The potyviral protein 6K1 reduces plant protease activity during Turnip mosaic virus infection

Potyviral genomes encode just 11 major proteins and multifunctionality is associated to most of these proteins at different stages of virus life cycle. The potyviral protein 6K1 is required for potyvirus replication at the early stages of viral infection and may mediate cell-to-cell movement at later stages.Our study demonstrates that the 6K1 protein from Turnip mosaic virus (TuMV) reduces the abundance of transcripts related to jasmonic acid biosynthesis and transcripts that encode cysteine protease inhibitors when expressed in trans in Nicotiana benthamiana relative to controls. Furthermore, 6K1 stability increases when lipoxygenase and cysteine protease activity is inhibited chemically, linking a mechanism to the rapid turnover of 6K1 when expressed in trans. Using transient expression, we show 6K1 is degraded rapidly at early time points in the infection process, whereas at later stages of infection protease activity is reduced and 6K1 becomes more stable, resulting in higher TuMV accumulation in systemic leaves. There was no impact of 6K1 transient expression on TuMV accumulation in local leaves. Together, these results suggest a novel function for the TuMV 6K1 protein which has not been reported previously and enhances our understanding of the complex interactions occurring between plants and potyviruses.

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Light Quantity and Photosystem Function Mediate Host Susceptibility to Turnip mosaic virus Via a Salicylic Acid–Independent Mechanism

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-10-0191 ◽

2011 ◽

Vol 24 (3) ◽

pp. 315-327 ◽

Cited By ~ 24

Author(s):

A. Manfre ◽

M. Glenn ◽

A. Nuñez ◽

R. A. Moreau ◽

C. Dardick

Keyword(s):

Salicylic Acid ◽

Mosaic Virus ◽

Cell Movement ◽

Time Lapse ◽

Defense Responses ◽

Turnip Mosaic Virus ◽

Host Susceptibility ◽

Systemic Movement ◽

Underlying Mechanisms ◽

Light Deficiency

Evidence going as far back as the early part of the 20th century suggests that both light and chloroplast function may play key roles in host susceptibility to viruses. Despite the long history of such work, confirmation of these phenomena and a determination of the underlying mechanisms remain elusive. Here, we revisited these questions using modern imaging technologies to study the susceptibility of Nicotiana benthamiana to Turnip mosaic virus (TuMV). We found that both light deficiency and photosystem impairment increased the susceptibility of N. benthamiana to TuMV infection. Time-lapse photography studies indicated that, under these conditions, rub-inoculated plants exhibited greater numbers of infection foci and more rapid foci development. The rate of systemic movement was also accelerated though cell-to-cell movement appeared unchanged. Inhibition of salicylic acid (SA)-mediated defense responses is not likely responsible for changes in susceptibility because SA and pathogen response-1 gene induction were not affected by light deficiency or chloroplast impairment and treatment of plants with SA had no measureable impact on TuMV infection. Taken together, these data suggest that both light and optimal chloroplast function influence virus infection either by limiting the cellular resources needed by TuMV to establish replication complexes or the host's ability to activate SA-independent defenses.

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The Vesicle-Forming 6K2Protein of Turnip Mosaic Virus Interacts with the COPII Coatomer Sec24a for Viral Systemic Infection

Journal of Virology ◽

10.1128/jvi.00503-15 ◽

2015 ◽

Vol 89 (13) ◽

pp. 6695-6710 ◽

Cited By ~ 37

Author(s):

Jun Jiang ◽

Camilo Patarroyo ◽

Daniel Garcia Cabanillas ◽

Huanquan Zheng ◽

Jean-François Laliberté

Keyword(s):

Arabidopsis Thaliana ◽

Endoplasmic Reticulum ◽

Mosaic Virus ◽

Viral Protein ◽

Cell Movement ◽

Systemic Infection ◽

Turnip Mosaic Virus ◽

Tryptophan Residue ◽

Content Type ◽

Er Export

ABSTRACTPositive-sense RNA viruses remodel host cell endomembranes to generate quasi-organelles known as “viral factories” to coordinate diverse viral processes, such as genome translation and replication. It is also becoming clear that enclosing viral RNA (vRNA) complexes within membranous structures is important for virus cell-to-cell spread throughout the host. In plant cells infected by turnip mosaic virus (TuMV), a member of the familyPotyviridae, peripheral motile endoplasmic reticulum (ER)-derived viral vesicles are produced that carry the vRNA to plasmodesmata for delivery into adjacent noninfected cells. The viral protein 6K2is responsible for the formation of these vesicles, but how 6K2is involved in their biogenesis is unknown. We show here that 6K2is associated with cellular membranes. Deletion mapping and site-directed mutagenesis experiments defined a soluble N-terminal 12-amino-acid stretch, in particular a potyviral highly conserved tryptophan residue and two lysine residues that were important for vesicle formation. When the tryptophan residue was changed into an alanine in the viral polyprotein, virus replication still took place, albeit at a reduced level, but cell-to-cell movement of the virus was abolished. Yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitation experiments showed that 6K2interacted with Sec24a, a COPII coatomer component. Appropriately, TuMV systemic movement was delayed in anArabidopsis thalianamutant line defective in Sec24a. Intercellular movement of TuMV replication vesicles thus requires ER export of 6K2, which is mediated by the interaction of the N-terminal domain of the viral protein with Sec24a.IMPORTANCEMany plant viruses remodel the endoplasmic reticulum (ER) to generate vesicles that are associated with the virus replication complex. The viral protein 6K2of turnip mosaic virus (TuMV) is known to induce ER-derived vesicles that contain vRNA as well as viral and host proteins required for vRNA synthesis. These vesicles not only sustain vRNA synthesis, they are also involved in the intercellular trafficking of vRNA. In this investigation, we found that the N-terminal soluble domain of 6K2is required for ER export of the protein and for the formation of vesicles. ER export is not absolutely required for vRNA replication but is necessary for virus cell-to-cell movement. Furthermore, we found that 6K2physically interacts with the COPII coatomer Sec24a and that anArabidopsis thalianamutant line with a defective Sec24a shows a delay in the systemic infection by TuMV.

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Ultrastructural Characterization of Turnip Mosaic Virus-Induced Cellular Rearrangements Reveals Membrane-Bound Viral Particles Accumulating in Vacuoles

Journal of Virology ◽

10.1128/jvi.02138-15 ◽

2015 ◽

Vol 89 (24) ◽

pp. 12441-12456 ◽

Cited By ~ 18

Author(s):

Juan Wan ◽

Kaustuv Basu ◽

Jeannie Mui ◽

Hojatollah Vali ◽

Huanquan Zheng ◽

...

Keyword(s):

Mosaic Virus ◽

Viral Particle ◽

Rna Replication ◽

Turnip Mosaic Virus ◽

Infection Process ◽

Viral Rna ◽

Particle Assembly ◽

Induced Membrane ◽

Positive Strand Rna ◽

Viral Particle Assembly

ABSTRACTPositive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.IMPORTANCEPositive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The ultrastructure of turnip mosaic virus (TuMV)-induced membrane remodeling was investigated over several days of infection. The first change that was observed involved endoplasmic reticulum-connected convoluted membrane accumulation. This was followed by the formation of single-membrane tubules, which were shown to be viral RNA replication sites. Later in the infection process, double-membrane tubular structures were observed and were associated with viral particle bundles. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. This work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation.

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P3N-PIPO Interacts with P3 via the Shared N-Terminal Domain To Recruit Viral Replication Vesicles for Cell-to-Cell Movement

Journal of Virology ◽

10.1128/jvi.01898-19 ◽

2020 ◽

Vol 94 (8) ◽

Cited By ~ 6

Author(s):

Mengzhu Chai ◽

Xiaoyun Wu ◽

Jiahui Liu ◽

Yue Fang ◽

Yameng Luan ◽

...

Keyword(s):

Viral Replication ◽

Mosaic Virus ◽

Essential Role ◽

Movement Protein ◽

Cell Movement ◽

Plant Viruses ◽

Turnip Mosaic Virus ◽

Limited Information ◽

Intercellular Movement ◽

Model Virus

ABSTRACT 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.

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