Host-Specific Involvement of the HC Protein in the Long-Distance Movement of Potyviruses

Plum pox virus (PPV) is a member of the Potyvirus genus that, in nature, infects trees of the Prunus genus. Although PPV infects systemically several species of the Nicotiana genus, such as N. clevelandii and N. benthamiana, and replicates in the inoculated leaves of N. tabacum, it is unable to infect systemically the last host. The long-distance movement defect of PPV was corrected in transgenic tobacco plants expressing the 5"-terminal region of the genome of tobacco etch virus (TEV), a potyvirus that infects systemically tobacco. The fact that PPV was unable to move to upper noninoculated leaves in tobacco plants transformed with the same TEV transgene, but with a mutation in the HC protein (HC-Pro)-coding sequences, identifies the multifunctional HC-Pro as the complementing factor, and strongly suggests that a defect in an HC-Pro activity is responsible for the long-distance movement defect of PPV in tobacco. Whereas PPV HC-Pro strongly intensifies the symptoms caused by potato virus X (PVX) in the PPV systemic hosts N. clevelandii and N. benthamiana, it has no apparent effect on PVX pathogenicity in tobacco, supporting the hypothesis that long-distance movement and pathogenicity enhancement are related activities of the potyviral HC proteins. The movement defect of PPV in tobacco could also be complemented by cucumber mosaic virus in a mixed infection, demonstrating that at least some components of the long-distance machinery of the potyviruses are not strictly virus specific. A general conclusion of this work is that the HC-Pro might be a relevant factor for controlling the host range of the potyviruses.     

Gene C2 of the monopartite geminivirus tomato yellow leaf curl virus-China encodes a pathogenicity determinant that is localized in the nucleus

Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent protein (GFP) fused to the C2 protein (C2-GFP) in Nicotiana benthamiana from a Potato virus X (PVX) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. The localization of GFP fluorescence in plant cells infected with PVX/C2-GFP and in insect cells transfected with Baculovirus expressing C2-GFP indicates that the TYLCV-C C2 protein is capable of shuttling GFP into plant and insect cell nuclei. Our data demonstrate that the TYLCV-C C2 protein may contribute to viral pathogenicity in planta and is nuclear localized.     

The nuclear shuttle protein of Tomato leaf curl New Delhi virus is a pathogenicity determinant

The role of the movement protein (MP) and nuclear shuttle protein (NSP) in the pathogenicity of Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, was studied. Both genes were expressed in Nicotiana benthamiana, Nicotiana tabacum, and Lycopersicon esculentum plants with the Potato virus X (PVX) expression vector or by stable transformation of gene constructs under the control of the 35S promoter in N. tabacum. No phenotypic changes were observed in any of the three species when the MP was expressed from the PVX vector or constitutively expressed in transgenic plants. Expression of the ToLCNDV NSP from the PVX vector in N. benthamiana resulted in leaf curling that is typical of the disease symptoms caused by ToLCNDV in this species. Expression of NSP from PVX in N. tabacum and L. esculentum resulted in a hypersensitive response (HR), demonstrating that the ToLCVDV NSP is a target of host defense responses in these hosts. The NSP, when expressed as a transgene under the control of the 35S promoter, resulted in necrotic lesions in expanded leaves that initiated from a point and then spread across the leaf. The necrotic response was systemic in all the transgenic plants. Deletion of 100 amino acids from the C terminus did not compromise the HR response, suggesting that this region has no role in HR. Deletion of 60 or 100 amino acids from the N terminus of NSP abolished the HR response, suggesting that these sequences are required for the HR response. These findings demonstrate that the ToLCNDV NSP is a pathogenicity determinant as well as a target of host defense responses.     

Identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus X vector.

In this study, we analyzed the influence of two nested genes (p19 and p22) of tomato bushy stunt virus (TBSV) on disease symptoms in systemically infected plants and in local lesion hosts. The contribution of individual genes was determined by bioassays with an infectious clone of wild-type TBSV, with p19/p22 mutant derivatives, and by expression of individual TBSV genes from a heterologous potato virus X (PVX) vector. Our results showed that TBSV genes could be expressed at high levels from the PVX vector. The subcellular localization of these proteins as well as the ability of PVX-expressed p22 to trans complement TBSV cell-to-cell movement defective mutants indicate that the exogenously expressed proteins are functionally active. Inoculation studies with TBSV mutants and the PVX derivatives demonstrated that p19 induced a generalized necrosis upon systemic infection of Nicotiana benthamiana and N. clevelandii. In addition, p19 elicited the formation of local necrotic lesions in N. tabacum; however, in N. glutinosa and N. edwardsonii, the local lesion response was activated by p22. These results show that the p19 and p22 proteins of TBSV are important symptom determinants and that closely related plant species may contain different resistance genes that selectively respond to individual TBSV proteins.     

Potyviral 6K2 protein long-distance movement and symptom-induction functions are independent and host-specific

Deletion of various portions, or insertion of six histidine residues (6xHis) into various positions of the membrane-bound 6K2 protein (53 amino acids) of Potato virus A (PVA, genus Potyvirus), inhibited systemic infection in Nicotiana tabacum and N. benthamiana plants. However, a spontaneous mutation (Gly2Cys) that occurred in 6K2 adjacent to the 6xHis insert placed between Ser1 and Gly2 enabled systemic infection in a single N. benthamiana plant. No symptoms were observed, but virus titers were similar to the symptom-inducing wild-type (wt) PVA. N. tabacum plants were not systemically infected, albeit virus propagation was observed in inoculated protoplasts. The 6xHis/Gly2Cys mutant was reconstructed in vitro and serially propagated by mechanical inoculation in N. benthamiana. Following the third passage, a novel viral mutant appeared, lacking the last four His residues of the insert, as well as the Gly2 and Thr3 of 6K2. It infected N. tabacum plants systemically, and in the systemically infected N. benthamiana leaves, vein chlorosis and mild yellowing symptoms were observed, typical of wt PVA infection. The mutant virus accumulated to titers similar to wt PVA in both hosts. These results show that the PVA 6K2 protein affects viral long-distance movement and symptom induction independently and in a host-specific manner.     

Efficient transient expression of human GM-CSF protein in Nicotiana benthamiana using potato virus X vector

The human granulocyte macrophage colony-stimulating factor (GM-CSF) is a glycoprotein with important clinical applications for the treatment of neutropenia and aplastic anemia and reducing infections associated with bone marrow transplants. We evaluated the potential for using a potato virus X (PVX) viral vector system for efficient expression of the biologically functional GM-CSF protein in Nicotiana benthamiana leaves. The GM-CSF gene was cloned into PVX viral expression vector, driven with the CaMV 35S promoter. Gene transfer was accomplished by inoculating N. benthamiana leaves with the plasmid DNA of PVX vector containing the GM-CSF gene. The expression level of the recombinant GM-CSF protein was determined with ELISA and its size was confirmed by Western blot analysis. The results showed that: (1) leaf age significantly affects GM-CSF protein concentration with younger leaves accumulating 19.8 mg g⁻¹ soluble protein which is 2.6 times the concentration in older leaves, (2) recombinant protein accumulation within a given leaf declined slightly over time but was not significantly different between 7 and 11 days post-inoculation (dpi), and (3) the two leaves immediately above the inoculated leaves play an important role for GM-CSF accumulation in the younger leaves. Protein extracts of infected N. benthamiana leaves contained recombinant human GM-CSF protein in concentrations of up to 2% of total soluble protein, but only when the pair of leaves immediately above the inoculated leaves remained intact. The recombinant protein actively stimulated the growth of human TF-1 cells suggesting that the recombinant human GM-CSF expressed via PVX viral vector was biologically active.     

The 5' noncoding region of grapevine chrome mosaic nepovirus RNA-2 triggers a necrotic response on three Nicotiana spp

The 5' noncoding region (NCR) of grapevine chrome mosaic nepovirus (GCMV) was cloned in a viral vector derived from potato virus X (PVX). The recombinant virus obtained was inoculated to Nicotiana benthamiana, N. clevelandii, and N. tabacum plants. Infected plants developed necrotic symptoms in place of the vein clearing and mosaic typically observed after inoculation with PVX. Northern (RNA) blot analysis showed that the replication of PVX was not specifically altered by the presence of the GCMV 5' NCR. Inoculation of recombinant PVX harboring deleted forms of the GCMV 5' NCR showed that the three stem-loop structures at the 3' end of the 5' NCR (nucleotides 153 to 206) are dispensable for the induction of necrosis. Further deletion analysis indicated that neither the 5'-most 70 nucleotides of the 5' NCR nor the downstream region (nucleotides 71 to 217) alone is able to induce the necrotic symptoms. In the presence of both the sequence encoding the GCMV coat protein and the GCMV 3' NCR, the GCMV 5' NCR failed to induce necrosis in the PVX background. The mechanisms by which the expression of the 5' NCR might modify PVX symptoms are discussed.     

Properties of a resistance-breaking strain of potato virus X

During indexing of a potato germplasm collection from Bolivia, a strain of potato virus X (PVX), X_HB, which failed to cause local lesions in inoculated leaves of Gomphrena globosa was found in 7% of the clones. X_HB was transmitted by inoculation of sap to 56 species from 11 families out of 64 species from 12 families tested. It was best propagated in Nicotiana glutinosa or N. debneyi; Montia perfolia and Petunia hybrida were useful as local lesion hosts. Inoculated leaves of G. globosa plants kept at 10°, 14°, 18°, 22°, or 26 °C after inoculation were always infected symptomlessly. X_HB caused a mild mosaic, systemic chlorotic blotching or symptomless infection in 16 wild potato species and eight Andean potato cultivars, systemic necrotic symptoms in clone A6 and cultivar Mi Peru, and bright yellow leaf markings in cultivar Renacimiento. It caused necrotic local lesions in inoculated leaves of British potato cultivars with the PVX hypersensitivity gene Nb but then invaded the plants systemically without causing further necrosis; with gene Nx systemic invasion occurred but no necrotic symptoms developed. These reactions resemble those of PVX strain group four. X_HB differed from other known strains of PVX in readily infecting PVX-immune clones 44/1016/10, G. 4298.69 and USDA 41956, cultivars Saphir and Saco, and Solanum acaule PI 230554. X_HB had slightly flexuous filamentous particles with a normal length of 516 nm. It was transmitted readily by plant contact and it partially protected G. globosa leaves from infection with X_CP, a group two strain of PVX. Sap from infected N. glutinosa was infective after dilution to 10^--⁶ but not 10^--⁷ after 10 min at 75° but not 80 °C and after 1 yr at 20 °C. X_HB was readily purified from infected N. debneyi leaves by precipitation with polyethylene glycol followed by differential centrifugation. Microprecipitin tests showed that X_HB and X_CP are closely related serologically.     

Potato virus X as a vector for gene expression in plants

The suitability of potato virus X (PVX) as a gene vector in plants was tested by analysis of two viral constructs. In the first, the GUS gene of Escherichia coli was substituted for the viral coat protein gene. In the second, GUS was added into the viral genome coupled to a duplicated copy of the viral promoter for the coat protein mRNA. The viral construct with the substituted coat protein gene accumulated poorly in inoculated protoplasts and failed to spread from the site of infection in plants. These results suggest a role for the viral coat protein in key stages of the viral infection cycle and show that gene replacement constructs are not suitable for the production of PVX-based gene vector. The construct with GUS coupled to the duplicated promoter for coat protein mRNA also accumulated less well in protoplasts than the unmodified PVX, but did infect systemically and directed high level synthesis of GUS in inoculated and systemically infected tissue. Although there was some genome instability in the PVX construct, much of the viral RNA in the systemically infected tissue had retained the foreign gene insertion, especially in infected Nicotiana clevelandii plants. These data point to a general utility of PVX as a vector for unregulated gene expression in plants.     

Jellyfish green fluorescent protein as a reporter for virus infections

The gene encoding green fluorescent protein (GFP) of Aequorea victoria was introduced into the expression cassette of a virus vector based on potato virus X (PVX). Host plants of PVX inoculated with PVX.GFP became systemically infected. Production of GFP in these plants was detected initially between 1 and 2 days postinoculation by the presence of regions on the inoculated leaf that fluoresced bright green under UV light. Subsequently, this green fluorescence was evident in systemically infected tissue. The fluorescence could be detected by several methods. The simplest of these was by looking at the UV-illuminated plants in a darkened room. The PVX.GFP-infected tissue has been analysed either by epifluorescence or confocal laser scanning microscopy. These microscopical methods allow the presence of the virus to be localized to individual infected cells. It was also possible to detect the green fluorescence by spectroscopy or by electrophoresis of extracts from infected plants. To illustrate the potential application of this reporter gene in virological studies a derivative of PVX.GFP was constructed in which the coat protein gene of PVX was replaced by GFP. Confocal laser scanning microscopy of the inoculated tissue showed that the virus was restricted to the inoculated cells thereby confirming earlier speculation that the PVX coat protein is essential for cell-to-cell movement. It is likely that GFP will be useful as a reporter gene in transgenic plants as well as in virus-infected tissue.     

Selection for wild type size derivatives of tomato golden mosaic virus during systemic infection.

A chimeric tomato golden mosaic virus (TGMV) A component DNA, which results from replacement of the coding region of the viral coat protein gene (CP) with the larger bacterial beta-glucuronidase coding sequence (GUS), can replicate in agroinoculated leaf discs but is unstable in systemically infected plants (1). We have made similar replacements of the TGMV CP gene with the GUS coding sequence in both the sense and antisense orientations. Both derivatives replicated in leaf discs inoculated via Agrobacterium. However, systemic movement of the GUS substituted vectors was not detected in agroinoculated Nicotiana benthamiana plants. The only TGMV A derivatives detected in systemically infected leaves of inoculated plants were similar in size to the wild type viral component. Sequence analysis of derivatives from six independently inoculated plants revealed that they did not result from internal deletions of the larger replicons detected in leaf discs but, instead, were generated by fusion events occuring within the original T-DNA insert. These results indicate that systemic movement of TGMV in N. benthamiana plants provides a strong selective pressure favoring viral derivatives similar in size to the wild type virus components.     

Agroinoculation of Citrus tristeza virus causes systemic infection and symptoms in the presumed nonhost Nicotiana benthamiana

Citrus tristeza virus (CTV) naturally infects only some citrus species and relatives and within these it only invades phloem tissues. Failure to agroinfect citrus plants and the lack of an experimental herbaceous host hindered development of a workable genetic system. A full-genome cDNA of CTV isolate T36 was cloned in binary plasmids and was used to agroinfiltrate Nicotiana benthamiana leaves, with or without coinfiltration with plasmids expressing different silencing-suppressor proteins. A time course analysis in agroinfiltrated leaves indicated that CTV accumulates and moves cell-to-cell for at least three weeks postinoculation (wpi), and then, it moves systemically and infects the upper leaves with symptom expression. Silencing suppressors expedited systemic infection and often increased infectivity. In systemically infected Nicotiana benthamiana plants, CTV invaded first the phloem, but after 7 wpi, it was also found in other tissues and reached a high viral titer in upper leaves, thus allowing efficient transmission to citrus by stem-slash inoculation. Infected citrus plants showed the symptoms, virion morphology, and phloem restriction characteristic of the wild T36 isolate. Therefore, agroinfiltration of Nicotiana benthamiana provided the first experimental herbaceous host for CTV and an easy and efficient genetic system for this closterovirus.     

Rice dwarf phytoreovirus segment S6-encoded nonstructural protein has a cell-to-cell movement function

Rice dwarf virus (RDV) is a member of the genus Phytoreovirus, which is composed of viruses with segmented double-stranded RNA genomes. Proteins that support the intercellular movement of these viruses in the host have not been identified. Microprojectile bombardment was used to determine which open reading frames (ORFs) support intercellular movement of a heterologous virus. A plasmid containing an infectious clone of Potato virus X (PVX) defective in cell-to-cell movement and expressing either beta-glucuronidase or green fluorescent protein (GFP) was used for cobombardment with plasmids containing ORFs from RDV gene segments S1 through S12 onto leaves of Nicotiana benthamiana. Cell-to-cell movement of the movement-defective PVX was restored by cobombardment with a plasmid containing S6. In the absence of S6, no other gene segment supported movement. Identical results were obtained with Nicotiana tabacum, a host that allows fewer viruses to infect and spread within its tissue. S6 supported the cell-to-cell movement of the movement-defective PVX in sink and source leaves of N. benthamiana. A mutant S6 lacking the translation start codon did not complement the cell-to-cell movement of the movement-defective PVX. An S6 protein product (Pns6)-enhanced GFP fusion was observed near or within cell walls of epidermal cells from N. tabacum. By immunocytochemistry, unfused Pns6 was localized to plasmodesmata in rice leaves infected with RDV. S6 thus encodes a protein with characteristics identical to those of other viral proteins required for the cell-to-cell movement of their genome and therefore is likely required for the cell-to-cell movement of RDV.