Rapid, high-level expression of glycosylated rice alpha-amylase in transfected plants by an RNA viral vector.

Tobamoviral vectors have been developed for the heterologous expression of glycoproteins in plants. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of a tobamovirus subgenomic promoter in a RNA viral vector. One to two weeks after inoculation, transfected Nicotiana benthamiana plants accumulated glycosylated alpha-amylase to levels of at least 5% total soluble protein. The 46kDa recombinant enzyme was purified, and its structural and biological properties were analyzed. Post-translational modifications of the secreted protein were compared to rice alpha-amylase expressed in amylolytic strains of Pichia pastoris and Saccharomyces cerevisiae. Endo-H analysis revealed that the alpha-amylase was moderately glycosylated in transfected plants and hyperglycosylated in yeast.

Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors.

A series of tobacco mosaic virus (TMV)-based hybrid vectors for transient gene expression were constructed with similar designs but differing in the source of heterologous tobamovirus sequence: Odontoglossum ringspot virus, tobacco mild green mosaic virus variants U2 and U5, tomato mosaic virus, and sunn-hemp mosaic virus. These vectors contained a heterologous coat protein subgenomic mRNA promoter and coat protein open reading frame (ORF) and either TMV or heterologous 3' nontranslated region. The foreign ORF, from the jellyfish green fluorescent protein (GFP) gene, was transcribed from the native TMV coat protein subgenomic mRNA promoter, which extended into the coat protein ORF. The presence of an in-frame stop codon within the GFP mRNA leader and the choice of sequence of GFP ORFs substantially affected translational efficiency. However, the major regulatory component of gene expression in these vectors appeared to be transcriptional rather than translational. There was an inverse relationship between expression of GFP and the heterologous coat protein genes that was reflected in accumulation of the respective mRNAs and proteins. The most effective vector in this series (30B) contained sequences encoding the coat protein subgenomic mRNA promoter, coat protein ORF, and 3' nontranslated region from tobacco mild green mosaic virus U5. Expressed from 30B, GFP accumulated up to 10% of total soluble protein in leaves.

Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA.

The carotenoid biosynthetic pathway in higher plants was manipulated by using an RNA viral vector. A cDNA encoding phytoene synthase and a partial cDNA encoding phytoene desaturase (PDS) were placed under the transcriptional control of a tobamovirus subgenomic promoter. One to two weeks after inoculation, systemically infected Nicotiana benthamiana plants were analyzed for phytoene. Leaves from transfected plants expressing phytoene synthase developed a bright orange phenotype and accumulated high levels of phytoene. Cytoplasmic inhibition of plant gene expression by viral RNA was demonstrated with an antisense RNA transcript to a partial PDS cDNA derived from tomato. The leaves of the plants transfected with the antisense PDS sequence developed a white phenotype and also accumulated high levels of phytoene. A partial cDNA to the corresponding N. benthamiana PDS gene was isolated and found to share significant homology with the tomato antisense PDS transcript. This work demonstrates that an episomal RNA viral vector can be used to deliberately manipulate a major, eukaryotic biosynthetic pathway. In addition, our results indicate that an antisense transcript generated in the cytoplasm of a plant cell can turn off endogenous gene expression.

Broad resistance to tobamoviruses is mediated by a modified tobacco mosaic virus replicase transgene.

Tobacco plants made transgenic to express the wild type tobacco mosaic virus (TMV) 183-kDa replicase gene were not resistant to TMV. However, transgenic plants containing essentially the same sequences, but with an additional insertion that would terminate translation in the middle of the 183-kDa gene, were highly resistant to systemic infection by TMV and other tobamoviruses. The 1.4-kbp insertion in the replicase open reading frame (ORF) of the resistant plants was shown by DNA sequencing to be an IS10-like transposable element, which apparently inserted itself into the TMV sequence at nucleotide 2875 sometime during the propagation of this replicase ORF plasmid (pREP21). Because of four stop codons, in frame with the TMV replicase ORF on the immediate 5' border of the IS insertion, REP21 effectively represents domain 1 (putative methylase domain) and a portion of domain 2 (putative helicase domain) of the TMV replicase ORF. REP21 Xanthi tobacco plants had a level of resistance to TMV similar to other reported transgenic replicase plants. No TMV was detected in upper leaves of these plants at 1-mo postinoculation. In addition, REP21 plants were resistant to an unusually broad range of tobamoviruses including tomato mosaic virus, tobacco mild green mosaic virus, TMV-U5, green tomato atypical mosaic virus, and ribgrass mosaic virus. These plants were not resistant to cucumber mosaic cucumovirus. The lack of systemic infection by TMV was due to reduced multiplication in inoculated leaves rather than complete prevention of replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Low level of genetic drift in foreign sequences replicating in an RNA virus in plants.

The accumulation of mutations was measured in foreign sequences constituting a portion of a hybrid virus derived from the 6.4-kb (+) RNA virus, tobacco mosaic tobamovirus (TMV). Neither of the two foreign sequences tested (dihydrofolate reductase and neomycin phosphotransferase II) are functionally required by the virus, so they should be free of selective pressures and should be a true measure of viral sequence drift in whole plants. Four hybrid virus populations, two of each foreign sequence, were taken through 9-10 passages in whole plants of Nicotiana benthamiana. Sequences were sampled from these populations by conversion to cDNA, amplification by the polymerase chain reaction, and sequencing resulting bacterial clones. The background mutation rate contributed by the enzymes of this assay system allowed viral mutation rates greater than 10(-4) mutations per base per passage to be measured. Surprisingly, all native and foreign genes accumulated mutations at a very low rate, lower than could be detected by the assay procedure. This low mutation accumulation rate of < or = 10(-4) mutations per base per passage may be due to replicase fidelity or populational "bottlenecking." Sequence drift should not be a practical limitation to most uses of TMV as a vector, although deletion phenomena observed in this study may present difficulties.

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.

Role of monocyte fucose-receptors in T-cell fibronectin activity.

T-cell fibronectin (FN) is a lymphokine produced by antigen- and mitogen-activated T cells that agglutinates human monocytes at femtomolar concentrations. This extreme degree of activity derives from co-operative interactions between multiple FN domains and multiple monocyte integrin protein receptors. T-cell FN, like other FN, is a glycoprotein. The role interactions between T-cell FN carbohydrate and lectin-like monocyte surface receptors play in mediating T-cell FN activity was studied by determining the ability of monosaccharides to inhibit T-cell FN activity. L-Fucose and L-rhamnose significantly inhibited T-cell FN-mediated monocyte agglutination at concentrations as low as 0.01 mM; D-glucose, D- or L-galactose, D- or L-mannose and D-fucose were not inhibitory at 10-100 mM. This inhibition appeared to be due to interference with the binding of T-cell FN fucose residues to monocyte fucose receptors since: (i) treatment of T-cell FN with alpha-L-fucosidase abolished its agglutinating activity for human monocytes, while treatment with beta-D-galactosidase or with alpha-L-fucosidase in the presence of L-fucose had no effect; (ii) treatment of monocytes with alpha-L-fucosidase did not affect their response to T-cell FN; and (iii) L-fucose or L-rhamnose did not alter the expression of monocyte integrin FN receptors under conditions where T-cell FN-mediated monocyte agglutination was completely inhibited. In vivo, 1 mumol intracutaneous L-fucose inhibited expression of delayed hypersensitivity by 30% (P much less than 0.001); similar doses of L-rhamnose inhibited responses by 10% (P less than 0.02). These data implicate a fucose receptor in monocyte response to T-cell FN, and suggest that T-cell FN is only one of the mediators involved in initiating delayed hypersensitivity reactions in vivo.

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.

Point substitution in a promoter-like region and the V1 gene affect the host range and symptoms of maize streak virus.

The nucleotide sequences of full-length infectious clones of two symptomatic and host range variants (MSV-Ns and MSV-Nm) of the Nigerian strain of maize streak virus (MSV) have been determined and shown to differ by only three nucleotides. MSV-Ns produced symptoms in infected maize plants sooner and the streaks were wider and more chlorotic than those of MSV-Nm; variant MSV-Ns also had a wider host range within the Gramineae. None of the three nucleotide differences resulted in amino acid changes. Site-directed mutagenesis showed that a substitution at nucleotide (nt) 40 in the V1 gene affected streak width, while severity of chlorosis, length of streaks, latency, and host range was determined by a single base change at nt 2473 in the large intergenic region. The nt 2473 change altered a potential promoter sequence (TATA box) in MSV-Ns 101 nucleotides upstream of the initiation codon of the C1 gene. Mutagenesis of TATA sequences located downstream of TATA -101 showed that TATA -101 alone was sufficient to confer a wide host range phenotype on MSV-Ns and suggested that it might function as a promoter for the expression of complementary-sense open reading frames. When compared with an updated promoter consensus derived from genes of the Gramineae, the promoter context around TATA -101 in MSV-Ns was not more favorable than those found at -57 and -62 in MSV-Nm.

Mutational analysis of the virion-sense genes of maize streak virus.

Insertion and deletion mutagenesis of the two virion-sense genes, V1 and V2, of maize streak virus (MSV) prevents symptomatic infections following Agrobacterium-mediated 'agroinoculation' of maize seedlings. These genes code for an Mr 10900 protein and for coat protein, respectively. Mutants containing insertions or deletions in the coat protein gene, V2, were able to replicate to low levels, producing dsDNA although virion ssDNA was not detected and symptoms were not observed. Hence, unlike the bipartite geminiviruses, MSV requires coat protein to produce symptomatic systemic infection. Mutations in gene V1 which considerably shortened the Mr 10900 protein (V1 gene) resulted either in low levels of replication, in which all the DNA forms associated with a wild-type infection were produced, or in no infection, in which case coat protein production may also have been affected. A V1 mutant generated in vivo with 11 of the 14 N-terminal amino acids altered, was viable and produced symptoms typical of a wild-type infection. Infectivity, assessed by replication and symptom expression, was restored by co-inoculating constructs containing single mutations in different open reading frames, thus rescue can occur by trans-complementation of gene products. The experiments showed that the mutations did not affect the nucleotide sequence requirements for replication and that in all cases intermolecular recombination eventually resulted in dominant wild-type virus.

Mapping of Digitaria streak virus transcripts reveals different RNA species from the same transcription unit.

All, except 19 [corrected] bp, of the Digitaria streak virus (DSV) genome is transcribed. Two RNA transcripts (1+ and 2+) are encoded by the virion DNA strand and up to five (1- to 5-) by the complementary DNA strand [corrected]. Detailed mapping of these RNAs has revealed evidence for splicing in one species (RNA 4-), which together with its more abundant unspliced counterpart (RNA 2-) could synthesize both a 30.5 and 41 kd polypeptide from the same transcription unit. This extensive overlapping of spliced and unspliced RNAs could indicate that the initiation and splicing of transcripts is temporally regulated. At least one transcript (RNA 1-) may have a non-translational role. Transcription of the DSV genome shows similarities to some animal DNA viruses, particularly the papovaviruses.

Agrobacterium-mediated infectivity of cloned digitaria streak virus DNA.

A monomeric clone of double-stranded DNA synthesized in vitro DNA of the geminivirus Digitaria streak (DSV) was subcloned as a tandem dimeric unit into a binary vector of Agrobacterium tumefaciens, creating a plasmid pDS2. Inoculation of digitaria sanguinalis with A. tumefaciens carrying pDS2 resulted in viral infection. The symptoms, virus particles, and DNA forms obtained were indistinguishable from those of a natural DSV infection of D. sanguinalis. Inoculations have also induced infections in Zea mays and Avena sativa. The sequence of the Agrobacterium-mediated infectious clone of DSV has been determined.

Detection of a non-structural protein of M r 11 000 encoded by the virion DNA of maize streak virus.

A polypeptide of approximately 11 000 daltons (11 kDa protein) encoded by an open reading frame (10.9 ORF) from the virion sense of maize streak virus (MSV) DNA has been detected among the products of in vitro translation reactions programmed with RNA from infected maize plants and also in total protein extracts from infected leaves. The 11 kDa protein has not been detected in virions and is therefore proposed to have a nonstructural role.Viral DNA with an additional in-frame translation stop codon in the 10.9 ORF was not infectious when transmitted to maize plants via Agrobacterium tumefaciens "agroinfection", suggesting that the 10.9 ORF may be essential for virus function. Computer comparison data show that equivalent ORFs in wheat dwarf virus (WDV) and digitaria streak virus (DSV) have some sequences in common with the 10.9 ORF of MSV. Further-more, the absence of similar sequences in geminiviruses which infect dicotyledonous plants suggests that the 11 kDa protein and its putative homologs in WDV and DSV have a function necessary only for those geminiviruses which infect the Gramineae.The significance of the 11 kDa protein in relation to expression of the virion sense DNA of MSV is discussed.

The nucleotide sequence of a geminivirus from Digitaria sanguinalis.

The encapsidated single-stranded circular DNA of a geminivirus isolated from Digitaria sanguinalis has been sequenced. The data obtained are consistent with there being one DNA circle of 2701 nucleotides. Comparison of the nucleotide sequence with those of maize streak virus (MSV) and wheat dwarf virus showed 64 and 47% DNA homology, respectively. The sequence has four potential coding regions for proteins of greater than 10 kDa, two in the viral (+) sense and two in the complementary (-) sense. Each of these potential coding regions has a highly homologous counterpart among the seven open reading frames previously described for MSV. Virion DNA contained, in addition to the circular single-stranded DNA, a population of small DNA molecules similar to those associated with MSV particles. A comparison with MSV DNA of the region complementary to these small DNA molecules revealed conserved sequences, which may have a role in defining the limits of these primer-like molecules.

Structure and replication of geminivirus genomes.

The geminiviruses are a group of plant viruses containing single-stranded (ss) DNA in particles comprising two quasi-icosahedral units. Some are transmitted by whiteflies, others by leafhoppers. Comparisons were made of the genome organization and expression of cassava latent virus (CLV) and maize streak virus (MSV) and beet curly top virus (BCTV), each with distinct host range and insect vector species characteristics. From these studies, several indications as to the replication mechanism(s) are suggested.

Bidirectional transcription of maize streak virus DNA and identification of the coat protein gene.

Three RNA transcripts encoded by maize streak virus DNA were detected in polyadenylated RNA from virus-infected maize leaves. Two of the transcripts, a major 0.9 kb and a minor 1.05 kb RNA, were mapped on the virion (+) sense DNA and the other minor transcript of 1.2 kb was mapped on the complementary (-) sense DNA, demonstrating that transcription of MSV DNA was bidirectional. The two virion sense transcripts were 3' coterminal at nucleotide 1114 but had 5' termini at nucleotides 2682 and 163 respectively. Virus-specific polyadenylated RNA translated in vitro to produce a 28,000 MW polypeptide, specifically immunoprecipitable by antiserum raised against whole virus. The mRNA for this protein was mapped by hybrid-arrested translation to the long open reading frame in virion sense DNA whose potential amino acid composition, calculated from nucleotide sequence data, closely agreed with that determined experimentally for the coat protein.

Computer analysis identifies sequence homologies between potential gene products of Maize Streak Virus and those of Cassava Latent Virus and Tomato Golden Mosaic Virus.

The amino acid sequences of the putative polypeptides of maize streak virus (MSV) have been systematically compared with those of cassava latent virus (CLV) and tomato golden mosaic virus (TGMV) using the programme DIAGON (8).Conserved sequences have been detected between peptides encoded by the complementary (-) sense of MSV and those of CLV and TGMV, viz; the 40 200 Mr polypeptide of CLV-1 (3) and the 40 285 Mr polypeptide of TGMV-A (4) show extensive homologies with the 17 768 Mr and 31 388 Mr polypeptides of MSV (6).Distant and variable homologies have been detected between the putative coat protein of MSV when compared with those of CLV and TGMV. No other relationships between the potential gene products of MSV and those of CLV and TGMV have been detected.The extensive homologies detected between the complementary sense encoded peptides suggest that they are derived from functional genes, and that the directly conserved sequences may contain amino acids essential to the function of these proteins. The less extensive homologies among the putative coat proteins are considered in relation to their possible structures and functions.

The nucleotide sequence of maize streak virus DNA.

The nucleotide sequence of the DNA of maize streak virus (MSV) has been determined. The data were accommodated into one DNA circle of 2687 nucleotides, in contrast to previously characterised geminiviruses which have been shown to possess two circles of DNA. Comparison of the nucleotide sequences of the DNA of MSV with those of cassava latent virus (CLV) and tomato golden mosaic virus (TGMV) showed no detectable homology. Analysis of open reading frames revealed seven potential coding regions for proteins of mol. wt. greater than or equal to 10 000, three in the viral (+) sense and four in the complementary (-) sense. The position of likely transcription signals on the MSV DNA sequence would suggest a bidirectional strategy of transcription as proposed for CLV and TGMV. Nine inverted repeat sequences which have a potential of forming hairpin structures of delta G greater than or equal to -14 kcal/mol have been detected. Three of these hairpin structures are in non-coding regions and could be involved in the regulation of transcription and/or replication.

A putative primer for second-strand DNA synthesis of maize streak virus is virion-associated.

We have isolated, from maize streak virus (MSV) preparations, a population of 'nested' DNA molecules. These molecules have ribonucleotides covalently linked to the DNA species' discrete 5' deoxyribonucleotide terminus. The major species has a DNA sequence of 80 nucleotides which is complementary to a region 5' of two hairpin structures on the MSV genome, almost exclusively in an intergenic region. These molecules have been used to prime the synthesis in vitro of a complementary strand to virion DNA, initiating this reaction at one site on the genome.

The application of spot hybridization to the detection of DNA and RNA viruses in plant tissues.

A solid-phase nucleic acid hybridization technique for the detection of DNA and RNA viruses in plant tissues is described. The method involves spotting crude samples onto nitrocellulose and using 12P-labelled DNA hybridization probes. The limit of sensitivity is 5-20 pg virus/spot or approximately 5 micrograms/g leaf tissue. The method is quantitative for DNA viruses in crude homogenates, but not for RNA viruses. The amount of cauliflower mosaic virus in infected leaves and protoplasts was estimated. The amplitude of spot hybridization to screening plant material from glasshouses and field is discussed.