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Free Content Identification of plant cytoskeleton-interacting proteins by screening for actin stress fiber association in mammalian fibroblasts

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Summary

Taking advantage of the high conservation of the cytoskeleton building blocks actin and tubulin between plant and animal kingdoms, we developed a functional genomic screen for the isolation of new plant cytoskeleton-binding proteins that uses a mammalian cell expression system. A yellow fluorescent protein (YFP)-fusion cDNA library from Arabidopsis was inserted into rat fibroblasts and screened for fluorescent chimeras localizing to cytoskeletal structures. The high-throughput screen was performed by an automated microscope. An initial set of candidate genes identified in the screen was isolated, sequenced, the full-length cDNAs were synthesized by RT-PCR and tested by biochemical approaches to verify the ability of the genes to bind actin directly. Alternatively, indirect binding via interaction with other actin-binding proteins was studied. The full-length cDNAs were transferred back to plants as YFP chimeras behind the CAMV-35S promoter. We give here two examples of new plant cytoskeletal proteins identified in the pilot screen. ERD10, a member of the dehydrin family of proteins, was localized to actin stress fibers in rat fibroblasts. Its direct binding to actin filaments was confirmed by several biochemical approaches. Touch-induced calmodulin-like protein, TCH2, was also localized to actin stress fibers in fibroblasts, but was unable to bind actin filaments directly in vitro. Nevertheless, it did bind to the IQ domains of Arabidopsis myosin VIII in a calcium-dependent manner. Further evidence for a cytoskeletal function of ERD10 was obtained in planta; GFP-ERD10 was able to protect the actin cytoskeleton from latrunculin-mediated disruption in Nicotiana benthamiana leaves.
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Keywords: cDNA library; cytoskeleton; high-throughput; screen; yellow fluorescent protein (YFP)

Document Type: Research Article

Affiliations: 1: The Institute of Plant Sciences, The Volcani Center, Bet-Dagan 50250 Israel, 2: Department of Biological Sciences and the Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907-2064, USA, and 3: The Department of Cell Molecular Biology, The Weizmann Institute of Science, Rehovot 76100, Israel

Publication date: November 1, 2006

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