The intercellular movement of plant viruses requires both viral and host proteins. Previous studies have demonstrated that the frame-shift protein P3N-PIPO (for the protein encoded by the open reading frame [ORF] containing 59-terminus of P3 and a +2 frame-shift ORF called Pretty Interesting Potyviridae ORF and embedded in the P3) and CYLINDRICAL INCLUSION (CI) proteins were required for potyvirus cell-to-cell movement. Here, we provide genetic evidence showing that a Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) mutant carrying a truncated PIPO domain of 58 amino acid residues could move between cells and induce systemic infection in Nicotiana benthamiana plants; mutants carrying a PIPO domain of seven, 20, or 43 amino acid residues failed to move between cells and cause systemic infection in this host plant. Interestingly, the movementdefective 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 of N. benthamiana (referred to as NbDREPP) interacted with both P3N-PIPO and CI of the movement-competent TVBMV. The knockdown of NbDREPP gene expression in N. benthamiana impeded the cell-to-cell movement of TVBMV. NbDREPP was shown to colocalize with TVBMV P3N-PIPO and CI at plasmodesmata (PD) and traffic to PD via the early secretory pathway and the actomyosin motility system. We also show that myosin XI-2 is specially required for transporting NbDREPP to PD. In conclusion, NbDREPP is a key host protein within the early secretory pathway and the actomyosin motility system that interacts with two movement proteins and influences virus movement.

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BackgroundCross protection is a promising alternative to control plant viral diseases. One critical factor limiting the application of cross protection is the availability of attenuated mutants or mild strains. Potato virus X (PVX) infects many crops and induces huge economic losses to agricultural production. However, researches on the variability and mechanism of PVX virulence are scarce.MethodsThe mutants were obtained by introducing mutations into the RNA dependent RNA polymerase (RdRp) gene of PVX via site-directed mutagenesis. Attenuated mutants were screen according to their symptoms in Nicotiana benthamiana plants. The protection efficacy against severe infection were evaluated with interval of 5, 10 and 15 days.ResultsAmong the 40 mutants obtained, four mutants carrying substitutions of either Glu46, Asn863, Asn968 or Glu1001 to Ala in PVX RdRp showed drastically attenuated symptom, accompanying with reduced accumulation levels of coat protein, plus- and minus-sense RNAs. When the interval between protective and challenging inoculations was 15 days, mutant E1001A (with substitution of Glu1001 to Ala in RdRp) provided complete protection against severe infection in both Nicotiana benthamiana and tomato, while E46A (Glu46 mutated to Ala) provided incomplete protection. To reduce the risk of reverse mutation, we constructed mutant dM which carries double mutations of both Glu46 and Glu1001 to Ala in RdRp. The mutant dM could provide effective protection against severe PVX infection.ConclusionMutations of Glu46, Asn863, Asn968 or Glu1001 to Ala in PVX RdRp significantly reduced the viral symptoms. Mutants E1001A and E46A could provide effective protection against wild type PVX in both Nicotiana benthamiana and tomato. These results provide theoretical and practical bases for the control of PVX via cross protection.

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