Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Abstract

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation 1 . To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation 2,3 . Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies 3,4 . As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development 6 , and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves 7 , providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

Dissecting Functions of KATANIN and WRINKLED1 in Cotton Fiber Development by Virus-Induced Gene Silencing

“…Because cotton transformation is laborious and technically challenging, the development of a rapid method for gene functional analysis would greatly extend the power of molecular analysis. Gao et al (2011aGao et al ( , 2011b have shown that TRV-VIGS functions in cotton leaves with a 100% silencing efficiency. Extending the utility of this method, we showed here that sTRV-VIGS can also be effective in suppressing gene expression in reproductive organs.…”

“…Our method for agroinfection of cotton plants was different from that of Gao et al (2011aGao et al ( , 2011b, who used a syringe to infiltrate Agrobacterium tumefaciens culture into two fully expanded cotyledons. By contrast, we used cotton plants with two to four true leaves, vacuum infiltrated the leaves with A. tumefaciens culture, and subsequently drenched the root system using the same culture.…”

“…Using the geminivirus Cotton leaf crumple virus (CLCrV) as a vector, Tuttle et al (2008) silenced two visible marker genes encoding magnesium chelatase subunit I and phytoene desaturase with an efficiency of 70%. Gao et al (2011aGao et al ( , 2011b, on the other hand, used the Tobacco rattle virus (TRV) as a vector to silence another marker gene, chloroplastos alterados1, with an efficiency of 100%. The TRV-VIGS system appears to be better than the CLCrV-VIGS system because the former has a higher silencing efficiency and a more homogenous silencing phenotype with a milder virus symptom.…”

Characterization, Expression, and Functional Analysis of a Novel NAC Gene Associated with Resistance to Verticillium Wilt and Abiotic Stress in Cotton

“…The VIGS transient expression methods followed Gao et al (2011). VIGS-infiltrated seedlings were allowed to grow for 2 wk until the two-true-leaf stage and then leaves were collected and stored at −80°.…”

Development of Agrobacterium-Mediated Virus-Induced Gene Silencing and Performance Evaluation of Four Marker Genes in Gossypium barbadense

“…Tuttle et al [15] reported the silencing of two visible marker genes, magnesium chelatase subunit I and phytoene desaturase ( PDS ), using the geminivirus Cotton leaf crumple virus (CLCrV) as a vector. Gao et al [16,17] used the Tobacco rattle virus (TRV) as a vector to silence another marker gene, cloroplastos alterados 1 ( CLA1 ). Although both VIGS systems were shown to work in G.…”