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Hans D. Vanetten - One of the best experts on this subject based on the ideXlab platform.
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Pisatin biosynthesis from enantiomeric intermediates via an achiral 7 2 dihydroxy 4 5 methylenedioxyisoflav 3 ene
Phytochemistry, 2014Co-Authors: Rhodesia M Celoy, Hans D. VanettenAbstract:(+)-Pisatin, produced by peas (Pisum sativum L.), is an isoflavonoid derivative belonging to the pterocarpan family. It was the first chemically identified phytoalexin, and subsequent research has demonstrated that most legumes produce pterocarpans with the opposite stereochemistry. Studies on the biosynthesis of (+)-Pisatin have shown that (-) enantiomeric compounds are intermediates in (+)-Pisatin synthesis. However, the steps from the (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanone [(-)-sophorol] intermediate to (+)-6a-hydroxymaackiain intermediate are undetermined. Chemical reduction of (-)-sophorol using sodium borohydride (NaBH4) produced two isomers of (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol [(-)-DMDI] with optimal UV absorbance at 299.3 and 300.5 nm, respectively. In contrast, enzymatic reduction of (-)-sophorol by the pea enzyme sophorol reductase (SOR) produced only the 299.3 nm (-)-DMDI isomer. Proton nuclear magnetic resonance ((1)H NMR) analysis of the 299.3 nm (-)-DMDI isomer demonstrated that this isomer had the same NMR spectrum as previously reported for cis-isoflavanol isomers, indicating that cis-(-)-DMDI is an intermediate in (+)-Pisatin biosynthesis. Enzyme assays using protein extracts from pea tissue treated with CuCl2 as an elicitor converted the cis-(-)-DMDI isomer into an achiral isoflavene, 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and the trans-(-)-DMDI isomer was not metabolized by the same protein preparation. A comparison of the enzyme activities on cis-(-)-DMDI with protein preparations from elicited tissue versus non-elicited tissue showed a threefold increase in the amount of activity in the proteins from the elicited tissue. Proteins from the elicited tissues of alfalfa, bean, and chickpea converted cis-(-)-DMDI into either (-)-maackiain and/or (-)-sophorol, while proteins from the elicited tissues of broccoli and pepper produced no detectable product. These results are consistent with the involvement of cis-(-)-DMDI and the achiral DMDIF as intermediates in (+)-Pisatin biosynthesis.
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An ABC transporter and a cytochrome P450 of Nectria haematococca MPVI are virulence factors on pea and are the major tolerance mechanisms to the phytoalexin Pisatin.
Molecular plant-microbe interactions : MPMI, 2011Co-Authors: Jeffrey J Coleman, Gerard J. White, Marianela Rodriguez-carres, Hans D. VanettenAbstract:The fungal plant pathogen Nectria haematococca MPVI produces a cytochrome P450 that is responsible for detoxifying the phytoalexin Pisatin, produced as a defense mechanism by its host, garden pea. In this study, we demonstrate that this fungus also produces a specific ATP-binding cassette (ABC) transporter, NhABC1, that enhances its tolerance to Pisatin. In addition, although both mechanisms individually contribute to the tolerance of Pisatin and act as host-specific virulence factors, mutations in both genes render the fungus even more sensitive to Pisatin and essentially nonpathogenic on pea. NhABC1 is rapidly induced after treatment with Pisatin in vitro and during infection of pea plants. Furthermore, NhABC1 was able to confer tolerance to the phytoalexin rishitin, produced by potato. NhABC1 appears to be orthologous to GpABC1 of the potato pathogen Gibberella pulicaris and, along with MoABC1 from Magnaporthe oryzae, resides in a phylogenetically related clade enriched with ABC transorters involved in virulence. We propose that NhABC1 and the cytochrome P450 may function in a sequential manner in which the energy expense from Pisatin efflux by NhABC1 releases the repression of the cytochrome P450, ultimately allowing Pisatin tolerance by two mechanisms. These results demonstrate that a successful pathogen has evolved multiple mechanisms to overcome these plant antimicrobial compounds.
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inactivation of pea genes by rnai supports the involvement of two similar o methyltransferases in the biosynthesis of Pisatin and of chiral intermediates with a configuration opposite that found in Pisatin
Phytochemistry, 2008Co-Authors: Evans Kaimoyo, Hans D. VanettenAbstract:(+)-Pisatin, the major phytoalexin of pea (Pisum sativum L.), is believed to be synthesized via two chiral intermediates, (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanone [(-)-sophorol] and (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol [(-)-DMDI]; both have an opposite C-3 absolute configuration to that found at C-6a in (+)-Pisatin. The expression of isoflavone reductase (IFR), which converts 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone (DMD) to (-)-sophorol, sophorol reductase (SOR), which converts (-)-sophorol to (-)-DMDI, and hydroxymaackiain-3-O-methyltransferase (HMM), believed to be the last step of (+)-Pisatin biosynthesis, were inactivated by RNA-mediated genetic interference (RNAi) in pea hairy roots. Some hairy root lines containing RNAi constructs of IFR and SOR accumulated DMD or (-)-sophorol, respectively, and were deficient in (+)-Pisatin biosynthesis supporting the involvement of chiral intermediates with a configuration opposite to that found in (+)-Pisatin in the biosynthesis of (+)-Pisatin. Pea proteins also converted (-)-DMDI to an achiral isoflavene suggesting that an isoflavene might be the intermediate through which the configuration is changed to that found in (+)-Pisatin. Hairy roots containing RNAi constructs of HMM also were deficient in (+)-Pisatin biosynthesis, but did not accumulate (+)-6a-hydroxymaackiain, the proposed precursor to (+)-Pisatin. Instead, 2,7,4'-trihydroxyisoflavanone (TIF), daidzein, isoformononetin, and liquiritigenin accumulated. HMM has a high amino acid similarity to hydroxyisoflavanone-4'-O-methyltransferase (HI4'OMT), an enzyme that methylates TIF, an early intermediate in the isoflavonoid pathway. The accumulation of these four compounds is consistent with the blockage of the synthesis of (+)-Pisatin at the HI4'OMT catalyzed step resulting in the accumulation of liquiritigenin and TIF and the diversion of the pathway to produce daidzein and isoformononetin, compounds not normally made by pea. Previous results have identified two highly similar "HMMs" in pea. The current results suggest that both of these O-methyltransferases are involved in (+)-Pisatin biosynthesis and that one functions early in the pathway as HI4'OMT and the second acts at the terminal step of the pathway.
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studies on the late steps of Pisatin biosynthesis evidence for enantiomeric intermediates
Phytochemistry, 2006Co-Authors: Gregory L Dicenzo, Hans D. VanettenAbstract:Abstract Pisatin, a 6a-hydroxyl-pterocarpan phytoalexin from pea (Pisum sativum L.), is relatively unique among naturally occurring pterocarpans by virtue of the (+) stereochemistry of its 6a–11a C–C bond. However, Pisatin synthesizing pea tissue has an isoflavone reductase, first identified in alfalfa, which acts on the (−) antipode. In order to establish the natural biosynthetic pathway to (+) Pisatin, and to evaluate the possible involvement of intermediates with a (−) chirality in its biosynthesis, we administered chiral, tritium-labeled, isoflavanones and pterocarpans to Pisatin-synthesizing pea cotyledons and compared the efficiency of their incorporation. Pea incorporated the isoflavanone, (−) sophorol, more efficiently than either its (+) antipode, or the pterocarpans (+) or (−) maackiain. (−) Sophorol was also metabolized by protein extracts from Pisatin-synthesizing pea seedlings in a NADPH-dependent manner. Three products were produced. One was the isoflavene (7,2′-dihydroxy-4′,5′-methylenedioxyisoflav-3-ene), and another had properties consistent with the isoflavanol (7,2′-dihydroxy-4′,5′-methylenedioxyisoflavanol), the expected product for an isoflavanone reductase. A cDNA encoding sophorol reductase was also isolated from a cDNA library made from Pisatin-synthesizing pea. The cloned recombinant sophorol reductase preferred (−) sophorol over (+) sophorol as a substrate and produced 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavanol. Although no other intermediates in (+) Pisatin biosynthesis were identified, the results lend additional support to the involvement of intermediates of (−) chirality in (+) Pisatin synthesis.
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introduction of plant and fungal genes into pea pisum sativum l hairy roots reduces their ability to produce Pisatin and affects their response to a fungal pathogen
Molecular Plant-microbe Interactions, 2004Co-Authors: Hans D. VanettenAbstract:Pisatin is an isoflavonoid phytoalexin synthesized by pea (Pisum sativum L.). Previous studies have identified two enzymes apparently involved in the synthesis of this phytoalexin, isoflavone reductase (IFR), which catalyzes an intermediate step in Pisatin biosynthesis, and (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM), an enzyme catalyzing the terminal step. To further evaluate the involvement of these enzymes in Pisatin biosynthesis, sense- and antisense-oriented cDNAs of Ifr and Hmm fused to the 35s CaMV promoter, and Agrobacterium rhizogenes, were used to produce transgenic pea hairy root cultures. PDA, a gene encoding Pisatin demethylating activity (pda) in the pea-pathogenic fungus Nectria haematococca, also was used in an attempt to reduce Pisatin levels. Although hairy root tissue with either sense or antisense Ifr cDNA produced less Pisatin, the greatest reduction occurred with sense or antisense Hmm cDNA. The reduced Pisatin production in these lines was associated with reduced amounts of Hmm transcripts, HMM protein, and HMM enzyme activity. Hairy roots containing the PDA gene also produced less Pisatin. To evaluate the role of Pisatin in disease resistance, the virulence of N. haematococca on the transgenic roots that produced the lowest levels of Pisatin was tested. Hairy roots expressing antisense Hmm were more susceptible than the control hairy roots to isolates of N. haematococca that are either virulent or nonvirulent on wild-type pea plants. This appears to be the first case of producing transgenic plant tissue with a reduced ability to produce a phytoalexin and demonstrating that such tissue is less resistant to fungal infection: these results support the hypothesis that phytoalexin production is a disease resistance mechanism.
David C. Straney - One of the best experts on this subject based on the ideXlab platform.
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Analysis of determinants of binding and transcriptional activation of the Pisatin-responsive DNA binding factor of Nectria haematococca.
Molecular plant-microbe interactions : MPMI, 1996Co-Authors: He J, Ruan Y, David C. StraneyAbstract:Pisatin is a fungistatic isoflavonoid produced by garden pea. Field isolates of the ascomycete Nectria haematococca mating population VI (anamorph: Fusarium solani) that are highly virulent on pea have been found to possess the PDA1 gene encoding a Pisatin detoxifying activity. Expression of PDA1 is specifically and highly induced by exposure of mycelia to Pisatin. A Pisatin-responsive DNA-binding activity has previously been identified with properties suggestive of a transcriptional regulator of PDA1. In this study, the sequence determinants for binding this Pisatin-responsive factor (PRF) were localized to a 14-bp region through analysis of sequence alterations that reduced PRF binding. Using a homologous in vitro transcription system, a transcriptional activator of PDA1 was shown to be present in mycelial extracts that shared the sequence specificity characteristic of the PRF, indicating function of the DNA-binding protein in transcriptional control. A 70-kDa protein was shown to be a DNA-binding component of PRF by three independent assays for DNA-binding proteins: Southwestern (DNA-protein) blotting, UV-crosslinking, and binding to immobilized DNA. These results characterize a transcriptional activator acting on the PDA1 promoter that is responsive to a host-specific compound and provide insight into the regulation of fungal genes in response to plant flavonoids.
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In vitro transcription from the Nectria haematococca PDA1 promoter in an homologous extract reflects in vivo Pisatin-responsive regulation.
Current genetics, 1994Co-Authors: Yijun Ruan, David C. StraneyAbstract:The PDA1 gene of Nectria haematococca MP VI (anamorph: Fusarium solani) encodes Pisatin demethylase. This enzyme detoxifies the isoflavanoid phytoalexin Pisatin produced by the plant on which this fungus is pathogenic. Expression of Pisatin demethylase activity is induced in a mycelium by pretreatment with Pisatin. We have developed homologous in vitro system which accurately initiates transcription from the PDA1 promoter. Transcription levels in vitro reflect the same Pisatin-responsive stimulation as measured for PDA1 mRNA in vivo, and are dependent upon sequences in the 5' upstream region of PDA1. Pisatin-responsive transcription from the PDA1 promoter indicates that initiation of transcription is a major regulatory step in the Pisatin induction of Pisatin demethylase expression.
Vipan Kumar - One of the best experts on this subject based on the ideXlab platform.
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1h 1 2 3 triazole tethered nitroimidazole isatin conjugates synthesis docking and anti proliferative evaluation against breast cancer
ACS omega, 2018Co-Authors: Sumit Kumar, Parvesh Singh, Sourav Taru Saha, Gabriella Palma, Shanen Perumal, Ashona Singhpillay, Amit Anand, Mandeep Kaur, Vipan KumarAbstract:1H-1,2,3-Triazole tethered imidazole–isatin and imidazole–isatin–thiosemicarbazone conjugates were synthesized and evaluated against MCF-7 and MDA-MB-231 cell lines. Antiproliferative activities of the synthesized conjugates revealed an optimum combination of longer alkyl chain length as spacer and a halogen-substituent on the isatin ring as a pre-requisite for good activity. The compound 6g with an optimum combination of chloro-substituent at C-5 position of isatin ring and a butyl chain length proved to be most active and noncytotoxic with IC50s of 54.25 and 26.12 μM against MCF-7 and MDA-MB-231 cell lines, respectively.
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azide alkyne cycloaddition en route to 1h 1 2 3 triazole tethered 7 chloroquinoline isatin chimeras synthesis and antimalarial evaluation
European Journal of Medicinal Chemistry, 2013Co-Authors: Raghu Raj, Pardeep Singh, Parvesh Singh, Jiri Gut, Philip J Rosenthal, Vipan KumarAbstract:We describe the synthesis and antimalarial activities of 1H-1,2,3-triazole tethered 7-chloroquinoline-isatin hybrids. Activity against cultured parasites was dependent on the C-5 substituent of the isatin ring as well as the alkyl chain length between the isatin and 7-chloroquinoline moieties. Compound 8h, with an optimum alkyl chain length (n = 3) and a chloro substituent at the C-5 position of the isatin ring, displayed the best activity among the test compounds, with IC50 value of 1.21 μM against cultured W2-strain Plasmodium falciparum.
Durgadas P Kasbekar - One of the best experts on this subject based on the ideXlab platform.
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effects of Pisatin on dictyostelium discoideum its relationship to inducible resistance to nystatin and extension to other isoflavonoid phytoalexins
Archives of Microbiology, 1998Co-Authors: Bhavani T Prasanna, M Vairamani, Durgadas P KasbekarAbstract:Dictyostelium discoideum amoebae can acquire resistance to otherwise inhibitory concentrations of Pisatin, an isoflavonoid phytoalexin of pea, and nystatin, a polyene antibiotic, following pretreatment with sublethal concentrations of these compounds. Additionally, growth on medium containing Pisatin can induce nystatin resistance. We show here that distinct mechanisms mediate the inducible resistance to these two compounds because it is possible to isolate mutations that specifically block the induction of nystatin resistance but do not affect the induction of Pisatin resistance. Pisatin did not affect wild-type sterol biosynthesis; therefore, the induction of nystatin resistance by Pisatin is probably not via an alteration of membrane sterols. The inducible Pisatin resistance phenotype was shown to extend to the isoflavonoid phytoalexins maackiain and biochanin A, and all three compounds inhibited the aggregation of amoebae that is normally triggered by starvation.
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dictyostelium caveatum mutants selected for a nystatin resistant phenotype are cross resistant to Pisatin and other isoflavonoid phytoalexins
Journal of Genetics, 1998Co-Authors: Durgadas P Kasbekar, Bhavani T Prasanna, M VairamaniAbstract:Cellular slime mould amoebae can be induced to become resistant to an otherwise inhibitory concentration of Pisatin, an isoflavonoid phytoalexin of pea, if they are first treated with a subinhibitory concentration. We report here the serendipitous isolation of Pisatin-resistant mutants in the cellular slime mouldDictyostelium caveatum. However, the Pisatin resistance phenotype of the mutants appears to have a different basis than the inducible Pisatin resistance phenotype of the wild type.
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nondegradative Pisatin resistance in dictyostelium discoideum neurospora crassa and nectria haematococca similarities and differences
Journal of Biosciences, 1994Co-Authors: Durgadas P KasbekarAbstract:Paradoxically, on Pisatin-medium (150 μg/ml) the cellular slime mouldDictyostelium discoideum grows only when plated as spores but not when plated as amoebae. The recent discovery of inducible nondegradativc Pisatin resistance in amoebae has allowed us to formulate a model that resolves this paradox. In this model, the germinating amoeba is postulated to acquire a Pisatin-resistance phenotype while ensconced within the spore wall. This article reviews the findings on which this model is based and extends it to also account for the differences in Pisatin sensitivity phenotype that result from sterol alteration in cellular slime moulds and fungi.
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Nondegradative Pisatin-resistance inDictyostelium discoideum, Neurospora crassa andNectria haematococca: Similarities and differences
J Biosci, 1994Co-Authors: Durgadas P KasbekarAbstract:Paradoxically, on Pisatin-medium (150 μg/ml) the cellular slime mould Dictyostelium discoideum grows only when plated as spores but not when plated as amoebae. The recent discovery of inducible nondegradativc Pisatin resistance in amoebae has allowed us to formulate a model that resolves this paradox. In this model, the germinating amoeba is postulated to acquire a Pisatin-resistance phenotype while ensconced within the spore wall. This article reviews the findings on which this model is based and extends it to also account for the differences in Pisatin sensitivity phenotype that result from sterol alteration in cellular slime mould^s and fungi.
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haematococca: Similarities and differences
1994Co-Authors: Nondegradative Pisatin-resistance In Dictyostelium, Durgadas P KasbekarAbstract:Abstract. Paradoxically, on Pisatin-medium (150 µg/ml) the cellular slime mould Dictyos-telium discoideum grows only when plated as spores but not when plated as amoebae. The recent discovery of inducible nondegradativc Pisatin resistance in amoebae has allowed us to formulate a model that resolves this paradox. In this model, the germinating amoeba is postulated to acquire a Pisatin-resistance phenotype while ensconced within the spore wall. This article reviews the findings on which this model is based and extends it to also account for the differences in Pisatin sensitivity phenotype that result from sterol alteration in cellular slime moulds and fungi
He J - One of the best experts on this subject based on the ideXlab platform.
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Analysis of determinants of binding and transcriptional activation of the Pisatin-responsive DNA binding factor of Nectria haematococca.
Molecular plant-microbe interactions : MPMI, 1996Co-Authors: He J, Ruan Y, David C. StraneyAbstract:Pisatin is a fungistatic isoflavonoid produced by garden pea. Field isolates of the ascomycete Nectria haematococca mating population VI (anamorph: Fusarium solani) that are highly virulent on pea have been found to possess the PDA1 gene encoding a Pisatin detoxifying activity. Expression of PDA1 is specifically and highly induced by exposure of mycelia to Pisatin. A Pisatin-responsive DNA-binding activity has previously been identified with properties suggestive of a transcriptional regulator of PDA1. In this study, the sequence determinants for binding this Pisatin-responsive factor (PRF) were localized to a 14-bp region through analysis of sequence alterations that reduced PRF binding. Using a homologous in vitro transcription system, a transcriptional activator of PDA1 was shown to be present in mycelial extracts that shared the sequence specificity characteristic of the PRF, indicating function of the DNA-binding protein in transcriptional control. A 70-kDa protein was shown to be a DNA-binding component of PRF by three independent assays for DNA-binding proteins: Southwestern (DNA-protein) blotting, UV-crosslinking, and binding to immobilized DNA. These results characterize a transcriptional activator acting on the PDA1 promoter that is responsive to a host-specific compound and provide insight into the regulation of fungal genes in response to plant flavonoids.