J. Roosien

Evidence that alfalfa mosaic virus infection starts with three RNA-protein complexes

The tripartite genome of alfalfa mosaic virus (AMV) needs to be activated by its coat protein. To establish whether coat protein exerts its role by interacting structurally with one, two, or all three AMV-RNA species, the infectivity of mixtures of RNA-protein complexes with free RNAs were studied (Smit and Jaspars, Virology 104, 454-461, 1980). These studies were not fully conclusive since some redistribution of coat protein does occur as soon as RNAs are brought into contact with RNA-protein complexes. This problem was overcome by the use of ts mutants of AMV. Free ts coat protein subunits were not able to activate the wt genomic RNAs at 30 degrees in tobacco. Once complexed to the genomic RNAs at 0 degrees , the biological activity of the ts coat protein remained when assayed at the nonpermissive temperature. Apparently, the ts coat protein is inactivated during attempted redistribution at 30 degrees . Studies of mixtures of RNA-protein complexes and free RNA show that coat protein has to be present on at least two of the three RNA species. This result in combination with previous results (Smit and Jaspars, 1980) warrants the conclusion that the alfalfa mosaic virus infection starts with three RNA-protein complexes.

Description and Complementation Analysis of 13 Temperaturesensitive Mutants of Alfalfa Mosaic Virus

Summary Thirteen thermosensitive (ts) mutants of alfalfa mosaic virus (AMV), a virus with a tripartite genome, are described. Eight of these mutants were spontaneous, one was induced with HNO2 and four were induced by u.v. irradiation of one purified component. Using supplementation tests six ts mutations were located on top component b (Tb), three on middle component (M) and four on bottom component (B). Complementation tests with mutants with ts defects on the same component showed that none of the ts mutants on Tb could complement each other; the three ts mutants on M could be subdivided into two complementation groups, while only one pair of mutants showed complementation from the four ts mutants on B. It was demonstrated that the coat proteins from the six ts mutants on Tb were not able to activate the AMV genome at 30 °C in tobacco.

Complementation and Interference of Ultraviolet-induced Mts Mutants of Alfalfa Mosaic Virus

Summary Ten new thermosensitive (ts) mutants of alfalfa mosaic virus (AMV), a plant virus with a tripartite genome, are described. Stable ts mutants were induced by u.v. irradiation of purified middle (M) component. Mutants were isolated by subculturing in bean plants (instead of in the usual plant host, tobacco), in which unstable ts mutants apparently arise spontaneously. As well as ts mutants, we found a mutant which in contrast to the parent strain (AMV 425) was able to infect bean plants systemically. As expected, all stable mutations mapped on M component. Complementation analysis confirmed the presence of two complementation groups on M component. This complementation is probably intracistronic. Furthermore, we found that mutants belonging to the same complementation group often interfered with the multiplication of each other, even at the permissive temperature. Taken together, these results could suggest that the product directed by AMV RNA 2 (the RNA inside M component) has two functional domains and is active in a multimeric form.

Replication of temperature-sensitive mutants of alfalfa mosaic virus in protoplasts.

Six mutants of alfalfa mosaic virus (AIMV), previously found to have conditionally lethal defects when inoculated to tobacco leaf discs, were assayed for a temperature-sensitive (ts) phenotype in cowpea protoplasts. At 30 degrees the virus production of four mutants was less than 10% of that obtained at 25 degrees , whereas wild-type AIMV multiplied with similar efficiencies at both temperatures. Supplementation experiments performed in protoplasts with two mutants indicated the presence of mutations on both RNA 1 and RNA 2. Viral plus- and minus-strand RNA synthesis induced by these mutants was monitored by the Northern blotting technique under various supplementation conditions. The ts defect was mainly in the production of viral minus-strand RNA whereas transcription of minus-strand into genomic and subgenomic RNAs was much less affected. In addition, results indicate that replication of RNA 1 requires a function not essential for the synthesis of the other RNAs. One of the mutants showed a host-dependent expression of ts mutations, probably reflecting that host-viral interactions play an important role in A1MV replication.

A mutant of alfalfa mosaic virus with an unusual structure

A spontaneous mutant of alfalfa mosaic virus (AMV) with an altered structure is described. By analysis of pseudorecombinants the mutation(s) responsible for the altered structure were assigned to RNA 3. By in vitro translation and serology it is shown that both proteins (35K protein and coat protein) encoded by RNA 3 are changed. The mutation(s) present in RNA 3 also have an effect on the ratio of the three genomic RNAs, both in virion as well as in double-stranded RNA preparations. Compared to wild-type particles mutant particles (1) have a lower electrophoretic mobility in polyacrylamide gels, (2) have a greater sedimentation velocity in sucrose density gradients, (3) do contain the same percentage of RNA, (4) are more prone to aggregation, (5) are somewhat less stable during storage, and (6) are less sensitive to uncoating by AMV-RNA. Electron micrographs show that the mutant preparations contain some very long bacilliform particles, however the majority of the particles is spheroidal and bear a strong resemblance to the particles of the ilarviruses. The structural properties of this mutant support the classification of AMV as an ilarvirus.

An alfalfa mosaic virus RNA 2 mutant, which does not induce a hypersensitive reaction in cowpea plants, is multiplied to a high concentration in cowpea protoplasts

A mutant of alfalfa mosaic virus (AMV), which in contrast to wild type (wt) can invade cowpea plants systemically, is replicated more efficiently in cowpea protoplasts than the wt. Mutant preparations isolated from infected cowpea protoplasts contained a higher amount of middle component (M, containing RNA 2) than wt preparations. Both in cowpea plants and in cowpea protoplasts a wt phenotype is obtained upon addition of wt M to this mutant, suggesting a correlation between the type of plant reaction evoked by the virus infection and the regulation of viral RNA synthesis.

Towards Plantibody-Mediated Resistance Against Nematodes

The lack of available resistance genes severely hampers the introduction of durable disease resistance in plants. Therefore we explored the feasibility to obtain resistance by the expression in plants of monoclonal antibodies. The rational behind this idea is that by binding to its antigen a monoclonal antibody is capable to inactivate the biological activity of that antigen. As a model system we have chosen the interaction between potato and potato cyst nematodes while saliva proteins of nematodes served as antigen. These proteins are thought to play an important role in this interaction. Inhibition of the biological activity of these proteins might interupt the interaction which results in resistance.

Competition between the RNA 3 molecules of wildtype alfalfa mosaic virus and the temperature-sensitive mutant Tbts 7(uv)

SummaryIn mixed infections of wildtype (wt) alfalfa mosaic virus (AMV) and a temperature-sensitive mutant Tbts 7(uv), which carries a thermosensitive defect in the early function of the coat protein, the mutant symptoms were not found at 30°C. In the progeny from these mixed infections almost no mutant coat protein and no mutant RNA 3 could be detected. Even at 23°C there was some loss of mutant RNA 3 and coat protein from the progeny of the mixed infections. Analysis and comparison of mutant and wt ds RNA preparations revealed a lower ds RNA 3 content for the mutant preparation at 23°C. Also the amount of RNA 3 in virion preparations was lower for the mutant than for wt. These results point to a mutation in the RNA 3 of Tbts 7(uv) which diminishes its affinity for the viral replicase.

Flow cytometric analysis of tobacco and cowpea protoplasts infected in vivo and in vitro with alfalfa mosaic virus

Immunofluorescence flow cytometry was used to study the distribution of viral antigen in protoplast populations. Protoplasts were isolated from healthy and alfalfa mosaic virus (AMV) infected tobacco leaves (designated in vivo infected). Furthermore isolated tobacco and cowpea protoplasts were infected in vitro with AMV. The FITC-conjugated antibodies could penetrate formaldehyde fixed protoplasts. The flow cytometric measurements were rapid and reproducible. Comparable immunofluorescence patterns were found for all infected samples (per sample 104 protoplasts were measured). Infectious virus could only be detected in in vivo infected tobacco protoplasts and in in vitro infected cowpea protoplasts.

Structure and Function of Plant Virus Genomes

When the presently classified viruses are grouped according to their genetic material (DNA, RNA, double-stranded, single-stranded, plus — or minus-type) we see (Table 1) that the majority of plant viruses has a genome consisting of single-stranded RNA of the plus polarity (virion RNA has the same polarity as mRNA). Within this group both the structural features (number of genome parts, structure present at the 5’ or the 3’ termini of the RNA) as well as the strategy of expression are extremely diverse (Table 2)2,3 (and references therein).