Genome organizations of HLSV and TMV and detection of viral RNA and protein levels in cross protection.

<p>(<b>A</b>) Genome organization of HLSV and TMV. Transcriptional level of HLSV (<b>B</b>) or TMV (<b>C</b>) gRNA/total viral RNA determined by quantitative real-time RT-PCR and translational level of CPs by western blot (<b>B</b> and <b>C</b>). Significant differences were calculated using the Student’s <i>t</i>-test, * and ** indicate significance at the 0.05 and 0.01 levels of confidence, respectively.</p

<i>NbTOM1</i> transcript levels and virus accumulation with overexpression or silencing of <i>NbTOM1</i> in <i>Nicotiana</i><i>benthamiana</i>.

<p>(A) The transcriptional levels of <i>NbTOM1</i> were detected in mock inoculation buffer, HLSV, TMV, HLSV+TMV (100:1) and HLSV+TMV (1:1) co-infected plants. The viral RNA levels of HLSV and TMV were determined using quantitative real-time RT-PCR with primer sequences corresponding to the coat protein genes in HLSV, or TMV or co-infected leaves. (B and C) The transcriptional levels of <i>NbTOM1</i> were detected in <i>NbTOM1</i>overexpressed or silenced leaves. The viral RNA levels were detected in plants first infiltrated with pGreen orpGreen-<i>NbTOM1</i> (for overexpression), and pGreen or pGreen-<i>NbTOM1</i>(nt1-581) (for silencing), followed by single virus (HLSV or TMV) infection or co-infection(HLSV+TMV) at 40 h post inoculation (hpi). (D) The coat proteins of HLSV and TMV were detected by western blot in <i>NbTOM1</i>overexpressed or silenced leaves which were subsequently infected with single virus (HLSV or TMV) or co-infected with HLSV+TMV at 5 dpi (details see Materials & Methods). Total protein from mock buffer inoculated <i>N</i><i>. benthamiana</i> leaves was used as the negative control, while the total protein from HLSV or TMV infected leave samples which were confirmed earlier were used as positive controls.</p

Cross protection between HLSV and TMV and its symptom expressions in <i>Nicotiana</i><i>benthamiana</i>.

<p>(A) The number of surviving <i>N</i><i>. benthamiana</i> plants inoculated with mock buffer, HLSV, TMV and HLSV+TMV at different time points. All plants survived mock and HLSV inoculation. No plants survived inoculation with TMV. Half of the TMV infected <i>N</i><i>. benthamiana</i> (pre-inoculated with HLSV 12 days earlier) survived at 40 dpi. (B) The top, middle and bottom rows showed the top, close-ups and the side views of mock buffer (M), HLSV (H), TMV (T) and HLSV+TMV (H+T) inoculated <i>N</i><i>. benthamiana</i> plants, respectively. The red dotted line boxes in the top panels highlighted the close-up areas shown in the middle panels. Typical symptoms (red arrows pointing to) of inoculated <i>N</i><i>. benthamiana</i> plants are shown in the middle row. Among them, panel M, no symptom; panel H, mild leaf puckering at 20 dpi; panel T, systemic necrosis (plant death) at 8 dpi; panel H+T, mild mosaic symptoms at 20 dpi, plant height was shorter than H but taller than T inoculated plants. All scale bars represent 1 cm.</p

Transcriptional levels of selected genes in cross protection.

<p>The transcriptional levels of <i>NbARP1</i>, <i>NbCaM3</i>, <i>NbCP2</i>, <i>NbPI</i>, <i>NbVPE1α</i>, <i>NbACO</i>, <i>NbSAR8.2m</i>, <i>NbWIPK</i>, <i>NbWRKY8</i>, <i>NbTOM1, NbHsp101</i> and <i>NbAGO4-2</i>.</p

Hierarchical cluster analysis of transcriptional profiles and gene ontology analysis.

<p>(A) A hierarchical cluster analysis of transcriptional profiles for 24237 genes or ESTs of <i>Nicotiana</i><i>benthamiana</i> inoculated with inoculation buffer (mock), HLSV, HLSV+TMV (plants cross protected by pre-inoculation of HLSV 12 days prior to TMV challenge inoculation). The 3 columns correspond to biological repeats after inoculation with buffer (mock), HLSV, HLSV+TMV and TMV (at 12 dpi for mock and HLSV, and 3 dpi for HLSV+TMV and TMV which is equal to 15 dpi of HLSV). The clustering on the top of hierarchical map represents the differences among samples and biological repeats, while the left clustering is based on the expression levels of different genes. (B) Venn diagram of genes in response to HLSV infection and cross protection. (C) HLSV infection. (D) Gene ontology (GO) analysis of genes in response to HLSV+TMV infection (cross protection).</p