Hans D Vanetten

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• Celoy, R. M., & VanEtten, H. D. (2014). (+)-Pisatin biosynthesis: From (-) enantiomeric intermediates via an achiral 7,2′-dihydroxy-4′,5′-methylenedioxyisoflav-3-ene. Phytochemistry, 98, 120-127.
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  Abstract: (+)-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 (1H 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. © 2013 Elsevier Ltd. All rights reserved.
• Curlango-Rivera, G., Pew, ,., VanEtten, H., Zhongguo, X., Yu, N., & Hawes, M. (2013). Measuring root disease suppression in response to a compost water extract. Phytopathol.
• Geiser, D. M., Aoki, T., Bacon, C. W., Baker, S. E., Bhattacharyya, M. K., Brandt, M. E., Brown, D. W., Burgess, L. W., Chulze, S., Coleman, J. J., Correll, J. C., Covert, S. F., Crous, P. W., Cuomo, C. A., Sybren, G., Pietro, A. D., Elmer, W. H., Epstein, L., Frandsen, R. J., , Freeman, S., et al. (2013). One fungus, one name: Defining the genus Fusarium in a scientifically robust way that preserves longstanding use. Phytopathology, 103(5), 400-408.
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  PMID: 23379853;Abstract: In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice. © 2013 The American Phytopathological Society.
• Hawes, M., Curlango-Rivera, G., VanEtten, H., Xiong, Z., & Kessler, J. (2012). Extracellular DNA in defense of plant cells: rooting out novel discoveries. International Innovation, 3, 15.
• Milani, N., Daniel, A., Arnold, A., & VanEtten, H. (2012). Origin of pisatin demethylase (PDA) in the genus Fusarium. Fungal Genetics and Biology, 49, 933-942.
• Coleman, J., Wasmann, C., Usami, T., White, G., Temporini, E., McCluskey, K., & VanEtten, H. (2011). Characterization of the Gene Encoding Pisatin Demethylase (FoPDA1) in Fusarium oxysporum. Mol. Plant-Microbe Inter, 24, 1482-1491.
• Coleman, J., White, G., Rodriguez-Carres, M., & VanEtten, H. (2011). 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. Mol. Plant-Microbe Inter, 24, 368-376.
• Hawes, M., Curlango, G., Wen, F., White, G., VanEtten, H., & Xiong, Z. (2011). Extracellular DNA: The tip of root defenses?. Plant Science, 180, 741-745.
• Coleman, J. J., Rounsley, S. D., Rodriguez-Carres, M., Kuo, A., Wasmann, C. C., Grimwood, J., Schmutz, J., Taga, M., White, G. J., Zhou, S., Schwartz, D. C., Freitag, M., Ma, L., G., E., Henrissat, B., Coutinho, P. M., Nelson, D. R., Straney, D., Napoli, C. A., , Barker, B. M., et al. (2009). The genome of Nectria haematococca: Contribution of supernumerary chromosomes to gene expansion. PLoS Genetics, 5(8).
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  PMID: 19714214;PMCID: PMC2725324;Abstract: The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the "Fusarium solani species complex". Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 kb to 6.52 Mb and that the physical size of the genome, 54.43 Mb, and the number of predicted genes, 15,707, are among the largest reported for ascomycetes. Two classes of genes have contributed to gene expansion: specific genes that are not found in other fungi including its closest sequenced relative, Fusarium graminearum; and genes that commonly occur as single copies in other fungi but are present as multiple copies in N. haematococca MPVI. Some of these additional genes appear to have resulted from gene duplication events, while others may have been acquired through horizontal gene transfer. The supernumerary nature of three chromosomes, 14, 15, and 17, was confirmed by their absence in pulsed field gel electrophoresis experiments of some isolates and by demonstrating that these isolates lacked chromosome-specific sequences found on the ends of these chromosomes. These supernumerary chromosomes contain more repeat sequences, are enriched in unique and duplicated genes, and have a lower G+C content in comparison to the other chromosomes. Although the origin(s) of the extra genes and the supernumerary chromosomes is not known, the gene expansion and its large genome size are consistent with this species' diverse range of habitats. Furthermore, the presence of unique genes on supernumerary chromosomes might account for individual isolates having different environmental niches.
• Kaimoyo, E., & VanEtten, H. D. (2008). 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, 69(1), 76-87.
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  PMID: 17707445;Abstract: (+)-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. © 2007 Elsevier Ltd. All rights reserved.
• Kaimoyo, E., Farag, M. A., Sumner, L. W., Wasmann, C., Cuello, J. L., & VanEtten, H. (2008). Sub-lethal levels of electric current elicit the biosynthesis of plant secondary metabolites. Biotechnology Progress, 24(2), 377-384.
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  PMID: 18331050;Abstract: Many secondary metabolites that are normally undetectable or in low amounts in healthy plant tissue are synthesized in high amounts in response to microbial infection. Various abiotic and biotic agents have been shown to mimic microorganisms and act as elicitors of the synthesis of these plant compounds. In the present study, sub-lethal levels of electric current are shown to elicit the biosynthesis of secondary metabolites in transgenic and non-transgenic plant tissue. The production of the phytoalexin (+)-pisatin by pea was used as the main model system. Non-transgenic pea hairy roots treated with 30-100 mA of electric current produced 13 times higher amounts of (+)-pisatin than did the non-elicited controls. Electrically elicited transgenic pea hairy root cultures blocked at various enzymatic steps in the (+)-pisatin biosynthetic pathway also accumulated intermediates preceding the blocked enzymatic step. Secondary metabolites not usually produced by pea accumulated in some of the transgenic root cultures after electric elicitation due to the diversion of the intermediates into new pathways. The amount of pisatin in the medium bathing the roots of electro-elicited roots of hydroponically cultivated pea plants was 10 times higher 24 h after elicitation than in the medium surrounding the roots of non-elicited control plants, showing not only that the electric current elicited (+)-pisatin biosynthesis but also that the (+)-pisatin was released from the roots. Seedlings, intact roots or cell suspension cultures of fenugreek (Trigonella foenum-graecum), barrel medic, (Medicago truncatula), Arabidopsis thaliana, red clover (Trifolium pratense) and chickpea (Cicer arietinum) also produced increased levels of secondary metabolites in response to electro-elicitation. On the basis of our results, electric current would appear to be a general elicitor of plant secondary metabolites and to have potential for application in both basic and commercial research. © 2008 American Chemical Society and American Institute of Chemical Engineers.
• Rodriguez-Carres, M., White, G., Tsuchiya, D., Taga, M., & VanEtten, H. D. (2008). The supernumerary chromosome of Nectria haematococca that carries pea-pathogenicity-related genes also carries a trait for pea rhizosphere competitiveness. Applied and Environmental Microbiology, 74(12), 3849-3856.
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  PMID: 18408061;PMCID: PMC2446569;Abstract: Fungi are found in a wide range of environments, and the ecological and host diversity of the fungus Nectria haematococca has been shown to be due in part to unique genes on different supernumerary chromosomes. These chromosomes have been called "conditionally dispensable" (CD) since they are not needed for axenic growth but are important for expanding the host range of individual isolates. From a biological perspective, the CD chromosomes can be compared to bacterial plasmids that carry unique genes that can define the habits of these microorganisms. The current study establishes that the N. haematococca PDA1-CD chromosome, which contains the genes for pea pathogenicity (PEP cluster) on pea roots, also carries a gene(s) for the utilization of homoserine, a compound found in large amounts in pea root exudates. Competition studies demonstrate that an isolate that lacks the PEP cluster but carries a portion of the CD chromosome which includes the homoserine utilization (HUT) gene(s) is more competitive in the pea rhizosphere than an isolate without the CD chromosome. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
• Akashi, T., VanEtten, H. D., Sawada, Y., Wasmann, C. C., Uchiyama, H., & Ayabe, S. (2006). Catalytic specificity of pea O-methyltransferases suggests gene duplication for (+)-pisatin biosynthesis. Phytochemistry, 67(23), 2525-2530.
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  PMID: 17067644;Abstract: S-adenosyl-l-methionine: 2-hydroxyisoflavanone 4′-O-methyltransferase (HI4′OMT) methylates 2,7, 4′-trihydroxyisoflavanone to produce formononetin, an essential intermediate in the synthesis of isoflavonoids with methoxy or methylenedioxy groups at carbon 4′ (isoflavone numbering). HI4′OMT is highly similar (83% amino acid identity) to (+)-6a-hydroxymaackiain 3-O-methyltransferase (HMM), which catalyzes the last step of (+)-pisatin biosynthesis in pea. Pea contains two linked copies of HMM with 96% amino acid identity. In this report, the catalytic activities of the licorice HI4′OMT protein and of extracts of Escherichia coli containing the pea HMM1 or HMM2 protein are compared on 2,7,4′-trihydroxyisoflavanone and enantiomers of 6a-hydroxymaackiain. All these enzymes produced radiolabelled 2,7-dihydroxy-4′-methoxyisoflavanone or (+)-pisatin from 2,7,4′-trihydroxyisoflavanone or (+)-6a-hydroxymaakiain when incubated with [methyl-14C]-S-adenosyl-l-methionine. No product was detected when (-)-6a-hydroxymaackiain was used as the substrate. HI4′OMT and HMM1 showed efficiencies (relative Vmax/Km) for the methylation of 2,7,4′-trihydroxyisoflavanone 20 and 4 times higher than for the methylation of (+)-6a-hydroxymaackiain, respectively. In contrast, HMM2 had a higher Vmax and lower Km on (+)-6a-hydroxymaackiain, and had a 67-fold higher efficiency for the methylation of (+)-6a-hydroxymaackiain than that for 2,7,4′-trihydroxyisoflavanone. Among the 15 sites at which HMM1 and HMM2 have different amino acid residues, 11 of the residues in HMM1 are the same as found in HI4′OMTs from three plant species. Modeling of the HMM proteins identified three or four putative active site residues responsible for their different substrate preferences. It is proposed that HMM1 is the pea HI4′OMT and that HMM2 evolved by the duplication of a gene encoding a general biosynthetic enzyme (HI4′OMT). © 2006 Elsevier Ltd. All rights reserved.
• DiCenzo, G. L., & VanEtten, H. D. (2006). Studies on the late steps of (+) pisatin biosynthesis: Evidence for (-) enantiomeric intermediates. Phytochemistry, 67(7), 675-683.
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  PMID: 16504226;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. © 2006 Elsevier Ltd. All rights reserved.
• Jian, H. e., M., E., Bashyal, B. P., Zhan, J., Seliga, C. J., Liu, M. X., Pierson, E. E., S., L., VanEtten, H. D., & A., A. (2004). Cytotoxic and other metabolites of Aspergillus inhabiting the rhizosphere of sonoran desert plants. Journal of Natural Products, 67(12), 1985-1991.
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  PMID: 15620238;Abstract: In a study to discover potential anticancer agents from rhizosphere fungi of Sonoran desert plants cytotoxic EtOAc extracts of four Aspergillus strains have been investigated. Two new metabolites, terrequinone A (1) and terrefuranone (2), along with Nα-acetyl aszonalemin (LL-S490β) (3) were isolated from As. terreus occurring in the rhizosphere of Ambrosia ambrosoides, whereas As. terreus inhabiting the rhizosphere of an unidentified Brickellia sp. afforded dehydrocurvularin (4), 11-methoxycurvularin (5), and 11-hydroxycurvularin (6). As. cervinus isolated from the rhizosphere of Anicasanthus thurberi contained two new compounds, 4R*,5S*- dihydroxy-3-methoxy-5-methylcyclohex-2-enone (7) and 6-methoxy-5(6)- dihydropenicillic acid (8), in addition to penicillic acid (9). Penicillic acid was also isolated from As. wentii occurring in the rhizosphere of Larrea tridentata. The structures of 1-9 were elucidated by spectroscopic methods and chemical derivatizations. Acetylation of 2 afforded 14-acetylterrefuranone (13) and 14-deoxy-13(14)-dehydroterrefuranone (14). Metabolites 1-9, the dienone 14, and 5(6)-dihydropenicillic acid (16) were evaluated for cytotoxicity in a panel of four human cancer cell lines and in normal human primary fibroblast cells. Compounds 4 and 5 displayed considerable cytotoxicity, whereas 1, 6, 9, and 14 were found to be moderately active, with 6 and 9 exhibiting selective cytotoxicity against cancer cell lines compared with the normal fibroblast cells.
• Qindong, W. u., & VanEtten, H. D. (2004). 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, 17(7), 798-804.
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  PMID: 15242174;Abstract: 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, senseand 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.
• Temporini, E. D., & VanEtten, H. D. (2004). An analysis of the phylogenetic distribution of the pea pathogenicity genes Nectria haematococca MPVI supports the hypothesis of their origin by horizontal transfer and uncovers a potentially new pathogen of garden pea: Neocosmospora boniensis. Current Genetics, 46(1), 29-36.
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  PMID: 15118835;Abstract: The filamentous fungus Nectria haematococca mating population VI (MPVI) contains a cluster of genes required to cause disease on pea. This cluster of pea pathogenicity genes (the PEP cluster) is located on a supernumerary chromosome that is dispensable for normal growth in culture. The genes in the PEP cluster have a different G + C content and codon usage compared with the genes located on the other chromosomes and a non-homogeneous distribution within the species. These features suggest that the PEP cluster may have been acquired by N. haematococca MPVI through horizontal gene transfer (HGT). In this work, we show that homologues of the PEP genes are present in another pea pathogen, Fusarium oxysporum f. sp. pisi, but are not common among fungi that are phylogenetically closely related to N. haematococca MPVI. This phylogenetic discontinuity supports the hypothesis that the PEP cluster originated by HGT. Our analysis has also determined that homologues for all the PEP genes are present in Neocosmospora boniensis. A molecular characterization of the PEP homologues in this fungus shows that they are organized as a cluster, which has a different physical organization from the PEP cluster in N. haematococca. In addition, although no reports have been found to show that N. boniensis is a naturally occurring pea pathogen, we show here that this species is able to cause disease on pea. © Springer-Verlag 2004.
• Wijeratne, E. K., Carbonezi, C. A., Takahashi, J. A., Seliga, C. J., Turbyville, T. J., Pierson, E. E., S., L., VanEtten, H. D., Whitesell, L., da, V., & Gunatilaka, A. L. (2004). Isolation, optimization of production and structure-activity relationship studies of monocillin I, the cytotoxic constituent of Paraphaeosphaeria quadriseptata. Journal of Antibiotics, 57(8), 541-546.
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  PMID: 15515894;
• Zhan, J., Wijeratne, E. K., Seliga, C. J., Zhang, J., Pierson, E. E., S., L., Vanetten, H. D., & Gunatilaka, A. L. (2004). A new anthraquinone and cytotoxic curvularins of a Penicillium sp. from the rhizosphere of Fallugia paradoxa of the Sonoran desert. Journal of Antibiotics, 57(5), 341-344.
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  PMID: 15317106;
• Zhou, G., M., E., Bigelow, D., S., L., VanEtten, H. D., & A., A. (2004). Aspochalasins I, J, and K: Three New Cytotoxic Cytochalasans of Aspergillus flavipes from the Rhizosphere of Ericameria laricifolia of the Sonoran Desert. Journal of Natural Products, 67(3), 328-332.
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  PMID: 15043404;Abstract: Bioassay-guided fractionation of a cytotoxic EtOAc extract of Aspergillus flavipes occurring in the rhizosphere of Ericameria laricifolia resulted in the isolation of three new cytochalasans, namely, aspochalasins I (1), J (2), and K (3), and four known cytochalasans, aspochalasins C (4), D (5), and E (6) and TMC-169 (7). The structures of compounds 1-3 were established on the basis of extensive 1D and 2D NMR spectroscopic analysis. All compounds exhibited weak to moderate cytotoxicity against NCI-H460, MCF-7, and SF-268 cancer cell lines, but none showed significant selectivity.
• Liu, X., Inlow, M., & VanEtten, H. D. (2003). Expression profiles of pea pathogenicity (PEP) genes in vivo and in vitro, characterization of the flanking regions of the PEP cluster and evidence that the PEP cluster region resulted from horizontal gene transfer in the fungal pathogen Nectria haematococca. Current Genetics, 44(2), 95-103.
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  PMID: 12925899;Abstract: A cluster of pathogenicity genes (PEP1, PEP2, PDA1, PEP5), termed the pea pathogenicity (PEP) cluster and located on a 1.6-Mb conditionally dispensable (CD) chromosome, was identified in the fungal pathogen Nectria haematococca. Studies determined that the expression of PDA1 is induced in both infected pea tissues and in vitro by the phytoalexin pisatin. The present study reports the use of real-time quantitative RT-PCR to monitor the expression of each PEP gene and PDA1. In mycelia actively growing in culture, the mRNA levels of PEP1, PEP5 and PDA1 were very low and the PEP2 transcript was undetectable. In planta, PDA1 and PEP2 were strongly induced, while PEP1 and PEP5 were moderately induced. Starvation slightly enhanced the expression of PEP1, PDA1 and PEP5, while the expression of PEP2 remained undetectable. Exposure to pisatin in culture stimulated the expression of PDA1 and each PEP gene to a similar level as occurred in planta. In addition, all four pathogenicity genes displayed similar temporal patterns of expression in planta and in vitro, consistent with a coordinated regulation of these genes by pisatin during pea pathogenesis. In the flanking regions of the PEP cluster, six open reading frames (ORFs) were identified and all were expressed during infection of pea. Comparison of the codon preferences of these ORFs and seven additional genes from CD chromosomes with the codon preferences of 21 genes from other chromosomes revealed there is a codon bias that correlates with the source of the genes. This difference in codon bias is consistent with the hypothesis that genes on the CD chromosome have a different origin from genes of normal chromosomes, suggesting that horizontal gene transfer may have played a role in the evolution of pathogenesis in N. haematococca.
• M., E., Turbyville, T. J., Zhang, Z., Bigelow, D., S., L., VanEtten, H. D., Whitesell, L., Canfield, L. M., & A., A. (2003). Cytotoxic Constituents of Aspergillus terreus from the Rhizosphere of Opuntia versicolor of the Sonoran Desert. Journal of Natural Products, 66(12), 1567-1573.
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  PMID: 14695798;Abstract: A novel cyclopentenedione, asterredione (1), two new terrecyclic acid A derivatives, (+)-5(6)-dihydro-6-methoxyterrecyclic acid A (2) and (+)-5(6)-dihydro-6-hydroxyterrecyclic acid A (3), and five known compounds, (+)-terrecyclic acid A (4), (-)-quadrone (5), betulinan A (6), asterriquinone D (7), and asterriquinone C-1 (8), were isolated from Aspergillus terreus occurring in the rhizosphere of Opuntia versicolor, using bioassay-guided fractionation. Acid-catalyzed reaction of 2 under mild conditions afforded 4, whereas under harsh conditions 2 yielded 5 and (-)-isoquadrone (9). Catalytic hydrogenation and methylation of 4 afforded 5(6)-dihydro-terrecyclic acid A (10) and (+)-terrecyclic acid A methyl ester (11), respectively. The structures of 1-11 were elucidated by spectroscopic methods. All compounds were evaluated for cytotoxicity in a panel of three sentinel cancer cell lines, NCI-H460 (non-small cell lung cancer), MCF-7 (breast cancer), and SF-268 (CNS glioma), and were found to be moderately active. Cell cycle analysis of 2, 4, and 5 using the NCI-H460 cell line indicated that 4 is capable of disrupting the cell cycle through an apparent arrest to progression at the G1 and G 2/M phases in this p53 competent cell line. A pathway for the biosynthetic origin of asterredione (1) from asterriquinone D (7) is proposed.
• Funnell, D. L., & VanEtten, H. D. (2002). Pisatin demethylase genes are on dispensable chromosomes while genes for pathogenicity on carrot and ripe tomato are on other chromosomes in Nectria haematococca. Molecular Plant-Microbe Interactions, 15(8), 840-846.
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  PMID: 12182342;Abstract: Studies on the wide-host-range fungus Nectria haematococca MP VI have shown a linkage between virulence on pea and five of nine PDA genes that encode the ability to detoxify the pea phytoalexin, pisatin. Most of the PDA genes are on chromosomes of approximately 1.6 megabases (Mb) and two of these genes, PDA1-2 and PDA6-1, have been demonstrated to reside on approximately 1.6-Mb chromosomes that can be lost during meiosis. Prior studies also have shown that the dispensable chromosome carrying PDA6-1 contains a gene (MAK1) necessary for maximum virulence on chickpea. The present study evaluated whether the other approximately 1.6-Mb chromosomes that carry PDA genes also are dispensable, their relationship to each other, and whether they contain genes for pathogenicity on hosts other than pea or chickpea. DNA from the PDA1-1 chromosome (associated with virulence on pea) and the PDA6-1 chromosome (associated with virulence on chickpea) were used to probe blots of contour-clamped homogeneous electric field (CHEF) gels of isolates carrying different PDA genes and genetically related Pda-isolates. All of the approximately 1.6-Mb PDA-bearing chromosomes hybridized with both probes, indicating that they share significant similarity. Genetically related Pda- progeny lacked chromosomes of approximately 1.6 Mb and there was no significant hybridization of any chromosomes to the PDA1-1 and PDA6-1 chromosome probes. When isolates carrying different PDA genes and related Pda- isolates were tested for virulence on carrot and ripe tomato, there was no significant difference in lesion sizes produced by Pda+ and Pda- isolates, indicating that genes for pathogenicity on these hosts are not on the PDA-containing chromosomes. These results support the hypothesis that the chromosomes carrying PDA genes are dispensable and carry host-specific virulence genes while genes for pathogenicity on other hosts are carried on other chromosomes.
• Funnell, D. L., Matthews, P. S., & VanEtten, H. D. (2002). Identification of new pisatin demethylase genes (PDA5 and PDA7) in Nectria haematococca and non-Mendelian segregation of pisatin demethylating ability and virulence on pea due to loss of chromosomal elements. Fungal Genetics and Biology, 37(2), 121-133.
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  PMID: 12409098;Abstract: Previous studies have shown that high virulence on pea in Nectria haematococca Mating Population VI is linked to the ability to detoxify the pea phytoalexin, pisatin, via demethylation (Pda). To test this linkage further, a highly virulent Pda+ isolate (34-18) was used as the recurrent parent in backcrosses to Pda- isolates, but most of the progeny were low in virulence on pea, and tetrad analysis gave conflicting ratios for the genetic control of Pda. Southern analysis of 34-18 and progeny showed that 34-18 carries a gene similar to PDA1 (PDA1-2), two new PDA genes, PDA5 and PDA7, and that all three genes can be lost during meiosis. Southern analysis of electrophoretic karyotypes showed that PDA1-2 is on a 1.5-Mb dispensable chromosome in 34-18 and that PDA5 and PDA7 are on a 4.9-Mb chromosome in 34-18 but are found on variably sized chromosomes in progeny. Loss of PDA5 or PDA7 in progeny was not generally associated with morphological phenotypes, except in progeny from some crosses between PDA5 parents. Loss of PDA5 was associated with growth abnormalities in these crosses, suggesting that in some genetic backgrounds at least a portion of the PDA5/PDA7 chromosome is essential for normal growth. All highly virulent progeny had PDA1-2 or a combination of PDA5 and PDA7 while isolates that lacked the three genes were low in virulence, supporting the hypothesis that Pda, or genes linked to PDA genes, are necessary for virulence on pea. However, low virulence isolates with PDA genes were also identified, suggesting that there are pathogenicity genes that can segregate independently of PDA genes. © 2002 Elsevier Science (USA). All rights reserved.
• Temporini, E. D., & VanEtten, H. D. (2002). Distribution of the pea pathogenicity (PEP) genes in the fungus Nectria haematococca mating population VI. Current Genetics, 41(2), 107-114.
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  PMID: 12073092;Abstract: Previous studies identified a cluster of six genes that are expressed in the fungus Nectria haematococca mating population VI during infection of pea. Four of these genes were shown to contribute to pathogenicity on pea and were called PEP genes for pea pathogenicity. The cluster is located on a "conditionally dispensable" (CD) chromosome and has features similar to bacterial pathogenicity islands. In this study, the occurrence and location of members of the PEP cluster were analyzed in laboratory strains and nine pea pathogenic and 16 non-pea pathogenic field isolates of N. haematococca. Our results indicate that all pea-pathogenic isolates have homologues for all six genes present in the PEP cluster and the homologues appear to be clustered. PEP homologues are also present in isolates that are not pathogenic on pea, although none of these isolates have homologues of all six genes. In addition, PEP homologues are found in CD chromosomes and in other chromosomes. Isolates without PEP homologues are virulent on ripe tomato fruits and carrot roots, indicating that PEP genes are not required for pathogenicity on these hosts.
• Funnell, D. L., Matthews, P. S., & VanEtten, H. D. (2001). Breeding for highly fertile isolates of Nectria haematococca MPVI that are highly virulent on pea and in planta selection for virulent recombinants. Phytopathology, 91(1), 92-101.
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  PMID: 18944283;Abstract: Funnell, D. L., Matthew, P. S., and VanEtten, H. D. 2001. Breeding for highly fertile isolates of Nectria haematococca MPVI that are highly virulent on pea and in planta selection for virulent recombinants. Phytopathology 91:92-101. The heterothallic ascomycete Nectria haematococca mating population VI (anamorph Fusarium solani) is a broad host range pathogen. Field isolates of this fungus that are pathogenic on pea tend to be female sterile, of low fertility, and the same mating type (MAT-1), whereas female fertile isolates of either mating type that are highly fertile tend to be nonpathogenic on this plant. To facilitate genetic analysis of traits that may be important in the ability of N. haematococca to parasitize peas, a breeding project was undertaken to produce hermaphroditic isolates of each mating type that are highly fertile and highly virulent on peas. Although the association of high virulence on peas with female sterility and the MAT-1 mating type was not completely broken, isolates with high fertility and high virulence on peas were bred within two generations. Highly virulent progeny were also isolated by an alternative method in which pea plants were inoculated with a mixture of ascospores from a cross between two moderately virulent parents. Whereas all ascospores isolated without selection in planta had lower virulence than the parents, many isolates recovered from diseased tissue were more, virulent than the parental isolates. Some of the recovered isolates were shown by restriction fragment length polymorphism analysis to be genetic recombinants of the parents, demonstrating that the pea tissue selected virulent recombinants. All highly virulent isolates tested had the ability to detoxify the pea phytoalexin pisatin, again showing a link between this trait and pathogenicity on the pea.
• George, H. L., & VanEtten, H. D. (2001). Characterization of pisatin-inducible cytochrome p450s in fungal pathogens of pea that detoxify the pea phytoalexin pisatin. Fungal Genetics and Biology, 33(1), 37-48.
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  PMID: 11407884;Abstract: Many fungi that are pathogenic on pea have the ability to demethylate and thus detoxify the pea phytoalexin pisatin. This detoxification reaction has been studied most thoroughly in Nectria haematococca MP VI where it functions as a virulence trait. The enzyme catalyzing this reaction [pisatin demethylase (pda)] is a cytochrome P450. In the current study, the induction of whole-cell pda activity and the biochemical properties of pda in microsomal preparations from the pea pathogens Ascochyta pisi, Mycosphaerella pinodes, and Phoma pinodella are compared to the pda produced by N. haematococca. Based on cofactor requirements and their inhibition by carbon monoxide, cytochrome P450 inhibitors, and antibodies to NADPH:cytochrome P450 reductase, we conclude that the pdas from the other pea pathogens also are cytochrome P450s. All of the enzymes show a rather selective induction by pisatin, have a low Km toward pisatin, and have a fairly high degree of specificity toward pisatin as a substrate, suggesting that each pathogen may have a specific cytochrome P450 for detoxifying this plant antibiotic. Since the pdas in these fungi differ in their pattern of sensitivity to P450 inhibitors and display other minor biochemical differences, we suggest that these fungi may have independently evolved a specialized cytochrome P450 as a virulence trait for a common host. © 2001 Academic Press.
• Han, Y., Liu, X., Benny, U., Kistler, H. C., & VanEtten, H. D. (2001). Genes determining pathogenicity to pea are clustered on a supernumerary chromosome in the fungal plant pathogen Nectria haematococca. Plant Journal, 25(3), 305-314.
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  PMID: 11208022;Abstract: Three genes that contribute to the ability of the fungus Nectria haematococca to cause disease on pea plants have been identified. These pea pathogenicity (PEP) genes are within 25 kb of each other and are located on a supernumerary chromosome. Altogether, the PEP gene cluster contains six transcriptional units that are expressed during infection of pea tissue. The biochemical function of only one of the genes is known with certainty. This gene, PDA1, encodes a specific cytochrome P450 that confers resistance to pisatin, an antibiotic produced by pea plants. The three new PEP genes, in addition to PDA1, can independently increase the ability of the fungus to cause lesions on pea when added to an isolate lacking the supernumerary chromosome. Based on predicted amino acid sequences, functions for two of these three genes are hypothesized. The deduced amino acid sequence of another transcribed portion of the PEP cluster, as well as four other open reading frames in the cluster, have a high degree of similarity to known fungal transposases. Several of the features of the PEP cluster - a cluster of pathogenicity genes, the presence of transposable elements, and differences in codon usage and GC content from other portions of the genome - are shared by pathogenicity islands in pathogenic bacteria of plants and animals.
• Jaime-Garcia, R., Orum, T. V., Felix-Gastelum, R., Trinidad-Correa, R., VanEtten, H. D., & Nelson, M. R. (2001). Spatial analysis of Phytophthora infestans genotypes and late blight severity on tomato and potato in the Del Fuerte Valley using geostatistics and geographic information systems. Phytopathology, 91(12), 1156-1165.
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  PMID: 18943330;Abstract: Genetic structure of Phytophthora infestans, the causal agent of potato and tomato late blight, was analyzed spatially in a mixed potato and tomato production area in the Del Fuerte Valley, Sinaloa, Mexico. Isolates of P. infestans were characterized by mating type, allozyme analysis at the glucose-6-phosphate isomerase and peptidase loci, restriction fragment length polymorphism with probe RG57, metalaxyl sensitivity, and aggressiveness to tomato and potato. Spatial patterns of P. infestans genotypes were analyzed by geographical information systems and geostatistics during the seasons of 1994-95, 1995-96, and 1996-97. Spatial analysis of the genetic structure of P. infestans indicates that geographic substructuring of this pathogen occurs in this area. Maps displaying the probabilities of occurrence of mating types and genotypes of P. infestans, and of disease severity at a regional scale, were presented. Some genotypes that exhibited differences in epidemiologically important features such as metalaxyl sensitivity and aggressiveness to tomato and potato had a restricted spread and were localized in isolated areas. Analysis of late blight severity showed recurring patterns, such as the earliest onset of the disease in the area where both potato and tomato were growing, strengthening the hypothesis that infected potato tubers are the main source of primary inoculum. The information that geostatistical analysis provides might help improve management programs for late blight in the Del Fuerte Valley.
• Sandrock, R. W., & VanEtten, H. D. (2001). The relevance of tomatinase activity in pathogens of tomato: Disruption of the β₂-tomatinase gene in Colletotrichum coccodes and Septoria lycopersici and heterologous expression of the Septoria lycopersici β₂-tomatinase in Nectria haematococca, a pathogen of tomato fruit. Physiological and Molecular Plant Pathology, 58(4), 159-171.
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  Abstract: Many fungal pathogens of tomato have the ability to enzymatically detoxify the toxic steroidal glycoalkaloid α-tomatine, synthesized by Lycopersicon and some Solanum species. However, it is unclear whether detoxification of this phytoanticipin is involved in the ability of these fungi to parasitize tomato. We have taken two approaches to address this question. For one approach, we have reduced or eliminated this enzymatic function by transformation-mediated disruption of the gene encoding β2-tomatinase in Colletotrichum coccodes and Septoria lycopersici. Gene disruption of the S. lycopersici β2-tomatinase gene in an albino strain resulted in both a loss of β2-tomatinase activity and a loss of tolerance to α-tomatine. The results of pathogenicity tests with this mutant were inconclusive due to the lack of pathogenicity of the albino mutant. Disruption of the β2-tomatinase gene in C. coccodes resulted in a loss of β2-tomatinase activity but these mutants retained their tolerance to α-tomatine and their abilities to degrade α-tomatine via other enzyme(s). The gene-disrupted mutants were as pathogenic as wild type isolates on green tomato fruit, an organ containing high levels of α-tomatine. For our other approach, we have expressed the S. lycopersici β2-tomatinase gene in a tomatine tolerant mutant of Nectria haematococca MPVI, a fungus that normally can infect ripe but not green tomato fruit. Expression of β2-tomatinase in this N. haematococca mutant enabled it to detoxify α-tomatine and resulted in its ability to parasitize green tomato fruit. © 2001 Academic Press.
• VanEtten, H., Temporini, E., & Wasmann, C. (2001). Phytoalexin (and phytoanticipin) tolerance as a virulence trait: Why is it not required by all pathogens?. Physiological and Molecular Plant Pathology, 59(2), 83-93.
• Delserone, L. M., McCluskey, K., Matthews, D. E., & Vanetten, H. D. (1999). Pisatin demethylation by fungal pathogens and nonpathogens of pea: Association with pisatin tolerance and virulence. Physiological and Molecular Plant Pathology, 55(6), 317-326.
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  Abstract: Previous studies have indicated that detoxification of their hosts' phytoalexins is a tolerance mechanism for some true fungi, but not the fungus-like Oomycota, and may be involved in determining the virulence of a pathogen. In the present study, the associations between demethylation of the pea phytoalexin pisatin, tolerance to pisatin, and virulence on pea were examined for 50 fungal isolates which represent 17 species of pathogens and nonpathogens of pea. All isolates of Pythium coloratum and P. irregulare failed to metabolize and were sensitive to pisatin, consistent with previous observations that members of the Oomycota generally lack the ability to metabolize and are sensitive to their hosts' phytoalexins. Among true fungi tested, the ability to demethylate pisatin was common, regardless of whether the particular isolate was pathogenic on pea or not. However, when the rate of pisatin demethylation was compared to virulence, all but one of the moderate to highly virulent isolates rapidly demethylated pisatin. In addition, the more rapidly demethylating isolates were generally more tolerant of pisatin. These results suggest that a specialized enzyme system for quickly detoxifying pisatin might be present in most pea pathogens. In previous studies a specific cytochrome P450 enzyme for demethylating pisatin was identified in the pea pathogen Nectria haematococca mating population VI, and genes (PDA genes) encoding that enzyme have been cloned from this fungus. When DNA specific for these genes was used to probe genomic DNA from other fungi that demethylate pisatin, significant hybridization was detected with only one fungus, the pea pathogen Fusarium oxysporum f. sp. pisi. If the other pea pathogens possess a specific cytochrome P450 system for detoxification of pisatin, the genes encoding these enzymes apparently share limited nucleotide similarity with N. haematococca PDA genes.
• Taga, M., Murata, M., & Vanetten, H. D. (1999). Visualization of a conditionally dispensable chromosome in the filamentous ascomycete Nectria haematococca by fluorescence in situ hybridization. Fungal Genetics and Biology, 26(3), 169-177.
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  PMID: 10361031;Abstract: Supernumerary chromosomes, termed 'conditionally dispensable' (CD) chromosomes, are known in Nectria haematococca. Because these CD chromosomes had been revealed solely by pulsed-field gel electrophoresis, their morphological properties were unknown. In this study, we visualized a 1.6-Mb CD chromosome of this fungus by three different types of fluorescence in situ hybridization. The CD chromosome at mitotic metaphase was similar in its appearance to the other chromosomes in the genome. Heterochromatic condensation was not distinct in the CD chromosome, suggesting that it is primarily euchromatic. It was also evident that the CD chromosome is unique and not a duplicate of other chromosomes in the genome. At interphase and prophase, the CD chromosome was not dispersed throughout the nucleus, but occupied a limited domain. Occasionally, occurrence of two distinct unattached copies of the CD chromosome were observed during interphase and metaphase.
• Wang, P., Sandrock, R. W., & Vanetten, H. D. (1999). Disruption of the cyanide hydratase gene in Gloeocercospora sorghi increases its sensitivity to the phytoanticipin cyanide but does not affect its pathogenicity on the cyanogenic plant sorghum. Fungal Genetics and Biology, 28(2), 126-134.
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  PMID: 10587474;Abstract: The release of hydrogen cyanide (HCN) from preformed cyanogenic compounds in plants such as sorghum is thought to provide a protective barrier against infection by microorganisms. Gloeocercospora sorghi, a fungal pathogen of sorghum, produces the enzyme cyanide hydratase (CHT) which converts HCN to the less toxic compound formamide. There is considerable prior evidence indicating that this mechanism for detoxifying HCN plays an important role in the pathogenicity of G. sorghi on sorghum. In the present study, the CHT gene was made nonfunctional in G. sorghi through transformation-mediated gene disruption. The transformant lacked CHT activity and no reacting polypeptides were detected with CHT-specific antibodies. This CHT mutant was highly sensitive to HCN, confirming that CHT is an HCN detoxifying mechanism, but it retained virulence on sorghum, causing lesions indistinguishable from those caused by the wild-type strain. This result indicates that G. sorghi does not require CHT for pathogenicity on cyanogenic lines of sorghum and suggests that cyanogenic compounds in plants may serve functions other than providing a mechanism of disease resistance.
• George, H. L., Hirschi, K. D., & VanEtten, H. D. (1998). Biochemical properties of the products of cytochrome P450 genes (PDA) encoding pisatin demethylase activity in Nectria haematococca. Archives of Microbiology, 170(3), 147-154.
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  Abstract: Pea plants produce the antibiotic (+)pisatin in response to infection by the fungus Nectria haematococca, which can detoxify pisatin utilizing a cytochrome P450 monooxygenase called pisatin demethylase. Genes (PDA) have been identified that encode different whole-cell Pda phenotypes that can be distinguished by the length of the lag period and the resulting amount of enzyme activity produced: Pda(SH) = short lag, high activity; Pda(SM) = short lag, moderate activity; and Pda(LL) = long lag, low activity. Only the Pda(SH) and Pda(SM) phenotypes have been correlated with pathogenicity on pea. In this study, we utilize heterologous expression of the PDA(LL) gene PDA6-1 in Aspergillus nidulans to compare the biochemical properties of the product of this gene with the products of the PDA(SH) gene PDA1 expressed in N. haematococca. Preliminary measurements were also done on the PDA(SM) gene PDA5 expressed in N. haematococca. The PDA gene products differed somewhat in their substrate specificity and in their sensitivity to a few cytochrome P450 inhibitors. However, the enzymes produced by PDA6-1 and PDA1 both had low apparent K(m) values toward (+)pisatin (
• Sandrock, R. W., & VanEtten, H. D. (1998). Fungal sensitivity to and enzymatic degradation of the phytoanticipin α-tomatine. Phytopathology, 88(2), 137-143.
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  PMID: 18944982;Abstract: α-Tomatine, synthesized by Lycopersicon and some Solanum species, is toxic to a broad range of fungi, presumably because it binds to 3β-hydroxy sterols in fungal membranes. Several fungal pathogens of tomato have previously been shown to be tolerant of this glycoalkaloid and to possess enzymes thought to be involved in its detoxification. In the current study, 23 fungal strains were examined for their ability to degrade α-tomatine and for their sensitivity to this compound and two breakdown products, β2-tomatine and tomatidine. Both saprophytes and all five nonpathogens of tomato tested were sensitive, while all but two tomato pathogens (Stemphylium solani and Verticillium dahliae) were tolerant of α-tomatine (50% effective dose > 300 μM). Except for an isolate of Botrytis cinerea isolated from grape, no degradation products were detected when saprophytes and nonpathogens were grown in the presence of α-tomatine. All tomato pathogens except Phytophthora infestans and Pythium aphanidermatum degraded α-tomatine. There was a strong correlation between tolerance to α-tomatine, the ability to degrade this compound, and pathogenicity on tomato. However, while β2-tomatine and tomatidine were less toxic to most tomato pathogens, these breakdown products were inhibitory to some of the saprophytes and nonpathogens of tomato, suggesting that tomato pathogens may have multiple tolerance mechanisms to α-tomatine.
• VanEtten, H., Jorgensen, S., Enkerli, J., & Covert, S. F. (1998). Inducing the loss of conditionally dispensable chromosomes in Nectria haematococca during vegetative growth. Current Genetics, 33(4), 299-303.
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  PMID: 9560438;Abstract: A procedure for inducing and detecting the loss of conditionally dispensable (CD) chromosomes in filamentous fungi during vegetative growth was developed using Nectria haematococca mating population VI as a model. CD chromosomes in two different isolates of N. haematococca were tagged via integrative transformation with a gene conferring resistance to hygromycin B. In each case the transformation vector included chromosome-specific DNA in order to direct its homologous recombination with the desired chromosome. Chromosome loss was induced by exposing tagged isolates to inhibitory concentrations of benomyl either for protracted periods of time on solid medium or for short periods of time in liquid medium. After exposure to benomyl, isolates that lost the tagged chromosome were identified by their loss of resistance to hygromycin B. Electrophoretic karyotyping was used to verify that isolates which failed to grow on hygromycin B lacked an intact CD chromosome. Ten other chemicals known to interfere with mitotic events or cell development in other organisms did not induce CD chromosome loss in N. haematococca.
• Qindong, W. u., Preisig, C. L., & VanEtten, H. D. (1997). Isolation of the cDNAs encoding (+)6a-hydroxymaackiain 3-O-methyltransferase, the terminal step for the synthesis of the phytoalexin pisatin in Pisum satvium. Plant Molecular Biology, 35(5), 551-560.
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  PMID: 9349277;Abstract: Pisatin is the major phytoalexin produced by pea upon microbial infection. The enzyme that catalyzes the terminal step in the pisatin biosynthetic pathway is (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM). We report here the isolation and characterization of two HMM cDNA clones (PHMM1 and pHMM2) made from RNA obtained from Nectria haematococca-infected pea tissue. The two clones were confirmed to encode HMM activity by heterologous expression in Escherichia coli. The substrate specificity of the methyltransferases in E. coli was similar to the activity detected in CuCl2-treated pea tissue. Nucleotide sequence analysis of Hmm1 and Hmm2 revealed an open reading frame of 1080 bp and 360 amino acid residues which would encode 40.36 kda and 40.41 kDa polypeptides, respectively. The deduced amino acid sequence of HMM1 has 95.8% identity to HMM2, 40.6% identity to Zrp4, a putative O-methyltransferase (OMT) in maize root, and 39.1% to pBH72-F1, a putative OMT induced in barley by fungal pathogens or UV light. Comparison of the deduced amino acid sequences of the cDNA clones to OMTs from other higher plants identified the binding sites of S-adenosylmethionine (AdoMet). Southern blot analysis showed two closely linked genes with strong homology to Hmm in the pea genome.
• Ciuffetti, L. M., & VanEtten, H. D. (1996). Virulence of a pisatin demethylase-deficient Nectria haematococca MPVI isolate is increased by transformation with a pisatin demethylase gene. Molecular Plant-Microbe Interactions, 9(9), 787-792.
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  Abstract: Previous studies have shown that all isolates of the fungus Nectria haematococca mating population (MP) VI pathogenic on pea are able to detoxify the pea phytoalexin pisatin by demethylation. This demethylation is catalyzed by pisatin demethylase (pdm), a cytochrome P450, and genes (PDA) encoding this cytochrome P450 have been cloned. Naturally occurring isolates of N. haematococca that lack the ability to demethylate pisatin (Pda-) normally lack PDA genes and are not pathogenic on pea. In this study, the effect of Pda on virulence was evaluated by introducing a palm-encoding gene, PDAT9, into a Pda- isolate. Of the 146 Pda+ transformants obtained, 52 were significantly more virulent on pea than the Pda- recipient and control transformants but none was highly virulent on pea. An analysis of three Pda+ transformants revealed that all three transformants, though differing in virulence, were equally tolerant of pisatin and produced similar pdm activity. The results suggest that ability to detoxify pisatin can increase the virulence of N. haematococca MPVI but that for high virulence on pea additional genes are needed.
• Covert, S. F., Enkerli, J., P., V., & VanEtten, H. D. (1996). A gene for maackiain detoxification from a dispensable chromosome of Nectria haematococca. Molecular and General Genetics, 251(4), 397-406.
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  PMID: 8709942;Abstract: In Nectria haematococca the MAK1 gene product converts a chick-pea (Cicer arietinum) phytoalexin, maackiain, into a less toxic compound. The presence of MAK1 in this fungal pathogen is also correlated with high virulence on chick-pea. Previous genetic analysis suggested that MAK1 is located on a meiotically unstable, dispensable chromosome. The unstable nature of this chromosome facilitated MAK1 cloning by allowing us to identify a subset of genomic cosmid clones likely to contain MAK1. Truncated forms of the chromosome, generated during meiosis, were isolated from strains either able (Mak+) or unable (Mak-) to metabolize maackiain and used to probe a chromosome-specific cosmid library. Only clones that hybridized exclusively to the chromosome from the Mak+ strain were then screened for their ability to transform a Mak- isolate to the Mak+ phenotype. A 2.7 kb HindIII-PstI fragment was subcloned from a cosmid conferring MAK1 activity, and its nucleotide sequence determined. Because MAK1 transcription is not induced strongly by maackiain, a reverse transcriptase-polymerase chain reaction was required to detect MAK1 transcription in a Mak+ strain, and to isolate MAK1 cDNA fragments. Comparison of the genomic and cDNA sequences of MAK1 revealed the presence of three introns and an open reading frame encoding a protein 460 amino acids in length. Two diagnostic domains in its deduced amino acid sequence suggest MAK1 encodes a flavin-containing mono-oxygenase. MAK1 is the first gene encoding maackiain detoxification to be cloned, and is the second functional gene cloned from this dispensable chromosome. Southern analysis of genomic DNA from ascospore isolates containing MAK2, MAK3, and MAK4 indicated that MAK1 is not homologous to other known maackiain-detoxifying genes.
• Hirschi, K., & VanEtten, H. (1996). Expression of the pisatin detoxifying genes (PDA) of Nectria haematococca in vitro and in planta. Molecular Plant-Microbe Interactions, 9(6), 483-491.
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  PMID: 8755624;Abstract: The phytopathogenic fungus Nectria haematococca detoxities pisatin, a phytoalexin produced by pea. Pisatin demethylating ability (a phenotype called Pda) is due to pisatin demethylase (pdm) and the genes encoding this enzyme are called PDA. Some isolates rapidly acquire a high to moderate rate of pisatin demethylating activity in culture in response to pisatin (phenotypes Pda(SH) and Pda(SM)), while other isolates only slowly demethylate pisatin (phenotype Pda(LL)). Here we report that PDA-specific RNA levels increased more quickly in response to pisatin in isolates with PDA genes confering a Pda(SH) or Pda(SM) phenotype than in isolates with genes confering a Pda(LL) phenotype. In addition, the pdm activity of transformants of N. haematococca containing chimeric constructs of PDA(SH) and PDA(LL) genes in which the 5' regulatory regions of these genes had been switched supports the conclusion that differential expression of PDA genes is responsible for the different Pda phenotypes detected in vitro. Northern analysis of pea tissue infected with isolates carrying PDA(SH) or PDA(LL) genes indicated that differential induction of these genes also occurred in planta. Only PDA(SH)-specific RNA is readily detected in tissue infected with isolates containing PDA(SH) and PDA(LL) genes. Recently a pisatin biosynthetic gone, isoflavone reductase (IFR), has been identified. Using the polymerase chain reaction, qualitative detection of IFR and PDA(SH) transcripts in infected tissue were made to assess the relative tinting of these genes' expression. No transcripts were detected 6 h after inoculation, but transcripts of both genes were detected at 12 h, suggesting an interplay between the regulatory systems controlling the plants's defense response and the pathogen's counter response.
• Soby, S., Caldera, S., Bates, R., & VanEtten, H. (1996). Detoxification of the phytoalexins maackiain and medicarpin by fungal pathogens of alfalfa. Phytochemistry, 41(3), 759-765.
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  Abstract: Nine fungal pathogens of alfalfa (Medicago sativa) (lucerne) were assayed for their ability to metabolize the pterocarpanoid phytoalexins (-)maackiain and (-)medicarpin. All of the alfalfa fungal isolates were able to metabolize both (-)maackiain and (-)medicarpin. Six different initial reaction products were observed, and often a single isolate produced multiple metabolic products. All the products have been previously described, except for those produced by Cercospora medicaginis. This fungus hydroxylated the pterocarpans at the 1a carbon to form a 1a[R]OH-dienone, in which the hydroxyl group is trans to the bridgehead protons, and is the 1a epimer of the previously described cis form of the compound. (-)Maackiain and both of the 1a hydroxylated epimers were tested for toxicity on isolates of Nectria haematococca mating population I and Saccharomyces cerevisiae that do not degrade maackiain or its 1aOH-dienone products. Both epimers were less toxic than maackiain, and the trans epimer was less toxic than the cis form. Copyright © 1996 Elsevier Science Ltd. All rights reserved.
• Wasmann, C. C., & VanEtten, H. D. (1996). Transformation-mediated chromosome loss and disruption of a gene for pisatin demethylase decrease the virulence of Nectria haematococca on pea. Molecular Plant-Microbe Interactions, 9(9), 793-803.
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  Abstract: Isolates of the fungus Nectria haematococca pathogenic on pea are able to detoxify the phytoalexin pisatin via cytochrome P450-mediated demethylation. To examine the role of pisatin demethylating ability (Pda) in pathogenicity, mutants of N. haematococca deficient in Pda were created by transformation-mediated gene disruption (gdr). Eleven Pda- transformants were obtained as determined biochemically by their inability to demethylate pisatin. The transformants were of two types. Seven of the Pda-transformants were gdr mutants, while the other four were Pda- because they had lost the 1.6-Mb chromosome which carries the gene (PDA1) for Pda in the recipient isolates. All of the Pda- mutants were more sensitive to pisatin and less virulent on pea than the Pda+ recipient isolates. However, only the four chromosome-deletion transformants were reduced to virulence equivalent to naturally occurring Pda- isolates of N. haematococca. There was no evidence that the Pda- gdr mutants contained a cryptic PDA gene induced in planta. Lesion tissue produced by these mutants did not demethylate pisatin while that produced by the wild-type isolates did. In addition, the four chromosome-deletion transformants were as virulent on ripe tomato fruit os the wild-type isolate. These results support the hypothesis that (i) lack of Pda reduces but does not eliminate the virulence of N. haematococca on pea and (ii) an additional gene(s) required for high virulence on pea but not tomato is located on the dispensable chromosome that contains PDA1.
• Sandrock, R. W., DellaPenna, D., & VanEtten, H. D. (1995). Purification and characterization of beta2-tomatinase, an enzyme involved in the degradation of alpha-tomatine and isolation of the gene encoding beta2-tomatinase from Septoria lycopersici. Molecular Plant-Microbe Interactions, 8(6), 960-970.
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  PMID: 8664504;
• Maloney, A. P., & VanEtten, H. D. (1994). A gene from the fungal plant pathogen Nectria haematococca that encodes the phytoalexin-detoxifying enzyme pisatin demethylase defines a new cytochrome P450 family. MGG Molecular & General Genetics, 243(5), 506-514.
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  PMID: 8208242;Abstract: The gene PDAT9 from the fungus Nectria haematococca encodes pisatin demethylase, an enzyme that detoxifies the phytoalexin pisatin, an antimicrobial compound produced by pea in response to infection by this plant pathogen. PDAT9 was found to contain an open reading frame (ORF) encoding 515 amino acids and four introns of 52-58 nucleotides each within its coding region. The amino acid sequence F-G-A-G-S-R-S-C-I-G, indicative of the "fifth ligand binding site" present in all cytochrome P454s, occurs as residues 446 to 455, confirming that PDAT9 is a cytochrome P450. The deduced amino acid sequence is distinct from all other reported cytochrome P-450s, and PDAT9 has been assigned to a new cytochrome P450 family, CYP57. A 1.3 kb SacI fragment of the PDAT9 ORF that lacked the fifth ligand binding site, hybridized to unique DNA fragments in N. haematococca isolates known to possess PDA genes that encode different whole cell phenotypes for pisatin demethylating activity. These genes were also tentatively identified as cytochrome P450s by the hybridization of the same fragments to separate subclones of PDAT9, one of which contained the fifth ligand sequence. That probe also hybridized to DNA other than that attributed to pisatin demethylase genes; these other DNAs are presumed to represent other cytochrome P450s. © 1994 Springer-Verlag.
• Paiva, N. L., Sun, Y., Dixon, R. A., VanEtten, H. D., & Hrazdina, G. (1994). Molecular cloning of isoflavone reductase from pea (Pisum sativum L.): Evidence for a 3R-isoflavanone intermediate in (+)-pisatin biosynthesis. Archives of Biochemistry and Biophysics, 312(2), 501-510.
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  PMID: 8037464;Abstract: Isoflavone reductase (IFR) reduces achiral isoflavones to chiral isoflavanones during the biosynthesis of chiral pterocarpan phytoalexins. A cDNA clone for IFR from pea (Pisum sativum) was isolated using the polymerase chain reaction and expressed in Escherichia coli. Analysis of circular dichroism (CD) spectra of the reduction product sophorol obtained using the recombinant enzyme indicated that the isoflavanone possessed the 3R stereochemistry, in contrast to previous reports indicating a 3S-isoflavanone as the product of the pea IFR. Analysis of CD spectra of sophorol produced using enzyme extracts of CuCl2-treated pea seedlings confirmed the 3R stereochemistry. Thus, the stereochemistry of the isoflavanone intermediate in (+)-pisatin biosynthesis in pea is the same as that in (-)-medicarpin biosynthesis in alfalfa, although the final pterocarpans have the opposite stereochemistry. At the amino acid level the pea IFR cDNA was 91.8 and 85.2% identical to the IFRs from alfalfa and chickpea, respectively. IFR appears to be encoded by a single gene in pea. Its transcripts are highly induced in CuCl2-treated seedlings, consistent with the appearance of IFR enzyme activity and pisatin accumulation.
• Reimmann, C., & VanEtten, H. D. (1994). Cloning and characterization of the PDA6-1 gene encoding a fungal cytochrome P-450 which detoxifies the phytoalexin pisatin from garden pea. Gene, 146(2), 221-226.
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  PMID: 8076822;Abstract: The ability to detoxify pisatin, a phytoalexin produced by garden pea (Pisum sativum), is controlled by a family of PDA (pisatin demethylating ability) genes in the phytopathogenic fungus Nectria haematococca, MP (mating population) VI. Six known PDA genes each encode characteristic levels of inducible enzyme activity and are associated with different degrees of virulence on pea. To elucidate the phenotypic differences associated with these genes, we have cloned and characterized the PDA6-1 gene which encodes a pisatin-detoxifying enzyme and we compare it to another PDA gene, PDAT9. Pisatin demethylation was measured in PDA6-1 transformants of Aspergillus nidulans and shown to be regulated by glucose. The deduced amino acid (aa) sequence of PDA6-1 was 90% identical to that of the cytochrome P-450 encoded by PDAT9, but lacked nine aa at the C terminus, which has been postulated to be a site involved in substrate binding. A 35-bp sequence present upstream of a third PDA gene, PDA1, which appears to be important for induction of PDA1 by pisatin, was conserved in PDAT9, but not in PDA6-1. We conclude that PDA6-1, which does not appear to contribute to the virulence of N. haematococca on pea, differs significantly from PDAT9, which is associated with high virulence. © 1994.
• Straney, D. C., & VanEtten, H. D. (1994). Characterization of the PDA1 promoter of Nectria haematococca and identification of a region that binds a pisatin-responsive DNA binding factor. Molecular Plant-Microbe Interactions, 7(2), 256-266.
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  PMID: 8012044;
• VanEtten, H., Funnell-Baerg, D., Wasmann, C., & McCluskey, K. (1994). Location of pathogenicity genes on dispensable chromosomes in Nectria haematococca MPVI. Antonie van Leeuwenhoek, 65(3), 263-267.
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  PMID: 7847894;Abstract: Nectria haematococca MPVI can be found in many different biological habitats but has been most studied as a pathogen of pea (Pisum sativum). Genetic analyses of isolates obtained from a variety of biological sources has indicated that a number of genes control pathogenicity on pea but that one important PEa Pathogenicity (PEP) gene is PDA, which confers the ability to detoxify the pea phytoalexin pisatin. In these studies, all naturally occurring isolates that lacked PDA (i.e. Pda- isolates) and all Pda- progeny were essentially non-pathogenic on pea. However, we have demonstrated recently that Pda- mutants created by transformation-mediated gene disruptions, while having a modest reduction in virulence, and more virulent than any naturally occurring Pda- isolates. In addition we know that PDA genes are on dispensable (DS) chromosomes in this fungus. We believed that the gene disruption mutants have allowed the detection of other PEP genes that are present on the DS chomosomes along with PDA and that naturally occuring Pda- isolates usually lack this DS chromosome. This would explain why naturally occurring Pda- isolates are always low in virulence. We propose that the DS chromosomes in fungi are analogous to bacterial plasmids which allow those microorganisms to colonise different habitats, i.e. the DS chromosomes of Nectria haematococca contain genes that allow individual isolates of this broad host range pathogen to occupy different biological niches. © 1994 Kluwer Academic Publishers.
• Delserone, L. M., Matthews, D. E., & VanEtten, H. D. (1992). Differential toxicity of enantiomers of maackiain and pisatin to phytopathogenic fungi. Phytochemistry, 31(11), 3813-3819.
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  Abstract: Enantiomers of the isoflavonoid phytoalexins maackiain and pisatin were tested for toxicity to 36 fungal isolates representing 19 species. Nine of these species were pathogens of red clover, which synthesizes (-)maackiain; seven species were reported to be pathogenic on garden pea, which synthesizes predominantly (+)pisatin and minor amounts of (-)maackiain. In general, non-host phytoalexins were more inhibitory than host phytoalexins to growth of these pathogens. In addition, the opposite enantiomer of the host phytoalexin was often more inhibitory to a pathogenic fungus than the normally-occurring enantiomer. There was little correlation between the tolerance of fungi to (+) versus (-)pisatin and differential ability to metabolize the pisatin enantiomers. Nevertheless the data suggest that the production of non-host phytoalexins in transgenic plants might increase resistance of red clover and garden pea to some fungal pathogens. © 1992.
• Miao, V. P., & Vanetten, H. D. (1992). Genetic analysis of the role of phytoalexin detoxification in virulence of the fungus Nectria haematococca on chickpea (Cicer arietinum). Applied and Environmental Microbiology, 58(3), 809-814.
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  PMID: 16348672;PMCID: PMC195338;Abstract: Chickpea (Cicer arietium L.) produces the antimicrobial compounds (phytoalexins) medicarpin and maackiain in response to infection by microorganisms. Nectria haematococca mating population (MP) VI, a fungus pathogenic on chickpea, can metabolize maackiain and medicarpin to less toxic products. These reactions are thought to be detoxification mechanisms in N. haematococca MP VI and required for pathogenesis by this fungus on chickpea. In the present study, these hypotheses were tested by examining the phenotypes of progeny from crosses of the fungus that segregated for genes (Mak genes) controlling phytoalexin metabolism. Mak1 and Mak2, two genes that individually confer the ability to convert maackiain to its 1a-hydroxydienone derivative, were linked to higher tolerance of the phytoalexins and high virulence on chickpea. These results indicate that this metabolic reaction is a mechanism for increased phytoalexin tolerance in the fungus, which thereby allows a higher virulence on chickpea. Mak3, a gene conferring the ability to convert maackiain to its 6a-hydroxypterocarpan derivative, also increased tolerance to maackiain in strains which carried it; however, the contribution of Mak3 to the overall level of pathogenesis could not be evaluated because most progeny from the cross segregating for this gene were low in virulence. Thus, metabolic detoxification of phytoalexins appeared to be necessary, as demonstrated in the Mak1 and Mak2 crosses, but not sufficient by itself, as in the Mak3 cross, for high virulence of N. haematococca MP VI on chickpea.
• Miao, V. P., & Vanetten, H. D. (1992). Three genes for metabolism of the phytoalexin maackiain in the plant pathogen Nectria haematococca: Meiotic instability and relationship to a new gene for pisatin demethylase. Applied and Environmental Microbiology, 58(3), 801-808.
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  PMID: 16348671;PMCID: PMC195337;Abstract: Some isolates of the plant-pathogenic fungus Nectria haematococca mating population (MP) VI metabolize maackiain and medicarpin, two antimicrobial compounds (phytoalexins) synthesized by chickpea (Cicer arietinum L.). The enzymatic modifications by the fungus convert the phytoalexins to less toxic derivatives, and this detoxification has been proposed to be important for pathogenesis on chickpea. In the present study, loci controlling maackiain metabolism (Mak genes) were identified by crosses among isolates of N. haematococca MP VI that differed in their ability to metabolize the phytoalexin. Strains carrying Mak1 or Mak2 converted maackiain to 1a- hydroxymaackiain, while those with Mak3 converted it to 6a-hydroxymaackiain. Mak1 and Mak2 were unusual in that they often failed to be inherited by progeny. Mak1 was closely linked to Pda6, a new member in a family of genes in N. haematococca MP VI that encode enzymes for detoxification of pisatin, the phytoalexin synthesized by garden pea. Like Mak1, Pda6 was also transmitted irregularly to progeny. Although the unusual meiotic behaviors of some Mak genes complicate genetic analysis, identification of these genes should afford a more thorough evaluation of the role of phytoalexin detoxification in the pathogenesis of N. haematococca MP VI on chickpea.
• Wang, P., & VanEtten, H. D. (1992). Cloning and properties of a cyanide hydratase gene from the phytopathogenic fungus Gloeocercospora sorghi. Biochemical and Biophysical Research Communications, 187(2), 1048-1054.
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  PMID: 1382413;Abstract: The Cht gene encoding cyanide hydratase (CHT, EC 4.2.1.66), which detoxifies HCN and is thought to be important in fungal infection of cyanogenic plants, has been cloned from the phytopathogenic fungus Gloeocercospora sorghi. The gene was isolated by screening an expression library of G. sorghi using a CHT-specific antibody and using one of the positive cDNA clones as a probe in Southern hybridization to identify a 3.1 kb PstI genomic fragment. This PstI fragment expressed CHT activity when transformed into Aspergillus nidulans, a fungus that normally lacks CHT activity. Sequence analysis identified a single open reading frame of 1,107 base pairs which encodes a polypeptide of 40,904 daltons. The deduced amino acid sequence of CHT shares 36.5% identity to a nitrilase from the bacterium Klebsiella pneumoniae subsp. ozaenae. © 1992.
• Wang, P., Matthews, D. E., & VanEtten, H. D. (1992). Purification and characterization of cyanide hydratase from the phytopathogenic fungus Gloeocercospora sorghi. Archives of Biochemistry and Biophysics, 298(2), 569-575.
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  PMID: 1416986;Abstract: Previous studies have demonstrated that fungal pathogens of cyanogenic plants produce cyanide hydratase (CHT, EC 4.2.1.66), which converts HCN to formamide. Production of CHT in these fungi is thought to be a means of circumventing cyanide toxicity, and CHT is thus believed to be an important pathogenicity trait. In the present study, 13 species of fungi were assayed for CHT production, and all 7 species that were pathogens of sorghum, a cyanogenic plant, produced this enzyme. CHT was purified to apparent homogeneity from one of these sorghum pathogens, Gloeocercospora sorghi. The enzyme had a Km of 12 mm for KCN. Enzymatically functional CHT was obtained only as a large molecular entity of greater than 300 kDa. However, a polypeptide of approximately 45 kDa was identified as the only component of purified CHT detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 45-kDa polypeptide band could be resolved into three isozymes of pI 6.1, 6.3, and 6.5. Antibodies raised against the 45-kDa polypeptide inhibited the G. sorghi CHT activity and showed high specificity in Western blots to a polypeptide of approximately the same size. The evidence suggests that functional G. sorghi CHT is an aggregated protein that consists of 45-kDa polypeptides. A CHT with similar properties was also found in the fungus Colletotrichum graminicola, another pathogen of sorghum. © 1992.
• Miao, V. P., Covert, S. F., & Vanetten, H. D. (1991). A fungal gene for antibiotic resistance on a dispensable ("B") chromosome. Science, 254(5039), 1773-1776.
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  PMID: 1763326;Abstract: A family of cytochrome P-450 (Pda) genes in the pathogenic fungus Nectria haematococca is responsible for the detoxification of the phytoalexin pisatin, an antimicrobial compound produced by garden pea (Pisum sativum L.). The Pda6 gene was mapped by electrophoretic karyotype analysis to a small meiotically unstable chromosome that is dispensable for normal growth. Such traits are typical of B chromosomes. The strains of Nectria studied here have no sequences that are homologous to the Pda family other than Pda6 and therefore demonstrate that unique, functional genes can be found on B chromosomes. Unstable B chromosomes may be one mechanism for generating pathogenic variation in fungi.
• Miao, V. P., Matthews, D. E., & VanEtten, H. D. (1991). Identification and chromosomal locations of a family of cytochrome P-450 genes for pisatin detoxification in the fungus Nectrla haematococca. MGG Molecular & General Genetics, 226(1-2), 214-223.
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  PMID: 2034215;Abstract: The ability to detoxify the phytoalexin, pisatin, an antimicrobial compound produced by pea (Pisum sativum L.), is one requirement for pathogenicity of the fungus Nectria haematococca on this plant. Detoxification is mediated by a cytochrome P-450, pisatin demethylase, encoded by any one of six Pda genes, which differ with respect to the inducibility and level of pisatin demethylase activity they confer, and which are associated with different levels of virulence on pea. A previously cloned Pda gene (PdaT9) was used in this study to characterize further the known genes and to identify additional members of the Pda family in this fungus by Southern analysis. DNA from all isolates which demethylate pisatin (Pda+ isolates) hybridized to PdaT9, while only one Pda- isolate possessed DNA homologous to the probe. Hybridization intensity and, in some cases, restriction fragment size, were correlated with enzyme inducibility. XhoI/BamHI restricted DNA from reference strains with a single active Pda allele had only one fragment with homology to PdaT9; no homology attributable to alleles associated with the Pda- phenotype was found. Homology to this probe was also limited to one or two restriction fragments in most of the 31 field isolates examined. Some unusual progeny from laboratory crosses that failed to inherit demethylase activity also lost the single restriction fragment homologous to PdaT9. At the chromosome level, N. haematococca is highly variable, each isolate having a unique electrophoretic karyotype. In most instances, PdaT9 hybridized to one or two chromosomes containing 1.6-2 million bases of DNA, while many Pda- isolates lacked chromosomes in this size class. The results from this study of the Pda family support the hypothesis that deletion of large amounts of genomic DNA is one mechanism that reduces the frequency of Pda genes in N. haematococca, while simultaneously increasing its karyotypic variation. © 1991 Springer-Verlag.
• Preisig, C. L., VanEtten, H. D., & Moreau, R. A. (1991). Induction of 6a-hydroxymaackiain 3-O-methyltransferase and phenylalanine ammonia-lyase mRNA translational activities during the biosynthesis of pisatin. Archives of Biochemistry and Biophysics, 290(2), 468-473.
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  PMID: 1929414;Abstract: The isoflavonoid phytoalexin pisatin is synthesized by pea (Pisum sativum L.) in response to microbial infection and certain other forms of stress. The terminal step in the biosynthesis of pisatin is catalyzation by the (+)-6ahydroxymaackiain 3-O-methyltransferase (HMKMT). This enzyme, identified as a protein of Mr 43,000 by photoaffinity labeling (Preisig et al. (1989) Plant Physiol. 91, 559-566), was purified 280-fold from CuCl2-stressed pea seedlings and subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies were raised in rabbit against this protein cut from the polyacrylamide gels. The antiserum against the purified enzyme inhibited HMKMT enzyme activity and showed high specificity for the Mr 43,000 protein on Western blots and in immunoprecipitations. This enzyme, present almost exclusively in the 95,000g supernatant after differential centrifugation, was induced in pea from a low constitutive level by treatment with CuCl2, suggesting that the HMKMT is newly synthesized in response to stress. HMKMT mRNA translational activity increased in peas with time after treatment with CuCl2. Peak translational activity occurred about 12 h after treatment, preceding peak enzyme activity by a few hours. Phenylalanine ammonia-lyase (PAL) mRNA abundance increased coordinately with that of HMKMT. The increase in PAL mRNA translational activity in response to stress is known to reflect transcriptional activation of PAL genes. Thus, the induction by stress of enzyme activity both at an early step and at the terminal step in the phenylpropanoid/isoflavonoid biosynthetic pathway appears to be at the transcriptional level. © 1991.
• Preisig, C. L., Bell, J. N., Sun, Y., Hrazdina, G., Matthews, D. E., & Vanetten, H. D. (1990). Biosynthesis of the phytoalexin pisatin: Isoflavone reduction and further metabolism of the product sophorol by extracts of Pisum sativum. Plant Physiology, 94(3), 1444-1448.
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  PMID: 16667851;PMCID: PMC1077396;Abstract: NADPH-dependent reduction of 2′,7-dihydroxy-4′,5′-methylenedioxyisoflavone to the isoflavanone sophorol, a proposed intermediate step in pisatin biosynthesis, was detected in extracts of Pisum sativum. This isoflavone reductase activity was inducible by treatment of pea seedlings with CuCl2. The timing of induction coincided with that of the 6a-hydroxymaackiain 3-O-methyltransferase, which catalyzes the terminal biosynthetic step. Neither enzyme was light inducible. Further NADPH-dependent metabolism of sophorol by extracts of CuCl2-treated seedlings was also observed; three products were radiolabeled when [3H]sophorol was the substrate, one of which is tentatively identified as maackiain.
• Matthews, D. E., Plattner, R. D., & VanEtten, H. D. (1989). The 6a oxygen of the pterocarpan glycinol is derived from molecular oxygen. Phytochemistry, 28(1), 113-115.
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  Abstract: Previous studies on the biosynthesis of pterocarpan phytoalexins in soybean, Glycine max, suggest that one of the late steps in the pathway is aliphatic hydroxylation of 3,9-dihydroxypterocarpan to 3, 6a, 9-trihydroxypterocarpan (glycinol). We have confirmed this hypothesis by 18O labelling and tandem mass spectrometry. Glycinol synthesized by UV-irradiated cotyledons in the presence of 18O2 contained up to three labelled oxygen atoms per molecule; H2, 18O labelled the remaining two oxygens. One of the atoms labelled by 18O2 was the 6a oxygen. The pathway for biosynthesis of glycinol in soybean thus differs significantly from that for the 6α-hydroxypterocarpan pisatin in pea. © 1989.
• Lucy, M. C., Matthews, P. S., & VanEtten, H. D. (1988). Metabolic detoxification of the phytoalexins maackiain and medicarpin by Nectria haematococca field isolates: relationship to virulence on chickpea. Physiological and Molecular Plant Pathology, 33(2), 187-199.
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  Abstract: Previous studies on isolates of Nectria haematococca mating population (MP) VI demonstrated that this fungus could detoxify the phytoalexins maackiain and medicarpin via three different metabolic routes. In the present study, 130 field isolates of N. haematococca MP VI were tested for ability to metabolize maackiain, tolerance of maackiain, and virulence on chickpea, Cicer arietinum, a plant that produces maackiain and medicarpin as phytoalexins. The isolates differed in these traits, including which of the metabolic routes were used to metabolize maackiain as well as whether maackiain was metabolized at all. Most of the isolates were quite tolerant of maackiain, as they were inhibited less than 25% at 50 μg ml-1. The more sensitive isolates were all low in virulence on chickpea, suggesting that some level of tolerance to maackiain is required for high virulence on this plant. All isolates that were able to metabolize maackiain were tolerant of it, and most isolates lacking this ability were sensitive. However, several maackiain-tolerant isolates failed to metabolize maackiain, and one of these isolates was virulent on chickpea, suggesting that phytoalexin tolerance mechanisms other than detoxification may also be important. Tolerance of medicarpin was strongly correlated with maackiain tolerance in the 53 isolates examined for both traits. © 1988.
• Scala, F., Matthews, D., Costa, M., & VanEtten, H. D. (1988). Immunochemical relatedness of fungal NADPH-cytochrome P-450 reductases and their ability to reconstitute pisatin demethylase activity. Experimental Mycology, 12(4), 377-385.
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  Abstract: NADPH-cytochrome P-450 reductase was purified from isolate T9 of the pathogenic fungus Nectria haematococca, whose virulence on Pisum sativum is due in part to its ability to metabolize and detoxify the pea phytoalexin pisatin via a cytochrome P-450-dependent demethylation. Polyclonal antibodies specific for the reductase were prepared and used to help compare this reductase with the corresponding enzyme from a range of other fungi, including plant pathogenic and nonpathogenic ascomycetes, a basidiomycete, and an oomycete. Some similarities were detected in the reductases from all eight ascomycete species examined and even from the oomycete Pythium coloratum, whereas Western blots were able to distinguish differences among some filamentous ascomycetes. The ability to participate in pisatin demethylation in a reconstituted system differed strongly among the reductases, but also extended as far as the reductase from P. coloratum. © 1988.
• Weltring, K., Turgeon, B., Yoder, O. C., & VanEtten, H. D. (1988). Isolation of a phytoalexin-detoxification gene from the plant pathogenic fungus Nectria haematococca by detecting its expression in Aspergillus nidulans. Gene, 68(2), 335-344.
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  PMID: 3065148;Abstract: Detoxification of the pea phytoalexin pisatin via demethylation, mediated by a cytochrome P-450 monooxygenase, is thought to be important for pathogenicity of the fungus Nectria haematococca on pea. To isolate a fungal gene encoding pisatin demethylating activity (pda), we transformed Aspergillus nidulans with a genomic library of N. haematococca DNA constructed in a cosmid which carried the A. nidulans trpC gene. Transformants were selected for Trp+ and then screened for pda. One transformant among 1250 tested was Pda+ and was less sensitive to pisatin in culture than Pda- A. nidulans. The cosmid containing the gene (PDA) conferring this activity was recovered by phage λ packaging of transformant genomic DNA. When A. nidulans was transformed with the cloned cosmid, 98% of the Trp+ transformants were Pda+. RNA blots probed with a 3.35 kb subclone carrying PDA indicated that the gene is expressed constitutively in A. nidulans but is inducible by pisatin in N. haematococca. © 1988.
• Denny, T. P., Matthews, P. S., & VanEtten, H. D. (1987). A possible mechanism of nondegradative tolerance of pisatin in Nectria haematococca MP VI. Physiological and Molecular Plant Pathology, 30(1), 93-107.
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  Abstract: Nectria haematococca MP VI, a pathogen of pea, is tolerant of pisatin, the major phytoalexin that accumulates in pea during pathogenesis. One mode of pisatin tolerance is the detoxification of this phytoalexin. In previous research, we identified and partially characterized a second mode of tolerance that occurs independently of detoxification ("nondegradative" tolerance). In the current study, we attempted to determine a likely mechanism for nondegradative pisatin tolerance. Pisatin was taken up and retained by mycelium of isolate 126-80 during the time that growth was inhibited by the phytoalexin, but a decrease in the amount of pisatin retained was associated with development of nondegradative tolerance. The evidence was consistent with nondegradative tolerance being the result of a mechanism that excludes pisatin from its site of action, although other mechanisms could not be ruled out. The initial accumulation of pisatin did not appear to require metabolic energy, but energy and protein synthesis were needed for the expression of reduced pisatin retention. However, it was unclear whether metabolic energy was necessary for the maintenance of reduced levels of pisatin. In the absence of any significant changes in pisatin efflux, we deduced that reduced pisatin retention probably occurs when the rate of pisatin influx decreases, and that membrane modifications may be occurring simultaneously. © 1987.
• Desjardins, A. E., & VanEtten, H. D. (1986). Partial purification of pisatin demethylase, a cytochrome P-450 from the pathogenic fungus Nectria haematococca. Archives of Microbiology, 144(1), 84-90.
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  Abstract: The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and ω-aminohexyl-agarose. The reductase was further purified by chromatography on 2′,5′-ADP-agarose to a specific activity of about 16 μmoles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after ω-aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts. © 1986 Springer-Verlag.
• Kistler, H. C., & Vanetten, H. D. (1984). Regulation of pisatin demethylation in Nectria haematococca and its influence on pisatin tolerence and virulence. Journal of General Microbiology, 130(10), 2605-2613.
• Kistler, H. C., & Vanetten, H. D. (1984). Three non-allelic genes for pisatin demethylation in the fungus Nectria haematococca. Journal of General Microbiology, 130(10), 2595-2603.
• VanEtten, H. D., & Matthews, P. S. (1984). Naturally occurring variation in the inducibility of pisatin demethylating activity in Nectria haematococca mating population VI. Physiological Plant Pathology, 25(2), 149-160.
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  Abstract: Fifty-nine field isolates of Nectria haematococca mating population VI, all of which were able to demethylate pisatin, were assayed for the rate of demethylation over a short time interval after a prior treatment with pisatin. Isolates showed a continuous gradation in their demethylation rates, but all isolates highly virulent on pea demonstrated a measurable rate of pisatin demethylation in response to pisatin. This suggested that regulatory control of pisatin demethylation in N. haematococca might influence the virulence of this organism on pea. An attempt to identify further differences in regulatory phenotypes, based on differential induction of pisatin demethylating activity by pisatin versus 6-methoxy-1-tetralone (a known inducer of pisatin demethylation), was unsuccessful. However, the time course of pisatin demethylation by freshly harvested mycelium in the absence of a previous pisatin treatment did differ among the isolates tested. The length of the lag period before initiation of pisatin demethylation might be used as an additional parameter to distinguish different regulatory phenotypes in N. haematococca. © 1984.
• Denny, T. P., & Vanetten, H. D. (1983). Characterization of an inducible, nondegradative tolerance of Nectria haematococca MP VI to phytoalexins. Journal of General Microbiology, 129(9), 2903-2913.
• Denny, T. P., & Vanetten, H. D. (1983). Tolerance of Nectria haematococca MP VI to the phytoalexin Pisatin in the absence of detoxification. Journal of General Microbiology, 129(9), 2893-2901.
• VanEtten, H. D., Matthews, P. S., & Mercer, E. H. (1983). (+)-Maackiain and (+)-medicarpin as phytoalexins in Sophora Japonica and identification of the (-) isomers by biotransformation. Phytochemistry, 22(10), 2291-2295.
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  Abstract: Leaves of Sophora japonica produced the pterocarpanoid phytoalexins medicarpin and maackiain in response to inoculation with the fungus Helminthosporium carbonum. The optical rotation of the phytoalexin samples indicated that the (+) enantiomers were the major optical isomers produced along with a small amount of the (-) enantiomers. This appears to be the first case in which (+) enantiomers of 6aH pterocarpans have been reported as phytoalexins. The presence of (-) enantiomers in the phytoalexin samples was substantiated by stereospecific biotransformations with the fungus Nectria haematococca. The fungus converted optically pure standards of (-)-medicarpin and (-)-maackiain to their (-)-6a-hydroxy derivatives but no 6a-hydroxy derivatives accumulated when (+)-medicarpin and (+)-maackiain were used as the substrates. When the phytoalexin samples from inoculated S. japonica leaves were metabolized by N. haematococca, (-)-6a-hydroxypterocarpans were produced confirming the presence of the (-)enantiomers of medicarpin and maackiain in the phytoalexin samples. Similar analysis of preformed maackiain in root tissue of S. japonica indicated the presence of both optical isomers, as has been reported previously. © 1983.
• Willeke, U., Weltring, K. M., Barz, W., & VanEtten, H. D. (1983). Degradation of the isoflavone biochanin a by isolates of Nectria haematococca (Fusarium solani). Phytochemistry, 22(7), 1539-1541.
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  Abstract: Twelve isolates of Nectria haematococca, mating population VI (Fusarium solani) previously characterized for their virulence on pea plants and their ability to degrade the phytoalexin pisatin were assayed for the catabolism of the isoflavone biochanin A (5,7-dihydroxy-4′-methoxyisoflavone). Eleven isolates catabolized the isoflavone along the pathway: biochanin A → dihydrobiochanin A → 3-(p-methoxyphenyl)-6-hydroxy-γ-pyrone → p-methoxyphenylacetic acid → p-hydroxyphenylacetic acid → 3,4-dihydroxyphenylacetic acid. © 1983.
• Denny, T. P., & VanEtten, H. D. (1982). Metabolism of the phytoalexins medicarpin and maackiain by Fusarium solani. Phytochemistry, 21(5), 1023-1028.
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  Abstract: Non-inhibitory concentrations of the pterocarpan phytoalexin medicarpin were completely metabolized by isolates of Fusarium solani f. sp. pisi, f. sp. cucurbitae, f. sp. phaseoli and two other F. solani isolates genetically related to f. sp. pisi during 24 hr of growth in liquid medium. The major metabolic products accumulated without significant further degradation. Medicarpin was modified at one of three adjacent carbon atoms to form either an isoflavanone derivative, a 1a-hydroxydienone derivative or 6a-hydroxymedicarpin. Whereas each isolate degraded medicarpin to one or more metabolises, the isolates varied as to which metabolise they produced. Maackiain, another pterocarpan phytoalexin, was also metabolized by all the isolates to products analogous to those formed from medicarpin. The ability to metabolize medicarpin and maackiain was not always associated with the ability to metabolize pisatin and phaseollin, two other pterocarpan phytoalexins that were degraded by several of the isolates. Tolerance of medicarpin and maackiain was similarly not always associated with tolerance to pisatin. © 1982.
• VanEtten, H. D., Matthews, P. S., Tegtmeier, K. J., Dietert, M. F., & Stein, J. I. (1980). The association of pisatin tolerance and demethylation with virulence on pea in Nectria haematococca. Physiological Plant Pathology, 16(2), 257-268.
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  Abstract: Fifty-seven field isolates and two ascospore isolates of Nectria haematococca mating population (MP) VI were tested for sensitivity to pisatin, ability to demethylate pisatin, and virulence on pea. There was a range of ability to express each attribute. All of the highly virulent isolates were tolerant of pisatin and were able to demethylate pisatin. All of the isolates most sensitive to pisatin apparently lacked the ability to demethylate pisatin and were of low virulence on pea. An additional group of low virulence isolates was tolerant of pisatin and/or able to metabolize this phytoalexin. Tolerance of and/or the ability to metabolize pisatin appears to be required for high virulence on pea by N. haematococca MP VI. Genetic analysis of the diversity present in this collection of isolates would provide a more rigorous assessment of this hypothesis. © 1980.
• Pueppke, S. G., & VanEtten, H. D. (1976). Accumulation of pisatin and three additional antifungal pterocarpans in Fusarium solani-infected tissues of Pisum sativum. Physiological Plant Pathology, 8(1), 51-61.
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  Abstract: Pisatin and three new pterocarpans accumulated in pea epicotyls and root crowns infected with Fusarium solani f. sp. pisi. Pisatin was first detected 2 days after inoculation and reached a maximum concentration of 78μg g-1 (fresh weight) of diseased tissues 15 days after inoculation. 4-Hydroxy-2,3,9-trimethoxypterocarpan, 3-hydroxy-2,9-dimethoxypterocarpan and 2,3,9-trimethoxypterocarpan first appeared 6 days after inoculation. Although concentrations of the latter two compounds remained low, the concentration of 4-hydroxy-2,3,9-trimethoxypterocarpan was greater than that of the other three antifungal pterocarpans combined by 21 days after inoculation. The three new pterocarpans were not detected in liquid cultures of F. solani f. sp. pisi or in pea tissues induced to produce pisatin by treatment with NaF or by irradiation with u.v. light. Antifungal activity of the new compounds was comparable to that of pisatin. F. solani f. sp. pisi did not interconvert the three new pterocarpans in vitro, but 3-hydroxy-2,9-dimethoxypterocarpan was rapidly degraded to the corresponding isoflavan, 4′,6-dimethoxyisoflavan-2′,7-diol. © 1976.
• VanEtten, H. D. (1976). Antifungal activity of pterocarpans and other selected isoflavonoids. Phytochemistry, 15(5), 655-659.
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  Abstract: Six pterocarpans and 11 structurally related isoflavonoids were tested for antifungal activity against Fusarium solani f. sp. cucurbitae and Aphanomyces euteiches. Representatives from the pterocarpan, isoflavan, and 6a, 11a-dehydropterocarpan classes of isoflavonoids were found that were antifungal. The activity of the antifungal isoflavonoids does not appear to be dependent on a common 3-dimensional shape. © 1976.
• Pueppke, S. G., & VanEtten, H. D. (1975). Identification of three new pterocarpans (6a,11a-dihydro-6H-benzofuro[3,2-c][1]benzopyrans) from Pisum sativum infected with Fusarium solani f. sp. pisi. Journal of the Chemical Society, Perkin Transactions 1, 946-948.
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  Abstract: The isolation of three antifungal pterocarpans from Pisum sativum epicotyls infected with Fusarium solani f. sp. pisi is described. By a combination of spectroscopic and degradative techniques the compounds were identified as 4-hydroxy-2,3,9-trimethoxy- (1), 3-hydroxy-2,9-dimethoxy- (2), and 2,3,9-trimethoxy-pterocarpan (3).
• Smith, D. A., VanEtten, H. D., & Bateman, D. F. (1975). Accumulation of phytoalexins in Phaseolus vulgaris hypocotyls following infection by Rhizoctonia solani. Physiological Plant Pathology, 5(1), 51-64.
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  Abstract: Phaseollin, kievitone, phaseollidin and phaseollinisoflavan were isolated from Rhizoctonia solani-infected bean hypocotyls bearing lesions of different ages. The four phytoalexins were concentrated in the lesion areas per se and accumulated with time. Kievitone occurred at 183 μg/g fresh weight in young lesions and at 638 μg/g fresh weight in mature lesions. Phaseollin concentrations were 18 and 782 μg/g fresh weight, respectively, at comparable stages of lesion maturity. Phaseollidin and phaseollinisoflavan never exceeded 50 μg/g fresh weight. The ED50 values for inhibition of the radial growth of R. solani on an agar medium were 18, 36, 20 and 27 μg/ml, respectively, for phaseollin, kievitone, phaseollidin and phaseollinisoflavan. Phaseollin and kievitone exerted additive fungistatic effects. The concentrations of phaseollin and kievitone present in young lesions caused 85% inhibition of the radial growth of R. solani on a solid medium and a 40% loss of dry weight of R. solani mycelium in shake culture. The phytoalexins, primarily kievitone, may contribute a functional role in disease resistance by restricting the spread of R. solani in invaded host tissues. Bioassays of a number of fungi indicated that there was no correlation between fungal sensitivity to kievitone and pathogenicity to bean. © 1975.
• VanEtten, H. D., & Smith, D. A. (1975). Accumulation of antifungal isoflavonoids and 1a-hydroxyphaseollone, a phaseollin metabolite, in bean tissue infected with Fusarium solani f. sp. phaseoli. Physiological Plant Pathology, 5(3), 225-237.
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  Abstract: 1a-Hydroxyphaseollone, a metabolic product of phaseollin produced in vitro by Fusarium solani f. sp. phaseoli, was isolated from bean pod "drop diffusates" and infected bean hypocotyls 24 and 48 h, respectively, after inoculation with F. solani f. sp. phaseoli. Bioassays of phaseollin and 1a-hydroxyphaseollone against eight fungal species indicated that the latter was less inhibitory, representing a detoxification product of phaseollin. The occurrence of 1a-hydroxy-phaseollone in F. solani f. sp. phaseoli-infected tissue provided evidence for an in situ interaction between phaseollin and F. solani f. sp. phaseoli. While phaseollin, phaseollinisoflavan and 2′-methoxyphaseollinisoflavan accumulated to high concentrations (as high as 5% of the dry weight) in F. solani f. sp. phaseoli-infected tissue, only trace amounts of phaseollidin occurred and no kievitone was detected. F. solani f. sp. phaseoli metabolized phaseolinsoflavan in vitro, but 2′-methoxyphaseollinisoflavan was not detected as a product. This methylated analogue of phaseollinisoflavan, also isolated from Thielaviopsis basicola-infected bean tissue, may be synthesized by the host. © 1975.
• Smith, D. A., VanEtten, H. D., Serum, J. W., Jones, T. M., Bateman, D. F., Williams, T. H., & Coffen, D. L. (1973). Confirmation of the structure of kievitone, an antifungal isoflavanone isolated from Rhizoctonia-infected bean tissues. Physiological Plant Pathology, 3(3), 293-297.
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  Abstract: Hypocotyls of Phaseolus vulgaris L. infected with Rhizoctonia solani Kühn yield the antifungal isoflavanone, kievitone (C20H20O6). On the basis of high and low resolution mass spectrometry, nuclear magnetic resonance spectrometry, infrared and ultraviolet spectroscopy and chemical transformation kievitone was identified as 5,7,2′,4′-tetrahydroxy-8-isopentenylisoflavanone. © 1973.
• VanEtten, H. D. (1973). Identification of a second antifungal isoflavan from diseased Phaseolus vulgaris tissue. Phytochemistry, 12(7), 1791-1792.
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  Abstract: A second antifungal isoflavan has been isolated from diseased bean hypocotyls and identified as 2′-methoxyphascollinisoflavan. © 1973.
• van, J., & VanEtten, H. D. (1973). Detoxification of phaseollin by Fusarium solani f. sp. phaseoli. Physiological Plant Pathology, 3(3), 327-339.
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  Abstract: Evidence is presented indicating that the bean pathogen Fusarium solani f. sp. phaseoli is able to metabolize the phytoalexin phaseollin in vitro. Phaseollin is metabolized in shake cultures of actively growing mycelium exposed to a concentration non-inhibitory to growth. An inhibitory concentration of phaseollin is likewise rapidly metabolized, provided the fungus is first allowed to adapt during exposure to a non-inhibitory dosage. Germinating spores can also metabolize phaseollin. Disappearance of phaseollin is accompanied by the appearance of several metabolic products. The most prominent of these, compound B (C20H18O5), differs from phaseollin in that its mass is 16 units higher and it has a carbonyl group, which suggested that it was an oxidation product of phaseollin. Compound B is less fungitoxic than phaseollin. It is suggested that F. solani f. sp. phaseoli is able to detoxify phaseollin by an inducible oxidizing system. © 1973.

Presentations

• Vanetten, H. D. (2012, January). Habitat defining genes on the supernumerary chromosomes of Nectria haematococcaUMass.
• Vanetten, H. D. (2011, January). Habitat defining genes on the supernumerary chromosomes of Nectria haematococcaUMass.