Abstract
Zingiber zerumbet (Zingiberaceae) is a wild, tropical medicinal herb that shows a high degree of resistance to diseases affecting cultivated ginger. Barley stripe mosaic virus (BSMV) silencing vectors containing an endogenous phytoene desaturase (PDS) gene fragment were agroinfiltrated into young leaves of Z. zerumbet under controlled growth conditions to effect virus-induced gene silencing (VIGS). Infiltrated leaves as well as newly emerged leaves and tillers showed visual signs of PDS silencing after 30 days. Replication and systemic movement of the viral vectors in silenced plants were confirmed by RT-PCR. Real-time quantitative PCR analysis verified significant down-regulation of PDS transcripts in the silenced tissues. Label-free proteomic analysis was conducted in leaves with established PDS transcript down regulation and buffer-infiltrated (mock) leaves. A total of 474 proteins were obtained, which were up-regulated, down-regulated or modulated de novo during VIGS. Most of these proteins were localized to the chloroplast, as revealed by UniprotKB analysis, and among the up-regulated proteins there were abiotic stress responsive, photosynthetic, metabolic and membrane proteins. Moreover, the demonstration of viral proteins together with host proteins proved successful viral infection. We report for the first time the establishment of a high-throughput gene functional analysis platform using BSMV-mediated VIGS in Z. zerumbet, as well as proteomic changes associated with VIGS.
Highlights
Zingiber zerumbet Smith (Zingiberaceae) or ‘shampoo ginger’ is a rhizomatous perennial herb native to Southeast Asia
Barley stripe mosaic virus (BSMV) silencing vectors containing an endogenous phytoene desaturase (PDS) gene fragment were agroinfiltrated into young leaves of Z. zerumbet under controlled growth conditions to effect virus-induced gene silencing (VIGS)
We report for the first time the establishment of a high-throughput gene functional analysis platform using BSMV-mediated VIGS in Z. zerumbet, as well as proteomic changes associated with VIGS
Summary
Zingiber zerumbet Smith (Zingiberaceae) or ‘shampoo ginger’ is a rhizomatous perennial herb native to Southeast Asia. Ongoing efforts in our group have identified an ensemble of genes in Z. zerumbet that is regulated in response to Pythium inoculation [3,4] These designated defense-gene candidates need to be functionally validated to assess their potential in future improvement programs for cultivated ginger. We combined the BSMV-VIGS technique with Agrobacterium-mediated transient transformation of Z. zerumbet to down-regulate PDS, an endogenous marker gene widely used for VIGS studies in plants [12]. To our knowledge, this is the first report of such an attempt in Z. zerumbet. Our protocol could potentially accelerate the functional validation and characterization of candidate defense genes in Z. zerumbet
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