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.     

Different Lepidopteran Elicitors Account for Cross-Talk in Herbivory-Induced Phytohormone Signaling

Salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and their interactions mediate plant responses to pathogen and herbivore attack. JA-SA and JA-ET cross-signaling are well studied, but little is known about SA-ET cross-signaling in plant-herbivore interactions. When the specialist herbivore tobacco hornworm (Manduca sexta) attacks Nicotiana attenuata, rapid and transient JA and ET bursts are elicited without significantly altering wound-induced SA levels. In contrast, attack from the generalist beet armyworm (Spodoptera exigua) results in comparatively lower JA and ET bursts, but amplified SA bursts. These phytohormone responses are mimicked when the species' larval oral secretions (OS_Se and OS_Ms) are added to puncture wounds. Fatty acid-amino acid conjugates elicit the JA and ET bursts, but not the SA burst. OS_Se had enhanced glucose oxidase activity (but not β-glucosidase activity), which was sufficient to elicit the SA burst and attenuate the JA and ET levels. It is known that SA antagonizes JA; glucose oxidase activity and associated hydrogen peroxide also antagonizes the ET burst. We examined the OS_Ms-elicited SA burst in plants impaired in their ability to elicit JA (antisense [as]-lox3) and ET (inverted repeat [ir]-aco) bursts and perceive ET (35s-etr1b) after fatty acid-amino acid conjugate elicitation, which revealed that both ET and JA bursts antagonize the SA burst. Treating wild-type plants with ethephone and 1-methylcyclopropane confirmed these results and demonstrated the central role of the ET burst in suppressing the OS_Ms-elicited SA burst. By suppressing the SA burst, the ET burst likely facilitates unfettered JA-mediated defense activation in response to herbivores that otherwise would elicit SA.     

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.     

Parasitism by Cuscuta pentagona sequentially induces JA and SA defence pathways in tomato

While plant responses to herbivores and pathogens are well characterized, responses to attack by other plants remain largely unexplored. We measured phytohormones and C₁₈ fatty acids in tomato attacked by the parasitic plant Cuscuta pentagona, and used transgenic and mutant plants to explore the roles of the defence‐related phytohormones salicylic acid (SA) and jasmonic acid (JA). Parasite attachment to 10‐day‐old tomato plants elicited few biochemical changes, but a second attachment 10 d later elicited a 60‐fold increase in JA, a 30‐fold increase in SA and a hypersensitive‐like response (HLR). Host age also influenced the response: neither Cuscuta seedlings nor established vines elicited a HLR in 10‐day‐old hosts, but both did in 20‐day‐old hosts. Parasites grew larger on hosts deficient in SA (NahG) or insensitive to JA [jasmonic acid‐insensitive1 (jai1) ], suggesting that both phytohormones mediate effective defences. Moreover, amounts of JA peaked 12 h before SA, indicating that defences may be coordinated via sequential induction of these hormones. Parasitism also induced increases in free linolenic and linoleic acids and abscisic acid. These findings provide the first documentation of plant hormonal signalling induced by a parasitic plant and show that tomato responses to C. pentagona display characteristics similar to both herbivore‐ and pathogen‐induced responses.     

Elevated ozone induces jasmonic acid defense of tomato plants and reduces midgut proteinase activity in Helicoverpa armigera

As a consequence of membrane lipid peroxidation, foliar defense compounds are changed by elevated ozone (O₃), which in turn affects the palatability and performance of insect herbivores. The induced defense of two tomato [Solanum esculentum L. (Solanaceae)] genotypes, namely jasmonic acid (JA) pathway‐deficient mutant spr2 and its wild‐type control, was studied in response to cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae), as well as the digestive adaptation of these insects under elevated O₃ in open‐top field chambers. Our data indicated that elevated O₃ increased foliar JA and salicylic acid (SA) levels simultaneously and up‐regulated proteinase inhibitors (PIs) and lipoxidase activities in wild‐type plants, regardless of H. armigera infestation. In contrast, only the O₃+H. armigera treatment increased free SA levels in spr2 plants, but did not affect JA level or PI activities. Additionally, the lower activity of midgut digestive enzymes, including active alkaline trypsin‐like enzyme and chymotrypsin‐like enzyme, was observed in the midgut of cotton bollworms after they consumed wild‐type plants treated for 2 h with elevated O₃. With temporary increases at 8 h, all four digestive enzymes of interest in the insect midgut dropped when they were fed with wild‐type plants under elevated O₃ treatment. Increases in atmospheric O₃ are thought to increase JA signaling and consequently reduce the activities of midgut digestive enzymes in H. armigera, therefore enhancing plant resistance against insect herbivores.     

The maize lipoxygenase,ZmLOX10, mediates green leaf volatile,jasmonate and herbivore‐induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, major regulatory genes and the signals that modulate these defense metabolites are vastly understudied, especially in important agro‐economic monocot species. Here we show that products and signals derived from a single Zea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbivory. We provide genetic evidence that two 13‐LOXs, ZmLOX10 and ZmLOX8, specialize in providing substrate for the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the specialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indicating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression of JA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10‐derived signaling is required for LOX8‐mediated JA. The possible role of GLVs in JA signaling is supported by their ability to partially restore wound‐induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produce GLVs and JA led to dramatic reductions in herbivore‐induced plant volatiles (HIPVs) and attractiveness to parasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic link to the diurnal regulation of GLVs and HIPVs. GLV‐, JA‐ and HIPV‐deficient lox10 mutants display compromised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence that LOX10‐dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene to agro‐ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.     

Effects of jasmonate-induced defenses in tomato on the potato aphid, Macrosiphum euphorbiae

Jasmonates such as jasmonic acid (JA) are plant‐signaling compounds that trigger induced resistance (IR) to a broad range of arthropod herbivores. JA‐dependent defenses are known to reduce the growth and survivorship of many chewing insects, but their impact on piercing–sucking insects such as aphids has not been extensively investigated. In this study, induced resistance was activated in tomato (Lycopersicon esculentum Mill) (Solanaceae) using a foliar application of synthetic JA, and control plants were treated with carrier solution. The life parameters of individual potato aphids and their progeny (Macrosiphum euphorbiae Thomas) (Hemiptera: Aphididae) were evaluated on the unsprayed leaves of plants in order to access the systemic effects of the foliar treatments. IR significantly reduced the longevity and net reproduction of adult aphids, as well as the percentage of juveniles to survive to maturity. These results indicate that JA application induces systemic defenses in tomato that have a direct negative impact on aphid survivorship. This study also examined aphid honeydew excretion, in order to evaluate the potential influence of induced resistance on aphid feeding behavior. The average honeydew production per aphid was comparable on plants with or without JA treatment, indicating that JA‐dependent defenses did not deter feeding. This suggests that the observed effects of JA on aphid survivorship were due to antibiotic rather than antixenotic factors. In addition to studying the effects of JA treatment on a tomato cultivar that is susceptible to aphids, this study also examined the effects of exogenous application of JA on tomato plants that carry the aphid resistance gene, Mi‐1.2. JA application did not significantly enhance or inhibit aphid control on resistant tomato. These findings expand our understanding of the effects of JA‐dependent defenses on piercing–sucking insects, and of the potential interactions between induced resistance and R‐gene mediated aphid resistance in tomato.     

Insect eggs suppress plant defence against chewing herbivores

Plants activate direct and indirect defences in response to insect egg deposition. However, whether eggs can manipulate plant defence is unknown. In Arabidopsis thaliana, oviposition by the butterfly Pieris brassicae triggers cellular and molecular changes that are similar to the changes caused by biotrophic pathogens. In the present study, we found that the plant defence signal salicylic acid (SA) accumulates at the site of oviposition. This is unexpected, as the SA pathway controls defence against fungal and bacterial pathogens and negatively interacts with the jasmonic acid (JA) pathway, which is crucial for the defence against herbivores. Application of P. brassicae or Spodoptera littoralis egg extract onto leaves reduced the induction of insect‐responsive genes after challenge with caterpillars, suggesting that egg‐derived elicitors suppress plant defence. Consequently, larval growth of the generalist herbivore S. littoralis, but not of the specialist P. brassicae, was significantly higher on plants treated with egg extract than on control plants. In contrast, suppression of gene induction and enhanced S. littoralis performance were not seen in the SA‐deficient mutant sid2‐1, indicating that it is SA that mediates this phenomenon. These data reveal an intriguing facet of the cross‐talk between SA and JA signalling pathways, and suggest that insects have evolved a way to suppress the induction of defence genes by laying eggs that release elicitors. We show here that egg‐induced SA accumulation negatively interferes with the JA pathway, and provides an advantage for generalist herbivores.     

Jasmonoyl‐l‐isoleucine hydrolase 1 (JIH1) regulates jasmonoyl‐l‐isoleucine levels and attenuates plant defenses against herbivores

For most plant hormones, biological activity is suppressed by reversible conjugation to sugars, amino acids and other small molecules. In contrast, the conjugation of jasmonic acid (JA) to isoleucine (Ile) is known to enhance the activity of JA. Whereas hydroxylation and carboxylation of JA‐Ile permanently inactivates JA‐Ile‐mediated signaling in plants, the alternative deactivation pathway of JA‐Ile by its direct hydrolysis to JA remains unstudied. We show that Nicotiana attenuata jasmonoyl‐l ‐isoleucine hydrolase 1 (JIH1), a close homologue of previously characterized indoleacetic acid alanine resistant 3 (IAR3) gene in Arabidopsis, hydrolyzes both JA‐Ile and IAA‐Ala in vitro. When the herbivory‐inducible NaJIH1 gene was silenced by RNA interference, JA‐Ile levels increased dramatically after simulated herbivory in irJIH1, compared with wild‐type (WT) plants. When specialist (Manduca sexta) or generalist (Spodoptera littoralis) herbivores fed on irJIH1 plants they gained significantly less mass compared with those feeding on wild‐type (WT) plants. The poor larval performance was strongly correlated with the higher accumulation of several JA‐Ile‐dependent direct defense metabolites in irJIH1 plants. In the field, irJIH1 plants attracted substantially more Geocoris predators to the experimentally attached M. sexta eggs on their leaves, compared with empty vector plants, which correlated with higher herbivory‐elicited emissions of volatiles known to function as indirect defenses. We conclude that NaJIH1 encodes a new homeostatic step in JA metabolism that, together with JA and JA‐Ile‐hydroxylation and carboxylation of JA‐Ile, rapidly attenuates the JA‐Ile burst, allowing plants to tailor the expression of direct and indirect defenses against herbivore attack in nature.