TuMV genome map (9,835 nt). In the plasmid p35STuMV-GFP, GFP is fused between P1 and HC-Pro. The position of the overlapping CDS, pipo, and the G₂A₆ motif are indicated.

Detection of TuMV accumulation in plants by RT-PCR. Primers specific to p35STuMV-GFP were used to amplify cDNAs from RNA isolated from plants infected with WT TuMV (p35STuMV-GFP; lane 2), the two PIPO knockout mutants, p41 and p68 (lanes 2 and 3 respectively), and both negative (water; lane 5) and nonbombarded (lane 6) controls, all at 12 dpi. The PCR controls were plasmid p35STuMV-GFP for the positive control (lane 7) and water for the negative control (lane 8). Marker DNAs are in lane 1. TuMV RNA was present only in WT-infected plants.

Immunoblot detection of PIPO products. (A) An approximately 25-kDa PIPO fusion product. AS 2–15, antiserum against PIPO N-terminal peptide (amino acids 2–15). AS 39–52, antiserum against PIPO C-terminal peptide (amino acids 39–52). −, lysate from noninfected plants. +, lysate from plants infected with WT TuMV (p35STuMV-GFP). Lanes were loaded with equal amounts of tissue; similar results were obtained when lanes were loaded with equal total protein (data not shown). (B) +, detection of an ≈7-kDa product with AS 39–52 from in vitro translation of pipo nucleotide sequence preceded by T7 promoter and followed by FLAG epitope, demonstrating that the antiserum would detect such a product if it were expressed in vivo. −, negative control (no RNA).

N. benthamiana 12 days postinoculation (dpi). (Upper) Natural light. (Lower) UV light. From left: plants bombarded with (1) no DNA; (2) WT p35STuMVGFP virus (i.e., TuMV with GFP fused between P1 and HC-Pro); (3) and (4) p35STuMV-GFP PIPO knockout mutants p41 and p68; and (5) nonbombarded plants. In each of (1–5), only one of four replicates is depicted. Only WT-infected plants showed any sign of infection (three of four replicates) with extensive GFP fluorescence, stunted growth, and wilting by 12 dpi. Note that the small yellowish-green patches visible on some leaves under UV—e.g., Lower (1) and (4), are necrosis caused by bombardment (cf. natural light photos). Some patches on the soil also appear green under UV. However, GFP fluorescence is clearly distinguishable from both of these. High resolution photos are in Fig. S9 and Fig. S10.

Pipo sequence. (A) Extract of 9 RefSeqs from an alignment of the 48 Potyvirus GenBank RefSeqs, showing the region around the 5′ end of pipo, which coincides with the annotated highly conserved G_1-2A_6-7 motif (see Fig. S8 for the full 48-RefSeq alignment). Viruses: NC_001445, plum pox; NC_001555, tobacco etch; NC_001616, potato Y; NC_001671, pea seed-borne mosaic; NC_002509, turnip mosaic; NC_002634, soybean mosaic; NC_003397, bean common mosaic; NC _004039, potato A; NC_003398, sugarcane mosaic. (B) Similar [GA]-rich tracts from the 5′ end of pipo in representative RefSeqs from other Potyviridae genera (see Fig. S8 for other RefSeqs). Viruses: NC_001814, ryegrass mosaic; NC_002350, wheat yellow mosaic; NC_001886, wheat streak mosaic; NC_003797, sweet potato mild mottle. (C) The TuMV/p35STuMV-GFP PIPO peptide and nucleotide sequences, with the two antigen sites underlined in the peptide sequence and the two knockout point mutation sites underlined in the nucleotide sequence.

(Upper) MLOGD sliding-window plot for an alignment of the 48 GenBank Potyvirus RefSeqs (polyprotein region only; CLUSTALW (10) amino acid alignment back-translated to nucleotide sequence). Reference sequence, NC_002509 (TuMV); window size, 40 codons; step size, 20 codons. Each window is represented by a small circle (showing the likelihood ratio score for that window), and gray bars show the width (ends) of the window. The null model, in each window, is that only the polyprotein frame is coding, whereas the alternative model is that both the polyprotein frame and the window frame are coding. Positive scores (i.e., above the horizontal dashed line) favor the alternative model. The positions of stop codons in each frame are shown below the likelihood scores and the positions of alignment gaps (green) in all 48 RefSeqs are shown at the top. Only the +1 (red) and +2 (purple) frames are shown because the +0 frame is the polyprotein frame that is included in the null model. Scores are generally negative with occasional random scatter into low positive scores, except for the pipo region, which has consecutive high-positively scoring windows and a corresponding ORF present across the alignment. Note that other apparent “gaps” in the stop codon annotation mainly correspond to alignment gaps. See ref. 3 for details of MLOGD software. (Lower) Zoom-in showing pipo and the flanking ±400 nt. Window size, 10 codons; step size, 5 codons. Note that pipo contains at least five nonoverlapping and hence completely independent high-positively scoring windows. Formally, and within the MLOGD model, P < 10^–100.