Data_Sheet_1.docx

<p>The tomato resistance gene Tm-2² encodes a coiled coil-nucleotide binding site-leucine rich repeat type resistance protein and confers effective immune response against tobamoviruses by detecting the presence of viral movement proteins (MPs). In this study, we show that the Nicotiana benthamiana Heat shock protein 90-kD (Hsp90) interacts with Tm-2². Silencing of Hsp90 reduced Tm-2²-mediated resistance to Tobacco mosaic virus (TMV) and the steady-state levels of Tm-2² protein. Further, Hsp90 associates with SGT1 in yeast and in plant cells. These results suggest that Hsp90-SGT1 complex takes part in Tm-2²-mediated TMV resistance by functioning as chaperone to regulate Tm-2² stability.</p

Type I J-Domain NbMIP1 Proteins Are Required for Both <i>Tobacco Mosaic Virus</i> Infection and Plant Innate Immunity

<div><p>Tm-2² is a coiled coil-nucleotide binding-leucine rich repeat resistance protein that confers durable extreme resistance against <i>Tomato mosaic virus</i> (ToMV) and <i>Tobacco mosaic virus</i> (TMV) by recognizing the viral movement protein (MP). Here we report that the <i>Nicotiana benthamiana</i> J-domain MIP1 proteins (NbMIP1s) associate with tobamovirus MP, Tm-2² and SGT1. Silencing of <i>NbMIP1s</i> reduced TMV movement and compromised <i>Tm-2²</i>-mediated resistance against TMV and ToMV. Furthermore, silencing of <i>NbMIP1s</i> reduced the steady-state protein levels of ToMV MP and Tm-2². Moreover, NbMIP1s are required for plant resistance induced by other <i>R</i> genes and the nonhost pathogen <i>Pseudomonas syringae pv. tomato</i> (<i>Pst</i>) DC3000. In addition, we found that SGT1 associates with Tm-2² and is required for <i>Tm-2²</i>-mediated resistance against TMV. These results suggest that NbMIP1s function as co-chaperones during virus infection and plant immunity.</p></div

OPN is responsible for the HBx-mediated upregulation of Capn4.

<p>(A, B and C) HepG2-X (or H7402-X) cells were transfected with the indicated doses of siRNA targeting OPN mRNA (Si-OPN) and control siRNA for 48 h. (A) The promoter activity of Capn4 was detected by luciferase reporter gene assay (**P<0.01, *P<0.05 Student's <i>t</i> test). (B) The mRNA levels of Capn4 were detected by RT-PCR. (C) The protein levels of Capn4 were examined by western blot analysis. (D, E and F) HepG2 (or H7402) cells were transiently transfected with the indicated doses of pcDNA3.0-OPN for 48 h. (D) The promoter activity of Capn4 was measured by luciferase reporter gene assay (*P<0.05 Student's <i>t</i> test). (E, F) The mRNA and protein levels of Capn4 were examined by RT-PCR and western blot analysis, respectively.</p

The subcellular localization of NbMIP1.1a in <i>N. benthamiana</i> cells.

<p>(<b>A</b>) Confocal image of the subcellular localization of NbMIP1.1a in leaf epidermal cells. YFP-NbMIP1.1a was transiently expressed in leaves of <i>N. benthamiana</i> via agroinfiltration and imaged at 48 hpi using a Zeiss LSM 710 laser scanning microscope. YFP signal revealed that NbMIP1.1a is present in the cell membrane, cytoplasm and nucleus. PCD3-1002: a CFP-tagged plasma membrane marker <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003659#ppat.1003659-Nelson1" target="_blank">[81]</a>. DAPI: staining for nuclei. Scale bar represents 20 µm. (<b>B</b>) YFP-NbMIP1.1a was found in both the soluble fraction and the membrane fraction (upper panel). Protein extracts were centrifuged at 100,000×g to produce crude soluble (S100) and microsomal (P100) fractions. Fractions were analyzed by western blot following separation by SDS-PAGE. The gels were probed using anti-GFP, anti-V-H-ATPase (vacuolar H-ATPase subunit, a vacuolar membrane marker) and anti-PEPC (phosphoenolpyruvate carboxylase, a cytosolic marker) antibodies as indicated.</p

<i>Tm-2²</i>-mediated resistance against TMV requires <i>NbMIP1s</i>.

<p>(<b>A</b>) Phenotype of <i>NbMIP1s</i>-silenced and TRV control <i>N. benthamiana</i> plants. <i>NbMIP1s</i>-silenced <i>N. benthamiana</i> plants developed dwarfed stems and crinkled leaves compared to TRV-infected control plants. (<b>B</b>) Leaves of <i>NbMIP1</i>-silenced and TRV control <i>N. benthamiana</i> plants. The leaf edge of <i>NbMIP1s</i>-silenced plants curled downward but TRV-infected control leaves looked normal (right side). Photos were taken at 14 days post agroinfiltration for VIGS. (<b>C</b>) Real time RT-PCR to confirm the suppression of <i>NbMIP1s</i>, and the <i>Actin</i> mRNA levels were used as internal controls. <i>NbMIP1</i> VIGS: silencing using pTRV2-<i>NbMIP1</i>. (<b>D</b>) Silencing of <i>NbMIP1s</i> caused the appearance of TMV-GFP infection foci and visible HR lesions in the inoculated leaves of <i>NbMIP1s</i>-silenced <i>Tm-2²</i>-containing TM#1 plants. (<b>E</b>) Silencing of <i>NbMIP1s</i> compromised <i>Tm-2²</i>-mediated resistance against TMV, and TMV-GFP spread from the inoculated leaves into the upper non-inoculated leaves of <i>NbMIP1s</i>-silenced TM#1 plants. TRV-infected TM#1 plants were used as negative controls. Photos were taken at 10 days post TMV-GFP infection (dpi). Scale bars represent 1 cm. (<b>F</b>) RT-PCR was performed to confirm the presence of TMV-GFP in systemic leaves of <i>NbMIP1s</i>-silenced TM#1 plants.</p

Tm-2² (VAALLA) mutant, NbMIP1.1a and ToMV MP could exist in the same complex in plants.

<p>(<b>A</b>) The Tm-2² (V<u>AA</u>LL<u>A</u>) mutant did not induce HR when co-expressed with ToMV MP-Myc (upper), and failed to induce resistance against TMV-GFP (lower). Tm-2²-HA or Tm-2² (V<u>AA</u>LL<u>A</u>)-HA were agroinfiltrated into wild-type <i>N. benthamiana</i> leaves with either ToMV MP-Myc (upper) or TMV-GFP (lower). Photos were taken at 4 dpi under UV light. (<b>B</b>) Tm-2² (V<u>AA</u>LL<u>A</u>)-HA co-immunoprecipitated with both ToMV MP and NbMIP1.1a (top). YFP-NbMIP1.1a co-immunoprecipitated with both ToMV MP and Tm-2² (V<u>AA</u>LL<u>A</u>) mutant (middle). Loading controls were also analyzed by western blot (bottom). Tm-2² (V<u>AA</u>LL<u>A</u>)-HA and YFP-NbMIP1.1a were co-expressed with ToMV MP-Myc or empty Myc vector in <i>N. benthamiana</i> leaves through agroinfiltration. At 60 hpi, leaf lysates were immunoprecipitated with anti-HA or anti-GFP beads, then the immunoprecipitates were assessed by western blotting using anti-HA, anti-Myc or anti-GFP antibodies as indicated.</p

Capn4 can regulate OPN in a positive feedback manner.

<p>(A, B and C) HepG2-X (or H7402-X) cells were transfected for 48 h with the indicated doses of siRNA targeting Capn4 mRNA (Si-Capn4). (A) The promoter activity of OPN was measured by luciferase reporter gene assay (*P<0.05, Student's <i>t</i> test). (B, C) The mRNA and protein levels of OPN were detected by RT-PCR and western blot analysis, respectively. (D, E and F) HepG2 cells were transiently transfected with the indicated doses of pcDNA3.0-Capn4 for 48 h. (D) The promoter activity of OPN was measured by luciferase reporter gene assay (*P<0.05 Student's <i>t</i> test). (E, F) The mRNA and protein levels of OPN were examined by RT-PCR and western blot analysis, respectively.</p

<i>NbMIP1s</i> are involved in TMV movement.

<p>(<b>A</b>) Silencing of <i>NbMIP1s</i> reduced cell-to-cell movement of TMV. TMV-GFP formed much smaller infection foci in <i>NbMIP1s</i>-silenced plants (right) compared to control plants (left). Photos were taken at 3 dpi. Scale bars represent 1 cm. (<b>B</b>) Average sizes of TMV-GFP foci at 3 dpi are shown. All values in bar graphs represent means with standard deviation. **: p<0.01 (Student's <i>t</i>-test). Data are from 3 independent experiments, and 9 leaves for each construct per experiment. (<b>C</b>) RT-PCR to confirm that the suppression of <i>NbMIP1s</i> reduced TMV-GFP vRNA levels in local inoculated leaves at 3 dpi. (<b>D</b>) Silencing of <i>NbMIP1s</i> delayed systemic TMV movement. At 5 dpi, TMV-GFP had already spread into the upper non-inoculated leaves in control plants (left), but not into the systemic leaves in <i>NbMIP1s</i>-silenced plants (right). Scale bars represent 1 cm. (<b>E</b>) RT-PCR to confirm the delay of TMV-GFP systemic movement in <i>NbMIP1s</i>-silenced plants at 5 dpi. For each RT-PCR, <i>Actin</i> was used as an internal control.</p

NbMIP1.1a interacts with ToMV MP and Tm-2²<i>in vivo</i>.

<p>Firefly luciferase complementation imaging assays for <i>in vivo</i> interaction of NbMIP1.1a with ToMV MP (<b>A</b>) and Tm-2² (<b>B</b>). Panels show luminescence images of <i>N. benthamiana</i> leaves agro-infiltrated with nLUC-NbMIP1.1a and ToMV MP-cLUC or Tm-2²-cLUC. The combinations of nLUC-NbMIP1.1a and cLUC, nLUC and ToMV MP-cLUC, nLUC and Tm-2²-cLUC were included as negative controls. (<b>C</b>) NbMIP1.1a co-immunoprecipitated (co-IP) with ToMV MP and Tm-2². NbMIP1.1a-Myc was co-expressed with ToMV MP-HA or Tm-2²-HA in <i>N. benthamiana</i> leaves by agroinfiltration. NbMIP1.1a-Myc co-expressed with HA-nLUC was introduced as a negative control. At 48 hours post infiltration (hpi), leaf lysates were immunoprecipitated with anti-HA beads, then the immunoprecipitates were assessed by western blotting using anti-Myc (upper panel) and anti-HA antibodies (middle panel). In addition to immunoblotting for co-IP, presence of NbMIP1.1a-Myc, ToMV MP-HA, Tm-2²-HA and HA-nLUC in the cell lysates were also analyzed (lower panel).</p

5-LOX is responsible for the upregulation of OPN mediated by HBx.

<p>(A) The promoter activities of OPN were detected by luciferase reporter gene assay in HepG2-X and HepG2 cells treated with MK886 or Indo, respectively (**P<0.01, ns, not significant, Student's <i>t</i> test). (B) The promoter activity of OPN was detected by luciferase reporter gene assay in HepG2-X (or H7402-X) cells treated with the indicated doses of siRNA targeting 5-LOX mRNA (Si-5-LOX) (**P<0.01, Student's <i>t</i> test). (C) The protein levels of OPN, 5-LOX and HBx were examined in the cells by western blot analysis.</p