ecophysiology and sustainability · 10/09/2013 · 18 type feeding, which are characteristic of...

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Running head: 3

Plant defense and herbivore suitability 4

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Hiroshi ABE, 6

Experimental Plant Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 7

305-0074, Japan 8

Tel.: +81-29-836-9189 9

Fax: +81-29-836-9053 10

E-mail: [email protected] 11

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Research category: Ecophysiology and Sustainability 13

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Plant Physiology Preview. Published on September 10, 2013, as DOI:10.1104/pp.113.222802

Copyright 2013 by the American Society of Plant Biologists

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Disarming the jasmonate-dependent plant defense makes non-host 2

Arabidopsis plants accessible to the American serpentine leafminer, 3

Liriomyza trifolii 4

Hiroshi ABE 1*, Ken TATEISHI 2, Shigemi SEO 2, Soichi KUGIMIYA 3, Masami Yokota 5

HIRAI 4, Yuji SAWADA 4, Yoshiyuki MURATA 5, Kaori YARA 6, Takeshi SHIMODA 7, 6

Masatomo KOBAYASHI 1 7

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1 Experimental Plant Division, RIKEN BioResource Center, Tsukuba 305-0074, Japan 9

2 National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan 10

3 National Institute for Agro-Environmental Sciences, Tsukuba 305-8604, Japan 11

4 RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan 12

5 Okayama University, Okayama 700-7530, Japan 13

6 NARO Institute of Vegetable and Tea Science, Shimada 428-8501, Japan 14

7 National Agricultural Research Center, Tsukuba 305-8666, Japan 15

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Abbreviations: Brassica rapa, B. rapa; chiB, β-chitinase; coi1-1, coronatine-insensitive 1-1; 17

JA, jasmonate; LOX2, lipoxygenase 2; PDF1.2, plant defensin 1.2; VSP2, vegetative storage 18

protein 2; WT, wild-type. 19

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This work was financed by a Grant-in-Aid for Scientific Research (B) to T.A., H.A., and S.K. 6

from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 7

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*Corresponding author: Hiroshi ABE 9

E-mail: [email protected] 10

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Abstract 1

Here, we analyzed the interaction between Arabidopsis and the American serpentine 2

leafminer (Liriomyza trifolii), an important and intractable herbivore of many cultivated 3

plants. We examined the role of the immunity-related plant hormone jasmonate (JA) in the 4

plant response and resistance to leafminer feeding to determine whether JA affects host 5

suitability for leafminers. The expression of marker genes for the JA-dependent plant defense 6

was induced by leafminer feeding on Arabidopsis wild-type (WT) plants. Analyses of JA-7

insensitive coi1-1 mutants suggested the importance of JA in the plant response to leafminer 8

feeding. The JA content of WT plants significantly increased after leafminer feeding. 9

Moreover, coi1-1 mutants showed lower feeding resistance against leafminer attack than did 10

WT plants. The number of feeding scars caused by inoculated adult leafminers in JA-11

insensitive coi1-1 mutants was higher than that in WT plants. In addition, adults of the 12

following generation appeared only from coi1-1 mutants and not from WT plants, suggesting 13

that the loss of the JA-dependent plant defense converted non-host plants to accessible host 14

plants. Interestingly, the glucosinolate-myrosinase defense system may play at most a minor 15

role in this conversion, indicating that this major anti-herbivore defense of Brassica plants 16

probably does not have a major function in plant resistance to leafminer. Application of JA to 17

WT plants before leafminer feeding enhanced feeding resistance in Chinese cabbage 18

(Brassica rapa), tomato (Solanum lycopersicum), and garland chrysanthemum 19

(Chrysanthemum coronarium). Our results indicate that JA plays an important role in the 20

plant response and resistance to leafminers, and in so doing affects host plant suitability for 21

leafminers.22



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Introduction 1

Insect attack is one of the most important factors affecting agricultural production, together 2

with pathogen infections and abiotic stress conditions such as dehydration, high salinity, and 3

heat. Insect attacks retard plant growth and decrease harvest levels (Hendrix, 1988). Plants 4

exhibit many types of defenses to insect attack, which are classified into two major classes: 5

constitutive defense and induced defense (Kessler and Baldwin, 2002; Howe and Jander, 6

2008; Howe and Schaller, 2008). Plant defenses have been well analyzed at the molecular, 7

metabolic, and physiological levels (Van Poecke, 2007; Howe and Schaller, 2008). However, 8

we currently understand only some aspects of plant defenses to herbivore attacks. Insects 9

comprise the most numerous species on Earth, with approximately 920,000 insect species 10

having been described (Matthews and Matthews, 2010). Among them, more than 400,000 11

herbivorous insect species exist (Schoonhoven et al., 2005). Insect herbivores have various 12

feeding styles, and those of agriculturally important insect pests have been well studied. The 13

chewing-type feeding by lepidopteran larvae (caterpillars) and the sucking-type feeding by 14

aphids and whiteflies are the best understood feeding mechanisms from the viewpoint of the 15

plant response to herbivore attacks (Rossi et al., 1998; Kahl et al., 2000; Reymond et al., 16

2000; Winz and Baldwin, 2001; Li et al., 2003; Nombela et al., 2003). Piercing and sucking-17

type feeding, which are characteristic of thrips and spider mites, respectively, has also been 18

extensively analyzed (Parrella, 1995; Abe et al., 2008; Abe et al., 2009). The other important 19

feeding manner with agricultural impact is mining-type feeding by leafminer larvae. 20

Leafminers are the larvae of various beetles, flies, and moths. The adult lays its eggs on the 21

leaf, and the larvae feed inside the leaf and stem tissues, creating tunnels (Connor and 22

Taverner, 1997; Yamazaki, 2010). 23

Numerous studies analyzing the plant response to various insect herbivores indicate the 24

importance of the plant hormone jasmonate (JA). JA mediates many processes in plant 25

growth and development and regulates part of the plant’s basal defense system, such as plant 26

responses to insect feeding (Browse and Howe, 2008; Schaller and Stintzi, 2008), pathogen 27

attack, mechanical wounding, UV irradiation, ozone exposure, and osmotic stress (Thomma 28

et al., 1998; Sasaki-Sekimoto et al., 2005). Reymond et al. (2004) reported the importance of 29

JA in plant resistance to cabbage butterfly (Pieris rapae), whereas Ellis et al. (2002) reported 30

that the JA-dependent plant defense is also involved in aphid resistance. JA-dependent plant 31

defense also has a role in the response and resistance to thrips attack (Abe et al., 2008; Abe et 32

al., 2009). Interestingly, several reports indicate that application of JA can reduce the feeding, 33

oviposition, and population growth of herbivores (Thaler et al., 2001; Lu et al., 2004; 34



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Rodriguez-Saona and Thaler, 2005; Abe et al., 2009). However, the effect of the JA-1

dependent plant defense on host suitability for herbivores is unknown. 2

The American serpentine leafminer (Liriomyza trifolii), a member of the family 3

Agromyzidae, is a highly polyphagous pest insect that causes serious damage to many crops, 4

vegetables, fruits, and flower plants, including members of the Brassicaceae, Solanaceae, 5

Asteraceae, Fabaceae, Cucurbitaceae, and Apiaceae (Parrella, 1987). Leafminer larvae are 6

mining-type feeders. In addition to the American serpentine leafminer, the tomato leafminers 7

(Liriomyza sativae and L. bryoniae) and pea leafminer (L. huidobrensis) cause major 8

problems worldwide. Because of the frequent emergence of insecticide resistance, it is 9

difficult to control leafminers with insecticides (Parrella, 1987). Therefore, elucidation of the 10

molecular mechanisms responsible for the plant response and resistance to leafminers is 11

important to contribute to the development of new methods to prevent damage. 12

Here, we analyzed the role of JA in the plant response to American serpentine leafminer 13

attack and the function of the JA-dependent plant defense in leafminer resistance and host 14

suitability by using Arabidopsis. 15

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Results 18

Expression of marker genes for the JA response 19

The larvae of American serpentine leafminers have a unique feeding style in that they make 20

tunnels in plant leaves (mining-type feeding; Fig. 1); the adults of American serpentine 21

leafminers have a piercing-type feeding style, which may also cause serious problems for 22

some agricultural crops. To characterize the plant response to leafminer feeding, we analyzed 23

the jasmonate (JA)-, ethylene (ET)-, and salicylic acid (SA)-dependent marker genes for the 24

plant defense response in Arabidopsis. In this analysis, five adult females were allowed to 25

feed on each whole plant in a cylindrical acryl chamber with air ventilation windows covered 26

with a fine mesh. We sampled the aboveground portions of the plants after 3 days to analyze 27

adult feeding, because the larvae did not hatch within these 3 days. We also sampled at 7 days 28

after the start of the assay to analyze larval feeding in addition to adult feeding. Expression of 29

the vegetative storage protein 2 (VSP2) and lipoxygenase 2 (LOX2) genes, markers for the 30

JA-dependent plant defense, was induced in WT plants by leafminer feeding after 3 and 7 31

days (Fig. 2A, B). Similarly, expression of the JA- and ET-dependent marker genes β-32

chitinase (chiB) and plant defensin1.2 (PDF1.2) was also induced by leafminer feeding after 33



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both 3 and 7 days (Fig. 2C, D). However, expression of the SA-dependent marker genes 1

pathogenesis-related protein 1 (PR1) and β-1,3-glucanase 2 (BGL2) was not induced (Fig. 2

2E, F). These results indicate the possible involvement of JA in the plant response to 3

leafminer feeding. Therefore, we subsequently used the JA-insensitive coi1-1 mutant (Xie et 4

al., 1998) to analyze the expression of these marker genes in response to leafminer feeding. 5

Induction of all four JA-related marker genes, VSP2, LOX2, PDF1.2, and chiB, by leafminer 6

feeding was notably decreased or abolished in the coi1-1 mutants after 3 and 7 days (Fig. 2A–7

D). Expression of SA-dependent PR1 and BGL2 was not affected in the coi1-1 mutant, as was 8

found with the WT plants (Fig. 2E, F). 9

To further analyze the function of JA in the response to leafminer feeding, we measured the 10

contents of JA in WT plants that were fed upon by both larval and adult leafminers for 7 days. 11

The JA content in these plants after leafminer feeding was about twice that in the uninfested 12

control plants (Fig. 3). These data support the importance of JA in the plant response to 13

leafminer feeding. 14

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Effect of the JA-dependent plant defense on leafminer attack 16

We then analyzed the role of the JA-dependent plant defense in resistance to leafminers. We 17

compared the feeding damage between WT plants and JA-insensitive coi1-1 mutant plants. 18

Each line was inoculated with five adult female leafminers in a cylindrical acryl chamber with 19

air ventilation windows covered with a fine mesh. The feeding scars by the adult female 20

leafminers were found in both WT and coi1-1 plants after 7 days (Fig. 4A, C). We found only 21

tiny feeding scars, probably caused by the feeding of first-instar larvae, on the WT and coi1-1 22

plants at 7 days. These tiny feeding scars on the WT plants did not diffuse even after 14 days 23

(Fig. 4B). On the other hand, the coi1-1 mutants showed many huge, drawn-out feeding scars, 24

which were probably produced by third-instar larvae at 14 days (Fig. 4D). We found pupae 25

only on coi1-1 mutant leaves (Fig. 4D, G). 26

To further analyze the role of the JA-dependent plant defense on resistance to leafminers, we 27

compared the feeding scar areas of WT plants to those of coi1-1 mutants after each plant had 28

been inoculated with five adult female leafminers. Injury from these leafminers was 29

significantly lower in WT plants than in coi1-1 mutants at the 3-day time point (Fig. 5). The 30

differences in adult and larval feeding were even more pronounced after 7 days. These 31

findings suggest that the JA-dependent plant defense has a role in plant resistance to 32

leafminers and affects the degree of damage caused by these insects. 33



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Effect of the JA-dependent plant defense on the leafminer population and host 1

suitability 2

Because we observed leafminer pupae on coi1-1 mutants but not on WT plants, we analyzed 3

the effect of the JA-dependent plant defense on the progeny of the adult leafminers used for 4

inoculation. We put five adult females on WT and coi1-1 plants and then counted the number 5

of feeding scars left by adults and larvae and also the number of first-instar larvae after 3 6

days, and second- and third-instar larvae after 9 days. The number of feeding scars on the 7

coi1-1 mutants left by adults and larvae was significantly larger than the number on WT 8

plants (Fig. 6A, B). It was difficult to distinguish first-instar larvae from eggs; therefore, we 9

classified them all as first-instar larvae. We did not find any significant difference in the 10

number of first-instar larvae between WT plants and coi1-1 mutants (Fig. 6C). However, we 11

did find a significant difference in the number of second-instar larvae between WT plants and 12

coi1-1 mutants (Fig. 6D). Interestingly, third-instar larvae were found only in coi1-1 plants 13

(Fig. 6E). These results clearly indicate that all of the first- and second-instar larvae died 14

before they could become third-instar larvae in WT plants. On the other hand, most of the 15

larvae in the coi1-1 plants matured to third-instar larvae. These findings were supported by 16

the presence of dead leafminer larvae in WT plants only (Fig. 6F–H). 17

We then determined the number of next-generation adult leafminers. This experiment was 18

carried out in independent cylindrical acryl chambers for WT plants and coi1-1 mutants, as 19

described in Materials and Methods. The next-generation adult leafminers only appeared from 20

the coi1-1 mutants. This result clearly indicates that the leafminer larvae could grow into 21

adults only in the coi1-1 mutants and not in the WT plants (Fig. 7). Finally, we performed 22

loss-of-function analyses of the glucosinolate-myrosinase defense system, one of the best 23

understood anti-herbivore factors in Brassica species, to test its effects on host suitability for 24

leafminers. To assess the role of the glucosinolate-myrosinase defense system, we looked for 25

next-generation adult leafminers on inoculated tgg1/tgg2 double-knockout mutants of the 26

myrosinase genes, which encode for proteins that catalyze isothiocyanate production (Barth 27

and Jander, 2006), and on inoculated myb28/myb29 and cyp79B2/cyp79B3 double-knockout 28

mutants, in which biosynthesis of methionine-derived aliphatic glucosinolates (Hirai et al., 29

2007) and tryptophan-derived indole glucosinolates (Zhao et al., 2002; Celenza et al., 2005; 30

Sugawara et al., 2009), respectively, is defective. As with the WT plants, no second-31

generation adult leafminers appeared on the tgg1/tgg2 or myb28/myb29 double-knockout 32

mutants. However, a few second-generations adult leafminers appeared on the 33



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cyp79b2/cyp79b3 double-knockout mutants (Fig. 7). 1

To assess the importance of the glucosinolate-myrosinase defense system in more detail, we 2

analyzed the contents of glucosinolates in WT, coi1-1, tgg1/tgg2, myb28/myb29, and 3

cyp79B2/cyp79B3 plants. The contents of aliphatic glucosinolates such as glucoiberin 4

(3MSOP), glucoraphanin (4MSOB), glucoalyssin (5MSOP), glucohesperin (6MSOH), and 5

glucoibarin (7MSOH) were increased after leafminer attack (Fig. 8A-F). In addition, the 6

contents of indole glucosinolates such as glucobrassicin (I3M), 1-methoxyglucobrassicin 7

(1MO-I3M), and 4-methoxyglucobrassicin (4MO-I3M) were significantly increased after 8

leafminer attack (Fig. 8G-I). On the other hand, the contents of both aliphatic and indole 9

glucosinolates were decreased in coi1-1 mutants as compared to WT plants under normal 10

conditions (Fig. 8A-C, D-I), and they were not increased after leafminer attack (Fig. 8A-I). 11

The contents of aliphatic glucosinolates and indole glucosinolates were significantly 12

decreased in myb28/myb29 and cyp79B2/cyp79B3 plants, respectively (Fig. 8A-I), as reported 13

previously (Zhao et al., 2002; Celenza et al., 2005; Hirai et al., 2007; Sugawara et al., 2009). 14

The glucosinolate contents in tgg1/tgg2 plants were similar to the contents in WT plants (Fig. 15

8A-I). 16

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JA-dependent plant resistance to leafminers in Chinese cabbage, tomato, and garland 18

chrysanthemum 19

To determine whether the JA-dependent resistance to leafminers extended to other plant 20

species, we analyzed the effect of JA application to Chinese cabbage (Brassica rapa ssp. 21

pekinensis), one of the most important Brassica crops; tomato (Solanum lycopersicum), an 22

important solanaceous crop; and garland chrysanthemum (Chrysanthemum coronarium), a 23

major composite crop. In all cases, each plant was grown in a single pot that was immersed in 24

a 100 μM JA solution for 2 days before inoculation with five adult female leafminers. Injury 25

from leafminer attack in Chinese cabbage was dramatically lower in plants pretreated with JA 26

than in untreated plants (Fig. 8A, B). Similar results were obtained with tomato and garland 27

chrysanthemum plants (Fig. 8C, D). These results indicate that JA has an important role in 28

resistance to leafminer attack in Chinese cabbage, tomato, and garland chrysanthemum. 29

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Discussion 1

Various responses at the molecular, metabolic, and physiologic levels are induced in plants 2

when they undergo insect attack, and these responses contribute to plant resistance to those 3

insects (Van Poecke, 2007; Howe and Schaller, 2008). Such resistance is generically termed 4

induced plant resistance. Here, we analyzed the JA-dependent plant response and resistance to 5

American serpentine leafminer feeding and assessed host suitability for leafminers. 6

Interestingly, adult and larval leafminers have different feeding styles, namely, piercing-type 7

and mining-type feeding, respectively. Both feeding styles induced the expression of the JA-8

related marker genes VSP2, LOX2, chiB, and PDF1.2 (Fig. 2). Note that the induction of 9

VSP2 and LOX2 was higher 3 days after the start of the leafminer attack than at 7 days, 10

whereas the induction of PDF1.2 and chiB was lower at 3 days than it was at 7 days. On day 3 11

after the leafminer release, feeding was limited to adults, which laid eggs in the plant 12

mesophyll tissue. On day 7 after the leafminer release, both adults and hatched larvae were 13

feeding on the plants. We cannot distinguish between the plant responses to adult feeding and 14

larval feeding accurately because of this sequential process. However, differences in the 15

feeding styles of the adults and larvae may be reflected in the different patterns of gene 16

induction 3 and 7 days post-leafminer release. We also found that induction of the marker 17

genes for the JA-dependent plant defense was repressed or decreased in JA-insensitive coi1-1 18

mutants after both 3 and 7 days. In addition, the JA contents in Arabidopsis WT plants 19

increased 7 days after the leafminer release (Fig. 3). These results indicate that JA has 20

important roles in the plant response to both adult and larval feeding of leafminers. JA is 21

known to have a role in the plant response to insect herbivores such as lepidopteran 22

caterpillars, thrips, and spider mites (Arimura et al., 2000; Reymond et al., 2004; Abe et al., 23

2008; Howe and Jander, 2008). This study shows that JA also functions in the plant response 24

to leafminers. 25

When we analyzed the role of the JA-dependent plant-induced defense in plant resistance to 26

leafminer attack, we found that JA-insensitive coi1-1 mutants were fed on much more than 27

WT plants (Fig. 4). The increased damage to coi1-1 mutants was confirmed by both the areas 28

of feeding scars (Fig. 5) and the number of feeding scars left by adults and larvae (Fig. 6A, B). 29

In addition to affecting the plant response to feeding, the JA-dependent plant defense thus has 30

a role in plant resistance to both leafminer adults and larvae. 31

Interestingly, our results suggest that coi1-1 mutants of Arabidopsis plants are a suitable food 32

source for American serpentine leafminers, whereas the WT plants used in this study are not. 33

Neither adults nor larvae fed much on the WT plants (Fig. 4, 5), and all of the first- and 34



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second-instar larvae died before they could become third-instar larvae (Fig. 6). In contrast, we 1

found that some of the leafminer larvae successfully developed to the pupal stage and 2

emerged to the adult stage of the next generation in coi1-1 mutants (Fig. 7). These results 3

mean that the loss of the JA-dependent plant defense changes non-host plants to accessible 4

host plants for leafminers. Thus, the JA-dependent plant defense may affect host suitability 5

for American serpentine leafminers. In Brassicaceae species, including Arabidopsis, 6

glucosinolate breakdown products such as isothiocyanate are well-known defense 7

components that exert plant resistance to herbivores (Hopkins et al., 2009). Glucosinolates are 8

hydrolyzed by myrosinase to form isothiocyanates. Loss of isothiocyanate in the Arabidopsis 9

tgg1/tgg2 double knock-out mutant of myrosinase genes improves the growth of tobacco 10

hornworm (Manduca sexta) and cabbage looper (Trichoplusia ni) (Barth and Jander, 2006). In 11

addition, Muller et al. (2010) performed experiments with aliphatic glucosinolate-deficient 12

myb28/myb29 and indole glucosinolate-deficient cyp79B2/cyp79B3 mutants and reported a 13

positive function of indole and aliphatic glucosinolates in resistance against several 14

lepidopteran larvae. Glucosinolates are constitutively expressed in plant tissues; however, 15

their levels clearly increase upon insect attack, such as lepidopteran larval feeding (Mewis et 16

al., 2006). Because of these findings, we analyzed whether leafminer larvae could 17

successfully develop to the pupal stage and ultimately emerge in the adult stage from 18

tgg1/tgg2, myb28/myb29, and cyp79B2/cyp79B3 mutants. The leafminer larvae in tgg1/tgg2 19

and myb28/myb29 mutants did not develop to the pupal stage. On the other hand, the 20

leafminer larvae in cyp79B2/cyp79B3 mutants developed to the pupal stage and then became 21

adults, indicating a possible function of indole glucosinolates in determining host suitability 22

for leafminers (Fig. 7). However, the effect of indole glucosinolates is likely to be limited 23

because the number of next-generation adult leafminers in cyp79B2/cyp79B3 mutants was 24

much lower than the number in coi1-1 mutants (Fig. 7). Importantly, we detected increased 25

contents of aliphatic and indole glucosinolates after leafminer feeding (Fig. 8). As reported by 26

Mewis et al. (2006), both aliphatic and indole glucosinolate contents were decreased in coi1-1 27

mutants not exposed to leafminer feeding in our study. In addition, these glucosinolate 28

compounds were not increased in coi1-1 mutants after leafminer attack. Interestingly, the 29

amount of increase of indole glucosinolate contents after leafminer feeding was much higher 30

than that of aliphatic glucosinolate contents (Fig. 8). It is well understood that 31

cyp79B2/cyp79B3 mutants are also defective in the biosynthesis of the Arabidopsis 32

phytoalexin, camalexin (Glawischnig et al., 2004). However, camalexin contents were not 33

increased by leafminer attack (data not shown). Muller et al. (2010) reported a clear 34



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differential effect of aliphatic and indole glucosinolates on resistance to several herbivores 1

between cyp79B2/cyp79B3 and myb28/myb29 double-knockout mutants and 2

cyp79B2/cyp79B3/myb28/myb29 quadruple-knockout mutants, in which biosynthesis of both 3

aliphatic and indole glucosinolates is defective. Further advanced analyses using this 4

quadruple-knockout mutant would reveal the function of these defense compounds for host 5

plant suitability of leafminer resistance in Arabidopsis plants. 6

Plant defenses against insect pests are classified into two groups: constitutive defenses and 7

induced defenses (Kessler and Baldwin, 2002; Howe and Jander, 2008; Howe and Schaller, 8

2008). The constitutive defense against herbivores is often considered as a host-determining 9

factor in the plant–herbivore interaction (Schoonhoven et al., 2005). Anatomical traits, such 10

as leaf trichomes, and plant surface texture, such as cuticle texture, are important constitutive 11

defense components that affect host suitability (Schoonhoven et al., 2005). The existence of 12

secondary metabolites is also important for host determination (Konno et al. (2006). 13

Schoonhoven et al. (2005) reviewed the mechanism of host-plant selection and discovered 14

that the importance of the feeding stimulants of host plants and the feeding deterrents of non-15

host plants lies with the balance between the stimulants and the deterrents. 16

The relationship between induced plant defenses and host-determining factors is poorly 17

understood, although increasing the level of the JA-dependent plant defense enhances plant 18

resistance to various herbivores (Browse and Howe, 2008; Schaller and Stintzi, 2008). The 19

JA-dependent plant defense is multifaceted; it involves increasing herbivore avoidance and 20

shortening the herbivore’s life cycle by decreasing egg production, hatching rates, etc. (Thaler 21

et al., 2001; Lu et al., 2004; Rodriguez-Saona and Thaler, 2005; Abe et al., 2009). It is 22

noteworthy that these facets may also play a part in host suitability for herbivores. Zarate et al. 23

(2006) reported that the JA-dependent plant defense functions in resistance to silverleaf 24

whitefly. They suggested that feeding by the silverleaf whitefly induces the SA-dependent 25

plant defense, which is antagonistic to the JA-dependent plant defense. The presence of such a 26

system in herbivores for deactivating the JA-dependent plant defense may explain why this 27

plant defense is an important target of host plant determination and co-evolution of plants and 28

herbivores. Kessler et al. (2004) performed a field experiment that suggested a relationship 29

between the JA-dependent plant defense and host suitability for herbivores. They found that 30

transgenic Nicotiana attenuata plants that overexpressed the gene for lipoxygenase, an 31

enzyme involved in JA biosynthesis, in the antisense orientation suffered feeding damage 32

caused by a herbivore that does not usually feed on N. attenuata. Here, for the first time, on 33

the basis experimental results obtained with Arabidopsis coi1-1 mutants, we report in detail 34



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the conversion of non-host plants to possible candidate host plants as a result of the loss of the 1

JA-dependent plant-induced defense. The larvae of the leafminers fed inside the leaf 2

mesophyll tissue and could not escape from the leaf. This specific feeding mode thus 3

prevented the leafminer larvae from escaping from the plant defense system. The specific 4

feeding mode of the leafminer larvae may make this herbivore particularly sensitive to plant-5

induced defenses. 6

The American serpentine leafminer is among the most problematic of herbivores, being 7

difficult to control with insecticides and having a wide host range. We found that JA 8

application to activate JA-dependent plant resistance is an effective way to decrease the 9

effects of leafminers in a brassicaceous crop (Chinese cabbage), a solanaceous crop (tomato), 10

and a composite crop (garland chrysanthemum) (Fig. 9). The effect of JA application in 11

Chinese cabbage might be slightly explained by the role of glucosinolate. However, JA 12

treatment was also effective in tomato and garland chrysanthemum, which do not contain a 13

glucosinolate-myrosinase defense system. There should be a common mechanism to provide 14

leafminer resistance by JA application. Our next goal should be the identification of the main 15

defensive compounds that function in leafminer resistance and affect host suitability for 16

leafminer. Many candidate compounds exist. For example, many defensive compounds 17

contain phenolics, terpenoids, and alkaloids, which are regulated by JA (Howe and Schaller, 18

2008). In addition, a VSP encoded by the JA-inducible marker gene VSP2 has also been 19

reported to have anti-insect activity (Liu et al., 2005). Further efforts to understand in detail 20

the JA-dependent plant-induced defense against leafminers are warranted. 21

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Materials and Methods 24

Plant materials and cultivation 25

Wild-type (ecotype Col-0) Arabidopsis plants, JA-insensitive coi1-1 mutants (Feys et al., 26

1994), and tgg1-1/tgg2-1 (Barth and Jander, 2006), myb28/myb29 (Hirai et al., 2007), and 27

cyp79B2/cyp79B3 double-knockout mutants (Zhao et al., 2002; Celenza et al., 2005; 28

Sugawara et al., 2009) were grown in soil as described previously (Weigel and Glazebrook, 29

2002). Seeds were sown on sterile soil in pots, moistened, and held at 4°C for 7 d in the dark 30

to synchronize germination. The pots were then transferred to 22°C with a long-day 31

photoperiod (16 h light / 8 h dark). Plants at the four-leaf stage were transferred to individual 32

pots and grown to the rosette stage. Chinese cabbage (B. rapa subsp. pekinensis ‘Kyoto No. 33



14

3’, Takii Seed Co. Ltd., Kyoto, Japan) plants, garland chrysanthemum (C. coronarium ‘Ohba 1

shungiku’, Sakata Seed Co. Ltd., Yokohama, Japan) plants, and tomato (S. lycopersicum 2

‘Momotaro’, Takii Seed Co. Ltd., Kyoto, Japan) plants were similarly grown in soil, except 3

the tomato plants were grown at 25°C. 4

5

Identification of coi1-1 plants 6

Homozygous coi1-1 plants were selected by using TaqMan SNP Genotyping Assays (Applied 7

Biosystems, Tokyo, Japan). Nucleotide sequences of the primers used were as follows: 8

forward primer, 5′-CTTAAGCTACATCGGACAGTACAGT-3′; reverse primer, 5′-9

CCTTCATCTGATTCACCTACGTAACC-3′; reporter primers, 5′-10

CAGCAGCATCCATCTC-3′, 5′-CAGCAGCATTCATCTC-3′. 11

12

Leafminer attack 13

Laboratory colonies of American serpentine leafminers (Liriomyza trifolii) were maintained 14

in a closed environmental chamber as described previously (Amano et al., 2008). Only adult 15

females were used in this study. The mated adult females were starved for 2–3 h before being 16

allowed to feed on the test plants. Five females were allowed to feed on each whole plant in a 17

cylindrical acryl chamber with air ventilation windows covered with a fine mesh. 18

19

Jasmonate treatment 20

Pots holding 2-week-old Chinese cabbage plants or 3-week-old tomato or garland 21

chrysanthemum plants grown on soil were transferred into a cylindrical acryl chamber 22

containing a 100 μM JA solution. JA treatment was carried out for 2 days before the 23

leafminer attack was initiated. 24

25

Assessment of the leafminer population 26

Three-week-old Arabidopsis plants grown in soil were placed in a cylindrical acryl chamber. 27

Three plants were placed in each chamber. Five adult female leafminers were then put in each 28

chamber. After 12 h, these adults were removed. The numbers of first-, second-, and third-29

instar larvae were observed under a stereoscopic microscope, and the number of adults was 30

counted with the unaided eye. 31



15

RNA extraction and transcript measurements 1

Five adult female leafminers were fed on three 2-week-old Arabidopsis plants at the rosette 2

stage for 7 or 14 d in a closed container with air vents. Experiments were repeated twice. 3

After feeding, the plants were frozen in liquid nitrogen. Total RNA (2 µg), isolated with 4

Trizol reagent (Invitrogen, Carlsbad, CA, USA) and an RNeasy MinElute Cleanup Kit 5

(Qiagen, Valencia, CA, USA), was treated with RNase-free DNase (Takara) to eliminate 6

genomic DNA. First-strand cDNA was synthesized with random oligo-hexamers and 7

Superscript III reverse transcriptase according to the manufacturer’s instructions (Invitrogen). 8

Quantitative real-time PCR was carried out with the Power SYBR Green PCR Master Mix 9

(Applied Biosystems) by using the first-strand cDNA as a template on a sequence detector 10

(ABI Prism 7900HT, Applied Biosystems). Expression of CBP20 was used for normalization 11

as a standard control gene. Nucleotide sequences of the gene-specific primers used were as 12

follows: VSP2 (At5g24770; forward primer, 5′-GTTAGGGACCGGAGCATCAA-3; reverse 13

primer, 5′-AACGGTCACTGAGTATGATGGGT-3′); LOX2 (At3g45140; forward primer, 5′-14

TTGCTCGCCAGACACTTGC-3′; reverse primer, 5′-GGGATCACCATAAACGGCC-3′); 15

chiB (At3g12500; forward primer, 5′-ACGGAAGAGGACCAATGCAA -3′; reverse primer, 16

5′-GTTGGCAACAAGGTCAGGGT-3′); PDF1.2 (At5g44420; forward primer, 5′-17

CCATCATCACCCTTATCTTCGC-3′; reverse primer, 5′-TGTCCCACTTGGCTTCTCG-3′); 18

BGL2 (At3g57260; forward primer, 5′-GCCGACAAGTGGGTTCAAGA-3′; reverse primer, 19

5′-AACCCCCCAACTGAGGGTT-3′); PR1 (At2g14610; forward primer, 5′-20

GTTGCAGCCTATGCTCGGAG-3′; reverse primer, 5′-CCGCTACCCCAGGCTAAGTT-3′); 21

and CBP20 (At5g44200; forward primer, 5′-CCTTGTGGCTTTTGTTTCGTC-3′; reverse 22

primer, 5′-ACACGAATAGGCCGGTCATC-3′) 23

24

Jasmonate quantification 25

JA and its methyl ester were quantified as described previously (Seo et al., 1995), except that 26

an HP6890 gas chromatograph fitted to a quadrupole mass spectrometer (Hewlett-Packard, 27

Wilmington, DE, USA) was used. 28

29

Glucosinolate quantification 30

GSLs were analyzed by liquid chromatography–mass spectrometry using 10-camphorsulfonic 31

acid as an internal standard for relative quantification (Sawada et al., 2012) 32

33



16

Feeding scar area measurements 1

The areas of leafminer feeding scars on the surface of each Arabidopsis, Chinese cabbage, 2

tomato, and garland chrysanthemum leaf were measured by using WinROOF software, 3

version 5.8.1 (Mitani Corporation, Tokyo, Japan). The areas were analyzed with JMP 4

software, ver. 5.1 (SAS Institute, Inc., Cary, NC, USA). 5

6

Accession numbers 7

The Arabidopsis Genome Initiative gene codes (AGI codes) and GenBank accession numbers, 8

respectively, for the genes mentioned in this article are as follows: VSP2 (At5g24770, 9

AB006778), LOX2 (At3g45140, AYO62611), chiB (At3g12500, AY054628), PDF1.2 10

(At5g44420, AY063779), BGL2 (At3g57260, AY099668), PR1 (At2g14610, AY064023), 11

and CBP20 (At5g44200, AF140219). 12

13

Acknowledgments 14

We thank F. Mori, S. Kawamura, and I. Sasaki of RIKEN BRC for their excellent technical 15

assistance. We are grateful to Dr. H. Kasahara for providing cyp79B2/cyp79B3 double-16

knockout mutants. The metabolome analysis was supported by the Japan Advanced 17

Plant Science Network. 18

19

20

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11

12

13

14

Figure legends 15

Figure 1. Life cycle of the American serpentine leafminer 16

(A) An adult female American serpentine leafminer. The scale bar indicates 500 μm. 17

(B) Diagram of the life cycle of the American serpentine leafminer. Adult females oviposit 18

their eggs into the leaf mesophyll, and the eggs subsequently hatch into first-instar larvae. The 19

first-instar larvae feed inside the mesophyll tissue; the feeding scars resemble mining tunnels. 20

The first-instar larvae mature to second-instar larvae, and subsequently to third-instar larvae. 21

The third-instar larvae become pupae, which appear on the leaf surface and develop into 22

adults. 23

24

Figure 2. Expression of marker genes for JA, ET, and SA signals induced by leafminer 25

feeding on Arabidopsis. 26

(A, B) VSP2 and LOX2, marker genes for the JA pathway; (C, D) chiB and PDF1.2, marker 27

genes for the JA/ET pathway; and (E, F) PR1 and BGL2, marker genes for the SA pathway. 28

Five 3-week-old plants were grown in a single pot, and five adult female leafminers were 29

allowed to feed on them. After 0, 3, and 7 d, total RNA was prepared from the plants with 30

(+Feeding) or without (-Feeding) leafminer feeding, and first-strand cDNA was synthesized 31

for PCR analysis. Primer sequences used in this analysis are described in Materials and 32

Methods. The expression level of each gene was normalized to the expression of CBP20 33

(control) and is shown as a relative value. Each value represents the average ± SD of three 34

replications of 10 plants each. 35

36



21

Figure 3. Effect of leafminer feeding on JA biosynthesis in Arabidopsis. 1

Endogenous levels of JA (JA + methyl JA; A) were measured. Five adult female leafminers 2

were allowed to feed on a 3-week-old WT plant in a closed container with air vents (Feeding). 3

The control plant was kept in a container without leafminers (Control). The JA content of 4

plant tissue (1 g) was measured 10 d after the start of feeding. The results shown are means ± 5

SD of at least four independent measurements. Asterisks indicate significant differences 6

(Student’s t-test), **P < 0.01. 7

8

Figure 4. Effect of the JA-dependent plant defense on leafminer attack. 9

The effects of a leafminer attack on WT plants and coi1-1 mutants were compared. Three-10

week-old WT plants (upper) and coi1-1 mutants (lower) were grown. Five adult female 11

leafminers were allowed to feed on each plant. The photo shows plants after 7 (A, C) and 14 d 12

(B, D). A part of a leaf in (A), (C), and (D) is magnified in (E), (F), and (G), respectively. The 13

arrow indicates a pupa emerging from the leaf mesophyll tissue. 14

15

Figure 5. Effect of the JA-dependent plant defense on leafminer feeding. 16

The feeding scars left by leafminers on WT plants and coi1-1 mutants were compared. Three-17

week-old WT plants and coi1-1 mutants were grown. Five adult female leafminers were 18

allowed to feed on each plant. The feeding scars were measured after 3 and 7 d. The results 19

shown are means ± SD of at least six independent measurements. The different letters indicate 20

a statistically significant difference between treatments (Tukey–Kramer HSD test; P < 0.05). 21

22

Figure 6. Effect of the JA-dependent plant defense on the leafminer larval population. 23

Three-week-old WT plants and coi1-1 mutants were grown. Five adult female leafminers 24

were allowed to feed on each plant. The number of feeding scars left by adults (A) and larvae 25

(B) on WT plants and coi1-1 mutants was determined after 7 d. The number of first- (C), 26

second- (D), and third-instar larvae (E) was determined after 2 weeks. Means ± SD of the 27

feeding scar areas are based on at least 30 independent determinations. Asterisks indicate 28

significant differences (Student’s t-test), ***P < 0.001. The photo shows a live second-instar 29

larva in a coi1-1 mutant (F, G). For clarity, epidermal tissue was removed in (G). A dead 30

second-instar larva in a WT plant is shown in (H). 31

32

33

34



22

Figure 7. Effect of the JA-dependent plant defense on the population of adult leafminers. 1

Five adult female leafminers were allowed to feed on a 3-week-old WT plant, coi1-1 mutants, 2

and tgg1-1/tgg2-1, myb28/myb29, and cyp79B2/cyp79B3 double mutants in a closed container 3

with air vents for 1 day. The number of adult leafminers was counted after 2 weeks. The 4

results shown are means ± SD of five independent measurements. The different letters 5

indicate a statistically significant difference between treatments (Tukey–Kramer HSD test; P 6

< 0.05). 7

8

Figure 8. Effect of leafminer feeding on glucosinolate contents. 9

Five adult female leafminers were allowed to feed on three-week-old WT plants, coi1-1 10

mutants, and tgg1-1/tgg2-1, myb28/myb29, and cyp79B2/cyp79B3 double-knockout mutants 11

in a closed container with air vents for 3 days (gray bars). A control treatment was performed 12

without leafminer feeding for 3 days (white bars). The concentrations of the individual 13

glucosinolates are shown as relative values: (A) 3MSOP, (B) 4MSOB, (C) 5MSOP, (D) 14

6MSOH, (E) 7MSOH, (F) 8MSOD, (G) I3M, (H) 1MO-I3M, and (I) 4MO-I3M. Different 15

letters indicate statistically significant differences between treatments (Tukey–Kramer HSD 16

test; P < 0.05). Asterisks indicate significant differences (Student’s t-test), * P < 0.05. 17

18

Figure 9. Effect of JA application on plant resistance to leafminers. 19

Five adult females fed on 2-week-old Chinese cabbage (A, B) and 3-week-old tomato (C) and 20

garland chrysanthemum plants (D) for 2 weeks. Plants were immersed in water or a 100 µM 21

JA solution 2 days before leafminers were introduced. (B) Typical images of Chinese cabbage 22

after leafminer feeding. The results shown are means ± SD of the areas of the feeding scars 23

based on at least 20 independent determinations. Asterisks indicate significant differences 24

(Student’s t-test), ***P < 0.001. 25



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