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.

Botrytis cinerea detoxifies the sesquiterpenoid phytoalexin rishitin through multiple metabolizing pathways.

Botrytis cinerea is a necrotrophic pathogen that infects across a broad range of plant hosts, including high-impact crop species. Its generalist necrotrophic behavior stems from its ability to detoxify structurally diverse phytoalexins. The current study aims to provide evidence of the ability of B. cinerea to tolerate the sesquiterpenoid phytoalexin rishitin, which is produced by potato and tomato. While the growth of potato pathogens Phytophthora infestans (late blight) and Alternaria solani (early blight) was severely inhibited by rishitin, B. cinerea was tolerant to rishitin. After incubation of rishitin with the mycelia of B. cinerea, it was metabolized to at least six oxidized forms. Structural analysis of these purified rishitin metabolites revealed a variety of oxidative metabolism including hydroxylation at C7 or C12, ketone formation at C5, and dihydroxylation at the 10,11-olefin. Six rishitin metabolites showed reduced toxicity to P. infestans and A. solani, indicating that B. cinerea has at least 5 distinct enzymatic reactions to detoxify rishitin. Four host-specialized phytopathogenic Botrytis species, namely B. elliptica, B. allii, B. squamosa, and B. tulipae also had at least a partial ability to metabolize rishitin as B. cinerea, but their metabolic capacity was significantly weaker than that of B. cinerea. These results suggest that the ability of B. cinerea to rapidly metabolize rishitin through multiple detoxification mechanisms could be critical for its pathogenicity in potato and tomato.

Induction of plant disease resistance by mixed oligosaccharide elicitors prepared from plant cell wall and crustacean shells.

Basal plant immune responses are activated by the recognition of conserved microbe-associated molecular patterns (MAMPs), or breakdown molecules released from the plants after damage by pathogen penetration, so-called damage-associated molecular patterns (DAMPs). While chitin-oligosaccharide (CHOS), a primary component of fungal cell walls, is most known as MAMP, plant cell wall-derived oligosaccharides, cello-oligosaccharides (COS) from cellulose, and xylo-oligosaccharide (XOS) from hemicellulose are representative DAMPs. In this study, elicitor activities of COS prepared from cotton linters, XOS prepared from corn cobs, and chitin-oligosaccharide (CHOS) from crustacean shells were comparatively investigated. In Arabidopsis, COS, XOS, or CHOS treatment triggered typical defense responses such as reactive oxygen species (ROS) production, phosphorylation of MAP kinases, callose deposition, and activation of the defense-related transcription factor WRKY33 promoter. When COS, XOS, and CHOS were used at concentrations with similar activity in inducing ROS production and callose depositions, CHOS was particularly potent in activating the MAPK kinases and WRKY33 promoters. Among the COS and XOS with different degrees of polymerization, cellotriose and xylotetraose showed the highest activity for the activation of WRKY33 promoter. Gene ontology enrichment analysis of RNAseq data revealed that simultaneous treatment of COS, XOS, and CHOS (oligo-mix) effectively activates plant disease resistance. In practice, treatment with the oligo-mix enhanced the resistance of tomato to powdery mildew, but plant growth was not inhibited but rather tended to be promoted, providing evidence that treatment with the oligo-mix has beneficial effects on improving disease resistance in plants, making them a promising class of compounds for practical application.

Safety and efficacy of manual vacuum suction compared with conventional dilatation and sharp curettage and electric vacuum aspiration in surgical treatment of miscarriage: a randomized controlled trial.

BACKGROUND: The World Health Organization does not recommend dilatation and sharp curettage (D&C) for the surgical treatment of miscarriage during the first trimester because this may cause Asherman's syndrome due to endometrial damage; therefore, suction remains the primary treatment option. While manual vacuum aspiration (MVA) has been widely used since the 1990s outside Japan, the use of an MVA device (Women's MVA system) was approved in Japan in October 2015. Here, we examined the efficacy of the MVA kit in women surgically treated for miscarriage. METHODS: This retrospective cohort study was conducted between 2014 and 2018 at the International University of Health and Welfare Hospital in Japan. Women who underwent surgical treatment for miscarriage within 12 weeks of pregnancy were identified and enrolled in the study. A total of 404 women were included who underwent the following procedures: 121 D&C, 123 electric vacuum aspiration (EVA), and 160 MVA. For each participant, the duration of surgery, amount of bleeding, amount of anesthetic used, incomplete abortion requiring repeat procedures, and intraoperative/postoperative complications were evaluated. RESULTS: The duration of surgery was 13.7 ± 7.2, 11.2 ± 4.2, and 6.9 ± 4.3 min in the D&C, EVA, and MVA groups, respectively (p = 1.00). The amount of anesthetic used was not significantly different among all groups. Bleeding of ≥ 100 mL was confirmed in three (2.4%), one (0.8%), and one (0.6%) patient(s) in the D&C, EVA, and MVA groups, respectively (p = 0.50). Incomplete abortion was identified in three (2.4%), two (1.6%), and one (0.6%) patient(s) in the D&C, EVA, and MVA groups, respectively (p = 0.61). However, severe intraoperative/postoperative complications were not observed in any group. CONCLUSIONS: Surgical treatment for miscarriage performed using the MVA kit has safety and efficacy similar to those of conventional methods, such as D&C and EVA.

Draft genome sequences of Epichloë bromicola strains isolated from Elymus ciliaris.

Epichloë species are endophytic fungi that systemically colonize grass species. Here, we report the genome sequences of Epichloë bromicola strains HS and DP isolated for the first time from Elymus ciliaris in Nagoya, Japan.

Status of Cassava Witches' Broom Disease in the Philippines and Identification of Potential Pathogens by Metagenomic Analysis.

Cassava witches' broom disease (CWBD) is one of the most devastating diseases of cassava (Manihot esculenta Crantz), and it threatens global production of the crop. In 2017, a phytoplasma, Candidatus Phytoplasma luffae (Ca. P. luffae), was reported in the Philippines, and it has been considered as the causal agent, despite unknown etiology and transmission of CWBD. In this study, the nationwide occurrence of CWBD was assessed, and detection of CWBD's pathogen was attempted using polymerase chain reaction (PCR) and next-generation sequencing (NGS) techniques. The results showed that CWBD has spread and become severe, exhibiting symptoms such as small leaf proliferation, shortened internodes, and vascular necrosis. PCR analysis revealed a low phytoplasma detection rate, possibly due to low titer, uneven distribution, or absence in the CWBD-symptomatic cassava. In addition, NGS techniques confirm the PCR results, revealing the absence or extremely low phytoplasma read counts, but a surprisingly high abundance of fastidious and xylem-limited fungus, Ceratobasidium sp. in CWBD-symptomatic plants. These findings cast doubt over the involvement of phytoplasma in CWBD and instead highlight the potential association of Ceratobasidium sp., strongly supporting the recent findings in mainland Southeast Asia. Further investigations are needed to verify the etiology of CWBD and identify infection mechanisms of Ceratobasidium sp. to develop effective diagnostic and control methods for disease management.

Bacterial cytochrome P450 system catabolizing the Fusarium toxin deoxynivalenol.

Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (k(cat)/K(m)) of 6.4 mM(-1) s(-1). The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat.

Inter-Day Variation in the Fasting Plasma Lipopolysaccharide Concentration in the Morning Is Associated with Inter-Day Variation in Appetite in Japanese Males: A Short-Term Cohort Study.

Injection of lipopolysaccharide (LPS), a product of gut bacteria, into the blood increases blood triglycerides and cortisol, an appetite-stimulating hormone. Meanwhile, small amounts of LPS derived from gut bacteria are thought to enter the bloodstream from the gut in daily basis. This study aimed to investigate the effect of LPS influx on appetite or lipid metabolism in humans in everyday life. We measured the fasting plasma LPS concentration before breakfast and the corresponding days' appetite and fat-burning markers for 10 days in four Japanese males (28-31 years) and analyzed the correlation of their inter-day variation. The LPS concentration was negatively correlated with fullness, and positively correlated with the carbohydrate intake. Against our hypothesis, the LPS concentration was positively correlated with the fasting breath acetone concentration, a fat-burning marker. There was a positive correlation between the LPS concentration and fasting body mass index (BMI), but the inter-day variation in BMI was slight. The results suggest that the LPS influx in everyday life is at least associated with appetite in the day.

Neoxanthin is undetectable in human blood after ingestion of fresh young spinach leaf.

In a previous study, we demonstrated that the carotenoid neoxanthin was contained in young leafy vegetables including spinach and showed a fat accumulation inhibitory effect in vitro. To evaluate the bioavailability of neoxanthin, a raw young spinach leaf (100 g day-1 for 4 weeks) intake test was performed on 14 participants (36.5 ± 8.0 years; male:female ratio = 9:5). Neoxanthin, neochrome, ß-carotene, and lutein concentration in the spinach and blood of participants (before and after the test) was measured using high performance liquid chromatography. Neither neoxanthin nor neochrome was detected in the blood samples, whereas ß-carotene and lutein concentration significantly increased (1.4- and 1.9-fold, respectively) during testing. Neoxanthin bioavailability in humans is low; thus, it is unlikely to have a fat accumulation inhibitory effect in vivo, contrary to the result in vitro. Ingesting the leafy vegetables raw can help maintain high neoxanthin levels, but it is not beneficial for neoxanthin bioavailability.

Draft Genome Sequences of Neofusicoccum parvum Strains Isolated from Mango and Rice-Paper Plant (Tetrapanax papyrifer).

Neofusicoccum parvum is a polyxenous phytopathogenic fungus that infects important fruits, such as grapes and mangoes. Here, we report the genome sequences of N. parvum strains that were isolated from mango in Okinawa, Japan (strain PPO83), and an invasive weed (rice-paper plant [Tetrapanax papyrifer]) in Nagoya, Japan (strain NSSI1).

Botrytis cinerea tolerates phytoalexins produced by Solanaceae and Fabaceae plants through an efflux transporter BcatrB and metabolizing enzymes.

Botrytis cinerea, a plant pathogenic fungus with a wide host range, has reduced sensitivity to fungicides as well as phytoalexins, threatening cultivation of economically important fruits and vegetable crops worldwide. B. cinerea tolerates a wide array of phytoalexins, through efflux and/or enzymatic detoxification. Previously, we provided evidence that a distinctive set of genes were induced in B. cinerea when treated with different phytoalexins such as rishitin (produced by tomato and potato), capsidiol (tobacco and bell pepper) and resveratrol (grape and blueberry). In this study, we focused on the functional analyses of B. cinerea genes implicated in rishitin tolerance. LC/MS profiling revealed that B. cinerea can metabolize/detoxify rishitin into at least 4 oxidized forms. Heterologous expression of Bcin08g04910 and Bcin16g01490, two B. cinerea oxidoreductases upregulated by rishitin, in a plant symbiotic fungus Epichloë festucae revealed that these rishitin-induced enzymes are involved in the oxidation of rishitin. Expression of BcatrB, encoding an exporter of structurally unrelated phytoalexins and fungicides, was significantly upregulated by rishitin but not by capsidiol and was thus expected to be involved in the rishitin tolerance. Conidia of BcatrB KO (ΔbcatrB) showed enhanced sensitivity to rishitin, but not to capsidiol, despite their structural similarity. ΔbcatrB showed reduced virulence on tomato, but maintained full virulence on bell pepper, indicating that B. cinerea activates BcatrB by recognizing appropriate phytoalexins to utilize it in tolerance. Surveying 26 plant species across 13 families revealed that the BcatrB promoter is mainly activated during the infection of B. cinerea in plants belonging to the Solanaceae, Fabaceae and Brassicaceae. The BcatrB promoter was also activated by in vitro treatments of phytoalexins produced by members of these plant families, namely rishitin (Solanaceae), medicarpin and glyceollin (Fabaceae), as well as camalexin and brassinin (Brassicaceae). Consistently, ΔbcatrB showed reduced virulence on red clover, which produces medicarpin. These results suggest that B. cinerea distinguishes phytoalexins and induces differential expression of appropriate genes during the infection. Likewise, BcatrB plays a critical role in the strategy employed by B. cinerea to bypass the plant innate immune responses in a wide variety of important crops belonging to the Solanaceae, Brassicaceae and Fabaceae.

Glutathione reductase/glutathione is responsible for cytotoxic elemental sulfur tolerance via polysulfide shuttle in fungi.

Fungi that can reduce elemental sulfur to sulfide are widely distributed, but the mechanism and physiological significance of the reaction have been poorly characterized. Here, we purified elemental sulfur-reductase (SR) and cloned its gene from the elemental sulfur-reducing fungus Fusarium oxysporum. We found that NADPH-glutathione reductase (GR) reduces elemental sulfur via glutathione as an intermediate. A loss-of-function mutant of the SR/GR gene generated less sulfide from elemental sulfur than the wild-type strain. Its growth was hypersensitive to elemental sulfur, and it accumulated higher levels of oxidized glutathione, indicating that the GR/glutathione system confers tolerance to cytotoxic elemental sulfur by reducing it to less harmful sulfide. The SR/GR reduced polysulfide as efficiently as elemental sulfur, which implies that soluble polysulfide shuttles reducing equivalents to exocellular insoluble elemental sulfur and generates sulfide. The ubiquitous distribution of the GR/glutathione system together with our findings that GR-deficient mutants derived from Saccharomyces cerevisiae and Aspergillus nidulans reduced less sulfur and that their growth was hypersensitive to elemental sulfur indicated a wide distribution of the system among fungi. These results indicate a novel biological function of the GR/glutathione system in elemental sulfur reduction, which is distinguishable from bacterial and archaeal mechanisms of glutathione- independent sulfur reduction.

Iodide oxidation by a novel multicopper oxidase from the alphaproteobacterium strain Q-1.

Alphaproteobacterium strain Q-1 is able to oxidize iodide (I(-)) to molecular iodine (I(2)) by an oxidase-like enzyme. One of the two isoforms of the iodide-oxidizing enzyme (IOE-II) produced by this strain was excised from a native polyacrylamide gel, eluted, and purified. IOE-II appeared as a single band (51 kDa) and showed significant in-gel iodide-oxidizing activity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis without heat treatment. However, at least two bands with much higher molecular masses (150 and 230 kDa) were observed with heat treatment (95°C, 3 min). IOE-II was inhibited by NaN(3), KCN, EDTA, and a copper chelator, o-phenanthroline. In addition to iodide, IOE-II showed significant activities toward phenolic compounds such as syringaldazine, 2,6-dimethoxy phenol, and p-phenylenediamine. IOE-II contained copper atoms as prosthetic groups and had UV/VIS absorption peaks at 320 and 590 nm. Comparison of several internal amino acid sequences obtained from trypsin-digested IOE-II with a draft genome sequence of strain Q-1 revealed that the products of two open reading frames (IoxA and IoxC), with predicted molecular masses of 62 and 71 kDa, are involved in iodide oxidation. Furthermore, subsequent tandem mass spectrometric analysis repeatedly detected peptides from IoxA and IoxC with high sequence coverage (32 to 40%). IoxA showed homology with the family of multicopper oxidases and included four copper-binding regions that are highly conserved among various multicopper oxidases. These results suggest that IOE-II is a multicopper oxidase and that it may occur as a multimeric complex in which at least two proteins (IoxA and IoxC) are associated.

A novel actinomycete derived from wheat heads degrades deoxynivalenol in the grain of wheat and barley affected by Fusarium head blight.

Deoxynivalenol (DON) is a hazardous and globally prevalent mycotoxin in cereals. It commonly accumulates in the grain of wheat, barley and other small grain cereals affected by Fusarium head blight (caused by several Fusarium species). The concept of reducing DON in naturally contaminated grain of wheat or barley using a DON-degrading bacterium is promising but has not been accomplished. In this study, we isolated a novel DON-utilising actinomycete, Marmoricola sp. strain MIM116, from wheat heads through a novel isolation procedure including an in situ plant enrichment step. Strain MIM116 had background degradation activity, and the activity was enhanced twofold by the consumption of DON. Among Tween 20, Triton X-100 and Tween 80, we selected Tween 80 as a spreading agent of strain MIM116 because it promoted DON degradation and the growth of strain MIM116 in the presence of DON. The inoculation of MIM116 cell suspension plus 0.01% Tween 80 into 1,000 harvested kernels of wheat and barley resulted in a DON decrease from approximately 3 mg kg(-1) to less than 1 mg kg(-1) of dry kernels, even when cells had only basal levels of DON-degrading activity. To the best of our knowledge, this is the first report that describes (1) the isolation of a DON-degrading bacterium from wheat heads, (2) the effects of surfactants on the biodegradation of DON and (3) the decrease of DON levels in naturally contaminated wheat and barley grain using a DON-degrading bacterium.

Draft Genome Sequences of Prototrophic and Biotin-Auxotrophic Fusarium langsethiae Strains Isolated from an Oat Grain in the Northern Region of Russia.

Fusarium langsethiae is a suspected plant-pathogenic fungus causing cereal contamination with trichothecene mycotoxins. Here, we report the genome sequences of two F. langsethiae strains, MFG217701 (a prototroph) and MFG217702 (a biotin auxotroph), isolated from a grain of oat harvested in Russia.

Botrytis cinerea identifies host plants via the recognition of antifungal capsidiol to induce expression of a specific detoxification gene.

The gray mold pathogen Botrytis cinerea has a broad host range, causing disease in >400 plant species, but it is not known how this pathogen evolved this polyxenous nature. Botrytis cinerea can metabolize a wide range of phytoalexins, including the stilbenoid resveratrol in grape, and the sesquiterpenoids capsidiol in tobacco and rishitin in potato and tomato. In this study, we analyzed the metabolism of sesquiterpenoid phytoalexins by B. cinerea. Capsidiol was dehydrogenated to capsenone, which was then further oxidized, while rishitin was directly oxidized to epoxy- or hydroxyrishitins, indicating that B. cinerea has separate mechanisms to detoxify structurally similar sesquiterpenoid phytoalexins. RNA-seq analysis revealed that a distinct set of genes were induced in B. cinerea when treated with capsidiol or rishitin, suggesting that B. cinerea can distinguish structurally similar phytoalexins to activate appropriate detoxification mechanisms. The gene most highly upregulated by capsidiol treatment encoded a dehydrogenase, designated Bccpdh. Heterologous expression of Bccpdh in a capsidiol-sensitive plant symbiotic fungus, Epichloë festucae, resulted in an acquired tolerance of capsidiol and the ability to metabolize capsidiol to capsenone, while B. cinerea Δbccpdh mutants became relatively sensitive to capsidiol. The Δbccpdh mutant showed reduced virulence on the capsidiol producing Nicotiana and Capsicum species but remained fully pathogenic on potato and tomato. Homologs of Bccpdh are found in taxonomically distant Ascomycota fungi but not in related Leotiomycetes species, suggesting that B. cinerea acquired the ancestral Bccpdh by horizontal gene transfer, thereby extending the pathogenic host range of this polyxenous pathogen to capsidiol-producing plant species.

Nocardioides sp. strain WSN05-2, isolated from a wheat field, degrades deoxynivalenol, producing the novel intermediate 3-epi-deoxynivalenol.

The mycotoxin deoxynivalenol (DON) causes serious problems worldwide in the production of crops such as wheat and barley because of its toxicity toward humans and livestock. A bacterial culture capable of degrading DON was obtained from soil samples collected in wheat fields using an enrichment culture procedure. The isolated bacterium, designated strain WSN05-2, completely removed 1,000 µg/mL of DON from the culture medium after incubation for 10 days. On the basis of phylogenetic studies, WSN05-2 was classified as a bacterium belonging to the genus Nocardioides. WSN05-2 showed significant growth in culture medium with DON as the sole carbon source. High-performance liquid chromatography analysis indicated the presence of a major initial metabolite of DON in the culture supernatant. The metabolite was identified as 3-epi-deoxynivalenol (3-epi-DON) by mass spectrometry and (1)H and (13)C nuclear magnetic resonance analysis. The amount of DON on wheat grain was reduced by about 90% at 7 days after inoculation with WSN05-2. This is the first report of a Nocardioides sp. strain able to degrade DON and of the yet unknown 3-epi-DON as an intermediate in the degradation of DON by a microorganism.

Geographic Distribution, Genetic Variability and Biological Properties of Rice Orange Leaf Phytoplasma in Southeast Asia.

Rice orange leaf phytoplasma (ROLP) causes clear orange to yellowish leaf discoloration and severe stunting in rice seedlings. The ecological and biological characteristics of ROLP are largely unknown because the disease has not widely caused serious problems in rice cultivated areas, thereby leading to the low accumulation of research data. However, in the past decade, the disease became a threat to rice production, particularly in South China and India; it has also been recognised in other Asian countries, such as Vietnam, Thailand and the Philippines. Here, we observed the occurrence of ROLP in paddies of the Southeast Asian counties (Cambodia, Vietnam and the Philippines) and found that the isolates in the Philippines and Vietnam were monophyletic, while those in India, Thailand and Cambodia were more diverse, suggesting their potential origins. In Cambodia, it was revealed that following polymerase chain reaction (PCR) detection, the known ROLP-insect vectors, N. virescens Distant and Recilia dorsalis Motchulsky, were ROLP-positive, indicating their roles in pathogen dispersal. Moreover, fluorescent and scanning electron microscopy revealed the intensive accumulation of the phytoplasma in phloem tissues and massive accumulation of storage starch in vascular bundle sheath and parenchyma. Altogether, this study illustrated the genetic variability of global ROLP isolates and the pathogen's biological impact on rice tissue.

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.