Antagonistic Plant Defense System Regulated by Phytohormones Assists Interactions Among Vector Insect, Thrips and a Tospovirus

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1 Regular Paper Antagonistic Plant Defense System Regulated by Phytohormones Assists Interactions Among Vector Insect, Thrips and a Tospovirus Hiroshi Abe, *, Yasuhiro Tomitaka, Takeshi Shimoda, Shigemi Seo, Tamito Sakurai, Soichi Kugimiya, Shinya Tsuda, * and Masatomo Kobayashi Experimental Plant Division, RIKEN BioResource Center, Tsukuba, -7 Japan National Agricultural Research Center, Tsukuba, Japan National Institute of Agrobiological Sciences, Tsukuba, Japan National Agricultural Research Center for Tohoku Region, Morioka, -98 Japan National Institute for Agro-Environmental Sciences, Tsukuba, -86 Japan *Corresponding authors: Hiroshi Abe, , ahiroshi@rtc.riken.jp; Fax: ; Shinya Tsuda, , shinyat@affrc.go.jp; Fax: (Received September, ; Accepted November 6, ) The western flower thrips (Frankliniella occidentalis) is a polyphagous herbivore that causes serious damage to many agricultural plants. In addition to causing feeding damage, it is also a vector insect that transmits tospoviruses such as Tomato spotted wilt virus (). We previously reported that thrips feeding on plants induces a jasmonate (JA)-regulated plant defense, which negatively affects both the performance and preference (i.e. host plant attractiveness) of the thrips. The antagonistic interaction between a JA-regulated plant defense and a salicylic acid (SA)-regulated plant defense is well known. Here we report that infection allows thrips to feed heavily and multiply on Arabidopsis plants. infection elevated SA contents and induced SA-regulated gene expression in the plants. On the other hand, infection decreased the level of JA-regulated gene expression induced by thrips feeding. Importantly, we also demonstrated that thrips significantly preferred -infected plants to uninfected plants. In JA-insensitive coi- mutants, however, thrips did not show a preference for -infected plants. In addition, SA application to wild-type plants increased their attractiveness to thrips. Our results suggest the following mechanism: infection suppresses the anti-herbivore response in plants and attracts its vector, thrips, to virus-infected plants by exploiting the antagonistic SA JA plant defense systems. Keywords: Host plant defense Insect vector Thrips Tospovirus Tritrophic interaction. Abbreviations: BGL, -,-glucanase ; CBP, cap-binding protein ; ChiB, chitinase; coi-, coronatine insensitive -; JA, jasmonate; LOX, lipoxygenase ; PR, pathogenesis-related gene ; PR, pathogenesis-related gene ; SA, salicylic acid;, Tomato spotted wilt virus; VSP, vegetative storage protein ; WT, wild type. Introduction Tospovirus is a type species in the family Bunyaviridae that consists of three RNA segments (L, M and S) (German et al. 99), and it is the only group in the family Bunyaviridae that infects plants. Tomato spotted wilt virus (), a member of the genus Tospovirus (Murphy 99), infects many agricultural and horticultural crops and is a typical insect-transmitted plant virus (Sherwood et al. ). Interestingly, is transmitted exclusively by thrips (Jones, Whitfield et al. ). Among thrips, the western flower thrips [Frankliniella occidentalis (Pergande)] is the main species that transmits (Inoue et al., Whitfield et al. ). This thrips is one of the most important insect herbivores, with a wide host range that includes many crops, vegetables, fruits and flowers (Parrella 99, Reitz 9). The worldwide emergence of insecticide-resistant thrips makes it difficult to them (Jensen ). Both the feeding damage caused by this thrips and the tospovirus disease it transmits are serious problems. Most plant viruses are transmitted by insect vectors that include aphids, whiteflies, leafhoppers, planthoppers and thrips (Hogenhout et al. 8). Because the plant virus cannot move from infected to healthy host plants by itself, an effective means of attracting vector insects is a life or death issue for viruses. Several studies have demonstrated the existence of such attraction mechanisms. Mauck et al. () Plant Cell Physiol. (): () doi:.9/pcp/pcr7, available online at The Author. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please journals.permissions@oup.com Plant Cell Physiol. (): () doi:.9/pcp/pcr7! The Author. Downloaded from on February 8

2 Plant vector virus tritrophic interactions reported that infection with Cucumber mosaic virus (CMV) increases the host plant attractiveness to vector aphids. Maris et al. () also reported that -infected pepper plants attract thrips more effectively than uninfected plants. Similar findings have been reported for plant pathogens other than viruses, such as phytoplasmas (Mayer et al. 8a, Mayer et al. 8b). However, it is not well understood how pathogen infection triggers changes in host traits that attract the insect vector. infection is also reported to affect thrips performance. Maris et al. () showed that thrips grow faster and better on -infected pepper plants than on healthy plants. Therefore, infection is likely to affect many physiological properties of host plants, some of which may attract or benefit the vector insect. Our previous studies have shown that jasmonate (JA) plays an important role in a plant s response and resistance to thrips, and that the JA-regulated plant defense negatively affects the performance of thrips and their preference (Abe et al. 8, Abe et al. 9). Numerous studies have examined the functions of JA in a plant s defense against pathogen attack, insect feeding and mechanical wounding (Howe and Jander 8, Thomma et al. 998). On the other hand, salicylic acid (SA) regulates a different set of basal defense responses in plants and functions in a plant s response to attack by pathogens such as fungi, bacteria and plant viruses (Pieterse et al. 6). Importantly, negative interaction between the JA and SA pathways has been clearly shown in previous studies (Niki et al. 998). In this study, we investigated the mechanism of a tritrophic (three-level) interaction among, western flower thrips and Arabidopsis. We suggest that the antagonistic JA- and SA-regulated defense systems in host plants increase both the attractiveness and the food quality of -infected plants for the vector thrips. Results Effect of infection on the performance of thrips in Arabidopsis To analyze the effect of infection on thrips feeding, we analyzed the difference in feeding damage between viruliferous (virus-containing) thrips and non-viruliferous thrips. We put seven female adults of either viruliferous or non-viruliferous thrips on non-infected Arabidopsis plants and compared the feeding damage between the two types of insects. We did not find a notable difference within the first 6 d (Fig. A, D). However, we found a remarkable difference after d (Fig. B, E), and the magnitude of this difference increased subsequently (Fig. C, F). We also found that plants that had been mechanically inoculated with suffered more thrips feeding than did mock-infected plants (data not shown). Next, we analyzed the numbers of adult and larval thrips to examine the effect of infection on the thrips population. After d, few adult females were found due to the adult life span of this species (Fig. ). However, whereas an average of 8 larvae were found on each of the plants treated with viruliferous thrips, only about nine were found on the plants treated with non-viruliferous thrips (Fig. ). Effect of infection on plant defense mechanisms To analyze the mechanism by which infection enhanced the thrips performance, we analyzed the defense response of the host plants. Plant defenses are regulated by several complex signaling pathways (Van der Ent et al. 8). SA and JA are global signals in plant defense systems, and, through cross-talk mechanisms, they are antagonistic to one another (Reymond and Farmer 998, Pieterse et al. 6). Therefore, we examined the expression of several SA- and JA-regulated marker genes in 6 days days nonviruliferous thrips viruliferous thrips Fig. Comparison of feeding damage caused by viruliferous and non-viruliferous thrips. The symptoms caused by attacks by viruliferous thrips and non-viruliferous thrips were compared. WT plants were grown for 6 d in each pot. Seven adult females with or without were then allowed to feed on each plant. The photo shows plants 6 (A, D), (B, E) and d (C, F) after the beginning of feeding. Plant Cell Physiol. (): () doi:.9/pcp/pcr7! The Author. Downloaded from on February 8

3 H. Abe et al. Number of larvae per plant 6 8 larvae nonviruliferous adults nonviruliferous viruliferous Arabidopsis, which are used to indicate regulation by these two signaling pathways. Plants were mechanically inoculated with. We monitored accumulation by using the expression of the NSs gene, which encodes the. kda non-structural protein of. In mechanically infected plants, could be detected after 7 d and had increased dramatically by d (Fig. B). We also visually confirmed the accumulation of after mechanical infection by performing tissue blot analyses of whole rosette plants (data not shown); the results were consistent with those of the expression analyses of the viral NSs gene. The expression of the SA-regulated marker genes PR (pathogenesis-related gene ) and BGL (b-,-glucanase ) was up-regulated in association with virus proliferation (Fig. C, D). The expression of the JA-regulated marker genes VSP (vegetative storage protein ) and LOX (lipoxygenase ) was also up-regulated by infection (Fig. E, F); however, the magnitude of their expression was much lower compared with that of the SA-regulated marker genes. To analyze the effect of infection on the gene expression induced by thrips feeding, we put non-viruliferous female adults on plants d after mechanical inoculation or mock treatment and analyzed gene expression after and d. Thrips feeding did not change the expression of the SA-regulated marker genes induced by infection (Fig. C, D). Importantly, thrips feeding on mock-treated plants dramatically increased the expression of JA-regulated marker genes VSP, LOX, ChiB (b chitinase) and PR (pathogenesis-related gene ), but the increase in the expression of these genes caused by thrips feeding on -infected plants was much smaller (Fig. E H). Thrips feeding did not affect the level of accumulation (Fig. B). We then analyzed the SA and JA contents of the plants d after mechanical infection. infection clearly increased the endogenous levels of both SA and JA ** viruliferous Fig. Effect of infection on the number of thrips. WT plants were grown for 6 d in each pot. Seven adult females with or without were allowed to feed on each plant. The number of adults and larvae was measured after d, and is shown as the mean ± SD of five independent determinations. Asterisks indicate significant differences (Student s t-test), **P <.. (Fig. A, B), but the increase in the level of SA was much greater than that in the level of JA. We previously reported that thrips feeding increased the endogenous level of JA but not of SA (Abe et al. 8), and we observed the same result here (Fig. A, B). Thrips feeding plus infection seemed to increase the JA level over that seen with infection alone (Fig. A). However, the SA levels in -infected plants were similar, with or without thrips feeding (Fig. B). Effect of infection on host plant attractiveness Finally, we analyzed the effect of infection on the thrips host plant preference (i.e. host plant attractiveness to thrips). Maris et al. () reported that thrips prefer -infected pepper plants over mock-treated pepper plants. To examine the thrips preference in the Arabidopsis plant system, we performed a choice test by using -infected and mock-treated wild-type (WT) Arabidopsis plants (Fig. A); the thrips clearly preferred the -infected plants (replicated G-test, G p =.7, df =, P <.; Fig. A). To analyze the importance of JA- and SA-regulated plant defense systems to this preference, we performed the same assay using JA-insensitive coi- (coronatine insensitive -) mutant plants; interestingly, thrips were not attracted to -infected coi- mutants (G p =.67, df =, P =.; Fig. B). We also compared the accumulation between WT plants and coi- mutants and found no significant difference in accumulation between them (Fig. C). To analyze further the effect of elevated SA levels after infection on the attraction of -infected plants to thrips, we applied SA directly to WT plants with an ink brush and compared the thrips preference for SA-treated plants over water-treated plants. The SA-treated plants were significantly more attractive to thrips than the water-treated plants (G p = 9.67, df =, P <.; Fig. D). We also found many more thrips feeding scars on the SA-treated plants than on the water-treated plants (G p =., df =, P <.; Fig. E). Discussion Here, we report that infection indirectly promotes attacks by thrips through the plant defense system. We found that infection enhanced the feeding of its thrips vector on Arabidopsis (Fig. ) and also increased the population density of thrips of the next generation (Fig. ). Maris et al. () reported that infection of pepper plants also increases thrips performance. The enhancement of thrips performance by infection in both pepper (Solanaceae) and Arabidopsis (Brassicaceae) indicates that this phenomenon is not restricted to a particular plant family. Many previous reports have described the importance of the immunity-related plant phytohormones JA and SA in both the response and the resistance of plants to biotic stresses (Pieterse et al. 6, Bari et al. 9). Numerous studies have examined 6 Plant Cell Physiol. (): () doi:.9/pcp/pcr7! The Author. Downloaded from on February 8

4 Plant vector virus tritrophic interactions A h (days) B NSs gene infection or treament thrips input h C PR gene D BGL gene h h E VSP gene F LOX gene h h G ChiB gene H PR gene h h Fig. Expression of JA- and SA-regulated marker genes. (A) Time course of treatments used for expression analyses. At time, plants were mechanically inoculated with (). treatment was performed on other plants as a (). The plants were sampled after 7 and d. At d, adult female non-viruliferous thrips were allowed to feed on inoculated and mock-treated plants (thrips). A treatment without thrips was also performed (). After and d, the plants were sampled. Each value represents the average of three replications of three plants each ± SD. (B) The NSs gene; (C, D) PR and BGL, marker genes of the SA-regulated plant defense; (E H) VSP, LOX, ChiB and PR, marker genes of the JA-regulated plant defense. The expression level of each gene was normalized to the expression of CBP and the relative value is shown. Plant Cell Physiol. (): () doi:.9/pcp/pcr7! The Author. 7 Downloaded from on February 8

5 H. Abe et al. A JA contents (ng/gfw) JA Control C C AB +Thr B +Thr A B SA contents (ng/gfw) SA Control B B B +Thr A A +Thr Fig. Effect of infection and thrips feeding on the biosynthesis of JA and SA. Endogenous levels of JA (JA + methyl JA; A) and SA (B) were measured. Mechanical inoculation of was done on 6-day-old WT plants (). treatment was also performed as a (). Plants were sampled after d. Ten adult female non-viruliferous thrips were allowed to feed on -inoculated or mock-treated plants for d, 7 d after inoculation/treatment ( + Thr, + Thr). Control plants were maintained for the same amount of time without inoculation or thrips feeding (Control). The results shown are means ± SD of at least four independent measurements. Different letters indicate statistically significant differences between treatments (Tukey Kramer HSD test; P <.). the functions of JA in plant defenses against pathogen attack and insect feeding (Thomma et al. 998, Howe and Schaller 8). We previously reported that JA functions to increase a plant s resistance to thrips and that a reduction in the JA-regulated plant defense enhanced the thrips performance characteristics such as feeding and oviposition, thus leading to an increase in the thrips population under virus-free conditions (Abe et al. 9). On the other hand, SA regulates a different set of basal immune system responses in plants and functions in the plant s response to attack by pathogens such as fungi, bacteria and plant viruses (Pieterse et al. 6). The JA and SA pathways have different effects on plant resistance to pathogens (Uknes et al. 99, Thomma et al. 998) and insect herbivores (Cipollini et al., Howe and Jander 8). A negative interaction between the JA and SA pathways has been clearly shown in previous studies (Niki et al. 998). Leon-Reyes et al. (9) also noted the antagonistic relationship between JA and SA in the context of plant resistance to thrips, reporting that JA-induced resistance to a thrips attack is decreased by SA. Importantly, our results here clearly indicate that infection activated SA biosynthesis and the SA-regulated plant defense (Figs., ). The inducibility of SA-regulated gene expression depended on the accumulation of (Fig. B). We also analyzed the antagonistic effect of infection on feeding-induced JA-regulated gene expression, and found that JA-regulated gene expression in -infected plants was lower than that in mock-treated plants (Fig. E H). It is possible that the increased performance of thrips on -infected plants was caused by a reduction of the JA-regulated plant defense, which was antagonistically regulated by an increase in SA-regulated plant defense responses. In this model, infection indirectly enhances the performance of its vector, thrips, by means of the antagonistic JA- and SA-regulated host plant defense systems. Viruses are immobile and thus need to be transported by vectors such as insects. We further examined the attractiveness of -infected Arabidopsis plants to thrips. -infected plants attracted more thrips than did mock-treated plants (Fig. A). These results are consistent with the findings of Maris et al. () in pepper. Interestingly, we found here that the attraction to thrips observed in -infected WT Arabidopsis plants disappeared in -infected, JA-insensitive coi- mutants (Fig. B). This result indicates the importance of JA signaling to the attractiveness to thrips of -infected plants. We previously reported that non-infected JA-insensitive coi- mutants showed a dramatically increased attraction (or decreased avoidance) to thrips compared with WT plants (>7 thrips per coi- mutant vs. thrips per WT plant) (Abe et al. 9). Taken together, these data indicate that infection affects thrips preference by interrupting the JA-regulated plant defense. To analyze the effect of SA on the attractiveness of a plant to thrips, we applied SA directly to WT plants. SA-treated plants were clearly preferred to untreated plants and suffered much more feeding by thrips than did untreated plants (Fig. D, E). These results suggest that the increased level of SA in a plant after infection increased its attraction to thrips. When considering in combination the importance of JA signaling and the increased SA levels, SA JA antagonism in the plant defense system can explain the enhanced performance of thrips on TSWS-infected plants and their preference for such plants. In this tritrophic interaction, thrips benefit from being viruliferous because of the response of the host plant defense system to. Feeding by adult -viruliferous thrips 8 Plant Cell Physiol. (): () doi:.9/pcp/pcr7! The Author. Downloaded from on February 8

6 Plant vector virus tritrophic interactions A Number of thrips WT *** B Number of thrips coil-l NS infected other infected other C D Number of thrips Relative TSVV NSs expression..... infected infected *** WT SA other SA Fig. Effect of infection on host plant preference of thrips. (A, B) Attractiveness of non-infected plants and infected plants to thrips was compared using WT (A) and coi- mutant plants (B). Non-infected and infected plants were grown at each end of a rectangular pot. Fifty adult females were collected in a ml tube, which was laid between the plants. The number of adult thrips on each plant was counted after d. Other represents the number of adult thrips found somewhere other than on the plants. Bars indicate the mean ± SD based on more than three independent determinations. Asterisks indicate a significant difference between the two plants, replicated G-test, ***P <.. NS designates not significant at P <.. (C) Virus accumulation as indicated by the level of NSs expression on WT plants and coi- mutant plants. (D, E) Effect of SA treatment on the attraction of plants to thrips. SA-treated ( mm) and water-treated plants were grown at each end of a rectangular pot. The number of adult thrips on each plant was counted after d (D). The relative area of feeding scars on the SA-treated and water-treated plants is shown in (E). Bars indicate the mean ± SD based on more than three independent determinations. Asterisks indicate a significant difference between the two plants, replicated G-test, ***P <.. E Relative area of feeding scars coil-l..... *** results in infection of the host plant, which reduces the JA-regulated host plant defense. Both the adult thrips and their progeny can be expected to benefit from this lowering of the host plant s defense. Note that is not transmitted transovarially (Wijkamp et al. 996): thrips acquire only at the larval stage from infected host plants (Whitfield et al. ). We found more larvae on plants inoculated with viruliferous adult females than on those inoculated with non-viruliferous females (Fig. B). These results indicate that infection facilitates the production of candidate thrips of the next generation, which will spread the. In addition, the reduction in the JA-regulated plant defense is also beneficial to because it increases the attractiveness of the plants to thrips and thus assists in the spread of. Plant Cell Physiol. (): () doi:.9/pcp/pcr7! The Author. 9 Downloaded from on February 8

7 H. Abe et al. Our next topic is to clarify the possible effects of plant chemicals induced by infection on the behavior of thrips. infection probably changes the emission levels of volatile plant chemicals that can act as attractants or repellents of thrips, as reported for other plant herbivore interactions (de Vos et al. ). For example, overexpression of the dual linalool/nerolidol synthase (FaNES) gene causes Arabidopsis to release these two terpenes, which act as repellents of green peach aphids (Myzus persicae) (Aharoni et al. ). Further, it is possible that a change of the composition of plant volatiles after infection decreases the attraction of natural enemies to thrips-infested plants, the efficiency of their foraging success, or both. Note that infection increased the JA level as well as the SA level, and also induced JA-regulated marker gene expression. Several types of plant virus infection have been reported to elevate endogenous JA levels as well as SA levels (Jameson and Clarke ); Conconi et al. (996) proposed that the JA elevation is caused by disruption of the plant cell membrane. Two opposing arguments have been made about the function of elevated JA after plant virus infection, namely that JA either inhibits plant virus replication or assists in the multiplication of the plant virus (Jameson and Clarke ). The biological function of elevated JA after infection is still unknown. Further analyses will be needed to reveal the details of the relationships among, thrips and host plants. In this study, we demonstrated the importance of the antagonistic effects of JA- and SA-regulated plant defense systems in enhanced thrips performance and in the attractiveness of -infected plants to thrips. induces the host plant SA-regulated defense system, which leads to a decrease in the JA-regulated defense. Many remaining questions need to be answered before we will understand the entire picture of this tritrophic interaction at the molecular level. Most recently, Stafford et al. () reported that infection directly influences the feeding behavior of male thrips, but not that of female thrips, and enhances the transmission efficiency of the virus. This observation raises the question as to how plants respond to attacks by thrips of different sex. Moreover, infection seems to have more wide-reaching effects on multitrophic interactions. For example, infection via thrips may affect the survival and development of other, non-vector herbivores (e.g. spider mites; Belliure et al., ). Further studies, including transcriptome and metabolome analyses, are required to improve the molecular basis of our understanding of additional multitrophic interactions, based on the tritrophic plant herbivore virus interactions. Materials and Methods Plants Plants (Arabidopsis thaliana ecotype Col-) were grown in soil as described previously (Weigel and Glazebrook ). JA-insensitive coi- mutants (Col- genetic background) were also used in this study. Arabidopsis seeds were sown on sterile soil in pots, moistened and placed in a cold room at C in the dark to synchronize germination. The pots were then transferred to C with a long-day photoperiod (6 h light/8 h dark). Plants at the four-leaf stage were transferred individually to pots and grown to the rosette stage. Virus isolation The O isolate, originating from green pepper (Capsicum annuum) in Japan, was used in this study (Tsuda et al. 99). The isolate was first maintained by mechanical transmission on Nicotiana rustica and later by thrips transmission on Arabidopsis plants. Mechanical inoculation of the virus was performed as described previously (Tsuda et al. 99). Thrips maintenance and virus acquisition A colony of western flower thrips, Frankliniella occidentalis, was maintained as described previously (Murai and Loomans ). Datura stramonium infected with was used as the virus acquisition source. First-instar larvae were confined with D. stramonium leaves infected with in plastic dishes for an acquisition access period of h. After this period, the larvae were reared to adulthood. Viruliferous and nonviruliferous thrips were distinguished by using the petunia leaf disk assay (Wijkamp and Peters 99). Thrips determined to be viruliferous or non-viruliferous were used for the following analyses. Thrips attack assay The thrips attack assay was performed with female adults d after their emergence from the pupal stage. The insects were starved for h before being placed on test plants. Adult females were grouped and allowed to feed on each whole plant in an acryl cylinder chamber with air ventilation windows covered with a fine mesh. Expression analyses Total RNA ( mg), 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 cdna was synthesized with random oligohexamers and Superscript III reverse transcriptase according to the manufacturer s instructions (Invitrogen). Quantitative real-time PCR analysis was performed as described previously (Abe et al. 8). Expression ofcbp (cap-binding protein ) was used for normalization as a standard gene. Nucleotide sequences of the gene-specific primers were as follows: VSP (Atg77; forward primer, -GTTAGGGACCGGA GCATCAA- ; reverse primer, -AACGGTCACTGAGTATGAT GGGT- ); LOX (Atg; forward primer, -TTGCTCGCC AGACACTTGC- ; reverse primer, -GGGATCACCATAAACG GCC- ); ChiB (Atg; forward primer, -ACGGAAGAGG ACCAATGCAA- ; reverse primer, -GTTGGCAACAAGGTCA GGGT- ); PR (Atg7; forward primer, -GTGGGATGCT GATAAGCCGT- ; reverse primer, -AACACTTGCCGCAAGA Plant Cell Physiol. 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8 Plant vector virus tritrophic interactions AGCT- ); BGL (Atg76; forward primer, -GCCGACAAG TGGGTTCAAGA- ; reverse primer, -AACCCCCCAACTGAG GGTT- ); PR (Atg6; forward primer, -GTTGCAGCCT ATGCTCGGAG- ; reverse primer, -CCGCTACCCCAGGCTA AGTT- ); CBP (Atg; forward primer, -CCTTGTGGC TTTTGTTTCGTC- ; reverse primer, -ACACGAATAGGCCG GTCATC- ); and NSs (forward primer, -AGTGAAAT CTCTGCTCATGTCAGC- ; reverse primer, -GCCAGAGGCT CAGCTTGAAAT- ). Quantification of JA and SA Quantification of JA and its methyl ester was performed as described previously (Seo et al. 99) except that an HP689 gas chromatograph fitted to a quadrupole mass spectrometer (Hewlett-Packard) was used for the analysis. Quantification of free SA was performed as described previously (Seo et al. 99). Counting of the thrips population Arabidopsis plants were grown in soil covered with fine zirconia beads (. mm diameter; Nikkato Co.) to make it easy to find the thrips. At the time of the assay, 6-day-old plants were placed in a cylindrical acryl chamber. Seven adult females with or without were put on non-infected WT plants. After weeks, the adults and larvae were counted. Choice assay The choice assay was described previously (Abe et al. 9). Fifty adult females were deposited halfway between the plants and allowed to move freely. After d, the thrips on each plant were counted. The results from 6 replications of each experiment were subjected to a replicated G-test; the null hypothesis was that the thrips would be distributed equally between the two pots (Sokal and Rohlf 99). We confirmed that there was no significant heterogeneity among replications in each experiment (P >. for each G h ), suggesting good reproducibility of the tests. Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant-in-Aid for Scientific Research (C) to H.A.]. Acknowledgments We thank F. Mori, S. Kawamura and I. Sasaki of RIKEN BRC, and Y. Matsumura of the National Agricultural Research Center for their excellent technical assistance. References Abe, H., Ohnishi, J., Narusaka, M., Seo, S., Narusaka, Y., Tsuda, S. et al. (8) Function of jasmonate in response and tolerance of Arabidopsis to thrip feeding. Plant Cell Physiol. 9: Abe, H., Shimoda, T., Ohnishi, J., Kugimiya, S., Narusaka, M., Seo, S. et al. (9) Jasmonate-dependent plant defense restricts thrips performance and preference. BMC Plant Biol. 9: 97. Aharoni, A., Giri, A.P., Deuerlein, S., Griepink, F., de Kogel, W.J., Verstappen, F.W.A. et al. () Terpenoid metabolism in wild-type and transgenic Arabidopsis plants. Plant Cell : Bari, R. and Jones, J. (9) Role of plant hormones in plant defence responses. Plant. Mol. Biol. 69: Belliure, B., Sabelis, M.W. and Janssen, A. () Vector and virus induce plant responses that benefit a non-vector herbivore. Basic Appl. Ecol. : Cipollini, D., Enright, S., Traw, M.B. and Bergelson, J. 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