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Time course of H2A.X phosphorylation after SAHA and Actinomycin D treatment. LA1-55n cells were treated (+) or not (-) with 0.1 nM of actinomycin D (ActD) in the presence (+) or in the absence (-) of 1 μ M SAHA. Indicated protein expression was determined by Western blot analysis at the indicated times after the treatment. (DOCX 710 kb

Organelle distributions of <i>Z</i>. <i>zerumbet</i> leaf proteins as predicted by UniProtKB.

<p>Data indicate that the majority of the proteins (283 proteins) were localized to the chloroplast compared to other organelles. The observation supports the hypothesis that BSMV infection directly regulates chloroplast-specific proteins and results in their upregulation.</p

Venn diagram showing the number of proteins identified in the leaf proteome of <i>Z</i>. <i>zerumbet</i> in response to BSMV infection and <i>PDS</i> silencing.

<p>Total leaf protein was isolated by following the method of Isaacson et al. (2006) with some modifications, and proteomic analysis was carried out using LC/MS/MS. Among the474 proteins identified, 156 were expressed <i>de novo</i> in the silenced (tester) sample, 45 proteins were up-regulated and 9 down-regulated in response to VIGS. Proteomes of mock and <i>ZzPDS</i>-infiltrated leaves were profiled at 30 dpi.</p

Young plant of <i>Zingiber zerumbet</i>.

<p>Young plant of <i>Zingiber zerumbet</i>.</p

Detection of BSMV in silenced leaves of <i>Z</i>. <i>zerumbet</i> by RT-PCR.

<p>RNA was isolated from newly emerged leaves 30 days after infiltration with empty vector (control) and <i>ZzPDS</i>. Leaves were collected from the newly formed tillers as well. cDNA was prepared and RT-PCR was carried out using BSMV γ-specific primers. An amplicon of 309 bp representing BSMV γ transcript was obtained in all samples. M- 100bp ladder (NEB); Lane 1- Empty vector control; Lanes 2 & 3- Newly formed leaves of BSMV: PDS infiltrated plant 15dpi and 30dpi respectively; Lane-4- Leaf from new tiller 30dpi.</p

Real-time PCR analysis of PDS transcript accumulation in control and silenced leaves.

<p>Total RNA from control (empty vector) and <i>PDS</i>-silenced leaves of <i>Z</i>. <i>zerumbet</i> were isolated by the method of Salzmann et al. (1999) 15 and 30dpi. Quantitative real-time PCR was conducted using gene-specific primers of the <i>ZzPDS</i> gene to analyze its relative expression in leaf samples. <i>ZzGAPDH</i> was used as an internal control to normalize the samples. Values are means of two biological replicates performed in triplicate. *<i>p</i> < 0.05, **<i>p</i> < 0.01. X-axis- Leaf samples analyzed (from extreme left) 1- Empty vector-infiltrated (control); 2- Newly formed leaves of <i>ZzPDS</i>-infiltrated plant (15 dpi); 3- Newly formed leaf of <i>ZzPDS</i>-infiltrated plant (30dpi); 4- Leaf from new tiller developed from a BSMV:<i>ZzPDS</i>-infiltrated plant (30dpi). All the three silenced leaf samples showed significant downregulation of <i>PDS</i> transcripts and the values were found to be statistically significant. Y-axis- Relative expression levels of <i>ZzPDS</i></p

Development of an Efficient Virus Induced Gene Silencing Strategy in the Non-Model Wild Ginger-<i>Zingiber zerumbet</i> and Investigation of Associated Proteome Changes

<div><p><i>Zingiber zerumbet</i> (Zingiberaceae) is a wild, tropical medicinal herb that shows a high degree of resistance to diseases affecting cultivated ginger. <i>Barley stripe mosaic virus</i> (BSMV) silencing vectors containing an endogenous phytoene desaturase (<i>PDS</i>) gene fragment were agroinfiltrated into young leaves of <i>Z</i>. <i>zerumbet</i> 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 <i>PDS</i> 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 <i>PDS</i> transcripts in the silenced tissues. Label-free proteomic analysis was conducted in leaves with established <i>PDS</i> transcript down regulation and buffer-infiltrated (mock) leaves. A total of 474 proteins were obtained, which were up-regulated, down-regulated or modulated <i>de novo</i> 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 <i>Z</i>. <i>zerumbet</i>, as well as proteomic changes associated with VIGS.</p></div

Agroinfiltration in <i>Z</i>. <i>zerumbet</i> leaves.

<p><b>Fig 3a</b>. Control leaf of infiltrated <i>Z</i>. <i>zerumbet</i>. Agroinfiltration was carried out by mixing equal amounts of the three <i>Agrobacterium</i> (GV3103) cultures harboring pCaBS-α, pCaBS-β and pCa-γb: 00.The cultures were infiltrated into 2–4 leaves of 3-month <i>Z</i>. <i>zerumbet</i> plants using a 1-ml needleless syringe. The agroinfiltrated plants were maintained in a growth chamber for 30 dpi. Control leaf showed no visual signs of viral infection. <b>Fig 3b.</b> Leaves infiltrated with equal amounts of <i>Agrobacterium</i> cultures harboring pCaBS-α, pCaBS-β and pCaγb:<i>ZzPDS</i>. After 30dpi, yellow stripes were visible along the parallel veins, indicating sites of <i>PDS</i> down-regulation. <b>Fig 3c.</b> A closer view of a <i>PDS-</i>silenced leaf of <i>Z</i>. <i>zerumbet</i>.</p

Categorization of the different leaf proteins of <i>Z</i>. <i>zerumbet</i> according to their predicted molecular functions based on annotations in UniProtKB.

<p>Nearly half of the total proteins identified (46%) were predicted to be associated with binding activity, closely followed by proteins showing catalytic functions (37%).</p

Distribution of the total proteins in the leaf proteome of <i>Z</i>. <i>zerumbet</i> identified by LC-MS/MS into biological processes according to annotations in UniProtKB.

<p>A major proportion of the identified proteins were predicted to be involved in metabolic (32%) or cellular processes (30%).</p