The research, led by Guowei Yang, Yinlu Deng, and colleagues at Zhejiang University, focused on two cytorhabdoviruses: rice stripe mosaic virus (RSMV), a pathogen of rice in the genus Betacytorhabdovirus, and tomato yellow mottle-associated virus (TYMaV), an Alphacytorhabdovirus studied in the model plant Nicotiana benthamiana. Plant rhabdoviruses are a substantial group, comprising at least 269 species across 12 genera in the family Rhabdoviridae. Most produce bullet-shaped or bacilliform virions wrapped in a lipid envelope and are transmitted by sap-feeding hemipteran insects such as leafhoppers, planthoppers, aphids, and whiteflies in a persistent-propagative manner. Their genomes encode five conserved structural proteins—nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and the large polymerase (L)—plus a movement protein belonging to the 30K superfamily.

During replication, the viral RNA is encapsidated by N in association with P and L to form nucleocapsids, which previous work has established as the minimal infectious units for cell-to-cell movement. The movement protein interacts with N and P to redirect these nucleocapsids through plasmodesmata. Separately, the M protein condenses nucleocapsids into tightly coiled complexes and drives budding through G-enriched host endomembranes, producing enveloped particles that accumulate in the lumen of the nuclear envelope in nucleorhabdoviruses or the endoplasmic reticulum in cytorhabdoviruses. Whether these endomembrane-trapped virions are simply terminal assembly products or active participants in vascular trafficking has been a long-standing open question, particularly because plant rhabdovirus particles have never been observed within or around desmotubules, the ER-derived tubes that thread through plasmodesmata.

To address this, the team first mapped the spatial distribution of RSMV in infected rice using immunofluorescence staining of cryosections with an N protein-specific monoclonal antibody. In both leaf and stem sections, viral antigens were predominantly localized to vascular tissues, including xylem and phloem-associated regions, surrounding parenchyma and bundle sheath cells, and, in leaves, hypodermal sclerenchyma. Strong fluorescence appeared in infected samples while healthy controls showed none, confirming assay specificity. However, immunofluorescence could not resolve the structural form of the virus at these sites, so the researchers turned to transmission electron microscopy (TEM) on ultrathin resin-embedded sections.

The TEM results were striking. Mature enveloped RSMV virions, roughly 50 to 60 nanometers in diameter, accumulated abundantly in xylem parenchyma cells surrounding mature vessel elements, often arranged in orderly arrays within enclosing vesicle-like membranes that occupied a substantial portion of the cytoplasmic space. More remarkably, bacilliform particles with or without an enclosing membrane were detected within the lumens of mature xylem vessels themselves—hollow, dead conduits that have undergone programmed cell death and lost all cytoplasmic contents. Such particles appeared in 5 of 46 vessel-containing fields examined. Because fixing and detecting material inside these large lumens is technically difficult, the observation is all the more significant. The team also found membrane-enclosed mature virions in sclerenchyma cells that retain little cytoplasm, suggesting that RSMV particles can persist through advanced stages of cellular autolysis.

To corroborate these ultrastructural findings, the researchers collected xylem sap from the stems and roots of infected rice plants using vacuum infiltration and centrifugation, taking care to exclude cytoplasmic contamination with a glucose-6-phosphate dehydrogenase activity assay. Immunoblotting with antisera raised against purified RSMV virions detected four major protein bands in stem sap whose migration patterns matched the N, P, M, and G structural proteins of purified virions; root sap showed distinct G and M signals. Negative-stain TEM of concentrated sap then revealed bacilliform particles approximately 65 nanometers in diameter with morphology characteristic of plant rhabdovirus virions, the slightly larger apparent size reflecting differences in sample preparation and staining. Together, these data demonstrate that intact enveloped virions, not just soluble proteins or nucleocapsids, are present in the xylem translocation stream.

The phloem told a parallel story. In infected rice stems, the team identified sieve element–companion cell complexes containing virus particles, using sieve element plastids and pore-plasmodesmal units as diagnostic landmarks. Bacilliform particles consistent with mature enveloped RSMV virions were observed within both companion cells and sieve elements, with most virions in sieve elements occurring in clusters enclosed by vesicle-like membranes. Virions were found in 8 of 62 sieve element-containing fields. The same pattern held in the second pathosystem: in TYMaV-infected N. benthamiana, enveloped virions were detected in immature vessel elements that still retained residual cytoplasm, in fully differentiated vessel elements with patterned wall thickening and no protoplasmic contents, and in phloem sieve elements with or without an evident surrounding vesicle. Across the observations, enveloped virions appeared in at least 3 immature vessels, 7 mature vessels, and 6 sieve element-containing fields.

The decisive experiment came from reverse genetics. The researchers constructed TYMaV-ΔM:RFP, an infectious clone in which the matrix protein gene was replaced with a red fluorescent protein reporter while a green fluorescent protein reporter was retained. In diluted agroinoculation assays designed to test cell-to-cell movement, the mutant behaved essentially like the wild type: isolated fluorescent single cells appeared at 6 to 7 days post inoculation and foci expanded to roughly 12 cells on average by 10 days for both viruses. Immunoblotting showed GFP accumulating to slightly higher levels in mutant-infected tissues, likely because the M protein normally suppresses viral RNA synthesis. Yet negative-stain and immunogold TEM revealed that the mutant failed to assemble any detectable mature virions; gold labeling was distributed over viral material lacking recognizable virion morphology rather than decorating intact particles.

When the researchers inoculated plants under conditions permitting systemic infection, the phenotypes diverged dramatically. Wild-type TYMaV spread extensively, producing systemic GFP fluorescence from about 10 days post inoculation followed by typical mottling and leaf distortion in upper leaves, and systemically infecting 59 of 60 plants by 21 days. The M deletion mutant, by contrast, produced no systemic fluorescence or symptoms in any of 60 inoculated plants throughout the observation period. Deletion of the matrix gene thus abolished long-distance movement completely while leaving local cell-to-cell propagation intact—a phenotype that directly links virion morphogenesis to vascular dissemination.

Integrating these results, the authors propose a model in which enveloped virions serve as a major mobile entity for rhabdovirus systemic trafficking through both vascular conduits. For xylem-mediated movement, nucleocapsids colonize metabolically active immature xylem precursor cells or xylem parenchyma, where replication and envelopment occur; subsequent programmed cell death and autolysis then release assembled virions into the hollow vessel lumen for upward transport in the transpiration stream. For phloem-dependent movement, virions assembled in companion cells may be loaded into sieve elements through dilated pore-plasmodesmal units, or nucleocapsids may invade undifferentiated sieve element precursors before maturation, with the abundant membrane-enclosed virion bundles in sieve elements favoring in situ envelopment. The envelope may also protect nucleocapsids from hydrolytic enzymes abundant in vascular sap, mirroring the reliance of many positive-sense RNA viruses on intact coat proteins for stable phloem transport. The model does not exclude nucleocapsids as a less-efficient alternative—M or G deletion mutants of the nucleorhabdovirus sonchus yellow net virus retain limited systemic infectivity at roughly 9 to 14 percent of wild-type levels—but the complete block in TYMaV-ΔM:RFP underscores the importance of mature virions. The findings carry implications beyond the plant as well: because hemipteran vectors feed on both phloem and xylem sap, and successful insect acquisition requires intact virions whose surface glycoprotein mediates entry into midgut cells, virions resident in both conduits may provide dual sources for transmission between plants.

Subject of Research: Vascular long-distance movement mechanisms of enveloped plant rhabdoviruses

Article Title: Potential contribution of enveloped virions to long-distance movement of plant rhabdoviruses

Article References: Yang, G., Deng, Y., Liu, Q., Lin, W., Wang, J., Xie, L., & Li, Z. (2026). Potential contribution of enveloped virions to long-distance movement of plant rhabdoviruses. Frontiers in Microbiology, 17, Article 1994490. https://doi.org/10.3389/fmicb.2026.1994490

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1994490

Keywords: plant rhabdoviruses, enveloped virions, xylem, phloem, sieve elements, vessel elements, rice stripe mosaic virus, tomato yellow mottle-associated virus, matrix protein, systemic movement, transmission electron microscopy, reverse genetics