Molecular characterization of cucumber mosaic virus isolates infecting tomato in Hamedan and Tehran provinces of Iran.

Here we identified four isolates, MS, 3H, 50A, and 2K of cucumber mosaic virus (CMV) infecting tomato, on the basis of their non-coding intergenic region and a part of the coat protein (CP) sequence in the CMV genomic RNA3. The sequences from the four isolates were compared with other previously characterized isolates of CMV isolated from different plant species across the globe. Sequence comparisons revealed that the two CMV isolates from Hamedan province (MS and 3H) had the highest sequence identity with CMV-G10 (98%), which was previously reported as a severe Hellenic tomato isolate of CMV, while the CMV isolates from Tehran province, including CMV-2K (isolated from Karaj region) and CMV-50A (isolated from Varamin region), had the highest sequence identity with that of CMV-ALF (99%). Phylogenetic analysis of the nucleotide sequences showed that CMV-MS and CMV-3H belong to group IB, while CMV-2K and CMV-50A belong to group IA. This is the first report on the molecular characterization of novel isolates of CMV infecting tomato plants in Iran.

The movement protein of cucumber mosaic virus traffics into sieve elements in minor veins of nicotiana clevelandii

The location of the 3a movement protein (MP) of cucumber mosaic virus (CMV) was studied by quantitative immunogold labeling of the wild-type 3a MP in leaves of Nicotiana clevelandii infected by CMV as well as by using a 3a-green fluorescent protein (GFP) fusion expressed from a potato virus X (PVX) vector. Whether expressed from CMV or PVX, the 3a MP targeted plasmodesmata and accumulated in the central cavity of the pore. Within minor veins, the most extensively labeled plasmodesmata were those connecting sieve elements and companion cells. In addition to targeting plasmodesmata, the 3a MP accumulated in the parietal layer of mature sieve elements. Confocal imaging of cells expressing the 3a-GFP fusion protein showed that the 3a MP assembled into elaborate fibrillar formations in the sieve element parietal layer. The ability of 3a-GFP, expressed from PVX rather than CMV, to enter sieve elements demonstrates that neither the CMV RNA nor the CMV coat protein is required for trafficking of the 3a MP into sieve elements. CMV virions were not detected in plasmodesmata from CMV-infected tissue, although large CMV aggregates were often found in the parietal layer of sieve elements and were usually surrounded by 3a MP. These data suggest that CMV traffics into minor vein sieve elements as a ribonucleoprotein complex that contains the viral RNA, coat protein, and 3a MP, with subsequent viral assembly occurring in the sieve element parietal layer.

Spontaneous change of a benign satellite RNA of cucumber mosaic virus to a pathogenic variant.

Plant satellite RNAs generally reduce the level of helper virus accumulation and attenuate the disease symptoms induced by the helper virus that they depend upon for replication and packaging. As such, satellite RNAs could be used as biocontrol agents to reduce the level of disease in field crops, either by the application of a viral vaccine to healthy plants, or by the transgenic expression of satellite RNA in transformed plants. One such virus/satellite RNA system already under use in field tests is cucumber mosaic virus (CMV) and its satellite RNAs. However, in this system, some satellite RNAs also intensify viral disease in particular host plants. We passaged a satellite RNA of CMV with its helper virus to determine whether a satellite RNA that attenuates CMV-induced disease on tobacco plants could mutate to a pathogenic form, which might then be selected. In several experiments involving strains of CMV from each of the two subgroups, the satellite rapidly mutated to a pathogenic form, which was selected. This demonstrates an inherent risk associated with the use of attenuating satellite RNAs as a form of biocontrol of CMV.

Pathogenicity regulation by satellite RNAs of cucumber mosaic virus: minor nucleotide sequence changes alter host responses.

Six satellite RNAs of cucumber mosaic virus (CMV) could be differentiated on the basis of symptom expression they elicit in tomato and tobacco, and all but two could be differentiated by gel electrophoretic migration. Three of the satellite RNAs (B2-sat, G-sat, and WL1-sat RNA) ameliorated the symptoms induced by CMV on tomato, whereas three others (B1-sat, B3-sat, and WL2-sat RNA) induced chlorosis on tomato, the extent and nature of which was CMV-strain dependent. By contrast, B2-sat RNA induced chlorosis in tobacco, whereas WL1-sat and G-sat RNAs did not. Thus, the symptoms observed were dependent on the host species, the particular satellite RNA, and also the strain of helper virus, suggesting that a complex association of at least three factors is involved in symptom elicitation. Comparisons of the nucleotide sequences of pairs of satellite RNAs inducing the various chlorotic responses suggest that only a few nucleotide changes in specific domains are required for the elicitation of different host responses.

Mapping determinants within cucumber mosaic virus and its satellite RNA for the induction of necrosis in tomato plants.

A mutant of the WL47 satellite (WL47-sat) RNA of cucumber mosaic virus (CMV), constructed in vitro, induces lethal necrosis in tomato plants when associated with either of two subgroup II strains of CMV: LS- or WL-CMV (D. E. Sleat and P. Palukaitis, Proc. Natl. Acad. Sci. USA 87:2946-2950, 1990). The phenotype of this mutant (WLM2-sat RNA) has been examined on tomato plants, after co-inoculation with a wider range of CMV strains. Necrosis was observed when WLM2-sat RNA was associated with any of the subgroup II CMV strains tested. However, WLM2-sat RNA ameliorated the symptoms induced by subgroup I CMV strains without inducing necrosis. Inoculation with WLM2-sat RNA with pseudorecombinants formed between subgroup I and subgroup II CMV strains showed an association of the necrosis induction phenotype with RNA 2 of the subgroup II strains. In contrast to WLM2-sat RNA, infectious transcripts of the naturally necrogenic D4-sat RNA induced lethal necrosis in tomato with all CMV strains tested. Experiments involving the exchange of sequences between WLM2- and D4-sat RNAs indicated that nucleotides sequences in either of two separate regions could influence the necrosis phenotype. Thus, the subgroup-specific necrosis-inducing phenotype may be due to subtle alterations in secondary and/or tertiary structure in the satellite RNA, as well as the presence or absence of particular nucleotide sequences.

The chlorosis-induction domain of the satellite RNA of cucumber mosaic virus: identifying sequences that affect accumulation and the degree of chlorosis.

A number of satellite RNAs of cucumber mosaic virus (CMV) can induce chlorosis in either tobacco or tomato. A region containing the chlorosis domain was delimited, and one nucleotide position (153) regulating chlorosis induction as well as the nucleotide position (149) controlling the host specificity for chlorosis were identified. A cDNA clone of the B5-sat RNA was modified by site-directed mutagenesis to identify other nucleotides affecting chlorosis itself, as well as the extent of chlorosis and the helper virus-strain specificity for chlorosis. Four nucleotides conserved in chlorosis-inducing satellite RNAs (positions 127, 148, 149, 158) as well as two nucleotides within the chlorosis-induction domain that vary among chlorosis-inducing satellite RNAs (170, 171) were altered. Only substitutions at nucleotide positions 127 and 171 did not affect expression of the chlorosis phenotype. Alteration of position 148 affected satellite RNA accumulation. The pleiotropic effects of various sequences within the chlorosis-induction domain are discussed relative to particular sequence contexts in different chlorosis-inducing RNAs.

Cucumber mosaic virus-plant interactions: identification of 3a protein sequences affecting infectivity, cell-to-cell movement, and long-distance movement.

Mutants of the Cucumber mosaic virus (CMV) movement protein (MP) were generated and analyzed for their effects on virus movement and pathogenicity in vivo. Similar to the wild-type MP, mutants M1, M2, and M3, promoted virus movement in eight plant species. Mutant M3 showed some differences in pathogenicity in one host species. Mutant M8 showed some host-specific alterations in movement in two hypersensitive hosts of CMV. Mutant M9 showed altered pathogenicity on three hosts and was temperature sensitive for long-distance movement, demonstrating that cell-to-cell and long-distance movement are distinct movement functions for CMV. Four mutants (M4, M5, M6, and M7) were debilitated from movement in all hosts tested. Mutants M4, M5, and M6 could be complemented in trans by the wild-type MP expressed transgenically, although not by each other or by mutant M9 (at the restrictive temperature). Mutant M7 showed an inability to be complemented in trans. From these mutants, different aspects of the CMV movement process could be defined and specific roles for particular sequence domains assigned. The broader implications of these functions are discussed.

Replicase-mediated resistance to potato virus Y in transgenic tobacco plants.

Nicotiana tabacum 'Turkish Samsun NN' plants were transformed with nuclear inclusion b (NIb) gene sequences of potato virus Y, O strain (PVYO). The full-length construct included an additional in-frame initiation codon contiguous to the putative N-terminal amino acid codon and a stop codon replacing the C-terminal amino acid codon. Of 13 independently transformed lines, four yielded 37 (out of 100) plants in the R1 generation that were resistant to PVYO infection. Progeny of 13 out of 15 of R1 plants tested expressed resistance in the R2 generation. Conversely, 30 independently transformed tobacco lines expressing essentially the same sequence but deleted for the Gly-Asp-Asp (GDD) motif were not resistant. Two other constructs encoding either the 5'-deleted or 3'-truncated NIb gene, but harboring the GDD motif, conferred resistance to PVYO in some tobacco plants. Despite the high level of nucleotide and amino acid identity shared by strains PVYO and PVYN for the NIb gene, PVYN replication was found in all PVYO-resistant plants. However, plants of one R2 line showed reduced PVYN replication.

Oligonucleotide probes in infectious bronchitis virus diagnosis and strain identification.

Genomic RNA fingerprints of infectious bronchitis virus (IBV) strains M41 and Conn46 were prepared to identify T1 RNase-resistant oligonucleotides 'unique' to each of the two IBV strains. Such oligonucleotides were subsequently eluted from the gels and their nucleotide sequences determined. When oligonucleotide probes of those sequences were synthesized and used in a dot-blot hybridization assay, the probes lacked IBV strain-specificity and reacted with the RNAs of homologous as well as heterologous IBV strains. Based on these results, the methods used in this study need to be applied to a large number of oligonucleotide probes, to find one or a few that might be suitable as IBV strain- or serotype-specific oligonucleotide probes.

Serological and molecular characterization of three enteric isolates of infectious bronchitis virus of chickens.

Three coronaviruses isolated from the intestines of laying chickens were partially characterized. Serological and molecular assays indicated that the enteric coronaviruses are infectious bronchitis virus (IBV) isolates. Although the three isolates were recovered from three unrelated chicken flocks, their RNase T1-resistant oligonucleotide fingerprints were almost identical. The three isolates were not neutralized by antisera specific to IBV serotype Connecticut, but their RNase T1-resistant oligonucleotide fingerprints closely matched the fingerprints of strain Conn-46, a Connecticut serotype. This and the co-fingerprinting data suggested that the three field isolates may have emerged from the Connecticut virus through mutation(s). The mutation(s) apparently involved the S1 protein gene that determines the virus serotype.

Potato spindle tuber viroid: Investigation of the long-distance, intra-plant transport route.

Potato spindle tuber viroid (PSTV) could be detected in tomato plants by dot-blot hybridization as early as 6-7 days postinoculation (p.i.), approximately 1 week before the appearance of symptoms. The viroid was always first detectable in the shoot tip of the plant and in leaves that were very close to the tip, followed temporally by detection in leaves further from the tip down to and eventually including the inoculated leaf. Although PSTV also moved down to the roots, it did not move into leaves below the inoculated leaf, unless such leaves were shaded to reverse the normal direction of movement of photosynthetic materials from these leaves. Shading the inoculated leaf had the effect of delaying the movement of PSTV out of the inoculated leaf. These results are all consistent with the rapid systemic movement of PSTV from the inoculated leaf to actively growing tissue via the phloem, a route also utilized by most plant viruses.

Characterization of cucumber mosaic virus. I. Molecular heterogeneity mapping of RNA 3 in eight CMV strains.

RNAs from 13 strains of cucumber mosaic virus (CMV) were divided into two groups on the basis of their ability to hybridize to cDNA of either Fny-CMV RNA or WL-CMV RNA. The extent of the cross-hybridization within one of these groups was analyzed by an RNA protection assay. A cDNA clone of RNA 3 of the Fny strain of CMV was placed in a transcription vector between bacterial promoters T3 and T7. Labeled, minus-sense RNA transcripts prepared from all or part of the cDNA to RNA 3 of Fny-CMV were annealed to the genomic RNA of each of a number of cucumoviruses and digested with RNases. The patterns of RNA fragments protected from digestion were specific for each CMV strain and revealed the extent and location of heterogeneity among the viruses as well as within the Fny-CMV natural population. This approach will allow the differences in host range and disease processes to be correlated with variations in genomic RNAs.

The nature and biological significance of linear potato spindle tuber viroid molecules.

"Naturally occurring" linear potato spindle tuber viroid (PSTVL) was shown to be as infectious as circular PSTV (PSTVc). The occurrence of PSTVL was shown not to be (a) an artifact of the extraction procedure per se; (b) due to the presence of metal ions in extraction buffers; or (c) related to the host species used for propagation, to a particular PSTV strain, or to the duration of infection. From labeling and blot-hybridization experiments, it was concluded that in infected tissue PSTVc appears first followed by PSTVL, which is produced as the result of cleavage of PSTVc at specific sites. One of the sites of cleavage of PSTVc was delineated by 5'-end labeling PSTVL, digesting it with RNase T1, separating the fragments by two-dimensional gel electrophoresis, and sequencing the major RNase T1-resistant fragments. The major site of in vitro labeling was shown to be variable, but was confined to the right-hand loop of PSTVc, i.e., between nucleotides 177 and 182. Furthermore, the right-hand end stem and loop of PSTVc contains sequences which are similar to a rDNA gene promoter sequence, and thus we suggest that DNA-dependent RNA polymerase I may be involved in viroid biosynthesis. Other cleavage sites in PSTV were determined by primer extension cDNA synthesis and by dideoxynucleotide chain termination, and were shown to correspond to nucleotides 113-114 and 80-81 and to sequences in the region of 334-340, 300-312, and 271-275. The significance of these cleavage sites is discussed.

Biological activity of T7 transcripts of a prototype clone and a sequence variant clone of a satellite RNA of cucumber mosaic virus.

Molecular cloning of the (D)CARNA 5 (previously known as n-CARNA 5) necrosis-inducing satellite RNA of cucumber mosaic virus produced a prototype clone (pDsat4) and a sequence variant clone (pDsat1). pDsat1 contained 10 nucleotide changes between positions 70 and 160 which rendered that region identical to the corresponding region of a satellite RNA which does not induce necrosis. T7 RNA polymerase transcripts of each clone replicated in both tobacco and tomato, and the progeny satellite RNAs did not retain the 57-nucleotide non-satellite sequence at the 5' ends of the T7 transcripts. RNase T1 fingerprint analysis of both T7 transcripts and progeny satellite RNAs proved that the satellite sequence portion of each transcript was faithfully replicated in tobacco, and the variations in pDsat1 relative to pDsat4 were maintained. Replication of transcripts of either pDsat4 or pDsat1 in tomato resulted in lethal necrosis, suggesting that the determinant of necrosis induction lies outside the region between nucleotides 70 and 160.

RNA sequence heterogeneity in natural populations of three satellite RNAs of cucumber mosaic virus.

Sequence heterogeneity within populations of three satellite RNAs of cucumber mosaic virus (CMV) was assessed using two different approaches. Comparison of the nucleotide sequences of several cDNA clones of each satellite RNA revealed microsequence heterogeneity, which is often seen in populations of RNA genomes. RNase protection assays using minus-sense satellite RNA probes were used to detect major sites of heterogeneity within natural populations of each satellite RNA. In RNase protection assays of WL1-sat RNA populations, no major sites of heterogeneity were detected within seven different populations, including preparations from four different host plant species. In contrast, RNase protection assays of nine populations of B1-sat RNA showed three different patterns, which were most likely due to the existence of the B1-sat RNA populations as mixtures in which different sequence variants predominated in different preparations. Assays of five independent populations of D-sat RNA revealed a single major site of heterogeneity which was common to each population and was localized at approximately nucleotide 225 of the 335-nucleotide satellite sequence. This common site of heterogeneity is a feature of the D-sat RNA population structure which may represent an equilibrium between alternative nucleotides at a selectively neutral position, or may be actively selected for and maintained.

Satellite RNAs of cucumber mosaic virus: characterization of two new satellites.

Two new satellite RNAs of cucumber mosaic virus (CMV) which did not induce necrosis on tomato in the presence of CMV, B-sat RNA, and WL-sat RNA, were shown to be related by sequence to two well-characterized satellite RNAs of CMV: G-sat RNA (non-necrotic on tomato) and n-CARNA 5 (necrotic on tomato). Using the techniques of molecular hybridization analysis, RNA fingerprinting and partial RNA sequencing, B-sat RNA and WL-sat RNA were shown to be more closely related to each other (probably differing by only a small number of nucleotides) than to the other two satellite RNAs. Furthermore, B-sat RNA and WL-sat RNA showed greater sequence homology with G-sat RNA than with n-CARNA 5. WL-sat RNA, which induces a "white-leaf" disease on tomato in the presence of CMV [Gonsalves et al. (1982)., exhibited heterogeneity of sequence in at least one nucleotide position.

Serial passage of infectious transcripts of a cucumber mosaic virus satellite RNA clone results in sequence heterogeneity.

Populations of the D-satellite RNA (D-sat) of cucumber mosaic virus (CMV) have a major site of heterogeneity at which a significant proportion of the molecules vary from each other. The generation of this heterogeneity was investigated by using infectious transcript RNA of a prototype D-sat clone as an inoculum of uniform sequence to initiate serial passages in five different host plant species. RNase protection assays indicated that heterogeneity at the same site seen in natural D-sat RNA populations developed within two to four passages in most of the hosts tested, and the proportion of the population exhibiting this heterogeneity accumulated to a much higher level in tobacco than in the other hosts. Characterization of the heterogeneity which developed in tobacco showed it to be qualitatively different from that in the original D-sat population. This analysis of a CMV satellite RNA is the first example in which infectious transcript RNA from a clone has been used to investigate the rapid generation and selection of heterogeneity in a genomic RNA population replicating in plants.

Transgenically expressed cucumber mosaic virus RNA 1 simultaneously complements replication of cucumber mosaic virus RNAs 2 and 3 and confers resistance to systemic infection.

Tobacco plants transformed with a cDNA copy of RNA 1 of the Fny strain of cucumber mosaic virus (CMV) promoted the asymptomatic accumulation of inoculated viral RNAs 2 and 3, which could be detected in noninoculated leaves, suggesting that the transgene also permitted viral long-distance movement. Typical symptoms of infection appeared later and correlated with the appearance of viral RNA 1 regenerated from the transgenic mRNA. Although all R0-generation plants were susceptible to Fny-CMV, one line displaying variable susceptibility to the virus in R1-and R2-generations led to selected R3-generation lines with systemic resistance to Fny-CMV. In the inoculated leaves of resistant plants, a dramatic decrease in the accumulation of viral RNA 1 was observed, relative to susceptible plants. No viral RNAs were detected in noninoculated leaves of the resistant plants, but such leaves were susceptible to infection. Furthermore, these leaves could sustain replication of inoculated CMV RNAs 2 and 3, indicating that a complete transgene-silencing had not been induced. Although a transgene-mediated, CMV RNA 1-suppression occurred in the inoculated leaf of resistant plants, the absence of a complete systemically acquired silencing suggests the existence of additional interferences with viral infection that prevented systemic infection by viral RNAs 2 and 3.

Comparison of the nucleic acid- and NTP-binding properties of the movement protein of cucumber mosaic cucumovirus and tobacco mosaic tobamovirus.

The cucumber mosaic virus (CMV) 3a movement protein (MP) was compared directly to the well-characterized tobacco mosaic virus (TMV) 30K MP by cloning the genes encoding these proteins into Escherichia coli, isolating the E. coli-expressed MPs, and characterizing them with regard to RNA- and NTP-binding activities. The two MPs were shown to bind single-stranded RNA and DNA cooperatively, but with no sequence specificity. However, discrete lengths of CMV RNA 3 could be protected against RNase digestion by the CMV 3a protein, indicating that the RNA was not uniformly covered by the MP after cooperative binding. The TMV 30K:RNA complex was more stable in NaCl than the CMV 3a:RNA complex; about 50% of the corresponding complexes were stable in 0.6 and 0.4 M NaCl, respectively. Both MPs could bind GTP strongly and UTP weakly, but not ATP or CTP. The CMV 3a protein expressed either in E. coli or in planta from RNA 3 of CMV was tagged at its C-terminus with six histidine residues, which facilitated its purification by affinity chromatography on a matrix containing Ni(2+)-nitrilotriacetate. The soluble, His-tagged 3a proteins, affinity-purified from E. coli and zucchini squash, both were able bind CMV RNA 3 in vitro.