Sweet cherry leaves infected by PBNSPaV were collected from Shandong Province in 2021. Fluorescent expression vector 35S:GFP, Agrobacterium GV3101, Nicotiana benthamiana (N. benthamiana) seeds, and other plant materials were stored at our laboratory. The PVX vector (pGR107) was a gift from Professor Xiaofei Cheng at Northeast Agricultural University [12].

Total RNAs were extracted from 100 milligrams of cherry leaves using TRIzol reagent following the manufacturer’s instructions and stored at −80°C. Primers used to amplify the P6 gene were designed based on the PBNSPaV sequence (GenBank: MZ221026) to amplify the P6 gene. cDNAs were synthesized by first incubating primers, dNTPs, and total RNAs at 80°C for 5 min, then with PrimeScript reverse transcriptase in 1 × buffer (Takara Inc., Japan) at 42°C for 1.5 h, and subjected to PCR for P6 gene amplification. The PCR product was cloned into the pMD18-T vector and transformed into DH5α competent cells. Positive clones were sequenced at Boshang Biotech Company (Jinan, China) for validation. The sequencing results were aligned to the PBNSPaV genome sequences on the NCBI database. Multiple alignments and homology analyses were performed using ClustalX and BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/), and the transmembrane prediction was performed using the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM/).

A P6-GFP plasmid was constructed by cloning the P6 gene into the 35S:GFP vector via BamHI and sequencing validated by Boshang Biotech Company (Jinan, China). The P6-GFP and GFP plasmids were transformed into Agrobacterium tumefaciens GV3101 using the CaCl₂-mediated freeze-thaw method, as described previously. Single A. tumefaciens colonies were cultured in LB medium with 50 µg/mL kanamycin and 100 µg/mL rifampin at 28°C until OD₆₀₀ reached 1.0~2.0. After centrifugation, the pellets were resuspended in the infiltration solution containing 10 mM MgCl₂, 10 mM 2-(N-morpholino) and 0.15 mM acetosyringone to OD₆₀₀ of 1.2 and resuscitated at 28°C for 2 h. N. benthamiana at 5–7 leaf age was selected and treated on the back of leaves with resuscitated A. tumefaciens for 40–48 h using a 1 mL needless syringe. The infiltrated leaves were prepared as slides and observed under a laser confocal microscope (Zeiss, Germany) to check P6-GFP subcellular localization. The treatment was repeated by at least three times.

The P6 gene fragment was cloned via ligation using T4 ligase into the PVX expression vector using T4 ligase after the vector was digested with Sal I and Cla I. The resulting PVX heterologous expression vector and recombinant plasmid PVX-P6 and PVX heterologous expression vector were transformed into Agrobacterium GV3101 by the freeze-thaw method and cultured on plates containing 50 mg/L kanamycin and 25 mg/L rifampicin at 28°C. Single colonies were selected for colony PCR identification. After identification, they were propagated overnight in liquid medium containing kanamycin and rifampicin until the OD₆₀₀ reached 0.6–0.8. The cultures were then used to infiltrated into 5–7-leaf age N. benthamiana. After 7 and 14 days of infiltration, samples were collected, and their symptoms were recorded. Total RNAs were extracted from the upper new leaves (non-invasive leaves) inoculated with PVX and PVX-P6 which were fully ground after liquid nitrogen freezing and used to extract total RNAs. P6 and PVX expression were detected by RT-PCR using primer sets P6-ClF and P6-SlR and primer set PVX-F and PVX-R, respectively.

The obtained P6 gene is 174 bp long, encoding a protein of about 6.3 kD. BLAST analysis with 24 reference isolates in GenBank showed that the P6 protein is highly homologous to other reference proteins with only seven mutations (Fig. 1A). This indicates that P6 genes of PBNSPaV isolates are highly conservative in phylogeny. The P6 protein is a stable hydrophobic protein consisting of 57 amino acid residues and lacks a signal peptide. It has an isoelectric point (pI) of 7.81, and transmembrane prediction indicated that the P6 is a transmembrane protein with transmembrane region composed of amino acid residues located at position 15 to 37 (Fig. 1B). Structural analysis of the Swiss model showed that the P6 protein mainly consists of α-helix (Fig. 1C).

To determine P6 protein’s localization in plant cells, the P6 sequence was fused in-frame to the 5’ end of the GFP gene behind a 35S promoter to create p35S-P6:GFP vector. The expression vectors of GFP, P6-GFP, and CBL1-RFP (a membrane localization protein) were transformed into Agrobacterium GV3101 and used to infiltrate N. benthamiana. Confocal microscopic observation at 48 h of post-infiltration revealed green fluorescence signals in the cytomembrane and nuclear membrane of the epidermal cells of N. benthamiana infiltrated with P6-GFP and overlapped green and red fluorescence signals excited by P6-GFP and CBL1, respectively. These observations indicated that P6 proteins are presented in both cytomembrane and nuclear membrane (Fig. 2A). To further study the pathogenicity of p6 proteins, expression vectors PVX and PVX-P6 were transformed into Agrobacterium GV3101, which were then inoculated into N. benthamiana, respectively. After inoculation with PVX-P6 for 14 days, the upper leaves of N. benthamiana showed no significant changes, while the upper leaves of N. benthamiana inoculated with PVX only showed slight mosaic symptoms (Fig. 2B). RT-PCR analysis of viral gene expression (primers in Table S1) showed that PVX was detected in all N. benthamiana inoculated with PVX and PVX-P6, while P6 was only detected in N. benthamiana inoculated with PVX-P6 (Fig. 2C). These results suggested that heterologous expression of P6 in N. benthamiana could not enhanced the pathogenicity of PVX.