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Title: A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)

Abstract

Here, a method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. In conclusion, compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.

Authors:
 [1];  [1];  [1]
  1. Univ. of Rhode Island, Kingston, RI (United States). Dept. of Biological Sciences
Publication Date:
Research Org.:
Univ. of Rhode Island, Kingston, RI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1345832
Grant/Contract Number:  
SC0001090
Resource Type:
Accepted Manuscript
Journal Name:
Applications in Plant Science
Additional Journal Information:
Journal Volume: 3; Journal Issue: 7; Journal ID: ISSN 2168-0450
Publisher:
Botanical Society of America
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Physcomitrella patens; cellulose synthase; complementation assay; gametophore development; protein structure/function relationships

Citation Formats

Scavuzzo-Duggan, Tess R., Chaves, Arielle M., and Roberts, Alison W. A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae). United States: N. p., 2015. Web. doi:10.3732/apps.1500023.
Scavuzzo-Duggan, Tess R., Chaves, Arielle M., & Roberts, Alison W. A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae). United States. doi:10.3732/apps.1500023.
Scavuzzo-Duggan, Tess R., Chaves, Arielle M., and Roberts, Alison W. Tue . "A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)". United States. doi:10.3732/apps.1500023. https://www.osti.gov/servlets/purl/1345832.
@article{osti_1345832,
title = {A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)},
author = {Scavuzzo-Duggan, Tess R. and Chaves, Arielle M. and Roberts, Alison W.},
abstractNote = {Here, a method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. In conclusion, compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.},
doi = {10.3732/apps.1500023},
journal = {Applications in Plant Science},
number = 7,
volume = 3,
place = {United States},
year = {2015},
month = {7}
}

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Works referenced in this record:

Phytochrome Regulation of Cellulose Synthesis in Arabidopsis
journal, November 2011


Specialization of the Golden2-like regulatory pathway during land plant evolution
journal, July 2009


Mutations of cellulose synthase (CESA1) phosphorylation sites modulate anisotropic cell expansion and bidirectional mobility of cellulose synthase
journal, September 2010

  • Chen, S.; Ehrhardt, D. W.; Somerville, C. R.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 40
  • DOI: 10.1073/pnas.1012348107

The Moss Physcomitrella patens
journal, December 2005


Mosses as Model Systems for the Study of Metabolism and Development
journal, June 2006


A CELLULOSE SYNTHASE (CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella patens
journal, January 2012


What Do We Really Know about Cellulose Biosynthesis in Higher Plants?
journal, February 2010


The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration
journal, November 2006

  • Kamisugi, Yasuko; Schlink, Katja; Rensing, Stefan A.
  • Nucleic Acids Research, Vol. 34, Issue 21
  • DOI: 10.1093/nar/gkl832

Crystallographic snapshot of cellulose synthesis and membrane translocation
journal, December 2012

  • Morgan, Jacob L. W.; Strumillo, Joanna; Zimmer, Jochen
  • Nature, Vol. 493, Issue 7431
  • DOI: 10.1038/nature11744

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis
journal, October 2013

  • Omadjela, O.; Narahari, A.; Strumillo, J.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 44
  • DOI: 10.1073/pnas.1314063110

The role of ARPC4 in tip growth and alignment of the polar axis in filaments of Physcomitrella patens
journal, March 2006

  • Perroud, Pierre-François; Quatrano, Ralph S.
  • Cell Motility and the Cytoskeleton, Vol. 63, Issue 3
  • DOI: 10.1002/cm.20114

The cellulose synthase (CESA) gene superfamily of the moss Physcomitrella patens
journal, September 2006


Moss cell walls: structure and biosynthesis
journal, January 2012

  • Roberts, Alison W.; Roberts, Eric M.; Haigler, Candace H.
  • Frontiers in Plant Science, Vol. 3
  • DOI: 10.3389/fpls.2012.00166

Reversible Ponceau staining as a loading control alternative to actin in Western blots
journal, June 2010

  • Romero-Calvo, Isabel; Ocón, Borja; Martínez-Moya, Patricia
  • Analytical Biochemistry, Vol. 401, Issue 2
  • DOI: 10.1016/j.ab.2010.02.036

Tertiary model of a plant cellulose synthase
journal, April 2013

  • Sethaphong, L.; Haigler, C. H.; Kubicki, J. D.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 18
  • DOI: 10.1073/pnas.1301027110

Cellulose synthases: new insights from crystallography and modeling
journal, February 2014


Computational and genetic evidence that different structural conformations of a non-catalytic region affect the function of plant cellulose synthase
journal, September 2014

  • Slabaugh, Erin; Sethaphong, Latsavongsakda; Xiao, Chaowen
  • Journal of Experimental Botany, Vol. 65, Issue 22
  • DOI: 10.1093/jxb/eru383

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method
journal, June 2006

  • Zhang, Xiuren; Henriques, Rossana; Lin, Shih-Shun
  • Nature Protocols, Vol. 1, Issue 2
  • DOI: 10.1038/nprot.2006.97

Expression of a Mutant Form of Cellulose Synthase AtCesA7 Causes Dominant Negative Effect on Cellulose Biosynthesis
journal, June 2003

  • Zhong, Ruiqin; Morrison, W. Herbert; Freshour, Glenn D.
  • Plant Physiology, Vol. 132, Issue 2
  • DOI: 10.1104/pp.102.019331

    Works referencing / citing this record:

    Convergent evolution of hetero‐oligomeric cellulose synthesis complexes in mosses and seed plants
    journal, May 2019

    • Li, Xingxing; Speicher, Tori L.; Dees, Dianka C. T.
    • The Plant Journal
    • DOI: 10.1111/tpj.14366