Adaptation of an ecdysone-based genetic switch for transgene expression in soybean seeds.

Soybean was used as a model for studies of chemical induction of gene expression in seeds. A chimeric transcriptional activator, VGE, driven by the soybean seed glycinin G1 promoter, was used to induce the expression of an ER-targeted GFP(KDEL) reporter protein upon addition of the chemical ligand, methoxyfenozide. The chemical gene switch activated gene expression under in vitro conditions in somatic cotyledonary embryos and zygotic seed embryos cultured from transgenic soybean plants, as well as in seeds in planta under greenhouse conditions. The efficiency of induction of GFP expression under different growth conditions was strongly influenced by the developmental stage of the seed and availability of the inducer. The formation of ER-derived GFP-containing protein bodies in seed storage parenchyma cells was correlated with the level of induced expression.

Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA.

Little is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.

The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement.

The proposed role of the 30-kilodalton(kD) protein of tobacco mosaic virus is to facilitate cell-to-cell spread of the virus-during infection. To directly define the function of the protein, a chimeric gene containing a cloned complementary DNA of the 30-kD protein gene was introduced into tobacco cells via a Ti plasmid-mediated transformation system of Agrobacterium tumefaciens. Transgenic plants regenerated from transformed tobacco cells expressed the 30-kD protein messenger RNA and accumulated 30-kD protein. Seedlings expressing the 30-kD protein gene complemented the Lsl mutant of TMV, a mutant that is temperature-sensitive in cell-to-cell movement. In addition, enhanced movement of the Lsl virus at the permissive temperature was detected in seedlings that express the 30-kD protein gene. These results conclusively demonstrate that the 30-kD protein of tobacco mosaic virus potentiates the movement of the virus from cell to cell.

Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit.

The function of the 30-kilodalton movement protein (MP) of tobacco mosaic virus is to facilitate cell-to-cell movement of viral progeny in an infected plant. A novel method for delivering non-plasmalemma-permeable fluorescent probes to the cytosol of spongy mesophyll cells of tobacco leaves was used to study plasmodesmatal size exclusion limits in transgenic plants that express the MP gene. Movement of fluorescein isothiocyanate-labeled dextran (F-dextran) with an average molecular mass of 9400 daltons and an approximate Stokes radius of 2.4 nanometers was detected between cells of the transgenic plants, whereas the size exclusion limit for the control plants was 700 to 800 daltons. No evidence of F-dextran metabolism in the leaves of the transgenic plants was found. Thus, the tobacco mosaic virus movement protein has a direct effect on a plasmodesmatal function.

Interaction of tobamovirus movement proteins with the plant cytoskeleton.

The movement protein of tobacco mosaic tobamovirus and related viruses is essential for the cell-to-cell spread of infection and, in part, determines the host range of the virus. Movement protein (MP) was fused with the jellyfish green fluorescent protein (GFP), and a modified virus that contained this MP:GFP fusion protein retained infectivity. In protoplasts and leaf tissues, the MP:GFP fusion protein was detected as long filaments shortly after infection. Double-labeling fluorescence microscopy suggests that the MP interacts and coaligns with microtubules. The distribution of the MP is disrupted by treatments that disrupt microtubules, but not by cytochalasin B, which disrupts filamentous F-actin. Microtubules may target the MP to plasmodesmata, the intercellular channels that connect adjacent cells.

Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene.

A chimeric gene containing a cloned cDNA of the coat protein (CP) gene of tobacco mosaic virus (TMV) was introduced into tobacco cells on a Ti plasmid of Agrobacterium tumefaciens from which tumor inducing genes had been removed. Plants regenerated from transformed cells expressed TMV mRNA and CP as a nuclear trait. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for development of disease symptoms. The seedlings that expressed the CP gene were delayed in symptom development and 10 to 60 percent of the transgenic plants failed to develop symptoms for the duration of the experiments. Increasing the concentration of TMV in the inoculum shortened the delay in appearance of symptoms. The results of these experiments indicate that plants can be genetically transformed for resistance to virus disease development.

Regeneration of transgenic cassava plants (Manihot esculenta Crantz) from microbombarded embryogenic suspension cultures.

A protocol was established for the introduction of DNA into embryogenic suspension-derived tissues of cassava via microparticle bombardment, for the selection of genetically transformed cells, and for the regeneration of fully transgenic plants from these cells. The plasmid DNA used for bombardment contained a gene encoding neomycin phosphotransferase (nptII) and a gene encoding beta-glucuronidase (uidA). Selection of bombarded tissue with paromomycin resulted in the establishment of putative transgenic embryogenic calli. In most of these calli, beta-glucuronidase was detected histochemically. Molecular analysis of paromomycin-resistant embryogenic calli and of plants regenerated from these calli, confirmed the stable integration of bombarded DNA into the cassava genome.

Expression and inheritance of multiple transgenes in rice plants.

The ability to control integration, inheritance, and expression of multiple transgenes is a prerequisite for manipulating biosynthetic pathways and complex agronomic characteristics in plants. One hundred and twenty-five independent transgenic rice plants were regenerated after cobombarding embryogenic tissues with a mixture of 14 different pUC-based plasmids. Eighty-five percent of the R0 plants contained more than two, and 17% more than nine, of the target genes. Plants containing multiple transgenes displayed normal morphologies and 63% set viable seed. Multigene cotransformation efficiency was correlated with the ratio in which the plasmids were mixed with respect to the selectable marker. All target genes had an equal chance of integration, indicating that the nature of the coding region had no effect on the efficiency of integration. Three plant lines containing 11, 10, and 9 transgenes, respectively, were analyzed for patterns of integration and inheritance until the R3 generation. Integration of multiple transgenes occurred at either one or two genetic loci, with inheritance conforming to a 3:1 Mendelian ratio. Coexpression of four marker genes was investigated until the R2 generation.

Posttranscriptional Gene Silencing Does Not Play a Significant Role in Potato virus X Coat Protein-Mediated Resistance.

ABSTRACT The expression of a gene that encodes coat protein (CP) of Potato virus X (PVX) in transgenic tobacco plants confers a high level of CP-mediated rresistance (CP-MR) against PVX infection. To determine if posttranscriptional gene silencing (PTGS) plays a role in resistance, transgenic plants expressing PVX CP were challenged against PVX under conditions in which PTGS was suppressed by low temperatures or using viruses carrying PTGS suppressors. The data demonstrate that PTGS does not play a significant role in PVX CP-MR.

3D domain swapping modulates the stability of members of an icosahedral virus group.

BACKGROUND: Rice yellow mottle virus (RYMV) is a major pathogen that dramatically reduces rice production in many African countries. RYMV belongs to the genus sobemovirus, one group of plant viruses with icosahedral capsids and single-stranded, positive-sense RNA genomes. RESULTS: The structure of RYMV was determined and refined to 2.8 A resolution by X-ray crystallography. The capsid contains 180 copies of the coat protein subunit arranged with T = 3 icosahedral symmetry. Each subunit adopts a jelly-roll beta sandwich fold. The RYMV capsid structure is similar to those of other sobemoviruses. When compared with these viruses, however, the betaA arm of the RYMV C subunit, which is a molecular switch that regulates quasi-equivalent subunit interactions, is swapped with the 2-fold-related betaA arm to a similar, noncovalent bonding environment. This exchange of identical structural elements across a symmetry axis is categorized as 3D domain swapping and produces long-range interactions throughout the icosahedral surface lattice. Biochemical analysis supports the notion that 3D domain swapping increases the stability of RYMV. CONCLUSIONS: The quasi-equivalent interactions between the RYMV proteins are regulated by the N-terminal ordered residues of the betaA arm, which functions as a molecular switch. Comparative analysis suggests that this molecular switch can also modulate the stability of the viral capsids.

Display of epitopes on the surface of tobacco mosaic virus: impact of charge and isoelectric point of the epitope on virus-host interactions.

The biophysical properties of the tobacco mosaic tobamovirus (TMV) coat protein (CP) make it possible to display foreign peptides on the surface of TMV. The immunogenic epitopes G5-24 from the rabies virus (RV) glycoprotein, and 5B19 from murine hepatitis virus (MHV) S-glycoprotein were successfully displayed on the surface of TMV, and viruses accumulated to high levels in infected leaves of Nicotiana tabacum Xanthi-nn. The peptide RB19, which contains an arginine residue plus the 5B19 epitope fused to the CP (TMV-RB19), resulted in the induction of necrotic local lesions on inoculated leaves of N. tabacum Xanthi-nn and cell death of infected BY2 protoplasts. RNA dot blot assays confirmed that expression of the acidic and basic pathogenesis-related PR2 genes were induced in infected Xanthi-nn leaf tissue. TMV that carried epitope 31D from the RV nucleoprotein did not accumulate in inoculated tobacco leaves. Analysis of hybrid CPs predicted that the isoelectric points (pI):charge value was 5.31:-2 for wild-type CP, 5.64:-1 for CP-RB19, and 9.14:+2 for CP-31D. When acidic amino acids were inserted in CP-RB19 and CP-31D to bring their pI:charge to near that of wild-type CP, the resulting viruses TMV-RB19E and TMV-4D:31D infected N. tabacum Xanthi-nn plants and BY2 protoplasts without causing cell death. These data show the importance of the pI of the epitope and its effects on the hybrid CP pI:charge value for successful epitope display as well as the lack of tolerance to positively charged epitopes on the surface of TMV.

Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction.

Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 A resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca(2+) from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.

Coat protein-mediated resistance in transgenic tobacco expressing the tobacco mosaic virus coat protein from tissue-specific promoters.

Coat protein-mediated resistance (CP-MR) was studied in transgenic Nicotiana tabacum 'Xanthi nn' and 'Xanthi NN' that express chimeric tobacco mosaic virus (TMV) coat protein (CP) gene constructs using two different tissue-specific promoters. The Phaseolus vulgaris pal2 promoter leads to gene expression in the upper leaf epidermis and the xylem, while the rolC promoter from Agrobacterium rhizogenes leads to gene expression in pholem and leaf hair tip cells. Tissue-specific gene expression was verified using the gusA(uidA) reporter gene, while accumulation of TMV CP was verified by Western blot analysis. Transgenic Xanthi nn plants harboring the pal2-CP gene construct were partially resistant to TMV infection. On Xanthi NN plants that expressed the pal2-CP gene construct, fewer necrotic lesions were formed after TMV inoculation compared to nontransformed control plants. The level of resistance, however, was substantially less than in plant lines that expressed TMV CP from the cauliflower mosaic virus 35S promoter. By contrast, expression of the rolC-CP construct did not confer resistance in either Xanthi nn or Xanthi NN. The results provide further evidence that CP-MR to systemic TMV infection in tobacco is probably due to inhibition of infection rather than to effects on long-distance spread through the phloem.

Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses.

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.

Mutational analysis of the movement protein of odontoglossum ringspot virus to identify a host-range determinant.

Tobacco mosaic virus (TMV) which contains the movement protein (MP) of odontoglossum ringspot tobamovirus (ORSV) in place of the TMV MP systemically infects orchids but causes local infection in tobacco unless the carboxy-terminal 48 amino acids of the MP are deleted (C. A. Holt, C. A. Fenczik, S. J. Casper, and R. N. Beachy; Virology, in press, 1995). Frameshift mutations were created within the 3' ends of the MP gene that led to truncations of the ORSV MP by 11, 19, 28, 37, and 48 amino acids; each of the mutant MP genes was inserted into the cloned cDNA of TMV in place of the TMV MP and infectious transcripts were produced. Virus containing mutant MPs were used to infect vanilla orchids, a systemic host of ORSV, and tobacco plants. Removal of 11 amino acids from the ORSV MP prevented spread of the chimeric virus in orchids while restoring the ability to cause a systemic infection on tobacco. Further deletions of the MP affected the size of virus-induced necrotic local lesions on tobacco cv. Xanthi NN and the systemic spread and accumulation of virus in cv. Xanthi nn, a systemic host of TMV. However, each virus replicated to equivalent levels in protoplasts. A mechanism by which the ORSV MP limits the spread of the chimeric virus is proposed.

Characterization of the masked strain of tobacco mosaic virus: identification of the region responsible for symptom attenuation by analysis of an infectious cDNA clone.

A strain of tobacco mosaic virus (TMV) that produces mild (attenuated) symptoms on tobacco plants has been molecularly cloned to identify the region of the genome responsible for symptom attenuation. A full-length cDNA clone whose transcripts produce the parental disease phenotype on both systemic and hypersensitive host plants has been constructed. This infectious clone was sequenced, and 55 base changes relative to the published sequence of common TMV (strain U1) were identified. These changes resulted in 12 amino acid alterations in the open reading frames encoding the 126/183-kDa and 30-kDa movement proteins; two of these changes were determined not to be responsible for the attenuated phenotype. Exchange of restriction fragments between the infectious mild strain cDNA and an infectious U1 strain cDNA indicated that the determinants involved in symptom attenuation reside in the open reading frame encoding the 126/183-kDa proteins of TMV; these proteins are involved in viral replication.

A dysfunctional movement protein of tobacco mosaic virus interferes with targeting of wild-type movement protein to microtubules.

The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent.

Plant virus movement protein dynamics probed with a GFP-protein fusion.

A genetic fusion between the gene encoding green fluorescent protein (GFP) from the jellyfish Aequorea victoria, with that of the Ob-tobamovirus movement protein (MP) resulted in the expression of a fluorescent fusion protein (MP::GFP) that was fully biologically active in mediating the cell-to-cell spread of the Ob-virus. The MP::GFP fusion was used to follow in planta the subcellular trafficking of MP. GFP-tagged MP was transiently expressed and found to be associated with several subcellular compartments and structures including trans-wall structures, presumably plasmodesmata, and filament structures. The MP::GFP fusion can be used to monitor MP association with host proteins and structures, and for the isolation of interacting host components.

Selective inhibition of photosystem II in spinach by tobacco mosaic virus: an effect of the viral coat protein.

Leaves of Spinacia oleracea inoculated with tobacco mosaic virus (TMV) strain PV230 develop mild chlorotic and mosaic symptoms of infection. Thylakoid membranes isolated from these infected leaves showed a reduced Fv/Fm ratio for chlorophyll fluorescence kinetics, at 25 degrees C. The photosystem II (PS II)-mediated electron-transport rate was inhibited 50%, whereas PS I activity was unaffected by virus infection. Protein analysis indicated that TMV coat protein was associated with thylakoids, in particular with the PS II fraction. The results demonstrate that TMV-infected S. oleracea shows inhibition of photosynthetic electron transport through PS II. We propose that the inhibition of photosynthetic activity results from the association of viral coat protein with the PS II complex.