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Turnip mosaic virus (TuMV) reorganizes the endomembrane system of the infected cell to generate endoplasmic-reticulum-derived motile vesicles containing viral replication complexes. The membrane-associated viral protein 6K 2 plays a key role in the formation of these vesicles. Using confocal microscopy, we observed that this viral protein, a marker for viral replication complexes, localized in the extracellular space of infected Nicotiana benthamiana leaves. Previously, we showed that viral RNA is associated with multivesicular bodies (MVBs). Here, using transmission electron microscopy, we observed the proliferation of MVBs during infection and their fusion with the plasma membrane that resulted in the release of their intraluminal vesicles in the extracellular space. Immunogold labeling with a monoclonal antibody that recognizes double-stranded RNA indicated that the released vesicles contained viral RNA. Focused ion beam-extreme high-resolution scanning electron microscopy was used to generate a three-dimensional image that showed extracellular vesicles in the cell wall. The presence of TuMV proteins in the extracellular space was confirmed by proteomic analysis of purified extracellular vesicles from N benthamiana and Arabidopsis ( Arabidopsis thaliana ). Host proteins involved in biotic defense and in interorganelle vesicular exchange were also detected. The association of extracellular vesicles with viral proteins and RNA emphasizes the implication of the plant extracellular space in viral infection., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Like other positive-strand RNA viruses, the Turnip mosaic virus (TuMV) infection leads to the formation of viral vesicles at the endoplasmic reticulum (ER). Once released from the ER, the viral vesicles mature intracellularly and then move intercellularly. While it is known that the membrane-associated viral protein 6K2 plays a role in the process, the contribution of host proteins has been poorly defined. In this article, we show that 6K2 interacts with RHD3, an ER fusogen required for efficient ER fusion. When RHD3 is mutated, a delay in the development of TuMV infection is observed. We found that the replication of TuMV and the cell-to-cell movement of its replication vesicles are impaired in rhd3 This defect can be tracked to a delayed maturation of the viral vesicles from the replication incompetent to the competent state. Furthermore, 6K2 can relocate RHD3 from the ER to viral vesicles. However, a Golgi-localized mutated 6K2 GV is unable to interact and relocate RHD3 to viral vesicles. We conclude that the maturation of TuMV replication vesicles requires RHD3 for efficient viral replication and movement., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Plant viruses move from the initially infected cell to adjacent cells through plasmodesmata (PDs). To do so, viruses encode dedicated protein(s) that facilitate this process. How viral proteins act together to support the intercellular movement of viruses is poorly defined. Here, by using an infection-free intercellular vesicle movement assay, we investigate the action of CI (cylindrical inclusion) and P3N-PIPO (amino-terminal half of P3 fused to Pretty Interesting Potyviridae open reading frame), the two PD-localized potyviral proteins encoded by Turnip mosaic virus (TuMV), in the intercellular movement of the viral replication vesicles. We provide evidence that CI and P3N-PIPO are sufficient to support the PD targeting and intercellular movement of TuMV replication vesicles induced by 6K2, a viral protein responsible for the generation of replication vesicles. 6K2 interacts with CI but not P3N-PIPO. When this interaction is impaired, the intercellular movement of TuMV replication vesicles is inhibited. Furthermore, in transmission electron microscopy, vesicular structures are observed in connection with the cylindrical inclusion bodies at structurally modified PDs in cells coexpressing 6K2, CI, and P3N-PIPO. CI is directed to PDs through its interaction with P3N-PIPO. We hypothesize that CI serves as a docking point for PD targeting and the intercellular movement of TuMV replication vesicles. This work contributes to a better understanding of the roles of different viral proteins in coordinating the intercellular movement of viral replication vesicles., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Plant viruses move systemically in plants through the phloem. They move as virions or as ribonucleic protein complexes, although it is not clear what these complexes are made of. The approximately 10-kb RNA genome of Turnip mosaic virus (TuMV) encodes a membrane protein, known as 6K2, that induces endomembrane rearrangements for the formation of viral replication factories. These factories take the form of vesicles that contain viral RNA (vRNA) and viral replication proteins. In this study, we report the presence of 6K2-tagged vesicles containing vRNA and the vRNA-dependent RNA polymerase in phloem sieve elements and in xylem vessels. Transmission electron microscopy observations showed the presence in the xylem vessels of vRNA-containing vesicles that were associated with viral particles. Stem-girdling experiments, which leave xylem vessels intact but destroy the surrounding tissues, confirmed that TuMV could establish a systemic infection of the plant by going through xylem vessels. Phloem sieve elements and xylem vessels from Potato virus X-infected plants also contained lipid-associated nonencapsidated vRNA, indicating that the presence of membrane-associated ribonucleic protein complexes in the phloem and xylem may not be limited to TuMV. Collectively, these studies indicate that viral replication factories could end up in the phloem and the xylem., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein involved in viral genome translation and replication. Previous studies have shown that both eukaryotic translation initiation factor 4E (eIF4E) and eIF4G or their respective isoforms from the eIF4F complex, which modulates the initiation of protein translation, selectively interact with VPg and are required for potyvirus infection. Here, we report the identification of two DEAD-box RNA helicase-like proteins, PpDDXL and AtRH8 from peach (Prunus persica) and Arabidopsis (Arabidopsis thaliana), respectively, both interacting with VPg. We show that AtRH8 is dispensable for plant growth and development but necessary for potyvirus infection. In potyvirus-infected Nicotiana benthamiana leaf tissues, AtRH8 colocalizes with the chloroplast-bound virus accumulation vesicles, suggesting a possible role of AtRH8 in viral genome translation and replication. Deletion analyses of AtRH8 have identified the VPg-binding region. Comparison of this region and the corresponding region of PpDDXL suggests that they are highly conserved and share the same secondary structure. Moreover, overexpression of the VPg-binding region from either AtRH8 or PpDDXL suppresses potyvirus accumulation in infected N. benthamiana leaf tissues. Taken together, these data demonstrate that AtRH8, interacting with VPg, is a host factor required for the potyvirus infection process and that both AtRH8 and PpDDXL may be manipulated for the development of genetic resistance against potyvirus infections.

The initiation of the reproductive phase in winter cereals is delayed during winter until favorable growth conditions resume in the spring. This delay is modulated by low temperature through the process of vernalization. The molecular and genetic bases of the interaction between environmental factors and the floral transition in these species are still unknown. However, the recent identification of the wheat (Triticum aestivum L.) TaVRT-1 gene provides an opportunity to decipher the molecular basis of the flowering-time regulation in cereals. Here, we describe the characterization of another gene, named TaVRT-2, possibly involved in the flowering pathway in wheat. Molecular and phylogenetic analyses indicate that the gene encodes a member of the MADS-box transcription factor family that belongs to a clade responsible for flowering repression in several species. Expression profiling of TaVRT-2 in near-isogenic lines and different genotypes with natural variation in their response to vernalization and photoperiod showed a strong relationship with floral transition. Its expression is up-regulated in the winter genotypes during the vegetative phase and in photoperiod-sensitive genotypes during short days, and is repressed by vernalization to a level that allows the transition to the reproductive phase. Protein-protein interaction studies revealed that TaVRT-2 interacts with proteins encoded by two important vernalization genes (TaVRT-1/VRN-1 and VRN-2) in wheat. These results support the hypothesis that TaVRT-2 is a putative repressor of the floral transition in wheat.

Positive-sense single-stranded RNA viruses have developed strategies to exploit cellular resources at the expense of host mRNAs. The genomes of these viruses display a variety of structures at their 5' and 3' ends that differentiate them from cellular mRNAs. Despite this structural diversity, viral RNAs are still circularized by juxtaposition of their 5' and 3' ends, similar to the process used by cellular mRNAs. Also reminiscent of the mechanisms used by host mRNAs, translation of viral RNAs involves the recruitment of translation initiation factors. However, the roles played by these factors likely differ from those played by cellular mRNAs. In keeping with the general parsimony typical of RNA viruses, these host factors also participate in viral RNA replication. However, the dual use of host factors requires that viral RNA template utilization be regulated to avoid conflict between replication and translation. The molecular composition of the large ribonucleoprotein complexes that form the viral RNA replication and translation machineries likely evolves over the course of infection to allow for switching template use from translation to replication.

We have isolated, sequenced, and expressed a cold-specific cDNA clone, Wcs120, that specifically hybridizes to a major mRNA species of approximately 1650 nucleotides from cold-acclimated wheat (Triticum aestivum L.). The accumulation of this mRNA was induced in less than 24 hours of cold treatment, and remained at a high steady-state level during the entire period of cold acclimation in the two freezing-tolerant genotypes of wheat tested. The expression of Wcs120 was transient in a less-tolerant genotype even though the genomic organization of the Wcs120 and the relative copy number were the same in the three genotypes. The mRNA level decreased rapidly during deacclimation and was not induced by heat shock, drought, or abscisic acid. The Wcs120 cDNA contains a long open reading frame encoding a protein of 390 amino acids. The encoded protein is boiling stable, highly hydrophilic, and has a compositional bias for glycine (26.7%), threonine (16.7%), and histidine (10.8%), although cysteine, phenylalanine, and tryptophan were absent. The WCS120 protein contains two repeated domains. Domain A has the consensus amino acid sequence GEKKGVMENIKEKLPGGHGDHQQ, which is repeated 6 times, whereas domain B has the sequence TGGTYGQQGHTGTT, which is repeated 11 times. The two domains were also found in barley dehydrins and rice abscisic acid-induced protein families. The expression of this cDNA in Escherichia coli, using the T(7) RNA polymerase promoter, produced a protein of 50 kilodaltons with an isoelectric point of 7.3, and this product comigrated with a major protein synthesized in vivo and in vitro during cold acclimation.