Transmission of Citrus leprosis virus C by Brevipalpus phoenicis (Geijskes) to Alternative Host Plants Found in Citrus Orchards.

The equivalent of US$75 million is spent each year in Brazil to control Brevipalpus phoenicis, a mite vector of Citrus leprosis virus C (CiLV-C). In this study, we investigated the possibility that hedgerows and windbreaks normally found in citrus orchards could host CiLV-C. Mites confined by an adhesive barrier were reared on sweet orange fruit with leprosis symptoms then were transferred to leaves of Hibiscus rosa-sinensis, Malvaviscus arboreus, Grevilea robusta, Bixa orellana, and Citrus sinensis. Ninety days post infestation, the descendant mites were transferred to Pera sweet orange plants to verify the transmissibility of the virus back to citrus. Nonviruliferous mites which had no feeding access to diseased tissue were used as controls. Local chlorotic or necrotic spots and ringspots, symptoms of leprosis disease, appeared in most plants tested. Results generated by reversetranscription polymerase chain reaction with primers specific for CiLV-C and by electron microscope analyses confirmed the susceptibility of these plants to CiLV-C.

First Report of 'Candidatus Liberibacter asiaticus' Associated with Citrus Huanglongbing in the Dominican Republic.

In August 2008, unusual symptoms were observed in Mexican lime trees (Citrus aurantifolia (Christm.) Swing) in the municipality of Luperón, province of Puerto Plata on the north coast of the Dominican Republic. Symptoms observed in young and old trees included blotchy mottle on leaves, healthy-appearing larger branches with smaller side branches that displayed chlorotic leaves, abscised and lopsided fruit, and branch dieback, all symptoms similar to those of citrus huanglongbing associated with 'Candidatus Liberibacter' spp. (1). Symptoms were observed in an area of ~100 ha surrounding Luperón, where Mexican lime trees were grown as seedlings and no commercial plantings of other citrus were present. Symptomatic leaves were collected from 16 trees in September 2008, and DNA was extracted from petioles and midveins with a DNeasy kit (Qiagen, Gaithersburg, MD) or with chloroform/isoamyl alcohol (24:1). Real-time PCR with the16S rDNA primer/probe set specific to 'Ca. Liberibacter asiaticus' and performed as described (2) gave Ct values comparable with the positive control for five samples. Conventional PCR with the forward (5'-tcgagcgcgtatgcaatacg-3') and reverse (5'-ctacctttttctacgggataacgc-3') primers used in real-time PCR (2) amplified a 75-bp product from these five samples. Eleven to twelve clones were sequenced from each sample and BLAST analysis of a consensus sequence for each sample (GenBank Accession Nos. FJ489643-FJ489647) showed 98% (e.g., EU921622, EU921618) to 100% identity (e.g., FJ236554, FJ263702) with 16S rDNA sequences of 'Ca. Liberibacter asiaticus'. A larger 912-bp portion of the 16S rDNA gene was amplified from each sample by conventional PCR with sense (5'-gagcctaccaaggctacgat-3') and antisense (5'-gcgttatcccgtagaaaaaggtag-3') primers designed from the sequence of 'Ca. Liberibacter asiaticus' strain LJZ-4730 (GenBank Accession No. FJ263700). Nine to twelve clones were sequenced for each sample and BLAST analysis of a consensus sequence for each sample (GenBank Accession Nos. FJ811891-FJ811895) showed 100% identity to 'Ca. Liberibacter asiaticus' (DQ471900, DQ4719010) from Florida and Brazil, respectively. To confirm this identification, 693 bp of the outer membrane protein gene (omp) were amplified from each of the five positive samples by conventional PCR using sense (5'-gtgattctgagggtgagcg-3') and antisense (5'-cgaactcactgagaactgatc-3') primers designed from nucleotides 15 to 33 and 687 to 707, respectively, of the omp gene from 'Ca. Liberibacter asiaticus' strain MZ (GenBank Accession No. EF580135). The consensus sequences from 12 clones from each sample (GenBank Accession Nos. FJ489638-FJ489642) showed 99% (e.g., FJ236564, AY842429) to 100% (e.g., FJ236566, EF580135) nucleotide identity and the predicted translation product showed 100% amino acid identity (e.g., AAX47433, AAX47431) with the omp gene from 'Ca. Liberibacter asiaticus'. These results confirm the presence of and to our knowledge, this is the first report of 'Ca. Liberibacter asiaticus' in the Dominican Republic. Huanglongbing is a destructive disease of citrus (1) and its spread is expected to adversely affect citrus production in the Dominican Republic with subsequent negative effects predicted for the employment of thousands of people. References: (1) J. M. Bové, J. Plant Pathol. 88:7, 2006. (2) W. Li et al. J. Microbiol. Methods 66:104, 2006.

Amplification of Citrus tristeza virus from a cDNA clone and infection of citrus trees.

Isolates of the Closterovirus, Citrus tristeza virus (CTV), are populations of disparate genotypes and defective RNAs developed during long periods of vegetative propagation of citrus trees. Because it has not been possible to obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. We previously developed an infectious cDNA clone from which in vitro-produced RNA transcripts could infect protoplasts (Satyanarayana et al., 1999, Proc. Natl. Acad. Sci. USA 96, 7433-7438). However, neither the RNA transcripts nor virions from transcript-infected protoplasts were competent for infection of citrus trees. Using a green fluorescent protein-marked virus as inoculum, we found that the approximately 20-kb RNA from virions or transcripts of cDNA infected only a small percentage of protoplasts ( approximately 0.01%), but virions could infect more than 80% of the protoplasts. Based on this information, we amplified the virus from the cDNA clone (recombinant virus) by successive passages in protoplasts using virions in crude sap as inoculum. By the third to seventh passages in protoplasts, maximal amounts of recombinant progeny virus were produced, which were used for inoculation of small citrus trees by slashing stems in the presence of virion preparations. A relatively high percentage of plants became infected with the recombinant virus from protoplasts, resulting in the first defined pure culture of CTV in plants. The comparative biology of the pure culture of recombinant CTV with that of the parental population in planta demonstrated that the recombinant virus retained through all of the recombinant DNA manipulations the normal functions of replication, movement, and aphid transmissibility, and had a symptom phenotype indistinguishable from that of the parental population. Additionally, fulfilling Koch's postulates of the first pure culture of CTV in plants suggested that the major genotype of the CTV T36 population is the primary determinant of the symptom phenotype. We could distinguish no biological contributions resulting from the minor genotypes and defective RNAs of the parental population.

Sequences of Citrus tristeza virus separated in time and space are essentially identical.

The first Citrus tristeza virus (CTV) genomes completely sequenced (19.3-kb positive-sense RNA), from four biologically distinct isolates, are unexpectedly divergent in nucleotide sequence (up to 60% divergence). Understanding of whether these large sequence differences resulted from recent evolution is important for the design of disease management strategies, particularly the use of genetically engineered mild (essentially symptomless)-strain cross protection and RNA-mediated transgenic resistance. The complete sequence of a mild isolate (T30) which has been endemic in Florida for about a century was found to be nearly identical to the genomic sequence of a mild isolate (T385) from Spain. Moreover, samples of sequences of other isolates from distinct geographic locations, maintained in different citrus hosts and also separated in time (B252 from Taiwan, B272 from Colombia, and B354 from California), were nearly identical to the T30 sequence. The sequence differences between these isolates were within or near the range of variability of the T30 population. A possible explanation for these results is that the parents of isolates T30, T385, B252, B272, and B354 have a common origin, probably Asia, and have changed little since they were dispersed throughout the world by the movement of citrus. Considering that the nucleotide divergence among the other known CTV genomes is much greater than those expected for strains of the same virus, the remarkable similarity of these five isolates indicates a high degree of evolutionary stasis in some CTV populations.

An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication.

Citrus tristeza virus (CTV) populations in citrus trees are unusually complex mixtures of viral genotypes and defective RNAs developed during the long-term vegetative propagation of the virus and by additional mixing by aphid transmission. The viral replication process allows the maintenance of minor amounts of disparate genotypes and defective RNAs in these populations. CTV is a member of the Closteroviridae possessing a positive-stranded RNA genome of approximately 20 kilobases that expresses the replicase-associated genes as an approximately 400-kDa polyprotein and the remaining 10 3' genes through subgenomic mRNAs. A full-length cDNA clone of CTV was generated from which RNA transcripts capable of replication in protoplasts were derived. The large size of cDNA hampered its use as a genetic system. Deletion of 10 3' genes resulted in an efficient RNA replicon that was easy to manipulate. To investigate the origin and maintenance of the genotypes in CTV populations, we tested the CTV replicase for its acceptance of divergent sequences by creating chimeric replicons with heterologous termini and examining their ability to replicate. Exchange of the similar 3' termini resulted in efficient replication whereas substitution of the divergent (up to 58% difference in sequence) 5' termini resulted in reduced but significant replication, generally in proportion to the extent of sequence divergence.

Two paths of sequence divergence in the citrus tristeza virus complex.

ABSTRACT Comparison of a sampling of complementary DNA (cDNA) sequences from the Florida citrus tristeza virus (CTV) isolates T3 and T30 to the sequence of the genome of the Israeli isolate VT showed a relatively consistent or symmetrical distribution of nucleotide sequence identity in both the 5' and 3' regions of the 19.2-kb genome. In contrast, comparison of these sequences to the sequence of isolate T36 showed a dramatic decrease in sequence identity in the 5' proximal 11 kb of the genome. A cDNA probe derived from this region of the T36 genome hybridized to double-stranded RNA (dsRNA) of only 3 of 10 different Florida CTV isolates. In contrast, analogous probes from T3 and T30 hybridized differentially to the seven isolates not selected by the T36 probe. Primers designed from cDNA sequence for polymerase chain reaction (PCR) selectively amplified these 10 isolates, allowing them to be classified as similar to T3, T30, or T36. In contrast, individual cDNA probes derived from the 3' terminal open reading frames of the T3, T30, and T36 genomes all hybridized to dsRNA from all Florida CTV isolates tested, and PCR primers designed from the T36 capsid protein gene sequence amplified successfully from all isolates. Based on these data, we propose the creation of two groups of CTV, exemplified by the VT and T36 isolates, respectively. Isolates in the VT group, which include isolates VT, T3, and T30, have genomic sequence divergence that is relatively constant in proportion and distribution throughout the genome, and candidate isolates for that group could be considered strains of the same virus. The T36 group is differentiated from the VT group by the highly divergent 5' genomic sequence. This 5' region of the CTV genome, thus, can serve as a measure of the extent of sequence divergence and can be used to define new groups and group members in the CTV complex.

Transcriptional strategy of closteroviruses: mapping the 5' termini of the citrus tristeza virus subgenomic RNAs.

Citrus tristeza virus (CTV) induces formation of a nested set of at least nine 3' coterminal subgenomic RNAs (sgRNAs) in infected tissue. The organization and expression of the 19,296-nucleotide (nt) CTV genome resembles that of coronaviruses, with polyprotein processing, translational frameshifting, and multiple sgRNA formation, but phylogenetically the CTV polymerase, like polymerases of other closteroviruses, belongs to the Sindbis virus-like lineage of RNA virus polymerases. Both positive-strand RNA virus supergroups, coronaviruses and Sindbis-like viruses, utilize different mechanisms of transcription. To address the mechanism of CTV transcription, 5' termini for the two most abundant sgRNAs, 1.5 and 0.9 kb, respectively, were mapped by runoff reverse transcription. The two sgRNAs were demonstrated to have 48- and 38-nt 5' untranslated regions (5'-UTRs), respectively. The 5'-UTR for the 1.5-kb RNA was cloned, sequenced, and demonstrated to be colinear with the 48-nt genomic sequence upstream of the initiator codon of the respective open reading frame 10, i.e., to be of continuous template origin. The data obtained suggest that the sgRNA transcription of CTV is dissimilar from the coronavirus transcription and consistent with the transcriptional mechanism of other Sindbis-like viruses. Thus, the Sindbis virus-like mechanism of transcription of the positive-strand RNA genomes might be successfully utilized by the closterovirus genome of up to 19.3 kb with multiple sgRNAs.

Kinetics of accumulation of citrus tristeza virus RNAs.

Citrus tristeza virus (CTV), a member of the closterovirus group, is one of the more complex single-stranded RNA viruses. The 5' portion of its 19,296-nt, single-stranded RNA genome is expressed as an approximately 400-kDa polyprotein that is proteolytically processed, while the 10 3' open reading frames are expressed from 3'-coterminal subgenomic RNAs (sg RNAs). As an initial examination of the gene expression of this virus, we found that the kinetics of accumulation of genomic and sg RNAs and coat protein of the T36 isolate of CTV were similar in protoplasts of the natural host, citrus, and the experimental nonhost Nicotiana benthamiana. Newly synthesized genomic RNA was detected 2 days postinoculation and increased to a maximum at 3-5 days. The RNA complementary to the full-length virion RNA increased with similar kinetics, but at approximately one-tenth the concentration of genomic plus strands. Most of the abundant sg RNAs also accumulated in parallel to that of the genomic RNA. However, the smallest sg RNA, which corresponds to the p23 gene, increased earlier. The different sg RNAs accumulated in greatly differing amounts, in general with 3'-most sg RNAs accumulating to higher levels than 5' sg RNAs. However, some 3' sg RNAs (p13 and p18) accumulated to low levels. The two 3'-most sg RNAs (p23 and p20) accumulated to high levels approximately equal to that of the genomic RNA. The accumulation curve for coat protein paralleled that of its mRNA, suggesting that its regulation was transcriptional. Progeny virions from protoplasts were used to sequentially infect new protoplasts, serving as a potential source of virus that could evolve free from the genetic selection in intact plants for aphid transmission and movement.

Multiple species of defective RNAs in plants infected with citrus tristeza virus.

Alemow (Citrus macrophylla) and sweet orange (C. sinensis) plants infected, respectively, with several Israeli and Florida isolates of the citrus tristeza virus (CTV) were found to contain multiple species of RNA molecules with features similar to defective-interfering RNAs. Northern blot hybridizations of dsRNAs extracted from serial passages of the Israeli VT isolate (CTV-VT) and from different plants infected with a single source of inoculum showed considerable variation both in the presence and in the relative abundance of the defective RNA (D-RNA) bands. The D-RNA molecules were found to be encapsidated in the CTV particles. Sequence analysis of two VT D-RNA molecules of 2.7 and 4.5 kb revealed that they were composed of two regions corresponding to 1818 and 4036 nucleotides from the 5' and 938 and 442 nucleotides from the 3' termini of the CTV-VT genomic RNA, respectively. A short (ca. 0.8 kb) nonencapsidated single-stranded positive-sense RNA species was also found in infected plants. This ssRNA, which copurified with dsRNAs, was shown by hybridization to encompass the 5'-terminal part of the CTV genome and might have an extensive secondary structure.

Complete sequence of the citrus tristeza virus RNA genome.

The sequence of the entire genome of citrus tristeza virus (CTV), Florida isolate T36, was completed. The 19,296-nt CTV genome encodes 12 open reading frames (ORFs) potentially coding for at least 17 protein products. The 5'-proximal ORF 1a starts at nucleotide 108 and encodes a large polyprotein with calculated MW of 349 kDa containing domains characteristic of (from 5' to 3') two papain-like proteases (P-PRO), a methyltransferase (MT), and a helicase (HEL). Alignment of the putative P-PRO sequences of CTV with the related proteases of beet yellows closterovirus (BYV) and potyviruses allowed the prediction of catalytic cysteine and histidine residues as well as two cleavage sites, namely Val-Gly/Gly for the 5' proximal P-PRO domain and Met-Gly/Gly for the 5' distal P-PRO domain. The autoproteolytic cleavage of the polyprotein at these sites would release two N-terminal leader proteins of 54 and 55 kDa, respectively, and a 240-kDa C-terminal fragment containing MT and HEL domains. The apparent duplication of the leader domain distinguishes CTV from BYV and accounts for most of the size increase in the ORF 1a product of CTV. The downstream ORF 1b encodes a 57-kDa putative RNA-dependent RNA polymerase (RdRp), which is probably expressed via a +1 ribosomal frameshift. Sequence analysis of the frameshift region suggests that this +1 frameshift probably occurs at a rare arginine codon CGG and that elements of the RNA secondary structure are unlikely to be involved in this process. The complete polyprotein resulting from this frameshift event has a calculated MW of 401 kDa and after cleavage of the two N-terminal leaders would yield a 292-kDa protein containing the MT, HEL, and RdRp domains. Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate closterovirus lineage within the alpha-like supergroup of positive-strand RNA viruses. Two gene blocks or modules can be easily identified in the CTV genome. The first includes the replicative MT, HEL, and RdRp genes and is conserved throughout the entire alpha-like superfamily. The second block consists of five ORFs, 3 to 7, conserved among closteroviruses, including genes for the CTV homolog of HSP70 proteins and a duplicate of the coat protein gene. The 3'-terminal ORFs 8 to 11 encode a putative RNA-binding protein (ORF 11), and three proteins with unknown functions; this gene array is poorly conserved among closteroviruses.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of citrus tristeza virus subgenomic RNAs in infected tissue.

Citrus tristeza virus (CTV) specific RNAs extracted from infected citrus tissue were analyzed by Northern blot hybridization. RNAs were characterized by size and identified using cDNA probes specific to nine open reading frames (ORFs) identified by the analysis of sequence obtained from cDNA clones of the T36 isolate of CTV. Sequence specific cDNA probes identified the genomic RNA as well as subgenomic RNAs representing the p33, p65, p61, p27, p25, p18, p13, p20, and p23 ORFs in extracts of total or double-stranded RNA (dsRNA) isolated from infected tissue. A probe derived from the 3' terminal ORF (p23) hybridized to each of these subgenomic RNAs, indicating that the RNAs are 3' coterminal. The relative amounts of the different subgenomic RNAs varied widely. The RNAs for the p20 and p23 ORFs were the most abundant and surpassed the amount of the p25 or capsid protein specific subgenomic RNA. The number and sizes of the CTV subgenomic RNAs were the same in total RNA and dsRNA preparations. Propagation of T36 in seven different citrus hosts did not alter the pattern of subgenomic RNAs.

Nucleotide sequence and organization of eight 3' open reading frames of the citrus tristeza closterovirus genome.

The citrus tristeza closterovirus (CTV) RNA genome was cloned as cDNA generated from both CTV-specific double-stranded RNA and genomic RNA, and the sequence of the 3' 7292 nucleotides was determined. The sequenced portion contained eight open reading frames potentially encoding, in the 5' to 3' direction, proteins with the apparent molecular weights of 65, 61, 27, 25 (capsid protein, CP), 18, 13, 20, and 23 kDa, and a potential noncoding region of 277 nucleotides. The 65-kDa protein is a viral homolog of cellular hsp70 heat shock proteins (hsp), the 61-kDa protein is distantly related to the hsp90 proteins, and the 27-kDa protein is a diverged copy of the CP. Database searches did not identify any protein sequences of significant similarity to the remaining four ORFs downstream of the CP. A specific four-gene module consisting of the hsp70 protein, the hsp90-related protein, the diverged copy of the CP, and the CP itself was found to be common in organization between CTV and beet yellows closterovirus. All four proteins in this module were highly conserved, indicating that these viruses probably have evolved from a common ancestor.

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes.

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes.

The tobamovirus capsid protein functions as a host-specific determinant of long-distance movement.

The tobamoviruses, tobacco mosaic virus (TMV) and Odontoglossum ringspot virus (ORSV), differ in the range of plant species that each can systemically infect. Both viruses systemically infect Nicotiana benthamiana similarly, but differ in the ability to systemically infect tobacco (N. tabacum). ORSV is confined to the inoculated leaves of N. tabacum, whereas TMV causes a rapid systemic infection. Genetic chimeras were created to identify viral genes involved in specific interactions in long-distance movement in N. tabacum. In N. tabacum, a chimera expressing the ORSV 30-kDa protein exhibited a reduced rate of cell-to-cell spread, similar to that of ORSV, and, also like ORSV, was deficient in systemic invasion, implying a relationship between the rates of cell-to-cell and long-distance movement. However, a TMV chimera expressing the ORSV capsid protein gene spread cell to cell similarly to TMV, but was deficient in long-distance movement and systemic infection. These data suggest that tobamovirus capsid protein(s) may be required to interact with host components in a specific manner to allow efficient long-distance movement, and the ORSV capsid protein did not function in this manner in tobacco plants.

Rapid, high-level expression of biologically active alpha-trichosanthin in transfected plants by an RNA viral vector.

alpha-Trichosanthin, a eukaryotic ribosome-inactivating protein from Trichosanthes kirilowii, inhibits the replication of the human immunodeficiency virus (HIV) in vitro. The alpha-trichosanthin gene was placed under the transcriptional control of a tobamovirus subgenomic promoter in a plant RNA viral vector. Two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated alpha-trichosanthin to levels of at least 2% of total soluble protein. The recombinant alpha-trichosanthin was purified and its structural and biological properties were analyzed. The 23-amino acid signal peptide was recognized by N. benthamiana and the processed enzyme caused a concentration-dependent inhibition of protein synthesis in vitro. The high level of heterologous gene expression observed in these studies is due to the unique features of the RNA viral-based transfection system.

Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector.

Tobacco mosaic virus (TMV) produces large quantities of RNA and protein on infection of plant cells. This and other features, attributable to its autonomous replication, make TMV an attractive candidate for expression of foreign sequences in plants. However, previous attempts to construct expression vectors based on plant RNA viruses, such as TMV, have been unsuccessful in obtaining systemic and stable movement of foreign genes to uninoculated leaves in whole plants. A hybrid viral RNA (TB2) was constructed, containing sequences from two tobamoviruses (TMV-U1 and odontoglossum ringspot virus). Two bacterial sequences inserted independently into TB2 moved systemically in Nicotiana benthamiana, although they differed in their stability on serial passage. Systemic expression of the bacterial protein neomycin phosphotransferase was demonstrated. Hybrid RNAs containing both TMV-U1 and the inserted bacterial gene sequences were encapsidated by the odontoglossum ringspot virus coat protein, facilitating their transmission and amplification on passaging to subsequent plants. The vector TB2 provides a rapid means of expressing genes and gene variants in plants.

A tobacco mosaic virus-hybrid expresses and loses an added gene.

An additional open reading frame from the chloramphenicol acetyltransferase (CAT) gene was fused behind a tobacco mosaic virus (TMV) subgenomic RNA promoter and inserted into different positions in the complete TMV genome to examine how much this viral genome can be altered with continued replication. One hybrid virus, CAT-CP, with the insertion between the 30K and coat protein genes, replicated efficiently, produced an additional subgenomic RNA and CAT activity, and assembled into 350-nm virions, compared to 300-nm virions of wild-type TMV. However, during systemic infection of plants, the inserted sequences were deleted. This deletion was exact, resulting in progeny wild-type TMV. Another hybrid virus examined was CP-CAT, which had the insertion between the coat protein gene and the nontranslated 3' region. This virus replicated poorly, produced only minimal levels of CAT activity, and did not systemically invade infected plants. These data show that some extensive modifications of the TMV genome still allow efficient virus replication.