Celia Diezel

Priming of plant defense responses in nature by airborne signaling between Artemisia tridentata and Nicotiana attenuata

Plants release volatile organic compounds (VOCs) in response to wounding and herbivore attack, some of which trigger responses in neighboring unattacked plants in the laboratory under conditions that are not likely to occur in the real world. Whether plants ‘eavesdrop’ on the volatile emissions of their neighbors in nature is not known. The best documented field study of between-species signaling via above-ground VOCs involves increases in fitness parameters of native tobacco (Nicotiana attenuata) transplanted adjacent to clipped sagebrush (Artemesia tridentata tridentata). Clipped sagebrush releases many biologically active VOCs, including methyl jasmonate (MeJA), methacrolein and a series of terpenoid and green leaf VOCs, of which MeJA, while active under laboratory conditions, is not released in sufficient quantities to directly elicit induced resistance in the field. Here we demonstrate, with laboratory and field-based experiments, that priming (rather than direct elicitation) of native N. attenuata’s induced chemical defenses by a sagebrush-released VOC bouquet can account for earlier findings. With microarrays enriched in N. attenuata herbivore-regulated genes, we found transcriptional responses in tobacco growing adjacent to clipped sagebrush foliage, but failed to detect the direct elicitation of defensive chemicals or proteins. However, we observed an accelerated production of trypsin proteinase inhibitors when Manduca sexta caterpillars fed on plants previously exposed to clipped sagebrush. This readying of a defense response, termed priming, results in lower total herbivore damage to plants exposed to clipped sagebrush and in a higher mortality rate of young Manduca caterpillars. Our study demonstrates priming of plant defense responses as a mechanism of plant–plant signaling in nature, and provides an example for the analysis of between-plant signaling under ecologically realistic conditions. Although we describe priming as a potential mechanism for signaling between plants in nature, we critically discuss the ecological relevance of the particular interaction.

Arabidopsis WRKY33 Is a Key Transcriptional Regulator of Hormonal and Metabolic Responses toward Botrytis cinerea Infection

The Arabidopsis (Arabidopsis thaliana) transcription factor WRKY33 is essential for defense toward the necrotrophic fungus Botrytis cinerea. Here, we aimed at identifying early transcriptional responses mediated by WRKY33. Global expression profiling on susceptible wrky33 and resistant wild-type plants uncovered massive differential transcriptional reprogramming upon B. cinerea infection. Subsequent detailed kinetic analyses revealed that loss of WRKY33 function results in inappropriate activation of the salicylic acid (SA)-related host response and elevated SA levels post infection and in the down-regulation of jasmonic acid (JA)-associated responses at later stages. This down-regulation appears to involve direct activation of several jasmonate ZIM-domain genes, encoding repressors of the JA-response pathway, by loss of WRKY33 function and by additional SA-dependent WRKY factors. Moreover, genes involved in redox homeostasis, SA signaling, ethylene-JA-mediated cross-communication, and camalexin biosynthesis were identified as direct targets of WRKY33. Genetic studies indicate that although SA-mediated repression of the JA pathway may contribute to the susceptibility of wrky33 plants to B. cinerea, it is insufficient for WRKY33-mediated resistance. Thus, WRKY33 apparently directly targets other still unidentified components that are also critical for establishing full resistance toward this necrotroph.

Changing pollinators as a means of escaping herbivores.

All animal-pollinated plants must solve the problem of attracting pollinators while remaining inconspicuous to herbivores, a dilemma exacerbated when voracious larval-stage herbivores mature into important pollinators for a plant [1]. Herbivory is known to alter pollination rates, by altering flower number [2], size [3, 4], nectar production [5], seasonal timing of flowering [6], or pollinator behavior [7]. Nicotiana attenuata, a night-flowering tobacco that germinates after fires in the Southwestern United States, normally produces flowers that open at night and release benzyl acetone (BA) to attract night-active hawkmoth pollinators (Manduca quinquemaculata and M. sexta), which are both herbivores and pollinators. When plants are attacked by hawkmoth larvae, the plants produce flowers with reduced BA emissions that open in the morning and are preferentially pollinated by day-active hummingbirds. This dramatic change in flower phenology, which is elicited by oral secretions (OSs) from feeding hawkmoth larvae and requires jasmonate (JA) signal transduction, causes the majority of outcrossed seeds to be produced by pollinations from day-active hummingbirds rather than night-active hawkmoths. Because oviposition and nectaring are frequently coupled behaviors in hawkmoths, we propose that this OS-elicited, JA-mediated change in flower phenology complements similarly elicited responses to herbivore attack (direct defenses, indirect defenses, and tolerance responses) that reduce the risk and fitness consequences of herbivory to plants.

Petunia flowers solve the defence/apparency dilemma of pollinator attraction by deploying complex floral blends

Flowers recruit floral visitors for pollination services by emitting fragrances. These scent signals can be intercepted by antagonists such as florivores to locate host plants. Hence, as a consequence of interactions with both mutualists and antagonists, floral bouquets likely consist of both attractive and defensive components. While the attractive functions of floral bouquets have been studied, their defensive function has not, and field-based evidence for the deterrence of floral-scent constituents is lacking. In field and glasshouse experiments with five lines of transgenic Petunia x hybrida plants specifically silenced in their ability to release particular components of their floral volatile bouquet, we demonstrate that the emission of single floral-scent compounds can dramatically decrease damage from generalist florivores. While some compounds are used in host location, others prevent florivory. We conclude that the complex blends that comprise floral scents are likely sculpted by the selective pressures of both pollinators and herbivores.

Mechanisms of optimal defense patterns in Nicotiana attenuata: flowering attenuates herbivory-elicited ethylene and jasmonate signaling.

To defend themselves against herbivore attack, plants produce secondary metabolites, which are variously inducible and constitutively deployed, presumably to optimize their fitness benefits in light of their fitness costs. Three phytohormones, jasmonates (JA) and their active forms, the JA-isoleucine (JA-Ile) and ethylene (ET), are known to play central roles in the elicitation of induced defenses, but little is known about how this mediation changes over ontogeny. The Optimal Defense Theory (ODT) predicts changes in the costs and benefits of the different types of defenses and has been usefully extrapolated to their modes of deployment. Here we studied whether the herbivore-induced accumulation of JA, JA-Ile and ET changed over ontogeny in Nicotiana attenuata, a native tobacco in which inducible defenses are particularly well studied. Herbivore-elicited ET production changed dramatically during six developmental stages, from rosette through flowering, decreasing with the elongation of the first corollas during flower development. This decrease was largely recovered within a day after flower removal by decapitation. A similar pattern was found for the herbivore-induced accumulation of JA and JA-Ile. These results are consistent with ODT predictions and suggest that the last steps in floral development control the inducibility of at least three plant hormones, optimizing defense-growth tradeoffs.

An ecological analysis of the herbivory-elicited JA burst and its metabolism: plant memory processes and predictions of the moving target model.

Background Rapid herbivore-induced jasmonic acid (JA) accumulation is known to mediate many induced defense responses in vascular plants, but little is known about how JA bursts are metabolized and modified in response to repeated elicitations, are propagated throughout elicited leaves, or how they directly influence herbivores. Methodology/Principal Findings We found the JA burst in a native population of Nicotiana attenuata to be highly robust despite environmental variation and we examined the JA bursts produced by repeated elicitations with Manduca sexta oral secretions (OS) at whole- and within-leaf spatial scales. Surprisingly, a 2nd OS-elicitation suppressed an expected JA burst at both spatial scales, but subsequent elicitations caused more rapid JA accumulation in elicited tissue. The baseline of induced JA/JA-Ile increased with number of elicitations in discrete intervals. Large veins constrained the spatial spread of JA bursts, leading to heterogeneity within elicited leaves. 1st-instar M. sexta larvae were repelled by elicitations and changed feeding sites. JA conjugated with isoleucine (JA-Ile) translates elicitations into defense production (e.g., TPIs), but conjugation efficiency varied among sectors and depended on NaWRKY3/6 transcription factors. Elicited TPI activity correlated strongly with the heterogeneity of JA/JA-Ile accumulations after a single elicitation, but not repeated elicitations. Conclusions/Significance Ecologically informed scaling of leaf elicitation reveals the contribution of repeated herbivory events to the formation of plant memory of herbivory and the causes and importance of heterogeneity in induced defense responses. Leaf vasculature, in addition to transmitting long-distance damage cues, creates heterogeneity in JA bursts within attacked leaves that may be difficult for an attacking herbivore to predict. Such unpredictability is a central tenet of the Moving Target Model of defense, which posits that variability in itself is defensive.

How scent and nectar influence floral antagonists and mutualists.

Many plants attract and reward pollinators with floral scents and nectar, respectively, but these traits can also incur fitness costs as they also attract herbivores. This dilemma, common to most flowering plants, could be solved by not producing nectar and/or scent, thereby cheating pollinators. Both nectar and scent are highly variable in native populations of coyote tobacco, Nicotiana attenuata, with some producing no nectar at all, uncorrelated with the tobaccos main floral attractant, benzylacetone. By silencing benzylacetone biosynthesis and nectar production in all combinations by RNAi, we experimentally uncouple these floral rewards/attractrants and measure their costs/benefits in the plants native habitat and experimental tents. Both scent and nectar increase outcrossing rates for three, separately tested, pollinators and both traits increase oviposition by a hawkmoth herbivore, with nectar being more influential than scent. These results underscore that it makes little sense to study floral traits as if they only mediated pollination services. DOI: http://dx.doi.org/10.7554/eLife.07641.001

Pithy protection: Nicotiana attenuata's jasmonic acid-mediated defenses are required to resist stem-boring weevil larvae.

Folivory is the best studied plant-herbivore interaction, but it is unclear whether the signaling and resistance traits important for the defense of leaves are also important for other plant parts. Larvae of the tobacco stem weevil, Trichobaris mucorea, burrow into stems of Nicotiana attenuata and feed on the pith. Transgenic N. attenuata lines silenced in signaling and foliar defense traits were evaluated in a 2-year field study for resistance against attack by naturally occurring T. mucorea larva. Plants silenced in early jasmonic acid (JA) biosynthesis (antisense [as]-lipoxygenase3 [lox3]; inverted repeat [ir]-allene oxide cyclase), JA perception (as-coronatine insensitive1), proteinase inhibitors (ir-pi), and nicotine (ir-putrescine methyl-transferase) direct defenses and lignin (ir-cad) biosynthesis were infested more frequently than wild-type plants. Plants unable to emit C6 aldehydes (as-hpl) had lower infestation rates, while plants silenced in late steps in JA biosynthesis (ir-acyl-coenzyme A oxidase, ir-opr) and silenced in diterpene glycoside production (ir-geranylgeranyl pyrophosphate synthase) did not differ from wild type. Pith choice assays revealed that ir-putrescine methyl-transferase, ir-coronatine insensitive1, and ir-lox3 pith, which all had diminished nicotine levels, were preferred by larvae compared to wild-type pith. The lack of preference for ir-lox2 and ir-cad piths, suggest that oviposition attraction and vascular defense, rather than pith palatability accounts for the higher attack rates observed for these plants. We conclude that traits that influence a plant’s apparency, stem hardness, and pith direct defenses all contribute to resistance against this herbivore whose attack can be devastating to N. attenuata’s fitness.

Silencing NaTPI expression increases nectar germin, nectarins and hydrogen peroxide levels and inhibits nectar removal from plants in nature

Native flower visitors removed less nectar from trypsin proteinase inhibitor (TPI)-silenced Nicotiana attenuata plants (ir-pi) than from wild-type plants in four field seasons of releases, even when the nectar repellant, nicotine, was also silenced. Analysis of floral chemistry revealed no differences in the emission of the floral attractants benzylacetone and benzaldehyde or in the concentrations of nectar sugar and nicotine between wild-type and ir-pi flowers, suggesting that these two lines are equally able to attract insect visitors. TPI activity was found in all wild-type flower parts and was highest in anther heads, while TPI activity was not found in any parts of ir-pi flowers. The nectar of ir-pi flowers contained 3.6-fold more total proteins than the nectar of wild-type flowers. Proteomics analysis and hydrogen peroxide (H2O2) measurements revealed that ir-pi nectar contained more nectarins and nectar germin-like proteins and about 1.5-fold more H2O2 compared with wild-type nectar. Field experiments with wild-type flowers supplemented with a solution containing sugar and glucose oxidase demonstrated a causal association between the accumulation of H2O2 and the reduction in nectar removal. These results showed that silencing TPI expression increases the accumulation of nectar proteins and H2O2 levels, which in turn reduces nectar removal by native insect floral visitors. The effect of silencing TPIs on nectar protein accumulation suggests an endogenous regulatory function for TPIs in N. attenuata flowers. The repellency of H2O2 to floral visitors raises new questions about the qualities of nectar that make it attractive for pollinators.

Trichobaris weevils distinguish amongst toxic hostplants by sensing volatiles that do not affect larval performance

Herbivorous insects use plant metabolites to inform their host plant selection for oviposition. These host‐selection behaviours are often consistent with the preference–performance hypothesis; females oviposit on hosts that maximize the performance of their offspring. However, the metabolites used for these oviposition choices and those responsible for differences in offspring performance remain unknown for ecologically relevant interactions. Here, we examined the host‐selection behaviours of two sympatric weevils, the Datura (Trichobaris compacta) and tobacco (T. mucorea) weevils in field and glasshouse experiments with transgenic host plants specifically altered in different components of their secondary metabolism. Adult females of both species strongly preferred to feed on D. wrightii rather than on N. attenuata leaves, but T. mucorea preferred to oviposit on N. attenuata, while T. compacta oviposited only on D. wrightii. These oviposition behaviours increased offspring performance: T. compacta larvae only survived in D. wrightii stems and T. mucorea larvae survived better in N. attenuata than in D. wrightii stems. Choice assays with nicotine‐free, JA‐impaired, and sesquiterpene‐over‐produced isogenic N. attenuata plants revealed that although half of the T. compacta larvae survived in nicotine‐free N. attenuata lines, nicotine did not influence the oviposition behaviours of both the nicotine‐adapted and nicotine‐sensitive species. JA‐induced sesquiterpene volatiles are key compounds influencing T. mucorea females’ oviposition choices, but these sesquiterpenes had no effect on larval performance. We conclude that adult females are able to choose the best host plant for their offspring and use chemicals different from those that influence larval performance to inform their oviposition decisions.