Abstract
The coding sequence of P3N-PIPO was cloned by fusion PCR from Sugarcane mosaic virus (SCMV), a main causal agent of sugarcane (Saccharum spp. hybrid) mosaic disease. SCMV P3N-PIPO preferentially localized to the plasma membrane (PM) compared with the plasmodesmata (PD), as demonstrated by transient expression and plasmolysis assays in the leaf epidermal cells of Nicotiana benthamiana. The subcellular localization of the P3N-PIPO mutants P3N-PIPOT1 and P3N-PIPOT2 with 29 and 63 amino acids deleted from the C-terminus of PIPO, respectively, revealed that the 19 amino acids at the N-terminus of PIPO contributed to the PD localization. Interaction assays showed that the 63 amino acids at the C-terminus of PIPO determined the P3N-PIPO interaction with PM-associated Ca2+-binding protein 1, ScPCaP1, which was isolated from the SCMV-susceptible sugarcane cultivar Badila. Like wild-type P3N-PIPO, P3N-PIPOT1 and P3N-PIPOT2 could translocate to neighbouring cells and recruit the SCMV cylindrical inclusion protein to the PM. Thus, interactions with ScPCaP1 may contribute to, but not determine, SCMV Pm3N-PIPO’s localization to the PM or PD. These results also imply the existence of truncated P3N-PIPO in nature.
Highlights
PIPO, embedded within the P3 cistron, but translated from an RNA template generated by transcriptional slippage with a +1 A insertion in the motif GAAAAAA17–19
The P3 cistron was cloned from Sugarcane mosaic virus (SCMV) by RT-PCR, and its sequence was deposited into GenBank
When SCMV P3N-PIPO was co-expressed in N. benthamiana leaf cells with the plasma membrane (PM) localization protein CPK9-mRFP33, the green fluorescence of P3N-PIPO-green fluorescent protein (GFP) overlapped with the red fluorescence of CPK9-mRFP, indicating that SCMV P3N-PIPO localized to the PM (Fig. 1B)
Summary
PIPO, embedded within the P3 cistron, but translated from an RNA template generated by transcriptional slippage with a +1 A insertion in the motif GAAAAAA17–19. Among these 11 proteins, CP, CI, HC-Pro, VPg, 6K2 and P3N-PIPO are involved in viral cell-to-cell movement[20,21,22,23,24,25]. Introducing premature stop codons within pipo or mutations in the conserved G1–2A6–7 motif without altering the P3 amino acid sequence in the Soybean mosaic virus (SMV) genome restricted viral cell-to-cell movement[27]. The truncated P3N-PIPO with 63 amino acids deleted from the C-terminus of PIPO localized to PD, with some aggregates in the cytoplasm, independent of interactions with ScPCaP1. This truncated P3N-PIPO and the one with 29 amino acids deleted from the C-terminus of PIPO could move to neighbouring cells and recruited CIs to PM, like the wild-type SCMV P3N-PIPO
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