Three-dimensional reconstruction and comparison of vacuolar membranes in response to viral infection.

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV-AC ), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.

Virus-Induced Gene Silencing of the Eggplant Chalcone Synthase Gene during Fruit Ripening Modifies Epidermal Cells and Gravitropism.

Eggplant ( Solanum melongena L.) fruits accumulate flavonoids in their cuticle and epidermal cells during ripening. Although many mutants available in model plant species, such as Arabidopsis thaliana and Medicago truncatula, are enabling the intricacies of flavonoid-related physiology to be deduced, the mechanisms whereby flavonoids influence eggplant fruit physiology are unknown. Virus-induced gene silencing (VIGS) is a reliable tool for the study of flavonoid function in fruit, and in this study, we successfully applied this technique to downregulate S. melongena chalcone synthase gene ( SmCHS) expression during eggplant fruit ripening. In addition to the expected change in fruit color attributable to a lack of anthocyanins, several other modifications, including differences in epidermal cell size and shape, were observed in the different sectors. We also found that silencing of CHS gene expression was associated with a negative gravitropic response in eggplant fruits. These observations indicate that epidermal cell expansion during ripening is dependent upon CHS expression and that there may be a relationship between CHS expression and gravitropism during eggplant fruit ripening.

Influence of Epicuticular Physicochemical Properties on Porcine Rotavirus Adsorption to 24 Leafy Green Vegetables and Tomatoes.

Foodborne diseases are a persistent problem in the United States and worldwide. Fresh produce, especially those used as raw foods like salad vegetables, can be contaminated, causing illness. In this study, we determined the number of rotaviruses adsorbed on produce surfaces using group A porcine rotaviruses and 24 cultivars of leafy vegetables and tomato fruits. We also characterized the physicochemical properties of each produce's outermost surface layer, known as the epicuticle. The number of rotaviruses found on produce surfaces varied among cultivars. Three-dimensional crystalline wax structures on the epicuticular surfaces were found to significantly contribute to the inhibition of viral adsorption to the produce surfaces (p = 0.01). We found significant negative correlations between the number of rotaviruses adsorbed on the epicuticular surfaces and the concentrations of alkanes, fatty acids, and total waxes on the epicuticular surfaces. Partial least square model fitting results suggest that alkanes, ketones, fatty acids, alcohols, contact angle and surface roughness together can explain 60% of the variation in viral adsorption. The results suggest that various fresh produce surface properties need to be collectively considered for efficient sanitation treatments. Up to 10.8% of the originally applied rotaviruses were found on the produce surfaces after three washing treatments, suggesting a potential public health concern regarding rotavirus contamination.

Potato virus Y HCPro localization at distinct, dynamically related and environment-influenced structures in the cell cytoplasm.

Potyvirus HCPro is a multifunctional protein that, among other functions, interferes with antiviral defenses in plants and mediates viral transmission by aphid vectors. We have visualized in vivo the subcellular distribution and dynamics of HCPro from Potato virus Y and its homodimers, using green, yellow, and red fluorescent protein tags or their split parts, while assessing their biological activities. Confocal microscopy revealed a pattern of even distribution of fluorescence throughout the cytoplasm, common to all these modified HCPros, when transiently expressed in Nicotiana benthamiana epidermal cells in virus-free systems. However, in some cells, distinct additional patterns, specific to some constructs and influenced by environmental conditions, were observed: i) a small number of large, amorphous cytoplasm inclusions that contained α-tubulin; ii) a pattern of numerous small, similarly sized, dot-like inclusions distributing regularly throughout the cytoplasm and associated or anchored to the cortical endoplasmic reticulum and the microtubule (MT) cytoskeleton; and iii) a pattern that smoothly coated the MT. Furthermore, mixed and intermediate forms from the last two patterns were observed, suggesting dynamic transports between them. HCPro did not colocalize with actin filaments or the Golgi apparatus. Despite its association with MT, this network integrity was required neither for HCPro suppression of silencing in agropatch assays nor for its mediation of virus transmission by aphids.

The rubisco small subunit is involved in tobamovirus movement and Tm-2²-mediated extreme resistance.

The multifunctional movement protein (MP) of Tomato mosaic tobamovirus (ToMV) is involved in viral cell-to-cell movement, symptom development, and resistance gene recognition. However, it remains to be elucidated how ToMV MP plays such diverse roles in plants. Here, we show that ToMV MP interacts with the Rubisco small subunit (RbCS) of Nicotiana benthamiana in vitro and in vivo. In susceptible N. benthamiana plants, silencing of NbRbCS enabled ToMV to induce necrosis in inoculated leaves, thus enhancing virus local infectivity. However, the development of systemic viral symptoms was delayed. In transgenic N. benthamiana plants harboring Tobacco mosaic virus resistance-2² (Tm-2²), which mediates extreme resistance to ToMV, silencing of NbRbCS compromised Tm-2²-dependent resistance. ToMV was able to establish efficient local infection but was not able to move systemically. These findings suggest that NbRbCS plays a vital role in tobamovirus movement and plant antiviral defenses.

In planta localization and interactions of impatiens necrotic spot tospovirus proteins.

Impatiens necrotic spot tospovirus (INSV) is a significant pathogen of ornamentals. The tripartite negative- and ambi-sense RNA genome encodes six proteins that are involved in cytoplasmic replication, movement, assembly, insect transmission and defence. To gain insight into the associations of these viral proteins, we determined their intracellular localization and interactions in living plant cells. Nucleotide sequences encoding the nucleoprotein N, non-structural proteins NSs and NSm, and glycoproteins Gn and Gc of a Kentucky isolate of INSV were amplified by RT-PCR, cloned, sequenced and transiently expressed as fusions with autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana. All proteins accumulated at the cell periphery and co-localized with an endoplasmic reticulum marker. The Gc protein fusion also localized to the nucleus. N and NSm protein self-interactions and an NSm-N interaction were observed by using bimolecular fluorescence complementation. A tospovirus NSm homotypic interaction had not been reported previously.

Distribution of Potato virus Y in potato plant organs, tissues, and cells.

The distribution of Potato virus Y (PVY) in the systemically infected potato (Solanum tuberosum) plants of the highly susceptible cultivar Igor was investigated. Virus presence and accumulation was analyzed in different plant organs and tissues using real-time polymerase chain reaction and transmission electron microscopy (TEM) negative staining methods. To get a complete insight into the location of viral RNA within the tissue, in situ hybridization was developed and optimized for the detection of PVY RNA at the cellular level. PVY was shown to accumulate in all studied leaf and stem tissues, in shoot tips, roots, and tubers; however, the level of virus accumulation was specific for each organ or tissue. The highest amounts of viral RNA and viral particles were found in symptomatic leaves and stem. By observing cell ultrastructure with TEM, viral cytoplasmic inclusion bodies were localized in close vicinity to the epidermis and in trichomes. Our results show that viral RNA, viral particles, and cytoplasmic inclusion bodies colocalize within the same type of cells or in close vicinity.

Nicotiana benthamiana protein, NbPCIP1, interacting with Potato virus X coat protein plays a role as susceptible factor for viral infection.

The interactions of viral coat protein (CP) and host factors play an important role in viral replication and/or host defense mechanism. In this study, we constructed Nicotiana benthamiana cDNA library to find host factors interacting with Potato virus X (PVX) CP. Using yeast two-hybrid assay, we screened 3.3 x 10(6) independent yeast transformants from N. benthamiana cDNA library and identified six positive clones. One positive clone, named PVX CP-interacting protein 1 (NbPCIP1), is a plant-specific protein with homologue in N. tabacum (GenBank accession no. AB04049). We confirmed the PVX CP-NbPCIP1 interaction using yeast-two hybrid assay in yeast, protein-protein binding assay in vitro, and bimolecular fluorescent complementation assay in planta. Quantitative real-time RT-PCR analysis showed that the mRNA level of NbPCIP1 increased in PVX-infected N. benthamiana plants as compared to that of healthy plants. The green fluorescent protein (sGFP)-fused NbPCIP1 (NbPCIP1-sGFP) was localized in ER or ER-associated granular-like structure of cells. When we co-express NbPCIP1-sGFP and red fluorescent protein (RFP)-fused PVX CP (PVX CP-RFP), which were introduced by transiently expressing these proteins in N. benthamiana protoplasts and epidermal cells, however, we observed the co-localization of these proteins in the inclusion body-like complex in areas surrounding nucleus. Transient over-expression and transgene silencing of NbPCIP1 assay analysis indicated that NbPCIP1 plays a critical role in viral replication during PVX infection in host plant.

The arginine-rich motif of Bamboo mosaic virus satellite RNA-encoded P20 mediates self-interaction, intracellular targeting, and cell-to-cell movement.

Satellite RNA of Bamboo mosaic virus (satBaMV) has a single open reading frame for a nonstructural, RNA-binding protein, P20, which facilitates the long-distance movement of satBaMV in Nicotiana benthamiana. Here, we elucidate various biological properties of P20 and the involvement of a single domain in its activities. P20 displayed a strong self-interaction in vitro and in vivo, and cross-linking assays demonstrated its oligomerization. Domain mapping, using the bacterial two-hybrid system, indicated that the self-interacting domain overlaps the RNA-binding domain in the N-terminal arginine-rich motif (ARM) of P20. The deletion of the ARM abolished the self-interaction of P20 in vitro and in vivo and impaired its intracellular targeting and efficient cell-to-cell movement in N. benthamiana leaves. Moreover, RNA and protein accumulation of the ARM deletion mutant of satBaMV was significantly reduced in leaves systemically coinfected with Bamboo mosaic potexvirus and satBaMV. This is the first report of the involvement of ARM in various biological activities of a satellite RNA-encoded protein during infection of its host.

The 50-kDa protein of Apple chlorotic leaf spot virus interferes with intracellular and intercellular targeting and tubule-inducing activity of the 39-kDa protein of Grapevine berry inner necrosis virus.

To understand why transgenic Nicotiana occidentalis plants expressing a functional movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV) show specific resistance to Grapevine berry inner necrosis virus (GINV), the MPs of ACLSV (50KP) and GINV (39KP) were fused to green, yellow, or cyan fluorescent proteins (GFP, YFP, or CFP). These fusion proteins were transiently expressed in leaf cells of both transgenic (50KP) and nontransgenic (NT) plants, and the intracellular and intercellular trafficking and tubule-inducing activity of these proteins were compared. The results indicate that in epidermal cells and protoplasts from 50KP plant leaves, the trafficking and tubule-inducing activities of GINV-39KP were specifically blocked while those of ACLSV-50KP and Apple stem grooving virus MP (36KP) were not affected. Additionally, when 39KP-YFP and 50KP-CFP were coexpressed in the leaf epidermis of NT plants, the fluorescence of both proteins was confined to single cells, indicating that 50KP-CFP interferes with the cell-to-cell trafficking of 39KP-YFP and vice versa. Mutational analyses of 50KP showed that the deletion mutants that retained the activities described above still blocked cell-to-cell trafficking of 39KP, but the dysfunctional 50KP mutants could no longer impede cell-to-cell movement of 39KP. Transgenic plants expressing the functional 50KP deletion mutants showed specific resistance against GINV. In contrast, transgenic plants expressing the dysfunctional 50KP mutants did not show any resistance to the virus. From these results, we conclude that the specific resistance of 50KP plants to GINV is due to the ability of the 50KP to block intracellular and intercellular trafficking of GINV 39KP.

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement.

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.

Analysis of the N gene hypersensitive response induced by a fluorescently tagged tobacco mosaic virus.

The hypersensitive response (HR) triggered on Nicotiana edwardsonii by tobacco mosaic virus was studied using a modified viral genome that directed expression of the green fluorescent protein. Inoculated plants were initially incubated at 32 degrees C to inhibit the N gene-mediated HR. Transfer to 20 degrees C initiated the HR, and fluorescent infection foci were monitored for early HR-associated events. Membrane damage, which preceded visible cell collapse by more than 3 h, was accompanied by a transient restriction of the xylem within infection sites. Following cell collapse and the rapid desiccation of tissue undergoing the HR, isolated, infected cells were detected at the margin of necrotic lesions. These virus-infected cells were able to reinitiate infection on transfer to 32 degrees C, however, if maintained at 20 degrees C they eventually died. The results indicate that the tobacco mosaic virus-induced HR is a two-phase process with an early stage culminating in rapid cell collapse and tissue desiccation followed by a more extended period during which the remaining infected cells are eliminated.