Geminivirus-Mediated Gene Silencing fromCotton Leaf Crumple VirusIs Enhanced by Low Temperature in Cotton  

Abstract

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 impacts of BSMV on vegetative growth and water status in hulless barley (Hordeum vulgare var. nudum) in VIGS study

“…This severely limited the potential for the conventional BSMV-VIGS assay to analyze drought related candidate genes. It was reported that lower temperatures can lead to better silencing phenotypes and targeted gene silencing [32,[35][36][37]. Compared with the experimental condition of our previous BSMV-VIGS study [23], we found that by adjusting greenhouse temperatures from 24 ±1°C to 22 ±1°C, the time period for efficient BSMV-VIGS can be maintained to at least 40 days after inoculation.…”

Development and application of an efficient virus-induced gene silencing system in Nicotiana tabacum using geminivirus alphasatellite

“…By contrast, low temperatures often induce a rapid spread of virus diseases by the control of small interfering RNA (siRNA) generation (Szittya et al, 2003). For example, a growth temperature lower than 22 °C is required for efficient TRV-induced gene silencing in tomato, and endogenous gene silencing in cotton is more efficient by Cotton leaf crumple virus (CLCrV) at a relatively cool temperature (22/18 °C) for cotton growth (Liu et al, 2002b;Tuttle et al, 2008). We found that gene silencing based on 2mDNA1 and TbCSV can work in a wide range of temperatures, and no difference in silencing efficiency was observed between 18-32 °C.…”

“…Alphasatellites make no significant contribution to the pathogenicity of begomovirus, but most betasatellites are essential for begomovirus to induce typical disease symptoms in the host from which they were isolated (Cui et al, 2004;Wu and Zhou, 2005;Briddon and Stanley, 2006). Begomoviruses and their associated satellite molecules have been successfully modified into VIGS vectors which induce efficient gene silencing in diverse plant species such as Solanum lycopersicum, Nicotiana benthamiana, Arabidopsis thaliana, Manihot esculenta, and Gossypium hirsutum (Carrillo-Tripp et al, 2006;Tuttle et al, 2008).…”

<i>Peanut stunt virus</i>-induced gene silencing in white lupin (<i>Lupinus albus</i>)

“…The intensity of fluorescent light in the growth incubator was weaker in the former experiment, which could explain the longer duration. Growth conditions such as light and temperature promote good viral spread and effective silencing (Burch-Smith et al 2004Senthil-Kumar and Mysore 2011a;Tuttle et al 2008).…”