Abstract
For functional characterization of predicted genes encoding hypothetical proteins in fungal genomes, it is complementary to genetic studies to determine their expression and subcellular localization patterns in different developmental or infection stages. It is also important to identify and characterize other proteins that are physically associated with or functionally related to these genes in vivo by co-immunoprecipitation or affinity purification analyses. In this chapter, we described a set of yeast shuttle vectors and protocols to generate fusion constructs by the yeast gap repair approach. Because of the simplicity and efficiency of yeast gap repair, these vectors and the general methods described in this chapter are suitable for functional genomics studies in filamentous fungi.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Xu, J. R., Peng, Y. L., Dickman, M. B., and Sharon, A. (2006) The dawn of fungal pathogen genomics, Ann. Rev. Phytopathol. 44, 337–366.
Ebbole, D. J. (2007) Magnaporthe as a model for understanding host-pathogen interactions, Annu. Rev. Phytopathol. 45, 437–456.
Xu, J. R., Zhao, X., and Dean, R. A. (2007) From genes to genomes; a new paradigm for studying fungal pathogenesis in Magnaporthe oryzae, Adv. Genet. 57, 175–218.
Raymond, C. K., Sims, E. H., and Olson, M. V. (2002) Linker-mediated recombinational subcloning of large DNA fragments using yeast, Genome Res. 12, 190–197.
Raymond, C. K., Mugford, V. R., and Sexson, S. L. (1999) Plasmid topologies that enhance the transformation efficiency of yeast, Biotechniques 27, 892–894, 896.
Bourett, T. M., Sweigard, J. A., Czymmek, K. J., Carroll, A., and Howard, R. J. (2002) Reef coral fluorescent proteins for visualizing fungal pathogens, Fungal Genet. Biol. 37, 211–220.
Bruno, K. S., Tenjo, F., Li, L., Hamer, J. E., and Xu, J. R. (2004) Cellular localization and role of kinase activity of PMK1 in Magnaporthe grisea, Eukaryot. Cell 3, 1525–1532.
Ho, Y., Gruhler, A., Heilbut, A., Bader, G. D., et al. (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry, Nature 415, 180–183.
Zhao, X. H., and Xu, J. R. (2007) A highly conserved MAPK-docking site in Mst7 is essential for Pmk1 activation in Magnaporthe grisea, Mol. Microbiol. 63, 881–894.
Seong, K., Li, L., Hou, Z. M., Tracy, M., Kistler, H. C., and Xu, J. R. (2006) Cryptic promoter activity in the coding region of the HMG-CoA rediactase gene in Fusarium graminearum, Fungal Genet. Biol. 43, 34–41.
Xu, G., Jansen, G., Thomas, D. Y., Hollenberg, C. P., and Rad, M. R. (1996) Ste50p sustains mating pheromone-induced signal transduction in the yeast Saccharomyces cerevisiae, Mol. Microbiol. 20, 773–783.
Chao, C. C. T., and Ellingboe, A. H. (1991) Selection for mating competence in Magnaporthe grisea pathogenic to rice, Can. J. Bot. 69, 2130–2134.
Sweigard, J. A., Chumley, F. G., and Valent, B. (1992) Disruption of a Magnaporthe grisea cutinase gene, Mol. Gen. Genet. 232, 183–190.
Sambrook, J., and Russell, D. (2001) Molecular Cloning – A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Acknowledgments
The authors thank Dr. Flora Urmeev at Purdue University for assistance in constructing the yeast shuttle vectors. This work was supported by a USDA-NRI grant 2005-35319-16073 and grants from the USWBSI.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zhou, X., Li, G., Xu, JR. (2011). Efficient Approaches for Generating GFP Fusion and Epitope-Tagging Constructs in Filamentous Fungi. In: Xu, JR., Bluhm, B. (eds) Fungal Genomics. Methods in Molecular Biology, vol 722. Humana Press. https://doi.org/10.1007/978-1-61779-040-9_15
Download citation
DOI: https://doi.org/10.1007/978-1-61779-040-9_15
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-039-3
Online ISBN: 978-1-61779-040-9
eBook Packages: Springer Protocols