Simon Santa Cruz

Phloem Unloading in Sink Leaves of Nicotiana benthamiana: Comparison of a Fluorescent Solute with a Fluorescent Virus.

Using noninvasive imaging techniques, we compared phloem unloading of the membrane-impermeant, fluorescent solute carboxyfluorescein (CF) with that of potato virus X expressing the gene for the green fluorescent protein. Although systemic virus transport took considerably longer to occur than did CF transport, unloading of both solute and virus occurred predominantly from the class III vein network, a highly branched veinal system found between class II veins. The minor veins (classes IV and V) played no role in solute or virus import but were shown to be functional in xylem transport at the time of import by labeling with Texas Red dextran. After virus exit from the class III phloem, the minor veins eventually became infected by cell-to-cell virus movement from the mesophyll. During the sink/source transition, phloem unloading of CF was inhibited from class III veins before the cessation of phloem import through them, suggesting a symplastic isolation of the phloem in class III veins before its involvement in export. The progression of the sink/source transition for carbon was unaffected by the presence of the virus in the sink leaf. However, the virus was unable to cross the sink/source boundary for carbon that was present at the time of viral entry, suggesting a limited capacity for cell-to-cell virus movement into the apical (source) region of the leaf. A functional model of the sink/source transition in Nicotiana benthamiana is presented. This model provides a framework for the analysis of solute and virus movement in leaves.

Cell-to-Cell and Phloem-Mediated Transport of Potato Virus X: The Role of Virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.

The Movement Protein of Cucumber Mosaic Virus Traffics into Sieve Elements in Minor Veins of Nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a–green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a–GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a–GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.

Molecular Analysis of a Resistance-Breaking Strain of Potato Virus X

Full-length cDNA clones of potato virus X (PVX) strains PVXUK3 and PVXHB have been constructed in plasmid vectors to allow in vitro transcription of infectious PVX RNA. In both instances the transcript-derived virus infected tobacco and potato identically to the respective progenitor strains: in tobacco and susceptible potato cultivars both strains infected systemically, producing symptomless or mild mosaic symptoms. In potato carrying the Rx or Nx resistance genes, the virus derived from the PVXHB cDNA infected systemically, whereas the virus derived from the PVXUK3 cDNA failed to infect the Rx plants or induced apical necrosis, characteristic of a hypersensitive response of the Nx gene. Three hybrid viral genomes were constructed at the cDNA level to localize the resistance breaking determinants of PVXHB. Transcripts of all three hybrids were infectious on tobacco. On potato cultivars with either the Rx or Nx resistance genes, the hybrid viruses infected in the same way as PVXHB, rather than PVXUK3. The common feature of these hybrid viruses, the coat protein gene, is therefore the determinant of Nx and Rx resistance breaking of PVXHB.

Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A

Abstract. Potato virus X (PVX) has been used as an expression vector to target the green fluorescent protein (GFP) from the jellyfish Aequorea victoria to the endoplasmic reticulum (ER) of tobacco (Nicotiana clevelandii L.) leaves. Expression of free GFP resulted in strong cytoplasmic fluorescence with organelles being imaged in negative contrast. Translocation of GFP into the lumen of the ER was mediated by the use of the sporamin signal peptide. Retention of GFP in the ER was facilitated by the splicing of the ER retrieval/retention tetrapeptide, KDEL to the carboxy terminus of GFP. Fluorescence of GFP was restricted to a labile cortical network of ER tubules with occasional small lamellae and to streaming trans-vacuolar strands. Secretion of ER-targeted GFP was inhibited both by cold shock and low concentrations of the secretory inhibitor brefeldin A. However, both prolonged cold and prolonged incubation in brefeldin A resulted in the recovery of secretory capability. In leaves infected with the GFP-KDEL construct, high concentrations of brefeldin A induced the tubular network of cortical ER to transform into large lamellae or sheets which reverted to the tubular network on removal of the drug.

Perspective: phloem transport of viruses and macromolecules – what goes in must come out

Phloem transport of endogenous macromolecules and plant viruses remains poorly understood. Selective movement into and out of the phloem is tightly regulated, yet the mechanisms governing this selectivity have not been elucidated. Recent advances in identifying nonviral proteins and nucleic acids with the capacity for phloem transport will hopefully shed new light on the regulation of phloem loading and unloading.

Production of a functional single chain antibody attached to the surface of a plant virus

A potato virus X (PVX) vector was used to express a single chain antibody fragment (scFv) against the herbicide diuron, as a fusion to the viral coat protein. The modified virus accumulated in inoculated Nicotiana clevelandii plants and assembled to give virus particles carrying the antibody fragment. Electron microscopy was used to show that virus particles from infected leaf sap were specifically trapped on grids coated with a diuron‐BSA conjugate. The results demonstrate that the PVX vector can be used as a presentation system for functional scFv.

A novel strategy for the expression of foreign genes from plant virus vectors

Potato virus X (PVX)‐based vector constructs were generated to investigate the use of an internal ribosome entry site (IRES) sequence to direct translation of a viral gene. The 148‐nucleotide IREScp sequence from a crucifer‐infecting strain of tobacco mosaic virus was used to direct expression of the PVX coat protein (CP). The IRES was inserted downstream of the gene encoding green fluorescent protein (GFP) and upstream of the PVX CP, in either sense or antisense orientation, such that CP expression depended on ribosome recruitment to the IRES. Stem–loop structures were inserted at either the 3′‐ or 5′‐end of the IRES sequence to investigate its mode of action. In vitro RNA transcripts were inoculated to Nicotiana benthamiana plants and protoplasts: levels of GFP and CP expression were analysed by enzyme‐linked immunosorbent assay and the rate of virus cell‐to‐cell movement was determined by confocal laser scanning microscope imaging of GFP expression. PVX CP was expressed, allowing cell‐to‐cell movement of virus, from constructs containing the IRES sequence in either orientation, and from the construct containing a stem–loop structure at the 5′‐end of the IRES sequence. No CP was expressed from a construct containing a stem–loop at the 3′‐end of the IRES sequence. Our results suggest that the IRES sequence is acting in vivo to direct expression of the 3′‐proximal open reading frame in a bicistronic mRNA thereby demonstrating the potential of employing IRES sequences for the expression of foreign proteins from plant virus‐based vectors.

USING GFP TO STUDY VIRUS INVASION AND SPREAD IN PLANT TISSUES

GFP is beginning to revolutionize the study of virus movement in plants. Insertion of the gfpgene into the viral genome allows the virus to be tracked both in whole plants and also in single cells.

Rapid production of single-chain Fv fragments in plants using a potato virus X episomal vector

We have used a plant virus episomal vector, based on potato virus X (PVX) to transiently express a single-chain Fv (scFv) and its diabody derivative in plants. The scFv was directed against a continuous epitope (cryptotope) on the coat protein of potato virus V. A cloned, full-length PVX vector sequence, containing the scFv gene, was used to direct in vitro transcription and the resulting RNA was used to inoculate Nicotiana clevelandii plants. Within a few days, plants developed characteristic symptoms and immunoblot analysis showed that accumulation of scFv protein coincided with accumulation of PVX. Targeting of the scFv to the apoplast greatly increased protein accumulation compared with cytosolic scFv and produced more severe symptoms on infected plants. ELISA demonstrated that the scFv and diabody extracted from infected plants showed the same antigen-binding specificity as that of the parental monoclonal antibody. The PVX vector is a convenient, rapid, low-cost in planta expression system that can also be used for assessment of scFv production and function prior to stable plant transformation.