Name: agrobacterium-mediated virus-induced gene silencing assay in cotton
Uploaded: 2011-08-20T12:00:00
Description: Watch this Scientific Journal Video about Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton at JoVE.com

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.

1. Grow cotton seedlings
<ol>
 <li>Sow the seeds of the upland cotton (Gossypium hirsutum) variety Fibermax 832, Phytogen 425RF, Phytogen 480WR and Deltapine 90 in pots (7 cm in diameter) containing Metro Mix 700 (SunGR, Beavile, WA).</li>
 <li>Keep the pots in a tray covered with a plastic dome at 23&deg;C, 120 &mu;E m-2 S-1 light, with a 12 hour light/12 hour dark photoperiod in a growth room.</li>
 <li>Remove the dome when two cotyledons have emerged.</li>
 <li>Approximately two weeks later, us...

<ol>
	<li>Chen, Z. J., Scheffler, B. E., Dennis, E., Triplett, B. A., Zhang, T., Guo, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+sequencing+cotton+(Gossypium)+genomes.">Toward sequencing cotton (Gossypium) genomes.</a> Plant Physiol. 145, 1303-1310 (2007).</li><li>Dinesh-Kumar, S. P., Anandalakshmi, R., Marathe, R., Schiff, M., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing.">Virus-induced gene silencing.</a> Methods Mol Biol. 236, 287-294 (2003).</li><li>Hamilton, A. J., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+species+of+small+antisense+RNA+in+posttranscriptional+gene+silencing+in+plants.">A species of small antisense RNA in posttranscriptional gene silencing in plants.</a> Science. 286, 950-952 (1999).</li><li>Burch-Smith, T. M., Anderson, J. C., Martin, G. B., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+and+advantages+of+virus-induced+gene+silencing+for+gene+function+studies+in+plants.">Applications and advantages of virus-induced gene silencing for gene function studies in plants.</a> Plant J. 39, 734-746 (2004).</li><li>Liu, Y., Schiff, M., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing+in+tomato.">Virus-induced gene silencing in tomato.</a> Plant J. 31, 777-786 (2002).</li><li>Estevez, J. M., Cantero, A., Romero, C., Kawaide, H., Jimenez, L. F., Kuzuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+expression+of+CLA1,+a+gene+that+encodes+the+1-deoxyxylulose+5-phosphate+synthase+of+the+2-C-methyl-D-erythritol-4-phosphate+pathway+in+Arabidopsis.">Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis.</a> Plant Physiol. 124, 95-104 (2000).</li><li>Mandel, M. A., Feldmann, K. A., Herrera-Estrella, L., Rocha-Sosa, M., Leon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLA1,+a+novel+gene+required+for+chloroplast+development,+is+highly+conserved+in+evolution.">CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.</a> Plant J. 9, 649-658 (1996).</li><li>Gao, X., Wheeler, T., Li, Z., Kenerley, C., He, P., Shan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+GhNDR1+and+GhMKK2+compromised+cotton+resistance+to+Verticillium+wilt.">Silencing GhNDR1 and GhMKK2 compromised cotton resistance to Verticillium wilt.</a> Plant J. 66, 293-305 (2011).</li></ol>

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 f...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagents and equipments</th>
			<th>Company</th>
			<th>Catalogue/serial number</th>
			<th>Comments </th>
		</tr>
		<tr>
			<td>Roller drum</td>
			<td>Glas-Col, LLC.</td>
			<td>099A TC108</td>
			<td>Agro-bacterium culture</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Sheldon Manufacturing, Inc.</td>
			<td>01046209</td>
			<td>Agro-bacterium culture</td>
		</tr>
		<tr>
			<td>UV/Vis spectrophotometer</td>
			<td>Beckman Coulter</td>
			<td>Model: DU530</td>
			<td>Measuring OD</td>
		</tr>
		<tr>
			<td>Gene Pulser</td>
			<td>BioRad</td>
			<td>Model: 1652076; Serial: 154BR3880</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>Pulser Controller</td>
			<td>BioRad</td>
			<td>Model: 1652098; Serial: 232BR4833</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>Micropulser Electroporation cuvette</td>
			<td>BioRad</td>
			<td>165-2081</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>1 ml Syringe</td>
			<td>BD Biosciences</td>
			<td>30962</td>
			<td>Inoculation of agro-bacterium</td>
		</tr>
		<tr>
			<td>Metro Mix 700</td>
			<td>SUNGR</td>
			<td>SKU# 553001</td>
			<td>Growing seedling</td>
		</tr>
		<tr>
			<td>Terra Cotta pot</td>
			<td>T.O.Plastics</td>
			<td>GPS 3001B2</td>
			<td>Growing seedling</td>
		</tr>
		<tr>
			<td>MES monohydrate</td>
			<td>USB</td>
			<td>18886</td>
			<td>Infiltration buffer</td>
		</tr>
		<tr>
			<td>Acetosyringone</td>
			<td>Sigma</td>
			<td>D134406</td>
			<td>Infiltration buffer</td>
		</tr>
	</tbody>
</table>

agrobacterium-mediated virus-induced gene silencing assay in cotton

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.

Watch this Scientific Journal Video about Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton at JoVE.com

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

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