Abstract
Recently, the Lr67 resistance gene was identified as a hexose transporter variant which confers adult plant rust and mildew resistance in wheat. Methodologies used to characterize the protein encoded by Lr67 may be of use to non-transporter experts conducting similar experiments with other hexose transporters. Hence, in this chapter, we detail a protocol for the functional characterization of hexose transporter proteins in the Saccharomyces cerevisiae expression system. We also provide guidance on the use of metabolic inhibitors and competing sugars to probe transporter structural features, energization, and specificity.
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References
Ellis JG, Lagudah ES, Spielmeyer W et al (2014) The past, present and future of breeding rust resistant wheat. Front Plant Sci 5:641
Hulbert S, Pumphrey M (2014) A time for more booms and fewer busts? Unraveling cereal-rust interactions. Mol Plant-Microbe Interact 27:207–214
Moore JW, Herrera-Foessel S, Lan C et al (2015) A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat. Nat Genet 47:1494–1498
Hayes MA, Feechan A, Dry IB (2010) Involvement of Abscisic acid in the coordinated regulation of a stress-inducible hexose transporter (VvHT5) and a cell wall invertase in grapevine in response to Biotrophic fungal infection. Plant Physiol 153:211–221
Lemonnier P, Gaillard C, Veillet F et al (2014) Expression of Arabidopsis sugar transport protein STP13 differentially affects glucose transport activity and basal resistance to Botrytis cinerea. Plant Mol Biol 85:473–484
Ward JM (1997) Patch-clamping and other molecular approaches for the study of plasma membrane transporters demystified. Plant Physiol 114:1151–1159
Rentsch D, Boorer KJ, Frommer WB (1998) Structure and function of plasma membrane amino acid, oligopeptide and sucrose transporters from higher plants. J Membr Biol 162:177–190
Wieczorke R, Krampe S, Weierstall T et al (1999) Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett 464:123–128
Hayes MA, Davies C, Dry IB (2007) Isolation, functional characterization, and expression analysis of grapevine (Vitis vinifera L.) hexose transporters: differential roles in sink and source tissues. J Exp Bot 58:1985–1997
Mccurdy DW, Dibley S, Cahyanegara R et al (2010) Functional characterization and RNAi-mediated suppression reveals roles for hexose transporters in sugar accumulation by tomato fruit. Mol Plant 3:1049–1063
Nørholm MHH, Nour-Eldin HH, Brodersen P et al (2006) Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death. FEBS Lett 580:2381–2387
Toyofuku K, Kasahara M, Yamaguchi J (2000) Characterization and expression of monosaccharide transporters (OsMSTs) in rice. Plant Cell Physiol 41:940–947
Chen L-Q, Hou B-H, Lalonde S et al (2010) Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 468:527–532
Dohmen RJ, Strasser AW, Honer CB et al (1991) An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7:691–692
Rentsch D, Laloi M, Rouhara I et al (1995) NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis. FEBS Lett 370:264–268
Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272
Mumberg D, Muller R, Funk M (1994) Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res 22:5767–5768
Mumberg D, Muller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122
Gear ML, Mcphillips ML, Patrick JW et al (2000) Hexose transporters of tomato: molecular cloning, expression analysis and functional characterization. Plant Mol Biol 44:687–697
Acknowledgments
Funding support was provided by Grains Research and Development Corporation, Australia; The Bill and Melinda Gates Foundation; and the Office of the Chief Executive (OCE) Post-Doctoral Fellowship of the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia.
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Milne, R.J., Dibley, K.E., Lagudah, E.S. (2017). Yeast as a Heterologous System to Functionally Characterize a Multiple Rust Resistance Gene that Encodes a Hexose Transporter. In: Periyannan, S. (eds) Wheat Rust Diseases. Methods in Molecular Biology, vol 1659. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7249-4_23
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DOI: https://doi.org/10.1007/978-1-4939-7249-4_23
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-7249-4
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