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First published online August 7, 2009; 10.1104/pp.109.143727

Plant Physiology 151:506-514 (2009)
© 2009 American Society of Plant Biologists

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BREAKTHROUGH TECHNOLOGIES

Rapid Screening for Temperature-Sensitive Alleles in Plants1,[W],[OA]

Luis Vidali2, Robert C. Augustine, Scotty N. Fay, Paula Franco, Kelli A. Pattavina and Magdalena Bezanilla*

Biology Department, University of Massachusetts, Amherst, Massachusetts 01003

We developed a simple and fast method to identify temperature-sensitive alleles of essential plant genes. We used primary and tertiary structure information to identify residues in the core of the protein of interest. These residues were mutated and tested for temperature sensitivity, taking advantage of the exceptionally rapid 1-week complementation assay in the moss Physcomitrella patens. As test molecules, we selected the actin-binding proteins profilin and actin-depolymerizing factor, because they are essential and their loss-of-function phenotype can be fully rescued. Screening a small number of candidate mutants, we successfully identified temperature-sensitive alleles of both profilin and actin-depolymerizing factor. Plants harboring these alleles grew well at the permissive temperature of 20°C to 25°C but showed a complete loss of function at the restrictive temperature of 32°C. Notably, the profilin mutation identified in the moss gene can be transferred to profilins from other plant species, also rendering them temperature sensitive. The ability to routinely generate temperature-sensitive alleles of essential plant proteins provides a powerful tool for the study of gene function in plants.

1 This work was supported by the National Science Foundation (grant nos. MCB–0516702, MCB–0640530, and MCB–0747231), by the David and Lucille Packard Foundation (support to M.B.), by the National Science Foundation Integrative Graduate Education and Research Traineeship (grant no. DGE–065412) to the University of Massachusetts Institute for Cellular Engineering (fellowship support to R.C.A.), and by the Howard Hughes Medical Institutes Undergraduate Science Program (support to S.N.F. and K.A.P.).

2 Present address: Biology and Biotechnology Department, Worcester Polytechnic Institute, Worcester, MA 01609.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Magdalena Bezanilla (bezanilla{at}bio.umass.edu).

[W] The online version of this article contains Web-only data.

[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.143727

* Corresponding author; e-mail bezanilla{at}bio.umass.edu.

Received June 26, 2009; accepted August 6, 2009; published August 7, 2009.

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