The PVY cDNA clone PVY-N605(123) (51) was used for construction of PVY clones with mutant CP. This PVY clone was chosen as, among the few available clones, it is similar to PVY-NTN and it is easy to manipulate (52).

Deletion of 50 amino acid residues at the N terminus (ΔN50-CP) and deletions of 40 or 60 residues at the C terminus (ΔC40-CP and ΔC60-CP, respectively) of the CP protein were introduced into the plasmid using in vitro site-directed mutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit, Agilent Technologies) according to the manufacturer’s instructions with the following modifications: The plasmid was amplified in 25-μl polymerase chain reaction (PCR) reaction using a touch-down cycling program with annealing temperature from 65° to 55°C for 10 cycles followed by 8 cycles with constant annealing temperature of 55°C and an elongation step of 28 min. Dpn I (Agilent Technologies) digestion was performed by adding 40 U of enzyme to each amplification reaction and incubating it at 37°C for 2 hours. Afterward, 2 μl of the digested DNA was used to transform XL10-Gold Ultracompetent Cells (Agilent Technologies). The plasmids were isolated with a Monarch plasmid miniprep kit (New England BioLabs), and mutations were confirmed by automated Sanger sequencing (Eurofins Scientific).

The wild-type PVY clone and the three CP mutant clones (ΔN50-CP, ΔC40-CP, and ΔC60-CP) were used to infect N. clevelandii at the 6 to 10 leaf stage. For each construct, seven plants (five leaves per plant) were inoculated by biolistic bombardment using Helios Gen Gun system (Bio-Rad), as previously described (53). Bombardment with a yellow fluorescent protein–expressing plasmid that lacked any PVY-related sequence served as negative bombardment control to assess for any background signal due to PVY carry over during the bombardment procedure.

At 7 days postbombardment (dpb), samples of three inoculated leaves per plant were collected. Following the same procedure, three upper nonbombarded leaves were sampled at 14 dpb. TEM grids were prepared with sections of lower leaves for each treatment, at 7 dpb. The experiment was repeated twice for all three CP mutant clones. The homogenization, RNA isolation, deoxyribonuclease treatment and quality control of RNA were performed, as described (54). A one-step reverse transcription PCR (RT-PCR) kit (Qiagen) was used according to the manufacturer’s protocol to confirm that introduced mutations were maintained in the viral progeny. Relative PVY RNA concentration was analyzed by single-step quantitative RT-PCR (RT-qPCR; AgPath-ID One-Step RT-PCR, Thermo Fisher Scientific), and primers are shown in table S2. The cytochrome oxidase transcript was used as the endogenous control. All samples with Cq values above the background (Cq = 32) were considered negative. To check for the contribution of plasmid-derived signal to the concentration of PVY RNA, all samples were analyzed using the same assay and kit but omitting the RT step by preheating up the master mix (5 min, 95°C). The quantGenius software (55) was used for relative quantification of PVY RNA using the standard curve. The copy numbers calculated from qPCR without RT (presence of plasmid DNA in the sample) were subtracted from copy numbers calculated from RT-qPCR. One-way analysis of variance (ANOVA) with Bonferroni post hoc analysis was performed to statistically evaluate differences in virus accumulation between the PVY clones (data S1).

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