Abstract
Fungal cells are highly complex as their metabolism is compartmentalized harboring various types of subcellular organelles that are bordered by one or more membranes. Knowledge about the intracellular localization of transporter proteins is often required for the understanding of their biological function. Among different approaches available, the localization analysis based on the expression of GFP fusions is commonly used as a relatively fast and cost-efficient method that allows visualization of proteins of interest in both live and fixed cells. In addition, inactivation of transporter genes is an important tool to resolve their specific function. Here we provide a detailed protocol for the deletion and localization analysis of ABC transporters in the filamentous fungus Penicillium chrysogenum. It includes construction of expression plasmids, their transformation into fungal strains, cultivation of transformants, microscopy analysis, as well as additional protocols on staining of fungal cells with organelle-specific dyes like Hoechst 33342, MitoTracker DeepRed, and FM4-64.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Kuchler, K., Sterne, R. E., and Thorner, J. (1989) Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells, EMBO J. 8, 3973–3984.
McGrath, J. P. and Varshavsky, A. (1989) The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein, Nature 340, 400–404.
Hlavacek, O., Kucerova, H., Harant, K., Palkova, Z., and Vachova, L. (2009) Putative role for ABC multidrug exporters in yeast quorum sensing, FEBS Lett. 583, 1107–1113.
Smriti, Krishnamurthy, S., Dixit, B. L., Gupta, C. M., Milewski, S., and Prasad, R. (2002) ABC transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are general phospholipid translocators, Yeast 19, 303–318.
Kispal, G., Csere, P., Guiard, B., and Lill, R. (1997) The ABC transporter Atm1p is required for mitochondrial iron homeostasis, FEBS Lett. 418, 346–350.
Kispal, G., Csere, P., Prohl, C., and Lill, R. (1999) The mitochondrial proteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe/S proteins, EMBO J. 18, 3981–3989.
Leighton, J. and Schatz, G. (1995) An ABC transporter in the mitochondrial inner membrane is required for normal growth of yeast, EMBO J. 14, 188–195.
Gupta, A. and Chattoo, B. B. (2008) Functional analysis of a novel ABC transporter ABC4 from Magnaporthe grisea, FEMS Microbiol. Lett. 278, 22–28.
Sun, C. B., Suresh, A., Deng, Y. Z., and Naqvi, N. I. (2006) A multidrug resistance transporter in Magnaporthe is required for host penetration and for survival during oxidative stress, Plant Cell 18, 3686–3705.
Urban, M., Bhargava, T., and Hamer, J. E. (1999) An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease, EMBO J. 18, 512–521.
Barhoom, S., Kupiec, M., Zhao, X., Xu, J. R., and Sharon, A. (2008) Functional characterization of CgCTR2, a putative vacuole copper transporter that is involved in germination and pathogenicity in Colletotrichum gloeosporioides, Eukaryot. Cell 7, 1098–1108.
Callahan, T. M., Rose, M. S., Meade, M. J., Ehrenshaft, M., and Upchurch, R. G. (1999) CFP, the putative cercosporin transporter of Cercospora kikuchii, is required for wild type cercosporin production, resistance, and virulence on soybean, Mol. Plant Microbe Interact. 12, 901–910.
Chague, V., Maor, R., and Sharon, A. (2009) CgOpt1, a putative oligopeptide transporter from Colletotrichum gloeosporioides that is involved in responses to auxin and pathogenicity, BMC. Microbiol. 9, 173.
Fleissner, A., Sopalla, C., and Weltring, K. M. (2002) An ATP-binding cassette multidrug-resistance transporter is necessary for tolerance of Gibberella pulicaris to phytoalexins and virulence on potato tubers, Mol. Plant Microbe Interact. 15, 102–108.
Stefanato, F. L., bou-Mansour, E., Buchala, A., Kretschmer, M., Mosbach, A., Hahn, M., Bochet, C. G., Metraux, J. P., and Schoonbeek, H. J. (2009) The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana, Plant J. 58, 499–510.
Stergiopoulos, I., Zwiers, L. H., and De Waard, M. A. (2003) The ABC transporter MgAtr4 is a virulence factor of Mycosphaerella graminicola that affects colonization of substomatal cavities in wheat leaves, Mol. Plant Microbe Interact. 16, 689–698.
Wahl, R., Wippel, K., Goos, S., Kamper, J., and Sauer, N. (2010) A novel high-affinity sucrose transporter is required for virulence of the plant pathogen Ustilago maydis, PLoS. Biol. 8, e1000303.
Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold, EMBO J. 1, 945–951.
Bairoch, A. (1992) PROSITE: a dictionary of sites and patterns in proteins, Nucleic Acids Res. 20 Suppl, 2013–2018.
Kovalchuk, A. and Driessen, A. J. (2010) Phylo-genetic analysis of fungal ABC transporters, BMC. Genomics 11, 177.
Jungwirth, H. and Kuchler, K. (2006) Yeast ABC transporters-- a tale of sex, stress, drugs and aging, FEBS Lett. 580, 1131–1138.
Iwaki, T., Giga-Hama, Y., and Takegawa, K. (2006) A survey of all 11 ABC transporters in fission yeast: two novel ABC transporters are required for red pigment accumulation in a Schizosaccharomyces pombe adenine biosynthetic mutant, Microbiology 152, 2309–2321.
Snoek, I. S., van der Krogt, Z. A., Touw, H., Kerkman, R., Pronk, J. T., Bovenberg, R. A., van den Berg, M. A., and Daran, J. M. (2009) Construction of an hdfA Penicillium chrysogenum strain impaired in non-homologous end-joining and analysis of its potential for functional analysis studies, Fungal. Genet. Biol. 46, 418–426.
Nijland, J. G., Ebbendorf, B., Woszczysnka, M., Boer, R., Bovenberg, R. A. L., and Driessen, A. J. M. A nonlinear biosynthetic gene cluster dose effect on penicillin production by Penicillium chrysogenum.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual, second ed. Cold Spring Harbor Laboratory Press.
Walhout, A. J., Temple, G. F., Brasch, M. A., Hartley, J. L., Lorson, M. A., van den, H. S., and Vidal, M. (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes, Methods Enzymol. 328, 575–592.
Acknowledgments
This project was financially supported by the Netherlands Ministry of Economic Affairs and the B-Basic partner organizations (http://www.b-basic.nl) through B-Basic, a public–private NWO-ACTS programme (ACTS = Advanced Chemical Technologies for Sustai-nability) and by the Kluyver Centre for Genomics of Industrial Fermentation, which is part of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Kovalchuk, A., Weber, S.S., Nijland, J.G., Bovenberg, R.A.L., Driessen, A.J.M. (2012). Fungal ABC Transporter Deletion and Localization Analysis. In: Bolton, M., Thomma, B. (eds) Plant Fungal Pathogens. Methods in Molecular Biology, vol 835. Humana Press. https://doi.org/10.1007/978-1-61779-501-5_1
Download citation
DOI: https://doi.org/10.1007/978-1-61779-501-5_1
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-500-8
Online ISBN: 978-1-61779-501-5
eBook Packages: Springer Protocols