High-throughput virus-induced gene-silencing approach to assess the functional relevance of a moisture stress-induced cDNA homologous to lea4

Abstract

The abiotic stress-responsive cDNA database and their expression profiles suggest that stress genes are many and diverse. However, characterization and validation of their functional significance has been a constraint to assessing their role in imparting tolerance. Virus-induced gene silencing (VIGS) is a potential option for assessing the functional significance of stress genes. Here the effectiveness of VIGS to silence the expression of an ABA-responsive lea4 (late embryogenic abundant) gene involved in stress tolerance is documented. In the present study, low moisture-stress protocols were developed in such a way that the plants experienced the desired stress level when silencing the target stress gene using VIGS was at a maximum. The functional relevance of a groundnut (Arachis hypogaea) subtracted-stress cDNA clone putative lea4 was examined by VIGS in tomato. A 400 bp fragment of lea4 was cloned into tobacco rattle virus-based VIGS vector to trigger post-transcriptional gene silencing by Agrobacterium-mediated inoculation in tomato plants. In silenced plants only lea4 transcripts showed a substantial decline, whereas the expression of other known stress-responsive genes such as apx (ascorbate peroxidase) and elip (early light-induced protein) were unaltered. Under moderate moisture stress, the silenced plants showed enhanced susceptibility as measured by cell viability, superoxide radical activity, and cell osmotic adjustment. This approach illustrates the potential benefits of VIGS in identifying functional relevance of low moisture stress-responsive genes. It is also demonstrated that heterologous probes with a fairly high degree of homology to the native genes can be used to study the functional relevance of stress-responsive genes using VIGS.