Nicotiana attenuata is attacked by larvae of both specialist (Manduca sexta) and generalist (Spodoptera exigua) lepidopteran herbivores in its native habitat. Nicotine is one of N. attenuata's important defenses. M. sexta is highly nicotine tolerant; whether cytochrome P450 (CYP)-mediated oxidative detoxification and/or rapid excretion is responsible for its exceptional tolerance remains unknown despite five decades of study. Recently, we demonstrated that M. sexta uses its nicotine-induced CYP6B46 to efflux midgut-nicotine into the hemolymph, facilitating nicotine exhalation that deters predatory wolf spiders (Camptocosa parallela). S. exigua's nicotine metabolism is uninvestigated.
We compared the ability of these two herbivores to metabolize, tolerate and co-opt ingested nicotine for defense against the wolf spider. In addition, we analyzed the spider's excretion to gain insights into its nicotine metabolism. Contrary to previous reports, we found that M. sexta larvae neither accumulate the common nicotine oxides (cotinine, cotinine N-oxide and nicotine N-oxide) nor excrete them faster than nicotine. In M. sexta larvae, ingestion of nicotine as well as its oxides increases the accumulation of CYP6B46 transcripts. In contrast, S. exigua accumulates nicotine oxides and exhales less (66%) nicotine than does M. sexta. Spiders prefer nicotine-fed S. exigua over M. sexta, a preference reversed by topical or headspace nicotine supplementation, but not ingested or topically-coated nicotine oxides, suggesting that externalized nicotine but not the nicotine detoxification products deter spider predation. The spiders also do not accumulate nicotine oxides.
Nicotine oxidation reduces S. exigua's headspace-nicotine and renders it more susceptible to predation by spiders than M. sexta, which exhales unmetabolized nicotine. These results are consistent with the hypothesis that generalist herbivores incur costs of detoxification, which include the ecological costs of greater predation risks, in addition to the previously demonstrated energetic, physiological and metabolic costs.
From an herbivore's first bite, plants release herbivory-induced plant volatiles (HIPVs) which can attract enemies of herbivores. However, other animals and competing plants can intercept HIPVs for their own use, and it remains unclear whether HIPVs serve as an indirect defense by increasing fitness for the emitting plant. In a 2-year field study, HIPV-emitting N. attenuata plants produced twice as many buds and flowers as HIPV-silenced plants, but only when native Geocoris spp. predators reduced herbivore loads (by 50%) on HIPV-emitters. In concert with HIPVs, plants also employ antidigestive trypsin protease inhibitors (TPIs), but TPI-producing plants were not fitter than TPI-silenced plants. TPIs weakened a specialist herbivore's behavioral evasive responses to simulated Geocoris spp. attack, indicating that TPIs function against specialists by enhancing indirect defense.
As the population of the world continues to increase beyond 7 billion, and agricultural pests continue to rapidly evolve resistance to pesticides, it is becoming ever more important to cultivate arable land in a way that is sustainable for both humans and the environment. A better understanding of the different mechanisms used by wild plants to deter herbivores will help to increase crop production without harming the environment.
Plants use both direct and indirect methods to fend off herbivores. Direct defense methods include the production of chemicals that are toxic to herbivores or give them indigestion, and the growth of sticky prickles and spines that can injure or kill the herbivore. Indirect defense methods, on the other hand, generally rely on the plant attracting organisms that are either predators or parasites of the herbivore.
Plants produce odors known as herbivory-induced plant volatiles (HIPVs) that are thought to offer indirect defense against herbivores by betraying their location to predators and parasites. However, HIPVs also influence other members of the ecological community, sometimes in ways that are detrimental to plants. Moreover, despite 30 years of research, no study has demonstrated that HIPVs increase the fitness of a plant, so it is unclear what they have evolved to do.
Now, a 2-year field study by Schuman et al. has shown plants that emit green leaf volatiles (which are a type of HIPV) produce twice as many buds and flowers—a measure of fitness—as plants that have been genetically engineered not to emit green leaf volatiles. This study was conducted with Nicotiana attenuata, which is a wild tobacco plant that is often targeted by Manduca sexta, a type of moth that is also known as the tobacco hornworm. Green leaf volatiles only increased plants' fitness when various species of Geocoris—a bug that preys on Manduca sexta—reduced the number of herbivores by a factor of two. This is the first evidence that HIPVs offer indirect defense against herbivores.
Schuman et al. also studied the effects of molecules called protease inhibitors that are thought to function as direct defenses by making it difficult for herbivores to digest plants. They found that the ability to produce protease inhibitors did not increase the fitness of plants under herbivore attack; however, tobacco hornworms that had been fed plants containing protease inhibitors were found to be more sluggish in response to attack, which suggests that protease inhibitors can enhance the indirect defenses of plants. The results suggest that employing both direct and indirect defenses—such as a combination of biological pesticides and genetic engineering to produce both HIPVs and protease inhibitors—is the best approach for defending agricultural plants against pests.
Nicotiana attenuata; HIPV (herbivory-induced plant volatile); plant-predator interaction; GLV (green leaf volatile); TPI (trypsin protease inhibitor); indirect defense; Other
Herbivore feeding elicits dramatic increases in defenses, most of which require jasmonate (JA) signaling, and against which specialist herbivores are thought to be better adapted than generalist herbivores. Unbiased transcriptional analyses of how neonate larvae cope with these induced plant defenses are lacking.
We created cDNA microarrays for Manduca sexta and Heliothis virescens separately, by spotting normalized midgut-specific cDNA libraries created from larvae that fed for 24 hours on MeJA-elicited wild-type (WT) Nicotiana attenuata plants. These microarrays were hybridized with labeled probes from neonates that fed for 24 hours on WT and isogenic plants progressively silenced in JA-mediated defenses (N: nicotine; N/PI: N and trypsin protease inhibitors; JA: all JA-mediated defenses). H. virescens neonates regulated 16 times more genes than did M. sexta neonates when they fed on plants silenced in JA-mediated defenses, and for both species, the greater the number of defenses silenced in the host plant (JA > N/PI > N), the greater were the number of transcripts regulated in the larvae. M. sexta larvae tended to down-regulate while H. virescens larvae up- and down-regulated transcripts from the same functional categories of genes. M. sexta larvae regulated transcripts in a diet-specific manner, while H. virescens larvae regulated a similar suite of transcripts across all diet types.
The observations are consistent with the expectation that specialists are better adapted than generalist herbivores to the defense responses elicited in their host plants by their feeding. While M. sexta larvae appear to be better adapted to N. attenuata's defenses, some of the elicited responses remain effective defenses against both herbivore species. The regulated genes provide novel insights into larval adaptations to N. attenuata's induced defenses, and represent potential targets for plant-mediated RNAi to falsify hypotheses about the process of adaptation.
BAK1 is a co-receptor of brassinosteroid (BR) receptor BRI1, and plays a well-characterized role in BR signalling. BAK1 also physically interacts with the flagellin receptor FLS2 and regulates pathogen resistance. The role of BAK1 in mediating Nicotiana attenuata's resistance responses to its specialist herbivore, Manduca sexta, was examined here. A virus-induced gene-silencing system was used to generate empty vector (EV) and NaBAK1-silenced plants. The wounding- and herbivory-induced responses were examined on EV and NaBAK1-silenced plants by wounding plants or simulating herbivory by treating wounds with larval oral secretions (OS). After wounding or OS elicitation, NaBAK1-silenced plants showed attenuated jasmonic acid (JA) and JA-isoleucine bursts, phytohormone responses important in mediating plant defences against herbivores. However, these decreased JA and JA-Ile levels did not result from compromised MAPK activity or elevated SA levels. After simulated herbivory, NaBAK1-silenced plants had EV levels of defensive secondary metabolites, namely, trypsin proteinase inhibitors (TPIs), and similar levels of resistance to Manduca sexta larvae. Additional experiments demonstrated that decreased JA levels in NaBAK1-VIGS plants, rather than the enzymatic activity of JAR proteins or Ile levels, were responsible for the reduced JA-Ile levels observed in these plants. Methyl jasmonate application elicited higher levels of TPI activity in NaBAK1-silenced plants than in EV plants, suggesting that silencing NaBAK1 enhances the accumulation of TPIs induced by a given level of JA. Thus NaBAK1 is involved in modulating herbivory-induced JA accumulation and how JA levels are transduced into TPI levels in N. attenuata.
BAK1; defence; herbivory; jasmonic acid (JA); jasmonic acid-isoleucine (JA-Ile); Nicotiana attenuata; SERK; trypsin proteinase inhibitor
Pectin methylesterases (PMEs) catalyse the demethylation of pectin within plant cell walls, releasing methanol (MeOH) in the process. Thus far, PMEs have been found to be involved in diverse processes such as plant growth and development and defence responses against pathogens. Herbivore attack increases PME expression and activity and MeOH emissions in several plant species. To gain further insights into the role of PMEs in defence responses against herbivores, the expression of a Manduca sexta oral secretion (OS)-inducible PME in Nicotiana attenuata (NaPME1) was silenced by RNA interference (RNAi)-mediated gene silencing. Silenced lines (ir-pme) showed 50% reduced PME activity in leaves and 70% reduced MeOH emissions after OS elicitation compared with the wild type (WT), demonstrating that the herbivore-induced MeOH emissions originate from the demethylation of pectin by PME. In the initial phase of the OS-induced jasmonic acid (JA) burst (first 30 min), ir-pme lines produced WT levels of this hormone and of jasmonyl-isoleucine (JA-Ile); however, these levels were significantly reduced in the later phase (60–120 min) of the burst. Similarly, suppressed levels of the salicylic acid (SA) burst induced by OS elicitation were observed in ir-pme lines even though wounded ir-pme leaves contained slightly increased amounts of SA. This genotype also presented reduced levels of OS-induced trypsin proteinase inhibitor activity in leaves and consistently increased M. sexta larvae performance compared with WT plants. These latter responses could not be recovered by application of exogenous MeOH. Together, these results indicated that PME contributes, probably indirectly by affecting cell wall properties, to the induction of anti-herbivore responses.
Defence; herbivory; jasmonic acid; Manduca; methanol; Nicotiana; pectin methylesterase; proteinase inhibitor
The commonly invoked cost-benefit paradigm, central to most of functional biology, explains why one phenotype cannot be optimally fit in all environments; yet it is rarely tested. Trypsin proteinase inhibitors (TPIs) expression in Nicotiana attenuata is known to decrease plant fitness when plants compete with unattacked conspecifics that do not produce TPIs and also to decrease the performance of attacking herbivores.
In order to determine whether the putative benefits of TPI production outweigh its cost, we transformed N. attenuata to silence endogenous TPI production or restore it in a natural mutant that was unable to produce TPIs. We compared the lifetime seed production of N. attenuata genotypes of the same genetic background with low or no TPI to that of genotypes with high TPI levels on which M. sexta larvae were allowed to feed freely. Unattacked low TPI-producing genotypes produced more seed capsules than did plants with high TPI levels. Caterpillar attack reduced seed capsule production in all genotypes and reversed the pattern of seed capsule production among genotypes. M. sexta larvae attacking genotypes with high TPI activity consumed more TPI, less protein, and move later to the young leaves. Larval masses were negatively correlated (R2 = 0.56) with seed capsule production per plant.
Our results demonstrate that the fitness benefits of TPI production outweigh their costs in greenhouse conditions, when plants are attacked and that despite the ongoing evolutionary interactions between plant and herbivore, TPI-mediated decreases in M. sexta performance translates into a fitness benefit for the plant.
Plants produce metabolites that directly decrease herbivore performance, and as a consequence, herbivores are selected for resistance to these metabolites. To determine whether these metabolites actually function as defenses requires measuring the performance of plants that are altered only in the production of a certain metabolite. To date, the defensive value of most plant resistance traits has not been demonstrated in nature. We transformed native tobacco(Nicotiana attenuata) with a consensus fragment of its two putrescine N-methyl transferase (pmt) genes in either antisense or inverted-repeat (IRpmt) orientations. Only the latter reduced (by greater than 95%) constitutive and inducible nicotine. With D4-nicotinic acid (NA), we demonstrate that silencing pmt inhibits nicotine production, while the excess NA dimerizes to form anatabine. Larvae of the nicotine-adapted herbivore Manduca sexta (tobacco hornworm) grew faster and, like the beetle Diabrotica undecimpunctata, preferred IRpmt plants in choice tests. When planted in their native habitat, IRpmt plants were attacked more frequently and, compared to wild-type plants, lost 3-fold more leaf area from a variety of native herbivores, of which the beet armyworm, Spodoptera exigua, and Trimerotropis spp. grasshoppers caused the most damage. These results provide strong evidence that nicotine functions as an efficient defense in nature and highlights the value of transgenic techniques for ecological research.
Transgenic plants confirm the role that nicotine has in protecting tobacco plants from predators in nature and demonstrates the power of transgenic tools in studying ecological interactions in the field
Some plants distinguish mechanical wounding from herbivore attack by recognizing specific constituents of larval oral secretions (OS) which are introduced into plant wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of Manduca sexta OS and strong elicitors of herbivore-induced defense responses in Nicotiana attenuata plants.
The metabolism of one of the major FACs in M. sexta OS, N-linolenoyl-glutamic acid (18:3-Glu), was analyzed on N. attenuata wounded leaf surfaces. Between 50 to 70% of the 18:3-Glu in the OS or of synthetic 18:3-Glu were metabolized within 30 seconds of application to leaf wounds. This heat-labile process did not result in free α-linolenic acid (18:3) and glutamate but in the biogenesis of metabolites both more and less polar than 18:3-Glu. Identification of the major modified forms of this FAC showed that they corresponded to 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu. The formation of these metabolites occurred on the wounded leaf surface and it was dependent on lipoxygenase (LOX) activity; plants silenced in the expression of NaLOX2 and NaLOX3 genes showed more than 50% reduced rates of 18:3-Glu conversion and accumulated smaller amounts of the oxygenated derivatives compared to wild-type plants. Similar to 18:3-Glu, 13-oxo-13:2-Glu activated the enhanced accumulation of jasmonic acid (JA) in N. attenuata leaves whereas 13-hydroxy-18:3-Glu did not. Moreover, compared to 18:3-Glu elicitation, 13-oxo-13:2-Glu induced the differential emission of two monoterpene volatiles (β-pinene and an unidentified monoterpene) in irlox2 plants.
The metabolism of one of the major elicitors of herbivore-specific responses in N. attenuata plants, 18:3-Glu, results in the formation of oxidized forms of this FAC by a LOX-dependent mechanism. One of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of JA biosynthesis and differential monoterpene emission.
S-nitrosoglutathione reductase (GSNOR) reduces the nitric oxide (NO) adduct S-nitrosoglutathione (GSNO), an essential reservoir for NO bioactivity. In plants, GSNOR has been found to be important in resistance to bacterial and fungal pathogens, but whether it is also involved in plant–herbivore interactions was not known. Using a virus-induced gene silencing (VIGS) system, the activity of GSNOR in a wild tobacco species, Nicotiana attenuata, was knocked down and the function of GSNOR in defence against the insect herbivore Manduca sexta was examined. Silencing GSNOR decreased the herbivory-induced accumulation of jasmonic acid (JA) and ethylene, two important phytohormones regulating plant defence levels, without compromising the activity of two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK). Decreased activity of trypsin proteinase inhibitors (TPIs) were detected in GSNOR-silenced plants after simulated M. sexta feeding and bioassays indicated that GSNOR-silenced plants have elevated susceptibility to M. sexta attack. Furthermore, GSNOR is required for methyl jasmonate (MeJA)-induced accumulation of defence-related secondary metabolites (TPI, caffeoylputrescine, and diterpene glycosides) but is not needed for the transcriptional regulation of JAZ3 (jasmonate ZIM-domain 3) and TD (threonine deaminase), indicating that GSNOR mediates certain but not all jasmonate-inducible responses. This work highlights the important role of GSNOR in plant resistance to herbivory and jasmonate signalling and suggests the potential involvement of NO in plant–herbivore interactions. Our data also suggest that GSNOR could be a target of genetic modification for improving crop resistance to herbivores.
Defence; ethylene; insect herbivore; jasmonic acid; jasmonate signalling; Manduca sexta; Nicotiana attenuata; S-nitrosoglutathione reductase (GSNOR); secondary metabolites; trypsin proteinase inhibitor
Jasmonates are involved in plant defense, participating in the timely induction of defense responses against insect herbivores from different feeding guilds and with different degrees of host specialization. It is less clear to what extent the induction of plant defense is controlled by different members of the jasmonate family and how specificity of the response is achieved. Using transgenic plants blocked in jasmonic acid (JA) biosynthesis, we previously showed that JA is required for the formation of glandular trichomes and trichome-borne metabolites as constitutive defense traits in tomato, affecting oviposition and feeding behavior of the specialist Manduca sexta. In contrast, JA was not required for the local induction of defense gene expression after wounding. In JA-deficient plants, the JA precursor oxophytodienoic acid (OPDA) substituted as a regulator of defense gene expression maintaining considerable resistance against M. sexta larvae. In this study, we investigate the contribution of JA and OPDA to defense against the generalist herbivore Spodoptera exigua.
S. exigua preferred JA-deficient over wild-type tomato plants as a host for both oviposition and feeding. Feeding preference for JA-deficient plants was caused by constitutively reduced levels of repellent terpenes. Growth and development of the larvae, on the other hand, were controlled by additional JA-dependent defense traits, including the JA-mediated induction of foliar polyphenol oxidase (PPO) activity. PPO induction was more pronounced after S. exigua herbivory as compared to mechanical wounding or M. sexta feeding. The difference was attributed to an elicitor exclusively present in S. exigua oral secretions.
The behavior of M. sexta and S. exigua during oviposition and feeding is controlled by constitutive JA/JA-Ile-dependent defense traits involving mono- and sesquiterpenes in both species, and cis-3-hexenal as an additional chemical cue for M. sexta. The requirement of jasmonates for resistance of tomato plants against caterpillar feeding differs for the two species. While the OPDA-mediated induction of local defense is sufficient to restrict growth and development of M. sexta larvae in absence of JA/JA-Ile, defense against S. exigua relied on additional JA/JA-Ile dependent factors, including the induction of foliar polyphenol oxidase activity in response to S. exigua oral secretions.
Generalist and specialist herbivores; Glucose oxidase; Insect resistance; Jasmonic acid; Oxophytodienoic acid; Plant defense; Polyphenol oxidase; Oral secretions; Terpenes
Treatment with methyl jasmonate (MeJA) elicits herbivore resistance in many plant species and over-expression of JA carboxyl methyltransferase (JMT) constitutively increases JA-induced responses in Arabidopsis. When wild-type (WT) Nicotiana attenuata plants are treated with MeJA, a rapid transient endogenous JA burst is elicited, which in turn increases levels of nicotine and trypsin proteinase inhibitors (TPIs) and resistance to larvae of the specialist herbivore, Manduca sexta. All of these responses are impaired in plants silenced in lipoxygenase 3 expression (asLOX3) but are restored to WT levels by MeJA treatment. Whether these MeJA-induced responses are directly elicited by MeJA or by its cleavage product, JA, is unknown. Using virus-induced gene silencing (VIGS), we silenced MeJA-esterase (NaMJE) expression and found this gene responsible for most of the MeJA-cleaving activity in N. attenuata protein extracts. Silencing NaMJE in asLOX3, but not in WT plants, significantly reduced MeJA-induced nicotine levels and resistance to M. sexta, but not TPI levels. MeJA-induced transcript levels of threonine deaminase (NaTD) and phenylalanine ammonia lyase (NaPAL1) were also decreased in VIGS MJE (asLOX3) plants. Finally the performance of M. sexta larvae that fed on plants treated with JA or MeJA demonstrated that silencing NaMJE inhibited MeJA-induced but not JA-induced resistance in asLOX3 plants. From these results, we conclude that the resistance elicited by MeJA treatment is directly elicited not by MeJA but by its de-methylated product, JA.
Electronic supplementary material
The online version of this article (doi:10.1007/s00425-008-0690-8) contains supplementary material, which is available to authorized users.
MeJA esterase (NaMJE); Methyl jasmonate (MeJA); Jasmonate (JA); Nicotiana attenuata; Manduca sexta
A plant's inducible defenses against herbivores as well as certain developmental processes are known to be controlled by the jasmonic acid (JA) pathway. We have previously shown that ectopically expressing Arabidopsis thaliana JA O-methyltransferase in Nicotiana attenuata (35S-jmt) strongly reduces the herbivory-elicited jasmonate bursts by acting as metabolic sink that redirects free JA towards methylation; here we examine the consequences of this metabolic sink on N. attenuata's secondary metabolism and performance in nature. In the glasshouse, 35S-jmt plants produced fewer seed capsules due to shorter floral styles, which could be restored to wild type (WT) levels after hand-pollination, and were more susceptible to Manduca sexta larvae attack. When transplanted into the Great Basin Desert in Utah, 35S-jmt plants grew as well as WT empty vector, but were highly attacked by native herbivores of different feeding guilds: leaf chewers, miners, and single cell feeders. This greater susceptibility was strongly associated with reduced emissions of volatile organic compounds (hexenylesters, monoterpenes and sesquiterpenes) and profound alterations in the production of direct defenses (trypsin proteinase inhibitors [TPI], nicotine, diterpene glycosides [DTGs] and phenylpropanoid-polyamine conjugates) as revealed by a combination of targeted and metabolomics analyses of field collected samples. Complementation experiments with JA-Ile, whose formation is outcompeted in 35S-jmt plants by the methylation reaction, restored the local TPI activation to WT levels and partially complemented nicotine and DTG levels in elicited but not systemic leaves. These findings demonstrate that MeJA, the major JA metabolite in 35S-jmt plants, is not an active signal in defense activation and highlights the value of creating JA sinks to disrupt JA signaling, without interrupting the complete octadecanoid pathway, in order to investigate the regulation of plants' defense metabolism in nature.
The defensive effect of endogenous trypsin proteinase inhibitors (NaTPIs) on the herbivore Manduca sexta was demonstrated by genetically altering NaTPI production in M. sexta's host plant, Nicotiana attenuata. To understand how this defense works, we studied the effects of NaTPI on M. sexta gut proteinase activity levels in different larval instars of caterpillars feeding freely on untransformed and transformed plants.
Methodology/ Principal Findings
Second and third instars larvae that fed on NaTPI-producing (WT) genotypes were lighter and had less gut proteinase activity compared to those that fed on genotypes with either little or no NaTPI activity. Unexpectedly, NaTPI activity in vitro assays not only inhibited the trypsin sensitive fraction of gut proteinase activity but also halved the NaTPI-insensitive fraction in third-instar larvae. Unable to degrade NaTPI, larvae apparently lacked the means to adapt to NaTPI in their diet. However, caterpillars recovered at least part of their gut proteinase activity when they were transferred from NaTPI-producing host plants to NaTPI-free host plants. In addition extracts of basal leaves inhibited more gut proteinase activity than did extracts of middle stem leaves with the same protein content.
Although larvae can minimize the effects of high NaTPI levels by feeding on leaves with high protein and low NaTPI activity, the host plant's endogenous NaTPIs remain an effective defense against M. sexta, inhibiting gut proteinase and affecting larval performance.
Plant phenolics are generally thought to play significant roles in plant defense against herbivores and pathogens. Many plant taxa, including Solanaceae, are rich in phenolic compounds and some insect herbivores have been shown to acquire phenolics from their hosts to use them as protection against their natural enemies. Here we demonstrate that larvae of an insect specialist on Solanaceae, the tobacco hornworm, Manduca sexta L. (Lepidoptera: Sphingidae), acquire the plant phenolic chlorogenic acid (CA), and other caffeic acid derivatives as they feed on one of their hosts, Nicotiana attenuata L. (Solanaceae), and on artificial diet supplemented with CA. We test the hypothesis that larvae fed on CA-supplemented diet would have better resistance against bacterial infection than larvae fed on a standard CA-free diet by injecting bacteria into the hemocoel of fourth instars. Larvae fed CA-supplemented diet show significantly higher survival of infection with Enterococcus faecalis (Andrewes & Horder) Schleifer & Kilpper-Bälz, but not of infection with the more virulent Pseudomonas aeruginosa (Schroeter) Migula. Larvae fed on CA-supplemented diet possess a constitutively higher number of circulating hemocytes than larvae fed on the standard diet, but we found no other evidence of increased immune system activity, nor were larvae fed on CA-supplemented diet better able to suppress bacterial proliferation early in the infection. Thus, our data suggest an additional defensive function of CA to the direct toxic inhibition of pathogen proliferation in the gut.
chemical defense; acquired plant metabolite; immune defense; Lepidoptera; Solanaceae; Sphingidae; Nicotiana attenuata; tobacco hornworm; Enterococcus faecalis; Pseudomonas aeruginosa; chlorogenic acid
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.
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.
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.
The ability to decrypt volatile plant signals is essential if herbivorous insects are to optimize their choice of host plants for their offspring. Green leaf volatiles (GLVs) constitute a widespread group of defensive plant volatiles that convey a herbivory-specific message via their isomeric composition: feeding of the tobacco hornworm Manduca sexta converts (Z)-3- to (E)-2-GLVs thereby attracting predatory insects. Here we show that this isomer-coded message is monitored by ovipositing M. sexta females. We detected the isomeric shift in the host plant Datura wrightii and performed functional imaging in the primary olfactory center of M. sexta females with GLV structural isomers. We identified two isomer-specific regions responding to either (Z)-3- or (E)-2-hexenyl acetate. Field experiments demonstrated that ovipositing Manduca moths preferred (Z)-3-perfumed D. wrightii over (E)-2-perfumed plants. These results show that (E)-2-GLVs and/or specific (Z)-3/(E)-2-ratios provide information regarding host plant attack by conspecifics that ovipositing hawkmoths use for host plant selection.
Plants have developed a variety of strategies to defend themselves against herbivorous animals, particularly insects. In addition to mechanical defences such as thorns and spines, plants also produce compounds known as secondary metabolites that keep insects and other herbivores at bay by acting as repellents or toxins. Some of these metabolites are produced on a continuous basis by plants, whereas others—notably compounds called green-leaf volatiles—are only produced once the plant has been attacked. Green-leaf volatiles—which are also responsible for the smell of freshly cut grass—have been observed to provide plants with both direct protection, by inhibiting or repelling herbivores, and indirect protection, by attracting predators of the herbivores themselves.
The hawkmoth Manduca sexta lays its eggs on various plants, including tobacco plants and sacred Datura plants. Once the eggs have hatched into caterpillars, they start eating the leaves of their host plant, and if present in large numbers, these caterpillars can quickly defoliate and destroy it. In an effort to defend itself, the host plant releases green-leaf volatiles to attract various species of Geocoris, and these bugs eat the eggs.
One of the green-leaf volatiles released by tobacco plants is known as (Z)-3-hexenal, but enzymes released by M. sexta caterpillars change some of these molecules into (E)-2-hexenal, which has the same chemical formula but a different structure. The resulting changes in the ‘volatile profile’ alerts Geocoris bugs to the presence of M. sexta eggs and caterpillars on the plant.
Now Allmann et al. show that adult female M. sexta moths can also detect similar changes in the volatile profile emitted by sacred Datura plants that have been damaged by M. sexta caterpillars. This alerts the moths to the fact that Geocoris bugs are likely to be attacking eggs and caterpillars on the plant, or on their way to the plant, so they lay their eggs on other plants. This reduces competition for resources and also reduces the risk of newly laid eggs being eaten by predators. Allmann et al. also identified the neural mechanism that allows moths to detect changes in the volatile profile of plants—the E- and Z- odours lead to different activation patterns in the moth brain.
Manduca sexta; plant volatiles; oviposition; Ca imaging; Datura wrightii; Other
Plant fatty acid α-dioxygenases (α-DOX) are oxylipin-forming enzymes induced by biotic and abiotic stresses, which also participate in developmental processes. In Nicotiana attenuata, herbivory strongly induces the expression of an α-dox1 gene. To determine its role, we silenced its expression using Agrobacterium-mediated plant transformation with an inverted repeat construct. More than half of the transformed lines showed a severe dwarf growth phenotype that was very similar to the phenotype of tomato plants mutated at a second α-dox isoform. This led us to identify the corresponding α-dox2 gene in N. attenuata and examine the regulation of both α-dox genes as well as the consequences of their silencing in plant development and anti-herbivore defense.
The transformed lines exhibiting a dwarf growth phenotype are co-silenced for both α-dox genes resulting in a nearly complete suppression of α-DOX activity, which is associated with increases in ABA, JA and anthocyanin levels, all metabolic signatures of oxidative stress. The other lines, only silenced for α-dox1, developed similarly to wild-type plants, exhibited a 40% reduction of α-DOX activity resulting in a 50% reduction of its main product in planta (2-HOT) and showed no signs of oxidative stress. In contrast to α-dox1, the expression of α-dox2 gene is not induced by wounding or elicitors in the oral secretions of Manduca sexta. Instead, α-dox2 is expressed in roots and flowers which lack α-dox1 expression, but both genes are equally regulated during leaf maturation. We transiently silenced α-dox gene copies with gene-specific constructs using virus induced gene silencing and determined the consequences for plant development and phytohormone and 2-HOT levels. While individual silencing of α-dox1 or α-dox2 had no effects on plant growth, the co-suppression of both α-dox genes decreased plant growth. Plants transiently silenced for both α-dox genes had increased constitutive levels of JA and ABA but silencing α-dox1 alone resulted in lower M. sexta-induced levels of JA, 2-HOT and ABA.
Thus, both α-dox isoforms function in the development of N. attenuata. In leaf maturation, the two α-dox genes have overlapping functions, but only α-dox2 is involved in root and flower development and only α-dox1 functions in anti-herbivore defense.
RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant's lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata's dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene.
Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for two combinations of NaDCLs (DCL1/3/4 and DCL2/3/4) and contained higher concentrations of longer, undiced CYP6B46 dsRNA.
Both stable and transient expression of CYP6B46 dsRNA in host plants provides a specific and robust means of silencing this gene in M. sexta larvae, but the transient system is better suited for high throughput analyses. Transiently silencing NaDCLs in ir-CYP6B46 plants increased the silencing of MsCYP6B46, suggested that insect's RNAi machinery is more efficient with longer lengths of ingested dsRNA.
Pathogen infection of higher plants often induces a rapid production of phosphatic acid (PA) and changes in lipid profiles, but the enzymatic basis and the function of the lipid change in pathogen-plant interactions are not well understood.Infection of PLDβ1-deficient plants by Pseudomonas syringae pv. DC3000 resulted in less bacterial growth than in wild-type plants, and the effect was more profound in virulent Pst DC3000 than avirulent Pst DC3000 (avrRpt2) infection. The expression levels of salicylic acid (SA)-inducible genes were higher, but those inducible by jasmonic acid (JA) were lower in PLDβ1 mutants than in wild-type plants.However, PLDβ1-deficient plants were more susceptible than wild-type plants to the fungus Botrytis cinerea. The PLDβ1-deficient plants had lower levels of PA, JA and JA-related defense gene expression after B. cinerea inoculation.PLDβ1 plays a positive role in pathogen-induced JA production and plant resistance to necrotrophic fungal pathogen B. cinerea, but a negative role in the SA-dependent signaling pathway and plant tolerance to the infection of biotrophic Pst DC3000. PLDβ1 is responsible for the major part of PA increased in response to necrotrophic B. cinerea and virulent Pst DC3000 infection, but contributes less to the avirulent Pst DC3000 (avrRpt2)-induced PA production.
Botrytis cinerea; Pseudomonas syringae; Arabidopsis thaliana; phospholipase Dβ1; pathogeneses; phosphatidic acid; lysophospholipids; lipid signaling
To survive herbivore attack, plants have evolved potent mechanisms of mechanical or chemical defense that are either constitutively present or inducible after herbivore attack. Due to the costs of defense deployment, plants often regulate their biosynthesis using various transcription factors (TFs). MYC2 regulators belong to the bHLH family of transcription factors that are involved in many aspects of plant defense and development. In this study, we identified a novel MYC2 TF from N. attenuata and characterized its regulatory function using a combination of molecular, analytic and ecological methods.
The transcript and targeted metabolite analyses demonstrated that NaMYC2 is mainly involved in the regulation of the biosynthesis of nicotine and phenolamides in N. attenuata. In addition, using broadly-targeted metabolite analysis, we identified a number of other metabolite features that were regulated by NaMYC2, which, after full annotation, are expected to broaden our understanding of plant defense regulation. Unlike previous reports, the biosynthesis of jasmonates and some JA-/NaCOI1-dependent metabolites (e.g. HGL-DTGs) were not strongly regulated by NaMYC2, suggesting the involvement of other independent regulators. No significant differences were observed in the performance of M. sexta on MYC2-silenced plants, consistent with the well-known ability of this specialist insect to tolerate nicotine.
By regulating the biosynthesis of nicotine, NaMYC2 is likely to enhance plant resistance against non-adapted herbivores and contribute to plant fitness; however, multiple JA/NaCOI1-dependent mechanisms (perhaps involving other MYCs) that regulate separate defense responses are likely to exist in N. attenuata. The considerable variation observed amongst different plant families in the responses regulated by jasmonate signaling highlights the sophistication with which plants craft highly specific and fine-tuned responses against the herbivores that attack them.
bHLH; Manduca sexta; MYC2; Transcription factors; Nicotiana attenuata; Nicotine; Phenolamides; Plant-insect interactions; Transcriptional regulation
Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata), multiplication in the intercellular space (apoplast) of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR) is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst) DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord) mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA)-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA), and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA) biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC) protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to implicate an important role of stress-associated protein translation in stomatal guard cell signaling in response to microbe-associated molecular patterns and bacterial infection.
Pathogen entry into host tissue is a critical first step in causing infection. For foliar bacterial plant pathogens, natural surface openings, such as stomata, are important entry sites into the leaf apoplast (internal intercellular spaces). Recent studies have shown that plants respond to surface-inoculated bacterial pathogens by reducing stomatal aperture as part of the innate immune response to restrict bacterial invasion. Once inside plant tissue, bacteria encounter defenses in the apoplast. To counter host defenses during invasion and in the apoplast, bacterial pathogens produce a variety of virulence factors, such as the polyketide toxin coronatine produced by Pseudomonas syringae pv. tomato (Pst) DC3000. Coronatine-deficient Pst DC3000 mutants are compromised in virulence, especially when inoculated onto the plant surface. In this study, we conducted a random genetic screen to identify Arabidopsis mutants that could rescue the virulence of coronatine-deficient mutant bacteria and obtained three classes of Arabidopsis mutants: those that are defective in stomatal closure only, those defective in apoplastic defense only, and those compromised in both stomatal closure and apoplastic defenses. The isolation of these host mutants highlight the important role of COR, a molecular mimic of the plant hormone jasmonate, in overcoming both stomatal and apoplastic defenses during Pst DC3000 infection.
Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown.
We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC N-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the M. sexta-N. attenuata interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated ≥ 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the M. sexta-N. attenuata interaction.
The analysis of the early changes in the transcriptome of N. attenuata after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process.
Plants have evolved an array of constitutive and inducible defense strategies to restrict pathogen ingress. However, some pathogens still manage to invade plants and impair growth and productivity. Previous studies have revealed several key regulators of defense responses, and efforts have been made to use this information to develop disease resistant crop plants. These efforts are often hampered by the complexity of defense signaling pathways. To further elucidate the complexity of defense responses, we screened a population of T-DNA mutants in Colombia-0 background that displayed altered defense responses to virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000).
In this study, we demonstrated that the Arabidopsis Purple Acid Phosphatse5 (PAP5) gene, induced under prolonged phosphate (Pi) starvation, is required for maintaining basal resistance to certain pathogens. The expression of PAP5 was distinctly induced only under prolonged Pi starvation and during the early stage of Pst DC3000 infection (6 h.p.i). T-DNA tagged mutant pap5 displayed enhanced susceptibility to the virulent bacterial pathogen Pst DC3000. The pap5 mutation greatly reduced the expression of pathogen inducible gene PR1 compared to wild-type plants. Similarly, other defense related genes including ICS1 and PDF1.2 were impaired in pap5 plants. Moreover, application of BTH (an analog of SA) restored PR1 expression in pap5 plants.
Taken together, our results demonstrate the requirement of PAP5 for maintaining basal resistance against Pst DC3000. Furthermore, our results provide evidence that PAP5 acts upstream of SA accumulation to regulate the expression of other defense responsive genes. We also provide the first experimental evidence indicating the role PAP5 in plant defense responses.
Arabidopsis; Plant defense responses; PAP5; Pseudomonas syringae; Phosphate starvation
To defend themselves, plants activate inducible defense mechanisms that are effective against the invader that is encountered. There is partial overlap in the defense signaling pathways that are induced by insect herbivores and microbial pathogens that may result in cross-resistance. We have previously shown that infestation by tissue-chewing Pieris rapae larvae induces resistance in Arabidopsis thaliana against subsequent attack by the microbial pathogens Pseudomonas syringae pv. tomato (Pst), Xanthomonas campestris pv. armoraciae (Xca) and turnip crinkle virus (TCV). Phloem-feeding aphids, such as the generalist Myzus persicae, have a stealthy feeding strategy that is very different from chewing by lepidopteran larvae. Yet, M. persicae feeding results in a large transcriptomic change. Here, we report on the effectiveness of the defense response that is triggered by M. persicae infestation, as well as the sensitivity of M. persicae to microbially-induced resistance. M. persicae reproduction was not affected by prior conspecific feeding, nor was aphid-induced resistance effective against subsequent attack by Pst, Xca or TCV. Moreover, induced systemic resistance (ISR) triggered by beneficial Pseudomonas fluorescens rhizobacteria was not effective against M. persicae. However, systemic acquired resistance (SAR) induced by prior infection with avirulent Pst was associated with reduced aphid reproduction. These data provide insight into the effectiveness of pathogen and insect resistance and highlight the complexity of the defense responses that are triggered during multitrophic plant-attacker interactions.
Arabidopsis; induced resistance; defense signaling; Myzus persicae
The SR/CAMTA proteins represent a small family of transcription activators that play important roles in plant responses to biotic and abiotic stresses. Seven SlSR/CAMTA genes were identified in tomato as tomato counterparts of SR/CAMTA; however, the involvement of SlSRs/CAMTAs in biotic and abiotic stress responses is not clear. In this study, we performed functional analysis of the SlSR/CAMTA family for their possible functions in defense response against pathogens and tolerance to drought stress.
Expression of SlSRs was induced with distinct patterns by Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000. Virus-induced gene silencing (VIGS)-based knockdown of either SlSR1 or SlSR3L in tomato resulted in enhanced resistance to B. cinerea and Pst DC3000 and led to constitutive accumulation of H2O2, elevated expression of defense genes, marker genes for pathogen-associated molecular pattern-triggered immunity, and regulatory genes involved in the salicylic acid- and ethylene-mediated signaling pathways. Furthermore, the expression of SlSR1L and SlSR2L in detached leaves and whole plants was significantly induced by drought stress. Silencing of SlSR1L led to decreased drought stress tolerance, accelerated water loss in leaves, reduced root biomass and attenuated expression of drought stress responsive genes in tomato. The SlSR1 and SlSR3L proteins were localized in the nucleus of plant cells when transiently expressed in Nicotiana benthamiana and had transcriptional activation activity in yeast.
VIGS-based functional analyses demonstrate that both SlSR1 and SlSR3L in the tomato SlSR/CAMTA family are negative regulators of defense response against B. cinerea and Pst DC3000 while SlSR1L is a positive regulator of drought stress tolerance in tomato.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0286-3) contains supplementary material, which is available to authorized users.
Tomato (Solanum lycopersicum); SR/CAMTA; Disease resistance response; Drought stress