Alfalfa Mosaic Research Articles

The 5′ Terminal Sequence of Alfalfa Mosaic Virus RNA 3 Is Dispensable for Replication and Contains a Determinant for Symptom Formation

Transgenic P12 tobacco plants, transformed with the replicase genes P1 and P2 of alfalfa mosaic virus (AlMV), can be infected with RNA 3 of the tripartitite AlMV genome or with a DNA copy of RNA 3 fused to the CaMV 35S promoter andnosterminator. The effect of various modifications on the infectivity of the 35S/cDNA 3 construct to P12 plants was studied. When nonviral sequences ranging from 11 to 200 bp were inserted between the 35S promoter and cDNA 3, the infection became dependent on addition of coat protein (CP) to the inoculum. About 80% of the progeny RNAs resulting from these infections were full-length and had lost the nonviral sequence, whereas 20% were truncated by a deletion of the 5′ terminal 79 nucleotides (nt). When the sequence corresponding to the 5′ terminal 22 nt of RNA 3 was deleted from the 35S/cDNA 3 construct, the clone was as infectious as the wild type (wt), provided that CP was added to the inoculum, but only progeny RNA with a 5′ terminal deletion of 79 nt was produced. The 5′ truncated RNA 3 molecules induced necrotic ringspot-like symptoms on P12 tobacco plants, whereas wt RNA 3 did not induce detectable symptoms on these plants. It is proposed that in the infections with the modified 35S/cDNA 3 clones, CP is required in the inoculum to permit internal initiation of plus-strand RNA 3 synthesis on 3′-extended or 3′-truncated minus-strand RNA templates. Evidence was obtained that minus-strand RNA 3 synthesized under the control of the 35S promoter was not infectious to P12 plants.

Cis-Preferential Stimulation of Alfalfa Mosaic Virus RNA 3 Accumulation by the Viral Coat Protein

RNA 3 of alfalfa mosaic virus (AlMV) encodes the movement protein P3 and the viral coat protein (CP) which is translated from the subgenomic RNA 4. RNA 3 is able to replicate in tobacco plants transformed with the AlMV replicase genes P1 and P2 (P12 plants). Frameshifts or deletions in the P3 gene have little effect on RNA 3 accumulation in P12 protoplasts whereas such mutations in the CP gene result in a 100-fold reduction of plus-strand RNA 3 accumulation. When P12 protoplasts were inoculated with a mixture of a RNA 3 mutant with a deletion in the P3 gene and a mutant with a deletion in the CP gene, CP expressed by the P3 mutant was unable to upregulate plus-strand RNA accumulation of the CP mutant. However, when a wild-type CP gene and subgenomic promoter were inserted in a RNA 3 mutant with a defective CP gene, the mutant accumulated at wild-type levels. It is concluded that the function of CP in plus-strand RNA 3 accumulation actsin cisand cannot be complementedin trans.In P12 plants, P3 and CP mutants were able to complement each other at low and variable levels. This complementation in plants appeared to be correlated with the occurrence of recombination to wild-type RNA 3.

First Report of Alfalfa Mosaic Virus in Lupins in Australia

First Report of Alfalfa Mosaic Virus in Lupins in Australia

Transgenic plantlets of ‘Chancellor’ grapevine (Vitis sp.) from biolistic transformation of embryogenic cell suspensions

Transgenic plantlets of 'Chancellor' grapevine (Vitis L. complex interspecific hybrid) were produced via biolistic transformation. Embryogenic cell suspensions were bombarded with 1 μm tungsten particles coated with pBI426 which encodes a fusion peptide between β-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). The fusion peptide is under the control of a double 35S Cauliflower Mosaic Virus promoter and a leader sequence from Alfalfa Mosaic Virus. The cells were placed on kanamycin-containing media (10, 25 or 50 mg/l) 2 d after bombardment. Activated charcoal reduced cell browning. Embryos were first observed on selective media 14-29 weeks after bombardment. More than 1600 clusters of embryos were germinated and/or assayed for GUS. Of 621 embryos assayed for GUS expression, 182 (29.3%) were positive. PCR confirmed the presence of the NPTII gene in all 5 GUS-positive and 2 GUS-negative (bombarded) embryos tested. In germination experiments, 15% of the embryo clusters produced at least one plant with normal shoot growth. Of 164 normal plants assayed for GUS expression, 37 (22.6%) were positive. The NPTII gene was amplified by PCR in 1 (of 1) GUS-positive and 4 (of 5) GUS-negative bombarded plants, but not in non-bombarded control plants. Southern blotting confirmed integration of the NPTII gene in all 3 of the GUS and PCR-NPTII positive plants tested. Biolistics is an efficient method for transformation of 'Chancellor' and should be applicable to other important grape cultivars.

Interaction between RNA-Dependent RNA Polymerase of Alfalfa Mosaic Virus and Its Template: Oxidation of Vicinal Hydroxyl Groups Blocksin VitroRNA Synthesis

In the life cycle of a (+)-strand RNA plant virus the processes of template RNA recognition and initiation of the synthesis of a complementary strand by the viral RNA-dependent RNA polymerase (RdRp) are crucial early steps. Using a template-dependentin vitroRNA synthesizing system of alfalfa mosaic virus (AlMV) we were able to study the effect of small chemical modifications of the 3′ end of the template RNAs on product formation. After oxidation of the 3′-terminal nucleoside of the template no products could be detected. Presumably, RNA synthesis was blocked at the stage of initiation, since the promoter of the RdRp is internal (A. C. Van der Kuylet al., Virology176, 346–354, 1990). Blocking was probably due to an irreversible binding of the enzyme to the 3′ end of the modified RNA. Using this system it was shown that in template competition experiments the RdRp of AlMV displays a high specificity for its cognate template, either before or after the oxidation of the 3′-terminal nucleoside. From this it was concluded that periodate modification of the 3′-terminal nucleoside has little or no effect on template recognition. Furthermore, we showed that the viral coat protein, which forms a part of the viral polymerase (R. Quadtet al., Virology182, 309–315, 1991), was not the main target involved in the inhibition of RNA synthesis.

Characterization of Sequences Controlling the Synthesis of Alfalfa Mosaic Virus Subgenomic RNA in Vivo

RNA 3 of alfalfa mosaic virus encodes the movement protein P3 and the vital coat protein (CP) CP is translated from a subgenomic (sg) messenger, RNA 4. To characterize the sg promoter that is responsible for RNA 4 synthesis in vivo, putative sg promoter sequences were inserted in a unique Xhol site located between the initiation codon of the P3 gene and a second in-frame ATG codon in an infectious cDNA clone of RNA 3. Mutants with an active sg promoter insert expressed an N-terminally truncated P3 protein and were able to accumulate in plants, In addition, sg promoter activity was analyzed in protoplasts. When the transcription start site is taken as +1, the sequence of nucleotides -26/+1 was found to have a basal level of sg promoter activity, This activity was increased to near maximum levels when the sg promoter sequence was extended to -136/+12. The upstream positive regulatory element was mapped to nucleotides -136/-94. Engineering of point mutations and small deletions in RNA 3 around the transcription start site for RNA 4 synthesis revealed elements important for sg promoter activity with similarity to sequences conserved in sg promoters of alpha-like viruses. Some of these elements appeared to be required in cis for RNA 3 accumulation. A deletion of the C-terminal three amino acids of the P3 protein rendered this protein nonfunctional in cell-to-cell movement.

RNA duplex unwinding activity of alfalfa mosaic virus RNA-dependent RNA polymerase

An RNA-dependent RNA polymerase (RdRp) purified from alfalfa mosaic virus-infected tobacco is capable of synthesizing in vitro full-size RNAs of minus and plus polarities. However, the enzyme is not able to perform a complete replication cycle in vitro. The products were found to be completely base-paired to their templates. The enzyme was able to use double-stranded RNA as a template for RNA synthesis if it could initiate from a single-stranded promoter. The inability (of most) of our enzyme preparations to create a single-stranded initiation site could explain why they could not perform a complete replication cycle in vitro. This is the first report on duplex RNA unwinding activities by a plant viral RdRp.

In Vitro Evidence That the Coat Protein of Alfalfa Mosaic Virus Plays a Direct Role in the Regulation of Plus and Minus RNA Synthesis: Implications for the Life Cycle of Alfalfa Mosaic Virus

The coat protein of alfalfa mosaic virus has both structural and regulating functions. The latter is evident from the fact that the genomic RNAs of the virus, although they are of messenger polarity, cannot start an infection cycle in the absence of coat protein. The reason could be that the coat protein is needed for viral RNA synthesis. Indeed, the coat protein has been found in tight association with the viral RNA polymerase (R. Quadt et al., 1991, Virology 182, 309-315). To investigate the role of the coat protein, if any, in viral RNA synthesis, we have isolated the viral RNA polymerase (RNA-dependent RNA polymerase, RdRp) from mock-inoculated tobacco plants transformed with cDNAs 1 and 2, known as P12 plants (P. E M Taschner et al., 1991, Virology 181,687-693), which express the nonstructural proteins P1 and P2. Such an enzyme (called M-RdRp) will contain the viral subunits P1 and P2 but not the coat protein. As a comparison we also isolated the RdRp from virion-inoculated P12 plants (C-RdRp). This enzyme will contain the coat protein. We found that both M-RdRp and CRdRp could synthesize minus RNA, showing that coat protein is not needed for minus-strand synthesis. In contrast, minusstrand synthesis by both enzymes was inhibited by coat protein. Plus-strand synthesis was unaffected by coat protein in the case of C-RdRp, but strongly stimulated by coat protein in the case of M-RdRp. These data might explain why infected cells, which do not produce coat protein, display a very low accumulation of viral plus-strand RNA. They also give a possible explanation for the noninfectious character of the genomes of alfafa mosaic virus and ilarviruses in the absence of coat protein. The fact that an active enzyme could be isolated from the same membrane fraction in infected and noninfected P12 plants shows that coat protein is not needed for assembly and targeting of the viral RNA polymerase.

N-Terminal Basic Amino Acids of Alfalfa Mosaic Virus Coat Protein Involved in the Initiation of Infection

Alfalfa mosaic virus coat protein or its messenger RNA is required in the inoculum for virus infection. The N-terminus of the coat protein is required for activity; thus, changes were made in the amino acid sequence of this region. Six coat protein mutants were tested for activity in virus infection assays in protoplasts. A coat protein mutant in which N-terminal residues 3-19 were absent was inactive; whereas, a mutant in which residues 3-11 were absent (CPΔN9) still had 73% of wildtype activity. Substitution of alanine for the basic residues at positions 14, 17, and 15 in full-length coat protein and in CPΔN9 resulted in mutant proteins that were inactive in infection. Thus, one, two, or three of these basic residues in CP are required for activity.

The nucleotide sequence of RNA3 and RNA4 of olive latent virus 2.

Olive latent virus 2 (OLV2), a virus with particle shapes resembling those of alfalfa mosaic alfamovirus (AMV), has four major RNA species, two of which (RNA3 and RNA4) were completely sequenced. RNA3 was a bicistronic molecule containing two clear-cut ORFs, one of which (ORF1) coded for a 36.5 kDa polypeptide with conserved motifs of the '30K superfamily' movement proteins and the other (ORF2) encoded a 20 kDa polypeptide identified as the viral coat protein. RNA4, which was a little smaller than RNA3 (2078 nt versus 2438 nt), also differed from RNA3 in a few positions, but its in vitro translation did not produce any protein. By contrast, an additional RNA, 1042 nt in size with strong sequence homology with RNA3 and RNA4, was identified in RNA extracts from infected plants. This RNA, which may be a subgenomic form of RNA3 carrying the coat protein cistron, is apparently encapsidated to a very small extent, or not at all. Comparative computer-assisted analysis of virus-coded protein sequences disclosed homologies suggesting that OLV2 may belong to the family Bromoviridae, but as an entity separated from the currently known genera.

Replicase-Mediated Resistance to Alfalfa Mosaic Virus

Tobacco plants transformed with the P1 and P2 replicase genes of alfalfa mosaic virus (AIMV) have been shown to produce functional replicase proteins, permitting their infection with AIMV inocula lacking the genome segments encoding P1 and P2, respectively. To see whether expression of a mutant P2 protein would interfere with the assembly of a functional replicase complex, tobacco plants were transformed with modified P2 genes. When plants were transformed with a P2 gene encoding an N-terminally truncated protein which mimicked the tobacco mosaic virus 54K protein, no resistance was observed with 10 independent lines of transformants. Similarly, when the GDD motif in the full-length P2 protein was changed into VDD, no resistance was observed in 14 transgenic lines. However, when the GDD motif was changed into GGD (5 lines), GVD (15 lines), or DDD (13 lines), 20 to 30% of the transgenic lines showed a high level of resistance to AIMV infection. This resistance was effective to inoculum concentrations of 10 to 25 μg/ml of virus and 100 μg/ml of viral RNA, causing severe necrosis of control plants. For all transgenic lines, the expression of the transgenes was analyzed at the RNA level. With the GGD, GVD, and DDD mutants, resistance was generally observed in plants with a relatively high expression level. This indicates that the resistance is due to the mutant replicase rather than to an RNA-mediated cosuppression phenomenon.

Involvement of Residues Within Putative alpha Helix Motifs in the Behavior of the Alfalfa and Tobacco Mosaic Virus Movement Proteins

The movement proteins (MPs) of both alfalfa mosaic virus (AMV) and tobacco mosaic virus (TMV) have a 15-amino acid putative α helix motif that is part of a larger domain involved in the cell wall (CW) localization of these proteins. These α helices have common features since they are amphipathic and contain two clusters of acidic amino acids, EE and DE. Mutations were introduced into these helices to investigate their role in CW localization and in the activity of the MPs. Results showed that both these motifs are involved in the CW localization of the MPs, although through different mechanisms : whereas the helical structure and the EE cluster of the AMV-MP were required for optimum CW localization, the DE cluster of the TMV-MP, but not the helical structure, was involved in this process. Chimeric proteins resulting from an exchange of the α helices between MPs showed that these sequences did not function in the complementary background. Moreover, the region around aa 61 of the TMV-MP is necessary for protein stability. Transgenic tobacco plants expressing a mutated AMV-MP in which the α helix was deleted or had its structure destroyed exhibited an abnormal development and a modified morphology. In parallel, a similar mutation of the TMV-MP yielded a nonfunctional protein that still accumulated in the CW but that did not compete with the viral MP during infection.

Occurrence of alfalfa mosaic and subterranean clover red leaf viruses in legume pastures in Western Australia

When leaf samples were collected from 94 Trifolium subterraneum (subterranean clover) pastures from six districts in spring 1993 in the south-west of Western Australia and tested by enzyme-linked immunosorbent assay, no alfalfa mosaic virus (AMV) or subterranean clover red leaf virus (SCRLV) was detected. In contrast, when 21 irrigated T. repens (white clover) pastures from one district (Bunbury) were sampled and tested in January (summer) 1994, AMV was detected in 16, with eight having infection levels >86%, while SCRLV was found in seven at infection levels of <12%. When a further five T. repens pastures were tested for AMV in October (spring) 1994, the virus was found in all with incidences up to 100%. None of the T. repens pastures with high levels of AMV infection had been resown with T. repens within the last 20 years, whereas those resown within the last five years had little or no infection. AMV was detected in 9/91 annual medic (Medicago spp.) pastures from seven wheatbelt districts sampled in spring 1991 or 1993; a single pasture of M. polymorpha (burr medic) cv. Serena was 21% infected, but the other eight infected ones had <3%. AMV seed transmission was detected in 1/19 commercial seed stocks of M. polymorpha harvested in 1991-93. AMV infection was followed over a 12-year period in M. murex (murex medic) cv. Zodiac seed stocks. It persisted readily through successive seed harvests during this period. It is concluded that infection with AMV and SCRLV is currently not a threat to T. subterraneum pastures in the south-west of Western Australia and that AMV seems not to be one in wheatbelt annual medic pastures provided these are sown with healthy medic seed. In contrast, AMV poses a potential threat to the productivity of irrigated T. repens pastures. SCRLV is also sometimes present in T. repens pastures, but was not found at serious levels.

Studies on seed and pollen transmission of Alfalfa Mosaic, Cucumber Mosaic and Bean Yellow Mosaic Viruses in cultivars and accesions of annual Medicago species

Seed and pollen transmission of alfalfa mosaic (AMV), cucumber mosaic (CMV) and bean yellow mosaic (BYMV) viruses was investigated in annual medic species (Medicago spp.). For seed transmission studies with AMV, graft inoculation was used to establish early infection and maximize possible transmission rates to seedlings via seed, but with CMV and BYMV aphid and/or graft inoculation was used. For pollen transmission studies, pollen taken from virus-infected plants was used to pollinate healthy plants, the seed collected and seedlings tested. The rates of AMV isolate OUI-2 transmission to seedlings through seed produced on infected plants ranged from 6 to 53% for commercial cultivars and from 7 to 65% for accessions. Accession DZA 3181.1.1 of M. sphaerocarpos had the highest overall AMV transmission rate. Only two cultivars, cvv. Borung and Hannaford of M. truncatula, and accession SA 4268 of M. orbicularis, had transmission rates of less than 10%. The rates of CMV transmission to seedlings via seed produced on infected plants of the cultivars and accessions tested were 0.3 to 13%, the greatest being found in M. polymorpha cv. Serena, but 6 out of 11 had no detectable transmission. The rates of BYMV transmission to seedlings via seed of the cultivars and accessions tested were 0.3 to 1%, but in 12 out of 15 none was detected. AMV isolate OUI-2 was transmitted to 52% of seedlings via seed produced on healthy M. polymorpha cv. Circle Valley plants pollinated from infected plants. In contrast, no transmission to seedlings by either graft-inoculation or pollination of M. polymorpha plants was detected with a second AMV isolate, OUI-1, which appeared to have lost its ablilty to be seed transmitted. No CMV or BYMV transmission to seedlings via pollination of healthy plants with pollen from infected plants was detected in M. polymorpha cvv. Circle Valley or Santiago. When empty immature pods, and dissected seed coats and embryos from immature seeds produced on AMV-infected plants of M. polymorpha were tested, AMV isolates OUI-I and OUI-2 were detected in all pods and seed coats, but only in 59% of embryos with isolate OUI-2 and in none with isolate OUI-1. CMV was detected in 12% of embryos tested from immature seeds produced on CMV-infected M. polymorpha cv. Serena plants. Transmission of all three viruses through seed, and of AMV through pollen, is cause for concern in annual medic breeding and evaluation programs. Moreover, carry-over outside the growing season in medic pastures is possible through seed with all three viruses.

Mapping of the RNA-binding Domain of the Alfalfa Mosaic Virus Movement Protein

In-frame contiguous deletions were created in the movement protein gene of alfalfa mosaic virus by site-directed mutagenesis. The mutated movement proteins were expressed in Escherichia coli, extracted and then purified by denaturing gel electrophoresis and then renatured. Their binding ability with RNA was assayed by electrophoretic retardation and u.v.-crosslinking. Results indicated that a domain included within amino acids 36 to 81 was necessary for RNA binding.

A Chromatographic Analysis of Capsid Protein Isolated from Alfalfa Mosaic Virus: Zinc Binding and Proteolysis Cause Distinct Charge Heterogeneity

The capsid protein (CP) of alfalfa mosaic virus (AIMV) is required for viral replication when susceptible plants are inoculated with purified vital genomic RNA. The discovery of AIMV CP in the zinc activated RNA-dependent RNA polymerase complex prompted our further investigation of AIMV virions and the potential involvement of AIMV CP in metal binding. AIMV CP, isolated from nucleoprotein components, fractionated into four distinct ionic species when purified by cation exchange fast protein liquid chromatography. The CP existed as zinc complexed homodimers, metal-free homodimers, and two forms of proteolyzed heterodimers, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography, amino-terminal sequencing, and atomic absorption spectroscopy. Although the relative amounts of proteolyzed heterodimers varied, the ratio of zinc complexed homodimers to metal-free homodimers (1:10) was constant between virus and protein isolations for the strains 425 and WISC14. Purified metal-free and zinc-complexed homodimers could be interconverted in vitro by incubation with zinc chloride or with the metal chelator, sodium diethyldithiocarbamate (NaDDC). The potential role of zinc in AIMV nucleoprotein structure and infectivity was investigated by treatment of the virions with NaDDC. Electron microscopy and sucrose density gradient studies failed to detect any gross structural changes for zinc depleted virus; however, a decrease in infectivity was observed with local lesion leaf assays, suggesting a functional role for zinc in viral replication.

Plants Transformed with a Mutant Alfalfa Mosaic Virus Coat Protein Gene Are Resistant to the Mutant but Not to Wild-Type Virus

Transgenic tobacco plants expressing the wild-type (wt) coat protein (CP) gene of alfalfa mosaic virus (AIMV) have been shown to be resistant to infection with vital particles and RNAs or to infection with viral particles only. The difference in resistance of these plants to RNA inocula was found to correlate with a difference in the expression level of the transgene. Plants expressing a mutant AIMV CP with the N-terminal serine residue changed to glycine have been shown to be susceptible to infection with wt viral particles or RNAs. By site-directed mutagenesis of AIMV cDNA a viable mutant virus encoding CP with the same N-terminal mutation was obtained. Plants expressing wt or mutant CP were resistant to the mutant virus, demonstrating that a single amino acid substitution in CP did not permit the virus to overcome CP-mediated resistance. Although the mutant CP did not confer resistance to wt virus when expressed in transgenic plants, it was still effective in classical cross-protection: plants infected with the mutant virus were resistant to a severe strain of AIMV. A model to explain the data is discussed.

Early and Late Functions of Alfalfa Mosaic Virus Coat Protein Can Be Mutated Separately

To investigate the role of alfalfa mosaic virus coat protein (CP) in genome activation, asymmetric plus-strand RNA accumulation, and cell-to-cell spread of the virus, mutations were made in the CP gene and putative CP binding sites in the 3′-untranslated region (UTR) of RNA 3. Mutants that produced no CP-related peptide or CP with an N-terminal deletion of 20 amino acids were defective in all three functions. Insertion of several nonviral amino acids at position 85 of CP had little effect on genome activation and plus-strand RNA accumulation but abolished cell-to-cell spread. A mutant encoding CP with a C-terminal deletion of 21 amino acids was defective in plus-strand RNA accumulation but showed substantial levels of genome activation and cell-to-cell spread. Mutations in the 3′-UTR that interfered with CP binding affected plus-strand RNA accumulation and cell-to-cell spread. Neither CP nor CP binding sites at the 3′-end of RNA 3 were required for minus-strand RNA accumulation. The results demonstrate that early and late functions of CP can be mutated separately, indicating that different domains of CP are involved in the three functions investigated.

Genetics of Resistance to Peanut Stunt, Clover Yellow Vein, and Alfalfa Mosaic Viruses in White Clover

Peanut stunt virus (PSV), clover yellow vein virus (CYW), and alfalfa mosaic virus (AMV) reduce white clover (Trifolium repens L.) yield and persistence in the southeastern U.S. Southern regional virus resistant (SRVR) germplasm is the only white clover with resistance to these viruses, but little is known about the genetics of this resistance. Our objective was to determine the relative importance of general combining ability (GCA), specific combining ability (SCA), maternal effects, and nonmaternal reciprocal effects in the inheritance of resistance to PSV, CYW, and AMV in a diallel cross of one Tillman' and seven SRVR plants with differing virus susceptibilities. Progeny were grown in the greenhouse in three separate experiments and were inoculated with PSV, CYW, or AMV. Plants were evaluated for resistance by visual symptoms and either inoculation of ‘California Blackeye’ cowpeas, Vigna unguiculata (L.) Walp. subsp. unguiculata (for PSV and AMV) or enzyme‐linked immunosorbent assay (ELISA; for CYW). Differences among crosses for PSV, CYW, and AMV resistance were due to GCA, SCA, and nonmaternal reciprocal effects. For PSV and CYW resistance, additive genetic effects were more important than any other effects. For AMV resistance, nonadditive genetic effects and nonmaternal reciprocal effects were also important. Direction in which a cross is made is not important, because there were no consistent male or female effects of parents involved in more than one significant reciprocal effect. For this group of parents, breeding procedures utilizing additive genetic effects should be the most effective in improving the PSV, CYW, and possibly AMV resistance of white clover.

The role of the 3'-untranslated region of non-polyadenylated plant viral mRNAs in regulating translational efficiency

Tobacco mosaic virus (TMV) is a positive-sense RNA virus in which the single genomic RNA functions as a messenger RNA. It is a member of a class of plant viral RNAs that are the only known non-polyadenylated mRNAs in plants. The 3'-untranslated region ( UTR) of TMV genomic RNA is the functional equivalent ofapoly(A) tail in that it increases mRNA stability and regulates translational efficiency. To determine whether the 3'- UTR of other non-polyadenylated plant viral mRNAs regulate translation, those from turnip yellow mosaic (TYMV), brome mosaic (BMV), and alfalfa mosaic (A1MV) viruses were investigated. Chimeric gene constructs were made in which the viral 3'- UTRs were introduced immediately downstream from the reporter genes encoding β-glucuronidase (GUS) and luciferase (LUC), and were translated in plant protoplasts following delivery of the mRNA using electroporation. The 3'- UTR from BMV RNA3 regulated reporter gene expression in vivo to an extent comparable to that observed for the TMV 3'- UTR. The BMV 3'- UTR increased both message stability and translational efficiency. As regulators of translation, the BMV and TMV 3'- UTR were dependent on the presence of a cap at the 5' terminus for function. The 3' UTR of TYMV or A1MV RNA4 had little impact on translation or transcript stability. These data suggest that although the TMV 3'- UTR is not unique in regulating translation, the 3'- UTR of plant viral mRNAs do vary in their regulatory ability.