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Open Access Highly Accessed Methodology article

A high-throughput virus-induced gene silencing protocol identifies genes involved in multi-stress tolerance

Venkategowda Ramegowda123, Muthappa Senthil-kumar13, Makarla Udayakumar2 and Kirankumar S Mysore1*

Author Affiliations

1 Plant Biology Division, The Samuel Roberts Noble Foundation, 2510 Sam Noble Pkwy., Ardmore, OK 73402, USA

2 Department of Crop Physiology, University of Agricultural Sciences, GKVK, Bangalore 560 065Karnataka, India

3 Present address: VR: Department of Crop, Soil and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701 USA; MS: National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110 067, India

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BMC Plant Biology 2013, 13:193  doi:10.1186/1471-2229-13-193

Published: 1 December 2013

Abstract

Background

Understanding the function of a particular gene under various stresses is important for engineering plants for broad-spectrum stress tolerance. Although virus-induced gene silencing (VIGS) has been used to characterize genes involved in abiotic stress tolerance, currently available gene silencing and stress imposition methodology at the whole plant level is not suitable for high-throughput functional analyses of genes. This demands a robust and reliable methodology for characterizing genes involved in abiotic and multi-stress tolerance.

Results

Our methodology employs VIGS-based gene silencing in leaf disks combined with simple stress imposition and effect quantification methodologies for easy and faster characterization of genes involved in abiotic and multi-stress tolerance. By subjecting leaf disks from gene-silenced plants to various abiotic stresses and inoculating silenced plants with various pathogens, we show the involvement of several genes for multi-stress tolerance. In addition, we demonstrate that VIGS can be used to characterize genes involved in thermotolerance. Our results also showed the functional relevance of NtEDS1 in abiotic stress, NbRBX1 and NbCTR1 in oxidative stress; NtRAR1 and NtNPR1 in salinity stress; NbSOS1 and NbHSP101 in biotic stress; and NtEDS1, NbETR1, NbWRKY2 and NbMYC2 in thermotolerance.

Conclusions

In addition to widening the application of VIGS, we developed a robust, easy and high-throughput methodology for functional characterization of genes involved in multi-stress tolerance.

Keywords:
Stress tolerance; Drought; Salinity; Temperature stress; Nonhost resistance; Bacterial pathogens; VIGS; PTGS; Translational genomics