Two structurally different oomycete lipophilic microbe-associated molecular patterns induce distinctive plant immune responses.

Plants recognize a variety of external signals and induce appropriate mechanisms to increase their tolerance to biotic and abiotic stresses. Precise recognition of attacking pathogens and induction of effective resistance mechanisms are critical functions for plant survival. Some molecular patterns unique to a certain group of microbes, microbe-associated molecular patterns (MAMPs), are sensed by plant cells as nonself molecules via pattern recognition receptors. While MAMPs of bacterial and fungal origin have been identified, reports on oomycete MAMPs are relatively limited. This study aimed to identify MAMPs from an oomycete pathogen Phytophthora infestans, the causal agent of potato late blight. Using reactive oxygen species (ROS) production and phytoalexin production in potato (Solanum tuberosum) as markers, two structurally different groups of elicitors, namely ceramides and diacylglycerols, were identified. P. infestans ceramides (Pi-Cer A, B, and D) induced ROS production, while diacylglycerol (Pi-DAG A and B), containing eicosapentaenoic acid (EPA) as a substructure, induced phytoalexins production in potato. The molecular patterns in Pi-Cers and Pi-DAGs essential for defense induction were identified as 9-methyl-4,8-sphingadienine (9Me-Spd) and 5,8,11,14-tetraene-type fatty acid (5,8,11,14-TEFA), respectively. These structures are not found in plants, but in oomycetes and fungi, indicating that they are microbe molecular patterns recognized by plants. When Arabidopsis (Arabidopsis thaliana) was treated with Pi-Cer D and EPA, partially overlapping but different sets of genes were induced. Furthermore, expression of some genes is upregulated only after the simultaneous treatment with Pi-Cer D and EPA, indicating that plants combine the signals from simultaneously recognized MAMPs to adapt their defense response to pathogens.

The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2 Function in Pre- and Postinvasion Defense against Phytophthora infestans in Nicotiana benthamiana.

The sesquiterpenoid capsidiol is the major phytoalexin produced by Nicotiana and Capsicum species. Capsidiol is produced in plant tissues attacked by pathogens and plays a major role in postinvasion defense by inhibiting pathogen growth. Using virus-induced gene silencing-based screening, we identified two Nicotiana benthamiana (wild tobacco) genes encoding functionally redundant full-size ABCG (PDR-type) transporters, Nb-ABCG1/PDR1 and Nb-ABCG2/PDR2, which are essential for resistance to the potato late blight pathogen Phytophthora infestans Silencing of Nb-ABCG1/2 compromised secretion of capsidiol, revealing Nb-ABCG1/2 as probable exporters of capsidiol. Accumulation of plasma membrane-localized Nb-ABCG1 and Nb-ABCG2 was observed at the site of pathogen penetration. Silencing of EAS (encoding 5-epi-aristolochene synthase), a gene for capsidiol biosynthesis, reduced resistance to P. infestans, but penetration by P. infestans was not affected. By contrast, Nb-ABCG1/2-silenced plants showed reduced penetration defense, indicating that Nb-ABCG1/2 are involved in preinvasion defense against P. infestans Plastidic GGPPS1 (geranylgeranyl diphosphate synthase) was also found to be required for preinvasion defense, thereby suggesting that plastid-produced diterpene(s) are the antimicrobial compounds active in preinvasion defense. These findings suggest that N. benthamiana ABCG1/2 are involved in the export of both antimicrobial diterpene(s) for preinvasion defense and capsidiol for postinvasion defense against P. infestans.

Regional population differences of the brown planthopper (Nilaparvata lugens Stål) in Cambodia using genotyping-by-sequencing.

The brown planthopper Nilaparvata lugens Stål (BPH) can be found year-round in tropical region and causes severe damage to rice. Although there has been documented BPH damage to rice crops in the past decade in Cambodia, the extent of this epidemic is poorly understood. Here, we examined the time variation of BPH population in the abundance of morphotypes in 13 main rice-producing provinces (86 sites) by aspirator method and in the Takeo Province (five sites) by yellow sticky trap method. At least three generations were observed during the 3-month collection period in the rainy growing season. Regarding the occurrence of BPH morphotypes, in July the macropterous adults were restricted to south Cambodia and in August all morphotypes, adults (macropterous and brachypterous) and nymphs, appeared in all sampling sites. To explain the difference of regional distribution, the genetic differentiation was analyzed in south and northwest Cambodia (three sites) by using single nucleotide polymorphisms (SNP) analysis via genotyping-by-sequencing (GBS) using next-generation sequencing. The 2455 SNPs obtained by GBS clarified the three sub-populations and they corresponded to the expected dissemination patterns. These results provide a clue to understand the differentiation and epidemic of BPH in Cambodia.

Induced resistance in Solanum lycopersicum by algal elicitor extracted from Sargassum fusiforme.

Tomato (Solanum lycopersicum) production relies heavily on the use of chemical pesticides, which is undesired by health- and environment-concerned consumers. Environment-friendly methods of controlling tomato diseases include agroecological practices, organic fungicides, and biological control. Plants' resistance against pathogens is induced by applying agents called elicitors to the plants and would lead to disease prevention or reduced severity. We investigated the ability of a novel elicitor extracted from the brown sea algae (Sargassum fusiforme) to elicit induced resistance in tomato. The studied elicitor induced hypersensitive cell death and O2 (-) production in tomato tissues. It significantly reduced severities of late blight, grey mold, and powdery mildew of tomato. Taken together, our novel elicitor has not shown any direct antifungal activity against the studied pathogens, concluding that it is an elicitor of induced resistance.

Nucleoporin 75 is involved in the ethylene-mediated production of phytoalexin for the resistance of Nicotiana benthamiana to Phytophthora infestans.

Mature Nicotiana benthamiana shows stable resistance to the oomycete pathogen Phytophthora infestans. Induction of phytoalexin (capsidiol) production is essential for the resistance, which is upregulated via a mitogen-activated protein kinase (MAPK) cascade (NbMEK2-WIPK/SIPK) followed by ethylene signaling. In this study, NbNup75 (encodes a nuclear pore protein Nucleoporin75) was identified as an essential gene for resistance of N. benthamiana to P. infestans. In NbNup75-silenced plants, initial events of elicitor-induced responses such as phosphorylation of MAPK and expression of defense-related genes were not affected, whereas induction of later defense responses such as capsidiol production and cell death induction was suppressed or delayed. Ethylene production induced by either INF1 or NbMEK2 was reduced in NbNup75-silenced plants, whereas the expression of NbEAS (a gene for capsidiol biosynthesis) induced by ethylene was not affected, indicating that Nup75 is required for the induction of ethylene production but not for ethylene signaling. Given that nuclear accumulation of polyA RNA was increased in NbNup75-silenced plants, efficient export of mRNA from nuclei via nuclear pores would be important for the timely upregulation of defense responses. Collectively, Nup75 is involved in the induction of a later stage of defense responses, including the ethylene-mediated production of phytoalexin for the resistance of N. benthamiana to P. infestans.

Nicotiana benthamiana calreticulin 3a is required for the ethylene-mediated production of phytoalexins and disease resistance against oomycete pathogen Phytophthora infestans.

Mature Nicotiana benthamiana shows strong resistance to the potato late blight pathogen Phytophthora infestans. By screening using virus-induced random gene silencing, we isolated a gene for plant-specific calreticulin NbCRT3a as a required gene for resistance of N. benthamiana against P. infestans. NbCRT3a encodes an endoplasmic reticulum quality-control (ERQC) chaperone for the maturation of glycoproteins, including glycosylated cell-surface receptors. NbCRT3a-silenced plants showed no detectable growth defects but resistance to P. infestans was significantly compromised. Defense responses induced by the treatment with INF1 (a secretory protein of P. infestans), such as production of reactive oxygen species and accumulation of phytoalexins, were suppressed in NbCRT3a-silenced N. benthamiana. Expression of an ethylene-regulated gene for phytoalexin biosynthesis, NbEAS, was reduced in NbCRT3a-silenced plants, whereas the expression of salicylic acid-regulated NbPR-1a was not affected. Consistently, induction of ethylene production by INF1 was suppressed in NbCRT3a-silenced plants. Resistance reactions induced by a hyphal wall components elicitor prepared from P. infestans were also impaired in NbCRT3a-silenced plants. However, cell death induced by active mitogen-activated protein kinase kinase (NbMEK2(DD)) was not affected by the silencing of NbCRT3a. Thus, NbCRT3a is required for the initiation of resistance reactions of N. benthamiana in response to elicitor molecules derived from P. infestans.

Bis-aryl methanone compound is a candidate of nitric oxide producing elicitor and induces resistance in Nicotiana benthamiana against Phytophthora infestans.

Nitric oxide (NO) is important in some physiological responses of plants and plays a crucial role in the regulation of both defense responses and inducing resistance to fungal pathogens. NUBS-4190, a new bis-aryl-methanone compound elicited NO production and defense responses in Nicotiana benthamiana against Phytophthora infestans. NUBS-4190 induced resistance in N. benthamiana to P. infestans, without association of reactive oxygen generation and hypersensitive cell death. Callose induction was reduced in NUBS-4190-treated N. benthamiana leaves after challenge inoculation of P. infestans indicating the penetration resistance. Involvement of pathogenesis-related 1a (NbPR1a) and nitric oxide associated 1 (NbNOA1) genes in the induced resistance to N. benthamiana against P. infestans was found to be associated with resistance. Increased susceptibility in NbPR1a- and NbNOA1-silenced plants correlated with the constitutive accumulation of PR1a transcripts and NO associated salicylic acid. Moreover, reduced NO generation in NOA1 silenced N. benthamiana plants treated with NUBS-4190 indicated that NbNOA1 is involved in NUBS-4190-mediated NO production and is required for defense responses.

Proteomic analysis of S-nitrosylated proteins in potato plant.

Nitric oxide (NO) has various functions in physiological responses in plants, such as development, hormone signaling and defense. The mechanism of how NO regulates physiological responses has not been well understood. Protein S-nitrosylation, a redox-related modification of cysteine thiol by NO, is known to be one of the important post-translational modifications to regulate activity and interactions of proteins. To elucidate NO function in plants, proteomic analysis of S-nitrosylated proteins in potato (Solanum tuberosum) was performed. Detection and functional analysis of internal S-nitrosylated proteins is technically demanding because of the instability and reversibility of the protein S-nitrosylation. By using a modified biotin switch assay optimized for potato tissues, and nano liquid chromatography combined with mass spectrometry, approximately 80 S-nitrosylated candidate proteins were identified in S-nitrosoglutathione-treated potato leaves and tuber extracts. Identified proteins included redox-related enzymes, defense-related proteins and metabolic enzymes. Some of identified proteins were synthesized in Escherichia coli, and S-nitrosylation of recombinant proteins was confirmed in vitro. Dehydroascorbate reductase 1 (DHAR1, EC 1.8.5.1), one of the identified S-nitrosylated target proteins, showed glutathione-dependent dehydroascorbate-reducing activity. Either point mutation in a target cysteine of S-nitrosylation or treatment with an NO donor, S-nitroso-L-cysteine, significantly reduced the activity of DHAR1, indicating that DHAR1 is negatively regulated by S-nitrosylation of the cysteine residue essential for the enzymatic activity. These results show that the modified method developed in this study can be used to identify proteins regulated by S-nitrosylation in potato tissues.

Effect of flutianil on the morphology and gene expression of powdery mildew.

Flutianil, a fungicide effective only on powdery mildew, was previously reported to affect the host cell's haustorial formation and nutrient absorption. Studies were conducted to investigate flutianil's primary site of action on Blumeria graminis morphology using transmission electron microscope (TEM) observation and RNA sequencing (RAN-seq) techniques. TEM observation revealed that flutianil caused the extra-haustorial matrix and fungal cell wall to be obscured, without remarkable changes of other fungal organelles. RNA-seq analysis indicated that, unlike other powdery-mildew fungicides, flutianil did not significantly affect the constantly expressed genes for the survival of B. graminis. Genes whose expression is up- or downregulated by flutianil were found; these are the three sugar transporter genes and various effector genes, mainly expressed in haustoria. These findings indicate that the primary site of action of flutianil might be in the haustoria.

AP2/ERF Transcription Factor NbERF-IX-33 Is Involved in the Regulation of Phytoalexin Production for the Resistance of Nicotiana benthamiana to Phytophthora infestans.

Plants recognize molecular patterns unique to a certain group of microbes to induce effective resistance mechanisms. Elicitins are secretory proteins produced by plant pathogenic oomycete genera including Phytophthora and Pythium. Treatment of INF1 (an elicitin produced by P. infestans) induces a series of defense responses in Nicotiana species, including reactive oxygen species (ROS) production, transient induction of ethylene production, hypersensitive cell death and accumulation of the sesquiterpenoid phytoalexin capsidiol. In this study, we analyzed the expression profiles of N. benthamiana genes after INF1 treatment by RNAseq analysis. Based on their expression patterns, N. benthamiana genes were categorized into 20 clusters and 4,761 (8.3%) out of 57,140 genes were assigned to the clusters for INF1-induced genes. All genes encoding enzymes dedicated to capsidiol production, 5-epi-aristolochene (EA) synthase (NbEAS, 10 copies) and EA dehydrogenase (NbEAH, 6 copies), and some genes for ethylene production, such as 1-aminocyclopropane 1-carboxylate (ACC) synthase (NbACS) and ACC oxidase (NbACO), were significantly upregulated by INF1 treatment. Analysis of NbEAS1 and NbEAS4 promoters revealed that AGACGCC (GCC box-like motif) is the essential cis-element required for INF1-induced expression of NbEAS genes. Given that the GCC box is known to be targeted by ERF (ethylene-responsive factor) transcription factors, we created a complete list of N. benthamiana genes encoding AP2/ERF family transcription factors, and identified 45 out of 337 AP2/ERF genes in the clusters for INF1-induced genes. Among INF1-induced NbERF genes, silencing of NbERF-IX-33 compromised resistance against P. infestans and INF1-induced production of capsidiol. Recombinant NbERF-IX-33 protein can bind to the promoter sequence of NbEAS4, suggesting that NbERF-IX-33 is a transcription factor directly regulating the expression of genes for phytoalexin production.

Age-related resistance of Nicotiana benthamiana against hemibiotrophic pathogen Phytophthora infestans requires both ethylene- and salicylic acid-mediated signaling pathways.

Phytophthora infestans, the agent of late blight disease of potato, is a hemibiotrophic pathogen with biotrophic action during early infection and necrotrophic in the later stage of colonization. Mature Nicotiana benthamiana was resistant to P. infestans, whereas relatively young plants were susceptible to this pathogen. Young plants became resistant following a pretreatment with acibenzolar-S-methyl, a functional analog of salicylic acid (SA), indicating that susceptibility of young plants is due to a lack of induction of SA signaling. Further analysis with virus-induced gene silencing indicated that NbICS1 and NbEIN2, the genes for SA biosynthesis and ethylene (ET) signaling, respectively, are required for the resistance of mature N. benthamiana against P. infestans. Furthermore, these genes are required for the production of reactive oxygen species (ROS) induced by treatment of the INF1 elicitor. In NbICS1-silenced plants, cell death induced by either INF1 or necrosis-inducing protein NPP1.1 was significantly accelerated. Expression of genes for phytoalexin (capsidiol) biosynthesis, NbEAS and NbEAH, were regulated by ET, and gene silencing of either of them compromised resistance of N. benthamiana to P. infestans. Together, these results suggest that resistance of N. benthamiana against hemibiotrophic P. infestans requires both SA-regulated appropriate induction of cell death and ET-induced production of phytoalexin.

In Vitro Assessment of Biocontrol Effects on Fusarium Head Blight and Deoxynivalenol (DON) Accumulation by DON-Degrading Bacteria.

Fusarium head blight (FHB) of cereals is a severe disease caused by the Fusarium graminearum species complex. It leads to the accumulation of the mycotoxin deoxynivalenol (DON) in grains and other plant tissues and causes substantial economic losses throughout the world. DON is one of the most troublesome mycotoxins because it is a virulence factor to host plants, including wheat, and exhibits toxicity to plants and animals. To control both FHB and DON accumulation, a biological control approach using DON-degrading bacteria (DDBs) is promising. Here, we performed a disease control assay using an in vitro petri dish test composed of germinated wheat seeds inoculated with F. graminearum (Fg) and DDBs. Determination of both grown leaf lengths and hyphal lesion lengths as a measure of disease severity showed that the inoculation of seeds with the DDBs Devosia sp. strain NKJ1 and Nocardioides spp. strains SS3 or SS4 were protective against the leaf growth inhibition caused by Fg. Furthermore, it was as effective against DON accumulation. The inoculation with strains SS3 or SS4 also reduced the inhibitory effect on leaves treated with 10 µg mL-1 DON solution (without Fg). These results indicate that the DDBs partially suppress the disease by degrading DON.

Nicotiana benthamiana RanBP1-1 Is Involved in the Induction of Disease Resistance via Regulation of Nuclear-Cytoplasmic Transport of Small GTPase Ran.

Plant cells enhance the tolerances to abiotic and biotic stresses via recognition of the stress, activation and nuclear import of signaling factors, up-regulation of defense genes, nuclear export of mRNA and translation of defense proteins. Nuclear pore-mediated transports should play critical roles in these processes, however, the regulatory mechanisms of nuclear-cytoplasmic transport during stress responses are largely unknown. In this study, a regulator of nuclear export of RNA and proteins, NbRanBP1-1 (Ran-binding protein1-1), was identified as an essential gene for the resistance of Nicotiana benthamiana to potato blight pathogen Phytophthora infestans. NbRanBP1-1-silenced plants showed delayed accumulation of capsidiol, a sesquiterpenoid phytoalexin, in response to elicitor treatment, and reduced resistance to P. infestans. Abnormal accumulation of mRNA was observed in NbRanBP1-1-silenced plants, indicating that NbRanBP1-1 is involved in the nuclear export of mRNA. In NbRanBP1-1-silenced plants, elicitor-induced expression of defense genes, NbEAS and NbWIPK, was not affected in the early stage of defense induction, but the accumulation of NbWIPK protein was reduced. Nuclear export of the small G-protein NbRan1a was activated during the induction of plant defense, whereas this process was compromised in NbRanBP1-1-silenced plants. Silencing of genes encoding the nuclear pore proteins, Nup75 and Nup160, also caused abnormal nuclear accumulation of mRNA, defects in the nuclear export of NbRan1a, and reduced production of capsidiol, resulting in decreased resistance to P. infestans. These results suggest that nuclear export of NbRan is a key event for defense induction in N. benthamiana, and both RanBP1-1 and nucleoporins play important roles in the process.

Current status of insecticide susceptibility in the brown planthopper in Cambodia.

Little information exists about pesticide availability and its effect on pest control in rural regions of developing countries. The availability of different insecticidal ingredients in rural areas of Cambodia was determined by inspecting labels on products used by farmers and in retail shops. A large number of products available in markets and used by farmers contained abamectin, emamectin benzoate, cypermethrin, and chlorpyrifos. The effects on the brown planthopper (BPH; Nilaparvata lugens (Stål)) were investigated by comparing the susceptibility of three BPH populations in Cambodia to twelve active ingredients in 2015. All populations showed high susceptibility to abamectin and cypermethrin; however, regional differences in susceptibility were observed for the other ingredients. The implication was that farmers selected the most effective products based on sellers' opinions. It is important to monitor insecticide use and BPH susceptibility in each region of Cambodia in order to minimize the risk of high BPH population densities.

EIN2-mediated signaling is involved in pre-invasion defense in Nicotiana benthamiana against potato late blight pathogen, Phytophthora infestans.

Nicotiana benthamiana ABCG1 and ABCG2 are ABC transporters which are probably involved in the export of capsidiol, the major phytoalexin of Nicotiana species. While capsidiol export by these transporters plays an essential role in post-invasion defense against Phytophthora infestans, they also export unidentified antimicrobial compound(s) involved in pre-invasion defense. In this study, promoter activity of NbABCG2 (Pabcg2a) was analyzed using a GFP marker. Expression of GFP under the control of Pabcg2a was significantly increased by co-expression with the INF1 elicitor from P. infestans. Disruption of the ethylene-responsive GCC box in Pabcg2a compromised INF1-induced activation of Pabcg2a. Consistently, penetration by P. infestans was increased by gene-silencing of NbEIN2, the key ethylene-signaling component, suggesting the involvement of ethylene for pre-invasion defense of N. benthamiana.

Methanol extract of mycelia from Phytophthora infestans-induced resistance in potato.

Plants recognize certain microbial compounds as elicitors in their active defence mechanisms. It has been shown that a series of defence reactions are induced in potato plant cells after treatment with water-soluble hyphal wall components prepared from Phytophthora infestans. In this study, a methanol extract from mycelia of P. infestans (MEM), which contains lipophilic compounds, was used as another elicitor for the induction of the defence reactions in potato. MEM elicitor induced reactive oxygen species (ROS), especially O2(-) and H2O2 production, and nitric oxide (NO) generation in potato leaves and suspension-cultured cells. Hypersensitive cell death was detected in potato leaves within 6-8 h after MEM elicitor treatment. The accumulation of phytoalexins was detected by MEM elicitor treatment in potato tubers. In potato suspension-cultured cells, several defence-related genes were induced by MEM elicitors, namely Strboh, Sthsr203J, StPVS3, StPR1, and StNR5, which regulate various defence-related functions. Enhanced resistance against P. infestans was found in MEM-treated potato plants. These results suggested that MEM elicitor is recognized by host and enhances defence activities to produce substances inhibitory to pathogens.

Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase.

Reactive oxygen species (ROS) are implicated in plant innate immunity. NADPH oxidase (RBOH; for Respiratory Burst Oxidase Homolog) plays a central role in the oxidative burst, and EF-hand motifs in the N terminus of this protein suggest possible regulation by Ca(2+). However, regulatory mechanisms are largely unknown. We identified Ser-82 and Ser-97 in the N terminus of potato (Solanum tuberosum) St RBOHB as potential phosphorylation sites. An anti-phosphopeptide antibody (pSer82) indicated that Ser-82 was phosphorylated by pathogen signals in planta. We cloned two potato calcium-dependent protein kinases, St CDPK4 and St CDPK5, and mass spectrometry analyses showed that these CDPKs phosphorylated only Ser-82 and Ser-97 in the N terminus of St RBOHB in a calcium-dependent manner. Ectopic expression of the constitutively active mutant of St CDPK5, St CDPK5VK, provoked ROS production in Nicotiana benthamiana leaves. The CDPK-mediated ROS production was disrupted by knockdown of Nb RBOHB in N. benthamiana. The loss of function was complemented by heterologous expression of wild-type potato St RBOHB but not by a mutant (S82A/S97A). Furthermore, the heterologous expression of St CDPK5VK phosphorylated Ser-82 of St RBOHB in N. benthamiana. These results suggest that St CDPK5 induces the phosphorylation of St RBOHB and regulates the oxidative burst.

Subcellular localization of Strboh proteins and NADPH-dependent O2(-)-generating activity in potato tuber tissues.

Rapid generation of reactive oxygen species (ROS) at the cell surface has been implicated in plant defence responses. Genetic evidence indicates that a plant NADPH oxidase (Rboh; respiratory burst oxidase homologue) is associated with oxidative burst. However, there is not enough physiological evidence of Rboh localization available yet. Isozyme-specific antibodies against potato StrbohA and StrbohB (St; Solanum tuberosum) were prepared to investigate the localization of these proteins. Immunoblot analyses using potato microsomal proteins revealed that StrbohA was expressed constitutively at a low level, whereas the accumulation of StrbohB protein was induced by the cell wall elicitor of the potato pathogen Phytophthora infestans. It is demonstrated here that StrbohA and StrbohB are distributed in plasma membrane fractions which have been separated by sucrose density-gradient centrifugation using their specific antibodies. Green fluorescent protein-tagged Strboh proteins were also located on the plasma membrane by transient expression assay in onion epidermal cells. Additionally, NADPH-dependent O2(-)-generating activities in plasma membrane fractions were diphenylene iodonium-sensitive and NaN3-insensitive. These data suggest that StrbohA and StrbohB are predominantly localized on the plasma membrane and regulate ROS production in defence signalling.

Nitrate reductase is responsible for elicitin-induced nitric oxide production in Nicotiana benthamiana.

Recent works have established a key role for nitric oxide (NO) in activating disease resistance in plants. Nitrate reductase (NR) is one of the enzymes that are capable of producing NO in plants. In a previous study, we reported that pathogen signals induce expression of NR genes in potato, suggesting the involvement of NR in NO production induced by pathogen signals. In this study, we cloned NR genes from Nicotiana benthamiana and investigated their involvement in NO production induced by INF1, a major elicitin secreted by Phytophthora infestans. Treatment of protoplasts prepared from N. benthamiana leaves with INF1 elevated NO production to a maximum level 1-3 h after treatment. INF1-induced NO generation was suppressed completely by an NO-specific scavenger, but partially by a nitric oxide synthase inhibitor. To investigate the involvement of NR in INF1-induced NO production, NR genes were silenced by virus-induced gene silencing. The NR-silenced plants showed yellowish leaves which resemble the characteristic of Arabidopsis NR double mutants. Silencing of NR genes significantly decreased both NO(2) (-)-producing activity and INF1-induced NO production, indicating that NR is involved in INF1-induced NO production. In contrast, overexpression of NbNR1 encoding N. benthamiana NR by Agrobacterium-mediated transient expression elevated NO(2) (-)-producing activity nine times over the control; however, INF1-induced NO production in protoplasts overexpressing NbNR1 was comparable with that in control protoplasts. These results suggest that NR is involved in INF1-induced NO production, and post-translational modification of NR or availability of substrate NO(2) (-) may be a rate-limiting step of NO production by NR.