disarming the jasmonate-dependent plant defense makes ... · the number of feeding scars caused by...

Disarming the Jasmonate-Dependent Plant DefenseMakes Nonhost Arabidopsis Plants Accessible to theAmerican Serpentine Leafminer1

Hiroshi Abe*, Ken Tateishi, Shigemi Seo, Soichi Kugimiya, Masami Yokota Hirai, Yuji Sawada,Yoshiyuki Murata, Kaori Yara, Takeshi Shimoda, and Masatomo Kobayashi

Experimental Plant Division, RIKEN BioResource Center, Tsukuba 305–0074, Japan (H.A., M.K.); NationalInstitute of Agrobiological Sciences, Tsukuba 305–8602, Japan (K.T., S.S.); National Institute for Agro-Environmental Sciences, Tsukuba 305–8604, Japan (S.K.); RIKEN Center for Sustainable Resource Science,Yokohama 230–0045, Japan (M.Y.H., Y.S.); Okayama University, Okayama 700–7530, Japan (Y.M.); NAROInstitute of Vegetable and Tea Science, Shimada 428–8501, Japan (K.Y.); and National Agricultural ResearchCenter, Tsukuba 305–8666, Japan (T.S.)

ORCID IDs: 0000-0002-7563-0918 (H.A.); 0000-0003-0802-6208 (M.Y.H.).

Here, we analyzed the interaction between Arabidopsis (Arabidopsis thaliana) and the American serpentine leafminer (Liriomyzatrifolii), an important and intractable herbivore of many cultivated plants. We examined the role of the immunity-related planthormone jasmonate (JA) in the plant response and resistance to leafminer feeding to determine whether JA affects host suitabilityfor leafminers. The expression of marker genes for the JA-dependent plant defense was induced by leafminer feeding onArabidopsis wild-type plants. Analyses of JA-insensitive coi1-1 mutants suggested the importance of JA in the plant responseto leafminer feeding. The JA content of wild-type plants significantly increased after leafminer feeding. Moreover, coi1-1mutantsshowed lower feeding resistance against leafminer attack than did wild-type plants. The number of feeding scars caused byinoculated adult leafminers in JA-insensitive coi1-1 mutants was higher than that in wild-type plants. In addition, adults of thefollowing generation appeared only from coi1-1mutants and not from wild-type plants, suggesting that the loss of the JA-dependentplant defense converted nonhost plants to accessible host plants. Interestingly, the glucosinolate-myrosinase defense system mayplay at most a minor role in this conversion, indicating that this major antiherbivore defense of Brassica species plants probablydoes not have a major function in plant resistance to leafminer. Application of JA to wild-type plants before leafminer feedingenhanced 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 toleafminers and, in so doing, affects host plant suitability for leafminers.

Insect attack is one of the most important factorsaffecting agricultural production, together with path-ogen infections and abiotic stress conditions such asdehydration, high salinity, and heat. Insect attacksretard plant growth and decrease harvest levels(Hendrix, 1988). Plants exhibit many types of defensesto insect attack, which are classified into two majorclasses: constitutive defense and induced defense(Kessler and Baldwin, 2002; Howe and Jander, 2008;Howe and Schaller, 2008). Plant defenses have beenwell analyzed at the molecular, metabolic, and physi-ological levels (Van Poecke, 2007; Howe and Schaller,2008). However, we currently understand only some

aspects of plant defenses to herbivore attacks. Insectsconstitute the most numerous species on Earth, withapproximately 920,000 insect species having been de-scribed (Matthews and Matthews, 2010). Among them,more than 400,000 herbivorous insect species exist(Schoonhoven et al., 2005). Insect herbivores havevarious feeding styles, and those of agriculturallyimportant insect pests have been well studied. Thechewing-type feeding by lepidopteran larvae (cater-pillars) and the sucking-type feeding by aphids andwhiteflies are the best understood feeding mechanismsfrom the viewpoint of the plant response to herbivoreattacks (Rossi et al., 1998; Kahl et al., 2000; Reymondet 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, re-spectively, have also been extensively analyzed (Parrella,1995; Abe et al., 2008, 2009). The other important feedingmanner with agricultural impact is mining-type feedingby leafminer larvae. Leafminers are the larvae of variousbeetles, flies, and moths. The adult lays its eggs on theleaf, and the larvae feed inside the leaf and stem tissues,creating tunnels (Connor and Taverner, 1997; Yamazaki,2010).

1 This work was supported by a Grant-in-Aid for Scientific Re-search (B) from the Ministry of Education, Culture, Sports, Science,and Technology of Japan (to T.S., H.A., and S.K.) and by the JapanAdvanced Plant Science Network.

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Hiroshi Abe ([email protected]).

www.plantphysiol.org/cgi/doi/10.1104/pp.113.222802

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Numerous studies analyzing the plant response tovarious insect herbivores indicate the importance ofthe plant hormone jasmonate (JA). JA mediates manyprocesses in plant growth and development and reg-ulates part of the plant’s basal defense system, such asplant responses to insect feeding (Browse and Howe,2008; Schaller and Stintzi, 2008), pathogen attack,mechanical wounding, UV irradiation, ozone expo-sure, and osmotic stress (Thomma et al., 1998; Sasaki-Sekimoto et al., 2005). Reymond et al. (2004) reportedthe importance of JA in plant resistance to cabbagebutterfly (Pieris rapae), whereas Ellis et al. (2002)reported that the JA-dependent plant defense is alsoinvolved in aphid resistance. JA-dependent plant de-fense also has a role in the response and resistance tothrips attack (Abe et al., 2008, 2009). Interestingly,several reports indicate that application of JA can re-duce the feeding, oviposition, and population growthof 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 defenseon host suitability for herbivores is unknown.The American serpentine leafminer (Liriomyza trifolii),

a member of the family Agromyzidae, is a highlypolyphagous pest insect that causes serious damageto many crops, vegetables, fruits, and flower plants, in-cluding members of the Brassicaceae, Solanaceae, Aster-aceae, Fabaceae, Cucurbitaceae, and Apiaceae (Parrella,1987). Leafminer larvae are mining-type feeders. In ad-dition to the American serpentine leafminer, the tomatoleafminers (Liriomyza sativae and Liriomyza bryoniae) andpea leafminer (Liriomyza huidobrensis) cause majorproblems worldwide. Because of the frequent emer-gence of insecticide resistance, it is difficult to controlleafminers with insecticides (Parrella, 1987). Therefore,elucidation of the molecular mechanisms responsiblefor the plant response and resistance to leafminers isimportant to contribute to the development of newmethods to prevent damage.Here, we analyzed the role of JA in the plant re-

sponse to American serpentine leafminer attack andthe function of the JA-dependent plant defense in leaf-miner resistance and host suitability by using Arabi-dopsis (Arabidopsis thaliana).

RESULTS

Expression of Marker Genes for the JA Response

The larvae of American serpentine leafminers have aunique feeding style in that they make tunnels in plantleaves (mining-type feeding; Fig. 1); the adults ofAmerican serpentine leafminers have a piercing-typefeeding style, which may also cause serious problemsfor some agricultural crops. To characterize the plantresponse to leafminer feeding, we analyzed the JA-,ethylene (ET)-, and salicylic acid (SA)-dependentmarker genes for the plant defense response inArabidopsis. In this analysis, five adult females were

allowed to feed on each whole plant in a cylindricalacryl chamber with air ventilation windows coveredwith a fine mesh. We sampled the aboveground por-tions of the plants after 3 d to analyze adult feeding,because the larvae did not hatch within these 3 d. Wealso sampled at 7 d after the start of the assay to analyzelarval feeding in addition to adult feeding. Expressionof the VEGETATIVE STORAGE PROTEIN2 (VSP2) andLIPOXYGENASE2 (LOX2) genes, markers for theJA-dependent plant defense, was induced in wild-typeplants by leafminer feeding after 3 and 7 d (Fig. 2,A and B). Similarly, expression of the JA- andET-dependent marker genes b-CHITINASE (chiB) andPLANT DEFENSIN1.2 (PDF1.2) was also induced byleafminer feeding after both 3 and 7 d (Fig. 2, C and D).However, expression of the SA-dependent markergenes PATHOGENESIS-RELATED PROTEIN1 (PR1)and b-1,3-GLUCANASE2 (BGL2) was not induced(Fig. 2, E and F). These results indicate the possibleinvolvement of JA in the plant response to leafminerfeeding. Therefore, we subsequently used the JA-insensitivecoi1-1 mutant (Xie et al., 1998) to analyze the expres-sion of these marker genes in response to leafminerfeeding. Induction of all four JA-related marker genes,VSP2, LOX2, PDF1.2, and chiB, by leafminer feedingwas notably decreased or abolished in the coi1-1 mu-tants after 3 and 7 d (Fig. 2, A–D). Expression of SA-dependent PR1 and BGL2was not affected in the coi1-1mutant, as was found with the wild-type plants (Fig. 2,E and F).

To further analyze the function of JA in the responseto leafminer feeding, we measured the contents of JAin wild-type plants that were fed upon by both larvaland adult leafminers for 7 d. The JA content in theseplants after leafminer feeding was about twice that inthe uninfested control plants (Fig. 3). These data sup-port the importance of JA in the plant response toleafminer feeding.

Figure 1. Life cycle of the American serpentine leafminer. A, An adultfemale American serpentine leafminer. Bar = 500 mm. B, Diagram ofthe life cycle of the American serpentine leafminer. Adult femalesoviposit their eggs into the leaf mesophyll, and the eggs subsequentlyhatch into first-instar larvae. The first-instar larvae feed inside themesophyll 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 onthe leaf surface and develop into adults.

Plant Physiol. Vol. 163, 2013 1243

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Figure 2. Expression of marker genes for JA, ET, and SA signals induced by leafminer feeding on Arabidopsis. A and B, VSP2and 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

1244 Plant Physiol. Vol. 163, 2013

Abe et al.



Effect of the JA-Dependent Plant Defense onLeafminer Attack

We then analyzed the role of the JA-dependent plantdefense in resistance to leafminers. We comparedthe feeding damage between wild-type plants andJA-insensitive coi1-1 mutant plants. Each line was in-oculated with five adult female leafminers in a cylin-drical acryl chamber with air ventilation windowscovered with a fine mesh. The feeding scars by the adultfemale leafminers were found in both wild-type and coi1-1 plants after 7 d (Fig. 4, A and C). We found only tinyfeeding 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 didnot diffuse even after 14 d (Fig. 4B). On the otherhand, the coi1-1 mutants showed many huge, drawn-out feeding scars, which were probably produced bythird-instar larvae at 14 d (Fig. 4D). We found pupaeonly on coi1-1 mutant leaves (Fig. 4, D and G).To further analyze the role of the JA-dependent

plant defense on resistance to leafminers, we com-pared the feeding scar areas of wild-type plants withthose of coi1-1 mutants after each plant had been in-oculated with five adult female leafminers. Injury fromthese leafminers was significantly lower in wild-typeplants than in coi1-1mutants at the 3-d time point (Fig. 5).The differences in adult and larval feeding were evenmore pronounced after 7 d. These findings suggest thatthe JA-dependent plant defense has a role in plantresistance to leafminers and affects the degree of dam-age caused by these insects.

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

Because we observed leafminer pupae on coi1-1 mu-tants but not on wild-type plants, we analyzed theeffect of the JA-dependent plant defense on the prog-eny of the adult leafminers used for inoculation. Weput five adult females on wild-type and coi1-1 plantsand then counted the number of feeding scars left byadults and larvae and also the number of first-instarlarvae after 3 d and second- and third-instar larvaeafter 9 d. The number of feeding scars on the coi1-1 mu-tants left by adults and larvae was significantly largerthan the number on wild-type plants (Fig. 6, A and B).It was difficult to distinguish first-instar larvae fromeggs; therefore, we classified them all as first-instarlarvae. We did not find any significant difference inthe number of first-instar larvae between wild-typeplants 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 mu-tants (Fig. 6D). Interestingly, third-instar larvae werefound only in coi1-1 plants (Fig. 6E). These resultsclearly indicate that all of the first- and second-instarlarvae died before they could become third-instarlarvae in wild-type plants. On the other hand, mostof the larvae in the coi1-1 plants matured to third-instar larvae. These findings were supported by thepresence of dead leafminer larvae in wild-type plantsonly (Fig. 6, F–H).

We then determined the number of next-generationadult leafminers. This experiment was carried out inindependent cylindrical acryl chambers for wild-typeplants and coi1-1 mutants, as described in “Materialsand Methods.” The next-generation adult leafminersonly appeared from the coi1-1 mutants. This resultclearly indicates that the leafminer larvae could growinto adults only in the coi1-1 mutants and not in thewild-type plants (Fig. 7). Finally, we performed loss-of-function analyses of the glucosinolate-myrosinasedefense system, one of the best understood anti-herbivore factors in Brassica species, to test its effectson host suitability for leafminers. To assess the roleof the glucosinolate-myrosinase defense system, welooked for next-generation adult leafminers on inocu-lated tgg1/tgg2 double knockout mutants of the

Figure 2. (Continued.)BGL2, marker genes for the SA pathway. Five 3-week-old plants were grown in a single pot, and five adult female leafminerswere allowed to feed on them. After 0, 3, and 7 d, total RNAwas prepared from the plants with (+Feeding) or without (2Feeding)leafminer feeding, and first-strand cDNAwas synthesized for PCR analysis. Primer sequences used in this analysis are describedin “Materials and Methods.” The expression level of each gene was normalized to the expression of CBP20 (control) and isshown as a relative value. Each value represents the average 6 SD of three replications of 10 plants each. WT, Wild type.

Figure 3. Effect of leafminer feeding on JA biosynthesis in Arabidopsis.Endogenous levels of JA (JA + methyl JA) were measured. Five adultfemale leafminers were allowed to feed on a 3-week-old wild-typeplant in a closed container with air vents (Feeding). The control plantwas kept in a container without leafminers (Control). The JA content ofplant tissue (1 g) was measured 10 d after the start of feeding. Theresults shown are means 6 SD of at least four independent measure-ments. Asterisks indicate significant differences (Student’s t test),**P , 0.01. FW, Fresh weight.





myrosinase genes, which encode for proteins thatcatalyze isothiocyanate production (Barth and Jander,2006), and on inoculated myb28/myb29 and cyp79B2/cyp79B3 double knockout mutants, in which thebiosynthesis of Met-derived aliphatic glucosinolates(Hirai et al., 2007) and Trp-derived indole glucosino-lates (Zhao et al., 2002; Celenza et al., 2005; Sugawaraet al., 2009), respectively, is defective. As with the wild-type plants, no second-generation adult leafminersappeared on the tgg1/tgg2 or myb28/myb29 double knock-out mutants. However, a few second-generations adultleafminers appeared on the cyp79b2/cyp79b3 doubleknockout mutants (Fig. 7).

To assess the importance of the glucosinolate-myrosinase defense system in more detail, we analyzedthe contents of glucosinolates in wild-type, coi1-1,tgg1/tgg2, myb28/myb29, and cyp79B2/cyp79B3 plants.The contents of aliphatic glucosinolates such as glucoi-berin, glucoraphanin, glucoalyssin, glucohesperin, andglucoibarin were increased after leafminer attack (Fig. 8,A–F). In addition, the contents of indole glucosinolatessuch as glucobrassicin, 1-methoxyglucobrassicin, and4-methoxyglucobrassicin were significantly increasedafter leafminer attack (Fig. 8, G–I). On the other hand,the contents of both aliphatic and indole glucosinolateswere decreased in coi1-1 mutants as compared withwild-type plants under normal conditions (Fig. 8), andthey were not increased after leafminer attack (Fig. 8).The contents of aliphatic glucosinolates and indoleglucosinolates 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). Theglucosinolate contents in tgg1/tgg2 plants were similarto the contents in wild-type plants (Fig. 8).

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

To determine whether the JA-dependent resistanceto leafminers extended to other plant species, we an-alyzed the effect of JA application to Chinese cabbage(Brassica rapa subsp. pekinensis), one of the most impor-tant Brassica species crops; tomato (Solanum lycopersicum),an important solanaceous crop; and garland chrysan-themum (Chrysanthemum coronarium), a major compositecrop. In all cases, each plant was grown in a single potthat was immersed in a 100 mM JA solution for 2 d beforeinoculation with five adult female leafminers. Injuryfrom leafminer attack in Chinese cabbage was dramati-cally lower in plants pretreated with JA than in un-treated plants (Fig. 8, A and B). Similar results wereobtained with tomato and garland chrysanthemumplants (Fig. 8, C and D). These results indicate thatJA has an important role in resistance to leafminerattack in Chinese cabbage, tomato, and garlandchrysanthemum.

DISCUSSION

Various responses at the molecular, metabolic, andphysiological levels are induced in plants when theyundergo insect attack, and these responses contributeto plant resistance to those insects (Van Poecke, 2007;Howe and Schaller, 2008). Such resistance is generi-cally termed induced plant resistance. Here, we ana-lyzed the JA-dependent plant response and resistanceto American serpentine leafminer feeding and assessedhost suitability for leafminers. Interestingly, adult and

Figure 4. Effect of the JA-dependent plant defense on leafminer attack. Theeffects of a leafminer attack on wild-type plants (WT) and coi1-1 mutantswere compared. Three-week-old wild-type plants (top) and coi1-1 mutants(bottom) were grown. Five adult female leafminers were allowed to feedon 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 meso-phyll tissue.

Figure 5. Effect of the JA-dependent plant defense on leafminerfeeding. The feeding scars left by leafminers on wild-type plants (WT)and coi1-1 mutants were compared. Three-week-old wild-type plantsand coi1-1 mutants were grown. Five adult female leafminers wereallowed to feed on each plant. The feeding scars were measured after3 and 7 d. The results shown are means6 SD of at least six independentmeasurements. The different letters indicate statistically significant dif-ferences between treatments (Tukey-Kramer honestly significant dif-ference test; P , 0.05).


Abe et al.



larval leafminers have different feeding styles, namely,piercing-type and mining-type feeding, respectively.Both feeding styles induced the expression of theJA-related marker genes VSP2, LOX2, chiB, and PDF1.2(Fig. 2). Note that the induction of VSP2 and LOX2was higher 3 d after the start of the leafminer attackthan at 7 d, whereas the induction of PDF1.2 and chiBwas lower at 3 d than it was at 7 d. On day 3 after the

leafminer release, feeding was limited to adults, whichlaid eggs in the plant mesophyll tissue. On day 7 afterthe leafminer release, both adults and hatched larvaewere feeding on the plants. We cannot distinguishbetween the plant responses to adult feeding and lar-val feeding accurately because of this sequential pro-cess. However, differences in the feeding styles of theadults and larvae may be reflected in the different

Figure 6. 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 feedingscars left by adults (A) and larvae (B) on wild-type plants and coi1-1mutants 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 6 SD of the feeding scar areas are basedon 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 deadsecond-instar larva in a wild-type plant.





patterns of gene induction 3 and 7 d after leafminerrelease. We also found that induction of the markergenes for the JA-dependent plant defense was re-pressed or decreased in JA-insensitive coi1-1 mutantsafter both 3 and 7 d. In addition, the JA contents inArabidopsis wild-type plants increased 7 d after theleafminer release (Fig. 3). These results indicate that JAhas important roles in the plant response to both adultand larval feeding of leafminers. JA is known to havea role in the plant response to insect herbivores suchas 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 thatJA also functions in the plant response to leafminers.

When we analyzed the role of the JA-dependentplant-induced defense in plant resistance to leafminerattack, we found that JA-insensitive coi1-1 mutantswere fed on much more than wild-type plants (Fig. 4).The increased damage to coi1-1 mutants was con-firmed by both the areas of feeding scars (Fig. 5) andthe number of feeding scars left by adults and larvae(Fig. 6, A and B). In addition to affecting the plantresponse to feeding, the JA-dependent plant defensethus has a role in plant resistance to both leafmineradults and larvae.

Interestingly, our results suggest that coi1-1 mutantsof Arabidopsis plants are a suitable food source forAmerican serpentine leafminers, whereas the wild-type plants used in this study are not. Neither adultsnor larvae fed much on the wild-type plants (Figs. 4and 5), and all of the first- and second-instar larvaedied before they could become third-instar larvae(Fig. 6). In contrast, we found that some of the leafminerlarvae successfully developed to the pupal stage andemerged to the adult stage of the next generation in

coi1-1 mutants (Fig. 7). These results mean that the lossof the JA-dependent plant defense changes nonhostplants to accessible host plants for leafminers. Thus,the JA-dependent plant defense may affect host suit-ability for American serpentine leafminers. In Brassi-caceae species, including Arabidopsis, glucosinolatebreakdown products such as isothiocyanate are well-known defense components that exert plant resistanceto herbivores (Hopkins et al., 2009). Glucosinolates arehydrolyzed by myrosinase to form isothiocyanates.Loss of isothiocyanate in the Arabidopsis tgg1/tgg2double knockout mutant of myrosinase genes im-proves the growth of tobacco hornworm (Manducasexta) and cabbage looper (Trichoplusia ni; Barth andJander, 2006). In addition, Müller et al. (2010) performedexperiments with aliphatic glucosinolate-deficientmyb28/myb29 and indole glucosinolate-deficientcyp79B2/cyp79B3 mutants and reported a positivefunction of indole and aliphatic glucosinolates in re-sistance against several lepidopteran larvae. Glucosi-nolates are constitutively expressed in plant tissues;however, their levels clearly increase upon insect at-tack, such as lepidopteran larval feeding (Mewis et al.,2006).

Because of these findings, we analyzed whetherleafminer larvae could successfully develop to thepupal stage and ultimately emerge in the adult stagefrom tgg1/tgg2, myb28/myb29, and cyp79B2/cyp79B3mutants. The leafminer larvae in tgg1/tgg2 and myb28/myb29 mutants did not develop to the pupal stage. Onthe other hand, the leafminer larvae in cyp79B2/cyp79B3 mutants developed to the pupal stage andthen became adults, indicating a possible function ofindole glucosinolates in determining host suitabilityfor leafminers (Fig. 7). However, the effect of indoleglucosinolates is likely to be limited, because thenumber of next-generation adult leafminers incyp79B2/cyp79B3 mutants was much lower than thenumber in coi1-1 mutants (Fig. 7). Importantly, wedetected increased contents of aliphatic and indoleglucosinolates after leafminer feeding (Fig. 8). Asreported by Mewis et al. (2006), both aliphatic and indoleglucosinolate contents were decreased in coi1-1 mutantsnot exposed to leafminer feeding in our study. In addi-tion, these glucosinolate compounds were not increasedin coi1-1 mutants after leafminer attack. Interestingly, theamount of increase of indole glucosinolate contents afterleafminer feeding was much higher than that of aliphaticglucosinolate contents (Fig. 8). It is well understood thatcyp79B2/cyp79B3 mutants are also defective in the bio-synthesis of the Arabidopsis phytoalexin, camalexin(Glawischnig et al., 2004). However, camalexin contentswere not increased by leafminer attack (data not shown).Müller et al. (2010) reported a clear differential effect ofaliphatic and indole glucosinolates on resistance to sev-eral herbivores between cyp79B2/cyp79B3 and myb28/myb29 double knockout mutants and cyp79B2/cyp79B3/myb28/myb29 quadruple knockout mutants, in which thebiosynthesis of both aliphatic and indole glucosino-lates is defective. Further advanced analyses using this

Figure 7. Effect of the JA-dependent plant defense on the population ofadult leafminers. Five adult female leafminers were allowed to feed ona 3-week-old wild-type plant (WT), coi1-1mutants, and tgg1-1/tgg2-1,myb28/myb29, and cyp79B2/cyp79B3 double mutants in a closedcontainer with air vents for 1 d. The number of adult leafminers wascounted after 2 weeks. The results shown are means 6 SD of five in-dependent measurements. The different letters indicate statisticallysignificant differences between treatments (Tukey-Kramer honestlysignificant difference test; P , 0.05).


Abe et al.



Figure 8. Effect of leafminer feeding on glucosinolate contents. Five adult female leafminers were allowed to feed on 3-week-oldwild-type plants (WT), coi1-1mutants, and tgg1-1/tgg2-1,myb28/myb29, and cyp79B2/cyp79B3 double knockout mutants in aclosed container with air vents for 3 d (gray bars). A control treatment was performed without leafminer feeding for 3 d (whitebars). 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 sta-tistically significant differences between treatments (Tukey-Kramer honestly significant difference test; P , 0.05). Asterisksindicate significant differences (Student’s t test; *P , 0.05).





quadruple knockout mutant would reveal the functionof these defense compounds for host plant suitability ofleafminer resistance in Arabidopsis plants.

Plant defenses against insect pests are classified intotwo groups: constitutive defenses and induced de-fenses (Kessler and Baldwin, 2002; Howe and Jander,2008; Howe and Schaller, 2008). The constitutive de-fense against herbivores is often considered as a host-determining factor in the plant-herbivore interaction(Schoonhoven et al., 2005). Anatomical traits, such asleaf trichomes, and plant surface traits, such as cuticletexture, are important constitutive defense compo-nents that affect host suitability (Schoonhoven et al.,2005). The existence of secondary metabolites is alsoimportant for host determination (Konno et al., 2006).Schoonhoven et al. (2005) reviewed the mechanism ofhost-plant selection and discovered that the importanceof the feeding stimulants of host plants and the feedingdeterrents of nonhost plants lies with the balance be-tween the stimulants and the deterrents.

The relationship between induced plant defensesand host-determining factors is poorly understood,although increasing the level of the JA-dependentplant defense enhances plant resistance to variousherbivores (Browse and Howe, 2008; Schaller andStintzi, 2008). The JA-dependent plant defense ismultifaceted; it involves increasing herbivore avoidance

and shortening the herbivore’s life cycle by decreasingegg production, hatching rates, etc. (Thaler et al., 2001;Lu et al., 2004; Rodriguez-Saona and Thaler, 2005; Abeet al., 2009). It is noteworthy that these facets may alsoplay a part in host suitability for herbivores. Zarateet al. (2007) reported that the JA-dependent plant de-fense functions in resistance to silverleaf whitefly.They suggested that feeding by the silverleaf whiteflyinduces the SA-dependent plant defense, which isantagonistic to the JA-dependent plant defense. Thepresence of such a system in herbivores for deactivat-ing the JA-dependent plant defense may explain whythis plant defense is an important target of host plantdetermination and the coevolution of plants and her-bivores. Kessler et al. (2004) performed a field exper-iment that suggested a relationship between theJA-dependent plant defense and host suitability for her-bivores. They found that transgenic Nicotiana attenuataplants that overexpressed the gene for lipoxygenase,an enzyme involved in JA biosynthesis, in the anti-sense orientation suffered feeding damage caused byan herbivore that does not usually feed on N. attenuata.Here, on the basis experimental results obtained withArabidopsis coi1-1 mutants, we report in detail theconversion of nonhost plants to possible candidatehost plants as a result of the loss of the JA-dependentplant-induced defense. The larvae of the leafminers fed

Figure 9. Effect of JA application on plant resis-tance to leafminers. Five adult females were fedon 2-week-old Chinese cabbage (A and B) and3-week-old tomato (C) and garland chrysanthe-mum (D) plants for 2 weeks. Plants were immersedin water or a 100 mM JA solution 2 d before leaf-miners were introduced. B shows typical images ofChinese cabbage after leafminer feeding. The re-sults shown are means 6 SD of the areas of thefeeding scars based on at least 20 independentdeterminations. Asterisks indicate significant dif-ferences (Student’s t test; ***P , 0.001).


Abe et al.



inside the leaf mesophyll tissue and could not escapefrom the leaf. This specific feeding mode thus pre-vented the leafminer larvae from escaping from theplant defense system. The specific feeding mode of theleafminer larvae may make this herbivore particularlysensitive to plant-induced defenses.The American serpentine leafminer is among the

most problematic of herbivores, being difficult tocontrol with insecticides and having a wide host range.We found that JA application to activate JA-dependentplant resistance is an effective way to decrease theeffects of leafminers in a brassicaceous crop (Chinesecabbage), a solanaceous crop (tomato), and a com-posite crop (garland chrysanthemum; Fig. 9). The ef-fect of JA application in Chinese cabbage might beslightly explained by the role of glucosinolate. How-ever, JA treatment was also effective in tomato andgarland chrysanthemum, which do not contain aglucosinolate-myrosinase defense system. There shouldbe a common mechanism to provide leafminer resis-tance by JA application. Our next goal should be theidentification of the main defensive compounds thatfunction in leafminer resistance and affect host suit-ability for leafminer. Many candidate compounds exist.For example, many defensive compounds containphenolics, terpenoids, and alkaloids, which are regu-lated by JA (Howe and Schaller, 2008). In addition, aVSP encoded by the JA-inducible marker gene VSP2has also been reported to have anti-insect activity (Liuet al., 2005). Further efforts to understand in detail theJA-dependent plant-induced defense against leafminersare warranted.

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 andJander, 2006), myb28/myb29 (Hirai et al., 2007), and cyp79B2/cyp79B3 (Zhaoet al., 2002; Celenza et al., 2005; Sugawara et al., 2009) double knockout mu-tants 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 for7 d in the dark to synchronize germination. The pots were then transferred to22°C with a long-day photoperiod (16 h of light/8 h of dark). Plants at thefour-leaf stage were transferred to individual pots and grown to the rosettestage. Chinese cabbage (Brassica rapa subsp. pekinensis ‘Kyoto No. 3’; TakiiSeed) plants, garland chrysanthemum (Chrysanthemum coronarium ‘Ohbashungiku’; Sakata Seed) plants, and tomato (Solanum lycopersicum ‘Momotaro’;Takii Seed) plants were similarly grown in soil, except the tomato plants weregrown at 25°C.

Identification of coi1-1 Plants

Homozygous coi1-1 plants were selected by using TaqMan SNP GenotypingAssays (Applied Biosystems). Nucleotide sequences of the primers used were asfollows: forward primer, 59-CTTAAGCTACATCGGACAGTACAGT-39; reverseprimer, 59-CCTTCATCTGATTCACCTACGTAACC-39; reporter primers, 59-CAG-CAGCATCCATCTC-39 and 59-CAGCAGCATTCATCTC-39.

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 testplants. Five females were allowed to feed on each whole plant in a cylindricalacryl 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 orgarland chrysanthemum plants grown on soil were transferred into a cylin-drical acryl chamber containing a 100 mM JA solution. JA treatment was carriedout 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 cylin-drical acryl chamber. Three plants were placed in each chamber. Five adultfemale leafminers were then put in each chamber. After 12 h, these adults wereremoved. The numbers of first-, second-, and third-instar larvae were observedwith a stereoscopic microscope, and the number of adults was counted with theunaided eye.

RNA Extraction and Transcript Measurements

Five adult female leafminers were fed on three 2-week-old Arabidopsisplants at the rosette stage for 7 or 14 d in a closed container with air vents. Theexperimentswere repeated twice. After feeding, the plants were frozen in liquidnitrogen. Total RNA (2 mg), isolated with Trizol reagent (Invitrogen) and anRNeasy 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 re-verse transcriptase according to the manufacturer’s instructions (Invitrogen).Quantitative real-time PCR was carried out with the Power SYBR Green PCRMaster Mix (Applied Biosystems) by using the first-strand cDNA as a tem-plate on a sequence detector (ABI Prism 7900HT; Applied Biosystems). Ex-pression 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, 59-GTTAGGGACCGGAGCATCAA-3; reverseprimer, 59-AACGGTCACTGAGTATGATGGGT-39); LOX2 (At3g45140; forwardprimer, 59-TTGCTCGCCAGACACTTGC-39; reverse primer, 59-GGGATCAC-CATAAACGGCC-39); chiB (At3g12500; forward primer, 59-ACGGAA-GAGGACCAATGCAA-39; reverse primer, 59-GTTGGCAACAAGGTCAGGGT-39);PDF1.2 (At5g44420; forward primer, 59-CCATCATCACCCTTATCTTCGC-39;reverse primer, 59-TGTCCCACTTGGCTTCTCG-39); BGL2 (At3g57260; forwardprimer, 59-GCCGACAAGTGGGTTCAAGA-39; reverse primer, 59-AACCCCC-CAACTGAGGGTT-39); PR1 (At2g14610; forward primer, 59-GTTGCAGCC-TATGCTCGGAG-39; reverse primer, 59-CCGCTACCCCAGGCTAAGTT-39);and CBP20 (At5g44200; forward primer, 59-CCTTGTGGCTTTTGTTTCGTC-39;reverse primer, 59-ACACGAATAGGCCGGTCATC-39).

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 massspectrometer (Hewlett-Packard) was used.

Glucosinolate Quantification

Glucosinolates were analyzed by liquid chromatography-mass spectrom-etry using 10-camphorsulfonic acid as an internal standard for relative quanti-fication (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 byusingWinROOF software, version 5.8.1 (Mitani). The areas were analyzed withJMP software, version 5.1 (SAS Institute).

The Arabidopsis Genome Initiative gene codes and GenBank accessionnumbers, 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 RIKENBioResource Center for their excellent technical assistance. We are grateful toDr. Hiroyuki Kasahara of the RIKEN Center for Sustainable Resource Sciencefor providing cyp79B2/cyp79B3 double knockout mutants.

Received July 19, 2013; accepted August 31, 2013; published September 10,2013.

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