Indirect defence of Arabidopsis against herbivorous insects : combining parasitoid behaviour and chemical analyses with a molecular genetic approach

Many plant species are known to defend themselves against herbivorous insects indirectly, by producing volatiles in response to herbivory. These volatiles attract carnivorous enemies of the herbivores, such as parasitoids. Research on the model plant Arabidopsis thaliana (L.) Heynh. (Brassicaceae) has contributed considerably to the unravelling of signal transduction pathways involved in direct plant defence mechanisms against pathogens. In this thesis I demonstrate that Arabidopsis is also a good model plant for studying signal transduction pathways involved in indirect defence mechanisms, by showing that: (a) Arabidopsis plants infested by Pieris rapae caterpillars (Lepidoptera: Pieridae) emit volatiles from several major biosynthetic pathways, including terpenoids, methyl-salicylate (MeSA), nitriles and green leaf volatiles; (b) Adult females of Cotesia rubecula (Hymenoptera: Braconidae), a specialist parasitoid wasp of P. rapae , were attracted to P. rapae- infested Arabidopsis plants; and (c) Genes from major biosynthetic pathways involved in volatile production were induced by caterpillar feeding.Moreover, I show that the octadecanoid and the salicylic acid pathways are involved in the induced attraction of C. rubecula by P. rapae -infested Arabidopsis . Besides exogenous application of jasmonic acid (JA) or salicylic acid (SA), I used transgenic Arabidopsis that do not show induced JA levels after wounding (S-12) and transgenic Arabidopsis that do not accumulate SA (NahG). Treatment of Arabidopsis with JA resulted in an increased attraction of parasitoid wasps compared to untreated plants, whereas treatment with SA did not. Transgenic plants impaired in the octadecanoid or the salicylic acid pathway were less attractive than wild-type plants. Chemical analysis of the volatile blends emitted by JA-treated wild-type and P. rapae -infested wild-type, S-12 and NahG plants, demonstrate that JA and SA are indeed involved in the herbivory-induced production of volatiles. Moreover, these data suggest important roles for MeSA and the terpenoid (3 E,7E )-4,8,12-trimethyl-1,3,7,11-tridecate traene as parasitoid attractants . Additionally, these data suggest a signalling role of the plant hormone 12-oxo-phytodienoic acid in induced volatile emissions.Although direct defence mechanisms against pathogens and herbivores are often also regulated through JA and SA, these signal transduction pathways differ from those involved in indirect defence of Arabidopsis against P. rapae , as I demonstrated by using the signal transduction mutants npr1-1 and jar1-1 .In this thesis it is also shown that herbivore species with a different way of feeding compared to P. rapae caterpillars - aphids and spider mites - induced no or less attraction of C. rubecula when infesting Arabidopsis. This difference in parasitoid attraction may be the result of different induction of JA and SA signalling pathways by different herbivore species.In conclusion, this thesis demonstrates that combing parasitoid behaviour and chemical analysis with a molecular genetic approach can be highly valuable in unravelling signal-transduction pathways involved in indirect defence of plants, a method that so far has been under-exploited

Indirect defence of Arabidopsis against herbivorous insects : combining parasitoid behaviour and chemical analyses with a molecular genetic approach

Many plant species are known to defend themselves against herbivorous insects indirectly, by producing volatiles in response to herbivory. These volatiles attract carnivorous enemies of the herbivores, such as parasitoids. Research on the model plant Arabidopsis thaliana (L.) Heynh. (Brassicaceae) has contributed considerably to the unravelling of signal transduction pathways involved in direct plant defence mechanisms against pathogens. In this thesis I demonstrate that Arabidopsis is also a good model plant for studying signal transduction pathways involved in indirect defence mechanisms, by showing that: (a) Arabidopsis plants infested by Pieris rapae caterpillars (Lepidoptera: Pieridae) emit volatiles from several major biosynthetic pathways, including terpenoids, methyl-salicylate (MeSA), nitriles and green leaf volatiles; (b) Adult females of Cotesia rubecula (Hymenoptera: Braconidae), a specialist parasitoid wasp of P. rapae , were attracted to P. rapae- infested Arabidopsis plants; and (c) Genes from major biosynthetic pathways involved in volatile production were induced by caterpillar feeding.Moreover, I show that the octadecanoid and the salicylic acid pathways are involved in the induced attraction of C. rubecula by P. rapae -infested Arabidopsis . Besides exogenous application of jasmonic acid (JA) or salicylic acid (SA), I used transgenic Arabidopsis that do not show induced JA levels after wounding (S-12) and transgenic Arabidopsis that do not accumulate SA (NahG). Treatment of Arabidopsis with JA resulted in an increased attraction of parasitoid wasps compared to untreated plants, whereas treatment with SA did not. Transgenic plants impaired in the octadecanoid or the salicylic acid pathway were less attractive than wild-type plants. Chemical analysis of the volatile blends emitted by JA-treated wild-type and P. rapae -infested wild-type, S-12 and NahG plants, demonstrate that JA and SA are indeed involved in the herbivory-induced production of volatiles. Moreover, these data suggest important roles for MeSA and the terpenoid (3 E,7E )-4,8,12-trimethyl-1,3,7,11-tridecate traene as parasitoid attractants . Additionally, these data suggest a signalling role of the plant hormone 12-oxo-phytodienoic acid in induced volatile emissions.Although direct defence mechanisms against pathogens and herbivores are often also regulated through JA and SA, these signal transduction pathways differ from those involved in indirect defence of Arabidopsis against P. rapae , as I demonstrated by using the signal transduction mutants npr1-1 and jar1-1 .In this thesis it is also shown that herbivore species with a different way of feeding compared to P. rapae caterpillars - aphids and spider mites - induced no or less attraction of C. rubecula when infesting Arabidopsis. This difference in parasitoid attraction may be the result of different induction of JA and SA signalling pathways by different herbivore species.In conclusion, this thesis demonstrates that combing parasitoid behaviour and chemical analysis with a molecular genetic approach can be highly valuable in unravelling signal-transduction pathways involved in indirect defence of plants, a method that so far has been under-exploited

Signal transduction downstream of salicylic and jasmonic acid in herbivory-induced parasitoid attraction by Arabidopsis is independent of JAR1 and NPR1

Plants can defend themselves indirectly against herbivores by emitting a volatile blend upon herbivory that attracts the natural enemies of these herbivores, either predators or parasitoids. Although signal transduction in plants from herbivory to induced volatile production depends on jasmonic acid (JA) and salicylic acid (SA), the pathways downstream of JA and SA are unknown. Use of Arabidopsis provides a unique possibility to study signal transduction by use of signalling mutants, which so far has not been exploited in studies on indirect plant defence. In the present study it was demonstrated that jar1-1 and npr1-1 mutants are not affected in caterpillar (Pieris rapae)-induced attraction of the parasitoid Cotesia rubecula. Both JAR1 and NPR1 (also known as NIM1) are involved in signalling downstream of JA in induced defence against pathogens such as induced systemic resistance (ISR). NPR1 is also involved in signalling downstream of SA in defence against pathogens such as systemic acquired resistance (SAR). These results demonstrate that signalling downstream of JA and SA differs between induced indirect defence against herbivores and defence against pathogens such as SAR and ISR. Furthermore, it was demonstrated that herbivore-derived elicitors are involved in induced attraction of the parasitoid Cotesia rubecul

Indirect defence of plants against herbivores: using Arabidopsis thaliana as a model plant

In their defence against pathogens, herbivorous insects, and mites, plants employ many induced responses. One of these responses is the induced emission of volatiles upon herbivory. These volatiles can guide predators or parasitoids to their herbivorous prey, and thus benefit both plant and carnivore. This use of carnivores by plants is termed indirect defence and has been reported for many plant species, including elm, pine, maize, Lima bean, cotton, cucumber, tobacco, tomato, cabbage, and Arabidopsis thaliana. Herbivory activates an intricate signalling web and finally results in defence responses such as increased production of volatiles. Although several components of this signalling web are known (for example the plant hormones jasmonic acid, salicylic acid, and ethylene), our understanding of how these components interact and how other components are involved is still limited. Here we review the knowledge on elicitation and signal transduction of herbivory-induced volatile production. Additionally, we discuss how use of the model plant Arabidopsis thaliana can enhance our understanding of signal transduction in indirect defence and how cross-talk and trade-offs with signal transduction in direct defence against herbivores and pathogens influences plant responses

Inducible indirect defence of plants : from mechanisms to ecological functions

Inducible defences allow plants to be phenotypically plastic. Inducible indirect defence of plants by attracting carnivorous enemies of herbivorous arthropods can vary with plant species and genotype, with herbivore species or instar and potentially with other environmental conditions. So far, inducible indirect defence has mostly been studied for simple linear food chains. However, ultimately, ecologists should address inducible indirect defence in a food web context, where more than one organism (different herbivores and pathogens) may attack a plant and where a plant that emits herbivore-induced volatiles is surrounded by other plants that emit odours that can mix with the herbivore-induced volatiles from the attacked plant. Evolutionary ecologists are interested in the costs and benefits of interactions between plants and their attackers. These may be investigated by comparing different plant genotypes. The best comparison is between plant individuals that differ in only a single or restricted number of known traits. Such genotypes are difficult to obtain by conventional methods. However, rapid progress in the study of mechanisms of plant-attacker interactions and in the field of molecular genetics and genomics provides new tools that can be exploited by ecologists. For instance, genomic knowledge on Arabidopsis thaliana and the availability of characterized mutants and transgenes that are altered in one or a restricted number of genes can be exploited to address functional aspects of inducible indirect defence. In this paper we review progress in the knowledge of mechanisms of inducible indirect defence of plants and its importance for investigating the functional aspects of plant responses to herbivorous arthropods. Finally we identify some of the ecological questions that can be addressed by exploiting mechanistic aspects of inducible indirect defence

Signal signature in induced defense of Arabidopsis upon pathogen and insect attack

Three plant signaling molecules play a dominant role in the regulation of defenses in a number of plant-attacker model systems: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). In this study, the roles of these signaling compounds were determined in the induced defense responses of Arabidopsis thaliana upon attack by a set of microbial pathogens and herbivorous insects. The production of SA, JA and ET was activated in different combinations depending on the attacker encountered resulting in a specific signal signature. Analysis of the expression of SA-, JA-, and ET- responsive marker genes showed that the signal signature nicely correlates with the expression of the marker genes in each plant-attacker interaction. We hypothesize that the specific signal signature is involved in the activation of an optimal mix of defenses to counteract the intruder

Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium (Pseudomonas syringae pv. tomato), a pathogenic leaf fungus (Alternaria brassicicola), tissue-chewing caterpillars (Pieris rapae), cell-content-feeding thrips (Frankliniella occidentalis), or phloem-feeding aphids (Myzus persicae). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. Analysis of global gene expression profiles demonstrated that the signal signature characteristic of each Arabidopsis-attacker combination is orchestrated into a surprisingly complex set of transcriptional alterations in which, in all cases, stress-related genes are overrepresented. Comparison of the transcript profiles revealed that consistent changes induced by pathogens and insects with very different modes of attack can show considerable overlap. Of all consistent changes induced by A. brassicicola, Pieris rapae, and F occidentalis, more than 50% also were induced consistently by R syringae. Notably, although these four attackers all stimulated JA biosynthesis, the majority of the changes in JA-responsive gene expression were attacker specific. All together, our study shows that SA, JA, and ET play a primary role in the orchestration of the plant's defense response, but other regulatory mechanisms, such as pathway cross-talk or additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response

Signal signature of Arabidopsis induced upon pathogen and insect attack

Three plant signaling molecules play a dominant role in the regulation of defences in a number of plant-attacker model systems: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). In this study, the roles of these compounds were determined in the induced defense responses of Arabidopsis thaliana upon attack by a set of microbial pathogens and herbivorous insects. The production of SA, JA and ET was activated in different combinations depending on the attacker encountered resulting in a specific signal signature. Analysis of the expression of SA-,JA-,and ET responsive marker genes showed that the signal signature nicely correlates with the expression of the marker genes in each plant-attacker interaction. We hypothesize that the specific signal signature is involved in the activation of an optimal mix of defenses to counteract the intruder

Signal signature of Arabidopsis induced upon pathogen and insect attack

Three plant signaling molecules play a dominant role in the regulation of defences in a number of plant-attacker model systems: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). In this study, the roles of these compounds were determined in the induced defense responses of Arabidopsis thaliana upon attack by a set of microbial pathogens and herbivorous insects. The production of SA, JA and ET was activated in different combinations depending on the attacker encountered resulting in a specific signal signature. Analysis of the expression of SA-,JA-,and ET responsive marker genes showed that the signal signature nicely correlates with the expression of the marker genes in each plant-attacker interaction. We hypothesize that the specific signal signature is involved in the activation of an optimal mix of defenses to counteract the intruder