Tobacco Hornworm (Manduca sexta) Oral Secretion Elicits Reactive Oxygen Species in Isolated Tomato Protoplasts.

Plants are under constant attack by a suite of insect herbivores. Over millions of years of coexistence, plants have evolved the ability to sense insect feeding via herbivore-associated elicitors in oral secretions, which can mobilize defense responses. However, herbivore-associated elicitors and the intrinsic downstream modulator of such interactions remain less understood. In this study, we show that tobacco hornworm caterpillar (Manduca sexta) oral secretion (OS) induces reactive oxygen species (ROS) in tomato (Solanum lycopersicum) protoplasts. By using a dye-based ROS imaging approach, our study shows that application of plant-fed (PF) M. sexta OS generates significantly higher ROS while artificial diet-fed (DF) caterpillar OS failed to induce ROS in isolated tomato protoplasts. Elevation in ROS generation was saturated after ~140 s of PF OS application. ROS production was also suppressed in the presence of an antioxidant NAC (N-acetyl-L-cysteine). Interestingly, PF OS-induced ROS increase was abolished in the presence of a Ca2+ chelator, BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). These results indicate a potential signaling cascade involving herbivore-associated elicitors, Ca2+, and ROS in plants during insect feeding. In summary, our results demonstrate that plants incorporate a variety of independent signals connected with their herbivores to regulate and mount their defense responses.

Tomato PEPR1 ORTHOLOG RECEPTOR-LIKE KINASE1 Regulates Responses to Systemin, Necrotrophic Fungi, and Insect Herbivory.

Endogenous peptides regulate plant immunity and growth. Systemin, a peptide specific to the Solanaceae, is known for its functions in plant responses to insect herbivory and pathogen infections. Here, we describe the identification of the tomato (Solanum lycopersicum) PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the TOMATO PROTEIN KINASE1b (TPK1b) interacting protein and demonstrate its biological functions in systemin signaling and tomato immune responses. Tomato PORK1 RNA interference (RNAi) plants with significantly reduced PORK1 expression showed increased susceptibility to tobacco hornworm (Manduca sexta), reduced seedling growth sensitivity to the systemin peptide, and compromised systemin-mediated resistance to Botrytis cinerea. Systemin-induced expression of Proteinase Inhibitor II (PI-II), a classical marker for systemin signaling, was abrogated in PORK1 RNAi plants. Similarly, in response to systemin and wounding, the expression of jasmonate pathway genes was attenuated in PORK1 RNAi plants. TPK1b, a key regulator of tomato defense against B. cinerea and M. sexta, was phosphorylated by PORK1. Interestingly, wounding- and systemin-induced phosphorylation of TPK1b was attenuated when PORK1 expression was suppressed. Our data suggest that resistance to B. cinerea and M. sexta is dependent on PORK1-mediated responses to systemin and subsequent phosphorylation of TPK1b. Altogether, PORK1 regulates tomato systemin, wounding, and immune responses.

Over-expression of StZFP2 in Solanum tuberosum L. var. Kennebec (potato) inhibits growth of Tobacco Hornworm larvae (THW, Manduca sexta L.).

Tobacco hornworm (Manduca sexta, THW) is a voracious pest of tomato and potato. StZFP2 is a Q-type C2H2 zinc finger transcription factor (TF) that is induced upon wounding and infestation. Previous work has shown that Q-type C2H2 TFs are involved in stress responses and when over expressed, can enhance protection against drought, salinity or pathogen infection. Twelve transgenic lines (S1-S12) were tested that over-express StZFP2. Feeding S6 or S8 to THW significantly lowered larval weight (21-37%) as well as increased expression of StPIN2 in comparison to untransformed Kennebec. The increase in StPIN2, a classic marker for insect defense in potato, is consistent with the decreases in larval weight gain.

A Floral Fragrance, Methyl Benzoate, is An Efficient Green Pesticide.

Over-reliance on synthetic pesticides in insect pest control has caused widespread public and scientific concerns for human health and the environment, especially since many insect pests have already developed resistances to conventional pesticides and Bt products. For this reason, there is a considerable interest in development of alternative control methods for insect pest management. Based on laboratory studies, we report that methyl benzoate (MB), a naturally-occurring compound in many plants, may possess toxicity against various stages of a variety of insect pests, including the brown marmorated stinkbug, Halyomorpha halys, diamondback moth, Plutella xylostella, and tobacco hornworm, Manduca sexta, as well as the spotted wing drosophila, Drosophila suzukii. Based on our laboratory toxicity data, MB was at least 5 to 20 times more toxic than the conventional pyrethroid (ß-cyfluthrin), sulfur &pyrethrin mixture, and some organic commercial products available on the market against H. halys, P. xylostella, and M. sexta, eggs. Because MB is considered an environment-friendly, it has great potential to be used as an alternative tool to synthetic pesticide for insect pest management in crop production, thereby, reducing threats to natural ecosystems and human health caused by over-application of conventional synthetic pesticides.

Herbivory responsive C2H2 zinc finger transcription factor protein StZFP2 from potato.

While C2H2 zinc finger transcription factors (TF) are often regulated by abiotic stress, their role during insect infestation has been overlooked. This study demonstrates that the transcripts of the zinc finger transcription factors StZFP1 and StZFP2 are induced in potato (Solanum tuberosum L.) upon infestation by either the generalist tobacco hornworm (THW, Manduca sexta L.) or the specialist Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). StZFP1 has been previously characterized as conferring salt tolerance to transgenic tobacco and its transcript is induced by Phytophthora infestans and several abiotic stresses. StZFP2 has not been characterized previously, but contains the hallmarks of a C2H2 zinc finger TF, with two conserved zinc finger domains and DLN motif, which encodes a transcriptional repressor domain. Expression studies demonstrate that StZFP2 transcript is also induced by tobacco hornworm and Colorado potato beetle. These observations expand the role of the C2H2 transcription factor in potato to include the response to chewing insect pests.

Trichome-derived O-acyl sugars are a first meal for caterpillars that tags them for predation.

Plant glandular trichomes exude secondary metabolites with defensive functions, but these epidermal protuberances are surprisingly the first meal of Lepidopteran herbivores on Nicotiana attenuata. O-acyl sugars, the most abundant metabolite of glandular trichomes, impart a distinct volatile profile to the body and frass of larvae that feed on them. The headspace composition of Manduca sexta larvae is dominated by the branched chain aliphatic acids hydrolyzed from ingested O-acyl sugars, which waxes and wanes rapidly with trichome ingestion. In native habitats a ground-hunting predator, the omnivorous ant Pogonomyrmex rugosus, but not the big-eyed bug Geocoris spp., use these volatile aliphatic acids to locate their prey.

The COP9 signalosome controls jasmonic acid synthesis and plant responses to herbivory and pathogens.

The COP9 signalosome (CSN) is a multi-protein complex that regulates the activities of cullin-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate proteins in order to target them for proteasomal degradation. The CSN is required for proper plant development. Here we show that the CSN also has a profound effect on plant defense responses. Silencing of genes for CSN subunits in tomato plants resulted in a mild morphological phenotype and reduced expression of wound-responsive genes in response to mechanical wounding, attack by Manduca sexta larvae, and Prosystemin over-expression. In contrast, expression of pathogenesis-related genes was increased in a stimulus-independent manner in these plants. The reduced wound response in CSN-silenced plants corresponded with reduced synthesis of jasmonic acid (JA), but levels of salicylic acid (SA) were unaltered. As a consequence, these plants exhibited reduced resistance against herbivorous M. sexta larvae and the necrotrophic fungal pathogen Botrytis cinerea. In contrast, susceptibility to tobacco mosaic virus (TMV) was not altered in CSN-silenced plants. These data demonstrate that the CSN orchestrates not only plant development but also JA-dependent plant defense responses.

A phosphoketolase Mpk1 of bacterial origin is adaptively required for full virulence in the insect-pathogenic fungus Metarhizium anisopliae.

Pentose metabolism through the phosphoketolase pathway has been well characterized in bacteria. In this paper, we report the identification of a phosphoketolase homologue Mpk1 in the insect-pathogenic fungus Metarhizium anisopliae. Phylogenetic analysis showed that fungal phosphoketolases are of bacterial origin and diverged into two superfamilies. Frequent gene loss or lack of acquisition is evident in specific fungal lineages or species. The mpk1 gene is highly expressed when grown in trehalose-rich insect haemolymph but poorly induced by insect cuticle or carbohydrate-rich plant root exudate. In addition, mpk1 gene expression and enzyme activity could be upregulated by different sugars including xylose, trehalose, glucose or sucrose. mpk1 null mutants generated by homologous recombination grew similar to the wild type of M. anisopliae on medium amended with xylose as a sole carbon source. However, insect (tobacco hornworm, Manduca sexta) bioassays showed significantly reduced virulence in Deltampk1. The results of this study suggest that the horizontally transferred Mpk1 in M. anisopliae plays an important niche adaptation role for fungal propagation in insect haemocoel. Following the carbohydrate flux from plants to plant-feeding insects and insect pathogenic fungi, a tritrophic relationship is discussed in association with the requirement of fungal phosphoketolase pathway.

The expression of the hydroxyproline-rich glycopeptide systemin precursor A in response to (a)biotic stress and elicitors is indicative of its role in the regulation of the wound response in tobacco (Nicotiana tabacum L.).

Two hydroxyproline-rich glycopeptide systemin (TobHS) precursor proteins known as preproTobHypSys-A and B were recently discovered in tobacco (Nicotiana tabacum L.) [Pearce et al. in Nature 411:817-820, 2001]. In this work, the effect of elicitors, insect damage, and abiotic stress on the expression of preproTobHypSys-A ppTobHS-A) in tobacco plants was evaluated. Foliar application of methyl jasmonate preferentially induced the systemic expression of ppTobHS-A in leaves phyllotactically one position above-treated leaves. Abscisic acid strongly induced ppTobHS-A, but water-stress did not. Mechanical wound-induction of ppTobHS-A in young plantlets was rapidly (1 h) and simultaneously detected in wounded and upper unwounded leaves, whereas in older plants induction was slow (12 h) and localized. ppTobHS-A was induced in plants infested with Bemisia tabaci or damaged by herbivory with Manduca sexta larvae. Compared to mechanical wounding, larval herbivory induced a stronger and more stable expression of ppTobHS-A. Moreover, exposure to Manduca-damaged plants induced its expression in neighboring intact plants. In most treatments, the expression patterns of ppTobHS-A coincided with those of selected wound-responsive (WR) genes (e.g., PIOX, NtPI-I, TPI). This correlation was tighter in the wounded and MeJA-treated leaves, whereas in distal, undamaged leaves, it appeared to depend on the type of WR gene examined and on the type of damage sustained by the plant. These results are consistent with the perceived role of the TobHS in defense signaling.

Role of beta-oxidation in jasmonate biosynthesis and systemic wound signaling in tomato.

Jasmonic acid (JA) is a lipid-derived signal that regulates plant defense responses to biotic stress. Here, we report the characterization of a JA-deficient mutant of tomato (Lycopersicon esculentum) that lacks local and systemic expression of defensive proteinase inhibitors (PIs) in response to wounding. Map-based cloning studies demonstrated that this phenotype results from loss of function of an acyl-CoA oxidase (ACX1A) that catalyzes the first step in the peroxisomal beta-oxidation stage of JA biosynthesis. Recombinant ACX1A exhibited a preference for C12 and C14 straight-chain acyl-CoAs and also was active in the metabolism of C18 cyclopentanoid-CoA precursors of JA. The overall growth, development, and reproduction of acx1 plants were similar to wild-type plants. However, the mutant was compromised in its defense against tobacco hornworm (Manduca sexta) attack. Grafting experiments showed that loss of ACX1A function disrupts the production of the transmissible signal for wound-induced PI expression but does not affect the recognition of this signal in undamaged responding leaves. We conclude that ACX1A is essential for the beta-oxidation stage of JA biosynthesis and that JA or its derivatives is required both for antiherbivore resistance and the production of the systemic wound signal. These findings support a role for peroxisomes in the production of lipid-based signaling molecules that promote systemic defense responses.

Manipulation of endogenous trypsin proteinase inhibitor production in Nicotiana attenuata demonstrates their function as antiherbivore defenses.

Evidence for the in planta defensive function of trypsin protease inhibitors (TPIs) comes from observations of enhanced herbivore resistance after heterologous TPI expression or the manipulation of signal cascades that activate numerous defense responses, including TPI production; no studies have altered the expression of an endogenous pi gene to examine defensive function. We isolated two genes with seven- and six-repeat TPI domains from Nicotiana attenuata from the potato (Solanum tuberosum) PI-II family. To determine whether endogenous TPIs in N. attenuata function defensively against the native herbivores, hornworm (Manduca sexta) and mirids (Tupiocoris notatus), we expressed 175 bp of the seven-domain pi from N. attenuata in an antisense orientation in a TPI-producing genotype to reduce TPI expression and expressed the full-length seven-domain pi in a sense orientation under control of a constitutive promoter to restore TPI activity in a natural genotype from Arizona unable to produce TPIs. Constitutive and inducible TPI production in two antisense lines were diminished by 80% to 90% and 33% to 52%, respectively, and sense expression restored 67% of the activity found in the TPI-producing genotype after caterpillar attack in the TPI-deficient A genotype. Hornworm larvae fed on genotypes with low or no TPI activity grew faster, had higher survivorship, and produced heavier pupae than those that fed on genotypes with high TPI activity. T. notatus showed higher preference for genotypes with low or no TPI activity than for genotypes with high TPI levels. We conclude that endogenous TPIs are an effective defense against these native herbivores.

Pest and disease resistance enhanced by heterologous suppression of a Nicotiana plumbaginifolia cytochrome P450 gene CYP72A2.

The functional role of the Nicotiana plumbaginifolia cytochrome P450 gene CYP72A2 was investigated in transgenic plants. N. tabacum plants transformed with a sense or antisense CYP72A2 construct exhibited diminished heights, branched stems, smaller leaves and deformed flowers. Western blot analysis revealed reduced levels of a 58 kDa protein corresponding to CYP72A2, suggesting that the CYP72A2 homolog was suppressed in the sense and antisense plants. Transgenic plants had increased resistance to Manduca sexta larvae that consumed about 35 to 90 less of transgenic versus control leaves. A virulent strain of Pseudomonas syringae pv. tabaci induced a disease-limiting response followed by a delayed and decreased development of disease symptoms in the transgenics. CYP72A2 gene mediated resistance suggests that the plant-pest or -pathogen interactions may have been modified by changes in bioactive metabolite pools.

Manduca sexta recognition and resistance among allopolyploid Nicotiana host plants.

Allopolyploid speciation occurs instantly when the genomes of different species combine to produce self-fertile offspring and has played a central role in the evolution of higher plants, but its consequences for adaptive responses are unknown. We compare herbivore-recognition and -resistance responses of the diploid species and putative ancestral parent Nicotiana attenuata with those of the two derived allopolyploid species Nicotiana clevelandii and Nicotiana bigelovii. Manduca sexta larvae attack all three species, and in N. attenuata attack is recognized when larval oral secretions are introduced to wounds during feeding, resulting in a jasmonate burst, a systemic amplification of trypsin inhibitor accumulation, and a release of volatile organic compounds, which function as a coordinated defense response that slows caterpillar growth and increases the probability of their being attacked. Most aspects of this recognition response are retained with modifications in one allotetraploid (N. bigelovii) but lost in the other (N. clevelandii). Differences between diploid and tetraploid species were apparent in delays (maximum 1 and 0.5 h, respectively) in the jasmonate burst, the elicitation of trypsin inhibitors and release of volatile organic compounds, and the constitutive levels of nicotine, trypsin inhibitors, diterpene glycosides, rutin, and caffeoylputrescine in the leaves. Resistance to M. sexta larvae attack was most strongly associated with diterpene glycosides, which were higher in the diploid than in the two allotetraploid species. Because M. sexta elicitors differentially regulate a large proportion of the N. attenuata transcriptome, we propose that these species are suited for the study of the evolution of adaptive responses requiring trans-activation mechanisms.

Nonproteolytic serine proteinase homologs are involved in prophenoloxidase activation in the tobacco hornworm, Manduca sexta.

In insects, the prophenoloxidase activation system is a defense mechanism against parasites and pathogens. Recognition of parasites or pathogens by pattern recognition receptors triggers activation of a serine proteinase cascade, leading to activation of prophenoloxidase-activating proteinase (PAP). PAP converts inactive prophenoloxidase (proPO) to active phenoloxidase (PO), which then catalyzes oxidation of phenolic compounds that can polymerize to form melanin. Because quinone intermediates and melanin are toxic to both hosts and pathogens, activation of proPO must be tightly regulated and localized. We report here purification and cDNA cloning of serine proteinase homologs (SPHs) from the tobacco hornworm, Manduca sexta, which interact with PAP-1 in proPO activation. Two SPHs were co-purified from plasma of M. sexta larvae with immulectin-2, a C-type lectin that binds to bacterial lipopolysaccharide. They contain an amino-terminal clip domain connected to a carboxyl-terminal serine proteinase-like domain. PAP-1 alone cannot efficiently activate proPO, but a mixture of SPHs and PAP-1 was much more effective for proPO activation. Immulectin-2, proPO and PAP-1 in hemolymph bound to the immobilized recombinant proteinase-like domain of SPH-1, indicating that a complex containing these proteins may exist in hemolymph. Since immulectin-2 is a pattern recognition receptor that binds to surface carbohydrates on pathogens, such a protein complex may localize activation of proPO on the surface of pathogens. SPH, which binds to immulectin-2, may function as a mediator to recruit proPO and PAP to the site of infection.

Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. I. Large-scale changes in the accumulation of growth- and defense-related plant mRNAs.

Plants respond to herbivore attack with a dramatic functional reorganization that involves the activation of direct and indirect defenses and tolerance, which in turn make large demands on primary metabolism. Here we provide the first characterization of the transcriptional reorganization that occurs after insect attack in a model plant-herbivore system: Nicotiana attenuata Torr. ex Wats.-Manduca sexta. We used mRNA differential display to characterize one-twentieth of the insect-responsive transcriptome of N. attenuata and verified differential expression for 27 cDNAs. Northern analyses were used to study the effects of folivory and exposure to airborne methyl jasmonate and for kinetic analyses throughout a 16-h- light/8-h-dark cycle. Sequence similarity searches allowed putative functions to be assigned to 15 transcripts. Genes were related to photosynthesis, electron transport, cytoskeleton, carbon and nitrogen metabolism, signaling, and a group responding to stress, wounding, or invasion of pathogens. Overall, transcripts involved in photosynthesis were strongly down-regulated, whereas those responding to stress, wounding, and pathogens and involved in shifting carbon and nitrogen to defense were strongly up-regulated. The majority of transcripts responded similarly to airborne methyl jasmonate and folivory, and had tissue- and diurnal-specific patterns of expression. Transcripts encoding Thr deaminase (TD) and a putative retrotransposon were absent in control plants, but were strongly induced after herbivory. Full-length sequences were obtained for TD and the pathogen-inducible alpha-dioxygenase, PIOX. Effects of abiotic and biotic stimuli were investigated for transcripts encoding TD, importin alpha, PIOX, and a GAL83-like kinase cofactor.

Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. II. Accumulation of plant mRNAs in response to insect-derived cues.

The transcriptional changes in Nicotiana attenuata Torr. ex Wats. elicited by attack from Manduca sexta larvae were previously characterized by mRNA differential display (D. Hermsmeier, U. Schittko, I.T. Baldwin [2001] Plant Physiol 125: 683-700). Because herbivore attack causes wounding, we disentangled wound-induced changes from those elicited by M. sexta oral secretions and regurgitant (R) with a northern analysis of a subset of the differentially expressed transcripts encoding threonine deaminase, pathogen-induced oxygenase, a photosystem II light-harvesting protein, a retrotransposon homolog, and three unknown genes. R extensively modified wound-induced responses by suppressing wound-induced transcripts (type I) or amplifying the wound-induced response (type II) further down-regulating wound-suppressed transcripts (type IIa) or up-regulating wound-induced transcripts (type IIb). It is interesting that although all seven genes displayed their R-specific patterns in the treated tissues largely independently of the leaf or plant developmental stage, only the type I genes displayed strong systemic induction. Ethylene was not responsible for any of the specific patterns of expression. R collected from different tobacco feeding insects, M. sexta, Manduca quinquemaculata, and Heliothis virescens, as well as from different instars of M. sexta were equally active. The active components of M. sexta R were heat stable and active in minute amounts, comparable with real transfer rates during larval feeding. Specific expression patterns may indicate that the plant is adjusting its wound response to efficiently fend off M. sexta, but may also be advantageous to the larvae, especially when R suppress wound-induced plant responses.

Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses.

Feeding by the tobacco specialist Manduca sexta (Lepidoptera, Sphingidae) and application of larval oral secretions and regurgitant (R) to mechanical wounds are known to elicit: (a) a systemic release of mono- and sesquiterpenes, (b) a jasmonate burst, and (c) R-specific changes in transcript accumulation of putatively growth- and defense-related mRNAs in Nicotiana attenuata Torr. ex Wats. We identified several fatty acid-amino acid conjugates (FACs) in the R of M. sexta and the closely related species Manduca quinquemaculata which, when synthesized and applied to mechanical wounds at concentrations comparable with those found in R, elicited all three R-specific responses. Ion-exchange treatment of R, which removed all detectable FACs and free fatty acids (FAs), also removed all detectable activity. The biological activity of ion-exchanged R could be completely restored by the addition of synthetic FACs at R-equivalent concentrations, whereas the addition of FAs did not restore the biological activity of R. We conclude that the biological activity of R is not related to the supply of FAs to the octadecanoid cascade for endogenous jasmonate biosynthesis, but that FACs elicit the herbivore-specific responses by another mechanism and that the insect-produced modification of plant-derived FAs is necessary for the plant's recognition of this specialized herbivore.