Plant viruses must move through plasmodesmata (PD) to complete their life cycles. For viruses in the Potyviridae family (potyvirids), three viral factors (P3N-PIPO, CI, and CP) and few host proteins are known to participate in this event. Nevertheless, not all the proteins engaging in the cell-to-cell movement of potyvirids have been discovered. Here, we found that HCPro2 encoded by areca palm necrotic ring spot virus (ANRSV) assists viral intercellular movement, which could be functionally complemented by its counterpart HCPro from a potyvirus. Affinity purification and mass spectrometry identified several viral factors (including CI and CP) and host proteins that are physically associated with HCPro2. We demonstrated that HCPro2 interacts with both CI and CP in planta in forming PD-localized complexes during viral infection. Further, we screened HCPro2-associating host proteins, and identified a common host protein in Nicotiana benthamiana–Rubisco small subunit (NbRbCS) that mediates the interactions of HCPro2 with CI or CP, and CI with CP. Knockdown of NbRbCS impairs these interactions, and significantly attenuates the intercellular and systemic movement of ANRSV and three other potyvirids (turnip mosaic virus, pepper veinal mottle virus, and telosma mosaic virus). This study indicates that a nucleus-encoded chloroplast-targeted protein is hijacked by potyvirids as the scaffold protein to assemble a complex to facilitate viral movement across cells.

Potyviruses possess one positive-sense single-stranded RNA genome, mainly dependent on polyprotein processing as the expression strategy. The resulting polyproteins are proteolytically processed by three virus-encoded proteases into 11 or 12 mature proteins. One such factor, 6 kDa peptide 1 (6K1), is an understudied viral factor. Its function in viral infection remains largely mysterious. This study is to reveal part of its roles by using pepper veinal mottle virus (PVMV) as the model. Alanine substitution screening analysis revealed that 15 of 17 conserved residues across potyviral 6K1 sequences are essential for PVMV infection. However, 6K1 protein is less accumulated in virus-infected cells, although P3-6K1 and 6K1-CI junctions are efficiently processed by NIa-Pro for its release, indicating that 6K1 undergoes a self-degradation event. Mutating the cleavage site to prevent NIa-Pro processing abolishes viral infection, suggesting that the generation of 6K1 along with its degradation might be important for viral multiplication. We corroborated that cellular autophagy is engaged in 6K1’s degradation. Individual engineering of the 15 6K1 variants into PVMV allows their expression along with viral infection. Five of such variants, D30A, V32A, K34A, L36A, and L39A, significantly interfere with viral infection. The five residues are enclosed in a conserved lysine/arginine-rich motif; four of them appear crucial in engaging autophagy-mediated self-degradation. Based on these data, we envisaged a scenario which potyviral 6K1s interact with an unknown anti-viral component to be co-degraded by autophagy to promote viral infection. IMPORTANCE: Potyvirus is the largest genus of plant-infecting RNA viruses, which encompasses socio-economically important virus species, such as Potato virus Y , Plum pox virus , and Soybean mosaic virus . Like all picorna-like viruses, potyviruses express their factors mainly via polyprotein processing. Theoretically, viral factors P3 through CP, including 6K1, should share an equivalent number of molecules. The 6K1 is small in size (~6 kDa) and conserved across potyviruses but less accumulated in virus-infected cells. This study demonstrates that cellular autophagy is engaged in the degradation of 6K1 to promote viral infection. In particular, we found a conserved lysine/arginine-rich motif in 6K1s across potyviruses that is engaged in this degradation event. This finding reveals one facet of a small protein that helps understand the pro-viral role of cellular autophagy in viral infection.

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, woody, tropical vine. It produces edible round to oval fruit that is highly favored for its unique aroma and taste, as well as its richness in antioxidants, vitamins, and minerals. However, functional genomics studies of passion fruit are scarce, as simple and efficient genetic tools are lacking for this species. Here, we developed virus-mediated protein overexpression (VOX) and virus-induced gene silencing (VIGS) vectors based on the telosma mosaic virus (TelMV), an emerging potyvirus that infects passion fruit plants worldwide. This vector, designated pTelMV-GW, incorporates Gateway-compatible recombination sites for rapid gene cloning. Using this vector, we achieved systemic stable expression of two heterologous proteins in passion fruit: green fluorescent protein (GFP) and bacterial phytoene synthase (CrtB). Additionally, pTelMV-GW containing different GFP fragments also induced systemic gene silencing in GFP-transgenic Nicotiana benthamiana plants. Furthermore, we used this vector to trigger phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) silencing in passion fruit plants. The TelMV-based VIGS was enhanced using a mild TelMV strain encoding a mutated helper-component proteinase (HC-Pro) with impaired RNA silencing suppressor activity. This upgraded vector (pTelMV-R181K-GW), containing PDS or ChlI fragments, induced clear photobleaching or yellowing phenotypes in passion fruit plants. Overall, our work presents a set of VIGS and VOX vectors for use in passion fruit plants, a crucial step towards identifying horticulturally important genes for improving passion fruit production and quality.

A total of 164 viruses have been identified in peppers worldwide. To combat viruses, pathogen-derived resistance (PDR) has been employed by expressing a viral genomic segment or a viral protein in host plants. Unfortunately, peppers are recalcitrant to genetic transformation and regeneration. An alternative strategy is to generate PDR in pepper plants by using an attenuated virus-based expression vector. In a previous study on the function of pepper veinal mottle virus (PVMV) 6K1 protein, we mutated a conserved Ala with Arg (A15R), and a second mutation (R15G) was observed in virus progeny. In this study, we demonstrated that mutating Ala to Gly (A15G) in the 6K1 yields a stable attenuated strain (named PVMV-G-A15G). PVMV-G-A15G can cross-protect pepper plants against a severe strain of PVMV and its close relative, chilli veinal mottle virus. We then engineered PVMV-G-A15G into a viral expression vector that has the Golden Gate cloning feature. To examine if the PVMV vector can be used to mediate PDR, we inserted the complete CP sequence of potato virus X (PVX) into the engineered PVMV-G-A15G. Preinoculated plants with the recombinant PVMV-G-A15G that carries the PVX CP sequence are resistant to PVX infection. Taken together, our research demonstrates that the attenuated PVMV can be employed for delivering the target virus sequence to mediate PDR.