Daigo Takemoto

Nagoya University | Meidai · Graduate School of Bio-Agricultural Sciences

Regulated synthesis of reactive oxygen species (ROS) by membrane-bound fungal NADPH oxidases (Nox) plays a key role in fungal morphogenesis, growth, and development. Generation of reactive oxygen species (ROS) by the plant symbiotic fungus, Epichloë festucae, requires functional assembly of a multisubunit complex composed of NoxA, a regulatory comp...

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 post-invasion defense by inhibiting pathogen growth. Using virus-induced gene silencing (VIGS)-based screening, we identified two Nicotiana benthamiana (wild tobacc...

Agrobacterium tumefaciens is a bacterial pathogen that causes crown gall disease on a wide range of eudicot plants by genetic transformation. Besides T-DNA integrated by natural transformation in vegetative tissues of plants by pathogenic Agrobacterium, previous reports have indicated that T-DNA sequences originating from ancestral Agrobacterium sp...

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...

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-ass...

Social parasites employ diverse strategies to deceive and infiltrate their hosts in order to benefit from stable resources. Although escape behaviours are considered an important part of these multipronged strategies, little is known about the repertoire of potential escape behaviours and how they facilitate integration into the host colony. Here,...

Plants recognize a variety of environmental molecules, thereby triggering appropriate responses to biotic or abiotic stresses. Substances containing microbes-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs) are representative inducers of pathogen resistance and damage repair, thus treatment of healthy plants wi...

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.

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 tran...

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 phytoalex...

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...

Basal plant immune responses are activated by the recognition of the conserved pathogen-associated molecular patterns (PAMPs), or breakdown molecules released from plants after damage by pathogen infection, so-called danger-associated molecular patterns (DAMPs). While chitin-oligosaccharide (CHOS), a primary component of the fungal cell wall, is mo...

Synthesis of reactive oxygen species (ROS) by specific NADPH oxidases (Nox) can serve both defense and differentiation signalling roles in animals and plants. Fungi have three subfamilies of NADPH oxidase, NoxA, NoxB and NoxC. NoxA and NoxB have a structure very similar to the human gp91phox whereas NoxC has a Ca2+ binding motif similar to that fou...

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...

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).

Rice-paper plant, Tetrapanax papyrifer is a woody plant native to Taiwan and widely distributed in western Japan. Due to its low commercial value, little is known about its interactions with microorganisms. Here, we isolated Neofusicoccum parvum, a known causal agent of grape stem rot and mango stem-end rot, from leaf blight on T. papyrifer. Compar...

Phytophthora are plant pathogens that damage agricultural products. Lycosides (1a–d), found in vegetable juice, have the potential to curb the rapid outbreak and crop damage caused by the asexual reproduction of Phytophthora. Here, aglycones 2a, b with slightly higher activity than lycosides were synthesized as a diastereomeric mixture (mix-2) poss...

Botrytis cinerea, a generalist fungal pathogen of economically important crop species, has been shown to exhibit reduced sensitivity to fungicides and plant toxins. Specifically, previous reports indicate B. cinereas efficacy in tolerating a wide array of phytoalexins, toxic plant metabolites that play key role in plant immune defense strategies. P...

Phytophthora is a genus of fungus-like microorganisms that damages important crops, such as potatoes and tomatoes. Its asexual reproduction, which results in the production of numerous motile zoospores, is the cause of quick and severe outbreaks and crop damage. The search for substances that selectively inhibit the asexual reproduction of Phytopht...

Genome rearrangements in filamentous fungi are prevalent but little is known about the modalities of their evolution, in part because few complete genomes are available within a single genus. To address this, we have generated and compared 15 complete telomere-to-telomere ge-nomes across the phylogeny of a single genus of filamentous fungi, Epichlo...

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.

In plants, many invading microbial pathogens are recognized by cell-surface pattern recognition receptors, which induce defense responses. Here, we show that the ceramide Phytophthora infestans-ceramide D (Pi-Cer D) from the plant pathogenic oomycete P. infestans triggers defense responses in Arabidopsis. Pi-Cer D is cleaved by an Arabidopsis apopl...

The gray mold pathogen Botrytis cinerea has a broad host range, causing disease in over 400 plant species, but it is not known how this pathogen evolved this polyxenous nature. B. cinerea can metabolize a wide range of phytoalexins, including the stilbenoid, resveratrol, and the sesquiterpenoids capsidiol in tobacco, and rishitin in potato and toma...

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, in...

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,...

Partitiviruses are one of the most prevalent double-stranded RNA viruses that have been identified mostly in filamentous fungi and plants. Partitiviruses generally infect host fungi asymptomatically but infrequently exert significant effect(s) on morphology and virulence, thus being considered a potential source of biological control agents against...

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-ass...

In plants, many invading microbial pathogens are recognized by cell-surface pattern recognition receptors (PRRs), inducing defense responses; yet how PRRs perceive pathogen sphingolipids remains unclear. Here, we show that the ceramide Pi-Cer D from a plant pathogenic oomycete Phytophthora infestans triggers defense responses in Arabidopsis. Pi-Cer...

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) techni...

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. Howeve...

Filamentous fungal cells, unlike yeasts, fuse during vegetative growth. The orthologs of mitogen‐activated protein (MAP) kinase Fus3 and transcription factor Ste12 are commonly involved in the regulation of cell fusion. However, the specific regulatory mechanisms underlying cell fusion in filamentous fungi have not been revealed. In the present stu...

Epichloë festucae systemically colonizes the intercellular spaces of cool‐season grasses to establish a mutualistic symbiosis. A gene encoding a nuclear protein, NsiA, was isolated as an essential gene for symbiotic infection and hyphal cell fusion. Expression of genes required for cell fusion were down‐regulated in the nsiA mutant. However, the Ns...

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...

In Solanaceae plants, the major phytoalexins produced during the induction of plant defense are sesquiterpenoids, such as capsidiol for Nicotiana species and rishitin for Solanum species, which are produced via the mevalonate (MVA) pathway. Eight enzymes are involved in the production of farnesyl pyrophosphate (FPP), the common precursor of phytost...

AimsThe aim of this study was to provide a comprehensive understanding of the mechanisms underlying response of rice (Oryza sativa L.) to the endophytic N2-fixing bacterium Burkholderia vietnamiensis strain RS1 in growth and nitrogen (N) accumulation using transcriptome and hormonome analyses.Methods The effects of B. vietnamiensis RS1 on rice grow...

Pseudomonas syringae pv. tabaci 6605 has two multidrug resistance (MDR) efflux pump transporters, MexAB-OprM and MexEF-OprN. To understand the role of these MDR efflux pumps in virulence, we generated deletion mutants, ∆mexB, ∆mexF, and ∆mexB∆mexF, and investigated their sensitivity to plant-derived antimicrobial compounds, antibiotics, and virulen...

The endophytic fungus Epichloë festucae is known to produce bioactive metabolites, which consequently protect the host plants from biotic and abiotic stresses. We previously found that the overexpression of vibA (a gene for transcription factor) in E. festucae strain E437 resulted in the secretion of an unknown fungicide. In the present study, the...

Solanaceous plants produce sesquiterpenoid phytoalexins to defend themselves against a variety of pathogens. These toxic compounds are not only harmful to the pathogen but also to the plant, and thus need to be detoxified by the plant after the threat has been eliminated. We report that the detoxification of rishitin, the major phytoalexin in potat...

Background: Plants are exposed to various forms of environmental stress. Penetration by pathogens is one of the most serious environmental insults. Wounding caused by tissue damage or herbivory also affects the growth and reproduction of plants. Moreover, wounding disrupts physical barriers present at the plant surface and increases the risk of pa...

The oomycete Phytophthora infestans is the causal agent of potato late blight, one of the most devastating pathogens for potato. To investigate the plant mechanisms for resistance against P. infestans, the Solanaceae model plant Nicotiana benthamiana was employed in this study. Previously, we reported that NbNup75, a nuclear pore complex protein, i...

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 mecha...

Plant species produce a wide variety of antimicrobial metabolites to protect themselves against potential pathogens in natural environments. Phytoalexins are low molecular weight compounds produced by plants in response to attempted attacks of pathogens. Accumulation of phytoalexins in attacked plant tissues can inhibit the growth of penetrating pa...

The oomycete Phytophthora infestans is the causal agent of potato late blight, one of the most destructive and historically significant pathogens in agricultural production. A virus-induced gene silencing-based screening of the solanaceous model plant N. benthamiana resulted in revealing a wide range of resistance mechanisms of solanaceous plants a...

Epichloë festucae is an endophytic fungus which systemically colonizes temperate grasses to establish symbiotic associations. Maintaining symptomless infection is a key requirement for endophytes, a feature that distinguishes them from pathogenic fungi. While pathogenic fungi extend their hyphae by tip growth, hyphae of E. festucae systemically col...

Distribution of actin patches in hyphal tip of E. festucae wild type, cdc42 and racA mutants. Subcellular localization of actin patches visualized by Lifeact-GFP in hyphae of E. festucae wild type (WT), cdc42 (Δcdc42) and racA (ΔracA) mutants after growth on PDA for 11 days. Arrowheads indicate accumulation of actin patches near hyphal tips. Bars =...

Complementation of racA mutant by RacA under control of the TEF promoter. (A) Colony morphology and diameter of E. festucae wild type (WT), racA mutant and complemented strains grown on PDA for 12 days. Data are means ± standard error. n = 3. Data marked with asterisks are significantly different from wild type as assessed by two-tailed Student’s t...

Small GTPases of Ascomycota fungi, E. festucae, N. crassa, F. graminearum and M. oryzae. (XLSX)

Primers for sequencing, vector construction and quantitative PCR used in this study. (XLS)

Interactions of E. festucae Rho GTPases, RacA and Cdc42, with components of fungal Nox complex. Yeast strain AH109 was transformed with prey and bait vector as indicated and plated on to SD medium lacking leucine and tryptophan (-L/-T) or lacking leucine, tryptophan, histidine and adenine (-L/-T/-H/-A). Growth on the latter indicates an interaction...

Targeted gene replacement of the E. festucae cdc42 locus. (A) Physical map of the cdc42 wild-type (WT) genomic region and linear insert of Cdc42 replacement construct, showing restriction enzyme sites for EcoRV (EV), EcoRI (EI), BglII (Bg) and NdeI (N). The mutated genomic locus of cdc42 deletion mutant (Δcdc42) is depicted to show homologous recom...

Growth of perennial ryegrass infected with wild type E. festucae, racA mutant or cdc42 mutant. Photographs were taken approx. 2 months after inoculation. Bars = 10 cm. (PDF)

Plasmids for yeast two-hybrid assay used in this study. (XLSX)

Comparison of hyphal volume per compartment between wild type, cdc42 and racA mutants. Hyphae of endophyte strains were stained with calcofluor white and monitored with confocal laser microscopy. Bars = 20 μm. Area of cell compartments for each strain was measured using ImageJ software. Data are means ± standard error. n = 20. Data marked with aste...

Hyphal growth of E. festucae cdc42 and racA mutants on PDA. E. festucae wild type (WT), cdc42 and racA mutants were grown on PDA for 14 days. Hyphae of endophyte strains were stained with Calcofluor white and monitored with confocal laser microscopy. Bars = 30 μm. (PDF)

ROS production in colony of E. festucae. Hyphae of endophyte were grown on PDA for 12 days and O2- production of centeral, middle or growing edge (tip) part of colony was detected as L-012 mediated chemiluminescence. Chemiluminescence images were obtained using CCD camera. Data are means ± standard devision 15 sites from 5 colonies of each strain....

Alignment of deduced amino acid sequences of E. festucae and fungal Cdc42 and Rac. Amino acids specifically conserved among fungal Cdc42 and RacA are indicated by blue and red letters, respectively. An; Aspergillus nidulans (strain FGSC A4), Bc; Botrytis cinerea (T4), Bs; Bipolaris sorokiniana (ND90Pr), Co; Colletotrichum orbiculare (MAFF 240422),...

Identification of essential amino acids for specific interactions between Cdc42 and BemA or RacA and NoxR. (A) Yeast two-hybrid assays of the interactions between E. festucae NoxR and mutated RacA. (B) Yeast two-hybrid assays of the interactions between E. festucae BemA and chimeric or mutated Cdc42 and RacA. Rho GTPases have mutation in C-terminal...

Hyphal growth and symbiotic infection of E. festucae wild type, cdc42 and complemented strains. (A) E. festucae strains were grown on 3%water agar for 10 days. Hyphae of endophyte strains were stained with calcofluor white and monitored with confocal laser microscopy. Bars = 30 μm. (B) Colonization of E. festucae WT, cdc42 mutant and complemented s...

Plasmids for gene knock out and expression in E. festucae used in this study. (XLSX)

Yeast strains used in this study. (XLSX)

Phylogenetic analysis of small GTPase from E. festucae and Ascomycota fungi. The tree was prepared by the neighbor-joining method [64] using MEGA ver. 6.06 [65]. The scale bar corresponds to 5 estimated amino acid substitutions per site. Numbers at the nodes indicate the percentage of 1000 bootstrap replicates that supported each labeled interior b...

Expression of constitutively active form of Cdc42 (CA-Cdc42) or RacA (CA-RacA) induces swelling of E. festucae hyphae. CA-RacA or CA-Cdc42 were expressed in E. festucae wild type, noxR mutant (ΔnoxR) or bemA (ΔbemA) mutant under the control of the Tef promoter. Hyphae of endophyte strains were stained with Calcofluor white and monitored with confoc...

Three-dimensional structure models of E. festucae Cdc42 and RacA. Three-dimensional structure of Rho GTPases are predicted by iterative threading assembly refinement (I-TASSER [61]). Amino acid residues required for specific binding between Cdc42 and BemA, or RacA and NoxR are indicated blue and red letters, respectively. Conf. Score; Confidence sc...

Fungal strains used in this study. (XLSX)

Primers for construction of vectors for yeast two-hybrid assay. (XLS)

The hypersensitive response (HR) is a type of programmed cell death, which is part of the plant's defence response against pathogen attack. In the best of cases, this strategy isolates the pathogens from nutrients during the early steps of infection, causing them to starve before they are able to cause damage. The ability to suppress HR is a major...

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...

The oomycete genus Phytophthora includes some of the most destructive plant pathogens in the world. Plant diseases caused by Phytophthora species have an extremely significant impact on a wide range of agriculturally important crops and plants in natural ecosystems such as trees and shrubs in forests. In this chapter, we will describe the infection...

Cell-cell fusion in fungi is required for colony formation, nutrient transfer and signal transduction. Disruption of genes required for hyphal fusion in Epichloë festucae, a mutualistic symbiont of Lolium grasses, severely disrupts the host interaction phenotype. We examined whether symB and symC, the E. festucae homologs of Podospora anserina self...

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 app...

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), whi...

Phytophthora infestans is the causal agent of potato late blight. Genotypes of Japanese populations of P. infestans have been classified as US-1, JP-1, JP-2, JP-3, and JP-4 based on analyses of DNA polymorphisms. These populations may have been introduced to Japan by the migration of P. infestans from other countries and by domestic changes produce...

Mature Nicotiana benthamiana shows stable resistance to the oomycete pathogen Phytophthora infestans. Induction of phytoalexin (capsidiol) production is essential for the resistance, which is up-regulated via a MAPK cascade (NbMEK2-WIPK/SIPK) followed by ethylene signaling. In this study, NbNup75 (encodes a nuclear pore protein Nucleoporin75) was i...

Symbiotic association of epichloae endophytes (Epichloë/Neotyphodium species) with cool-season grasses of the subfamily Pooideae confers bioprotective benefits to the host plants against abiotic and biotic stresses. While the production of fungal bioprotective metabolites is a well-studied mechanism of host protection from insect herbivory, little...

The symbiotic association of epichloae endophytes (Epichloë/Neotyphodium species) with temperate grasses of the subfamily Pooideae is known to enhance plant host tolerance to abiotic and biotic stresses. While the protection of the host plant from insect herbivory by epichloae endophytes is well characterized, the mechanism by which they protect th...

Plant pathogens, including fungi, oomycetes, bacteria, aphids, and nematodes, produce a variety of effector proteins to counter plant disease resistance mechanisms. After delivery into the cytosol of the plant cell, effectors may target proteins localized to different compartments within the plant cell. Plants, in turn, have evolved disease resista...