Abstract
Plants produce low molecular weight compounds with antimicrobial activity in response to microbial attack termed phytoalexins. The first phytoalexin identified was (+) pisatin from pea, and several fungi are able to detoxify pisatin to a less inhibitory compound, including F. oxysporum f. sp. pisi. This detoxification is catalyzed by demethylation of the compound (termed pisatin demethylase activity, or PDA) by the cytochrome P450, Pda. Here we detail two procedures to assess PDA using radiolabeled [14C]pisatin as a substrate and monitoring activity using a scintillation counter.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cruickshank IAM, Perrin DR (1960) Isolation of a phytoalexin from Pisum sativum L. Nature 187:799–800
Cruickshank I (1962) Studies on phytoalexins IV. The antimicrobial spectrum of pisatin. Aust J Biol Sci 15:147–159
Delserone LM, McCluskey K, Matthews DE, Vanetten HD (1999) Pisatin demethylation by fungal pathogens and nonpathogens of pea: association with pisatin tolerance and virulence. Physiol Mol Plant Pathol 55:317–326
George HL, VanEtten HD (2001) Characterization of pisatin-inducible cytochrome P450s in fungal pathogens of pea that detoxify the pea phytoalexin pisatin. Fungal Genet Biol 33:37–48
Coleman JJ, Wasmann CC, Usami T, White GJ, Temporini ED, McCluskey K, VanEtten HD (2011) Characterization of the gene encoding pisatin demethylase (FoPDA1) in Fusarium oxysporum. Mol Plant-Microbe Interact 24:1482–1491
Vanetten H, Temporini E, Wasmann C (2001) Phytoalexin (and phytoanticipin) tolerance as a virulence trait: why is it not required by all pathogens? Physiol Mol Plant Pathol 59:83–93
Geiser DM, Al-Hatmi A, Aoki T, Arie T, Balmas V, Barnes I, Bergstrom GC, Bhattacharyya MKK, Blomquist CL, Bowden R, Brankovics B, Brown DW, Burgess LW, Bushley K, Busman M, Cano-Lira JF, Carrillo JD, Chang HX, Chen CY, Chen W, Chilvers MI, Chulze SN, Coleman JJ, Cuomo CA, de Beer ZW, de Hoog GS, Del Castillo-Múnera J, Del Ponte E, Diéguez-Uribeondo J, Di Pietro A, Edel-Hermann V, Elmer WH, Epstein L, Eskalen A, Esposto MC, Everts KL, Fernández-PavÃa SP, da Silva GF, Foroud NA, Fourie G, Frandsen RJN, Freeman S, Freitag M, Frenkel O, Fuller KK, Gagkaeva T, Gardiner DM, Glenn AE, Gold S, Gordon T, Gregory NF, Gryzenhout M, Guarro J, Gugino B, Gutiérrez S, Hammond-Kosack K, Harris LJ, Homa M, Hong CF, Hornok L, Huang JW, Ilkit M, Jacobs A, Jacobs K, Jiang C, Jimenez-Gasco MDM, Kang S, Kasson MT, Kazan K, Kennell JC, Kim H, Kistler HC, Kuldau GA, Kulik T, Kurzai O, Laraba I, Laurence MH, Lee TY, Lee YW, Lee YH, Leslie JF, Liew ECY, Lofton LW, Logrieco A, Sánchez López-Berges M, Luque AG, Lysøe E, Ma LJ, Marra RE, Martin FN, May SR, McCormick S, McGee CT, Meis JF, Migheli Q, Mohamed Nor NMI, Monod M, Moretti A, Mostert D, Mulé G, Munaut F, Munkvold GP, Nicholson P, Nucci M, O'Donnell K, Pasquali M, Pfenning LH, Prigitano A, Proctor R, Ranque S, Rehner S, Rep M, RodrÃguez-Alvarado G, Rose LJ, Roth MG, Ruiz-Roldán C, Saleh AA, Salleh B, Sang H, Scandiani M, Scauflaire J, Schmale D 3rd, Short DP, Å iÅ¡ić A, Smith J, Smyth CW, Son H, Spahr E, Stajich JE, Steenkamp E, Steinberg C, Subramaniam R, Suga H, Summerell BA, Susca A, Swett CL, Toomajian C, Torres-Cruz TJ, Tortorano AM, Urban M, Vaillancourt LJ, Vallad GE, van der Lee T, Vanderpool D, van Diepeningen AD, Vaughan M, Venter E, Vermeulen M, Verweij PE, Viljoen A, Waalwijk C, Wallace EC, Walther G, Wang J, Ward T, Wickes B, Wiederhold NP, Wingfield MJ, Wood AKM, Xu JR, Yang XB, Yli-Matilla T, Yun SH, Zakaria L, Zhang H, Zhang N, Zhang S, Zhang X (2020) Phylogenomic analysis of a 55.1 kb 19-gene dataset resolves a monophyletic Fusarium that includes the Fusarium solani Species Complex. Phytopathology 111:1064–1079
Wasmann CC, VanEtten HD (1996) Transformation-mediated chromosome loss and disruption of a gene for pisatin demethylase decrease the virulence of Nectria haematococca on pea. Mol Plant-Microbe Interact 9:793–803
Coleman JJ, White GJ, Rodriguez-Carres M, VanEtten HD (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 Interact 24:368–376
Ciuffetti LM, VanEtten HD (1996) Virulence of a pisatin demethylase-deficient Nectria haematococca MPVI isolate is increased by transformation with a pisatin demethylase gene. Mol Plant-Microbe Interact 9:787–792
Temporini ED, VanEtten HD (2004) An analysis of the phylogenetic distribution of the pea pathogenicity genes of Nectria haematococca MPVI supports the hypothesis of their origin by horizontal transfer and uncovers a potentially new pathogen of garden pea: Neocosmospora boniensis. Curr Genet 46:29–36
Van Etten HD, Barz W (1981) Expression of pisatin demethylating ability in Nectria haematococca. Arch Microbiol 129:56–60
Wang Q, Cobine PA, Coleman JJ (2018) Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein compleses. Fungal Genet Biol 117:21–29
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Wasmann, C.C., Coleman, J.J. (2022). Screening and Assessment of Pisatin Demethylase Activity (PDA ). In: Coleman, J. (eds) Fusarium wilt. Methods in Molecular Biology, vol 2391. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1795-3_15
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1795-3_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1794-6
Online ISBN: 978-1-0716-1795-3
eBook Packages: Springer Protocols