopsis. A and B, VSP2 and LOX2, marker genes for the JA pathway. C and D, chiB and PDF1.2, marker genes for the JA/ET pathway. E and F, PR1 and BGL2, marker genes for the SA pathway. Five 3-week-old plants were grown in a single pot, and five adult female leafminers were allowed to feed on them. After 0, 3, and 7 d, total RNA was prepared from the plants with (+Feeding) or without (−Feeding) leafminer feeding, and first-strand cDNA was synthesized for PCR analysis. Primer sequences used in this analysis are described in “Materials and Methods.” The expression level of each gene was normalized to the expression of CBP20 (control) and is shown as a relative value. Each value represents the average ± sd of three replications of 10 plants each. WT, Wild type.

To further analyze the function of JA in the response to leafminer feeding, we measured the contents of JA in wild-type plants that were fed upon by both larval and adult leafminers for 7 d. The JA content in these plants after leafminer feeding was about twice that in the uninfested control plants (Fig. 3). These data support the importance of JA in the plant response to leafminer feeding.

Figure 3.

Figure 3.

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Effect of leafminer feeding on JA biosynthesis in Arabidopsis. Endogenous levels of JA (JA + methyl JA) were measured. Five adult female leafminers were allowed to feed on a 3-week-old wild-type plant in a closed container with air vents (Feeding). The control plant was kept in a container without leafminers (Control). The JA content of plant tissue (1 g) was measured 10 d after the start of feeding. The results shown are means ± sd of at least four independent measurements. Asterisks indicate significant differences (Student’s t test), **P < 0.01. FW, Fresh weight.

Effect of the JA-Dependent Plant Defense on Leafminer Attack

We then analyzed the role of the JA-dependent plant defense in resistance to leafminers. We compared the feeding damage between wild-type plants and JA-insensitive coi1-1 mutant plants. Each line was inoculated with five adult female leafminers in a cylindrical acryl chamber with air ventilation windows covered with a fine mesh. The feeding scars by the adult female leafminers were found in both wild-type and coi1-1 plants after 7 d (Fig. 4, A and C). We found only tiny feeding scars, probably caused by the feeding of first-instar larvae, on the wild-type and coi1-1 plants at 7 d. These tiny feeding scars on the wild-type plants did not diffuse even after 14 d (Fig. 4B). On the other hand, the coi1-1 mutants showed many huge, drawn-out feeding scars, which were probably produced by third-instar larvae at 14 d (Fig. 4D). We found pupae only on coi1-1 mutant leaves (Fig. 4, D and G).

Figure 4.

Figure 4.

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Effect of the JA-dependent plant defense on leafminer attack. The effects of a leafminer attack on wild-type plants (WT) and coi1-1 mutants were compared. Three-week-old wild-type plants (top) and coi1-1 mutants (bottom) were grown. Five adult female leafminers were allowed to feed on each plant. The photograph shows plants after 7 d (A and C) and 14 d (B and D). A part of the leaf in A, C, and D is magnified in E, F, and G, respectively. The arrow indicates a pupa emerging from the leaf mesophyll tissue.

To further analyze the role of the JA-dependent plant defense on resistance to leafminers, we compared the feeding scar areas of wild-type plants with those of coi1-1 mutants after each plant had been inoculated with five adult female leafminers. Injury from these leafminers was significantly lower in wild-type plants than in coi1-1 mutants at the 3-d time point (Fig. 5). The differences in adult and larval feeding were even more pronounced after 7 d. These findings suggest that the JA-dependent plant defense has a role in plant resistance to leafminers and affects the degree of damage caused by these insects.

Figure 5.

Figure 5.

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Effect of the JA-dependent plant defense on leafminer feeding. The feeding scars left by leafminers on wild-type plants (WT) and coi1-1 mutants were compared. Three-week-old wild-type plants and coi1-1 mutants were grown. Five adult female leafminers were allowed to feed on each plant. The feeding scars were measured after 3 and 7 d. The results shown are means ± sd of at least six independent measurements. The different letters indicate statistically significant differences between treatments (Tukey-Kramer honestly significant difference test; P < 0.05).

Effect of the JA-Dependent Plant Defense on the Leafminer Population and Host Suitability

Because we observed leafminer pupae on coi1-1 mutants but not on wild-type plants, we analyzed the effect of the JA-dependent plant defense on the progeny of the adult leafminers used for inoculation. We put five adult females on wild-type and coi1-1 plants and then counted the number of feeding scars left by adults and larvae and also the number of first-instar larvae after 3 d and second- and third-instar larvae after 9 d. The number of feeding scars on the coi1-1 mutants left by adults and larvae was significantly larger than the number on wild-type plants (Fig. 6, A and B). It was difficult to distinguish first-instar larvae from eggs; therefore, we classified them all as first-instar larvae. We did not find any significant difference in the number of first-instar larvae between wild-type plants and coi1-1 mutants (Fig. 6C). However, we did find a significant difference in the number of second-instar larvae between wild-type plants and coi1-1 mutants (Fig. 6D). Interestingly, third-instar larvae were found only in coi1-1 plants (Fig. 6E). These results clearly indicate that all of the first- and second-instar larvae died before they could become third-instar larvae in wild-type plants. On the other hand, most of the larvae in the coi1-1 plants matured to third-instar larvae. These findings were supported by the presence of dead leafminer larvae in wild-type plants only (Fig. 6, F–H).

Figure 6.

Figure 6.

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Effect of the JA-dependent plant defense on the leafminer larval population. A to E, Three-week-old wild-type plants (WT) and coi1-1 mutants were grown. Five adult female leafminers were allowed to feed on each plant. The number of feeding scars left by adults (A) and larvae (B) on wild-type plants and coi1-1 mutants was determined after 7 d. The number of first-instar (C), second-instar (D), and third-instar (E) larvae was determined after 2 weeks. Means ± sd of the feeding scar areas are based on at least 30 independent determinations. Asterisks indicate significant differences (Student’s t test; ***P < 0.001). F and G, The photographs show a live second-instar larva in a coi1-1 mutant. For clarity, epidermal tissue was removed in G. H, A dead second-instar larva in a wild-type plant.

We then determined the number of next-generation adult leafminers. This experiment was carried out in independent cylindrical acryl chambers for wild-type plants and coi1-1 mutants, as described in “Materials and Methods.” The next-generation adult leafminers only appeared from the coi1-1 mutants. This result clearly indicates that the leafminer larvae could grow into adults only in the coi1-1 mutants and not in the wild-type plants (Fig. 7). Finally, we performed loss-of-function analyses of the glucosinolate-myrosinase defense system, one of the best understood antiherbivore factors in Brassica species, to test its effects on host suitability for leafminers. To assess the role of the glucosinolate-myrosinase defense system, we looked for next-generation adult leafminers on inoculated tgg1/tgg2 double knockout mutants of the myrosinase genes, which encode for proteins that catalyze isothiocyanate production (Barth and Jander, 2006), and on inoculated myb28/myb29 and cyp79B2/cyp79B3 double knockout mutants, in which the biosynthesis of Met-derived aliphatic glucosinolates (Hirai et al., 2007) and Trp-derived indole glucosinolates (Zhao et al., 2002; Celenza et al., 2005; Sugawara et al., 2009), respectively, is defective. As with the wild-type plants, no second-generation adult leafminers appeared on the tgg1/tgg2 or myb28/myb29 double knockout mutants. However, a few second-generations adult leafminers appeared on the cyp79b2/cyp79b3 double knockout mutants (Fig. 7).

Figure 7.

Figure 7.

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Effect of the JA-dependent plant defense on the population of adult leafminers. Five adult female leafminers were allowed to feed on a 3-week-old wild-type plant (WT), coi1-1 mutants, and tgg1-1/tgg2-1, myb28/myb29, and cyp79B2/cyp79B3 double mutants in a closed container with air vents for 1 d. The number of adult leafminers was counted after 2 weeks. The results shown are means ± sd of five independent measurements. The different letters indicate statistically significant differences between treatments (Tukey-Kramer honestly significant difference test; P < 0.05).

To assess the importance of the glucosinolate-myrosinase defense system in more detail, we analyzed the contents of glucosinolates in wild-type, coi1-1, tgg1/tgg2, myb28/myb29, and cyp79B2/cyp79B3 plants. The contents of aliphatic glucosinolates such as glucoiberin, glucoraphanin, glucoalyssin, glucohesperin, and glucoibarin were increased after leafminer attack (Fig. 8, A–F). In addition, the contents of indole glucosinolates such as glucobrassicin, 1-methoxyglucobrassicin, and 4-methoxyglucobrassicin were significantly increased after leafminer attack (Fig. 8, G–I). On the other hand, the contents of both aliphatic and indole glucosinolates were decreased in coi1-1 mutants as compared with wild-type plants under normal conditions (Fig. 8), and they were not increased after leafminer attack (Fig. 8). The contents of aliphatic glucosinolates and indole glucosinolates were significantly decreased in myb28/myb29 and cyp79B2/cyp79B3 plants, respectively (Fig. 8), as reported previously (Zhao et al., 2002; Celenza et al., 2005; Hirai et al., 2007; Sugawara et al., 2009). The glucosinolate contents in tgg1/tgg2 plants were similar to the contents in wild-type plants (Fig. 8).

Figure 8.

Figure 8.

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Effect of leafminer feeding on glucosinolate contents. Five adult female leafminers were allowed to feed on 3-week-old wild-type plants (WT), coi1-1 mutants, and tgg1-1/tgg2-1, myb28/myb29, and cyp79B2/cyp79B3 double knockout mutants in a closed container with air vents for 3 d (gray bars). A control treatment was performed without leafminer feeding for 3 d (white bars). The concentrations of the individual glucosinolates are shown as relative values: glucoiberin (3MSOP; A), glucoraphanin (4MSOB; B), glucoalyssin (5MSOP; C), glucohesperin (6MSOH; D), glucoibarin (7MSOH; E), 8MSOO (F), glucobrassicin (I3M; G), 1-methoxyglucobrassicin (1MO-I3M; H), and 4-methoxyglucobrassicin (4MO-I3M; I). Different letters indicate statistically significant differences between treatments (Tukey-Kramer honestly significant difference test; P < 0.05). Asterisks indicate significant differences (Student’s t test; *P < 0.05).

JA-Dependent Plant Resistance to Leafminers in Chinese Cabbage, Tomato, and Garland Chrysanthemum

To determine whether the JA-dependent resistance to leafminers extended to other plant species, we analyzed the effect of JA application to Chinese cabbage (Brassica rapa subsp. pekinensis), one of the most important Brassica species crops; tomato (Solanum lycopersicum), an important solanaceous crop; and garland chrysanthemum (Chrysanthemum coronarium), a major composite crop. In all cases, each plant was grown in a single pot that was immersed in a 100 μm JA solution for 2 d before inoculation with five adult female leafminers. Injury from leafminer attack in Chinese cabbage was dramatically lower in plants pretreated with JA than in untreated plants (Fig. 8, A and B). Similar results were obtained with tomato and garland chrysanthemum plants (Fig. 8, C and D). These results indicate that JA has an important role in resistance to leafminer attack in Chinese cabbage, tomato, and garland chrysanthemum.

DISCUSSION

Various responses at the molecular, metabolic, and physiological levels are induced in plants when they undergo insect attack, and these responses contribute to plant resistance to those insects (Van Poecke, 2007; Howe and Schaller, 2008). Such resistance is generically termed induced plant resistance. Here, we analyzed the JA-dependent plant response and resistance to American serpentine leafminer feeding and assessed host suitability for leafminers. Interestingly, adult and larval leafminers have different feeding styles, namely, piercing-type and mining-type feeding, respectively. Both feeding styles induced the expression of the JA-related marker genes VSP2, LOX2, chiB, and PDF1.2 (Fig. 2). Note that the induction of VSP2 and LOX2 was higher 3 d after the start of the leafminer attack than at 7 d, whereas the induction of PDF1.2 and chiB was lower at 3 d than it was at 7 d. On day 3 after the leafminer release, feeding was limited to adults, which laid eggs in the plant mesophyll tissue. On day 7 after the leafminer release, both adults and hatched larvae were feeding on the plants. We cannot distinguish between the plant responses to adult feeding and larval feeding accurately because of this sequential process. However, differences in the feeding styles of the adults and larvae may be reflected in the different patterns of gene induction 3 and 7 d after leafminer release. We also found that induction of the marker genes for the JA-dependent plant defense was repressed or decreased in JA-insensitive coi1-1 mutants after both 3 and 7 d. In addition, the JA contents in Arabidopsis wild-type plants increased 7 d after the leafminer release (Fig. 3). These results indicate that JA has important roles in the plant response to both adult and larval feeding of leafminers. JA is known to have a role in the plant response to insect herbivores such as lepidopteran caterpillars, thrips, and spider mites (Arimura et al., 2000; Reymond et al., 2004; Abe et al., 2008; Howe and Jander, 2008). This study shows that JA also functions in the plant response to leafminers.

When we analyzed the role of the JA-dependent plant-induced defense in plant resistance to leafminer attack, we found that JA-insensitive coi1-1 mutants were fed on much more than wild-type plants (Fig. 4). The increased damage to coi1-1 mutants was confirmed by both the areas of feeding scars (Fig. 5) and the number of feeding scars left by adults and larvae (Fig. 6, A and B). In addition to affecting the plant response to feeding, the JA-dependent plant defense thus has a role in plant resistance to both leafminer adults and larvae.

Interestingly, our results suggest that coi1-1 mutants of Arabidopsis plants are a suitable food source for American serpentine leafminers, whereas the wild-type plants used in this study are not. Neither adults nor larvae fed much on the wild-type plants (Figs. 4 and 5), and all of the first- and second-instar larvae died before they could become third-instar larvae (Fig. 6). In contrast, we found that some of the leafminer larvae successfully developed to the pupal stage and emerged to the adult stage of the next generation in coi1-1 mutants (Fig. 7). These results mean that the loss of the JA-dependent plant defense changes nonhost plants to accessible host plants for leafminers. Thus, the JA-dependent plant defense may affect host suitability for American serpentine leafminers. In Brassicaceae species, including Arabidopsis, glucosinolate breakdown products such as isothiocyanate are well-known defense components that exert plant resistance to herbivores (Hopkins et al., 2009). Glucosinolates are hydrolyzed by myrosinase to form isothiocyanates. Loss of isothiocyanate in the Arabidopsis tgg1/tgg2 double knockout mutant of myrosinase genes improves the growth of tobacco hornworm (Manduca sexta) and cabbage looper (Trichoplusia ni; Barth and Jander, 2006). In addition, Müller et al. (2010) performed experiments with aliphatic glucosinolate-deficient myb28/myb29 and indole glucosinolate-deficient cyp79B2/cyp79B3 mutants and reported a positive function of indole and aliphatic glucosinolates in resistance against several lepidopteran larvae. Glucosinolates are constitutively expressed in plant tissues; however, their levels clearly increase upon insect attack, such as lepidopteran larval feeding (Mewis et al., 2006).

Because of these findings, we analyzed whether leafminer larvae could successfully develop to the pupal stage and ultimately emerge in the adult stage from tgg1/tgg2, myb28/myb29, and cyp79B2/cyp79B3 mutants. The leafminer larvae in tgg1/tgg2 and myb28/myb29 mutants did not develop to the pupal stage. On the other hand, the leafminer larvae in cyp79B2/cyp79B3 mutants developed to the pupal stage and then became adults, indicating a possible function of indole glucosinolates in determining host suitability for leafminers (Fig. 7). However, the effect of indole glucosinolates is likely to be limited, because the number of next-generation adult leafminers in cyp79B2/cyp79B3 mutants was much lower than the number in coi1-1 mutants (Fig. 7). Importantly, we detected increased contents of aliphatic and indole glucosinolates after leafminer feeding (Fig. 8). As reported by Mewis et al. (2006), both aliphatic and indole glucosinolate contents were decreased in coi1-1 mutants not exposed to leafminer feeding in our study. In addition, these glucosinolate compounds were not increased in coi1-1 mutants after leafminer attack. Interestingly, the amount of increase of indole glucosinolate contents after leafminer feeding was much higher than that of aliphatic glucosinolate contents (Fig. 8). It is well understood that cyp79B2/cyp79B3 mutants are also defective in the biosynthesis of the Arabidopsis phytoalexin, camalexin (Glawischnig et al., 2004). However, camalexin contents were not increased by leafminer attack (data not shown). Müller et al. (2010) reported a clear differential effect of aliphatic and indole glucosinolates on resistance to several herbivores between cyp79B2/cyp79B3 and myb28/myb29 double knockout mutants and cyp79B2/cyp79B3/myb28/myb29 quadruple knockout mutants, in which the biosynthesis of both aliphatic and indole glucosinolates is defective. Further advanced analyses using this quadruple knockout mutant would reveal the function of these defense compounds for host plant suitability of leafminer resistance in Arabidopsis plants.

Plant defenses against insect pests are classified into two groups: constitutive defenses and induced defenses (Kessler and Baldwin, 2002; Howe and Jander, 2008; Howe and Schaller, 2008). The constitutive defense against herbivores is often considered as a host-determining factor in the plant-herbivore interaction (Schoonhoven et al., 2005). Anatomical traits, such as leaf trichomes, and plant surface traits, such as cuticle texture, are important constitutive defense components that affect host suitability (Schoonhoven et al., 2005). The existence of secondary metabolites is also important for host determination (Konno et al., 2006). Schoonhoven et al. (2005) reviewed the mechanism of host-plant selection and discovered that the importance of the feeding stimulants of host plants and the feeding deterrents of nonhost plants lies with the balance between the stimulants and the deterrents.

The relationship between induced plant defenses and host-determining factors is poorly understood, although increasing the level of the JA-dependent plant defense enhances plant resistance to various herbivores (Browse and Howe, 2008; Schaller and Stintzi, 2008). The JA-dependent plant defense is multifaceted; it involves increasing herbivore avoidance and shortening the herbivore’s life cycle by decreasing egg production, hatching rates, etc. (Thaler et al., 2001; Lu et al., 2004; Rodriguez-Saona and Thaler, 2005; Abe et al., 2009). It is noteworthy that these facets may also play a part in host suitability for herbivores. Zarate et al. (2007) reported that the JA-dependent plant defense functions in resistance to silverleaf whitefly. They suggested that feeding by the silverleaf whitefly induces the SA-dependent plant defense, which is antagonistic to the JA-dependent plant defense. The presence of such a system in herbivores for deactivating the JA-dependent plant defense may explain why this plant defense is an important target of host plant determination and the coevolution of plants and herbivores. Kessler et al. (2004) performed a field experiment that suggested a relationship between the JA-dependent plant defense and host suitability for herbivores. They found that transgenic Nicotiana attenuata plants that overexpressed the gene for lipoxygenase, an enzyme involved in JA biosynthesis, in the antisense orientation suffered feeding damage caused by an herbivore that does not usually feed on N. attenuata. Here, on the basis experimental results obtained with Arabidopsis coi1-1 mutants, we report in detail the conversion of nonhost plants to possible candidate host plants as a result of the loss of the JA-dependent plant-induced defense. The larvae of the leafminers fed inside the leaf mesophyll tissue and could not escape from the leaf. This specific feeding mode thus prevented the leafminer larvae from escaping from the plant defense system. The specific feeding mode of the leafminer larvae may make this herbivore particularly sensitive to plant-induced defenses.

The American serpentine leafminer is among the most problematic of herbivores, being difficult to control with insecticides and having a wide host range. We found that JA application to activate JA-dependent plant resistance is an effective way to decrease the effects of leafminers in a brassicaceous crop (Chinese cabbage), a solanaceous crop (tomato), and a composite crop (garland chrysanthemum; Fig. 9). The effect of JA application in Chinese cabbage might be slightly explained by the role of glucosinolate. However, JA treatment was also effective in tomato and garland chrysanthemum, which do not contain a glucosinolate-myrosinase defense system. There should be a common mechanism to provide leafminer resistance by JA application. Our next goal should be the identification of the main defensive compounds that function in leafminer resistance and affect host suitability for leafminer. Many candidate compounds exist. For example, many defensive compounds contain phenolics, terpenoids, and alkaloids, which are regulated by JA (Howe and Schaller, 2008). In addition, a VSP encoded by the JA-inducible marker gene VSP2 has also been reported to have anti-insect activity (Liu et al., 2005). Further efforts to understand in detail the JA-dependent plant-induced defense against leafminers are warranted.

Figure 9.

Figure 9.

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Effect of JA application on plant resistance to leafminers. Five adult females were fed on 2-week-old Chinese cabbage (A and B) and 3-week-old tomato (C) and garland chrysanthemum (D) plants for 2 weeks. Plants were immersed in water or a 100 µm JA solution 2 d before leafminers were introduced. B shows typical images of Chinese cabbage after leafminer feeding. The results shown are means ± sd of the areas of the feeding scars based on at least 20 independent determinations. Asterisks indicate significant differences (Student’s t test; ***P < 0.001).

MATERIALS AND METHODS

Plant Materials and Cultivation

Wild-type (ecotype Columbia) Arabidopsis (Arabidopsis thaliana) plants, JA-insensitive coi1-1 mutants (Feys et al., 1994), and tgg1-1/tgg2-1 (Barth and Jander, 2006), myb28/myb29 (Hirai et al., 2007), and cyp79B2/cyp79B3 (Zhao et al., 2002; Celenza et al., 2005; Sugawara et al., 2009) double knockout mutants were grown in soil as described previously (Weigel and Glazebrook, 2002). Seeds were sown on sterile soil in pots, moistened, and held at 4°C for 7 d in the dark to synchronize germination. The pots were then transferred to 22°C with a long-day photoperiod (16 h of light/8 h of dark). Plants at the four-leaf stage were transferred to individual pots and grown to the rosette stage. Chinese cabbage (Brassica rapa subsp. pekinensis ‘Kyoto No. 3’; Takii Seed) plants, garland chrysanthemum (Chrysanthemum coronarium ‘Ohba shungiku’; Sakata Seed) plants, and tomato (Solanum lycopersicum ‘Momotaro’; Takii Seed) plants were similarly grown in soil, except the tomato plants were grown at 25°C.

Identification of coi1-1 Plants

Homozygous coi1-1 plants were selected by using TaqMan SNP Genotyping Assays (Applied Biosystems). Nucleotide sequences of the primers used were as follows: forward primer, 5′-CTTAAGCTACATCGGACAGTACAGT-3′; reverse primer, 5′-CCTTCATCTGATTCACCTACGTAACC-3′; reporter primers, 5′-CAGCAGCATCCATCTC-3′ and 5′-CAGCAGCATTCATCTC-3′.

Leafminer Attack

Laboratory colonies of American serpentine leafminers (Liriomyza trifolii) were maintained in a closed environmental chamber as described previously (Amano et al., 2008). Only adult females were used in this study. The mated adult females were starved for 2 to 3 h before being allowed to feed on the test plants. Five females were allowed to feed on each whole plant in a cylindrical acryl chamber with air ventilation windows covered with a fine mesh.

JA Treatment

Pots holding 2-week-old Chinese cabbage plants or 3-week-old tomato or garland chrysanthemum plants grown on soil were transferred into a cylindrical acryl chamber containing a 100 μm JA solution. JA treatment was carried out for 2 d before the leafminer attack was initiated.

Assessment of the Leafminer Population

Three-week-old Arabidopsis plants grown in soil were placed in a cylindrical acryl chamber. Three plants were placed in each chamber. Five adult female leafminers were then put in each chamber. After 12 h, these adults were removed. The numbers of first-, second-, and third-instar larvae were observed with a stereoscopic microscope, and the number of adults was counted with the unaided eye.

RNA Extraction and Transcript Measurements

Five adult female leafminers were fed on three 2-week-old Arabidopsis plants at the rosette stage for 7 or 14 d in a closed container with air vents. The experiments were repeated twice. After feeding, the plants were frozen in liquid nitrogen. Total RNA (2 µg), isolated with Trizol reagent (Invitrogen) and an RNeasy MinElute Cleanup Kit (Qiagen), was treated with RNase-free DNase (Takara) to eliminate genomic DNA. First-strand complementary DNA (cDNA) was synthesized with random oligohexamers and SuperScript III reverse transcriptase according to the manufacturer’s instructions (Invitrogen). Quantitative real-time PCR was carried out with the Power SYBR Green PCR Master Mix (Applied Biosystems) by using the first-strand cDNA as a template on a sequence detector (ABI Prism 7900HT; Applied Biosystems). Expression of CBP20 was used for normalization as a standard control gene. Nucleotide sequences of the gene-specific primers used were as follows: VSP2 (At5g24770; forward primer, 5′-GTTAGGGACCGGAGCATCAA-3; reverse primer, 5′-AACGGTCACTGAGTATGATGGGT-3′); LOX2 (At3g45140; forward primer, 5′-TTGCTCGCCAGACACTTGC-3′; reverse primer, 5′-GGGATCACCATAAACGGCC-3′); chiB (At3g12500; forward primer, 5′-ACGGAAGAGGACCAATGCAA-3′; reverse primer, 5′-GTTGGCAACAAGGTCAGGGT-3′); PDF1.2 (At5g44420; forward primer, 5′-CCATCATCACCCTTATCTTCGC-3′; reverse primer, 5′-TGTCCCACTTGGCTTCTCG-3′); BGL2 (At3g57260; forward primer, 5′-GCCGACAAGTGGGTTCAAGA-3′; reverse primer, 5′-AACCCCCCAACTGAGGGTT-3′); PR1 (At2g14610; forward primer, 5′-GTTGCAGCCTATGCTCGGAG-3′; reverse primer, 5′-CCGCTACCCCAGGCTAAGTT-3′); and CBP20 (At5g44200; forward primer, 5′-CCTTGTGGCTTTTGTTTCGTC-3′; reverse primer, 5′-ACACGAATAGGCCGGTCATC-3′).

JA Quantification

JA and its methyl ester were quantified as described previously (Seo et al., 1995), except that an HP6890 gas chromatograph fitted to a quadrupole mass spectrometer (Hewlett-Packard) was used.

Glucosinolate Quantification

Glucosinolates were analyzed by liquid chromatography-mass spectrometry using 10-camphorsulfonic acid as an internal standard for relative quantification (Sawada et al., 2012).

Feeding Scar Area Measurements

The areas of leafminer feeding scars on the surface of each Arabidopsis, Chinese cabbage, tomato, and garland chrysanthemum leaf were measured by using WinROOF software, version 5.8.1 (Mitani). The areas were analyzed with JMP software, version 5.1 (SAS Institute).

The Arabidopsis Genome Initiative gene codes and GenBank accession numbers, respectively, for genes mentioned in this article are as follows: VSP2 (At5g24770, AB006778), LOX2 (At3g45140, AYO62611), chiB (At3g12500, AY054628), PDF1.2 (At5g44420, AY063779), BGL2 (At3g57260, AY099668), PR1 (At2g14610, AY064023), and CBP20 (At5g44200, AF140219).

Acknowledgments

We thank Fumie Mori, Setsuko Kawamura, and Issei Sasaki of the RIKEN BioResource Center for their excellent technical assistance. We are grateful to Dr. Hiroyuki Kasahara of the RIKEN Center for Sustainable Resource Science for providing cyp79B2/cyp79B3 double knockout mutants.

Glossary

JA

jasmonate

ET

ethylene

SA

salicylic acid

cDNA

complementary DNA

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