Igor B. Kaplan

Reduction of tobacco mosaic virus accumulation in transgenic plants producing non-functional viral transport proteins

Transgenic plants producing the 30K temperature-sensitive transport protein (TP) of tobacco mosaic virus (TMV) mutant Ni2519 (affecting cell-to-cell transport) were found to: (i) be susceptible to wild-type TMV U1 at 24 degrees C (a permissive temperature for Ni2519 TP), (ii) acquire a certain level of resistance to TMV U1 accumulation when maintained at 33 degrees C (a non-permissive temperature for Ni2519 TP) and (iii) lose the resistance to wild-type TMV after their transfer from 33 degrees C to 24 degrees C. It is suggested that reversible temperature-dependent conformational changes in Ni2519 TP are responsible for these phenomena and that production of a TP which is only partially functional in transgenic plants confers on these plants a resistance to the virus owing to reduction of the level of cell-to-cell transport. Transgenic tobacco plants producing the 32K TP of brome mosaic virus (BMV) acquired resistance to TMV U1 suggesting that BMV TP is partially functional in tobacco plants.

A Surface Loop of the Potato Leafroll Virus Coat Protein Is Involved in Virion Assembly, Systemic Movement, and Aphid Transmission

ABSTRACT Two acidic domains of the Potato leafroll virus (PLRV) coat protein, separated by 55 amino acids and predicted to be adjacent surface features on the virion, were the focus of a mutational analysis. Eleven site-directed mutants were generated from a cloned infectious cDNA of PLRV and delivered to plants by Agrobacterium-mediated mechanical inoculation. Alanine substitutions of any of the three amino acids of the sequence EWH (amino acids 170 to 172) or of D177 disrupted the ability of the coat protein to assemble stable particles and the ability of the viral RNA to move systemically in four host plant species. Alanine substitution of E109, D173, or E176 reduced the accumulation of virus in agrobacterium-infiltrated tissues, the efficiency of systemic infection, and the efficiency of aphid transmission relative to wild-type virus, but the mutations did not affect virion stability. A structural model of the PLRV capsid predicted that the amino acids critical for virion assembly were located within a depression at the center of a coat protein trimer. The other amino acids that affected plant infection and/or aphid transmission were predicted to be located around the perimeter of the depression. PLRV virions play key roles in phloem-limited virus movement in plant hosts as well as in transport and persistence in the aphid vectors. These results identified amino acid residues in a surface-oriented loop of the coat protein that are critical for virus assembly and stability, systemic infection of plants, and movement of virus through aphid vectors.

A study of TMV ts mutant Ni2519. I. Complementation experiments.

Two distinct virus-specific functions, i.e., virus assembly and spreading of infection from cell to cell (transport function), are temperature-sensitive (ts) in TMV mutant Ni2519. Assembly of Ni2519 cannot be complemented by the temperature-resistant TMV strains used: A14 (a wild type strain from which Ni2519 was derived) and dolichos enation mosaic virus (DEMV, or cowpea strain of TMV), a thermophilic strain. On the other hand, Ni2519 can serve as a donor of the coat protein to complement is strain Ni118, which has a mutation in the coat protein gene. The genomic RNA can be produced by Ni2519 at a nonpermissive temperature; functionally active Ni2519 coat protein (capable of coating Ni118 RNA upon mixed infection) is produced at a nonpermissive temperature as well. The is phenotype of Ni2519 upon virus assembly probably results not from the ts behavior of any virus-coded protein(s) but is due to the ts properties of the genomic RNA molecule itself, so the possibility of the complementation of assembly of Ni2519 is ruled out. Thus, Ni2519 appears to represent a novel class of virus mutants with is virion RNA. The second is function of Ni2519 (transport of infection) can be complemented by a helper virus. The experimental system used for complementation of the transport function allowed Ni2519 to spread from cell to cell at a nonpermissive temperature. Obviously, Ni2519 infection spreads under these conditions in a form different from that in the mature virions, since its assembly cannot be complemented by the helper virus. Some aspects of the transport function are discussed.

Site-Directed Mutagenesis and Generation of Chimeric Viruses by Homologous Recombination in Yeast to Facilitate Analysis of Plant-Virus Interactions

A yeast homologous recombination system was used to generate mutants and chimeras in the genome of Potato leafroll virus (PLRV). A yeast-bacteria shuttle vector was developed that allows mutants and chimeras generated in yeast to be transformed into Escherichia coli for confirmation of the mutations and transformed into Agrobacterium tumefaciens to facilitate agroinfection of plants by the mutant PLRV genomes. The advantages of the system include the high frequency of recovered mutants generated by yeast homologous recombination, the ability to generate over 20 mutants and chimeras using only two restriction endonuclease sites, the ability to introduce multiple additional sequences using three and four DNA fragments, and the mobilization of the same plasmid from yeast to E. coli, A. tumefaciens, and plants. The wild-type PLRV genome showed no loss of virulence after sequential propagation in yeast, E. coli, and A. tumefaciens. Moreover, many PLRV clones with mutations generated in the capsid protein and readthrough domain of the capsid protein replicated and moved throughout plants. This approach will facilitate the analysis of plant-virus interactions of in vivo-generated mutants for many plant viruses, especially those not transmissible mechanically to plants.

Biological activities of human interferon and 2'-5' oligoadenylates in plants.

Exogenous human interferon 2 (IFN) and 2′–5′ oligoadenylates (2–5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1–1 u/ml and 2–5A at 1–10 nM.

Production of the tobacco mosaic virus (TMV) transport protein in transgenic plants is essential but insufficient for complementing foreign virus transport: a need for the full-length TMV genome or some other TMV-encoded product

We have reported previously that tobamoviruses enable the transport of red clover mottle comovirus (RCMV) in tobacco plants normally resistant to RCMV. Here we show that RCMV transport does not take place in transgenic tobacco plants (line To-4) producing the 30K transport protein of tobacco mosaic virus (TMV), whereas the transport of the TMV Ls1 mutant, the cell-to-cell movement of which is temperature sensitive, is complemented in these plants. However, RCMV transport is observed when these transgenic plants are infected with both RCMV and TMV Ls1 at the non-permissive temperature (33 degrees C). It is suggested that (i) the hypothetical modification of transgenic plant plasmodesmata by the TMV 30K transport protein can specifically mediate the cell-to-cell movement of the homologous virus (TMV), but is insufficient to mediate RCMV transport; (ii) the presence of the full-length TMV genome or a certain TMV-encoded product(s) besides the 30K protein is essential for complementation of the RCMV transport function. The possibility that line To-4 might provide enough 30K protein to complement TMV Ls1 but not RCMV cannot be ruled out. During double infection the mutant 30K protein may, in concert with the wild-type 30K protein, provide the transport function for RCMV.

Subcellular localization and self-interaction of plant-specific Nt-4/1 protein

The Nicotiana tabacum Nt-4/1 protein is a plant-specific protein of unknown function. Analysis of bacterially expressed Nt-4/1 protein in vitro revealed that the protein secondary structure is mostly alpha-helical and suggested that it could consist of three structural domains. Earlier studies of At-4/1, the Arabidopsis thaliana-encoded ortholog of Nt-4/1, demonstrated that GFP-fused At-4/1 was capable of polar localization in plant cells, association with plasmodesmata, and cell-to-cell transport. Together with the At-4/1 ability to interact with a plant virus movement protein, these data supported the hypothesis of the At-4/1 protein involvement in viral transport through plasmodesmata. Studies of the Nt-4/1-GFP fusion protein reported in this paper revealed that the protein was localized to cytoplasmic bodies, which were co-aligned with actin filaments and capable of actin-dependent intracellular movement. The Nt-4/1-GFP bodies, being non-membrane structures, were found in association with the plasma membrane, the tubular endoplasmic reticulum and endosome-like structures. Bimolecular fluorescence complementation experiments and inhibition of nuclear export showed that the Nt-4/1 protein was capable of nuclear-cytoplasmic transport. The nuclear export signal (NES) was identified in the Nt-4/1 protein by site-directed mutagenesis. The Nt-4/1 NES mutant was localized to the nucleoplasm forming spherical bodies. Immunogold labeling and electron microscopy of cytoplasmic Nt-4/1-containing bodies and nuclear structures containing the Nt-4/1 NES mutant revealed differences in their fine structure. In mammalian cells, Nt-4/1-GFP formed cytoplasmic spherical bodies similar to those found for the Nt-4/1 NES mutant in plant cell nuclei. Using dynamic laser light scattering and electron microscopy, the Nt-4/1 protein was found to form multimeric complexes in vitro.

Synergy of Virus Accumulation and Pathology in Transgenic Plants Expressing Viral Sequences

Some viruses interact with other viruses resulting in an increase in the titre of one or both of the unrelated viruses, and an enhancement of the pathogencity induced by either virus. This interaction is called synergism. For one well-know type of synergism, it has been shown that a transgene containing particular sequences of one virus can enhance the accumulation and pathogenicity induced by a second virus. However, those transgenic viral sequences were not among the ones that have been used to date in studies on pathogen-mediated resistance.

A new method for producing biologically active nanocomplexes by a noncovalent conjugation of proteins with viral particles

A new method for noncovalent immobilization of a peptide epitope on the virion surface was developed to simplify and standardize the procedures for producing viral nanocomplexes. The efficacy of this approach was demonstrated by the example of a model system comprising the tobacco mosaic virus, synthetic cationic polymer poly-N-ethyl-4-vinylpyridinium bromide, and a model polypeptide. The principle of sequential adsorption, underlying production of the triple system virion-polycation-protein, was used for electrostatic immobilization of a recombinant hydrophilic fragment of the influenza virus hemagglutinin (Flu1-3) on the virion surface. The method provided for a significant increase in the immunogenic activity of this potential artificial vaccine protein.

Biocatalytic approach as alternative to chemical synthesis of polyaniline/carbon nanotube composite with enhanced electrochemical properties

A new approach is proposed to biocatalytical synthesis of a thin homogeneous layer of conducting polyaniline (PANI) on the surface of MWCNTs using the laccase Trametes hirsuta. Aniline dimer, an enhancer of laccase-catalyzed aniline polymerization, has been adsorbed on MWCNTs. Phytic acid has been used as acidic dopant and gelator. The PANI/MWCNT composite has a core shell structure and shows a high specific capacitance of 554 F g−1 and long-term cycling stability. The approach enables the production of nanostructured composite conducting hydrogel.