Visualization of the interaction between P0 and ASK1 in planta using bimolecular fluorescence complementation. Three-day-old dark-grown mustard seedlings were transformed by particle bombardment with combinations of plasmids expressing different YN- and YC-fusion proteins. To identify transformed cells, a plasmid expressing the CFP fused to the parsley common plant regulatory protein 2 (CPRF2), which localizes to the nucleus (nu), was included during bombardment. Images were recorded 5 h after bombardment by using CFP-specific and YFP-specific filters. Shown are typical cells bombarded with plasmids expressing P0^CA–YN and YC–ASK1 (A), P0^CALP1–YN and YC–ASK1 (B), P0^CA–YN and YC (C), and YN–ASK1 and YC–EID1 (D), a pair of proteins known to interact (17). A differential interference contrast (DIC) image is shown between each pair of fluorescent images. Regions of diffuse YFP fluorescence in A and B that are not confined to a single cell are background. (All images are at the same magnification; scale bar: 40 μm.)

The LP1 mutation inhibits the silencing suppressor activity of P0^BW but does not destabilize the protein. Leaves of N. benthamiana line 16c were infiltrated with a mixture of Agrobacteria strains containing pBin–GFP plus either empty vector (pBin61, lane 7) or pBin61 expressing the silencing suppressor protein(s) indicated at the top (lanes 1–6). Total RNA was extracted 5 days later from the agro-infiltrated patches and analyzed for the presence of GFP transcript (A), GFP transcript-derived siRNAs (B), and P0^BW transcript (C) by Northern blot using specific ³²P-labeled probes. Total protein was extracted from the same patches and analyzed for P0^BW protein by Western blot using a P0^BW-specific polyclonal antiserum (D). Loading controls are shown below each blot.

P0^CA interacts with ASK1 and ASK2 via an F-box domain. (A) Genetic organization of CABYV RNA. Labeled rectangles symbolize important genes. The genetic organization of BWYV RNA is identical. The blow-up image shows the positions of deletions (above) and point mutations (below) in P0. Shading indicates the notional position of the ≈60-residue F-box domain. (B) Three independent colonies of yeast transformed with bait and prey plasmids expressing the indicated fusion proteins were replicate-streaked on nonselective medium (Left) and on medium selective for a strong two-hybrid interaction (Right). Upper is a Western blot showing accumulation of the WT and mutant P0^CA fusion proteins in total yeast protein extracts. (C) Pull-down of [³⁵S]methionine-labeled P0^CA by glutathione S-transferase (GST) and GST–ASK1 and GST–ASK2 fusion proteins immobilized on glutathione-Sepharose beads. Proteins immobilized on the beads were visualized by Coomassie blue staining (Left) and ³⁵S-P0^CA by autoradiography (Right). An aliquot of the input ³⁵S–P0^CA was loaded in the leftmost lane of the gel. The minor bands marked by an asterisk in the two right-hand lanes of the autoradiogram comigrate with the GST–ASK1 and GST–ASK2 bands (Left) and may be ³⁵S–P0^CA, which has remained associated with the fusion proteins under denaturing conditions. (D) Sequences of P0 of CABYV, BWYV, Beet mild yellowing virus (RefSeq accession no. "NC 003491), Cereal yellow dwarf virus-RPV (NC 004751), and Potato leafroll virus (NC 001747) near the LPxxI/L motif and a downstream proline conserved in many plant F-box domains. (E) A yeast-bridging assay shows that interaction between P0^BW and P0^CA and AtCUL1 requires ASK1 or ASK2 as a bridging component. Three independent yeast AH109 colonies triply transformed with plasmids expressing the indicated BD-P0 and AD-CUL1 fusion proteins plus either empty pVT-U102 or pVT–U102 expressing ASK1 or ASK2 were replicate-streaked on a dropout plate under nonstringent (−UWL) and stringent (−AUWL) conditions selecting for interaction between BD–P0 and AD–CUL1. The plates were incubated at 21°C for 7 days.

Depletion of SKP1 in N. benthamiana by VIGS provokes resistance to BWYV. (A) Northern blot analysis of PVX (P) and PVX-NbSKP1 (P_SKP) progeny RNA in upper leaves of N. benthamiana at different times pi using a ³²P-labeled RNA probe complementary to the 3′-terminal PVX RNA sequence. Leaf positions are indicated in D. Lane H is RNA from a healthy plant. (B) Inhibition of NbSKP1 transcript accumulation by VIGS. Reverse transcription followed by PCR with primers specific for the 3′ noncoding region of NbSKP1 mRNA was carried out on total RNA from leaf 4 (16 and 23 days pi) or 5 (30 days pi) of P- or P_SKP-infected plants. In the dilution series, the amounts of cDNA template used for PCR were 1/10, 1/50, and 1/250 of that used in each left-hand lane. Products of RT-PCR amplification of a portion of the Elongation Factor 1α (EF-1α) sequence from the same RNA samples are shown as a control. (C) Inhibition of SKP1 accumulation by VIGS. NbSKP1 levels in leaf 3 were monitored at different times after inoculation with P or P_SKP by Western blot using an SKP1-specific antiserum. The left-hand lane (ASK) was loaded with a protein extract from A. thaliana and lane H with protein from healthy N. benthamiana. (D) BWYV titer in leaf 5 of plants that had been preinoculated with PVX or PVX–NbSKP1. The plants were aphid-inoculated with BWYV 16 days after infection with PVX or PVX–NbSKP1 and tested by ELISA for BWYV at 30 days. (Right) Bars represent the ELISA A₄₀₅ value for each plant. The ELISA background is indicated by the horizontal dashed line. The cartoon in Left shows the relative positions of the leaves subjected to the different treatments.