Co-immunoprecipitation of P3N-PIPO and PCaP1 expressed <i>in planta</i>.

<p>Proteins from crude extracts of <i>N. benthamiana</i> leaves (2 days post agroinfiltration) that co-expressed HA-P3N-PIPO and c-myc-PCaP1, or expressed HA-P3N-PIPO or c-myc-PCaP1 only were pulled-down using anti-HA (top panels) or anti-c-myc (bottom panels) antibodies, separated by 4–12% Novex Tris-Glycine PAGE, electroblotted onto PVDF membrane and probed with anti-HA or anti-c-myc antibody as indicated. Recognition of HA-P3N-PIPO and c-myc-PCAP1 are shown at right. Immunoblotting (IB).</p

Immunodetection of PCaP1 in wild-type and <i>PCaP1</i> knockout <i>Arabidopsis</i> plants.

<p>Total soluble proteins from leaves collected at 14 dpi were separated by 4–12% Novex Tris-glycine PAGE, blotted onto PVDF membrane, probed with anti-PCaP1 antibody or anti-GFP antibody and detected by ECL-Plus Western reagents. Samples were from mock inoculated or TuMV-GFP infected wild-type (WT) or <i>PCaP1</i> knockout (<i>pcap1</i>) plants. Equal loading of proteins was verified by similar levels of Coomassie staining of Rubisco protein (bottom panel).</p

TuMV-GFP infection in wild type and <i>pcap1 Arabidopsis</i> plants.

<p>(A) qRT-PCR quantification of TuMV-GFP RNA in inoculated leaves of indicated plant lines at 3 dpi. The values were normalized to the amounts of <i>Actin8</i> transcript in the same sample. Data are averages of three independent experiments, each consisting of five technical replicates and statistical significance was analyzed by the unpaired Student's <i>t</i>-test (P = 0.169). Error bars represent standard deviations. (B) TuMV-GFP infection foci in leaves analyzed by epifluorescence microscopy. Bars = 50 µm. (C) Quantification of size and number of TuMV-GFP infection foci. The data are averages of four independent experiments and each consisting of at least eight replicates. Error bars represent standard deviations. Statistical significance of difference in the size and number of the infection foci between WT and <i>pcap1</i> was analyzed by the unpaired Student's <i>t</i>-test and the calculated P values are indicated. Wild-type (WT), PCaP1 knocout (<i>pcap1</i>), inoculated leaf (I), uninoculated second cauline leaf (S).</p

Genome map of TuMV-GFP and expression of P3N-PIPO <i>in planta</i>.

<p>(A) Genome map of TuMV-GFP. Mature proteins processed from the proteolytic cleavage of the large polyprotein are indicated in boxes. <i>pipo</i> ORF is indicated above the polyprotein ORF with putative frameshift sequence indicated and bars showing codons in the polyprotein ORF (below) and <i>pipo</i> ORF (above). Sizes of P3N-PIPO and P3 proteins are indicated below the regions that code for them. P3-derived portion is shaded gray; PIPO-derived portion is in black. Position of inserted GFP coding insertion is indicated by dashed lines. Solid circle indicates VPg at 5′ end; (A)n indicates poly(A) tail. (B) Immunodetection of P3N-PIPO and P3 in TuMV-GFP-infected <i>Arabidopsis</i>. Total protein was extracted from the infected leaves at 14 days post inoculation (dpi), separated in either 4–12% NuPAGE Bis-Tris gel (Life Technologies) (for P3-N-PIPO and P3 detections) or in 4–12% Novex Tris-glycine gel (for GFP detection), blotted onto PVDF membrane and probed with anti-PIPO, anti-P3 or anti-GFP antibody and detected by ECL-plus Western reagents. Positions of protein mobility markers in kilodaltons (kDa) are indicated at right. Lanes indicate total protein from plants inoculated with wild-type (WT) TuMV-GFP, or <i>pipo</i> knockout mutants of TuMV-GFP (p41 and p68). These mutants differ from WT TuMV-GFP by single point mutations that introduce stop codons into the <i>pipo</i> ORF: CGA→<u>UGA</u> and <u>GGA</u>→<u>UGA</u> at bases 3103 and 3130 in mutants p41 and p68, respectively (<i>pipo</i>-frame codons shown). These mutations do not alter the amino acid sequence of the overlapping P3 region of the polyprotein. Note that putative P3N-PIPO migrates more slowly (∼28 kDa) than its predicted molecular weight (∼25 kDa).</p

ORMV infection of wild type and <i>pcap1 Arabidopsis</i> plants.

<p>(A) RT-PCR was used to determine the accumulation of plus-strand ORMV RNAs in upper uninoculated rosette leaves at 6 dpi (top panel). <i>Actin8</i> was amplified as control (bottom panel). (B) ORMV infected plants (21 dpi). Wild-type (WT), <i>PCaP1</i> knockout (<i>pcap1</i>).</p

TuMV-GFP RNA accumulation in <i>Arabidopsis</i> protoplasts.

<p>qRT-PCR was used to quantitate TuMV-GFP RNA accumulation in protoplasts at indicated time points. The values were normalized to the amounts of <i>Actin8</i> transcript in the same samples. Data are average of three independent experiments, each consisting of three replicates. Error bars represent standard deviations. Statistical significance between WT and <i>pcap1</i> was analyzed by the unpaired Student's <i>t</i>-test and calculated P values are indicated.</p

Hypothesis for the role of P3N-PIPO-PCaP1 interaction in potyvirus movement.

<p>We speculate that the virion-CI complex binds P3N-PIPO, via the CI protein. P3N-PIPO then transports the complex to the plasma membrane by binding PCaP1, which is anchored to the membrane via its myristoyl moiety. The PCaP1-bound complex accumulates in plasmodesmata possibly by the affinity of CI protein for the plasmodesmata, and/or via unidentified host proteins (question mark). Dashed arrow indicates possible route of inefficient movement of viral movement complex in <i>PCaP1</i> knockout plants. ER, endoplasmic reticulum; PD, plasmodesmata; PM, plasma membrane; CW, cell wall.</p

Bimolecular fluorescence complementation assay of P3N-PIPO, P3N, PIPO and P3 with wild-type and mutant PCaP1 in <i>N. benthamiana</i>.

<p>(A) Interactions of P3N-PIPO-YN, P3N-YN, PIPO-YN or P3-YN with PCaP1-YC, or P3N-PIPO-YN with PCaP1 containing a G to A amino acid substation at position two (G2A.PCaP1-YC) were analyzed at about 38 h post agroinfiltration (hpa) (panels a, d, g, j and m). P3N-PIPO-YN, P3N-YN, PIPO-YN or P3-YN interaction with GUS-YC are in panels b, e, h, k and n. Expression of GUS-YC was confirmed in all pair-wise interactions by histochemical staining (panels c, f, i, l and o). YN, N-terminal half of citrine; YC, C-terminal half of citrine; scale bar = 25 µm. (B) Co-localization of P3N-PIPO-YN, PCaP1-YC and PDCB1-mCherry in plasmodesmata (38 hpa). Interaction of P3N-PIPO-YN and PCaP1-YC (color changed digitally to green fluorescence, panel a). Localization of PDCB1-mCherry (panel b). Merged citrine signals and mCherry signals show colocalization of P3N-PIPO, PCaP1 and PDCB1-mCherry as orange spots (panel c). Nomarski DIC image of the same cell in panels a-c (panel d). All images are single confocal sections. Scale bar ∼10 µm.</p

TuMV-GFP infection of wild type and <i>pcap1 Arabidopsis</i> plants.

<p>Plants were mock inoculated or inoculated with TuMV-GFP and photographed under white light or UV light at 30 dpi. Wild-type (WT), <i>PCaP1</i> knockout (<i>pcap1</i>).</p

Cell-to-cell movement of P3N-PIPO-GFP in <i>N. benthamiana</i> leaves.

<p>(A) Images of epidermal cells expressing GFP alone or fused to P3N-PIPO (P3N-PIPO-GFP) at indicated hours post biolistic bombardment (hpb). Images are projections of single confocal section. (B) Quantification of cell-to-cell movement of GFP and P3N-PIPO-GFP. At least 160 GFP-expressing clusters in each bombarded sample were analyzed at 24 hpb and 48 hpb. At 48 hpb the number of cells in each cluster was counted, followed by statistical evaluation by the unpaired Student's <i>t</i>-test (P = 0.151). Error bars represent standard deviations.</p