Viruses belonging to the family Potyviridae represent the largest group of plant-infecting RNA viruses, including a variety of agriculturally and economically important viral pathogens. Like all picorna-like viruses, potyvirids employ polyprotein processing as the gene expression strategy.

The genomic 5′-terminal regions of viruses in the family Potyviridae (potyvirids) encode two types of leader proteases, serine-protease (P1) and cysteine-protease (HCPro), which differ greatly in the arrangement and sequence composition among inter-genus viruses. Most potyvirids have the same tandemly-arranged P1 and HCPro, whereas viruses in the genus Macluravirus encode single distinct leader protease - a truncated version of HCPro with yet-unknown functions. We investigated the RNA silencing suppression (RSS) activity and its underpinning mechanism of the distinct HCPro from alpinia oxyphylla mosaic macluravirus (aHCPro). Sequence analysis revealed that macluraviral HCPros have obvious truncations in the N-terminal and middle regions, when aligned to their counterparts in potyviruses (well-characterized viral suppressors of RNA silencing). Nearly all defined-elements essential for the RSS activity of potyviral counterparts are not discriminated in macluraviral HCPros. Here, we demonstrated that aHCPro exhibits the similar anti-silencing activity with potyviral counterpart. However, aHCPro fails to block both the local and systemic spreading of RNA silencing. In line, aHCPro interferes with the dsRNA synthesis, an upstream step in RNA silencing pathway. Affinity-purification and NanoLC-MS/MS analysis revealed that aHCPro has no association with core components or their potential interactors involving in dsRNA synthesis from the protein layer. Instead, the ectopic expression of aHCPro significantly reduces the transcript abundance of RDR2, RDR6, SGS3 and SDE5. This study represents the first report of the anti-silencing function of macluravirus-encoded HCPro and the underlying molecular mechanism.

Passion fruit (Passiflora edulis) is a perennial evergreen vine that grows mainly in tropical and subtropical regions due to its nutritional, medicinal and ornamental values. However, the molecular biology study of passion fruit is extremely hindered by the lack of an easy and efficient method for transformation. The protoplast transformation system plays a vital role in plant regeneration, gene function analysis and genome editing. Here, we present a new method (‘Cotyledon Peeling Method’) for simple and efficient passion fruit protoplast isolation using cotyledon as the source tissue. A high yield (2.3 × 107 protoplasts per gram of fresh tissues) and viability (76%) of protoplasts were obtained upon incubation in the enzyme solution [1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] for 2 hours. In addition, we achieved high transfection efficiency of 83% via the polyethylene glycol (PEG)-mediated transformation with a green fluorescent protein (GFP)-tagged plasmid upon optimization. The crucial factors affecting transformation efficiency were optimized as follows: 3 μg of plasmid DNA, 5 min transfection time, PEG concentration at 40% and protoplast density of 100 × 104 cells/ml. Furthermore, the established protoplast system was successfully applied for subcellular localization analysis of multiple fluorescent organelle markers and protein-protein interaction study. Taken together, we report a simple and efficient passion fruit protoplast isolation and transformation system, and demonstrate its usage in transient gene expression for the first time in passion fruit. The protoplast system would provide essential support for various passion fruit biology studies, including genome editing, gene function analysis and whole plant regeneration.

Passion fruit (Passiflora edulis) is a perennial evergreen vine that grows mainly in tropical and subtropical regions due to its nutritional, medicinal and ornamental values. However, the molecular biology study of passion fruit is extremely hindered by the lack of an easy and efficient method for transformation. The protoplast transformation system plays a vital role in plant regeneration, gene function analysis and genome editing. Here, we present a new method (Cotyledon Peeling Method) for simple and efficient passion fruit protoplast isolation using cotyledon as the source tissue. A high yield (2.3 X 107 protoplasts per gram of fresh tissues) and viability (76%) of protoplasts were obtained upon incubation in the enzyme solution [1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] for 2 hours. In addition, we achieved high transfection efficiency of 83% via the polyethylene glycol (PEG)-mediated transformation with a green fluorescent protein (GFP)-tagged plasmid upon optimization. The crucial factors affecting transformation efficiency were optimized as follows: 3 ug of plasmid DNA, 5 min transfection time, PEG concentration at 40% and protoplast density of 100 X 104 cells/ml. Furthermore, the established protoplast system was successfully applied for subcellular localization analysis of multiple fluorescent organelle markers and protein-protein interaction study. Taken together, we report a simple and efficient passion fruit protoplast isolation and transformation system, and demonstrate its usage in transient gene expression for the first time in passion fruit. The protoplast system would provide essential support for various passion fruit biology studies, including genome editing, gene function analysis and whole plant regeneration.