Phytoalexin Capsidiol Research Articles

NaWRKY70 is a key regulator of Nicotiana attenuata resistance to Alternaria alternata through regulation of phytohormones and phytoalexins biosynthesis.

Jasmonate (JA) and abscisic acid (ABA) are two major phytohormones involved in pathogen resistance. However, how their biosynthesis is regulated is not well understood. We silenced NaWRKY70 in wild tobacco Nicotiana attenuata and determined its role in regulating genes involved in the production of JA, ABA and the phytoalexin capsidiol in response to the fungal pathogen Alternaria alternata using techniques including electrophoretic mobility shift, chromatin immunoprecipitation, transient overexpression and virus-induced gene silencing. Silencing NaWRKY70 dramatically reduced both basal and A. alternata-induced jasmonoyl-isoleucine (JA-Ile) and ABA. Further evidence showed that NaWRKY70 directly binds to the W-boxes of the promoters of NaAOS and NaJAR4 (JA biosynthesis), NaNCED1 and NaXD1-like (ABA biosynthesis), and NaMPK4 (ABA signaling) to activate their expression, while binding but repressing the expression of NaCYP707A4-like3 (ABA degradation). Additionally, NaWRKY70 regulates capsidiol production through its key enzyme genes NaEASs and NaEAHs, and interacts with its regulator NaERF2-like to enhance their expression, whereas ABA negatively regulates capsidiol biosynthesis. Our results highlight the key role of NaWRKY70 in controlling both JA-Ile and ABA production, as well as capsidiol production, thus providing new insight into the defense mechanism of plant resistance to A. alternata.

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.

Nbnrp1 mediates Verticillium dahliae effector PevD1-triggered defense responses by regulating sesquiterpenoid phytoalexins biosynthesis pathway in Nicotiana benthamiana.

PevD1, a fungal effector secreted by Verticillium dahliae, could induce hypersensitive responses-like necrosis and systemic acquired resistance (SAR) in cotton and tobacco plants. PevD1 could drastically induce the expression of Nbnrp1, which is an asparagine-rich protein (NRP) of Nicotiana benthamiana. Our previous research indicated that Nbnrp1 positively regulated PevD1-induced cell necrosis and disease resistance. In this study, we further investigated PevD1-induced immune responses in both wild-type (WT) and Nbnrp1-RNAi lines through RNA-seq, in order to reveal the underlying mechanism of Nbnrp1-modulated PevD1-induced disease resistance in N. benthamiana. Results showed that Nbnrp1-RNAi lines exhibited reduced PevD1-induced immune responses, like inhibiting H2O2 accumulation and MAPK phosphorylation. To silence Nbnrp1 inhibited the expression of PevD1-induced differential expression genes (DEGs) involved in pathways associated with sesquiterpenoid and triterpenoid biosynthesis, flavone and flavonol biosynthesis, plant-pathogen interaction and phenylpropanoid biosynthesis, etc. It is worth noting that sesquiterpene phytoalexin capsidiol accumulation were obviously decreased in Nbnrp1-RNAi plants after PevD1 treatment, accompanied with the down-expression of EAS and EAH, which were two key genes related to capsidiol biosynthesis. These results suggested that Nbnrp1 mediates PevD1-induced defense responses by regulating sesquiterpenoid phytoalexins biosynthesis pathway.

Molecular Characterization of a Pathogen-Inducible Bidirectional Promoter from Hot Pepper (Capsicum annuum).

In hot pepper, the sesquiterpene phytoalexin capsidiol is catalyzed by the two final-step enzymes, a sesquiterpene cyclase (EAS) and a hydroxylase (EAH), which are genetically linked and present as head-to-head orientation in the genome. Transcriptomic analysis revealed that a subset of EAS and EAH is highly induced following pathogen infection, suggesting the coregulation of EAS and EAH by a potential bidirectional activity of the promoter (pCaD). A series of the nested deletions of pCaD in both directions verified the bidirectional promoter activity of the pCaD. Promoter deletion analysis revealed that the 226 bp of the adjacent promoter region of EAS and GCC-box in EAH orientation were determined as critical regulatory elements for the induction of each gene. Based on promoter analyses, we generated a set of synthetic promoters to maximize reporter gene expression within the minimal length of the promoter in both directions. We found that the reporter gene expression was remarkably induced upon infection with Phytophthora capsici, Phytophthora infestans, and bacterial pathogen Pseudomonas syringae pv. tomato DC3000 but not with necrotrophic fungi Botrytis cinerea. Our results confirmed the bidirectional activity of the pCaD located between the head-to-head oriented phytoalexin biosynthetic genes in hot pepper. Furthermore, the synthetic promoter modified in pCaD could be a potential tool for pathogen-inducible expression of target genes for developing disease-resistant crops.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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

ABSTRACT 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.

Nicotiana benthamiana exportin 1 is required for elicitor-induced phytoalexin production, cell death induction, and resistance against potato late blight pathogen Phytophthora infestans

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, is required for the resistance against P. infestans, suggesting that nuclear-pore-mediated transport is involved in the induction of defense responses. In this study, we investigated the role of N. benthamiana exportin 1 NbXpo1 in disease resistance. Mammalian and yeast exportin 1 proteins are known as a regulator for nuclear-pore-mediated export of proteins and RNAs. NbXpo1-silenced N. benthamiana showed minor growth defects and significantly decreased resistance to P. infestans. Gene silencing of NbXpo1 compromised defense responses induced by the elicitor INF1 (a secretory protein of P. infestans), such as production of the phytoalexin capsidiol and induction of cell death. In NbXpo1-silenced plants, the genes for capsidiol biosynthesis, NbEAS, and defense-related MAP kinase, NbWIPK, were significantly downregulated, while genes encoding the plant defensin, NbPDF, and anti-microbial protein thionin, NbTHI, were upregulated. These results indicate that N. benthamiana Xpo1 is involved in activating a specific group of defense-related genes.

Structural and biosynthetic studies on eremophilenols related to the phytoalexin capsidiol, produced by Botrytis cinerea

A thorough study of the fermentation broth of three strains of Botrytis cinerea which were grown on a modified Czapek-Dox medium supplemented with 5 ppm copper sulphate, yielded five undescribed metabolites. These metabolites possessed a sesquiterpenoid (+)-4-epi-eremophil-9-ene carbon skeleton which was enantiomeric to that of the phytoalexin, capsidiol. The isolation of these metabolites when the fungus was stressed, suggests that they may be potential effectors used by B. cinerea to circumvent plant chemical defences against phytopathogenic fungi. The biosynthesis of these compounds has been studied using 2H and 13C labelled acetate.

Expansion of sesquiterpene biosynthetic gene clusters in pepper confers nonhost resistance to the Irish potato famine pathogen.

Chemical barriers contribute to nonhost resistance, which is defined as the resistance of an entire plant species to nonadapted pathogen species. However, the molecular basis of metabolic defense in nonhost resistance remains elusive. Here, we report genetic evidence for the essential role of phytoalexin capsidiol in nonhost resistance of pepper (Capsicum spp.) to potato late blight Phytophthora infestans using transcriptome and genome analyses. Two different genes for capsidiol biosynthesis, 5-epi-aristolochene synthase (EAS) and 5-epi-aristolochene-1,3-dihydroxylase (EAH), belong to multigene families. However, only a subset of EAS/EAH gene family members were highly induced upon P.infestans infection, which was associated with parallel accumulation of capsidiol in P.infestans-infected pepper. Silencing of EAS homologs in pepper resulted in a significant decrease in capsidiol accumulation and allowed the growth of nonadapted P.infestans that is highly sensitive to capsidiol. Phylogenetic and genomic analyses of EAS/EAH multigene families revealed that the emergence of pathogen-inducible EAS/EAH genes in Capsicum-specific genomic regions rendered pepper a nonhost of P.infestans. This study provides insights into evolutionary aspects of nonhost resistance based on the combination of a species-specific phytoalexin and sensitivity of nonadapted pathogens.

Early signaling events induced by the peptide elicitor PIP-1 necessary for acetosyringone accumulation in tobacco cells.

A peptide elicitor PIP-1 induces defense-related secondary metabolites such as phytoalexin capsidiol in tobacco cells. In this study, we identified one of other metabolites induced by PIP-1 as acetosyringone. Unlike capsidiol accumulation that requires long-term stimulation with PIP-1, acetosyringone was induced by short-term stimulation with PIP-1. The importance of NADPH oxidase in the acetosyringone induction was also demonstrated.

Photocontrol of Elicitor Activity of PIP-1 to Investigate Temporal Factors Involved in Phytoalexin Biosynthesis.

The peptide elicitor PIP-1 can induce various immune responses in tobacco cells. Previously, we showed that types of responses induced by PIP-1 are different depending on its stimulation periods; short-term stimulation induces weak responses, whereas long-term stimulation leads to strong responses including production of the phytoalexin capsidiol. However, key components that directly regulate the initiation of capsidiol biosynthesis in response to continuous stimulation with PIP-1 remain unclear. In this study, we designed a photocleavable PIP-1 analog containing 3-amino-3-(2-nitrophenyl)propionic acid as a photocleavable residue. The activity of the analog can be "switched off" using ultraviolet (UV) irradiation without undesired side effects. This analog induced a significant level of capsidiol production unless UV-irradiated, whereas no capsidiol production was observed when tobacco cells were UV-irradiated 1 h after treatment. Using this analog, we found that the elicitor-inducible 3-hydroxy-3-methylglutaryl-CoA reductase activity is regulated based on the duration of the stimulation with PIP-1, which could be associated with the initiation of capsidiol biosynthesis.

Isolation of an elicitor-stimulated 5-epi-aristolochene synthase gene (gPEAS1) from chili pepper (Capsicum annuum)

Phytoalexins play an important role in the inducible defense responses of plants against diseases caused by fungi. Some enzymes, involved in the respective biosynthetic pathways, catalyze key steps. Farnesyl pyrophosphate (FPP) is a key intermediate in the biosynthesis of terpenes. It may be converted into several cyclic and some acyclic sesquiterpenic derivatives, sterol precursors, or to geranylgeranyl pyrophosphate according to the requirements of the plant cells. Specific cyclization reactions of FPP are catalyzed by particular sesquiterpene cyclases. The 5-epi-aristolochene synthase (EAS) enzyme of tobacco (Nicotiana tabacum) and pepper (Capsicum annuum) produce the 5-epi-aristolochene, which is the immediate precursor of the bicyclic phytoalexin capsidiol. Oligonucleotides homologous to 3′-end specific regions of tobacco and Hyoscyamus muticus inducible sesquiterpene cyclase genes were used with the system for rapid amplification of cDNA ends (3′-RACE) technique to prepare pepper EAS-cDNA fragments (PEAS-cDNA). Three specific PEAS-cDNAs were isolated (PEAS1, PEAS18, and PEAS55). Northern blots of total RNA samples from pepper plant tissues challenged with cellulase or Phytophthora capsici showed different expression levels of the respective transcripts. PEAS1 was used to identify the corresponding elicitor-stimulated gene (gPEAS1). The nucleotide sequence of the proposed gPEAS1 promoter showed putative stimuli- and tissue-specific responsive elements. A pepper 5-epi-aristolochene synthase gene family of 5–8 members was demonstrated by Southern blot.

Eremophilane Sesquiterpenes from Capsidiol

A series of eremophilane sesquiterpene alcohols and hydrocarbons was prepared from the phytoalexin capsidiol (1) for mechanistic studies with epiaristolochene synthase and epiaristolochene dihydroxylase. Among them, 3-deoxycapsidiol (10) was obtained through selective derivatization and reductive cleavage of the equatorial 3 alpha hydroxyl group. Two novel isomers of aristolochene and eremophilene were accessed from the 1- and 3-deoxycapsidiol isomers. 4-Epieremophilene (17) was obtained by conjugate reduction of epiaristolochen-1-one tosylhydrazone with catecholborane followed by sulfinate elimination and diimide rearrangement. Epimerization of epiaristolochen-3-one (27a) at the C4 methyl followed by reductions led to the previously unknown aristolochene isomer, eremophila-9(10),11(12)-diene (30). Optical rotations and characteristic (1)H NMR data for the related eremophilenols and dienes are collected in Tables 1 and 2. Finally, bioassays were used to assess the antifungal potencies of capsidiol and its synthetic derivatives. The minimum inhibitory concentration for capsidiol (3-10 ng) was at least 1 order of magnitude lower than that of any of the derivatives and considerably lower than those previously reported for ketoconazole, nystatin, and propiconazole.

Superoxide release is necessary for phytoalexin accumulation in Nicotiana tabacum cells during the expression of cultivar-race and non-host resistance towards Phytophthora spp.

The relationship between superoxide radical generation, the accumulation of the phytoalexin capsidiol and hypersensitive cell death has been examined in Nicotiana tabacum following challenges by compatible and incompatible races of Phytophthora nicotianae, and the non-host species Phytophthora palmivora. Challenging suspension cell cultures of N. tabacum with zoospores of incompatible isolates of P. nicotianae elicits a biphasic burst of superoxide release. The maximum rate of capsidiol accumulation between 9 and 12 h after challenge coincides with the second oxidative burst and the onset of hypersensitive cell death. Addition of superoxide dismutase or Mn (III) desferal, which scavenge superoxide anions and quench the superoxide burst, suppresses both phytoalexin accumulation and hypersensitive cell death. Mevastatin, an inhibitor of the sesquiterpenoid biosynthesis enzyme HMG-CoA reductase, has no effect on the oxidative burst or hypersensitive cell death, but abolishes capsidiol accumulation. Zoospores of the non-host pathogen P. palmivora also elicit superoxide release, but in a single, broad burst between 3 and 12 h after challenge. Capsidiol accumulates to levels similar to those seen in incompatible host reactions, although the onset of capsidiol accumulation is more rapid in the non-host interaction. As in the incompatible interaction, phytoalexin accumulation and hypersensitive cell death are both inhibited by superoxide scavengers, although scavenging does not render host cells susceptible to infection by non-host zoospores. Our findings indicate that phytoalexin accumulation and hypersensitive cell death in both incompatible and non-host interactions are regulated by pathways that diverge downstream of superoxide release. While hypersensitive cell death and phytoalexin accumulation appear to be necessary for gene-for-gene resistance, other factors cause non-host infection to fail.

Is accumulation of sesquiterpenoid phytoalexins induced in tobacco plants constitutively producing salicylic acid?

The accumulation of sesquiterpenoid phytoalexins was screened in constitutive salicylic acid producing (CSA)-tobacco leaves. Only after tobacco mosaic virus (TMV)-inoculation the sesquiterpenoid phytoalexin capsidiol was identified in CSA- and non-transgenic tobacco leaves. Capsidiol was not accumulated in CSA- and non-transgenic tobacco leaves after mechanical wounding.

Linear beta-1,3 glucans are elicitors of defense responses in tobacco.

Laminarin, a linear beta-1,3 glucan (mean degree of polymerization of 33) was extracted and purified from the brown alga Laminaria digitata. Its elicitor activity on tobacco (Nicotiana tabacum) was compared to that of oligogalacturonides with a mean degree of polymerization of 10. The two oligosaccharides were perceived by suspension-cultured cells as distinct chemical stimuli but triggered a similar and broad spectrum of defense responses. A dose of 200 microg mL(-1) laminarin or oligogalacturonides induced within a few minutes a 1.9-pH-units alkalinization of the extracellular medium and a transient release of H(2)O(2). After a few hours, a strong stimulation of Phe ammonia-lyase, caffeic acid O-methyltransferase, and lipoxygenase activities occurred, as well as accumulation of salicylic acid. Neither of the two oligosaccharides induced tissue damage or cell death nor did they induce accumulation of the typical tobacco phytoalexin capsidiol, in contrast with the effects of the proteinaceous elicitor beta-megaspermin. Structure activity studies with laminarin, laminarin oligomers, high molecular weight beta-1, 3-1,6 glucans from fungal cell walls, and the beta-1,6-1,3 heptaglucan showed that the elicitor effects observed in tobacco with beta-glucans are specific to linear beta-1,3 linkages, with laminaripentaose being the smallest elicitor-active structure. In accordance with its strong stimulating effect on defense responses in tobacco cells, infiltration of 200 microg mL(-1) laminarin in tobacco leaves triggered accumulation within 48 h of the four families of antimicrobial pathogenesis-related proteins investigated. Challenge of the laminarin-infiltrated leaves 5 d after treatment with the soft rot pathogen Erwinia carotovora subsp. carotovora resulted in a strong reduction of the infection when compared with water-treated leaves.

Cloning of a sesquiterpene cyclase and its functional expression by domain swapping strategy.

Sesquiterpene cyclase, the first committed step enzyme from the general isoprenoid building block farnesyl pyrophosphate (FPP) for the synthesis of phytoalexin capsidiol, was isolated from the UV-C treated leaves of Capsicum annuum. This sesquiterpene cyclase, termed as CASC2 showing 77% amino acid identity with the previously cloned sesquiterpene cyclase CASC1, was composed of 560 amino acids with a calculated molecular mass of 64,907. The mRNA expression pattern of CASC2 was very similar to that of CASC1 during the time course of UV-C irradiated leaves of pepper on RNA blot analysis by using each specific probe. The heterologous expression in Escherichia coli using the CASC2 full length failed; however the chimeric construct of CASC2 in which the amino terminal 164 amino acid substituted by the equivalent portion of either CASC1 or tobacco sesquiterpene cyclase was capable of expressing the functional sesquiterpene cyclase activities. The radio-labeled enzymatic products catalyzed by the partially purified chimeric CASC2 were comigrated with authentic radio-labeled sesquiterpene on thin layer chromatography.

Biochemical and molecular tools for the production of useful terpene products from pepper (Capsicum annuum).

Among other natural products such as colorants and flavorants, natural fungicides like the pepper phytoalexin capsidiol, and the related biochemical pathways, may be used for practical approaches. Key enzymes such as 3-hydroxy-3-methylglutaryl Coenzyme A: reductase, the farnesyl pyrophosphate synthase and and farnesyl pyrophosphate cyclases are known and some related genes have been isolated. However, specific enzymes for important and final modifications as methylation and others, are still to be studied. Construction of chimeric enzymes allowed already the synthesis of different products and the possibilities of designing new enzymes by gene manipulation to produce unknown and useful chemicals are open.

Characterization of the TAC box, acis‐element within an elicitor‐inducible sesquiterpene cyclase promoter

Summary The first unique step in the synthesis of the tobacco phytoalexin capsidiol is cyclization of farnesyl pyrophosphate catalyzed by 5‐epi‐aristolochene synthase (EAS), a sesquiterpene cyclase. Earlier work demonstrated that the elicitor‐inducibility of this enzyme activity corresponded to the transcriptional activation of at least one gene, EAS4 , of a rather complex gene family consisting of > 10 members. To investigate the mechanism(s) controlling expression of this gene, fragments of the EAS4 promoter were examined for binding by proteins in nuclear and whole‐cell extracts. A strong protein binding site (TAC box; ACTCTACAGTACTC) was identified between –245 and –232 by the electrophoretic mobility shift assay, and DNAase I and methylation interference footprinting. Several distinctly migrating bands representing protein–TAC box complexes were also observed in the mobility shift assays, and the relative abundance of these bands varied in extracts from cells at different stages of EAS induction. The TAC box binding factor (TacBBF) was purified > 450‐fold from crude whole‐cell extracts by a combination of DNA affinity and cation exchange chromatography. The purified fractions were enriched for polypeptides of 17 and 19 kDa and the DNA binding properties of these preparations were characterized. Mutation of 2 bp in the TAC box prevented protein binding in vitro and increased both basal and elicitor‐inducible gene expression 2.5‐fold in transgenic tobacco plants harboring promoter–GUS fusions, consistent with the notion that this cis ‐element functions as a silencer or repressor of EAS gene expression.

Cloning and bacterial expression of sesquiterpene cyclase, a key branch point enzyme for the synthesis of sesquiterpenoid phytoalexin capsidiol in UV-challenged leaves of Capsicum annuum.

Sesquiterpene cyclase, a branch point enzyme in the general isoprenoid pathway for the synthesis of phytoalexin capsidiol, was induced in detached leaves of Capsicum annuum (pepper) by UV treatment. The inducibility of cyclase enzyme activities paralleled the absolute amount of cyclase protein(s) of pepper immunodetected by monoclonal antibodies raised against tobacco sesquiterpene cyclase. A cDNA library was constructed with poly(A)+ RNA isolated from 24 h UV-challenged leaves of pepper. A cDNA clone for sesquiterpene cyclase in pepper was isolated by using a tobacco 5-epi aristolochene synthase gene as a heterologous probe. The predicted protein encoded by this cDNA was comprised of 559 amino acids and had a relative molecular mass of 65,095. The primary structural information from the cDNA clone revealed that it shared 77%, 72% and 49% identity with 5-epi aristolochene, vetispiradiene, and cadinene synthase, respectively. The enzymatic product catalyzed by the cDNA clone in bacteria was identified as 5-epi aristolochene, as judged by argentation TLC. RNA blot hybridization demonstrated the induction of an mRNA consistent with the induction of cyclase enzyme activity in UV-treated pepper.

Defence response of pepper (Capsicum annuum) suspension cells to Phytophthora capsici

Cell suspension cultures of three cultivars of Capsicum annuum L., with different degrees of sensibility to the fungus Phytophthora capsici, responded to elicitation by both lyophilized mycelium and fungus filtrate. They showed conductivity changes, browning, production of the phytoalexin capsidiol and synthesis or accumulation of pathogenesis‐related (PR) proteins with glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) activities. The cultivation medium was optimised for growth of both the plant and the fungus in order to avoid any stress during their combination. The resistant cv. Smith‐5, showed a more rapid and intense response to the elicitor preparations than the sensitive cvs Americano and Yolo Wonder. This was particularly evident when the cell suspensions were elicited with the filtrate, when differences became clearly visible after only 6 h incubation. The greatest rate of capsidiol accumulation occurred after 18 h in the mycelium‐elicited cells and after 12 h in those elicited with the filtrate. These times are the optimal for capsidiol accumulation, and the phytoalexin is produced much more rapidly than it can be excreted into the extracellular medium. The inhibition threshold of fungal growth (300 µg capsidiol [g dry weight]−1) was reached only in the resistant cultivar. The induction of an intracellular glucanase (pI 8.9 and Rf 0.18) and an extracellular chitinase (pI 5.4 and Rf 0.70) only in the resistant cultivar 24 h after elicitation suggests that these enzymes are involved in the resistance to Phytophthora capsici, while other hydrolases common to all three cultivars form part of a more general defence. The results indicate that elicitation of pepper cell suspension cultures by signal molecules from P. capsici exhibits properties of a multicomponent dynamic system in which different protective mechanisms play complementary roles in the overall expression of the defence reaction. We confirm that the differential responses of resistant and susceptible pepper cultivars to P. capsici previously seen in plant stem sections are retained in suspension culture.