P3N-PIPO (P3 N-terminal fused with Pretty Interesting Potyviridae ORF), the movement protein of potyviruses, is expressed as a translational fusion with the N-terminus of P3 in potyviruses. As reported in previous studies, P3N-PIPO is expressed via transcriptional slippage at a conserved G₂A₆ slippery site in the genus Potyvirus. However, it is still unknown whether a similar expression mechanism of P3N-PIPO is used in the other genera of the family Potyviridae. Moreover, due to the extremely low expression level of P3N-PIPO in natural virus-infected plants, the peptides spanning the slippery site which provide direct evidence of the slippage at the protein level, have not been identified yet. In this study, a potato virus X (PVX)-based expression vector was utilized to investigate the expression mechanism of P3N-PIPO. A high expression level of the P3N-PIPO(WT) of turnip mosaic virus (TuMV, genus Potyvirus) was observed based on the PVX expression vector. For the first time, we successfully identified the peptides of P3N-PIPO spanning the slippery site by mass spectrometry. Likewise, the P3N-PIPO(WT) of wheat yellow mosaic virus (WYMV, genus Bymovirus) was also successfully expressed using the PVX expression vector. Integrated proteome and transcriptome analyses revealed that WYMV P3N-PIPO was expressed at the conserved G₂A₆ site through transcriptional slippage. Moreover, as revealed by mutagenesis analysis, Hexa-adenosine of the G₂A₆ site was important for the frameshift expression of P3N-PIPO in WYMV. According to our results, the PVX-based expression vector might be used as an excellent tool to study the expression mechanism of P3N-PIPO in Potyviridae. To the best of our knowledge, this is the first experimental evidence dissecting the expression mechanism of a bymovirus P3N-PIPO in the experimental host Nicotiana benthamiana.

Similarly to other potyvirids, the bymovirus wheat yellow mosaic virus (WYMV) encodes a P3N-PIPO protein that is expressed by frameshifting occurring within the open reading frame of the P3 protein. P3N-PIPO is known to be essential for the cell-to-cell movement of several potyviruses, but this has not yet been confirmed for the WYMV. Here, we show that the WYMV P3N-PIPO protein influences disease symptom formation. Infection of Nicotiana benthamiana plants with a potato virus X (PVX)-based vector carrying the WYMV P3N-PIPO gene induced more severe disease symptoms and resulted in higher virus accumulation levels than did infection with PVX lacking the P3N-PIPO gene. N. benthamiana P3N-PIPO-interacting proteins were identified through co-immunoprecipitation (Co-IP) coupled with LC-MS/MS (mass spectrometry), and the interaction between P3N-PIPO and the N. benthamiana receptor-like kinase NbRLK6 was further verified by Co-IP and bimolecular fluorescence complementation (BiFC) of transiently-expressed proteins. Furthermore, our investigation showed that the disease symptom severity and accumulation level of PVX-P3N-PIPO were decreased in N. benthamiana plants when NbRLK6 expression was reduced by tobacco rattle virus-induced gene silencing.

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