Stemar-13-ene synthase, a diterpene cyclase involved in the biosynthesis of the phytoalexin oryzalexin S in rice

In suspension-cultured rice cells, diterpenoid phytoalexins are produced in response to exogenously applied elicitors. We isolated a cDNA encoding a diterpene cyclase, OsDTC2, from suspension-cultured rice cells treated with a chitin elicitor. The OsDTC2 cDNA was overexpressed in Escherichia coli as a fusion protein with glutathione S-transferase, and the recombinant OsDTC2 was indicated to function as stemar-13-ene synthase that converted syn-copalyl diphosphate to stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S. The level of OsDTC2 mRNA in suspension-cultured rice cells began to increase 3 h after addition of the elicitor and reached the maximum after 8 h. The expression of OsDTC2 was also induced in UV-irradiated rice leaves. In addition, we indicated that stemar-13-ene accumulated in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves.     

VdNEP, an elicitor from Verticillium dahliae, induces cotton plant wilting

Verticillium wilt is a vascular disease of cotton. The causal fungus, Verticillium dahliae, secretes elicitors in culture. We have generated approximately 1,000 5'-terminal expressed sequence tags (ESTs) from a cultured mycelium of V. dahliae. A number of ESTs were found to encode proteins harboring putative signal peptides for secretion, and their cDNAs were isolated. Heterologous expression led to the identification of a protein with elicitor activities. This protein, named V. dahliae necrosis- and ethylene-inducing protein (VdNEP), is composed of 233 amino acids and has high sequence identities with fungal necrosis- and ethylene-inducing proteins. Infiltration of the bacterially expressed His-VdNEP into Nicotiana benthamiana leaves resulted in necrotic lesion formation. In Arabidopsis thaliana, the fusion protein also triggered production of reactive oxygen species and induced the expression of PR genes. When added into suspension cultured cells of cotton (Gossypium arboreum), the fusion protein elicited the biosynthesis of gossypol and related sesquiterpene phytoalexins at low concentrations, and it induced cell death at higher concentrations. On cotton cotyledons and leaves, His-VdNEP induced dehydration and wilting, similar to symptoms caused by a crude preparation of V. dahliae elicitors. Northern blotting showed a low level of VdNEP expression in the mycelium during culture. These data suggest that VdNEP is a wilt-inducing factor and that it participates in cotton-V. dahliae interactions.     

Characterization of Novel Sesquiterpenoid Biosynthesis in Tobacco Expressing a Fungal Sesquiterpene Synthase

The gene encoding trichodiene synthase (Tri5), a sesquiterpene synthase from the fungus Fusarium sporotrichioides, was used to transform tobacco (Nicotiana tabacum). Trichodiene was the sole sesquiterpene synthase product in enzyme reaction mixtures derived from unelicited transformant cell-suspension cultures, and both trichodiene and 5-epi-aristolochene were observed as reaction products following elicitor treatment. Immunoblot analysis of protein extracts revealed the presence of trichodiene synthase only in transformant cell lines producing trichodiene. In vivo labeling with [3H]mevalonate revealed the presence of a novel trichodiene metabolite, 15-hydroxytrichodiene, that accumulated in the transformant cell-suspension cultures. In a trichodiene-producing transformant, the level of 15-hydroxytrichodiene accumulation increased after elicitor treatment. In vivo labeling with [14C]acetate showed that the biosynthetic rate of trichodiene and 15-hydroxytrichodiene also increased after elicitor treatment. Incorporation of radioactivity from [14C]acetate into capsidiol was reduced following elicitor treatment of a trichodiene-producing transformant as compared with wild type. These results demonstrate that sesquiterpenoid accumulation resulting from the constitutive expression of a foreign sesquiterpene synthase is responsive to elicitation and that the farnesyl pyrophosphate present in elicited cells can be utilized by a foreign sesquiterpene synthase to produce high levels of novel sesquiterpenoids.     

Inhibition of phytoalexin biosynthesis in elicitor-treated tobacco cell-suspension cultures by calcium/calmodulin antagonists

A role for calcium/calcium-binding proteins in a mechanism of signaling elicitor-inducible phytoalexin biosynthesis was investigated. Two classes of calcium/calmodulin antagonists, phenothiazines and naphthalenesulfonamides, inhibited sesquiterpene phytoalexin accumulation in tobacco (Nicotiana tabacum) cell-suspension cultures when added 1 h before elicitor. The antagonists also inhibited the induction of sesquiterpene cyclase enzyme activity, a key regulatory enzyme for sesquiterpene biosynthesis. The antagonists suppressed the induction of sesquiterpene cyclase only if added before or simultaneously with elicitor. Additionally, the antagonists inhibited (a) accumulation of the cyclase protein as measured in immunoblots; (b) the in vivo synthesis rate of the cyclase protein, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein; and (c) the cyclase mRNA translational activity, measured as the incorporation of [³⁵S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of RNA isolated from antagonist-treated, elicitor-induced cells. In contrast, elicitor-inducible phenylalanine ammonia lyase enzyme activity, the level of the enzyme protein, the in vivo synthesis rate, and the mRNA translational activity were not affected by any of the antagonist treatments. Uptake and incorporation of [³⁵S]methionine into total cellular proteins and total in vitro translation products were also not indiscriminately altered by the antagonist treatments. The current results suggest that calcium and/or calmodulin-like proteins may be elements of a signal transduction pathway mediating elicitor-induced accumulation of phytoalexins in tobacco.     

Inhibition of elicitor-induced phytoalexin formation in cotton and soybean cells by citrate

Addition of an elicitor preparation from Verticillium dahliae to soybean or cotton cell suspension cultures induces the formation of the phytoalexins, glycelollin or sesquiterpene aldehydes, respectively. Recent work (PS Low, PF Heinstein 1986 Arch Biochem Biophys 249: 472-479) has shown that the induction of phytoalexin biosynthesis in these cells is preceded by rapid changes in the plant cell membrane which can be conveniently monitored by membrane associated fluorescent probes. Using this elicitation assay, we have found that citrate, a common metabolite of higher plants, acts as a potent inhibitor of elicitation when added prior to treatment with elicitor. The citrate concentration required to obtain a 50% inhibition of the elicitor-induced fluorescence transition in cultured cotton cells was found to be about 2 millimolar, while the concentration of citrate observed to inhibit elicitor-induced sesquiterpene aldehyde formation in the same cell suspensions was also 2 millimolar. Curiously, in the presence of elicitor, citrate at less than ID₅₀ concentrations increased cell mass accumulation significantly above control incubations without elicitor. A similar inhibition of glyceollin formation with an increase in cell mass accumulation was also observed upon addition of 1 to 5 millimolar citrate to soybean cell suspension cultures. The physiological significance of the inhibition by citrate of phytoalexin formation in plant cell suspensions was supported by the observation that a similar inhibition of sesquiterpene aldehyde formation occurs in cotton plantlets elicited by cold shock or V. dahliae stress. The specificity of citrate as an inhibitor of phytoalexin formation was demonstrated by data showing that other di- and tricarboxylic-hydroxy acids did not inhibit, with the exception of malate which inhibited phytoalexin formation in soybean cells with roughly half the potency of citrate. These experiments not only demonstrate that citrate can act as a specific inhibitor of elicitation, but they further confirm the validity of monitoring elicitation and its modulation with fluorescent probes.     

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

Phytoalexin synthesis in soybean cells: elicitor induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs and correlation with phytoalexin accumulation

A glucan elicitor from cell walls of the fungus Phytophthora megasperma f. sp. glycinea, a pathogen of soybean (Glycine max), induced large and rapid increases in the activities of enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase, and of the flavonoid pathway, acetyl-CoA carboxylase and chalcone synthase, in suspension-cultured soybean cells. The changes in phenylalanine ammonia-lyase and chalcone synthase activities were correlated with corresponding changes in the mRNA activities encoding these enzymes, as determined by enzyme synthesis in vitro in a mRNA-dependent reticulocyte lysate. The time courses of the elicitor-induced changes in mRNA activities for both enzymes were very similar with respect to each other. Following the onset of induction, the two mRNA activities increased significantly at 3 h, reached highest levels at 5 to 7 h, and subsequently returned to low values at 10 h. Similar degrees of induction of mRNA activities and of the catalytic activities of phenylalanine ammonia-lyase and chalcone synthase were observed in response to three diverse microbial compounds, the glucan elicitor from P. megasperma, xanthan, an extracellular polysaccharide from Xanthomonas campestris, and endopolygalacturonase from Aspergillus niger. However, whereas the glucan elicitor induced the accumulation of large amounts of the phytoalexin, glyceollin, in soybean cells, endopolygalacturonase induced only low, albeit significant, amounts; xanthan did not enhance glyceollin accumulation under the conditions of this study. This result might imply that enzymes other than phenylalanine ammonia-lyase or chalcone synthase exert an important regulatory function in phytoalexin synthesis in soybean cells.     

Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor

Addition of elicitor, cell wall fragments of the fungus Phytophthora parasitica, to tobacco cell suspension cultures (Nicotiana tabacum) resulted in the rapid synthesis and secretion of large amounts of antibiotic sesquiterpenoids. Pulse-labeling experiments with [¹⁴C]acetate and [³H] mevalonate demonstrated that the induction of sesquiterpenoid biosynthesis, maximal by 6 to 9 hours after elicitor addition to the cell cultures, was paralleled by a rapid and large decline in the incorporation rate of radioactivity into sterols. Consequently, sterol accumulation was also inhibited upon addition of elicitor to the cell cultures. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 10 hours of elicitor addition to the cell cultures. The cyclase activity remained elevated for an additional 30 hours before declining. In contrast, squalene synthetase activity was suppressed to less than 15% of that found in control cells within 7 hours of elicitor addition. Our results suggest that the channeling of isoprenoid intermediates, and especially farnesyl diphosphate, into sesquiterpenoids occurred by a coordinated increase in the sesquiterpene cyclase and a decrease in the squalene synthetase enzyme activities. A reexamination of the data pertaining to the transient induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (EC 1.1.1.34) in elicitor-treated cells suggested that, while the reductase activity was necessary for sesquiterpenoid biosynthesis, it functioned more to maintain a sufficient level of intermediates between mevalonate and farnesyl diphosphate rather than as a rate limiting step controlling the synthesis rate of any one class of isoprenoids.     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.