A small open reading frame (ORF), pipo, overlaps with the P3 coding region of the potyviral polyprotein ORF. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. Here, we provide immunoblotting evidence that the protein PIPO is expressed as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO (P3N-PIPO). P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. Using a yeast two-hybrid screen, co-immunoprecipitation assays, and bimolecular fluorescence complementation (BiFC) assays, we found that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. BiFC revealed that it is the PIPO domain of P3N-PIPO that binds PCaP1 and that myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. In PCaP1 knockout mutants (pcap1) of Arabidopsis, accumulation of TuMV harboring a GFP gene (TuMV-GFP) was drastically reduced relative to the virus level in wild-type plants, only small localized spots of GFP were visible, and the plants showed few symptoms. In contrast, TuMV-GFP infection in wild-type Arabidopsis yielded large green fluorescent patches, and caused severe stunting. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. In contrast to TuMV, the tobamovirus Oilseed rape mosaic virus did not require PCaP1 to infect Arabidopsis plants. We conclude that potyviral P3N-PIPO interacts specifically with the host plasma membrane protein PCaP1 to participate in cell-to-cell movement. We speculate that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement. The PCaP1 knockout may contribute to a new strategy for recessive resistance to potyviruses.

Toxocara canis has a complex lifecycle including larval stages in the somatic tissue of dogs that tolerate macrocyclic lactones. In this study, we investigated T. canis permeability glycoproteins (P-gps, ABCB1) with a putative role in drug tolerance. Motility experiments demonstrated that while ivermectin failed to abrogate larval movement, the combination of ivermectin and the P-gp inhibitor verapamil induced larval paralysis. Whole organism assays revealed functional P-gp activity in larvae which were capable of effluxing the P-gp substrate Hoechst 33342 (H33342). Further investigation of H33342 efflux demonstrated a unique rank order of potency for known mammalian P-gp inhibitors, suggesting that one or more of the T. canis transporters has nematode-specific pharmacological properties. Analysis of the T. canis draft genome resulted in the identification of 13 annotated P-gp genes, enabling revision of predicted gene names and identification of putative paralogs. Quantitative PCR was used to measure P-gp mRNA expression in adult worms, hatched larvae, and somatic larvae. At least 10 of the predicted genes were expressed in adults and hatched larvae, and at least 8 were expressed in somatic larvae. However, treatment of larvae with macrocyclic lactones failed to significantly increase P-gp expression as measured by qPCR. Further studies are needed to understand the role of individual P-gps with possible contributions to macrocyclic lactone tolerance in T. canis.