1Department of Biochemistry and Biophysics, Institute of Plant Genomics and Biotechnology, Texas A&M University, 2Department of Plant Pathology and Microbiology, Institute of Plant Genomics and Biotechnology, Texas A&M University
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Gao, X., Britt Jr., R. C., Shan, L., He, P. Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton. J. Vis. Exp. (54), e2938, doi:10.3791/2938 (2011).
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 transformation1. 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 proliferation2,3. 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 studies3,4. 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 development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, 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.
1. Grow cotton seedlings
2. Construct VIGS vector carrying GrCLA1 gene
3. Perform VIGS inoculation
Note: VIGS works when plants are under either 23°C or glasshouse (25-28°C). When the temperature is relatively low (23 to 25°C), it is easier for inoculation and phenotype is more uniform compared with higher (28-30°C) or fluctuated temperature. Covering the plants with a dome also makes VIGS more efficient.
Note: The VIGS-ed plants should be kept under contained quarantine conditions and plants should be properly autoclaved and disposed after the assays as TRV has a broad host range and is a notifiable pathogen.
4. Representative Results:
Approximately two weeks after hand-infiltration with Agrobacterial mixture, the albino phenotype caused by GrCLA1 silencing was clearly observed on the true leaves. The silencing efficiency reaches almost 100% in multiple experiments with more than 50 plants. The silenced plants display a uniformly distributed and very strong albino phenotype on newly developing leaves with a mosaic on the elder leaves (Figure 4).
Figure 1. A cotton seedling at about two-week old stage with two fully expanded cotyledons used for Agrobacterial infiltration.
Figure 2. Punching tiny holes on the underside of cotyledons of cotton plants using a 25 G needle to facilitate Agrobacterial infiltration.
Figure 3. Hand infiltration of Agrobacterial mixture into the cotyledons of cotton through the wounding sites using a needleless syringe
Figure 4. Albino phenotype appeared on VIGS silenced cotton plants. Four cultivars are shown. A) Fibermax 832; B) Phytogen 480WR; C) Phytogen 425RF; D) Deltapine 90.
VIGS has been proven to be a powerful tool in functional genomics analysis by transiently knocking down the expression of endogenous genes. In this study, we developed an Agrobacterium-mediated VIGS by utilizing a TRV-based binary vector. The cotton CLA1 (GrCLA1) gene was developed as a visual marker to monitor the silencing efficiency. We have consistently obtained 100% of gene silencing efficiency, demonstrated by the albino phenotype appearing on the true leaves in all varieties tested, starting from about two weeks post infiltration. However, CLA1-silenced cotton plants hardly developed into the fibre or seed stage, which is likely due to strong defects on chloroplast development. The successful development of cotton VIGS provides an alternative to readily silence the genes of interest for loss-of-function assays and lays foundation for cotton functional genetics/genomics in the fast approaching post-genome era8.
No conflicts of interest declared.
We are grateful to Drs. S.P. Dinesh-Kumar and Yule Liu for TRV-VIGS vectors, and Drs. Chuck Kenerley, Terry Wheeler, Jim Starr and Bayer CropScience for providing cotton seeds. This work was funded by NSF to L.S. and NIH to P.H.
|Roller drum||Glas-Col||099A TC108||Agro-bacterium culture|
|Incubator||Sheldon Manufacturing, Inc.||01046209||Agro-bacterium culture|
|UV/Vis spectrophotometer||Beckman Coulter Inc.||Model: DU530||Measuring OD|
|Gene Pulser||Bio-Rad||Model: 1652076; Serial: 154BR3880||Electroporation|
|Pulser Controller||Bio-Rad||Model: 1652098; Serial: 232BR4833||Electroporation|
|Micropulser Electroporation cuvette||Bio-Rad||165-2081||Electroporation|
|1 ml Syringe||BD Biosciences||30962||Inoculation of agro-bacterium|
|Metro Mix 700||SUNGR||SKU# 553001||Growing seedling|
|Terra Cotta pot||T.O.Plastics||GPS 3001B2||Growing seedling|
|MES monohydrate||USB Corp., Affymetrix||18886||Infiltration buffer|