The p33 protein of Citrus tristeza virus affects viral pathogenicity by modulating a host immune response.

Accumulation of reactive oxygen species (ROS) is a general plant basal defense strategy against viruses. In this study, we show that infection by Citrus tristeza virus (CTV) triggered ROS burst in Nicotiana benthamiana and in the natural citrus host, the extent of which was virus-dose dependent. Using Agrobacterium-mediated expression of CTV-encoded proteins in N. benthamiana, we found that p33, a unique viral protein, contributed to the induction of ROS accumulation and programmed cell death. The role of p33 in CTV pathogenicity was assessed based on gene knockout and complementation in N. benthamiana. In the citrus-CTV pathosystem, deletion of the p33 open reading frame in a CTV variant resulted in a significant decrease in ROS production, compared to that of the wild type CTV, which correlated with invasion of the mutant virus into the immature xylem tracheid cells and abnormal differentiation of the vascular system. By contrast, the wild type CTV exhibited phloem-limited distribution with a minor effect on the vasculature. We conclude that the p33 protein is a CTV effector that negatively affects virus pathogenicity and suggest that N. benthamiana recognizes p33 to activate the host immune response to restrict CTV into the phloem tissue and minimize the disease syndrome.

The entry of cucumber mosaic virus into cucumber xylem is facilitated by co-infection with zucchini yellow mosaic virus.

We investigated the synergistic effects of co-infection by zucchini yellow mosaic virus (ZYMV) and cucumber mosaic virus (CMV) on viral distribution in the vascular tissues of cucumber. Immunohistochemical observations indicated that ZYMV was present in both the phloem and xylem tissues. ZYMV-RNA was detected in both the xylem wash and guttation fluid of ZYMV-inoculated cucumber. Steam treatment at a stem internode indicated that ZYMV enters the xylem vessels and moves through them but does not cause systemic infection in the plant. CMV distribution in singly infected cucumbers was restricted to phloem tissue. By contrast, CMV was detected in the xylem tissue of cotyledons in plants co-infected with CMV and ZYMV. Although both ZYMV-RNA and CMV-RNA were detected in the xylem wash and upper internodes of steam-treated, co-infected cucumbers grown at 24 °C, neither virus was detected in the upper leaves using an ELISA assay. Genetically modified CMV harboring the ZYMV HC-Pro gene was distributed in the xylem and phloem tissues of singly inoculated cucumber cotyledons. These results indicate that the ZYMV HC-Pro gene facilitates CMV entry into the xylem vessels of co-infected cucumbers.

Membrane-associated virus replication complexes locate to plant conducting tubes.

It is generally accepted that in order to establish a systemic infection in a plant, viruses move from the initially infected cell to the vascular tissues by cell-to-cell movement through plasmodesmata (PD), and load into the vascular conducting tubes (i.e. phloem sieve elements and xylem vessel elements) for long-distance movement. The viral unit in these movements can be a virion or a yet-to-be-defined ribonucleic protein (RNP) complex. Using live-cell imaging, our laboratory has previously demonstrated that membrane-bound replication complexes move cell-to-cell during turnip mosaic virus (TuMV) infection. Our recent study shows that these membrane-bound replication complexes end up in the vascular conducting tubes, which is likely the case for potato virus X (PVX) also. The presence of TuMV-induced membrane complexes in xylem vessels suggests that viral components could also be found in other apoplastic regions of the plant, such as the intercellular space. This possibility may have implications regarding how we approach the study of plant innate immune responses against viruses.

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.

Enhancement or attenuation of disease by deletion of genes from Citrus tristeza virus.

Stem pitting is a common virus-induced disease of perennial woody plants induced by a range of different viruses. The phenotype results from sporadic areas of the stem in which normal xylem and phloem development is prevented during growth of stems. These alterations interfere with carbohydrate transport, resulting in reduced plant growth and yield. Citrus tristeza virus (CTV), a phloem-limited closterovirus, induces economically important stem-pitting diseases of citrus. CTV has three nonconserved genes (p33, p18, and p13) that are not related to genes of other viruses and that are not required for systemic infection of some species of citrus, which allowed us to examine the effect of deletions of these genes on symptom phenotypes. In the most susceptible experimental host, Citrus macrophylla, the full-length virus causes only very mild stem-pitting symptoms. Surprisingly, we found that certain deletion combinations (p33 and p18 and/or p13) induced greatly increased stem-pitting symptoms, while other combinations (p13 or p13 plus p18) resulted in reduced stem pitting. These results suggest that the stem-pitting phenotype, which is one of more economically important disease phenotypes, can result not from a specific sequence or protein but from a balance between the expression of different viral genes. Unexpectedly, using green fluorescent protein-tagged full-length virus and deletion mutants (CTV9Δp33 and CTV9Δp33Δp18Δp13), the virus was found at pitted areas in abnormal locations outside the normal ring of phloem. Thus, increased stem pitting was associated not only with a prevention of xylem production but also with a proliferation of cells that supported viral replication, suggesting that at random areas of stems the virus can elicit changes in cellular differentiation and development.

Cocksfoot mottle sobemovirus establishes infection through the phloem.

Cocksfoot mottle virus (CfMV) localization in oat plants was analyzed during three weeks post infection by immunohistochemical staining to follow its spread through different tissues. In early stages of infection, the virus was first detectable in phloem parenchyma and bundle sheath cells of inoculated leaves. Bundle sheath and phloem parenchyma were also the cell types where the virus was first detected in stems and systemic leaves of infected plants. In later stages of infection, CfMV spread also into the mesophyll surrounding vascular bundles and was seldom detected in xylem parenchyma of inoculated leaves. In systemic leaves, CfMV was not detected from xylem. Moreover, sometimes it was found from phloem only. In straw and roots, CfMV was detected both from phloem and xylem. According to our observations, CfMV predominantly moves through phloem, which makes the systemic movement of CfMV different from that of another monocot-infecting sobemovirus, Rice yellow mottle virus (RYMV).