Turnip Mosaic Virus Components Are Released into the Extracellular Space by Vesicles in Infected Leaves.

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 6K2 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.

Turnip Mosaic Virus Uses the SNARE Protein VTI11 in an Unconventional Route for Replication Vesicle Trafficking.

Infection of plant cells by RNA viruses leads to the generation of organelle-like subcellular structures that contain the viral replication complex. During Turnip mosaic virus (TuMV) infection of Nicotiana benthamiana, the viral membrane protein 6K2 plays a key role in the release of motile replication vesicles from the host endoplasmic reticulum (ER). Here, we demonstrate that 6K2 contains a GxxxG motif within its predicted transmembrane domain that is vital for TuMV infection. Replacement of the Gly with Val within this motif inhibited virus production, and this was due to a relocation of the viral protein to the Golgi apparatus and the plasma membrane. This indicated that passage of 6K2 through the Golgi apparatus is a dead-end avenue for virus infection. Impairing the fusion of transport vesicles between the ER and the Golgi apparatus by overexpression of the SNARE Sec22 protein resulted in enhanced intercellular virus movement. Likewise, expression of nonfunctional, Golgi-located synaptotagmin during infection enhanced TuMV intercellular movement. 6K2 copurified with VTI11, a prevacuolar compartment SNARE protein. An Arabidopsis thaliana vti11 mutant was completely resistant to TuMV infection. We conclude that TuMV replication vesicles bypass the Golgi apparatus and take an unconventional pathway that may involve prevacuolar compartments/multivesicular bodies for virus infection.

Identification of host factors potentially involved in RTM-mediated resistance during potyvirus long distance movement.

The long distance movement of potyviruses is a poorly understood step of the viral cycle. Only factors inhibiting this process, referred to as "Restricted TEV Movement" (RTM), have been identified in Arabidopsis thaliana. On the virus side, the potyvirus coat protein (CP) displays determinants required for long-distance movement and for RTM-based resistance breaking. However, the potyvirus CP was previously shown not to interact with the RTM proteins. We undertook the identification of Arabidopsis factors which directly interact with either the RTM proteins or the CP of lettuce mosaic virus (LMV). An Arabidopsis cDNA library generated from companion cells was screened with LMV CP and RTM proteins using the yeast two-hybrid system. Fourteen interacting proteins were identified. Two of them were shown to interact with CP and the RTM proteins suggesting that a multiprotein complex could be formed between the RTM proteins and virions or viral ribonucleoprotein complexes. Co-localization experiments in Nicotiana benthamiana showed that most of the viral and cellular protein pairs co-localized at the periphery of chloroplasts which suggests a putative role for plastids in this process.

Turnip mosaic virus moves systemically through both phloem and xylem as membrane-associated complexes.

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.