Nicotiana Benthamiana Protoplasts Research Articles

Are Tubules Generated by the 3a Protein Necessary for Cucumber Mosaic Virus Movement?

The 3a movement protein of cucumber mosaic virus (CMV), fused to the jellyfish green fluorescent protein (3a-GFP) generated surface punctate aggregates as well as tubules protruding from infected tobacco and Nicotiana benthamiana protoplasts. Fluorescent tubules also appeared on the surface of protoplasts prepared from transgenic tobacco plants expressing 3a-GFP, indicating that the 3a protein is the only viral component required for the formation of the tubules. CMV with a mutation in the gene encoding the 3a protein, M8 CMV, could infect tobacco systemically, but tubules were not detected protruding from infected protoplasts when the mutated 3a protein was fused to the GFP [(M8)3a-GFP]. This indicates that the ability of the 3a protein to generate tubules in the surface of protoplasts is not a function required for the spread of CMV in tobacco. On the other hand, the (M8)3a-GFP did not traffic through plasmodesmata interconnecting tobacco epidermal cells, in contrast to the wild-type 3a-GFP. This suggests that there may be a correlation between the ability of the 3a protein to assemble tubules in protoplasts and its ability to promote movement within certain tissues.

Interactions between Tombusviruses and Satellite RNAs of Tomato Bushy Stunt Virus: A Defect in sat RNA B1 Replication Maps to ORF1 of a Helper Virus

The biological properties of two recently described satellite RNAs of tomato bushy stunt virus (TBSV) were analyzed in natural and experimental hosts. Full-length cDNA clones were constructed for sat RNAs B1(822 nt) and B10 (612 nt) and used in inoculations with satellite-free transcripts of different tombusviruses. In all virus-host combinations tested, TBSV sat RNA B10 drastically reduced the accumulation of viral genomic RNA and attenuated symptoms. In contrast, sat RNA B1 caused a less marked reduction of viral RNA level and did not have any effect on symptoms. Experiments with Nicotiana benthamiana protoplasts showed that the differential effects of sat RNAs B1 and B10 on TBSV titer were related to differential abilities to interfere with virus replication. Three tombusviruses tested were able to maintain both sat RNAs in N. benthamiana plants, although carnation Italian ringspot virus (CIRV) was a poor helper for sat RNA B1. Using chimeric viruses, a strong determinant for low sat RNA B1 accumulation was mapped to the 5′-terminal part of the genome of CIRV. The poor helper activity of CIRV was shown to be due to low sat RNA B1 replication. A single-nucleotide mutation in the start codon of CIRV ORF1 restored the ability to replicate sat RNA B1 to high levels. This mutant encodes an ORF1 that is 22 amino acids shorter at the N-terminus than the wild-type virus.

Evolution of bamboo mosaic virus in a nonsystemic host results in mutations in the helicase-like domain that cause reduced RNA accumulation

A mutant population of bamboo mosaic potexvirus (BaMV) was isolated after serial passage using Chenopodium quinoa plants. While the wild type inoculum induced indistinct chlorotic lesions, the mutant produced obvious lesions on C. quinoa although RNA accumulation of the mutant in Nicotiana benthamiana protoplasts was significantly reduced compared to wild type. Mutations were identified in the helicase-like domain. One RT-PCR-generated cDNA clone (designated pL1-33) representing the helicase-like region showed four nucleotide mutations encoding three amino acid changes that were shown to result in dramatically decreased viral accumulation. Independent analyses of the effects of these substitutions showed that nucleotide changes at position 1722 resulting in a leucine to proline switch and position 2129 resulting in a histidine to tyrosine switch had the greatest effect on viral accumulation. Combination of these two mutations resulted in a undetectable viral accumulation. We have identified that amino acids within the helicase domain but outside the universally conserved helicase-like motifs that play an important role in viral amplification.

Molecular Studies on Bromovirus Capsid Protein: V. Evidence for the Specificity of Brome Mosaic Virus Encapsidation Using RNA3 Chimera of Brome Mosaic and Cucumber Mosaic Viruses Expressing Heterologous Coat Proteins

Brome mosaic bromovirus (BMV) and cucumber mosaic cucumovirus (CMV) are structurally and genetically very similar. The specificity of the BMV and CMV coat proteins (CPs) during in vivoencapsidation was studied using two RNA3 chimera in which the respective CP genes were exchanged. The replicative competence of each chimera was analyzed in Nicotiana benthamianaprotoplasts, and their ability to cause infections was examined in two common permissive hosts, Chenopodium quinoaand N. benthamiana.Each RNA3 chimera replicated to near wild-type (wt) levels and synthesized CPs of expected parental origin when co-inoculated with their respective genomic wt RNAs 1 and 2. However, inoculum containing each chimera was noninfectious in the common permissive hosts tested. Encapsidation assays in N. benthamianaprotoplasts revealed that CMV CP expressed from chimeric BMV RNA3 was capable of packaging heterologous BMV RNA, however, at a lower efficiency than parental BMV CP. By contrast, BMV CP expressed from chimeric CMV RNA3 was unable to package heterologous CMV RNA. These observations demonstrate that BMV CP, but not CMV CP, exhibits a high degree of specificity during in vivopackaging. The reasons for the noninfectious nature of each chimera in the host plants tested and factors likely to affect encapsidation in vivoare discussed.

Specific inclusion bodies are associated with replication of lettuce infectious yellows virus RNAs in Nicotiana benthamiana protoplasts.

Nicotiana benthamiana mesophyll protoplasts, either mock-inoculated or inoculated using in vitro transcripts derived from lettuce infectious yellows virus (LIYV) RNA 1- and/or RNA 2-cloned cDNAs were analysed by transmission electron microscopy (TEM) and, in some cases, also by immunogold labelling. TEM revealed the main cytopathological effects of LIYV infections in N. benthamiana protoplasts infected with RNAs 1 and 2: (a) typical closterovirus-induced (beet yellows virus-type) accumulations of vesiculated cytoplasmic membranes as inclusion bodies, sometimes with associated virions; (b) scattered aggregations of virions within the cytoplasm; and (c) electron-dense plasmalemma deposits. These were not seen in mock-inoculated protoplasts. Protoplasts inoculated only with LIYV RNA 1 contained vesiculated cytoplasmic inclusion bodies, but not virions or plasmalemma deposits. Thus, infection by only LIYV RNA 1 is sufficient to induce characteristic closterovirus vesiculated cytoplasmic inclusion bodies. However, both LIYV RNAs 1 and 2 are needed for production of virions and plasmalemma deposits.

Specific sequence changes in the 5'-terminal region of the genome of satellite tobacco mosaic virus are required for adaptation to tobacco mosaic virus.

The genome of satellite tobacco mosaic virus (STMV) adapted to tobacco mosaic virus (TMV), tomato mosaic virus or green tomato atypical mosaic virus consistently had two single base deletions at positions 1 and 61, corresponding to bases A and G, respectively, as compared to the type-strain genome which is naturally adapted to tobacco mild green mosaic virus (TMGMV). Transcript RNAs (STMV(TMV)) from clone pSTMV(TMV) which captured the deletions at positions 1 and 61 were infectious when co-inoculated to tobacco plants with either TMV or TMGMV at infection frequencies of > 90%. Two new STMV variants were created to investigate whether both deletions were essential for adaptation to TMV. These were STMV(TMGMV) deltaA1, which had the A at position 1 (A1) deleted, and STMV(TMGMV) deltaG61, which lacked G61. STMV(TMGMV) deltaA1 was infectious (75% frequency) in the presence of either TMV or TMGMV. Virion RNA of STMV(TMGMV) deltaA1 lost G61 after one infection cycle with TMV. This deletion did not occur in co-infections with TMGMV. STMV(TMGMV) deltaG61, like the clone STMV(TMGMV), was infectious (100% frequency) with TMGMV but TMV did not support this clone. When Nicotiana benthamiana protoplasts were transfected with STMV(TMGMV), STMV(TMGMV) deltaA1 or STMV(TMV), STMV replicated when TMGMV was the helper virus. STMV(TMV) and STMV(TMGMV) deltaA1 replicated in the presence of helper TMV, but STMV(TMGMV) did not, the same result as in whole plants. The deletion of A1 is thus essential for initial STMV adaptation to TMV and the eventual deletion of G61 is a predicted additional change.

Identification of artichoke mottled crinkle virus (AMCV) proteins required for virus replication: complementation of AMCV p33 and p92 replication-defective mutants.

Mutagenesis of the artichoke mottled crinkle virus (AMCV) genome and complementation studies between replication-defective mutants were undertaken to identify viral protein(s) essential for AMCV replication. Inoculation of Nicotiana benthamiana protoplasts with mutant transcripts revealed that null mutations in ORFs 1 [tA33(-)], 2 [tA92(-)] and 6 [tA7(-)], as well as an ORF 2 mutation [tA92GED] in the GDD motif of the 92 kDa protein, the putative replicase, prevented accumulation of detectable levels of progeny RNA. Conversely, mutations of ORFs 3 [tA41(-)], 4 [tA21(-)] and 5 [tA19(-)] did not substantially affect the accumulation of AMCV genomic and subgenomic RNAs of both positive and negative polarity. Inoculation of N. benthamiana plants with transcripts impaired in replication revealed that tA92(-) and tA7(-) mutants lead to replicating pseudorevertants. Functional analysis of these pseudorevertants showed that: (i) the double stop codon introduced at the end of ORF 1 to prevent the translational readthrough of the 92 kDa protein reverted to a single amber, ochre or opal codon, giving rise to viable genomes; (ii) the putative 7 kDa protein is not essential for genome viability, although the RNA region spanning ORF 6 plays a role in cis in replication. Finally, the two replication-defective mutants tA33(-) and tA92(-) complemented when co-inoculated to N. benthamiana protoplasts, definitively proving that the 33 kDa protein is essential for tombusvirus genome replication. Analysis of viral RNAs from the coinfection experiments showed that tA92(-) was preferentially amplified over tA33(-).

Nonsense and missense translational suppression in plant cells mediated by tRNA(Lys).

A nuclear tRNA(Lys) gene from Arabidopsis thaliana was cloned and mutated so as to express tRNAs with altered anticodons which bind to a UAG nonsense (amber) codon and to the Arg (AGG), Asn (AAC,AAT), Gln (CAG) or Glu (GAG) codons. Concomitantly, a codon in the firefly luciferase gene for a functionally important Lys was altered to an amber codon, or to Arg, Asn, Gln, Glu, Thr and Trp codons, so as to construct reporter genes reliant upon incorporation of Lys. The altered tRNA(Lys) and luciferase genes were introduced into Nicotiana benthamiana protoplasts and expression of the mutated tRNAs was verified by translational suppression of the mutant firefly luciferase genes. Expression of the amber suppressor tRNA(LysCUA) from non-replicative vectors promoted 10-40% suppression of the luciferase nonsense reporters while expression of the amber and missense tRNA(Lys) suppressor genes from a geminivirus vector capable of replication promoted 30-80% suppression of the luciferase nonsense reporter and up to 10% suppression of the luciferase missense reporters with Arg, Asn, Gln and Glu codons.

Replication of wild-type and mutant clones of satellite tobacco mosaic virus in Nicotiana benthamiana protoplasts.

RNA transcribed from cloned satellite tobacco mosaic virus (STMV) cDNA replicated in Nicotiana benthamiana protoplasts when co-inoculated with tobacco mild green mosaic virus (TMGMV) genomic RNA, but degraded when inoculated alone. STMV genomic RNA extracted from wild-type virions replicated in protoplasts when co-inoculated with TMGMV, tobacco mosaic virus (TMV) or tomato mosaic virus (ToMV). Transcripts from clones of two STMV coat protein (CP) mutants accumulated to the same level as wild-type transcripts in protoplasts when co-inoculated with TMGMV, whereas a third mutant accumulated to detectable levels in some, but not all, experiments. These results confirm that STMV RNA requires helper virus for replication, and that the helper specificity exhibited by cloned STMV reflects a specific requirement for the TMGMV replicase. It also demonstrates that the low accumulation of STMV CP mutants observed previously in whole plants cannot be attributed to inefficient RNA replication.

Molecular analysis of the pothos latent virus genome.

Pothos latent virus (PoLV) is an isometric virus with a positive-sense, single-stranded RNA genome of 4415 nt. The genome contains five open reading frames (ORF), coding for five proteins with approximate molecular masses of 25, 84, 40, 27 and 14 kDa, respectively. In vitro synthesized PoLV RNA was infectious to Nicotiana benthamiana plants and protoplasts, but could not support replication of the defective interfering (DI) and satellite RNAs associated with Cymbidium ringspot tombusvirus. No DI RNA related to PoLV was generated after repeated passaging with infected sap. Mutagenesis studies defined the role of the ORF 4 product (27 kDa) as a movement protein, and the ORF 5 product (14 kDa) as being responsible for symptom severity. Moreover, it was shown that the coat protein (CP) is important in regulating synthesis of the 14 kDa protein, excess production of which is lethal to infected plants. CP mutants defective in capsid formation infected plants systemically and induced severe necrotic symptoms. Conversely, CP mutants able to form apparently normal virus particles induced symptoms indistinguishable from those elicited by wild-type virus.

In VitroTranscripts from Cloned cDNAs of the Lettuce Infectious Yellows Closterovirus Bipartite Genomic RNAs Are Competent for Replication inNicotiana benthamianaProtoplasts

Full-length cloned cDNAs of lettuce infectious yellows closterovirus (LIYV) RNAs 1 and 2 were constructed and fused to the bacteriophage T3 RNA polymerase promoter. To assess RNA replication,Nicotiana benthamianaprotoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-derivedin vitrotranscripts. Analysis of protoplasts inoculated with LIYV virion RNAs or capped (m7GpppG)in vitrotranscripts from LIYV RNA 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subgenomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions. Furthermore, protoplasts inoculated with only cappedin vitrotranscripts from LIYV RNA 1 cDNA showed accumulation of LIYV RNA 1 and its putative sgRNA, indicating that LIYV RNA 1 can replicate in the absence of LIYV RNA 2. Conversely, accumulation of LIYV RNA 2 was not detectable in protoplasts inoculated with only LIYV RNA 2 cDNA-derived cappedin vitrotranscripts. These data demonstrate that LIYV genomic RNAs are competent for replication in mesophyll protoplasts and that infectiousin vitrotranscripts can be derived from the cloned cDNAs of a closterovirus genome.

Simultaneous regulation of tomato golden mosaic virus coat protein and AL1 gene expression: expression of the AL4 gene may contribute to suppression of the AL1 gene.

The tomato golden mosaic virus (TGMV) coat protein and AL1 genes are located in opposite directions on either side of an intergenic region. To enable the effects of the AL1, AL2 and AL3 gene products on expression of the coat protein and AL1 genes to be studied simultaneously, a plasmid was constructed, containing the intergenic region linked on one side to a 5'-terminal portion of the AL1 gene fused to a beta-glucuronidase (GUS) reporter gene (to replace most of the AL1 gene) and on the other side to a neomycin phosphotransferase (NEO) reporter gene (to replace the coat protein gene). This GUS-NEO plasmid was mixed with plant expression plasmids containing the AL1, AL2 or AL3 coding regions, the DNA was transformed into Nicotiana benthamiana protoplasts and GUS activities and NEO protein levels were measured. Control transformations were carried out with the GUS-NEO plasmid mixed with the AL1, AL2 or AL3 plasmids in which mutations were introduced to prevent translation of the open reading frames (ORFs). The results showed that transactivation of the coat protein gene by the AL2 gene product and suppression of the AL1 gene by the expression of AL1 DNA (both reported previously) can occur simultaneously. It was also shown that expression of AL4, a small ORF contained within AL1 DNA but in a different reading frame, as well as expression of ORF AL1, can cause significant suppression of AL1 gene expression. Neither the AL1 nor the AL3 gene products affected the expression of the coat protein gene.

Association between the Particles of Raspberry Ringspot and Tobacco Rattle Viruses in Doubly Infected Nicotiana benthamiana Cells and Protoplasts

SUMMARY Electron microscopy of ultrathin sections indicated that nucleoprotein particles of raspberry ringspot (RRV) and tobacco rattle (strain CAM; TRV-CAM) viruses occurred in mixed aggregates in cells of doubly infected Nicotiana benthamiana leaves, and in protoplasts doubly infected by inoculation. RRV particles were attached to the sides and ends of TRV-CAM particles that were mostly bound to mitochondria. RRV particles attached to TRV-CAM particles linking mitochondria in clusters are considered to be the structures represented by the aggregates of RRV particle antigen found previously by fluorescent antibody staining in doubly infected protoplasts but not in those infected with RRV alone.