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Reverse genetics in Chlamydomonas: A platform for isolating insertional mutants
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Metadata
Document Title
Reverse genetics in Chlamydomonas: A platform for isolating insertional mutants
Author
Gonzalez-Ballester D.,Pootakham W.,Mus F.,Yang W.,Catalanotti C.,Magneschi L.,De Montaigu A.,Higuera J.J.,Prior M.,Galván A.,Fernandez E.,Grossman A.R.
Name from Authors Collection
Affiliations
Department of Plant Biology, The Carnegie Institution for Science, Stanford, CA 94305, United States; Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba 14071, Spain; National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani, 12120, Thailand; Montana State University, Department of Chemical and Biological Engineering, Department of Microbiology, Bozeman, MT 59171, United States; PlantLab, Scuola Superiore Sant'Anna, 56127 Pisa, Italy; Max Planck Insitute for Plant Breeding Research, Department of Plant Developmental Biology, D-50829, Köln, Germany
Type
Article
Source Title
Plant Methods
ISSN
17464811
Year
2011
Volume
7
Issue
1
Open Access
All Open Access, Gold, Green
DOI
10.1186/1746-4811-7-24
Abstract
Background: Transient expression of proteins in plants has become a favoured method over the production of stably transformed plants because, in addition to enabling high protein yields, it is both fast and easy to apply. An enhancement of transient protein expression can be achieved by plant virus-encoded RNA silencing suppressor proteins. Since viral suppressor proteins differ in their efficiency to enhance transient protein expression in plants, we developed a whole-leaf green fluorescent protein (GFP)-based imaging assay to quantitatively assess suppressor protein activity. Results: In a transient GFP-expression assay using wild-type and GFP-transgenic N. benthamiana, addition of the plant viral suppressors Beet mild yellowing virus (BMYV-IPP) P0 or Plum pox virus (PPV) HC-Pro was shown to increase fluorescent protein expression 3-4-fold, 7 days post inoculation (dpi) when compared to control plants. In contrast, in agroinfiltrated patches without suppressor activity, near complete silencing of the GFP transgene was observed in the transgenic N. benthamiana at 21 dpi. Both co-infiltrated suppressors significantly enhanced GFP expression over time, with HC-Pro co-infiltrations leading to higher short term GFP fluorescence (at 7 dpi) and P0 giving higher long term GFP fluorescence (at 21 dpi). Additionally, in contrast to HC-Pro co-infiltrations, an area of complete GFP silencing was observed at the edge of P0 co-infiltrated areas. Conclusions: Fluorescence imaging of whole intact leaves proved to be an easy and effective method for spatially and quantitatively observing viral suppressor efficiency in plants. This suppressor assay demonstrates that plant viral suppressors greatly enhanced transient GFP expression, with P0 showing a more prolonged suppressor activity over time than HC-Pro. Both suppressors could prove to be ideal candidates for enhancing target protein expression in plants. © 2011 Gonzalez-Ballester et al; licensee BioMed Central Ltd.
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License
CC BY
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Publication Source
Scopus