Trypsin Proteinase Inhibitors Research Articles

Herbivory-induced volatiles function as defenses increasing fitness of the native plant Nicotiana attenuata in nature

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.DOI:http://dx.doi.org/10.7554/eLife.00007.001.

Nicotiana attenuata α‐DIOXYGENASE1 through its production of 2‐hydroxylinolenic acid is required for intact plant defense expression against attack from Manduca sexta larvae

Nicotiana attenuata α-DIOXYGENASE1 (α-DOX1) is an oxylipin-forming gene elicited during herbivory by fatty acid amino acid conjugates (FACs) contained in oral secretions of Manduca sexta. To understand the roles of Naα-DOX1 and its major product, 2-hydroxylinolenic acid (2-hydroxylinolenic acid), in N.attenuata's anti-herbivore defenses, we used a transgenic line specifically silenced in Naα-DOX1 expression (ir-α-dox1) and monitored 2-HOT production in M.sexta-damaged tissues and its role in influencing the production of direct defense compounds and resistance to this insect. Attack by M.sexta larvae amplified 2-HOT formation at the feeding sites; a reaction probably facilitated by Naα-DOX1's high pH optimum which allows 2-HOT formation to occur in the more alkaline conditions at the feeding sites or potentially in the insect mouth parts after the leaf tissue is ingested. Manduca sexta larvae performed better on ir-α-dox1 plants than on wild-type (WT) plants as a result of attenuated herbivory-specific JA and 2-HOT bursts as well as JA-inducible well-established defenses (nicotine, caffeoylputrescine and trypsin proteinase inhibitors). Repeated applications of 2-HOT to wounds before insect feeding partly amplified JA-controlled defenses and restored the resistance of ir-α-dox1 plants. We conclude that 2-HOT, produced by attack-activated α-DOX1 activity, participates in defense activation during insect feeding.

OsNPR1 negatively regulates herbivore‐induced JA and ethylene signaling and plant resistance to a chewing herbivore in rice

NPR1 (a non-expressor of pathogenesis-related genes1) has been reported to play an important role in plant defense by regulating signaling pathways. However, little to nothing is known about its function in herbivore-induced defense in monocot plants. Here, using suppressive substrate hybridization, we identified a NPR1 gene from rice, OsNPR1, and found that its expression levels were upregulated in response to infestation by the rice striped stem borer (SSB) Chilo suppressalis and rice leaf folder (LF) Cnaphalocrocis medinalis, and to mechanical wounding and treatment with jasmonic acid (JA) and salicylic acid (SA). Moreover, mechanical wounding induced the expression of OsNPR1 quickly, whereas herbivore infestation induced the gene more slowly. The antisense expression of OsNPR1 (as-npr1), which reduced the expression of the gene by 50%, increased elicited levels of JA and ethylene (ET) as well as of expression of a lipoxygenase gene OsHI-LOX and an ACC synthase gene OsACS2. The enhanced JA and ET signaling in as-npr1 plants increased the levels of herbivore-induced trypsin proteinase inhibitors (TrypPIs) and volatiles, and reduced the performance of SSB. Our results suggest that OsNPR1 is an early responding gene in herbivore-induced defense and that plants can use it to activate a specific and appropriate defense response against invaders by modulating signaling pathways.

The rice hydroperoxide lyase OsHPL3 functions in defense responses by modulating the oxylipin pathway

As important signal molecules, jasmonates (JAs) and green leaf volatiles (GLVs) play diverse roles in plant defense responses against insect pests and pathogens. However, how plants employ their specific defense responses by modulating the levels of JA and GLVs remains unclear. Here, we describe identification of a role for the rice HPL3 gene, which encodes a hydroperoxide lyase (HPL), OsHPL3/CYP74B2, in mediating plant-specific defense responses. The loss-of-function mutant hpl3-1 produced disease-resembling lesions spreading through the whole leaves. A biochemical assay revealed that OsHPL3 possesses intrinsic HPL activity, hydrolyzing hydroperoxylinolenic acid to produce GLVs. The hpl3-1 plants exhibited enhanced induction of JA, trypsin proteinase inhibitors and other volatiles, but decreased levels of GLVs including (Z)-3-hexen-1-ol. OsHPL3 positively modulates resistance to the rice brown planthopper [BPH, Nilaparvata lugens (Stål)] but negatively modulates resistance to the rice striped stem borer [SSB, Chilo suppressalis (Walker)]. Moreover, hpl3-1 plants were more attractive to a BPH egg parasitoid, Anagrus nilaparvatae, than the wild-type, most likely as a result of increased release of BPH-induced volatiles. Interestingly, hpl3-1 plants also showed increased resistance to bacterial blight (Xanthomonas oryzae pv. oryzae). Collectively, these results indicate that OsHPL3, by affecting the levels of JA, GLVs and other volatiles, modulates rice-specific defense responses against different invaders.

Silencing COI1 in Rice Increases Susceptibility to Chewing Insects and Impairs Inducible Defense

The jasmonic acid (JA) pathway plays a key role in plant defense responses against herbivorous insects. CORONATINE INSENSITIVE1 (COI1) is an F-box protein essential for all jasmonate responses. However, the precise defense function of COI1 in monocotyledonous plants, especially in rice (Oryza sativa L.) is largely unknown. We silenced OsCOI1 in rice plants via RNA interference (RNAi) to determine the role of OsCOI1 in rice defense against rice leaf folder (LF) Cnaphalocrocis medinalis, a chewing insect, and brown planthopper (BPH) Nilaparvata lugens, a phloem-feeding insect. In wild-type rice plants (WT), the transcripts of OsCOI1 were strongly and continuously up-regulated by LF infestation and methyl jasmonate (MeJA) treatment, but not by BPH infestation. The abundance of trypsin protease inhibitor (TrypPI), and the enzymatic activities of polyphenol oxidase (PPO) and peroxidase (POD) were enhanced in response to both LF and BPH infestation, but the activity of lipoxygenase (LOX) was only induced by LF. The RNAi lines with repressed expression of OsCOI1 showed reduced resistance against LF, but no change against BPH. Silencing OsCOI1 did not alter LF-induced LOX activity and JA content, but it led to a reduction in the TrypPI content, POD and PPO activity by 62.3%, 48.5% and 27.2%, respectively. In addition, MeJA-induced TrypPI and POD activity were reduced by 57.2% and 48.2% in OsCOI1 RNAi plants. These results suggest that OsCOI1 is an indispensable signaling component, controlling JA-regulated defense against chewing insect (LF) in rice plants, and COI1 is also required for induction of TrypPI, POD and PPO in rice defense response to LF infestation.

NaJAZh regulates a subset of defense responses against herbivores and spontaneous leaf necrosis in Nicotiana attenuata plants.

The JASMONATE ZIM DOMAIN (JAZ) proteins function as negative regulators of jasmonic acid signaling in plants. We cloned 12 JAZ genes from native tobacco (Nicotiana attenuata), including nine novel JAZs in tobacco, and examined their expression in plants that had leaves elicited by wounding or simulated herbivory. Most JAZ genes showed strong expression in the elicited leaves, but NaJAZg was mainly expressed in roots. Another novel herbivory-elicited gene, NaJAZh, was analyzed in detail. RNA interference suppression of this gene in inverted-repeat (ir)JAZh plants deregulated a specific branch of jasmonic acid-dependent direct and indirect defenses: irJAZh plants showed greater trypsin protease inhibitor activity, 17-hydroxygeranyllinalool diterpene glycosides accumulation, and emission of volatile organic compounds from leaves. Silencing of NaJAZh also revealed a novel cross talk in JAZ-regulated secondary metabolism, as irJAZh plants had significantly reduced nicotine levels. In addition, irJAZh spontaneously developed leaf necrosis during the transition to flowering. Because the lesions closely correlated with the elevated expression of programmed cell death genes and the accumulations of salicylic acid and hydrogen peroxide in the leaves, we propose a novel role of the NaJAZh protein as a repressor of necrosis and/or programmed cell death during plant development.

DICER‐like Proteins and Their Role in Plant‐herbivore Interactions in Nicotiana attenuata

DICER-like (DCL) proteins produce small RNAs that silence genes involved in development and defenses against viruses and pathogens. Which DCLs participate in plant-herbivore interactions remains unstudied. We identified and stably silenced four distinct DCL genes by RNAi in Nicotiana attenuata (Torrey ex. Watson), a model for the study of plant-herbivore interactions. Silencing DCL1 expression was lethal. Manduca sexta larvae performed significantly better on ir-dcl3 and ir-dcl4 plants, but not on ir-dcl2 plants compared to wild type plants. Phytohormones, defense metabolites and microarray analyses revealed that when DCL3 and DCL4 were silenced separately, herbivore resistance traits were regulated in distinctly different ways. Crossing of the lines revealed complex interactions in the patterns of regulation. Single ir-dcl4 and double ir-dcl2 ir-dcl3 plants were impaired in JA accumulation, while JA-Ile was increased in ir-dcl3 plants. Ir-dcl3 and ir-dcl4 plants were impaired in nicotine accumulation; silencing DCL2 in combination with either DCL3 or DCL4 restored nicotine levels to those of WT. Trypsin proteinase inhibitor activity and transcripts were only silenced in ir-dcl3 plants. We conclude that DCL2/3/4 interact in a complex manner to regulate anti-herbivore defenses and that these interactions significantly complicate the already challenging task of understanding smRNA function in the regulation of biotic interactions.

The broad‐leaf herbicide 2,4‐dichlorophenoxyacetic acid turns rice into a living trap for a major insect pest and a parasitic wasp

Synthetic chemical elicitors of plant defense have been touted as a powerful means for sustainable crop protection. Yet, they have never been successfully applied to control insect pests in the field. We developed a high-throughput chemical genetics screening system based on a herbivore-induced linalool synthase promoter fused to a β-glucuronidase (GUS) reporter construct to test synthetic compounds for their potential to induce rice defenses. We identified 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin homolog and widely used herbicide in monocotyledonous crops, as a potent elicitor of rice defenses. Low doses of 2,4-D induced a strong defensive reaction upstream of the jasmonic acid and ethylene pathways, resulting in a marked increase in trypsin proteinase inhibitor activity and volatile production. Induced plants were more resistant to the striped stem borer Chilo suppressalis, but became highly attractive to the brown planthopper Nilaparvata lugens and its main egg parasitoid Anagrus nilaparvatae. In a field experiment, 2,4-D application turned rice plants into living traps for N. lugens by attracting parasitoids. Our findings demonstrate the potential of auxin homologs as defensive signals and show the potential of the herbicide to turn rice into a selective catch crop for an economically important pest.

Transcriptome analysis of resistant soybean roots infected by Meloidogyne javanica.

Soybean is an important crop for Brazilian agribusiness. However, many factors can limit its production, especially root-knot nematode infection. Studies on the mechanisms employed by the resistant soybean genotypes to prevent infection by these nematodes are of great interest for breeders. For these reasons, the aim of this work is to characterize the transcriptome of soybean line PI 595099-Meloidogyne javanica interaction through expression analysis. Two cDNA libraries were obtained using a pool of RNA from PI 595099 uninfected and M. javanica (J2) infected roots, collected at 6, 12, 24, 48, 96, 144 and 192 h after inoculation. Around 800 ESTs (Expressed Sequence Tags) were sequenced and clustered into 195 clusters. In silico subtraction analysis identified eleven differentially expressed genes encoding putative proteins sharing amino acid sequence similarities by using BlastX: metallothionein, SLAH4 (SLAC1 Homologue 4), SLAH1 (SLAC1 Homologue 1), zinc-finger proteins, AN1-type proteins, auxin-repressed proteins, thioredoxin and nuclear transport factor 2 (NTF-2). Other genes were also found exclusively in nematode stressed soybean roots, such as NAC domain-containing proteins, MADS-box proteins, SOC1 (suppressor of overexpression of constans 1) proteins, thioredoxin-like protein 4-Coumarate-CoA ligase and the transcription factor (TF) MYBZ2. Among the genes identified in non-stressed roots only were Ser/Thr protein kinases, wound-induced basic protein, ethylene-responsive family protein, metallothionein-like protein cysteine proteinase inhibitor (cystatin) and Putative Kunitz trypsin protease inhibitor. An understanding of the roles of these differentially expressed genes will provide insights into the resistance mechanisms and candidate genes involved in soybean-M. javanica interaction and contribute to more effective control of this pathogen.

Three MAPK Kinases, MEK1, SIPKK, and NPK2, are not Involved in Activation of SIPK after Wounding and Herbivore Feeding but Important for Accumulation of Trypsin Proteinase Inhibitors

We identified three Nicotiana attenuata mitogen-activated protein kinase (MAPK) kinases (MAPKKs), NaMEK1, NaSIPKK, and NaNPK2, whose transcript levels were up-regulated in a wild tobacco plant, N. attenuata, after wounding and simulated herbivore attack. A virus-induced gene silencing approach was used to analyze the functions of these three MAPKKs in plant defense against the tobacco hornworm Manduca sexta. NaMEK1 and NaSIPKK influenced the accumulation of the precursor of jasmonic acid (JA), 12-oxo-phytodienoic acid, and silencing NaSIPKK enhanced the levels of wounding- and herbivory-induced JA. In-gel kinase assays indicated that all three MAPKKs were not required for the activation of NaSIPK, an important MAPK in plant responses to wounding and herbivory. However, NaMEK1, NaSIPKK, and NaNPK2 appeared to regulate the levels of trypsin proteinase inhibitor activity. Bioassays revealed that M. sexta larval growth was not impaired on N. attenuata plants silenced in NaMEK1, NaSIPKK, or NaNPK2 expression. Our findings suggest that NaSIPKK is involved in JA biosynthesis after herbivore attack without activating NaSIPK, suggesting that the network of MAPK signaling in N. attenuata's defense responses against herbivore attack is more complicated than previously thought.

Diverting the Flux of the JA Pathway in Nicotiana attenuata Compromises the Plant's Defense Metabolism and Fitness in Nature and Glasshouse

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 Chloroplast-Localized Phospholipases D α4 and α5 Regulate Herbivore-Induced Direct and Indirect Defenses in Rice

The oxylipin pathway is of central importance for plant defensive responses. Yet, the first step of the pathway, the liberation of linolenic acid following induction, is poorly understood. Phospholipases D (PLDs) have been hypothesized to mediate this process, but data from Arabidopsis (Arabidopsis thaliana) regarding the role of PLDs in plant resistance have remained controversial. Here, we cloned two chloroplast-localized PLD genes from rice (Oryza sativa), OsPLDα4 and OsPLDα5, both of which were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis, mechanical wounding, and treatment with jasmonic acid (JA). Antisense expression of OsPLDα4 and -α5 (as-pld), which resulted in a 50% reduction of the expression of the two genes, reduced elicited levels of linolenic acid, JA, green leaf volatiles, and ethylene and attenuated the SSB-induced expression of a mitogen-activated protein kinase (OsMPK3), a lipoxygenase (OsHI-LOX), a hydroperoxide lyase (OsHPL3), as well as a 1-aminocyclopropane-1-carboxylic acid synthase (OsACS2). The impaired oxylipin and ethylene signaling in as-pld plants decreased the levels of herbivore-induced trypsin protease inhibitors and volatiles, improved the performance of SSB and the rice brown planthopper Nilaparvata lugens, and reduced the attractiveness of plants to a larval parasitoid of SSB, Apanteles chilonis. The production of trypsin protease inhibitors in as-pld plants could be partially restored by JA, while the resistance to rice brown planthopper and SSB was restored by green leaf volatile application. Our results show that phospholipases function as important components of herbivore-induced direct and indirect defenses in rice.

Protease inhibitor from Moringa oleifera leaves: Isolation, purification, and characterization

Protease inhibitors have great demand in medicine and biotechnology. We report here the purification and characterization of a protease inhibitor isolated from mature leaf extract of Moringa oleifera that showed maximum inhibitor activity. The protease inhibitor was purified to 41.4-fold by Sephadex G75 and its molecular mass was calculated as 23,600Da. Inhibitory activity was confirmed by dot-blot and reverse zymogram analyses. Glycine, glutamic acid, alanine, proline and aspartic acid were found as the major amino acids of the inhibitor protein. Maximal activity was recorded at pH 7 and at 40°C. The inhibitor was stable over pH 5–10; and at 50°C for 2h. Thermostability was promoted by CaCl2, BSA and sucrose. Addition of Zn2+ and Mg2+, SDS, dithiothreitol and β-mercaptoethanol enhanced inhibitory activity, while DMSO and H2O2 affected inhibitory activity. Modification of amino acids at the catalytic site by PMSF and DEPC led to an enhancement in the inhibitory activity. Stoichiometry of trypsin–protease inhibitor interaction was 1:1.5 and 0.6nM of inhibitor effected 50% inhibition. The low Ki value (1.5nM) obtained indicated scope for utilization of M. oliefera protease inhibitor against serine proteases.

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.

An EAR‐motif‐containing ERF transcription factor affects herbivore‐induced signaling, defense and resistance in rice

Ethylene responsive factors (ERFs) are a large family of plant-specific transcription factors that are involved in the regulation of plant development and stress responses. However, little to nothing is known about their role in herbivore-induced defense. We discovered a nucleus-localized ERF gene in rice (Oryza sativa), OsERF3, that was rapidly up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis. Antisense and over-expression of OsERF3 revealed that it positively affects transcript levels of two mitogen-activated protein kinases (MAPKs) and two WRKY genes as well as concentrations of jasmonate (JA), salicylate (SA) and the activity of trypsin protease inhibitors (TrypPIs). OsERF3 was also found to mediate the resistance of rice to SSB. On the other hand, OsERF3 was slightly suppressed by the rice brown planthopper (BPH) Nilaparvata lugens (Stål) and increased susceptibility to this piercing sucking insect, possibly by suppressing H(2)O(2) biosynthesis. We propose that OsERF3 affects early components of herbivore-induced defense responses by suppressing MAPK repressors and modulating JA, SA, ethylene and H(2)O(2) pathways as well as plant resistance. Our results also illustrate that OsERF3 acts as a central switch that gears the plant's metabolism towards an appropriate response to chewing or piercing/sucking insects.

Ectopic Expression ofAtJMTinNicotiana attenuata: Creating a Metabolic Sink Has Tissue-Specific Consequences for the Jasmonate Metabolic Network and Silences Downstream Gene Expression

To create a metabolic sink in the jasmonic acid (JA) pathway, we generated transgenic Nicotiana attenuata lines ectopically expressing Arabidopsis (Arabidopsis thaliana) jasmonic acid O-methyltransferase (35S-jmt) and additionally silenced in other lines the N. attenuata methyl jasmonate esterase (35S-jmt/ir-mje) to reduce the deesterification of methyl jasmonate (MeJA). Basal jasmonate levels did not differ between transgenic and wild-type plants; however, after wounding and elicitation with Manduca sexta oral secretions, the bursts of JA, jasmonoyl-isoleucine (JA-Ile), and their metabolites that are normally observed in the lamina, midvein, and petiole of elicited wild-type leaves were largely absent in both transformants but replaced by a burst of endogenous MeJA that accounted for almost half of the total elicited jasmonate pools. In these plants, MeJA became a metabolic sink that affected the jasmonate metabolic network and its spread to systemic leaves, with major effects on 12-oxo-phytodieonic acid, JA, and hydroxy-JA in petioles and on JA-Ile in laminas. Alterations in the size of jasmonate pools were most obvious in systemic tissues, especially petioles. Expression of threonine deaminase and trypsin proteinase inhibitor, two JA-inducible defense genes, was strongly decreased in both transgenic lines without influencing the expression of JA biosynthesis genes that were uncoupled from the wounding and elicitation with M. sexta oral secretions-elicited JA-Ile gradient in elicited leaves. Taken together, this study provides support for a central role of the vasculature in the propagation of jasmonates and new insights into the versatile spatiotemporal characteristics of the jasmonate metabolic network.

Salicylic acid and ethylene signaling pathways are involved in production of rice trypsin proteinase inhibitors induced by the leaf folder Cnaphalocrocis medinalis (Guenée)

The roles of signaling pathways in the production of trypsin proteinase inhibitors (TrypPIs) in rice infested by the leaf folder (LF) Cnaphalocrocis medinalis were studied. Infestation by LF increased TrypPI levels in the leaves of rice plants at the tillering, booting and flowering stages but decreased TrypPI levels at the ripening stage; TrypPI levels in rice stems did not increase at any developmental stage. Infestation by LF at the tillering stage systemically increased TrypPI levels in leaves but not in stems; it also enhanced salicylic acid (SA) levels in leaves and stems, and the ethylene level released from plants. However, LF infestation did not increase JA concentrations. Exogenous application of SA or ethylene enhanced TrypPI levels in the leaves and stems of plants at the tillering stage, whereas treatment with both SA and ethylene induced lower levels of TrypPIs than treatment with SA or ethylene alone, suggesting an antagonistic effect of SA and ethylene on TrypPIs induction. The results suggest that both SA and ethylene signaling pathways are involved in the production of TrypPIs in rice induced by LF; moreover, the antagonistic effect of SA and ethylene may explain the changes in TrypPI levels seen at different plant developmental stages and in different organs.

Genome‐wide transcriptional changes and defence‐related chemical profiling of rice in response to infestation by the rice striped stem borer Chilo suppressalis

How rice defends itself against pathogen infection is well documented, but little is known about how it defends itself against herbivore attack. We measured changes in the transcriptome and chemical profile of rice when the plant is infested by the striped stem borer (SSB) Chilo suppressalis. Infestation by SSBs resulted in changes in the expression levels of 4545 rice genes; this number accounts for about 8% of the genome and is made up of 18 functional groups with broad functions. The largest group comprised genes involved in metabolism, followed by cellular transport, transcription and cellular signaling. Infestation by SSBs modulated many genes responsible for the biosynthesis of plant hormones and plant signaling. Jasmonic acid (JA), salicylic acid (SA) and ethylene were the major hormones that shaped the SSB-induced defence responses of rice. Many secondary signal transduction components, such as those involved in Ca²⁺ signaling and G-protein signaling, receptor and non-receptor protein kinases, and transcription factors were involved in the SSB-induced responses of rice. Photosynthesis and ATP synthesis from photophosphorylation were restricted by SSB feeding. In addition, SSB infestation induced the accumulation of defence compounds, including trypsin proteinase inhibitors (TrypPIs) and volatile organic compounds. These results demonstrate that SSB-induced defences required rice to reconfigure a wide variety of its metabolic, physiological and biochemical processes.

S-Nitrosoglutathione reductase (GSNOR) mediates the biosynthesis of jasmonic acid and ethylene induced by feeding of the insect herbivore Manduca sexta and is important for jasmonate-elicited responses in Nicotiana attenuata.

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.

Two mitogen-activated protein kinase kinases, MKK1 and MEK2, are involved in wounding- and specialist lepidopteran herbivore Manduca sexta-induced responses in Nicotiana attenuata.

In a wild tobacco plant, Nicotiana attenuata, two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), play central roles in modulating herbivory-induced phytohormone and anti-herbivore secondary metabolites. However, the identities of their upstream MAPK kinases (MAPKKs) were elusive. Ectopic overexpression studies in N. benthamiana and N. tabacum suggested that two MAPKKs, MKK1 and MEK2, may activate SIPK and WIPK. The homologues of MKK1 and MEK2 were cloned in N. attenuata (NaMKK1 and NaMEK2) and a virus-induced gene silencing approach was used to knock-down the transcript levels of these MAPKK genes. Plants silenced in NaMKK1 and NaMEK2 were treated with wounding or simulated herbivory by applying the oral secretions of the specialist herbivore Manduca sexta to wounds. MAPK activity assay indicated that after wounding or simulated herbivory NaMKK1 is not required for the phosphorylation of NaSIPK and NaWIPK; in contrast, NaMEK2 and other unknown MAPKKs are important for simulated herbivory-elicited activation of NaSIPK and NaWIPK, and after wounding NaMEK2 probably does not activate NaWIPK but plays a minor role in activating NaSIPK. Consistently, NaMEK2 and certain other MAPKKs, but not NaMKK1, are needed for wounding- and simulated herbivory-elicited accumulation of jasmonic acid (JA), JA–isoleucine, and ethylene. Furthermore, both NaMEK2 and NaMKK1 regulate the levels of trypsin proteinase inhibitors. The findings underscore the complexity of MAPK signalling pathways and highlight the importance of MAPKKs in regulating wounding- and herbivory-induced responses.