Herbivore-induced plant vaccination. Part I. The orchestration of plant defenses in nature and their fitness consequences in the wild tobacco Nicotiana attenuata

A plant's responses to attack from particular pathogens and herbivores may result in resistance to subsequent attack from the same species, but may also affect different species. Such a cross-resistance, called immunization or vaccination, can benefit the plant, if the fitness consequences of attack from the initial attacker are less than those from subsequent attackers. Here, we report an example of naturally occurring vaccination of the native tobacco plant, Nicotiana attenuata, against Manduca hornworms by prior attack from the mirid bug, Tupiocoris notatus (Dicyphus minimus), which results from the elicitation of two categories of induced plant responses. First, attack from both herbivore species causes the plants in nature to release predator-attracting volatile organic compounds (VOCs), and the attracted generalist predator, Geocoris pallens, preferentially attacks the less mobile hornworm larvae. Second, attack from both mirids and hornworms increases the accumulation of secondary metabolites and proteinase inhibitors (PIs) in the leaf tissue, which is correlated with the slow growth of Manduca larvae. Mirid damage does not significantly reduce the fitness of the plant in nature, whereas attack from the hornworm reduces lifetime seed production. Consequently, plants that are attacked by mirids realize a significant fitness advantage in environments with both herbivores. The combination of growth-slowing direct defenses and predator-attracting indirect defenses results in greater hornworm mortality on mirid-attacked plants and provides the mechanism of the vaccination phenomenon.     

Seasonality of herbivory and communication between individuals of sagebrush

Seasonal changes in herbivore numbers and in plant defenses are well known to influence plant–herbivore interactions. Some plant defenses are induced in response to herbivore attack or cues correlated with risk of attack although seasonal variation in these defenses is relatively poorly known. We previously reported that sagebrush becomes more resistant to its herbivores when neighboring plants have been experimentally clipped with scissors. In this study we asked whether herbivory to leaves of sagebrush varied seasonally and whether there was seasonal variation in natural levels of damage when neighbors were clipped. We found that sagebrush accumulated most chewing damage early in the season, soon after the spring flush of new leaves. This damage was caused by generalist grasshoppers, deer, specialist caterpillars, beetles, gall makers, and other less common herbivores. Sagebrush showed no evidence of preferentially abscising leaves that had been experimentally clipped. Experimental clipping by Trirhabda pilosa beetle larvae caused neighbors to accumulate less herbivore damage later that season, similar to results in which clipping was done with scissors. Induced resistance caused by experimentally clipping a neighbor was affected by season; plants with neighbors clipped in May accumulated less damage throughout the season relative to plants with unclipped neighbors or neighbors clipped later in the summer. We found a correlation between seasonal herbivore pressure, damage accumulated by plants, and induced responses to experimentally clipping neighbors. The causal mechanisms responsible for this correlation are unknown although a strong seasonal effect was clear.     

Herbivore damage to sagebrush induces resistance in wild tobacco: evidence for eavesdropping between plants

Whether plants respond to cues produced by neighbors has been a topic of much debate. Recent evidence suggests that wild tobacco plants transplanted near experimentally clipped sagebrush neighbors suffer less leaf herbivory than tobacco controls with unclipped neighbors. Here we expand these results by showing evidence for induced resistance in naturally rooted tobacco when sagebrush neighbors are clipped either with scissors or damaged with actual herbivores. Tobacco plants with sagebrush neighbors clipped in both ways had enhanced activity levels of polyphenol oxidase (PPO), a chemical marker of induced resistance in many solanaceous plants. Eavesdropping was found for plants that were naturally rooted, although only when sagebrush and tobacco grew within 10 cm of each other. Although tobacco with clipped neighbors experienced reduced herbivory, tobacco that grew close to sagebrush had lower production of capsules than plants that grew far from sagebrush. When neighboring tobacco rather than sagebrush was clipped, neither levels of PPO nor levels of leaf damage to tobacco were affected. Eavesdropping on neighboring sagebrush, but not neighboring tobacco, may result from plants using a jasmonate signaling system. These results indicate that plants eavesdrop in nature and that this behavior can increase resistance to herbivory although it does not necessarily increase plant fitness.     

Priming by airborne signals boosts direct and indirect resistance in maize

Plants counteract attack by herbivorous insects using a variety of inducible defence mechanisms. The production of toxic proteins and metabolites that instantly affect the herbivore's development are examples of direct induced defence. In addition, plants may release mixtures of volatile organic compounds (VOCs) that indirectly protect the plant by attracting natural enemies of the herbivore. Recent studies suggest that these VOCs can also prime nearby plants for enhanced induction of defence upon future insect attack. However, evidence that this defence priming causes reduced vulnerability to insects is sparse. Here we present molecular, chemical and behavioural evidence that VOC-induced priming leads to improved direct and indirect resistance in maize. A differential hybridization screen for inducible genes upon attack by Spodoptera littoralis caterpillars identified 10 defence-related genes that are responsive to wounding, jasmonic acid (JA), or caterpillar regurgitant. Exposure to VOCs from caterpillar-infested plants did not activate these genes directly, but primed a subset of them for earlier and/or stronger induction upon subsequent defence elicitation. This priming for defence-related gene expression correlated with reduced caterpillar feeding and development. Furthermore, exposure to caterpillar-induced VOCs primed for enhanced emissions of aromatic and terpenoid compounds. At the peak of this VOC emission, primed plants were significantly more attractive to parasitic Cotesia marginiventris waSPS. This study shows that VOC-induced priming targets a specific subset of JA-inducible genes, and links these responses at the molecular level to enhanced levels of direct and indirect resistance against insect attack.     

Co(i)-ordinating defenses: NaCOI1 mediates herbivore- induced resistance in Nicotiana attenuata and reveals the role of herbivore movement in avoiding defenses

Arabidopsis and tomato plants mutated in the F-box protein COI1 mediating jasmonate (JA) responses are more susceptible to herbivores in laboratory trials, but the exact mechanisms of COI1-mediated resistance are not known. We silenced COI1 by transformation with an inverted repeat construct (ir-coi1) in Nicotiana attenuata, a plant the direct and indirect defenses of which against various herbivores have been well studied. ir-coi1 plants are male sterile and impaired in JA-elicited direct [nicotine, caffeoylputrescine and trypsin proteinase inhibitor (TPI) activity] and indirect (cis-alpha-bergamotene emission) defense responses; responses not elicited by JA treatment (ethylene production and flower TPI activity) were unaffected. Larvae of Manduca sexta, a common herbivore of N. attenuata, gained three times more mass feeding on ir-coi1 than on wild-type (WT) plants in glasshouse experiments. By regularly moving caterpillars to unattacked leaves of the same plant, we demonstrate that larvae on WT plants can grow and consume leaves as fast as those on ir-coi1 plants, a result that underscores the role of COI1 in mediating locally induced resistance in attacked leaves, and the importance of herbivore movement in avoiding the induced defenses of a plant. When transplanted into native habitats in the Great Basin Desert, ir-coi1 plants suffer greatly from damage by the local herbivore community, which includes herbivores not commonly found on N. attenuata WT plants. Choice assays with field-grown plants confirmed the increased attractiveness of ir-coi1 plants for both common and unusual herbivores. We conclude that NaCOI1 is essential for induced resistance in N. attenuata, and that ir-coi1 plants highlight the benefits of herbivore movement for avoiding induced defenses.     

Methyl jasmonate is blowing in the wind,but can it act as a plant–plant airborne signal?

Interplant communication in nature is beginning to look like a reality with the field demonstration that tobacco plants downwind of damaged sagebrush suffer less herbivory, a response that appears to be mediated by an airborne signal. Sagebrush constitutively releases methyl jasmonate (MeJA), a compound that is highly active in inducing a number of physiological responses in plants. Damage increases the absolute quantity of the MeJA released as well as the proportion of MeJA in the isomeric cis form. Several studies have shown that volatile MeJA, when released in sufficient quantities, can simulate responses elicited by direct MeJA applications. Additionally, the thermodynamically unstable cis isomer, which is responsible for the characteristic jasmine odor, is thought to be the biologically active form of MeJA. To examine the hypothesis that the cis-MeJA release is responsible for the apparent inter-plant communication, we developed methods to: (1) entrain sagebrush constituents in water which preserved the isomeric shift in the MeJA released after damage; (2) chemically manipulate the MeJA trans : cis ratio; and (3) isolate nearly pure cis-MeJA by HPLC. These treatments were applied as aqueous sprays to a natural population of tobacco plants, however, an outbreak of specialist herbivores consumed all treated plants and chemical analysis on previously harvested treated leaf material was inconclusive. The hypothesis is currently being carefully investigated with laboratory experiments.     

Communication between plants: induced resistance in wild tobacco plants following clipping of neighboring sagebrush

The possibility of communication between plants was proposed nearly 20 years ago, although previous demonstrations have suffered from methodological problems and have not been widely accepted. Here we report the first rigorous, experimental evidence demonstrating that undamaged plants respond to cues released by neighbors to induce higher levels of resistance against herbivores in nature. Sagebrush plants that were clipped in the field released a pulse of an epimer of methyl jasmonate that has been shown to be a volatile signal capable of inducing resistance in wild tobacco. Wild tobacco plants with clipped sagebrush neighbors had increased levels of the putative defensive oxidative enzyme, polyphenol oxidase, relative to control tobacco plants with unclipped sagebrush neighbors. Tobacco plants near clipped sagebrush experienced greatly reduced levels of leaf damage by grasshoppers and cutworms during three field seasons compared to unclipped controls. This result was not caused by an altered light regime experienced by tobacco near clipped neighbors. Barriers to soil contact between tobacco and sagebrush did not reduce the difference in leaf damage although barriers that blocked air contact negated the effect. Received: 15 February 2000 / Accepted: 1 April 2000     

Innovation in anti-herbivore defense systems during neopolypoloidy - the functional consequences of instantaneous speciation

Allopolyploid hybridization instantly merges two differentially adapted genomes into one individual. Allopolyploids are often evolutionarily successful, undergoing adaptive radiations despite the genetic and physiological challenges of merging genomes. We examine a suite of induced herbivore resistance traits in three independent lines of the synthetic allopolyploid Nicotianaxmierata (Nma) and its parent species, N. miersii (Nmi) and N. attenuata (Na), to determine how a complex polygenetic adaptation fares during the early stages of neoallopolyploid formation. All species responded to Manduca sexta oral secretions (OS) with a temporally prolonged jasmonate (JA) burst. In one parent (Na), the JA burst was additionally amplified and associated with the elicitation of direct and indirect defenses. In the other parent (Nmi), OS neither amplified the JA burst nor elicited defense responses, although applied MeJA confirmed the inducibility of the defense responses. All lines of Nma retained enough aspects of Na's JA signaling to recognize OS and to accumulate sufficient direct defenses to impair the growth of Manduca larvae. Most defense-related metabolites were retained in Nma even if inherited from only one parent; however, OS-elicited volatiles, trypsin protease inhibitors (TPIs) and chlorogenic acid were lost in some lines, even though MeJA treatment elicited similar responses in all lines. Herbivore defense systems are flexibly inherited in allopolyploids, causing individuals to diverge over only a few generations; for example, line 1 of Nma could not produce TPIs after OS elicitation, whereas lines 2 and 3 could. This flexible integration of defense signaling systems with a diversity of elicited responses may explain why adaptive radiations are commonly found in allopolyploid lineages.     

Ecophysiological comparison of direct and indirect defenses in Nicotiana attenuata

After herbivore attack, plants launch a suite of direct and indirect defense responses that must be coordinated if plants are to realize a fitness benefit from these responses. Here we characterize the volatile emissions in the native tobacco plant, Nicotiana attenuata Torr. ex Wats., that are elicited by tobacco hornworm (Manduca sexta L.) attack and are known to function as attractants for parasitoids. To provide the first ecophysiological comparison of examples of both types of defense in the same species, we characterize the elicitation and signaling mechanisms, the resources required, and the potential costs and benefits of the volatile release and compare these traits with those of the well-described induced direct defense in this species, nicotine production. The release of (E)-β-ocimene, cis-α-bergamotene and linalool is dramatically induced within 24 h by application of methyl jasmonate (MeJA), caterpillar feeding, and the treatment of mechanical wounds with larval oral secretions (OS), but not by mechanical damage alone. Plants from different geographic locations produce volatile blends that differ in composition. The most consistently released component from all genotypes, cis-α-berga-motene, is positively related to the amount of MeJA and the level of wounding if OS are applied to the wounds. The volatile release is strongly light dependent, dropping to undetectable quantities during dark periods, even when temperatures are elevated to match those of the light period. Inhibitors of wound-induced jasmonate accumulation (salicylates and auxins), which are known to inhibit wound-induced nicotine production, do not inhibit the release of volatiles. By individually inducing different leaf positions with OS and, on other plants, excising them after induction, we demonstrate that the emission is largely a systemic, whole-plant response, which is maximally triggered when the second fully expanded leaf is induced. We conclude that while both are whole-plant, systemic responses that utilize recently acquired resources for their production and are activated by the jasmonate cascade, the elicitation of the volatile release exhibits greater tissue sensitivity and utilizes additional signaling components than does nicotine production. In contrast to the large investment of fitness-limiting resources required for induced nicotine production or the resources used in benzyl acetone release from flowers for pollinator attraction, the resource requirements for the volatile release are minor. Hence the argument that the volatile release incurs comparatively large physiological costs cannot be supported in this system. Received: 4 November 1999 / Accepted: 1 March 2000     

Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata

Inhibition of jasmonic acid (JA) signaling has been shown to decrease herbivore resistance, but the responsible mechanisms are largely unknown because insect resistance is poorly understood in most model plant systems. We characterize three members of the lipoxygenase (LOX) gene family in the native tobacco plant Nicotiana attenuata and manipulate, by antisense expression, a specific, wound- and herbivory-induced isoform (LOX3) involved in JA biosynthesis. In three independent lines, antisense expression reduced wound-induced JA accumulation but not the release of green leaf volatiles (GLVs). The impaired JA signaling reduced two herbivore-induced direct defenses, nicotine and trypsin protease inhibitors (TPI), as well as the potent indirect defense, the release of volatile terpenes that attract generalist predators to feeding herbivores. All these defenses could be fully restored by methyl-JA (MeJA) treatment, with the exception of the increase in TPI activity, which was partially restored, suggesting the involvement of additional signals. The impaired ability to produce chemical defenses resulted in lower resistance to Manduca sexta attack, which could also be restored by MeJA treatment. Expression analysis using a cDNA microarray, specifically designed to analyze M. sexta-induced gene expression in N. attenuata, revealed a pivotal role for LOX3-produced oxylipins in upregulating defense genes (protease inhibitor, PI; xyloglucan endotransglucosylase/hydrolase, XTH; threonine deaminase, TD; hydroperoxide lyase, HPL), suppressing both downregulated growth genes (RUBISCO and photosystem II, PSII) and upregulated oxylipin genes (alpha-dioxygenase, alpha-DOX). By genetically manipulating signaling in a plant with a well-characterized ecology, we demonstrate that the complex phenotypic changes that mediate herbivore resistance are controlled by a specific part of the oxylipin cascade.     

Herbivore-specific plant volatiles prime neighboring plants for nonspecific defense responses

Plants produce species-specific herbivore-induced plant volatiles (HIPVs) after damage. We tested the hypothesis that herbivore-specific HIPVs prime neighboring plants to induce defenses specific to the priming herbivore. Since Manduca sexta (specialist) and Heliothis virescens (generalist) herbivory induced unique HIPV profiles in Nicotiana benthamiana, we used these HIPVs to prime receiver plants for defense responses to simulated herbivory (mechanical wounding and herbivore regurgitant application). Jasmonic acid (JA) accumulations and emitted volatile profiles were monitored as representative defense responses since JA is the major plant hormone involved in wound and defense signaling and HIPVs have been implicated as signals in tritrophic interactions. Herbivore species-specific HIPVs primed neighboring plants, which produced 2 to 4 times more volatiles and JA after simulated herbivory when compared to similarly treated constitutive volatile-exposed plants. However, HIPV-exposed plants accumulated similar amounts of volatiles and JA independent of the combination of priming or challenging herbivore. Furthermore, volatile profiles emitted by primed plants depended only on the challenging herbivore species but not on the species-specific HIPV profile of damaged emitter plants. This suggests that feeding by either herbivore species primed neighboring plants for increased HIPV emissions specific to the subsequently attacking herbivore and is probably controlled by JA.     

Induced Resistance in Wild Tobacco with Clipped Sagebrush Neighbors: The Role of Herbivore Behavior

Previous experiments showed that wild tobacco plants with experimentally clipped sagebrush neighbors experienced less damage by grasshoppers than tobacco plants with unclipped sagebrush neighbors. This result could have been caused by grasshoppers preferring not to feed near clipped sagebrush. This hypothesis was tested in field choice experiments using six grasshopper species feeding on an unresponsive and uniformly palatable food. When offered food that was either close to clipped sagebrush or close to unclipped sagebrush, grasshoppers showed no preference. When offered food that was either close to sagebrush (3 cm) or far from sagebrush (30 cm), grasshoppers preferred to feed far from sagebrush. However, this preference was similar whether or not the sagebrush had been clipped. Avoidance of feeding near clipped sagebrush, independent of changes in tobacco, was not found to contribute to our earlier result that tobacco near clipped sagebrush suffered less herbivory than tobacco near unclipped sagebrush.