Tobamoviral Movement Protein Transiently Expressed in a Single Epidermal Cell Functions Beyond Multiple Plasmodesmata and Spreads Multicellularly in an Infection-Coupled Manner
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Abstract:
Cell-to-cell movement of a plant virus requires expression of the movement protein (MP). It has not been fully elucidated, however, how the MP functions in primary infected cells. With the use of a microprojectile bombardment-mediated DNA infection system for Tomato mosaic virus (ToMV), we found that the cotransfected ToMV MP gene exerts its effects in the initially infected cells and in their surrounding cells to achieve multicellular spread of movement-defective ToMV. Five other tobamoviral MPs examined also transcomplemented the movement-defective phenotype of ToMV, but the Cucumber mosaic virus 3a MP did not. Together with the cell-to-cell movement of the mutant virus, a fusion between the MP and an enhanced green fluorescent protein variant (EGFP) expressed in trans was distributed multicellularly and localized primarily in plasmodesmata between infected cells. In contrast, in noninfected sites the MP-EGFP fusion accumulated predominantly inside the bombarded cells as irregularly shaped aggregates, and only a minute amount of the fusion was found in plasmodesmata. Thus, the behavior of ToMV MP is greatly modulated in the presence of a replicating virus and it is highly likely that the MP spreads in the infection sites, coordinating with the cell-to-cell movement of the viral genome.Keywords:
Plasmodesma
Movement protein
Tobamovirus
Cell fusion
Movement protein
Plasmodesma
Aequorea victoria
Tobamovirus
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Summary Previous micro‐injection studies showed that some recombinant viral movement proteins and plant proteins produced in and purified from Escherichia coli could traffic from cell to cell. However, the relevance of these findings obtained by micro‐injecting proteins produced in E. coli to the real functions of these proteins when produced in planta has been questioned. In this study, specific gene constructs were delivered by biolistic bombardment into tobacco ( Nicotiana tabacum var Samsun) leaf epidermis for in planta production of the green fluorescent protein (GFP) and various fusions between the cucumber mosaic virus 3a movement protein (3a MP) and GFP. Free GFP remained in cells producing it. In contrast, 3a MP:GFP fusion protein moved from approximately half of the cells producing it into neighboring cells. The movement also occurred at 4°C. A mutant 3a MP:GFP was incapable of cell‐to‐cell movement in all cases. A 3a MP:GUS fusion protein produced in this manner also moved from cell to cell. Our data provide direct evidence that specific viral proteins produced in planta can be transported between cells. Furthermore, our data suggest that the CMV 3a MP contains a signal for transport. Our approach is simple and efficient and has many potential applications in studying plasmodesma‐mediated macromolecular transport.
Movement protein
Plant cell
Epidermis (zoology)
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Tobamovirus
Protoplast
Movement protein
Potato virus X
Strain (injury)
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A chimeric gene encoding a dysfunctional tobacco mosaic virus (TMV) movement protein (MP) mutant lacking amino acids 3, 4 and 5 (MPΔ3–5), was expressed in transgenic Nicotiana tabacum Xanthi and Xanthi NN plants. Immunogold labeling studies of tissues from transgenic plants indicated that while wild‐type MP accumulated in the plasmodesmata, MPΔ3–5 did not. Tissue fractionation studies confirmed that only a low level of the mutant MP accumulated in the cell wall‐enriched fraction compared with the accumulation of the wild‐type MP. Dye coupling studies showed that MPΔ3–5 enabled the movement between leaf mesophyll cells of a fluorescently labeled dextran of 3 kDa, while 9.4 kDa molecules failed to move. In contrast, in transgenic plants expressing the wild‐type MP gene the 9.4 kDa probe did move from cell to cell. Seedlings from self‐fertilized transgenic plants were inoculated with TMV and observed for disease symptoms. Transgenic Xanthi NN plants that expressed the MPΔ3–5 gene developed fewer and smaller necrotic local lesions compared with control plants following inoculation with TMV. Transgenic Xanthi nn plants were delayed in the development of systemic symptoms. Inoculating the transgenic plants with TMV‐RNA, and the tobamo‐viruses TMGMV and SHMV, essentially produced the same results, i.e. inhibition of disease development. These results demonstrate that transgenic plants expressing an inactive MP can inhibit virus disease spread presumably by interfering with its cell‐to‐cell movement.
Movement protein
Plasmodesma
Tobamovirus
Wild type
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Abstract The tomato Tm‐2 2 gene was considered to be one of the most durable resistance genes in agriculture, protecting against viruses of the Tobamovirus genus, such as tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV). However, an emerging tobamovirus, tomato brown rugose fruit virus (ToBRFV), has overcome Tm‐2 2 , damaging tomato production worldwide. Tm‐2 2 encodes a nucleotide‐binding leucine‐rich repeat (NLR) class immune receptor that recognizes its effector, the tobamovirus movement protein (MP). Previously, we found that ToBRFV MP (MP ToBRFV ) enabled the virus to overcome Tm‐2 2 ‐mediated resistance. Yet, it was unknown how Tm‐2 2 remained durable against other tobamoviruses, such as TMV and ToMV, for over 60 years. Here, we show that a conserved cysteine (C68) in the MP of TMV (MP TMV ) plays a dual role in Tm‐2 2 activation and viral movement. Substitution of MP ToBRFV amino acid H67 with the corresponding amino acid in MP TMV (C68) activated Tm‐2 2 ‐mediated resistance. However, replacement of C68 in TMV and ToMV disabled the infectivity of both viruses. Phylogenetic and structural prediction analysis revealed that C68 is conserved among all Solanaceae‐infecting tobamoviruses except ToBRFV and localizes to a predicted jelly‐roll fold common to various MPs. Cell‐to‐cell and subcellular movement analysis showed that C68 is required for the movement of TMV by regulating the MP interaction with the endoplasmic reticulum and targeting it to plasmodesmata. The dual role of C68 in viral movement and Tm‐2 2 immune activation could explain how TMV was unable to overcome this resistance for such a long period.
Tobamovirus
Movement protein
Plasmodesma
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ABSTRACT The tomato Tm-2 2 gene was considered one of the most durable resistance genes in agriculture, protecting against viruses of the Tobamovirus genus, such as Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV). However, an emerging tobamovirus, Tomato brown rugose fruit virus (ToBRFV), has overcome Tm-2 2 , damaging tomato production worldwide. Tm-2 2 encodes a nucleotide-binding leucine-rich repeat (NLR) class immune receptor that recognizes its effector, the tobamovirus movement protein (MP). Previously, we found that ToBRFV MP (MP ToBRFV ) enabled the virus to overcome Tm-2 2 - mediated resistance. Yet, it was unknown how Tm-2 2 remained durable against other tobamoviruses, such as TMV and ToMV, for over 60 years. Here, we show that the presence of a conserved cysteine (C68) in the MP of TMV (MP TMV ) is both sufficient to trigger Tm-2 2 resistance and essential for viral movement. Substitution of MP ToBRFV amino acid H67 with the corresponding amino acid in MP TMV (C68) activated Tm-2 2 -medited resistance. However, replacement of C68 in TMV and ToMV disabled the infectivity of both viruses. Phylogenetic and structural prediction analysis revealed that C68 is conserved among all Solanaceae -infecting tobamoviruses except ToBRFV, and localizes to a predicted jelly-roll fold common to various MPs. Cell-to-cell, and subcellular movement analysis showed that C68 is required for the movement of TMV, by regulating the MP interaction with the endoplasmic reticulum and targeting it to plasmodesmata. The dual role of C68 in viral movement and Tm-2 2 immune activation could explain how TMV was unable to overcome this resistance for such a long period.
Tobamovirus
Movement protein
Plasmodesma
Mosaic virus
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Citations (1)
Plasmodesma
Movement protein
Plant cell
Tobamovirus
Intracellular transport
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Citations (19)
Cucumber mosaic virus (CMV, Cucumovirus, Bromoviridae) is an economically significant virus infecting important horticultural and field crops. Current knowledge regarding the specific functions of its movement protein (MP) is still incomplete. In the present study, potential post-translational modification sites of its MP were assayed with mutant viruses: MP/S28A, MP/S28D, MP/S120A and MP/S120D. Ser28 was identified as an important factor in viral pathogenicity on Nicotiana tabacum cv. Xanthi, Cucumis sativus and Chenopodium murale. The subcellular localization of GFP-tagged movement proteins was determined with confocal laser-scanning microscopy. The wild type movement protein fused to green fluorescent protein (GFP) (MP-eGFP) greatly colocalized with callose at plasmodesmata, while MP/S28A-eGFP and MP/S28D-eGFP were detected as punctate spots along the cell membrane without callose colocalization. These results underline the importance of phosphorylatable amino acids in symptom formation and provide data regarding the essential factors for plasmodesmata localization of CMV MP.
Plasmodesma
Movement protein
Cucumovirus
Cucumis
Potato virus X
Callose
Tobamovirus
Colocalization
Potato virus Y
Turnip mosaic virus
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Macromolecule and cytosolic signal distribution throughout the plant employs a unique cellular and intracellular mechanism called plasmodesmata (PD). Plant viruses spread throughout plants via PD using their movement proteins (MPs). Viral MPs induce changes in plasmodesmata’s structure and alter their ability to move macromolecule and cytosolic signals. The developmental distribution of a family member of proteins termed plasmodesmata located proteins number 5 (PDLP5) conjugated to GFP (PDLP5-GFP) is described here. The GFP enables the visual localization of PDLP5 in the cell via confocal microscopy. We observed that PDLP5-GFP protein is present in seed protein bodies and immediately after seed imbibition in the plasma membrane. The effect of three different plant viruses, the tobacco mosaic virus (TMV), tomato brown rugose fruit virus (ToBRFV, tobamoviruses), and tomato yellow leaf curl virus (TYLCV, begomoviruses), on PDLP5-GFP accumulation at the plasmodesmata was tested. In tobacco leaf, TMV and ToBRFV increased PDLP5-GFP amount at the plasmodesmata of cell types compared to control. However, there was no statistically significant difference in tomato leaf. On the other hand, TYLCV decreased PDLP5-GFP quantity in plasmodesmata in all tomato leaf cells compared to control, without any significant effect on plasmodesmata in tobacco leaf cells.
Plasmodesma
Movement protein
Plant cell
Tobamovirus
Cowpea mosaic virus
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Movement protein
Plasmodesma
Potato virus X
Tobamovirus
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Citations (78)