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.     

Herbivore-specific plant volatiles prime neighboring plants for nonspecific defense responses

Plants produce species-specific herbivore-induced plant volatiles (HIPVs) after damage. We tested the hypothesis that herbivore-specific HIPVs prime neighboring plants to induce defenses specific to the priming herbivore. Since Manduca sexta (specialist) and Heliothis virescens (generalist) herbivory induced unique HIPV profiles in Nicotiana benthamiana, we used these HIPVs to prime receiver plants for defense responses to simulated herbivory (mechanical wounding and herbivore regurgitant application). Jasmonic acid (JA) accumulations and emitted volatile profiles were monitored as representative defense responses since JA is the major plant hormone involved in wound and defense signaling and HIPVs have been implicated as signals in tritrophic interactions. Herbivore species-specific HIPVs primed neighboring plants, which produced 2 to 4 times more volatiles and JA after simulated herbivory when compared to similarly treated constitutive volatile-exposed plants. However, HIPV-exposed plants accumulated similar amounts of volatiles and JA independent of the combination of priming or challenging herbivore. Furthermore, volatile profiles emitted by primed plants depended only on the challenging herbivore species but not on the species-specific HIPV profile of damaged emitter plants. This suggests that feeding by either herbivore species primed neighboring plants for increased HIPV emissions specific to the subsequently attacking herbivore and is probably controlled by JA.     

斜纹夜蛾幼虫诱导的油菜抗虫性及其与茉莉酸信号途径的关系

植物在受植食性昆虫为害时能产生防御反应,并且植物的茉莉酸信号转导途径在这一过程中发挥着重要作用。然而, 迄今为止对于油菜Brassica campestris的诱导防御反应很少有研究报道。为此, 本实验通过测定油菜内茉莉酸和胰蛋白酶抑制剂含量研究了油菜在斜纹夜蛾Spodoptera litura幼虫为害后的抗虫性和胰蛋白酶抑制剂含量的变化,并分析这些变化在油菜诱导抗虫性与茉莉酸信号转导途径中的关系。结果表明：斜纹夜蛾幼虫取食能导致油菜体内茉莉酸和胰蛋白酶抑制剂含量系统性上升,外用茉莉酸甲酯处理也能系统性增加油菜的胰蛋白酶抑制剂含量,并且取食茉莉酸甲酯处理或斜纹夜蛾幼虫取食过的叶片能显著降低斜纹夜蛾幼虫的体重,两者的体重分别为对照植株上的67.5%和60.2%。机械损伤加斜纹夜蛾幼虫口腔分泌物处理能引起处理叶中茉莉酸和胰蛋白酶抑制剂含量的增加,但其诱导效果与机械损伤加水没有显著差异,并且两者明显低于虫害的诱导效果,两种处理的茉莉酸和胰蛋白酶抑制剂含量分别为虫害诱导的68.4%和24.4%及62.9%和36.9%;多次连续机械损伤的诱导效果与一次损伤的没有明显差异。结果说明斜纹夜蛾幼虫诱导的油菜抗虫性与茉莉酸信号转导途径有关,而其激活油菜抗虫反应的机理则可能与其特定的取食行为相关。     

The roles of jasmonate signalling in nitrogen uptake and allocation in rice (Oryza sativa L.)

Herbivore damage by chewing insects activates jasmonate (JA) signalling that can elicit systemic defense responses in rice. Few details are known, however, concerning the mechanism, whereby JA signalling modulates nutrient status in rice in response to herbivory. (¹⁵NH₄)₂SO₄ labelling experiments, proteomic surveys, and RT‐qPCR analyses were used to identify the roles of JA signalling in nitrogen (N) uptake and allocation in rice plants. Exogenous applications of methyl jasmonate (MeJA) to rice seedlings led to significantly reduced N uptake in roots and reduced translocation of recently‐absorbed ¹⁵N from roots to leaves, likely occurring as a result of down‐regulation of glutamine synthetase cytosolic isozyme 1–2 and ferredoxin–nitrite reductase. Shoot MeJA treatment resulted in a remobilization of endogenous unlabelled ¹⁴N from leaves to roots, and root MeJA treatment also increased ¹⁴N accumulation in roots but did not affect ¹⁴N accumulation in leaves of rice. Additionally, proteomic and RT‐qPCR experiments showed that JA‐mediated plastid disassembly and dehydrogenases GDH2 up‐regulation contribute to N release in leaves to support production of defensive proteins/compounds under N‐limited condition. Collectively, our results indicate that JA signalling mediates large‐scale systemic changes in N uptake and allocation in rice plants.     

JA but not JA‐Ile is the cell‐nonautonomous signal activating JA mediated systemic defenses to herbivory in Nicotiana attenuata

The whole‐plant activation of defense responses to wounding and herbivory requires systemic signaling in which jasmonates (JAs) play a pivotal role. To examine the nature of the slower cell‐nonautonomous as compared to the rapid cell‐autonomous signal in mediating systemic defenses in Nicotiana attenuata, reciprocal stem grafting‐experiments were used with plants silenced for the JA biosynthetic gene ALLENE OXIDE CYCLASE (irAOC) or plants transformed to create JA sinks by ectopically expressing Arabidopsis JA‐O‐methyltransferase (ovJMT). JA‐impaired irAOC plants were defective in the cell‐nonautonomous signaling pathway but not in JA transport. Conversely, ovJMT plants abrogated the production of a graft‐transmissible JA signal. Both genotypes displayed unaltered cell‐autonomous signaling. Defense responses (17‐hydroxygeranyllinalool diterpene glycosides, nicotine, and proteinase inhibitors) and metabolite profiles were differently induced in irAOC and ovJMT scions in response to graft‐transmissible signals from elicited wild type stocks. The performance of Manduca sexta larvae on the scions of different graft combinations was consistent with the patterns of systemic defense metabolite elicitations. Taken together, we conclude that JA and possibly MeJA, but not JA‐Ile, either directly functions as a long‐distance transmissible signal or indirectly interacts with long distance signal(s) to activate systemic defense responses.     

Herbivore-induced rice resistance against rice blast mediated by salicylic acid

In agro-ecosystems,plants are important mediators of interactions between their associated herbivorous insects and microbes,and any change in plants induced by one species may lead to cascading effects on interactions with other species.Often,such effects are regulated by phytohormones such as jasmonic acid(JA)and salicylic acid(SA).Here,we investigated the tripartite interactions among rice plants,three insect herbivores(Chilo suppressalis,Cnaphalocrocis medinalis or Nilapai-vata lugens),and the causal agent of rice blast disease,the fungus Magnaporthe oryzae.We found that pre-infestation of rice by C.suppressalis or N.lugens but not by C.medinalis conferred resistance to M.oryzae.For C.suppressalis and N.lugens,insect infestation without fungal inoculation induced the accumulation of both JA and SA in rice leaves.In contrast,infestation by C.medinalis increased JA levels but reduced SA levels.The exogenous application of SA but not of JA conferred resistance against M.oryzae.These results suggest that preinfestation by C suppressalis or N.lugens conferred resistance against M.oryzae by increasing SA accumulation.These findings enhance our understanding of the interactions among rice plant,insects and pathogens,and provide valuable information for developing an ecologically sound strategy for controlling rice blast.     

Spider mites (Tetranychus urticae) perform poorly on and disperse from plants exposed to methyl jasmonate

Jasmonates are plant hormones involved in wound and defense responses against herbivorous arthropods. Methyl jasmonate (MeJA) is used experimentally to induce defense responses in plants. In experiments outlined here we utilized a novel preference assay with unwounded plants that allowed us to study the impact of a MeJA spray on subsequent Tetranychus urticae Koch (Acari: Tetranychidae) proliferation and preference. Spraying plants with 100 μm MeJA 1 day before infestation caused mites to disperse within 2 days from treated impatiens [Impatiens wallerana Hook f., ‘Super Elfin Pink’ (Balsaminaceae)], pansy [Viola × wittrockiana Gams, ‘Imperial Beaconsfield’ (Violaceae)], and tomato [Solanum lycopersicum L., ‘Big Boy’ (Solanaceae)] plants. In addition, MeJA application reduced mite proliferation rate on impatiens and pansy by 60% (measured 22–34 days after infestation). Proteinase inhibitor (PI) assays suggested that MeJA‐induced PIs alone were not responsible for the observed results in pansy and impatiens but may have been a factor in tomato. Implications of these results in the context of MeJA‐induced resistance responses and possible directions for future research and application are discussed.     

Silicon amendment to rice plants contributes to reduced feeding in a phloem‐sucking insect through modulation of callose deposition

Silicon (Si) uptake by Poaceae plants has beneficial effects on herbivore defense. Increased plant physical barrier and altered herbivorous feeding behaviors are documented to reduce herbivorous arthropod feeding and contribute to enhanced plant defense. Here, we show that Si amendment to rice (Oryza sativa) plants contributes to reduced feeding in a phloem feeder, the brown planthopper (Nilaparvata lugens, BPH), through modulation of callose deposition. We associated the temporal dynamics of BPH feeding with callose deposition on sieve plates and further with callose synthase and hydrolase gene expression in plants amended with Si. Biological assays revealed that BPH feeding was lower in Si‐amended than in nonamended plants in the early stages post‐BPH infestation. Histological observation showed that BPH infestation triggered fast and strong callose deposition in Si‐amended plants compared with nonamended plants. Analysis using qRT‐PCR revealed that expression of the callose synthase gene OsGSL1 was up‐regulated more and that the callose hydrolase (β‐1,3‐glucanase) gene Gns5 was up‐regulated less in Si‐amended than in nonamended plants during the initial stages of BPH infestation. These dynamic expression levels of OsGSL1 and Gns5 in response to BPH infestation correspond to callose deposition patterns in Si‐amended versus nonamended plants. It is demonstrated here that BPH infestation triggers differential gene expression associated with callose synthesis and hydrolysis in Si‐amended and nonamended rice plants, which allows callose to be deposited more on sieve tubes and sieve tube occlusions to be maintained more thus contributing to reduced BPH feeding on Si‐amended plants.     

Jasmonic acid carboxyl methyltransferase regulates development and herbivory‐induced defense response in rice

Jasmonic acid(JA) and related metabolites play a key role in plant defense and growth. JA carboxyl methyltransferase(JMT) may be involved in plant defense and development by methylating JA to methyl jasmonate(Me JA) and thus influencing the concentrations of JA and related metabolites. However, no JMT gene has been well characterized in monocotyledon defense and development at the molecular level. After we cloned a rice JMT gene,Os JMT1, whose encoding protein was localized in the cytosol, we found that the recombinant Os JMT1 protein catalyzed JA to Me JA. Os JMT1 is up-regulated in response to infestation with the brown planthopper(BPH; Nilaparvata lugens). Plants in which Os JMT1 had been overexpressed(oeJMT plants) showed reduced height and yield. These oe-JMT plants also exhibited increased Me JA levels but reduced levels of herbivore-induced JA and jasmonoyl-isoleucine(JAIle). The oe-JMT plants were more attractive to BPH female adults but showed increased resistance to BPH nymphs,probably owing to the different responses of BPH female adults and nymphs to the changes in levels of H_2O_2 and Me JA in oe-JMT plants. These results indicate that Os JMT1,by altering levels of JA and related metabolites, plays a role in regulating plant development and herbivore-induced defense responses in rice.     

Innovation in anti-herbivore defense systems during neopolypoloidy - the functional consequences of instantaneous speciation

Allopolyploid hybridization instantly merges two differentially adapted genomes into one individual. Allopolyploids are often evolutionarily successful, undergoing adaptive radiations despite the genetic and physiological challenges of merging genomes. We examine a suite of induced herbivore resistance traits in three independent lines of the synthetic allopolyploid Nicotianaxmierata (Nma) and its parent species, N. miersii (Nmi) and N. attenuata (Na), to determine how a complex polygenetic adaptation fares during the early stages of neoallopolyploid formation. All species responded to Manduca sexta oral secretions (OS) with a temporally prolonged jasmonate (JA) burst. In one parent (Na), the JA burst was additionally amplified and associated with the elicitation of direct and indirect defenses. In the other parent (Nmi), OS neither amplified the JA burst nor elicited defense responses, although applied MeJA confirmed the inducibility of the defense responses. All lines of Nma retained enough aspects of Na's JA signaling to recognize OS and to accumulate sufficient direct defenses to impair the growth of Manduca larvae. Most defense-related metabolites were retained in Nma even if inherited from only one parent; however, OS-elicited volatiles, trypsin protease inhibitors (TPIs) and chlorogenic acid were lost in some lines, even though MeJA treatment elicited similar responses in all lines. Herbivore defense systems are flexibly inherited in allopolyploids, causing individuals to diverge over only a few generations; for example, line 1 of Nma could not produce TPIs after OS elicitation, whereas lines 2 and 3 could. This flexible integration of defense signaling systems with a diversity of elicited responses may explain why adaptive radiations are commonly found in allopolyploid lineages.     

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.     

Multiannual effects of induced plant defenses: Are defended plants good or bad neighbors?

Defenses induced by herbivore feeding or phytohormones such as methyl jasmonate (MeJA) can affect growth, reproduction, and herbivory, not only on the affected individual but also in its neighboring plants. Here, we report multiannual defense, growth, and reproductive responses of MeJA‐treated bilberry (Vaccinium myrtillus) and neighboring ramets. In a boreal forest in western Norway, we treated bilberry ramets with MeJA and water (control) and measured responses over three consecutive years. We observed the treatment effects on variables associated with herbivory, growth, and reproduction in the MeJA‐treated and untreated ramet and neighboring ramets distanced from 10 to 500 cm. MeJA‐treated ramets had fewer grazed leaves and browsed shoots compared to control, with higher effects in 2014 and 2015, respectively. In 2013, growth of control ramets was greater than MeJA‐treated ramets. However, MeJA‐treated ramets had more flowers and berries than control ramets 2 years after the treatment. The level of insect and mammalian herbivory was also lower in untreated neighboring ramets distanced 10–150 cm and, consistent with responses of MeJA‐treated ramets, the stronger effect was also one and 2 years delayed, respectively. The same neighboring ramets had fewer flowers and berries than untreated ramets, indicating a trade‐off between defense and reproduction. Although plant–plant effects were observed across all years, the strength varied by the distance between the MeJA‐treated ramets and its untreated neighbors. We document that induced defense in bilberry reduces both insect and mammalian herbivory, as well as growth, over multiple seasons. The defense responses occurred in a delayed manner with strongest effects one and 2 years after the induction. Additionally, our results indicate defense signaling between MeJA‐treated ramets and untreated neighbors. In summary, this study shows that induced defenses are important ecological strategies not only for the induced individual plant but also for neighboring plants across multiple years in boreal forests.     

Methyl jasmonate and cis-jasmone do not dispose of the herbivore-induced jasmonate burst in Nicotiana attenuata

The oxylipin pathway mediates wound- and herbivore-induced defense reactions in Nicotiana attenuata as evidenced by a transient jasmonic acid (JA)-burst that precedes these defense responses. The fate of this induced JA-burst remains unknown. Two derivatives of JA, its methylester, methyl jasmonate (MeJA) and cis -jasmone ( cis J), are thought to be a means of disposing of JA through volatilization at the plant surface. In N. attenuata, the headspace quantities of these compounds did not change over 3 days, although levels of MeJA and cis J increased 100- and 70-fold, respectively, in surface extracts of attacked leaves after feeding of Manduca sexta larvae or application of larval regurgitant to mechanical wounds. Inhibition of the wound-induced increase in JA with indole-3-acetic acid (IAA) revealed an association between the JA accumulation and subsequent increases in MeJA and cis J. Induced systemic increases of MeJA were not of local origin and therefore do not contribute to the inactivation of the JA-burst in the wounded leaf. The total amount of MeJA and cis J produced could only account for 9% of the JA-burst elicited by herbivore attack and therefore their production do not represent major disposal pathways of JA in N. attenuata .