Integrated Proteomics and Transcriptomics Analyses Reveal the Transcriptional Slippage of a Bymovirus P3N-PIPO Gene Expressed from a PVX Vector in Nicotiana benthamiana

Abstract

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 for the expression mechanism of P3N-PIPO in the genus Bymovirus, the only genus comprising bipartite virus species in the family Potyviridae.

Advances in understanding the soil-borne viruses of wheat: from the laboratory bench to strategies for disease control in the field

“…P3 targets the ER membrane to form inclusions and is transported along actin filaments to participate in the formation of replication vesicles (Cui et al 2017;Yang et al 2021). WYMV PIPO is usually expressed with a part of the P3 ORF as P3N-PIPO, which promotes the movement of viruses from cell to cell (Vijayapalani et al 2012;Chai et al 2020;Yu et al 2021).…”

The P3N-PIPO Protein Encoded by Wheat Yellow Mosaic Virus Is a Pathogenicity Determinant and Promotes Its Pathogenicity through Interaction with NbRLK6 in Nicotiana benthamiana

“…For the pathogenicity assessment of P3N-PIPO and Co-IP/MS and BiFC assays, the constructs were generated using Gateway technology (Invitrogen Life Technologies, Carlsbad, CA, USA). The full-length sequences of WYMV P3N-PIPO (contain G 2 A 7 motif) and P3N-PIPO-AS (antisense) were amplified with the corresponding primer pairs ( Table S1 ), from the constructed vector PVX-WYMV P3N-PIPO(FS-1)-GFP in our previous study [ 6 ]. The NbRLK6 gene was amplified with the corresponding primer pair ( Table S1 ), from N. benthamiana cDNA.…”

“…In recent years, a small overlapping coding sequence, which is termed PIPO (pretty interesting potyviridae ORF), was discovered by bioinformatics analysis within the genome sequences of viruses in the Potyviridae family [ 3 ]. A novel protein named P3N-PIPO, with the PIPO segment being encoded in a different frame within the P3 cistron, is expressed by a viral polymerase slippage mechanism [ 4 , 5 , 6 ]. In previous studies, P3N-PIPO was shown to be required for efficient cell-to-cell movement of several potyviruses [ 7 , 8 , 9 , 10 , 11 ].…”

The P1 protein of wheat yellow mosaic virus exerts RNA silencing suppression activity to facilitate virus infection in wheat plants

“…A study revealed that P3 is associated with virus infection (Luan et al., 2019). Protein P3N‐PIPO generated from P3 ORF by a +2 frameshift was shown to be essential for virus movement (Chung et al., 2008; Yu et al., 2021). Protein 7K binding protein P3 is involved in viral replication (Rodríguez‐Cerezo et al., 1993; Yu et al., 2021).…”

“…Protein P3N‐PIPO generated from P3 ORF by a +2 frameshift was shown to be essential for virus movement (Chung et al., 2008; Yu et al., 2021). Protein 7K binding protein P3 is involved in viral replication (Rodríguez‐Cerezo et al., 1993; Yu et al., 2021). The cylindrical inclusion protein (CI) forms intracellular windwheel inclusion bodies in cytoplasm during WYMV infection (Ohki et al., 2019; Rodríguez‐Cerezo et al., 1997).…”