D, Number ofTVBMV-GFP-infected cells in theNbDREPP-silenced leaves (black columns) at 2 and 5dpaior in theNbANK-silenced leaves (gray column) orTRV-infected leaves (light gray column) at 2dpai. inNicotiana benthamianaplants; mutants carrying a PIPO domain of seven, 20, or 43 amino acid residues failed to move between cells and cause systemic infection in this sponsor plant. Interestingly, the movement-defective mutants produced progeny that eliminated the previously introduced stop codons and thus restored their systemic movement ability. We also present evidence showing that a developmentally regulated plasma membrane protein ofN. benthamiana(referred to as NbDREPP) interacted with both P3N-PIPO and CI from the movement-competentTVBMV. The knockdown ofNbDREPPgene expression inN. benthamianaimpeded the cell-to-cell movement ofTVBMV. NbDREPP was shown to colocalize withTVBMVP3N-PIPO and CI at plasmodesmata (PD) and traffic toPDvia the early secretory pathway and the actomyosin motility system. We also show that myosin XI-2 is specially required for transporting NbDREPP toPD. In conclusion, NbDREPP is a key sponsor protein within the early secretory pathway and the actomyosin motility system that interacts with two movement proteins and influences virus movement. Guanosine The movement of viruses in plants can be divided into three stages: intracellular, intercellular, and long-distance movement (Nelson and Citovsky, 2005; Benitez-Alfonso et al., 2010). Plasmodesmata (PD) are plasma membrane-mediated channels in cell walls that control the intercellular trafficking of micromolecules and macromolecules, including plant viruses (Boevink and Oparka, 2005; Lucas et al., 2009). Plant viruses encode movement proteins (MPs) that can regulate the size exclusion limit (SEL) ofPDand mediate virus trafficking between cells (Lucas, 2006; Raffaele et al., 2009; Amari et al., 2010; Ueki et al., 2010). Based on the functions ofMPs during computer virus movement, the viralMPs are divided into three major groups. The first group ofMPs is represented by the 30-kD protein ofTobacco mosaic virus(TMV). The 30-kD proteins can interact with single-stranded RNAs and transport viral ribonucleoprotein complexes to cell walls, where they modify theSELofPDto allow viruses to traverse the cell walls (Olesinski et al., 1996; Tzfira et al., 2000; Kawakami et al., 2004). The second group ofMPs is known to form tubular structures that extend across thePDand allow virus to traverse. Viruses that encode this group ofMPs includeCowpea Guanosine mosaic computer virus, Grapevine fan leaf virus(GFLV), Cauliflower mosaic virus, andTomato spotted wilt virus(Ritzenthaler and Hofmann, 2007; Amari et al., 2011). The third group ofMPs is known as triple gene block proteins (TGBps), encoded by overlapping triple gene blocks. The threeTGBps(TGBp1, TGBp2, and TGBp3) function coordinately to transport viral genomes to and throughPD(Verchot-Lubicz, 2005; Jackson et al., 2009; Lim et al., 2009; Tilsner et al., 2013). Viruses that encodeTGBpsbelong to the generaPotexvirus, Hordeivirus, andPomovirus(Verchot-Lubicz et al., 2010). Potyviruses are different from the above viruses and lack a dedicatedMP. To date, multiple potyviral proteins, including COAT PROTEIN, CYLINDRICAL INCLUSION (CI), HELPER COMPONENT PROTEINASE (HC-Pro), and VIRAL GENOME-LINKED PROTEIN, have been shown to function in the cell-to-cell movement of potyviruses (Nicolas et al., 1997; Rojas et al., 1997; Carrington et al., 1998; Wei COG3 et al., 2010). Viruses ofPotyvirus(familyPotyviridae), the largest genus of plant-infecting viruses, cause great economic losses to world agriculture production (Fauquet et al., 2005). The potyviral genome is a positive sense, single-stranded RNA of approximately 10 kb in length. It contains a large open reading frame (ORF) encoding a polyprotein that is later processed into 10 mature proteins by three virus-encoded proteinases (Riechmann et al., 1992; Fauquet et al., 2005). A +2 frame-shiftPretty Interesting Potyviridae(PIPO)ORFthat is embedded within theP3ORFwas recently identified and proposed to produce a P3N-PIPO (for the protein encoded by 5-terminus ofP3and frame-shiftPIPO) fusion (Chung et al., 2008; Vijayapalani et al., 2012). The P3N-PIPOs ofTurnip mosaic virus(TuMV) andTobacco etch viruswere previously shown to localize atPD, interact with CI in planta, and transport CI toPDin a CI: P3N-PIPO ratio-dependent manner (Wei et al., 2010). Soybean mosaic viruswith a mutant PIPO domain failed to cause systemic infection in its sponsor plant (Wen and Hajimorad, 2010). Therefore , the potyvirus P3N-PIPO continues to be suggested as the classicalMP(Tilsner and Oparka, 2012; Vijayapalani et al., 2012). Viruses recruit sponsor factors for their movement in plants (Chen et al., 2000; Raffaele et al., 2009; Amari et al., 2010; Ueki et al., 2010). Compared with the progresses on viralMPcharacterization, identifications ofMP-interacting host proteins are much behind (Chen et al., 2000; Oparka, 2004; Raffaele et al., 2009; Amari et al., Guanosine 2010). To date, about 20 sponsor proteins have been identified to interact with specific viralMPs (Pallas and Garca, 2011). For example , the pectin methylesterase interacted with TMVMP, increased theSELofPD, and facilitated TMV movement between cells (Chen et al., 2000); an ankyrin repeat-containing protein (ANK).