Advertisement

Knocking Out the Wall: Protocols for Gene Targeting in Physcomitrella patens

  • Alison W. Roberts
  • Christos S. Dimos
  • Michael J. BudziszekJr.
  • Chessa A. Goss
  • Virginia Lai
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 715)

Abstract

The moss Physcomitrella patens has become established as a model for investigating plant gene function due to the feasibility of gene targeting. The chemical composition of the P. patens cell wall is similar to that of vascular plants and phylogenetic analyses of glycosyltransferase sequences from the P. patens genome have identified genes that putatively encode cell wall biosynthetic enzymes, providing a basis for investigating the evolution of cell wall polysaccharides and the enzymes that synthesize them. The protocols described in this chapter provide methods for targeted gene knockout in P. patens, from constructing vectors and maintaining cultures to transforming protoplasts and analyzing the genotypes and phenotypes of the resulting transformed lines.

Key words

Gene targeting Immunocytochemistry Physcomitrella patens Gateway® cloning 

Notes

Acknowledgements

We thank the members of the moss community for their collegiality and helpful suggestions. Magdalena Bezanilla and the members of her group provided invaluable advice and assistance. We thank Didier Schaefer for the gift of pBHSNR. This project was supported by the National Research Initiative Competitive Grant no. 2007-35318-18389 from the USDA National Institute of Food and Agriculture.

References

  1. 1.
    Schaefer, D. G. (2002) A new moss genetics: targeted mutagenesis in Physcomitrella patens. Annu Rev Plant Biol 53, 477–501.PubMedCrossRefGoogle Scholar
  2. 2.
    Strepp, R., Scholz, S., Kruse, S., Speth, V., and Reski, R. (1998) Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin. Proc Natl Acad Sci USA 95, 4368–4373.PubMedCrossRefGoogle Scholar
  3. 3.
    Schaefer, D. and Zrÿd, J. -P. (2004) Principles of targeted mutagenesis in the moss Physcomitrella patens. In: Wood, A.J., Oliver, M.J., and Cove, D.J., eds. New Frontiers in Bryology: Physiology, Molecular Biology and Functional Genomics. Dordrecht: Kluwer; 37–49.CrossRefGoogle Scholar
  4. 4.
    Perroud, P. -F. and Quatrano, R.S. (2006) The role of ARPC4 in tip growth and alignment of the polar axis in filaments of Physcomitrella patens. Cell Motil Cytoskeleton 63, 162–171.PubMedCrossRefGoogle Scholar
  5. 5.
    Sakakibara, K., Nishiyama, T., Sumikawa, N., Kofuji, R., Murata, T. and Hasebe, M. (2003) Involvement of auxin and a homeodomain-leucine zipper I gene in rhizoid development of the moss Physcomitrella patens. Development 130, 4835–4846.PubMedCrossRefGoogle Scholar
  6. 6.
    Cove, D. (2005) The moss Physcomitrella patens. Annu Rev Genet 39, 339–58.PubMedCrossRefGoogle Scholar
  7. 7.
    Richardt, S., Timmerhaus, G., Lang, D., Qudeimat, E., Corrêa, L.G.G., Reski, R., Rensing, S. A., and Frank, W. (2010) Microarray analysis of the moss Physcomitrella patens reveals evolutionarily conserved transcriptional regulation of salt stress and abscisic acid signalling. Plant Mol Biol 72, 27–45.PubMedCrossRefGoogle Scholar
  8. 8.
    Cuming, A. C., Cho, S. H., Kamisugi, Y., Graham, H., and Quatrano, R. S. (2007) Microarray analysis of transcriptional responses to abscisic acid and osmotic, salt, and drought stress in the moss, Physcomitrella patens. New Phytol 176, 275–287.PubMedCrossRefGoogle Scholar
  9. 9.
    Rensing, S. A., Lang, D., Zimmer, A. D., et al. (2008) The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants. Science 319, 64–69.PubMedCrossRefGoogle Scholar
  10. 10.
    Bezanilla, M., Perroud, P. -F., Pan, A., Klueh, P., and Quatrano, R. S. (2005) An RNAi system in Physcomitrella patens with an internal marker for silencing allows for rapid identification of loss of function phenotypes. Plant Biol 7, 251–257.PubMedCrossRefGoogle Scholar
  11. 11.
    Khraiwesh, B., Ossowski, S., Weigel, D., Reski, R., and Frank, W. (2008) Specific gene silencing by artificial MicroRNAs in Physcomitrella patens: an alternative to targeted gene knockouts. Plant Physiol 148, 684–693.PubMedCrossRefGoogle Scholar
  12. 12.
    Vidali, L., Augustine, R. C., Fay, S. N., Franco, P., Pattavina, K. A., and Bezanilla M. (2009) Rapid screening for temperature-sensitive alleles in plants. Plant Physiol 151, 506–514.PubMedCrossRefGoogle Scholar
  13. 13.
    Schumaker, K. S., and Dietrich, M. A. (1998) Hormone-induced signaling during moss development. Annu Rev Plant Physiol Plant Mol Biol 49, 501–523.PubMedCrossRefGoogle Scholar
  14. 14.
    Lee, K. J. D., Sakata, Y., Mau, S. -L., Pettolino, F., Bacic, A., Quatrano, R. S., Knight, C. D., Knox, J. P. (2005) Arabinogalactan proteins are required for apical cell extension in the moss Physcomitrella patens. Plant Cell 17, 3051–3065.PubMedCrossRefGoogle Scholar
  15. 15.
    Liepman, A. H., Nairn, C. J., Willats, W. G. T., Sørensen, I., Roberts, A. W., and Keegstra, K. (2007) Functional genomic analysis supports conservation of function among Cellulose synthase like A gene family members and suggests diverse roles of mannans in plants. Plant Physiol 143, 1881–1893.PubMedCrossRefGoogle Scholar
  16. 16.
    Fu, H., Yadav, M. P., and Nothnagel, E. A. (2007) Physcomitrella patens arabinogala­ctan proteins contain abundant terminal 3-O-methyl-l-rhamnosyl residues not found in angiosperms. Planta 226, 1511–1524.PubMedCrossRefGoogle Scholar
  17. 17.
    Moller, I., Sørensen, I., Bernal, A. J., Blaukopf, C., Lee, K., Øbro, J., Pettolino, F., Roberts, A., Mikkelsen, J. D., Knox, J. P., Bacic, A., and Willats,W. G. T. (2007) High-throughput mapping of cell-wall polymers within and between plants using novel microarrays. Plant J 50, 1118–1128.PubMedCrossRefGoogle Scholar
  18. 18.
    Peña, M. J., Darvill, A. G., Eberhard, S., York, W. S., and O’Neill, M. A. (2008) Moss and liverwort xyloglucans contain galacturonic acid and are structurally distinct from the xyloglucans synthesized by hornworts and vascular plants. Glycobiology 18, 891–904.PubMedCrossRefGoogle Scholar
  19. 19.
    Graham, L. E., Cook, M. E., and Busse, J. S. (2000) The origin of plants: Body plan changes contributing to a major evolutionary radiation. Proc Natl Acad Sci USA 97, 4535–4540.PubMedCrossRefGoogle Scholar
  20. 20.
    Niklas, K. J. (2004) The cell walls that bind the tree of life. BioScience 54, 831–841.CrossRefGoogle Scholar
  21. 21.
    Roberts, A. W., and Bushoven, J. T. (2007) The cellulose synthase (CESA) gene superfamily of the moss Physcomitrella patens. Plant Mol Biol 63, 207–219.PubMedCrossRefGoogle Scholar
  22. 22.
    Schaefer, D. G., and Zrÿd, J. -P. (2001) The moss Physcomitrella patens, now and then. Plant Physiol 127, 1430–1438.PubMedCrossRefGoogle Scholar
  23. 23.
    Lang, D., Zimmer, A. D., Rensing, S. A., and Reski, R. (2008) Exploring plant biodiversity: the Physcomitrella genome and beyond. Trends Plant Sci 13, 542–9.PubMedCrossRefGoogle Scholar
  24. 24.
    Schaefer, D. G. (2001) Gene targeting in Physcomitrella patens. Curr Opin Plant Biol 4, 143–150.PubMedCrossRefGoogle Scholar
  25. 25.
    Decker, E. L., Frank, W., Sarnighausen, E., and Reski, R. (2006) Moss systems biology en route: phytohormones in Physcomitrella development. Plant Biol 8, 397–405.PubMedCrossRefGoogle Scholar
  26. 26.
    Cove, D., Benzanilla, M., Harries, P., and Quatrano, R. (2006) Mosses as model systems for the study of metabolism and development. Annu Rev Plant Biol 57, 497–520.PubMedCrossRefGoogle Scholar
  27. 27.
    Frank, W., Decker, E. L., and Reski, R. (2005) Molecular tools to study Physcomitrella patens. Plant Biol 7, 220–227.PubMedCrossRefGoogle Scholar
  28. 28.
    Cove, D. J., Perroud, P. F., Charron, A. J., McDaniel, S. F., Khandelwal, A., and Quatrano, R. S. (2009) The moss Physcomitrella patens. A novel model system for plant development and genomic studies. In: Behringer, R.R., Johnson, A.D., Krumla\uf, R.E., eds. Emerging Model Organisms: A Laboratory Manual: Cold Spring Harbor Laboratory Press 69–104.Google Scholar
  29. 29.
    Knight, C. D., Cove, D. J., Cuming, A. C., and Quatrano, R. S. (2002) Moss gene technology. In: Gilmartin, P.M., C. B, eds. Molecular Plant Biology — A Practical Approach. Oxford, New York: Oxford Press; 285–301.Google Scholar
  30. 30.
    Vidali, L., Augustine, R. C., Kleinman, K. P., and Bezanilla, M. (2007) Profilin is essential for tip growth in the moss Physcomitrella patens Plant Cell 19, 3705–3722.Google Scholar
  31. 31.
    Cove, D. J., and Quatrano, R. S. (2004) The use of mosses for the study of cell polarity. In: Wood, A. J., Oliver, M. J., and Cove, D. J., eds. New Frontiers in Bryology: Physiology, Molecular Biology and Functional Genomics. Dordrecht: Kluwer, 189–203.CrossRefGoogle Scholar
  32. 32.
    Ashton, N. W., Grimsley, N. H., and Cove, D. J. (1979) Analysis of gametophytic development in the moss, Physcomitrella patens, using auxin and cytokinin resistant mutants Planta 144, 427–435.Google Scholar
  33. 33.
    Thelander, M., Nilsson, A., Olsson, T., Johansson, M., Girod, P. -A., Schaefer D. G., Zrÿd J. -P., and Ronne, H. (2007) The moss genes PpSKI1 and PpSKI2 encode nuclear SnRK1 interacting proteins with homolo­gues in vascular plants. Plant Mol Biol 64, 559–573.PubMedCrossRefGoogle Scholar
  34. 34.
    Kamisugi, Y., Cuming, A. C., and Cove, D. J. (2005) Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens. Nucleic Acids Res 33, e173.PubMedCrossRefGoogle Scholar
  35. 35.
    Kamisugi, Y., Schlink, K., Rensing, S. A., Schween, G., von Stackelberg, M., Cuming, A. C., Reski, R., and Cove, D. J. (2006) The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration. Nucleic Acids Res 34, 6205–6214.PubMedCrossRefGoogle Scholar
  36. 36.
    Nishiyama, T., Hiwatashi, Y., Sakakibara, I., Kato, M., and Hasebe, M. (2000) Tagged mutagenesis and gene-trap in the moss, Physcomitrella patens by shuttle mutagenesis. DNA Res 7, 9–17.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Alison W. Roberts
    • 1
  • Christos S. Dimos
    • 1
  • Michael J. BudziszekJr.
    • 1
  • Chessa A. Goss
    • 1
  • Virginia Lai
    • 1
  1. 1.Department of Biological SciencesCenter for Biotechnology and Life Sciences, University of Rhode IslandKingstonUSA

Personalised recommendations