OsGLP3-7 positively regulates rice immune response by activating hydrogen peroxide,jasmonic acid,and phytoalexin metabolic pathways

Although germin-like proteins (GLPs) have been demonstrated to participate in plant biotic stress responses, their specific functions in rice disease resistance are still largely unknown. Here, we report the identification and characterization of OsGLP3-7, a member of the GLP family in rice. Expression of OsGLP3-7 was significantly induced by pathogen infection, jasmonic acid (JA) treatment, and hydrogen peroxide (H₂O₂) treatment. OsGLP3-7 was highly expressed in leaves and sublocalized in the cytoplasm. Overexpression of OsGLP3-7 increased plant resistance to leaf blast, panicle blast, and bacterial blight, whereas disease resistance in OsGLP3-7 RNAi silenced plants was remarkably compromised, suggesting this gene is a positive regulator of disease resistance in rice. Further analysis showed that OsGLP3-7 has superoxide dismutase (SOD) activity and can influence the accumulation of H₂O₂ in transgenic plants. Many genes involved in JA and phytoalexin biosynthesis were strongly induced, accompanied with elevated levels of JA and phytoalexins in OsGLP3-7-overexpressing plants, while expression of these genes was significantly suppressed and the levels of JA and phytoalexins were reduced in OsGLP3-7 RNAi plants compared with control plants, both before and after pathogen inoculation. Moreover, we showed that OsGLP3-7-dependent phytoalexin accumulation may, at least partially, be attributed to the elevated JA levels observed after pathogen infection. Taken together, our results indicate that OsGLP3-7 positively regulates rice disease resistance by activating JA and phytoalexin metabolic pathways, thus providing novel insights into the disease resistance mechanisms conferred by GLPs in rice.     

Jasmonoyl-l-isoleucine is required for the production of a flavonoid phytoalexin but not diterpenoid phytoalexins in ultraviolet-irradiated rice leaves

Rice produces low-molecular-weight antimicrobial compounds known as phytoalexins, in response to not only pathogen attack but also abiotic stresses including ultraviolet (UV) irradiation. Rice phytoalexins are composed of diterpenoids and a flavonoid. Recent studies have indicated that endogenous jasmonyl-l-isoleucine (JA-Ile) is not necessarily required for the production of diterpenoid phytoalexins in blast-infected or CuCl₂-treated rice leaves. However, JA-Ile is required for the accumulation of the flavonoid phytoalexin, sakuranetin. Here, we investigated the roles of JA-Ile in UV-induced phytoalexin production. We showed that UV-irradiation induces the biosynthesis of JA-Ile and its precursor jasmonic acid. We also showed that rice jasmonate biosynthesis mutants produced diterpenoid phytoalexins but not sakuranetin in response to UV, indicating that JA-Ile is required for the production of sakuranetin but not diterpenoid phytoalexins in UV-irradiated rice leaves.     

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.     

Involvement of Jasmonic Acid in Elicitor-Induced Phytoalexin Production in Suspension-Cultured Rice Cells

It has been suggested that jasmonic acid (JA) could be an integral part of a general signal transduction system regulating inducible defense genes in plants. It was reported that treatment with an elicitor (N-acetylchitoheptaose) induced production of phytoalexin in suspension-cultured rice (Oryza sativa L.) cells. In this study, the role of JA in the induction of phytoalexin production by N-acetylchitoheptaose was investigated. Exogenously applied ([plus or minus])-JA (10-4 M) clearly induced the production of momilactone A, a major phytoalexin, in suspension-cultured rice cells. On the other hand, in rice cells treated with N-acetylchitoheptaose, endogenous JA was rapidly and transiently accumulated prior to accumulation of momilactone A. Treatment with ibuprofen, an inhibitor of JA biosynthesis, reduced production of momilactone A in the cells treated with N-acetylchitoheptaose, but the addition of ([plus or minus])-JA increased production of momilactone A to levels higher than those in the elicited rice cells. These results strongly suggest that JA functions as a signal transducer in the induction of biosynthesis of momilactone A by N-acetylchitoheptaose in suspension-cultured rice cells.     

Hydrogen peroxide is involved in methyl jasmonate-induced senescence of rice leaves

The role of H₂O₂ in the senescence of detached rice leaves induced by methyl jasmonate (MJ) was investigated. MJ treatment resulted in H₂O₂ production in detached rice leaves, which was prior to the occurrence of leaf senescence. Dimethylthiourea, a chemical trap of H₂O₂, was observed to be effective in inhibiting MJ‐induced senescence and MJ‐increased malondialdehyde (MDA) content in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, prevented MJ‐induced H₂O₂ production, suggesting that NADPH oxidase is a H₂O₂‐generating enzyme in MJ‐treated detached rice leaves. DPI and IMD also inhibited MJ‐promoted senescence and MJ‐increased MDA content in detached rice leaves. Phosphatidylinositol 3‐kinase inhibitors wortmannin (WM) or LY 294002 (LY) inhibited MJ‐induced H₂O₂ production and senescence of detached rice leaves. Exogenous H₂O₂ reversed the inhibitory effect of WM or LY. In terms of leaf senescence, it was observed that rice seedlings of cultivar Taichung Native 1 (TN1) are jasmonic acid (JA)‐sensitive and those of cultivar Tainung 67 (TNG67) are JA‐insensitive. On treatment with JA, H₂O₂ accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Evidence was also provided to show that MJ‐induced H₂O₂ production in detached rice leaves is abscisic acid (ABA)‐independent. Ethylene action inhibitor, silver thiosulfate, was observed to inhibit MJ‐ and ABA‐induced H₂O₂ production and senescence of detached rice leaves, suggesting that the action of MJ and ABA is ethylene‐dependent.     

Rice OsPAD4 functions differently from Arabidopsis AtPAD4 in host‐pathogen interactions

The extensively studied Arabidopsis phytoalexin deficient 4 (AtPAD4) gene plays an important role in Arabidopsis disease resistance; however, the function of its sequence ortholog in rice is unknown. Here, we show that rice OsPAD4 appears not to be the functional ortholog of AtPAD4 in host‐pathogen interactions, and that the OsPAD4 encodes a plasma membrane protein but that AtPAD4 encodes a cytoplasmic and nuclear protein. Suppression of OsPAD4 by RNA interference (RNAi) increased rice susceptibility to the biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo), which causes bacteria blight disease in local tissue. OsPAD4‐RNAi plants also show compromised wound‐induced systemic resistance to Xoo. The increased susceptibility to Xoo was associated with reduced accumulation of jasmonic acid (JA) and phytoalexin momilactone A (MOA). Exogenous application of JA complemented the phenotype of OsPAD4‐RNAi plants in response to Xoo. The following results suggest that OsPAD4 functions differently than AtPAD4 in response to pathogen infection. First, OsPAD4 plays an important role in wound‐induced systemic resistance, whereas AtPAD4 mediates systemic acquired resistance. Second, OsPAD4‐involved defense signaling against Xoo is JA‐dependent, but AtPAD4‐involved defense signaling against biotrophic pathogens is salicylic acid‐dependent. Finally, OsPAD4 is required for the accumulation of terpenoid‐type phytoalexin MOA in rice‐bacterium interactions, but AtPAD4‐mediated resistance is associated with the accumulation of indole‐type phytoalexin camalexin.     

Differential responses of rice to inoculation with wild-type and non-pathogenic mutants of Magnaporthe oryzae

We analyzed the response of rice to Magnaporthe oryzae infection using two mutant strains deficient in Mgb1 and Mst12, which are essential for the development of appresoria and penetration pegs. Both mutants induced the much lower levels of accumulation of phytoalexins than wild-type, suggesting that the massive production of phytoalexins requires the fungal invasion of rice cells. Intense accumulation of H₂O₂ in a single whole cell also required fungal penetration. Microarray analysis of rice gene expression revealed mutant-specific gene expression, indicating that signal exchange between rice and M. oryzae commence before fungal penetration of the rice cell. In situ detection of mRNAs for peroxidase and β-1,3-glucanase showed that expression of these genes also occurs after penetration as observed for phytoalexin production. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Tomoaki Kato, Shigeru Tanabe, and Marie Nishimura contributed equally to this work. Accession number of the original microarray data in NCBI is GSE9450.     

Accumulation of salicylic acid,jasmonic acid and phytoalexins in rice, <Emphasis Type="Italic">Oryza sativa</Emphasis>, infested by the white-backed planthopper, <Emphasis Type="Italic">Sogatella furcifera</Emphasis> (Hemiptera: Delphacidae)

In order to clarify the mechanism of induced resistance to blast disease in rice, Oryza sativa, that had been previously infested by the white-backed planthopper, Sogatella furcifera Horváth, we first investigated the accumulation of salicylic acid (SA) and jasmonic acid (JA) in rice plants infested by the planthopper. The results confirmed that infestation of S. furcifera strongly stimulates the production of SA and JA in rice. These results indicate that both salicylate- and jasmonate-mediated pathways (SA and JA pathways), which are involved in the general defense system in plants, were activated in rice infested by S. furcifera. Further results confirmed that S. furcifera infestation induces accumulation of a major rice diterpenoid phytoalexin, momilactone A, and a flavonoid phytoalexin, sakuranetin, which are well known as antimicrobial chemicals, particularly in blast disease caused by the blast fungus, Magnaporthe oryzae B. Couch. All these results strongly suggest the following hypothetical mechanism of induced-resistance to M. oryzae in rice infested by S. furcifera. First, S. furcifera releases some elicitor-active compounds, which might be produced in the salivary glands, into the rice plant during feeding. Next, the defense signal systems, SA- and JA-mediated pathways, are activated by the elicitor. Finally, phytoalexins are induced in rice as antimicrobial compounds mainly through activation of the JA-mediated pathway.     

Methionine-induced phytoalexin production in rice leaves

The application of methionine on wounded rice leaves induced the production of rice phytoalexins, sakuranetin and momilactone A. This induction resulted from stimulation of phenylalanine ammonia-lyase and naringenin 7-O-methyltransferase activity. Jasmonic acid, ethylene, and active oxygen species are important as signal transducers in disease resistance mechanisms. However, although the endogenous level of jasmonic acid rapidly increased in reaction to wound, methionine treatment could not induced endogenous JA production. Ethylene induced the production of the flavonoid phytoalexin, sakuranetin, but did not induce the production of a terpenoid phytoalexin, momilactone A. On the other hand, a free radical scavenger, Tiron, counteracted the induction of both sakuranetin and momilactone A production in methionine-treated leaves. Active oxygen species may be important in methionine-induced production of phytoalexins.     

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.     

Intracellular localization and physiological function of a rice Ca2+-permeable channel OsTPC1

Two-pore channels (TPCs) are cation channels with a voltage-sensor domain conserved in plants and animals. Rice OsTPC1 is predominantly localized to the plasma membrane (PM), and assumed to play an important role as a Ca²⁺-permeable cation channel in the regulation of cytosolic Ca²⁺ rise and innate immune responses including hypersensitive cell death and phytoalexin biosynthesis in cultured rice cells triggered by a fungal elicitor, xylanase from Trichoderma viride. In contrast, Arabidopsis AtTPC1 is localized to the vacuolar membrane (VM). To gain further insights into the intracellular localization of OsTPC1, we stably expressed OsTPC1-GFP in tobacco BY-2 cells. Confocal imaging and membrane fractionation revealed that, unlike in rice cells, the majority of OsTPC1-GFP fusion protein was targeted to the VM in tobacco BY-2 cells. Intracellular localization and functions of the plant TPC family is discussed.     

Temperature-related effects of treatments with jasmonic and salicylic acids on Arabidopsis infected with cucumber mosaic virus

Plant-virus interactions are affected by environmental factors, including temperature. Plant defenses are often inhibited by high or low temperature. In this study, oxidative damage and gene expression were detected in Arabidopsis thaliana infected with cucumber mosaic virus (CMV) at different temperatures. Before virus inoculation, plants were treated with jasmonic acid (JA) and salicylic acid (SA), both of which are important signaling molecules in plant defense responses. The levels of MDA and hydrogen peroxide (H₂O₂), and electrolyte leakage were significantly higher in CMV-infected leaves at 15 and 37°C. The accumulation of H₂O₂ and superoxide radical (O ₂ ^·? ) was obviously suppressed by spraying with JA followed by SA (JA → SA) at different temperatures. The CMV-CP expression analysis showed that virus replication was inhibited efficiently in the (JA → SA) treatment. Therefore, many JA- and SA-responsible resistance genes were quantified; MPK4 was expressed highly and steadily in the (JA → SA) treatment. To further confirm the role of MPK4, the CMV-CP gene expression was evaluated in wild-type Arabidopsis and its mpk4 mutant infected with CMV. The results suggested that MPK4 might play an important role in the antagonism between JA and SA at temperature fluctuation.     

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.