The nematicide Serratia plymuthica M24T3 colonizes Arabidopsis thaliana, stimulates plant growth, and presents plant beneficial potential.

Nine bacterial strains were previously isolated in association with pinewood nematode (PWN) from wilted pine trees. They proved to be nematicidal in vitro, and one of the highest activities, with potential to control PWN, was showed by Serratia sp. M24T3. Its ecology in association with plants remains unclear. This study aimed to evaluate the ability of strain M24T3 to colonize the internal tissues of the model plant Arabidopsis thaliana using confocal microscopy. Plant growth-promoting bacteria (PGPB) functional traits were tested and retrieved in the genome of strain M24T3. In greenhouse conditions, the bacterial effects of all nematicidal strains were also evaluated, co-inoculated or not with Bradyrhizobium sp. 3267, on Vigna unguiculata fitness. Inoculation of strain M24T3 increased the number of A. thaliana lateral roots and the confocal analysis confirmed effective bacterial colonization in the plant. Strain M24T3 showed cellulolytic activity, siderophores production, phosphate and zinc solubilization ability, and indole acetic acid production independent of supplementation with L-tryptophan. In the genome of strain M24T3, genes involved in the interaction with the plants such as 1-aminocyclopropane-1-carboxylate (ACC) deaminase, chitinolytic activity, and quorum sensing were also detected. The genomic organization showed ACC deaminase and its leucine-responsive transcriptional regulator, and the activity of ACC deaminase was 594.6 nmol α-ketobutyrate µg protein-1 µl-1. Strain M24T3 in co-inoculation with Bradyrhizobium sp. 3267 promoted the growth of V. unguiculata. In conclusion, this study demonstrated the ability of strain M24T3 to colonize other plants besides pine trees as an endophyte and displays PGPB traits that probably increased plant tolerance to stresses.

RNAi of the sesquiterpene cyclase gene for phytoalexin production impairs pre- and post-invasive resistance to potato blight pathogens.

Potato antimicrobial sesquiterpenoid phytoalexins lubimin and rishitin have been implicated in resistance to the late blight pathogen, Phytophthora infestans and early blight pathogen, Alternaria solani. We generated transgenic potato plants in which sesquiterpene cyclase, a key enzyme for production of lubimin and rishitin, is compromised by RNAi to investigate the role of phytoalexins in potato defence. The transgenic tubers were deficient in phytoalexins and exhibited reduced post-invasive resistance to an avirulent isolate of P. infestans, resulting in successful infection of the first attacked cells without induction of cell death. However, cell death was observed in the subsequently penetrated cells. Although we failed to detect phytoalexins and antifungal activity in the extract from wild-type leaves, post-invasive resistance to avirulent P. infestans was reduced in transgenic leaves. On the other hand, A. solani frequently penetrated epidermal cells of transgenic leaves and caused severe disease symptoms presumably from a deficiency in unidentified antifungal compounds. The contribution of antimicrobial components to resistance to penetration and later colonization may vary depending on the pathogen species, suggesting that sesquiterpene cyclase-mediated compounds participate in pre-invasive resistance to necrotrophic pathogen A. solani and post-invasive resistance to hemibiotrophic pathogen P. infestans.

Enhanced Production of δ-Guaiene, a Bicyclic Sesquiterpene Accumulated in Agarwood, by Coexpression of δ-Guaiene Synthase and Farnesyl Diphosphate Synthase Genes in Escherichia coli.

Two genes involved in δ-guaiene biosynthesis in Aquilaria microcarpa, δ-guaiene synthase (GS) and famesyl diphosphate synthase (FPS), were overexpressed in Escherichia coli cells. Immunoblot analysis revealed that the concentration of GS-translated protein was rather low in the cells transformed by solely GS while appreciable accumulation of the recombinant protein was observed when GS was coexpressed with FPS. GS-transformed cells liberated only a trace amount of δ-guaiene (0.004 µg/mL culture), however, the concentration of the compound elevated to 0.08 pg/mL culture in the cells transformed by GS plus FPS. δ-Guaiene biosynthesis was markedly activated when E. coli cells coexpressing GS and FPS were incubated in enriched Terrific broth, and the content of the compound increased to approximately 0.6 µg/mL culture. These results suggest that coexpression of FPS and GS in E. coli is required for efficient δ- guaiene production in the bacterial cells, and the sesquiterpene-producing activity of the transformant is appreciably enhanced in the nutrients-enriched medium.

Effect of heavy metals on acdS gene expression in Herbaspirillium sp. GW103 isolated from rhizosphere soil.

This study aimed to understand the influence of heavy metals on 1-aminocyclopropane-1-carboxylate deaminase activity (ACCD) and acdS gene expression in Herbaspirillium sp. GW103. The GW103 strain ACCD activity decreased in cells grown in a medium supplemented with Pb and As, whereas cells grown in medium supplemented with Cu showed increase in enzyme activity. The GW103 strain produced 262.2 ± 6.17 µmol of α-ketobutyrate per milligram of protein per hour during ACC deamination at 25 °C after 24 h incubation. Using a PCR approach, an acdS coding-gene of 1.06 kbp was amplified in isolate GW103, showing 92% identity with Herbaspirillum seropedicae SmR1 acdS gene. Real time quantitative polymerase chain reaction results indicate that the acdS expression rate was increased (7.1-fold) in the presence of Cu, whereas it decreased (0.2- and 0.1-fold) in the presence of As and Pb.

[Screening and identification of an endophytic bacterium with 1-aminocyclopropane-1-carboxylate deaminase activity from Panax ginseng and its effect on host growth].

OBJECTIVE: This study aimed to screen endophytic bacteria with 1-aminocyclopropane-1-carboxylate deaminase activity from Panax ginseng and test the capability of growth promotion to its host. METHODS: In total 120 endophytic bacterial strains isolated from Panax ginseng were screened for 1-aminocyclopropane-1-carboxylate deaminase activity using the qualitative and quantitative methods. The obtained strain was also tested for its ability of nitrogen fixation using the Ashby agar plates and the gene of nifH, for its ability of phosphate solubilization using the Pikovaskaia's plates and quantitative analysis of Mo-Sb-Ascrobiology acid colorimetry, for its ability of producing siderophores using the method of Chrome azurol S detecting, and its effect on promoting growth of Panax ginseng by laboratory and field experiments. The bacterial strain with ACC deaminase was identified based on morphology, physiological and biochemical traits, and 16S rRNA sequence analysis. RESULTS: The bacterial stain JJ8-3 with the ability of producing ACC deaminase activity was obtained through screening, which its ACC deaminase activity was alpha-ketobutyric acid 6.7 micromol/(mg x h). Strain JJ8-3 had other traits of phosphate solubilizing, nitrogen fixation, producing siderophores, and the ability of promoting growth of Panax ginseng. Strain JJ8-3 was identified as Pseudomonas fluorescens. CONCLUSIONS: Strain JJ8-3 of endophytic bacterium with ACC deaminase activity from Panax ginseng was obtained and would lay the foundation for its further study and application on plant growth promotion.

Induction, cloning and functional expression of a sesquiterpene biosynthetic enzyme, δ-guaiene synthase, of Aquilaria microcarpa cell cultures.

A homology-based cloning strategy yielded a cDNA clone presumably encoding δ-guaiene synthase, a sesquiterpene cyclase, from tissue cultures of Aquilaria microcarpa, which were treated with methyl jasmonate. Incubation of cell cultures of the plant with yeast extract also induced transcriptional activation of the sesquiterpene synthase gene. The translated protein of the gene obtained by heterologous expression in Escherichia coli catalyzed the cyclization of farnesyl diphosphate to liberate δ-guaiene with δ-guaiene and germacrene A as the minor products. The results obtained in the present study, together with the previously reported results, suggest that two classes of δ-guaiene synthase occur in Aquilaria; the enzyme proteins from A. microcarpa and A. sinensis liberate germacrene A as a minor product, while the protein from A. crassna generates α-humulene instead of germacrene A.

Metagenomic analysis of the 1-aminocyclopropane-1-carboxylate deaminase gene (acdS) operon of an uncultured bacterial endophyte colonizing Solanum tuberosum L.

Deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is a key plant-beneficial trait found in many plant growth-promoting bacteria. In this study, we analysed ACC deaminase genes (acdS) of bacterial endophytes colonizing field-grown potato plants. PCR analysis revealed the presence of two types of acdS genes, the dominant one showing high homology to an acdS gene derived from Pseudomonas fluorescens. Construction, functional screening and sequence analysis of metagenomic libraries revealed clones containing the acdS gene identified in the PCR library. Sequence analysis of one metagenomic clone identified the entire acdS operon of an uncultivated endophyte and revealed that the acdS gene is coupled upstream with an acdR transcriptional regulator gene as previously found in P. putida strain UW4 (Grichko and Glick 2000). However, in-silico analysis of 195 fully sequenced, acdS-containing bacterial genomes revealed that the majority of strains, including numerous strains belonging to the genus Pseudomonas, do not contain an acdR regulatory gene in the vicinity of the acdS gene or elsewhere in the genome. The acdR (+)-acdS (+) operon was exclusively found in several Alpha- and Betaproteobacteria most prominently in the genus Burkholderia.

DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment.

Global groundwater resources are constantly challenged by a multitude of contaminants such as aromatic hydrocarbons. Especially in anaerobic habitats, a large diversity of unrecognized microbial populations may be responsible for their degradation. Still, our present understanding of the respective microbiota and their ecophysiology is almost exclusively based on a small number of cultured organisms, mostly within the Proteobacteria. Here, by DNA-based stable isotope probing (SIP), we directly identified the most active sulfate-reducing toluene degraders in a diverse sedimentary microbial community originating from a tar-oil-contaminated aquifer at a former coal gasification plant. On incubation of fresh sediments with (13)C(7)-toluene, the production of both sulfide and (13)CO(2) was clearly coupled to the (13)C-labeling of DNA of microbes related to Desulfosporosinus spp. within the Peptococcaceae (Clostridia). The screening of labeled DNA fractions also suggested a novel benzylsuccinate synthase alpha-subunit (bssA) sequence type previously only detected in the environment to be tentatively affiliated with these degraders. However, carbon flow from the contaminant into degrader DNA was only ∼50%, pointing toward high ratios of heterotrophic CO(2)-fixation during assimilation of acetyl-CoA originating from the contaminant by these degraders. These findings demonstrate that the importance of non-proteobacterial populations in anaerobic aromatics degradation, as well as their specific ecophysiology in the subsurface may still be largely ungrasped.

Ethylene levels are regulated by a plant encoded 1-aminocyclopropane-1-carboxylic acid deaminase.

Control of the levels of the plant hormone ethylene is crucial in the regulation of many developmental processes and stress responses. Ethylene production can be controlled by altering endogenous levels of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene or by altering its conversion to ethylene. ACC is known to be irreversibly broken down by bacterial or fungal ACC deaminases (ACDs). Sequence analysis revealed two putative ACD genes encoded for in the genome of Arabidopsis thaliana (A. thaliana) and we detected ACD activity in plant extracts. Expression of one of these A. thaliana genes (AtACD1) in bacteria indicated that it had ACD activity. Moreover, transgenic plants harboring antisense constructs of the gene decreased ACD activity to 70% of wild-type (WT) levels, displayed an increased sensitivity to ACC and produced significantly more ethylene. Taken together, these results show that AtACD1 can act as a regulator of ACC levels in A. thaliana.

Quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases.

Throughout molecular evolution, organisms create assorted chemicals in response to varying ecological niches. Catalytic landscapes underlie metabolic evolution, wherein mutational steps alter the biosynthetic properties of enzymes. Here we report the first systematic quantitative characterization of the catalytic landscape underlying the evolution of sesquiterpene chemical diversity. On the basis of our previous discovery of a set of nine naturally occurring amino acid substitutions that functionally interconverted orthologous sesquiterpene synthases from Nicotiana tabacum and Hyoscyamus muticus, we created a library of all possible residue combinations (2(9) = 512) in the N. tabacum enzyme. The product spectra of 418 active enzymes revealed a rugged landscape where several minimal combinations of the nine mutations encode convergent solutions to the interconversions of parental activities. Quantitative comparisons indicated context dependence for mutational effects--epistasis--in product specificity and promiscuity. These results provide a measure of the mutational accessibility of phenotypic variability in a diverging lineage of terpene synthases.

Regulation of simultaneous synthesis of floral scent terpenoids by the 1,8-cineole synthase of Nicotiana suaveolens.

The white flowers of N. suaveolens emit a complex bouquet of fragrance volatiles. The dominant compounds are benzenoids (e.g. methyl benzoate, methyl salicylate, benzyl benzoate and benzyl salicylate), monoterpenes (1,8-cineole, limonene, sabinene, E-beta-ocimene, beta-beta-myrcene, alpha- and beta-pinene and alpha-terpineole) and sesquiterpenes (e.g. caryophyllene), which are all emitted at higher levels during the night. Here, we show that the simultaneous nocturnal emission of most monoterpenes is realized by a single floral-specific multi-product enzyme (1,8-cineole synthase, CIN), which synthesizes the monoterpenes of the "cineole cassette". Interestingly, N. suaveolens is the only known taxon of the Suaveolentes section to have a flower emitting "cineole cassette of monoterpenes" which is otherwise typical for the Alatae section. Gene sequence analysis of CIN has revealed the highest similarities to other angiosperm monoterpene synthases from Vitis vinifera, Quercus ilex, Citrus unshiu and C. limon, which cluster in the same branch of the terpene synthase B subfamily. However, based on its synthesized products, N. suaveolens CIN shares similarity with enzymes of the Arabidopsis thaliana root and Salvia officinalis leaf. The N. suaveolens CIN gene is only expressed in the stigma/style tissue and petals. Thin sections of petals present the enzyme primarily in the adaxial and abaxial epidermis; this facilitates the comprehensive emission of volatiles in all spacial directions. The oscillation of monoterpene emission is a consequence of the regulation of the CIN gene by the circadian clock, with oscillations occurring at the level of transcript and protein accumulations and of enzyme activity. Light/dark or dark/light transition signals synchronize the slow-running endogenous clock. Two strategies for synchronized scent emission have been established in N. suaveolens flowers: (i) the synthesis of volatile organic compounds by a multi-product enzyme and (ii) the coordination of biosynthetic pathways by a circadian clock.

Effect of biotin on activity and gene expression of biotin-dependent carboxylases in the liver of dairy cows.

Biotin is a cofactor of the gluconeogenic enzymes pyruvate carboxylase (PC) and propionyl-coenzyme A carboxylase (PCC). We hypothesized that biotin supplementation increases the activity and gene expression of PC and PCC and the gene expression of phosphoenol-pyruvate carboxykinase (PEPCK) in the liver of lactating dairy cows. Eight multiparous Holstein cows (40 +/- 2 kg/d of milk yield and 162 +/- 35 d in milk) were randomly assigned to 1 of 2 diet sequences in a crossover design with two 22-d periods. Treatments consisted of a basal diet (60% concentrate) containing 0 or 0.96 mg/kg of supplemental biotin. On d 21 of each period, liver tissue was collected by percutaneous liver biopsy. Activities of PC and PCC were determined by measuring the fixation of [14C]O2 in liver homogenates. Abundance of mRNA for PCC, PC, and PEPCK was determined by quantitative reverse-transcription PCR. Biotin supplementation did not affect milk production or composition. Biotin supplementation increased the activity of PC but had no effect on PCC activity. Biotin supplementation did not affect the gene expression of PC, PCC, and PEPCK. The increased activity of PC without changes in mRNA abundance may have been caused by increased activation of the apoenzymes by holocarboxylase synthetase. In conclusion, biotin supplementation affected the activity of PC in the liver of lactating dairy cows, but whether biotin supplementation increases glucose production in the liver remains to be determined.

Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale.

A cDNA clone encoding a sesquiterpene synthase, (+)-germacrene D synthase, has been isolated from ginger (Zingiber officinale). The full-length cDNA (AY860846) contains a 1650-bp open reading frame coding for 550 amino acids (63.8kDa) with a theoretical pI=5.59. The deduced amino acid sequence is 30-46% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a major product, (+)-germacrene D (50.2% of total sesquiterpenoids produced) and a co-product, germacrene B (17.1%) and a number of minor by-products. The optimal pH for the recombinant enzyme is around 7.5. Substantial (+)-germacrene D synthase activity is observed in the presence of Mg2+, Mn2+, Ni2+ or Co2+, while the enzyme is inactive when Cu2+ or Zn2+ is used. The Km- and kcat-values are 0.88 microM and 3.34 x 10(-3) s(-1), respectively. A reaction mechanism involving a double 1,2-hydride shift has been established using deuterium labeled substrates in combination with GC-MS analysis.

Polyunsaturated fats attenuate the dietary phytol-induced increase in hepatic fatty acid oxidation in mice.

The effects of dietary phytol and the type of dietary fat on hepatic fatty acid oxidation were examined in male ICR mice. Mice were fed diets containing 0 or 5 g/kg phytol and 100 g/kg palm, safflower, or fish oil for 21 d. Among the groups fed phytol-free diets, the activities and mRNA abundance of various enzymes involved in fatty acid oxidation were greater in mice fed fish oil than in those fed palm or safflower oil. Dietary phytol profoundly increased the activities and mRNA abundance of hepatic fatty acid oxidation enzymes in mice fed palm oil. However, safflower and fish oils, especially the latter, greatly attenuated the phytol-dependent increase in hepatic fatty acid oxidation. The hepatic concentration of phytanic acid, a metabolite of phytol that is the ligand and activator of retinoid X receptors and peroxisome proliferator-activated receptors, was higher in mice fed fish oil than safflower or palm oil, and in those administered safflower oil than palm oil. The hepatic mRNA abundance of sterol carrier protein-2, a lipid-binding protein involved in phytol metabolism, was inversely correlated with the hepatic concentration of phytanic acid. We demonstrated that polyunsaturated fats attenuate the enhancing effect of dietary phytol on hepatic fatty acid oxidation. Dietary fat-dependent changes in the hepatic phytanic acid concentration cannot account for this phenomenon.

Metabolic flux analysis of diterpene biosynthesis pathway in rice.

Relative transcript levels of eight rice diterpene cyclases at the branch points of gibberellins and phytoalexins biosynthesis pathway were measured by reverse transcription quantitative PCR. Metabolic flux analysis by the distribution ratio of common substrate showed that UV-irradiation of etiolated rice seedlings decreased the flux for primary metabolism of gibberellins biosynthesis by half (from 62 to 27%) and 41% of geranylgeranyl pyrophosphate was used for induction of pimaradiene intermediate as the major phytoalexin. In comparison, light-illumination used almost all geranylgeranyl pyrophosphate (96%) for gibberellin biosynthesis to stimulate the plant growth and strongly repressed the metabolic flux for phytoalexins biosynthesis.

Hydrolytic polyketide shortening by ayg1p, a novel enzyme involved in fungal melanin biosynthesis.

The pentaketide 1,3,6,8-tetrahydroxynaphthalene (T4HN) is a key precursor of 1,8-dihydroxynaphthalene-melanin, an important virulence factor in pathogenic fungi, where T4HN is believed to be the direct product of pentaketide synthases. We showed recently the involvement of a novel protein, Ayg1p, in the formation of T4HN from the heptaketide precursor YWA1 in Aspergillus fumigatus. To investigate the mechanism of its enzymatic function, Ayg1p was purified from an Aspergillus oryzae strain that overexpressed the ayg1 gene. The Ayg1p converted the naphthopyrone YWA1 to T4HN with a release of the acetoacetic acid. Although Ayg1p does not show significant homology with known enzymes, a serine protease-type hydrolytic motif is present in its sequence, and serine-specific inhibitors strongly inhibited the activity. To identify its catalytic residues, site-directed Ayg1p mutants were expressed in Escherichia coli, and their enzyme activities were examined. The single substitution mutations S257A, D352A, and H380A resulted in a complete loss of enzyme activity in Ayg1p. These results indicated that the catalytic triad Asp352-His380-Ser257 constituted the active-site of Ayg1p. From a Dixon plot analysis, 2-acetyl-1,3,6,8-tetrahydroxynaphthalene was found to be a strong mixed-type inhibitor, suggesting the involvement of an acyl-enzyme intermediate. These studies support the mechanism in which the Ser257 at the active site functions as a nucleophile to attack the YWA1 side-chain 1'-carbonyl and cleave the carbon-carbon bond between the naphthalene ring and the side chain. Acetoacetic acid is subsequently released from the Ser257-O-acetoacetylated Ayg1p by hydrolysis. An enzyme with activity similar to Ayg1p in melanin biosynthesis has not been reported in any other organism.

Maize genes induced by herbivory and volicitin.

In crop plants, both mechanical damage and insect attack trigger rapid changes in gene transcription. We investigated whether insect herbivory differs from a general wound response, and if so, is the induction specific to the pest/host plant interaction? Herbivory by beet armyworm (BAW; Spodoptera exigua) caterpillars on maize results in a unique pattern of volatile compounds not triggered by wounding alone that attracts the generalist parasitoid Cotesia marginiventris. Caterpillar-induced volatile emission can be mimicked when a component of the BAW oral secretions (N-(17-hydroxylinolenoyl)-L-glutamine) termed volicitin, is applied to wounded leaves. We identified genes that are affected by BAW feeding by comparing volicitin treatment with wounding alone. We compared cDNAs from these two populations by isolating genes from a subtractive library and using reverse northerns. Virtual northern blots confirmed these results and further showed that BAW infestation affected the expression of these genes. In some cases, BAW feeding inhibited the expression of volicitin-induced genes, suggesting the role of additional bioactive components in caterpillar regurgitate. Transcripts involved in volatile production are increased by volicitin and BAW infestation treatments, and are also detectable at low levels in mechanically wounded leaves. Finally, we identified three new sesquiterpene cyclase genes that are induced by volicitin.

The in vivo synthesis of plant sesquiterpenes by Escherichia coli.

Three plant genes encoding (+)-delta-cadinene, 5-epi-aristolochene, and vetispiradiene cyclases were expressed in Escherichia coli to evaluate the potential of this bacterium to synthesize sesquiterpenes in vivo. Various growth temperatures, carbon sources, and host strains were examined to optimize terpene production. The highest levels of sesquiterpene production occurred when the enzymes were expressed in strain DH5alpha from the trc promoter (Ptrc) of the high-copy plasmidpTrc99A in M9 medium supplemented with 0.2% (v/v) glycerol at 30 degrees C for 5-epi-aristolochene and vetispiradiene and 37 degrees C for (+)-delta-cadinene. The highest concentrations of sesquiterpenes observed were 10.3 microg of (+)-delta-cadinene, 0.24 microg of 5-epi-aristolochene (measured as (+)-delta-cadinene equivalents), and 6.4 microg of vetispiradiene (measured as (+)-delta-cadinene equivalents) per liter of culture. These sesquiterpene production levels are >500-fold lower than carotenoid production, both of which are synthesized from endogenous trans-farnesyl diphosphate (FDP) in E. coli. Based on these results, we conclude that the limiting factor for sesquiterpene synthesis in E. coli is the poor expression of the cyclase enzyme and not supply of the FDP precursor.

Effect of transferring 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA derivative CHA96 on their growth-promoting and disease-suppressive capacities.

Pseudomonas fluorescens strain CHA0, a root colonizing bacterium, has a broad spectrum of biocontrol activity against plant diseases. However, strain CHA0 is unable to utilize 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of plant ethylene, as a sole source of nitrogen. This suggests that CHA0 does not contain the enzyme ACC deaminase, which cleaves ACC to ammonia and alpha-ketobutyrate, and was previously shown to promote root elongation of plant seedlings treated with bacteria containing this enzyme. An ACC deaminase gene, together with its regulatory region, was transferred into P. fluorescens strains CHA0 and CHA96, a global regulatory gacA mutant of CHA0. ACC deaminase activity was expressed in both CHA0 and CHA96. Transformed strains with ACC deaminase activity increased root length of canola plants under gnotobiotic conditions, whereas strains without this activity had no effect. Introduction of ACC deaminase genes into strain CHA0 improved its ability to protect cucumber against Pythium damping-off, and potato tubers against Erwinia soft rot in small hermetically sealed containers. In contrast, ACC deaminase activity had no significant effect on the ability of CHA0 to protect tomato against Fusarium crown and root rot, and potato tubers against soft rot in large hermetically sealed containers. These results suggest that (i) ACC deaminase activity may have lowered the level of plant ethylene thereby increasing root length; (ii) the role of stress-generated plant ethylene in susceptibility or resistance depends on the host-pathogen system, and on the experimental conditions used; and (iii) the constructed strains could be developed as biosensors for the role of ethylene in plant diseases.

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.