Identification and study of tobacco mosaic virus movement function by complementation tests.

The phenomenon of trans-complementation of cell-to-cell movement between plant positive-strand RNA viruses is discussed with an emphasis on tobamoviruses. Attention is focused on complementation between tobamoviruses (coding for a single movement protein, MP) and two groups of viruses that contain the triple block of MP genes and require four (potato virus X) or three (barley stripe mosaic virus) proteins for cell-to-cell movement. The highlights of complementation data obtained by different experimental approaches are given, including (i) double infections with movement-deficient (dependent) and helper viruses; (ii) infections with recombinant viral genomes bearing a heterologous MP gene; (iii) complementation of a movement-deficient virus in transgenic plants expressing the MP of a helper virus; and (iv) co-bombardment of plant tissues with the cDNAs of a movement-dependent virus genome and the MP gene of a helper virus.

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.

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.

Plant virus-specific transport function. I. Virus genetic control required for systemic spread.

Complementation of tobacco mosaic virus (TMV) mutant LS1 which is temperature-sensitive (ts) in virus transport function was studied in two model systems. The first one was aimed at complementing LS1 cell-to-cell spread through the tissues of the plant systemically preinfected with a temperature-resistant (tr) virus. Two different experiments were performed in this model system. (a) Use was made of a host plant which reacted differently to LS1 (local lesions) and to the helper virus (systemic reaction) at a permissive (24 degrees) temperature. Complementation occurred in a mixedly infected plant at a temperature which is nonpermissive for LS1 (32 degrees), when the necrotic reaction of the host was switched off; the complementation could be revealed upon the temperature shift treatment (TST) (24 --> 32 --> 24 degrees ). (b) Transport of LS1 from cell to cell in the presence of the helper virus was tested directly by immunofluorescent microscopy. In the second model system, the effect of virus transport from the conducting tissues of the stem into the cells of the leaf mesophyll, preinfected with the helper virus, was studied. It was shown that ts mutant LS1, imbibed through the stem, can move into and replicate in the mesophyll cells of the leaf which was preinfected with a tr-helper virus. This was true both at 24 and 32 degrees . On the other hand, LS1 itself could serve as a helper virus only at 24, but not at 32 degrees . In both model systems, it was demonstrated that ts transport can be complemented not only by a related tr TMV strain, but also by an unrelated virus-potato virus X. It has been suggested that a virus-specific transport function performed by the helper virus "opens the gates" for the dependent virus by an unknown modification of host cells.

Plant virus-specific transport function. II. A factor controlling virus host range.

An experimental system allowing the transport of the dependent virus from the conducting tissues into the leaf mesophyll of a plant preinfected by the helper virus has been developed recently [Taliansky et al. (1982). Virology 122, 318-326]. The role of virus-specific transport function in the host range control of a plant virus was studied here by this technique. It was shown that Tm-2 tomato lines (in which the resistance to TMV is expressed only at the level of the intact plant, but not in protoplasts) became susceptible to TMV after preinfection with the helper virus (potato virus X, PVX). It is assumed that TMV accumulation in Tm-2 lines under these conditions becomes possible due to the transport function coded for by the helper virus (PVX), which allows TMV to spread over the resistant plant. Tm-1 tomato lines (whose protoplasts are resistant to TMV infection) remained resistant to TMV under the conditions outlined above. It was found that brome mosaic virus (BMV) can be transported from the conducting tissues and replicates in the mesophyll cells of tomato and bean plants preinfected by TMV and dolichos enation mosaic virus (DEMV), respectively. The same was true of TMV introduced into the conducting tissues of wheat plants preinfected with barley stripe mosaic virus (BSMV). It is concluded that a nonhost plant, whose resistance to a virus is due to the blockage of the transport function, can be infected systemically by this virus as a result of the complementation of transport with the helper virus.

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.

Homology in the 3'-terminal regions of different barley stripe mosaic virus RNA species.

Preparations of [3H]DNA complementary to the individual species of barley stripe mosaic virus (BSMV) RNA (cDNA) about 1000 nucleotides long were obtained using avian myeloblastosis virus reverse transcriptase and oligo(dT)10 primer. These cDNAs were used in kinetic hybridization experiments to study the sequence homology between the 3'-terminal regions (not including 150-200 nucleotides adjacent to the 3'-end) of individual BSMV RNA species. These regions in RNA 2 and 3 are highly homologous and have no sequences in common with the respective region in RNA 1.

A study of barley stripe mosaic virus (BSMV) genome. I. Determination of sequence homology between BSMV RNA species.

The sequence homology between individual RNA species of three-component Norwich strain of BSMV (BSMV-N) has been studied by kinetic hybridization analysis using complementary DNAs obtained for each of the three BSMV-N RNA species by the method of Taylor et al. (1976). No significant sequence homology could be detected between RNA 1, on the one hand, and RNA2 and RNA3, on the other, whereas RNA2 and RNA3 were found to be highly homologous.