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BREAKTHROUGH TECHNOLOGIES

Geminivirus-Mediated Gene Silencing from Cotton Leaf Crumple Virus Is Enhanced by Low Temperature in Cotton^1,[C],[OA]

Department of Plant Biology, North Carolina State University, Raleigh, North Carolina 27606 (J.R.T., C.H.H., D.R.); Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721 (A.M.I., J.K.B.); and Department of Crop Science, North Carolina State University, Raleigh, North Carolina 27695 (C.H.H.)

A silencing vector for cotton (Gossypium hirsutum) was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, the magnesium chelatase subunit I gene (ChlI) or the phytoene desaturase gene (PDS). Cotyledons of cotton cultivar ‘Deltapine 5415’ bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in approximately 70% of inoculated plants after 2 to 3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30°C/26°C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22°C/18°C. However, endogenous gene silencing decreased at 30°C/26°C. There was an approximately 7 d delay in the onset of gene silencing at 22°C/18°C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.

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: Dominique Robertson (niki_robertson{at}ncsu.edu).

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

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www.plantphysiol.org/cgi/doi/10.1104/pp.108.123869

^* Corresponding author; e-mail niki_robertson{at}ncsu.edu.

Received June 3, 2008; accepted June 25, 2008; published July 11, 2008.