Persistent lipedema pain in patients after bariatric surgery: a case series of 13 patients.

BACKGROUND: Lipedema often remains undiagnosed in patients with obesity, leading to mismanagement of treatment. Because of this, despite remarkable weight loss after bariatric surgery and decreases in hip and abdomen circumference, some patients show only small decreases in circumference of the extremities and report persistent limb pain. OBJECTIVE: The goal of this work is to raise awareness of lipedema coincident with obesity, mistakenly diagnosed as obesity alone, in order to ensure the correct diagnosis of the condition and to achieve better treatment outcomes for people with lipedema and coincident obesity. SETTING: CG Lympha Clinic, Cologne, and Ernst von Bergmann Clinic, Potsdam. METHODS: From clinical records, we identified 13 patients who were diagnosed with lipedema only after undergoing bariatric surgery. We describe the course of their pain before and after bariatric surgery, focusing on the long-term progression of symptoms accompanying the disease. RESULTS: Lipedema cannot be cured by bariatric surgery, and although the patients in this study lost an average of more than 50 kg of weight, they displayed no improvement in the pain symptoms typical of lipedema. CONCLUSIONS: Because of the different etiologies of lipedema and obesity, lipedema requires its own specific treatment. Patients suffering from obesity should always be assessed for pain and lipedema. If coincident lipedema is diagnosed, we suggest that bariatric surgery only be performed first if diet and exercise have failed, the patient's body mass index is >40 kg/m2, and the patient has been informed of the possible persistence of pain. Lipedema, like a coincident disease, must be additionally treated conservatively or preferably surgically. This optimized treatment may help to better manage patient expectations after weight loss.

The Addition of Hyaluronidase to Tumescent Local Anesthesia Supports Lymphological Liposculpture of Secondary Lymphedema.

Proteoglycans (PG) are essential for regulating water flow in the interstitium. From stage 1 of lymphostasis, there is an accumulation of interstitial PG, which regulate the increasing fluids. As the disease progresses, more PG are formed than degraded, resulting in proliferation, and increases in circumference and volume of solid tissue. The removal of this subcutaneous tissue, which is very rigid due to cross-linked PG, is a particular challenge in lymphedema surgery. Hyaluronidase has a lytic effect on these PG structures and, after subcutaneous infiltration, reduces the viscosity of the extracellular matrix, promoting diffusion and penetration of solutions into the surrounding tissue. By using hyaluronidase in our vascular-sparing surgical protocol (lymphological liposculpture), we have not observed any lymphedema recurrences even after 15 years.

The host jasmonic acid pathway regulates the transcriptomic changes of dodder and host plant under the scenario of caterpillar feeding on dodder.

BACKGROUND: Dodder (Cuscuta spp., Convolvulaceae) species are obligate leaf- and rootless parasites that totally depend on hosts to survive. Dodders naturally graft themselves to host stems to form vascular fusion, from which they obtain nutrients and water. In addition, dodders and their hosts also exchange various other molecules, including proteins, mRNAs, and small RNAs. It is very likely that vascular fusion also allows inter-plant translocation of systemic signals between dodders and host plants and these systemic signals may have profound impacts on the physiology of dodder and host plants. Herbivory is a common biotic stress for plants. When a dodder parasite is attacked by lepidopteran insects, how dodder responds to caterpillar feeding and whether there are inter-plant communications between the host plants and the parasites is still poorly understood. RESULTS: Here, wild-type (WT) tobacco and a tobacco line in which jasmonic acid (JA) biosynthesis was silenced (AOC-RNAi) were used as the hosts, and the responses of dodders and their host plants to herbivory by Spodoptera litura caterpillars on the dodders were investigated. It was found that after caterpillar attack, dodders grown on AOC-RNAi tobacco showed much a smaller number of differentially expressed genes, although the genotypes of the tobacco plants did not have an effect on the simulated S. litura feeding-induced JA accumulation in dodders. We further show that S. litura herbivory on dodder also led to large changes in transcriptome and defensive metabolites in the host tobacco, leading to enhanced resistance to S. litura, and the JA pathway of tobacco host is critical for these systemic responses. CONCLUSIONS: Our findings indicate that during caterpillar attack on dodder, the JA pathway of host plant is required for the proper transcriptomic responses of both dodder and host plants. This study highlights the importance of the host JA pathway in regulating the inter-plant systemic signaling between dodder and hosts.

Ultraviolet-B enhances the resistance of multiple plant species to lepidopteran insect herbivory through the jasmonic acid pathway.

Land plants protect themselves from ultraviolet-B (UV-B) by accumulating UV-absorbing metabolites, which may also function as anti-insect toxins. Previous studies have shown that UV-B enhances the resistance of different plant species to pierce-sucking pests; however, whether and how UV-B influences plant defense against chewing caterpillars are not well understood. Here we show that UV-B treatment increased Spodoptera litura herbivory-induced jasmonic acid (JA) production in Arabidopsis and thereby Arabidopsis exhibited elevated resistance to S. litura. Using mutants impaired in the biosynthesis of JA and the defensive metabolites glucosinolates (GSs), we show that the UV-B-induced resistance to S. litura is dependent on the JA-regulated GSs and an unidentified anti-insect metabolite(s). Similarly, UV-B treatment also enhanced the levels of JA-isoleucine conjugate and defense-related secondary metabolites in tobacco, rice, and maize after these plants were treated with simulated herbivory of lepidopteran insects; consistently, these plants showed elevated resistance to insect larvae. Using transgenic plants impaired in JA biosynthesis or signaling, we further demonstrate that the UV-B-enhanced defense responses also require the JA pathway in tobacco and rice. Our findings reveal a likely conserved JA-dependent mechanism by which UV-B enhances plant defense against lepidopteran insects.

Elevated CO2 differentially affects tobacco and rice defense against lepidopteran larvae via the jasmonic acid signaling pathway.

Atmospheric CO2 levels are rapidly increasing due to human activities. However, the effects of elevated CO2 (ECO2 ) on plant defense against insects and the underlying mechanisms remain poorly understood. Here we show that ECO2 increased the photosynthetic rates and the biomass of tobacco and rice plants, and the chewing lepidopteran insects Spodoptera litura and Mythimna separata gained less and more mass on tobacco and rice plants, respectively. Consistently, under ECO2 , the levels of jasmonic acid (JA), the main phytohormone controlling plant defense against these lepidopteran insects, as well as the main defense-related metabolites, were increased and decreased in insect-damaged tobacco and rice plants. Importantly, bioassays and quantification of defense-related metabolites in tobacco and rice silenced in JA biosynthesis and perception indicate that ECO2 changes plant resistance mainly by affecting the JA pathway. We further demonstrate that the defensive metabolites, but not total N or protein, are the main factors contributing to the altered defense levels under ECO2 . This study illustrates that ECO2 changes the interplay between plants and insects, and we propose that crops should be studied for their resistance to the major pests under ECO2 to predict the impact of ECO2 on future agroecosystems.

COI1-Regulated Hydroxylation of Jasmonoyl-L-isoleucine Impairs Nicotiana attenuata's Resistance to the Generalist Herbivore Spodoptera litura.

The phytohormone jasmonoyl-L-isoleucine (JA-Ile) is well-known as the key signaling molecule that elicits plant defense responses after insect herbivory. Oxidation, which is catalyzed by the cytochrome P450s of the CYP94 family, is thought to be one of the main catabolic pathways of JA-Ile. In this study, we identified four CYP94B3 homologues in the wild tobacco plant Nicotiana attenuata. Individually silencing the four homologues revealed that NaCYP94B3 like-1 and NaCYP94B3 like-2, but not NaCYP94B3 like-3 and NaCYP94B3 like-4, are involved in the C-12-hydroxylation of JA-Ile. Simultaneously silencing three of the NaCYP94B3 like genes, NaCYP94B3 like-1, -2, and -4, in the VIGS-NaCYP94B3s plants doubled herbivory-induced JA-Ile levels and greatly enhanced plant resistance to the generalist insect herbivore, Spodoptera litura. The poor larval performance was strongly correlated with the high concentrations of several JA-Ile-dependent direct defense metabolites in VIGS-NaCYP94B3s plants. Furthermore, we show that the abundance of 12-hydroxy-JA-Ile was dependent on JA-Ile levels as well as COI1, the receptor of JA-Ile. COI1 appeared to transcriptionally control NaCYP94B3 like-1 and -2 and thus regulates the catabolism of its own ligand molecule, JA-Ile. These results highlight the important role of JA-Ile degradation in jasmonate homeostasis and provide new insight into the feedback regulation of JA-Ile catabolism. Given that silencing these CYP94 genes did not detectably alter plant growth and highly increased plant defense levels, we propose that CYP94B3 genes can be potential targets for genetic improvement of herbivore-resistant crops.

Fatty acid-amino acid conjugates are essential for systemic activation of salicylic acid-induced protein kinase and accumulation of jasmonic acid in Nicotiana attenuata.

BACKGROUND: Herbivory induces the activation of mitogen-activated protein kinases (MAPKs), the accumulation of jasmonates and defensive metabolites in damaged leaves and in distal undamaged leaves. Previous studies mainly focused on individual responses and a limited number of systemic leaves, and more research is needed for a better understanding of how different plant parts respond to herbivory. In the wild tobacco Nicotiana attenuata, FACs (fatty acid-amino acid conjugates) in Manduca sexta oral secretions (OS) are the major elicitors that induce herbivory-specific signaling but their role in systemic signaling is largely unknown. RESULTS: Here, we show that simulated herbivory (adding M. sexta OS to fresh wounds) dramatically increased SIPK (salicylic acid-induced protein kinase) activity and jasmonic acid (JA) levels in damaged leaves and in certain (but not all) undamaged systemic leaves, whereas wounding alone had no detectable systemic effects; importantly, FACs and wounding are both required for activating these systemic responses. In contrast to the activation of SIPK and elevation of JA in specific systemic leaves, increases in the activity of an important anti-herbivore defense, trypsin proteinase inhibitor (TPI), were observed in all systemic leaves after simulated herbivory, suggesting that systemic TPI induction does not require SIPK activation and JA increases. Leaf ablation experiments demonstrated that within 10 minutes after simulated herbivory, a signal (or signals) was produced and transported out of the treated leaves, and subsequently activated systemic responses. CONCLUSIONS: Our results reveal that N. attenuata specifically recognizes herbivore-derived FACs in damaged leaves and rapidly send out a long-distance signal to phylotactically connected leaves to activate MAPK and JA signaling, and we propose that FACs that penetrated into wounds rapidly induce the production of another long-distance signal(s) which travels to all systemic leaves and activates TPI defense.

Virus-induced gene silencing in plant MAPK research.

Virus-induced gene silencing (VIGS) technology has become more and more widely used in various plant species for rapid screening of gene functions. VIGS does not require time-consuming tissue culture steps that are needed for stable transformation in most plant species and it can be used for studying gene function even in plants that are very difficult to stably transform. Furthermore, VIGS technology provides high gene silencing efficiency (up to 95 %) and specificity. Here, we describe a VIGS protocol that can be used for studying the functions of MAPKs and other genes in a wild tobacco species, Nicotiana attenuata. This method is also suitable for other Nicotiana species and tomato with minor modifications.

Scopoletin is a phytoalexin against Alternaria alternata in wild tobacco dependent on jasmonate signalling.

Alternaria alternata (tobacco pathotype) is a necrotrophic fungus causing severe losses in Nicotiana species by infection of mature leaves. Similar to what has been observed in cultivated tobacco, N. tabacum, young leaves of wild tobacco, N. attenuata, were more resistant to A. alternata than mature leaves, and this was correlated with stronger blue fluorescence induced after infection. However, the nature of the fluorescence-emitting compound, its role in defence, and its regulation were not clear. Silencing feruloyl-CoA 6'-hydroxylase 1 (F6'H1), the gene encoding the key enzyme for scopoletin biosynthesis, by virus-induced gene silencing (VIGS) revealed that the blue fluorescence was mainly emitted by scopoletin and its ß-glycoside form, scopolin. Further analysis showed that scopoletin exhibited strong antifungal activity against A. alternata in vitro and in vivo. Importantly, jasmonic acid (JA) levels were highly elicited in young leaves but much less in mature leaves after infection; and fungus-elicited scopoletin was absent in JA-deficient plants, but was largely restored with methyl jasmonate treatments. Consistent with this, plants strongly impaired in JA biosynthesis and perception were highly susceptible to A. alternata in the same way scopoletin/scopolin-depleted VIGS F6'H1 plants. Furthermore, silencing MYC2, a master regulator of most JA responses, reduced A. alternata-induced NaF6'H1 transcripts and scopoletin. Thus, it is concluded that JA signalling is activated in N. attenuata leaves after infection, which subsequently regulates scopoletin biosynthesis for the defence against A. alternata partly through MYC2, and higher levels of scopoletin accumulated in young leaves account for their strong resistance.

Nicotiana attenuata MPK4 suppresses a novel jasmonic acid (JA) signaling-independent defense pathway against the specialist insect Manduca sexta, but is not required for the resistance to the generalist Spodoptera littoralis.

How plants tailor their defense responses to attack from different insects remains largely unknown. Here, we studied the role of a mitogen-activated protein kinase (MAPK), MPK4, in the resistance of a wild tobacco Nicotiana attenuata to two herbivores, the specialist Manduca sexta and the generalist Spodoptera littoralis. Stably transformed N. attenuata plants silenced in MPK4 (irMPK4) were generated and characterized for traits important for defense against herbivores. Only the oral secretions (OS) from M. sexta, but not the OS from S. littoralis or mechanical wounding, induced elevated levels of jasmonic acid (JA) in irMPK4 plants relative to the wild-type plants. Moreover, silencing of MPK4 strongly increased the resistance of N. attenuata to M. sexta in a fashion that was independent of COI1 (CORONATINE INSENSITIVE1)-mediated JA signaling. Untargeted metabolomic screening identified several new MPK4-dependent putative defensive compounds against M. sexta. By contrast, silencing of MPK4 did not affect the growth of the generalist insect S. littoralis, and we propose that this was because of the very low levels of fatty acid-amino acid conjugates (FACs) in S. littoralis OS. Thus, MPK4 is likely to be a key signaling element that enables plants to tailor defense responses to different attackers.

The use of VIGS technology to study plant-herbivore interactions.

Plants employ a large variety of defense strategies to resist herbivores, which require transcriptional reprogramming of cells and profound changes in plant metabolism. Due to the large number of genes involved in defense processes, rapid screening strategies are essential for elucidating the contributions of individual genes in the responses of plants to herbivory. However, databases and seed banks of mutant plants which allow rapid retrieval of mutant genotypes are limited to a few model plant species, namely, Arabidopsis thaliana and Oryza sativa (rice). In other plants, virus-induced gene silencing (VIGS) offers an efficient alternative for screening the functions of individual genes in order to prioritize the allocations of the large time investments required to establish stably transformed RNAi-silenced lines. With VIGS, it is usually possible to achieve strong, specific silencing of target genes in the ecological models Nicotiana attenuata and Solanum nigrum, allowing the rapid assessment of gene silencing effects on phytohormone accumulation, signal transduction and accumulation of defense metabolites. VIGS plants are also useful in bioassays with specialist and generalist herbivores, allowing direct verification of gene function in plant resistance to herbivores.

High levels of jasmonic acid antagonize the biosynthesis of gibberellins and inhibit the growth of Nicotiana attenuata stems.

Hormones play pivotal roles in regulating plant development, growth, and stress responses, and cross-talk among different hormones fine-tunes various aspects of plant physiology. Jasmonic acid (JA) is important for plant defense against herbivores and necrotic fungi and also regulates flower development; in addition, Arabidopsis mutants over-producing JA usually have stunted stems and wound-induced jasmonates suppress Arabidopsis growth, suggesting that JA is also involved in stem elongation. Gibberellins (GAs) promote stem and leaf growth and modulate seed germination, flowering time, and the development of flowers, fruits, and seeds. However, little is known about the interaction between the JA and GA pathways. Two calcium-dependent protein kinases, CDPK4 and CDPK5, are important suppressors of JA accumulation in a wild tobacco species, Nicotiana attenuata. The stems of N. attenuata silenced in CDPK4 and CDPK5 (irCDPK4/5 plants) had dramatically increased levels of JA and exhibited stunted elongation and had very high contents of secondary metabolites. Genetic analysis indicated that the high JA levels in irCDPK4/5 stems accounted for the suppressed stem elongation and the accumulation of secondary metabolites. Supplementation of GA(3) to irCDPK4/5 plants largely restored normal stem growth to wild-type levels. Measures of GA levels indicated that over-accumulation of JA in irCDPK4/5 stems inhibited the biosynthesis of GAs. Finally, we show that JA antagonizes GA biosynthesis by strongly inhibiting the transcript accumulation of GA20ox and possibly GA13ox, the key genes in GA production, demonstrating that high JA levels antagonize GA biosynthesis in stems.

Calcium-dependent protein kinases, CDPK4 and CDPK5, affect early steps of jasmonic acid biosynthesis in Nicotiana attenuata.

Calcium-dependent protein kinases (CDPKs) modulate plant development and growth and are important regulators of biotic and abiotic stress responses. Recently it was found that simultaneously silencing Nicotiana attenuata NaCDPK4 and NaCDPK5 (IRcdpk4/5 plants) results in accumulation of exceptionally high JA levels after wounding or simulated herbivory treatments, which in turn induced high levels of defense metabolites that slowed the growth of Manduca sexta, a specialist insect herbivore. To investigate the mechanism by which NaCDPK4 and NaCDPK5 regulate JA accumulation, we analyzed the transcript levels of all important enzymes involved in JA biosynthesis, but these genes showed no differences between wild-type and IRcdpk4/5 plants. Moreover, the dynamics of JA were similar between these plants, excluding the possibility of decreased degradation rates in IRcdpk4/5 plants. To gain insight into the mechanism by which NaCDPK4 and NaCDPK5 regulate JA biosynthesis, free fatty acids, including C18:3, and (9S,13S)-12-oxo-phytodienoic acid (OPDA), two important precursors of JA were quantified at different times before and after wounding and simulated herbivore feeding treatments. We show that after these treatments, IRcdpk4/5 plants have decreased levels of C18:3, but have enhanced OPDA and JA levels, suggesting that NaCDPK4 and NaCDPK5 have a role in the early steps of JA biosynthesis. The possible role of NaCDPK4 and NaCDPK5 regulating AOS and AOC enzymatic activity is discussed.

Silencing Nicotiana attenuata calcium-dependent protein kinases, CDPK4 and CDPK5, strongly up-regulates wound- and herbivory-induced jasmonic acid accumulations.

The plant hormone jasmonic acid (JA) plays a pivotal role in plant-insect interactions. Herbivore attack usually elicits dramatic increases in JA concentrations, which in turn activate the accumulation of metabolites that function as defenses against herbivores. Although almost all enzymes involved in the biosynthesis pathway of JA have been identified and characterized, the mechanism by which plants regulate JA biosynthesis remains unclear. Calcium-dependent protein kinases (CDPKs) are plant-specific proteins that sense changes in [Ca(2+)] to activate downstream responses. We created transgenic Nicotiana attenuata plants, in which two CDPKs, NaCDPK4 and NaCDPK5, were simultaneously silenced (IRcdpk4/5 plants). IRcdpk4/5 plants were stunted and aborted most of their flower primordia. Importantly, after wounding or simulated herbivory, IRcdpk4/5 plants accumulated exceptionally high JA levels. When NaCDPK4 and NaCDPK5 were silenced individually, neither stunted growth nor high JA levels were observed, suggesting that NaCDPK4 and NaCDPK5 have redundant roles. Attack from Manduca sexta larvae on IRcdpk4/5 plants induced high levels of defense metabolites that slowed M. sexta growth. We found that NaCDPK4 and NaCDPK5 affect plant resistance against insects in a JA- and JA-signaling-dependent manner. Furthermore, IRcdpk4/5 plants showed overactivation of salicylic-acid-induced protein kinase, a mitogen-activated protein kinase involved in various stress responses, and genetic analysis indicated that the increased salicylic-acid-induced protein kinase activity in IRcdpk4/5 plants was a consequence of the exceptionally high JA levels and was dependent on CORONATINE INSENSITIVE1. This work reveals the critical roles of CDPKs in modulating JA homeostasis and highlights the complex duet between JA and mitogen-activated protein kinase signaling.

Silencing MPK4 in Nicotiana attenuata enhances photosynthesis and seed production but compromises abscisic acid-induced stomatal closure and guard cell-mediated resistance to Pseudomonas syringae pv tomato DC3000.

Mitogen-activated protein kinases (MAPKs) play pivotal roles in development and environmental interactions in eukaryotes. Here, we studied the function of a MAPK, NaMPK4, in the wild tobacco species Nicotiana attenuata. The NaMPK4-silenced N. attenuata (irNaMPK4) attained somewhat smaller stature, delayed senescence, and greatly enhanced stomatal conductance and photosynthetic rate, especially during late developmental stages. All these changes were associated with highly increased seed production. Using leaf epidermal peels, we demonstrate that guard cell closure in irNaMPK4 was strongly impaired in response to abscisic acid and hydrogen peroxide, and consistently, irNaMPK4 plants transpired more water and wilted sooner than did wild-type plants when they were deprived of water. We show that NaMPK4 plays an important role in the guard cell-mediated defense against a surface-deposited bacterial pathogen, Pseudomonas syringae pv tomato (Pst) DC3000; in contrast, when bacteria directly entered leaves by pressure infiltration, NaMPK4 was found to be less important in the resistance to apoplast-located Pst DC3000. Moreover, we show that salicylic acid was not involved in the defense against PstDC3000 in wild-type and irNaMPK4 plants once it had entered leaf tissue. Finally, we provide evidence that NaMPK4 functions differently from AtMPK4 and AtMPK11 in Arabidopsis (Arabidopsis thaliana), despite their sequence similarities, suggesting a complex functional divergence of MAPKs in different plant lineages. This work highlights the multifaceted functions of NaMPK4 in guard cells and underscores its role in mediating various ecologically important traits.

Nicotiana attenuata LECTIN RECEPTOR KINASE1 suppresses the insect-mediated inhibition of induced defense responses during Manduca sexta herbivory.

Nicotiana attenuata has the capacity to respond specifically to herbivory by its natural herbivore, Manduca sexta, through the perception of elicitors in larval oral secretions. We demonstrate that Lectin receptor kinase 1 (LecRK1) functions during M. sexta herbivory to suppress the insect-mediated inhibition of jasmonic acid (JA)-induced defense responses. Gene function analysis performed by reducing LecRK1 expression in N. attenuata by both virus-induced gene silencing and inverted repeated RNA interference (ir-lecRK1 plants) revealed that LecRK1 was essential to mount a full defense response against M. sexta folivory; larvae growing on ir-lecRK1 plants were 40 to 100% larger than those growing on wild-type plants. The insect-induced accumulation of nicotine, diterpene-glucosides, and trypsin protease inhibitors, as well as the expression of Thr deaminase, was severalfold reduced in ir-lecRK1 plants compared with the wild type. The accumulation of JA and JA-Ile was unaffected during herbivory in ir-lecRK1 plants; however, salicylic acid (SA) accumulation was increased by twofold. The expression of nahG in ir-lecRK1 plants prevented the increased accumulation of SA and restored the defense response against M. sexta herbivory. The results suggest that LecRK1 inhibits the accumulation of SA during herbivory, although other mechanisms may also be affected.

The multifaceted function of BAK1/SERK3: plant immunity to pathogens and responses to insect herbivores.

Almost a decade ago BRI1-associated kinase 1 (BAK1) was identified as a co-receptor of brassinosteroid (BR) insensitive 1 (BRI1), the receptor for BRs, which plays an essential role in transducing BR signaling to regulate plant development. BAK1 is also critical in resistance to various pathogens. BAK1 rapidly binds to certain receptors for pathogen/microbeassociated molecular patterns (PAMPs/MAMPs) after the perception of pathogen elicitors and is required for the full elicitation of pathogen-induced defense responses, such as the activation of the mitogen-activated protein kinase 6 (MPK6) and production of reactive oxygen species. Thus, BAK1 functions in both BR signaling and PAMP-triggered immunity (PTI). Recently BAK1 was also found to play an important role in mediating defense responses against an insect herbivore (Manduca sexta) of Nicotiana attenuata. In this interaction, BAK1 positively modulates wound- or herbivore feeding induced accumulation of jasmonic acid (JA) and JA-isoleucine (JA-Ile). This mini-review summarizes recent advances in our understanding of the functions of BAK1 in resistance to pathogens and herbivores.

BAK1 regulates the accumulation of jasmonic acid and the levels of trypsin proteinase inhibitors in Nicotiana attenuata's responses to herbivory.

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.

A comparison of two Nicotiana attenuata accessions reveals large differences in signaling induced by oral secretions of the specialist herbivore Manduca sexta.

Genetic variation within and among populations provides the raw material for evolution. Although many studies describe inter- and intraspecific variation of defensive metabolites, little is known about variation among plant populations within early signaling responses elicited by herbivory or by herbivore oral secretions (OS) introduced into wounds during feeding. In this study, we compare the OS-elicited early responses as well as the antiherbivore defensive metabolites in two accessions of the wild tobacco Nicotiana attenuata and show that, compared with an accession collected from Utah, an Arizona accession has lower herbivore-elicited activity of the salicylic acid-induced protein kinase, an important mitogen-activated protein kinase involved in herbivore resistance. These differences in salicylic acid-induced protein kinase activity were associated with substantially different levels of OS-elicited jasmonic acid, jasmonic acid-isoleucine conjugate, and ethylene bursts. Gene expression level polymorphism (ELP) determines phenotypic variation among populations, and we found the two accessions to have significantly different ELPs in the genes involved in early signaling responses to herbivory. In addition, we found differences between the Utah and the Arizona accessions in the concentrations of several secondary metabolites that contribute to N. attenuata's direct and indirect defenses. This study demonstrates significant natural variation in regulatory elements that mediate plant responses to herbivore attack, highlighting the role of ELP in producing a diversity of plant defense phenotypes.

Silencing geranylgeranyl diphosphate synthase in Nicotiana attenuata dramatically impairs resistance to tobacco hornworm.

In bioassays with artificial diets, the 17-hydroxygeranyllinalool diterpenoid glycosides (HGL-DTGs) of Nicotiana attenuata function as antifeedants for the plant's adapted herbivore, tobacco hornworm (Manduca sexta). To determine whether HGL-DTGs have a defensive function in planta, we suppressed HGL-DTG production by silencing the source of the geranylgeranyl diphosphates (GGPPs) required for geranyllinalool biosynthesis, a key intermediate. We used virus-induced gene silencing to suppress transcript levels of GGPP synthase gene (Naggpps) and farnesyl diphosphate (FPP) synthase gene (Nafpps), northern blotting and real-time polymerase chain reaction to quantify transcript accumulations, and radio gas chromatography to analyze prenyltransferase specificity. Silencing Nafpps had no effect on the accumulation of HGL-DTGs but decreased leaf steroid content, demonstrating that DTG-synthesizing enzymes do not use GGPP derived from FPP and confirming FPP's role as a steroid precursor. Unlike plants silenced in the phytoene desaturase gene (Napds), which rapidly bleached, Naggpps-silenced plants had reduced HGL-DTG but not carotenoids or chlorophyll contents, demonstrating that Naggpps supplies substrates for GGPP biosynthesis for HGL-DTGs, but not for phytoene or phytol. Expression of Naggpps in Escherichia coli revealed that the recombinant protein catalyzes the GGPP synthesis from isopentenyl diphosphate and dimethylallyl diphosphate. When fed on silenced plants, hornworm larvae gained up to 3 times more mass than those that fed on empty vector control plants or plants silenced in Nafpps, the trypsin protease inhibitor gene, or the putrescine N-methyltransferase gene. We conclude that HGL-DTGs or other minor undetected diterpenoids derived from GGPP function as direct defenses for N. attenuata and are more potent than nicotine or trypsin protease inhibitors against attack by hornworm larvae.