Dataset Information

Dissecting the Molecular Mechanism of the Subcellular Localization and Cell-to-cell Movement of the Sugarcane mosaic virus P3N-PIPO.

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.

SUBMITTER: Cheng G

PROVIDER: S-EPMC5575073 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Dissecting the Molecular Mechanism of the Subcellular Localization and Cell-to-cell Movement of the Sugarcane mosaic virus P3N-PIPO.

Cheng Guangyuan G Dong Meng M Xu Qian Q Peng Lei L Yang Zongtao Z Wei Taiyun T Xu Jingsheng J

Scientific reports 20170829 1

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 delete ...[more]

PMID: 28852157

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Project description:P3N-PIPO, the only dedicated movement protein (MP) of potyviruses, directs cylindrical inclusion (CI) protein from the cytoplasm to the plasmodesma (PD), where CI forms conical structures for intercellular movement. To better understand potyviral cell-to-cell movement, we further characterized P3N-PIPO using Turnip mosaic virus (TuMV) as a model virus. We found that P3N-PIPO interacts with P3 via the shared P3N domain and that TuMV mutants lacking the P3N domain of either P3N-PIPO or P3 are defective in cell-to-cell movement. Moreover, we found that the PIPO domain of P3N-PIPO is sufficient to direct CI to the PD, whereas the P3N domain is necessary for localization of P3N-PIPO to 6K2-labeled vesicles or aggregates. Finally, we discovered that the interaction between P3 and P3N-PIPO is essential for the recruitment of CI to cytoplasmic 6K2-containing structures and the association of 6K2-containing structures with PD-located CI inclusions. These data suggest that both P3N and PIPO domains are indispensable for potyviral cell-to-cell movement and that the 6K2 vesicles in proximity to PDs resulting from multipartite interactions among 6K2, P3, P3N-PIPO, and CI may also play an essential role in this process.IMPORTANCE Potyviruses include numerous economically important viruses that represent approximately 30% of known plant viruses. However, there is still limited information about the mechanism of potyviral cell-to-cell movement. Here, we show that P3N-PIPO interacts with and recruits CI to the PD via the PIPO domain and interacts with P3 via the shared P3N domain. We further report that the interaction of P3N-PIPO and P3 is associated with 6K2 vesicles and brings the 6K2 vesicles into proximity with PD-located CI structures. These results support the notion that the replication and cell-to-cell movement of potyviruses are processes coupled by anchoring viral replication complexes at the entrance of PDs, which greatly increase our knowledge of the intercellular movement of potyviruses.

Project description:Barley stripe mosaic virus (BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used Agrobacterium tumefaciens-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.

Dataset Information

Dissecting the Molecular Mechanism of the Subcellular Localization and Cell-to-cell Movement of the Sugarcane mosaic virus P3N-PIPO.

Publications

Dissecting the Molecular Mechanism of the Subcellular Localization and Cell-to-cell Movement of the Sugarcane mosaic virus P3N-PIPO.

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