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.

Substrate profiling of human transglutaminase 1 using cDNA display and next-generation sequencing.

Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences. The peptide sequence, AEQHKLPSKWPF, was identified showing high reactivity and specificity to TG1. The position weight matrix calculated from the per amino acid enrichment factors was employed to search human proteins using an in-house algorithm, revealing six known TG1 substrate proteins with high scores, alongside a list of candidate substrates currently under investigation. Our findings are expected to assist in future medical diagnoses and development of treatments for skin disorders.

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.

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.

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).

Comprehensive analysis of transglutaminase substrate preference by cDNA display coupled with next-generation sequencing and bioinformatics.

cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (1012) in a high throughput manner, based on their binding affinity. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions - 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position - 1 (Gln-Gln motif), and isoleucine or valine at position + 3. The highly enriched peptides indeed contained the indicated sequence and showed a higher reactivity as TG2 substrates than the peptide previously selected by phage display, thus representing the novel candidate peptide probes for TG2 research. Furthermore, the obtained information on substrate profiling can be used to identify potential TG2 protein targets. This platform will be further used for the substrate profiling of other TG isozymes, as well as for the selection and evolution of larger biomolecules.

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.

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.

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.

Detoxification of the solanaceous phytoalexins rishitin, lubimin, oxylubimin and solavetivone via a cytochrome P450 oxygenase.

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 potato tubers and tomato fruits, is mediated by a cytochrome P450 CYP76 family enzyme via the hydroxylation of the isopropenyl group resulting in the formation of 13-hydroxyrishitin, also known as rishitin-M1. We further observed hydroxylation of the potato phytoalexins solavetivone, lubimin and oxylubimin by the same enzyme. Constitutive expression of CYP76 in Nicotiana benthamiana also led to a reduction of the non-potato phytoalexins capsidiol and its derivative capsidiol 3-acetate. We therefore annotated this enzyme as sesquiterpenoid phytoalexins hydroxylase, SPH. This broad range of substrates indicates that SPH functions as a general phytoalexin detoxification enzyme in Solanaceae, and is therefore relevant for a better understanding of plant-pathogen interaction in solanaceous plants, which comprise many economically important crops, such as potato, tomato, eggplant and pepper.

Expression, Purification, and Enzyme Activity Assay of Phytoene Synthase In Vitro.

Phytoene synthase (PSY) is the rate-limiting step in carotenoid biosynthesis, and accordingly subjected to a number of regulatory mechanisms at various levels, including transcriptional, posttranscriptional, and posttranslational. Several PSY genes are present in most taxa and show various degrees of tissue and/or stress-specific responses providing an additional layer of regulating carotenogenesis. Moreover, only a small number of amino acid differences between paralogs or even single nucleotide polymorphisms distinguishing orthologs greatly affect enzyme properties, suggesting that different enzymatic parameters determined by intrinsic properties of PSY protein sequences also determine pathway flux. The characterization of enzyme properties of PSY variants from different origins requires in vitro enzyme assays with recombinant PSY. In this protocol, we present detailed instructions how to purify several milligrams of active PSY enzyme from bacterial lysates, which includes initial recombinant PSY enrichment through inclusion body purification, chaotropic unfolding, refolding in presence of detergents and purification through immobilized metal affinity chromatography. In addition, we provide a protocol to obtain active geranylgeranyl pyrophosphate (GGPP) synthase as active supply of GGPP substrate is a requirement for high in vitro PSY activity. The activity assay requires 14C-labeled substrate and allows to determine its incorporation into phytoene as well as GGPP. The protocol described here was successfully applied to a variety of PSY and GGPP synthase homologs from various plant species.

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.

Enzyme Fusion Removes Competition for Geranylgeranyl Diphosphate in Carotenogenesis.

Geranylgeranyl diphosphate (GGPP), a prenyl diphosphate synthesized by GGPP synthase (GGPS), represents a metabolic hub for the synthesis of key isoprenoids, such as chlorophylls, tocopherols, phylloquinone, gibberellins, and carotenoids. Protein-protein interactions and the amphipathic nature of GGPP suggest metabolite channeling and/or competition for GGPP among enzymes that function in independent branches of the isoprenoid pathway. To investigate substrate conversion efficiency between the plastid-localized GGPS isoform GGPS11 and phytoene synthase (PSY), the first enzyme of the carotenoid pathway, we used recombinant enzymes and determined their in vitro properties. Efficient phytoene biosynthesis via PSY strictly depended on simultaneous GGPP supply via GGPS11. In contrast, PSY could not access freely diffusible GGPP or time-displaced GGPP supply via GGPS11, presumably due to liposomal sequestration. To optimize phytoene biosynthesis, we applied a synthetic biology approach and constructed a chimeric GGPS11-PSY metabolon (PYGG). PYGG converted GGPP to phytoene almost quantitatively in vitro and did not show the GGPP leakage typical of the individual enzymes. PYGG expression in Arabidopsis resulted in orange-colored cotyledons, which are not observed if PSY or GGPS11 are overexpressed individually. This suggests insufficient GGPP substrate availability for chlorophyll biosynthesis achieved through GGPP flux redirection to carotenogenesis. Similarly, carotenoid levels in PYGG-expressing callus exceeded that in PSY- or GGPS11-overexpression lines. The PYGG chimeric protein may assist in provitamin A biofortification of edible plant parts. Moreover, other GGPS fusions may be used to redirect metabolic flux into the synthesis of other isoprenoids of nutritional and industrial interest.

Multiplexed antibody detection from blood sera by immobilization of in vitro expressed antigens and label-free readout via imaging reflectometric interferometry (iRIf).

The detection of antibodies from blood sera is crucial for diagnostic purposes. Miniaturized protein assays in combination with microfluidic setups hold great potential by enabling automated handling and multiplexed analyses. Yet, the separate expression, purification, and storage of many individual proteins are time consuming and limit applicability. In vitro cell-free expression has been proposed as an alternative procedure for the generation of protein assays. We report the successful in vitro expression of different model proteins from DNA templates with an optimized expression mix. His10-tagged proteins were specifically captured and immobilized on a Ni-NTA coated sensor surface directly from the in vitro expression mix. Finally, the specific binding of antibodies from rabbit-derived blood sera to the immobilized proteins was monitored by imaging reflectometric interferometry (iRIf). Antibodies in the blood sera could be identified by binding to the respective epitopes with minimal cross reactivity. The results show the potential of in vitro expression and label-free detection for binding assays in general and diagnostic purposes in specific.

Strigolactone Levels in Dicot Roots Are Determined by an Ancestral Symbiosis-Regulated Clade of the PHYTOENE SYNTHASE Gene Family.

Strigolactones (SLs) are apocarotenoid phytohormones synthesized from carotenoid precursors. They are produced most abundantly in roots for exudation into the rhizosphere to cope with mineral nutrient starvation through support of root symbionts. Abscisic acid (ABA) is another apocarotenoid phytohormone synthesized in roots, which is involved in responses to abiotic stress. Typically low carotenoid levels in roots raise the issue of precursor supply for the biosynthesis of these two apocarotenoids in this organ. Increased ABA levels upon abiotic stress in Poaceae roots are known to be supported by a particular isoform of phytoene synthase (PSY), catalyzing the rate-limiting step in carotenogenesis. Here we report on novel PSY3 isogenes from Medicago truncatula (MtPSY3) and Solanum lycopersicum (SlPSY3) strongly expressed exclusively upon root interaction with symbiotic arbuscular mycorrhizal (AM) fungi and moderately in response to phosphate starvation. They belong to a widespread clade of conserved PSYs restricted to dicots (dPSY3) distinct from the Poaceae-PSY3s involved in ABA formation. An ancient origin of dPSY3s and a potential co-evolution with the AM symbiosis is discussed in the context of PSY evolution. Knockdown of MtPSY3 in hairy roots of M. truncatula strongly reduced SL and AM-induced C13 α-ionol/C14 mycorradicin apocarotenoids. Inhibition of the reaction subsequent to phytoene synthesis revealed strongly elevated levels of phytoene indicating induced flux through the carotenoid pathway in roots upon mycorrhization. dPSY3 isogenes are coregulated with upstream isogenes and downstream carotenoid cleavage steps toward SLs (D27, CCD7, CCD8) suggesting a combined carotenoid/apocarotenoid pathway, which provides "just in time"-delivery of precursors for apocarotenoid formation.

Measuring Secretion of Capsidiol in Leaf Tissues of Nicotiana benthamiana.

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 pathogens. Thus phytoalexins play a major role in post-invasion defense against pathogens. Major phytoalexins produced by Solanaceous plants are sesquiterpenoids such as capsidiol produced by Nicotiana and Capsicum species, and rishitin produced by Solanum species, which are synthesized in the cytosol and secreted into the intercellular space of plant tissues. We previously reported that deficiency in capsidiol secretion causes enhanced susceptibility of Nicotiana benthamiana to potato late blight pathogen, Phytophthora infestans. Here, we describe a practical protocol to measure the secreted capsidiol in N. benthamiana.