Volume 2, Issue 5, Pages 1084-1094 (September 2009) Virus-Induced Gene Silencing in the Culinary Ginger (Zingiber officinale): An Effective Mechanism for Down-Regulating Gene Expression in Tropical Monocots  Renner Tanya , Bragg Jennifer , Driscoll Heather E. , Cho Juliana , Jackson Andrew O. , Specht Chelsea D.   Molecular Plant  Volume 2, Issue 5, Pages 1084-1094 (September 2009) DOI: 10.1093/mp/ssp033 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Photos of the Two Species Used in this Study, Clockwise from Upper Left. (A) Inflorescence of C. spicatus, showing current and past flowers. One flower opens per day from the conical inflorescence. (B) Close-up of C. spicatus flower (credit: Madelaine E. Bartlett). (C) Z. officinale inflorescence with flowers. (D, E) Z. officinale shoots and rhizomes showing facility for replication of greenhouse experiments. (D) Close-up of the three rhizome sections shown in (E). The growing end of the rhizome is towards the bottom of the photograph, seen as a small protrusion of the rhizome tissue. This protrusion represents a single apical meristem and will grow out as a new vegetative shoot. Lateral thickening at the base will form the rhizome, and roots will develop from axillary meristems along the rhizome. (E) Three rhizome sections with corresponding shoot and root systems, demonstrating 4 months of growth. Each rhizome section can be separated from adjoining sections and planted as an individual, complete with shoot and root system. Flowering, leafless shoots (C) form directly off the rhizome. Molecular Plant 2009 2, 1084-1094DOI: (10.1093/mp/ssp033) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Images of Z. officinale (A–D) and C. spicatus (E–G) demonstrating phenotypes associated with viral infection and gene silencing. (A) Whole Z. officinale plant with rhizome. (B) Leaf from BSMV-infected Z. officinale plant showing characteristic yellow stripes. (C) Leaf from a Z. officinale plant systemically infected with BSMV–ZoPDS. Photobleaching is evident in one sector of the single leaf located directly above the inoculated leaf and can be seen throughout the terminal leaves of plants 30 d post-innoculation. (D) Terminal leaf of Z. officinale plant with complete photobleaching characteristic of PDS silencing. (E) Whole C. spicatus stem produced from bulbil in the leaf axil of the parent flowering plant. The rhizome at the base of the shoot has rootlets that developed while this stem was still attached to the parent plant. (F) Close-up of uninfected C. spicatus leaves. (G) Close-up of C. spicatus leaf infected with BSMV, showing the infected phenotype of characteristic yellow stripes in a mosaic pattern. Molecular Plant 2009 2, 1084-1094DOI: (10.1093/mp/ssp033) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 Detection of BSMV in gingers. (A) Western blots showing antibody hybridization to BSMV CP. Detected CP in inoculated lanes indicates viral replication >3 cm from site of abrasion in three different Costus plants (a–c) and in Z. officinale (d). Negative control lanes use leaf material harvested from uninfected C. spicatus (left blot) or Z. officinale (right blot). Positive control lanes on both blots use leaf material from barley (Hordeum) known to be systemically infected with BSMV. (B) RT–PCR showing presence of viral constructs in plants post inoculation with either wt BSMV or BSMV RNA with the PDS insert. Primers were designed to amplify BSMVβ RNA. Control lanes contain RT–PCR from plants not infected with viral transcripts. (a) C. spicatus, (b) Z. officinale, (c) H. vulgare. Molecular Plant 2009 2, 1084-1094DOI: (10.1093/mp/ssp033) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Agarose Gel with RT–PCR Demonstrating Down-Regulation of PDS in Z. officinale Plants Infected with a BSMV Construct Containing a Z. officinale Endogenous PDS Gene Fragment. Actin was used as a positive RT–PCR control for RNA extractions from Z. officinale plants infected with BSMV containing no construct (WT), a fragment of the PDS gene from wheat (Ta), or the endogenous Z. officinale PDS fragment (Zo). In all cases, actin is consistently transcribed. Significantly less PDS is transcribed from plants infected with the BSMV vector containing the endogenous PDS gene fragment, indicating successful down-regulation of PDS (ZoPDS+). The negative control reaction lacks reverse transcriptase. Molecular Plant 2009 2, 1084-1094DOI: (10.1093/mp/ssp033) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Sequence Comparison of the Coding Region of PDS Amplified from Z. officinale (ZoPDS) to T. aestivum, H. vulgare, O. sativa, Z. mays, C. sativus, L. longiflorum, and H. verticillata (GenBank accession numbers EU854153, DQ270236, AF049356, AY062039, L39266, AY183118, AY500378, AY639658) utilizing the program GeneDoc version 2.06.02 (Nicholas et al., 1997). Nucleotides shaded by black boxes with white lettering (100% conserved), gray shading with white lettering (80% or greater conserved), gray shading with black lettering (60% or greater conserved), no shading with black lettering (less than 60% conserved). Molecular Plant 2009 2, 1084-1094DOI: (10.1093/mp/ssp033) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions