125 years of virology and ascentof biotechnologies based on viral expression.

The study of viruses lasts for more than a century since their discovery in 1892. In recent decades, viruses are also being actively exploited as a biotechnological tool. Plant-virus-driven transient expression of heterologous proteins is an actively developing production platform; it is the basis of several industrial processes that are currently being used for the production of multiple recombinant proteins. Viral vectors have also become useful tools for research. Viral vectors delivered by Agrobacterium (magnifection) provide for high pro-tein yield, rapid scale up and fast manufacturing. In this review, we explore modern approaches for bio technological production of recombinant proteins in plants using viral vectors.

Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma.

BACKGROUND: Animal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. DESIGN: We report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). RESULTS: Full idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. CONCLUSIONS: This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods.

Magnifection--a new platform for expressing recombinant vaccines in plants.

Today, plant biotechnology relies on two processes for delivery and expression of heterologous genes in plants: stable genetic transformation and transient infection with viral vectors. Although much faster, the transient route until recently was limited because of virus' low infectivity and its inability to carry average-size or larger transgenes. A recently developed new generation transfection technology overcomes these limitations by relying on Agrobacterium as an infective systemic agent that delivers viral replicons. This improved process is being used to simultaneously start transient gene amplification and high-level expression in all mature leaves of a plant, and such a transfection can be done on an industrial scale. This eclectic technology, called 'magnifection', combines advantages of three biological systems: vector efficiency and efficient systemic DNA delivery of Agrobacterium, speed and expression level/yield of a plant RNA virus, as well as posttranslational capabilities and low production costs of a plant. The proposed process allows for industrial production that does not require genetic modification of plants, that is much faster than previous methods, and that is biologically safe. Numerous applications in the area of vaccine manufacturing are being discussed.

Transposition patterns of unlinked transposed Ds elements from two T-DNA loci on tomato chromosomes 7 and 8.

We have previously reported that unlinked transposed Ds elements originating from chromosome 4 of tomato preferentially inserted in chromosome 2. This observation, together with data from other studies, suggested that there may be absolute preferences for transposition, irrespective of the chromosomal location of the donor site. The aim of the present work was to verify whether the distribution of transposed Ds elements on chromosome 2 was non-random and thus whether, unlike the case in maize, unlinked transpositions in tomato are not distributed randomly. To do this, unlinked acceptor sites of Ds elements originating from two donor T-DNA loci lying on chromosomes 7 and 8 were mapped. Receptor sites for tr Ds elements transposed from the 1601D locus on chromosome 8 exhibited a non-random distribution (P<0.01). Eleven out of 46 independent transpositions mapped to chromosome 2 and, as this was statistically significant (P<0.01), proves that receptor sites for this element are not randomly distribution on the chromosomes. In addition, deviation of the observed number from the expected number of tr Dss was close to being significant for chromosome 4 (P=0.05-0.1). In contrast, the distribution of unlinked receptor sites for tr Dss derived from the 1481J locus on chromosome 7 was random. Chi(2)tests were performed for each chromosome, and for chromosome 4 the difference between the observed and the expected number of tr Dss was very high but statistically non-significant (P=0.05-0.1). For chromosome 2 the difference was statistically negligible. Therefore, we conclude that chromosome 2 does not serve as a preferential receptor for the transposition of Ds elements independently of the location of the donor site.

Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA-insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics.

A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator (En/Spm) element was introduced into Arabidopsis. A single T-DNA construct carried a nonautonomous defective Spm (dSpm) element with a phosphinothricin herbicide resistance (BAR) gene, a transposase expression cassette, and a counterselectable gene. This construct was used to select for stable dSpm transpositions. Treatments for both positive (BAR) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale. To date, a total of 48,000 lines in pools of 50 have been recovered, of which approximately 80% result from independent insertion events. DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest. By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations.

A chromodomain protein encoded by the arabidopsis CAO gene is a plant-specific component of the chloroplast signal recognition particle pathway that is involved in LHCP targeting.

A recessive mutation in Arabidopsis, named chaos (for chlorophyll a/b binding protein harvesting-organelle specific; designated gene symbol CAO), was isolated by using transposon tagging. Characterization of the phenotype of the chaos mutant revealed a specific reduction of pigment binding antenna proteins in the thylakoid membrane. These nuclear-encoded proteins utilize a chloroplast signal recognition particle (cpSRP) system to reach the thylakoid membrane. Both prokaryotes and eukaryotes possess a cytoplasmic SRP containing a 54-kD protein (SRP54) and an RNA. In chloroplasts, the homolog of SRP54 was found to bind a 43-kD protein (cpSRP43) rather than to an RNA. We cloned the CAO gene, which encodes a protein identified as Arabidopsis cpSRP43. The product of the CAO gene does not resemble any protein in the databases, although it contains motifs that are known to mediate protein-protein interactions. These motifs include ankyrin repeats and chromodomains. Therefore, CAO encodes an SRP component that is unique to plants. Surprisingly, the phenotype of the cpSRP43 mutant (i.e., chaos) differs from that of the Arabidopsis cpSRP54 mutant, suggesting that the functions of the two proteins do not strictly overlap. This difference also suggests that the function of cpSRP43 is most likely restricted to protein targeting into the thylakoid membrane, whereas cpSRP54 may be involved in an additional process(es), such as chloroplast biogenesis, perhaps through chloroplast-ribosomal association with chloroplast ribosomes.

A novel signal recognition particle targets light-harvesting proteins to the thylakoid membranes.

The mechanisms involved in the posttranslational targeting of membrane proteins are not well understood. The light-harvesting chlorophyll proteins (LHCP) of the thylakoid membrane are a large family of hydrophobic proteins that are targeted in this manner. They are synthesized in the cytoplasm, translocated across the chloroplast envelope membranes into the stroma, bound by a stromal factor to form a soluble intermediate, "transit complex", and then integrated into the thylakoid membrane by a GTP dependent reaction. Signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, is known to mediate the GTP dependent cotranslational targeting of proteins to the endoplasmic reticulum. We show that chloroplasts contain an SRP consisting of, cpSRP54, a homologue of SRP54 and a previously undescribed 43-kDa polypeptide (cpSRP43) instead of an RNA. We demonstrate that both subunits of cpSRP are required for the formation of the transit complex with LHCP. Furthermore, cpSRP54, cpSRP43, and LHCP are sufficient to form a complex that appears to be identical to authentic transit complex. We also show that the complex formed between LHCP and cpSRP, together with an additional soluble factor(s) are required for the proper integration of LHCP into the thylakoid membrane. It appears that the expanded role of cpSRP in posttranslational targeting of LHCP has arisen through the evolution of the 43-kDa protein.

Transposon tagging of the Defective embryo and meristems gene of tomato.

The shoot and root apical meristems (SAMs and RAMs, respectively) of higher plants are mechanistically and structurally similar. This has led previously to the suggestion that the SAM and RAM represent modifications of a fundamentally homologous plan of organization. Despite recent interest in plant development, especially in the areas of meristem regulation, genes specifically required for the function of both the SAM and RAM have not yet been identified. Here, we report on a novel gene, Defective embryo and meristems (Dem), of tomato. This gene is required for the correct organization of shoot apical tissues of developing embryos, SAM development, and correct cell division patterns and meristem maintenance in roots. Dem was cloned using transposon tagging and shown to encode a novel protein of 72 kD with significant homology to YNV2, a protein of unknown function of Saccharomyces cerevisiae. Dem is expressed in root and shoot meristems and organ primordia but not in callus. The expression pattern of Dem mRNA in combination with the dem mutant phenotype suggests that Dem plays an important role within apical meristems.

AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression.

Based on homologies between the yeast DMC1 and the lily LIM15 meiosis-specific genes, degenerate PCR primers were designed that amplified the Arabidopsis DMC1 gene (AtDMC1). AtDMC1 genomic DNA (8 kb) was sequenced, and the transcript was characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) and by 5' and 3' RACE (rapid amplification of cDNA ends). The AtDMC1 gene contains 15 exons and 14 introns. RNA in situ hybridization analysis showed that expression of the AtDMC1 is restricted to pollen mother cells in anthers and to megaspore mother cells in ovules. The AtDMC1 promoter was fused to the GUS reporter gene, and conferred meiosis-associated expression in both male and female floral lineages. Comparison of AtDMC1 isolated from Landsberg erecta ecotype to its Columbia allele ArLIM15, revealed the presence of a 1874 bp transposon-like element within the promoter region of ArLIM15. RT-PCR analysis showed that the expression levels of AtDMC1 and ArLIM15 are similar. Possible uses for the AtDMC1 promoter are discussed.

Novel GUS expression patterns following transposition of an enhancer trap Ds element in Arabidopsis.

Enhancer trap derivatives of the maize Dissociation (Ds) transposon were introduced into Arabidopsis thaliana. The enhancer trap Ds was so designed that upon transposition to sites containing regulatory sequences in adjacent genomic DNA, transcription of a Ds-borne beta-glucuronidase (GUS) gene would be activated. Sixty percent of all transposition events were associated with GUS expression patterns including one linked to a mutant phenotype. Patterns of GUS expression were found in various organs and were stably inheritable in the F4 and F5 progenies. These results demonstrate the potential value of the technique as a means for detection of developmentally regulated genes and analysis of their function. The enhancer trap construct used in our experiments, as well as the seeds of primary transformants are publicly available.

Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator.

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3' Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.

Distribution of unlinked transpositions of a Ds element from a T-DNA locus on tomato chromosome 4.

In maize, receptor sites for unlinked transpositions of Activator (Ac) elements are not distributed randomly. To test whether the same is true in tomato, the receptor sites for a Dissociation (Ds) element derived from Ac, were mapped for 26 transpositions unlinked to a donor T-DNA locus on chromosome 4. Four independent transposed Dss mapped to sites on chromosome 4 genetically unlinked to the donor T-DNA, consistent with a preference for transposition to unlinked sites on the same chromosome as opposed to sites on other chromosomes. There was little preference among the nondonor chromosomes, except perhaps for chromosome 2, which carried seven transposed Dss, but these could not be proven to be independent. However, these data, when combined with those from other studies in tomato examining the distribution of transposed Acs or Dss among nondonor chromosomes, suggest there may be absolute preferences for transposition irrespective of the chromosomal location of the donor site. If true, transposition to nondonor chromosomes in tomato would differ from that in maize, where the preference seems to be determined by the spatial arrangement of chromosomes in the interphase nucleus. The tomato lines carrying Ds elements at known locations are available for targeted transposon tagging experiments.

Germinal transpositions of the maize element Dissociation from T-DNA loci in tomato.

We have analyzed the pattern of germinal transpositions of artificial Dissociation (Ds) transposons in tomato. T-DNA constructs carrying Ds were transformed into tomato, and the elements were trans-activated by crossing to lines transformed with a stabilized Activator (sAc) that expressed the transposase gene. The sAc T-DNA carried a GUS gene to monitor its segregation. The Ds elements were inserted in a marker gene so that excision from the T-DNA could be monitored. The Ds elements also carried a genetic marker that was intended to be used for reinsertion selection of the elements after excision. Unfortunately, this gene was irreversibly inactivated on crossing to sAc. Germinal excision frequencies of Ds averaged 15-40%, but there was large variation between and within plants. Southern hybridization analysis of stable transposed Ds elements indicated that although unique transpositions predominate, early transposition events can lead to large clonal sectors in the germline of developing plants and to sibling offspring carrying the same transposition event. Multiple germinal transpositions from three different loci were examined for uniqueness, and 15 different transpositions were identified from each of three T-DNA loci that carried a single independent Ds. These were mapped relative to the donor T-DNA loci, and for each locus 70-80% of the transposed elements were closely linked to the donor site.

Alkali treatment for rapid preparation of plant material for reliable PCR analysis.

For plant genetics, it would be useful to monitor easily the segregation of different alleles using the polymerase chain reaction (PCR). Preparation of DNA templates from individual plants needs to be rapid and reliable. A one tube protocol is described that involves subjecting plant tissue pieces to alkali, neutralization and heat denaturation prior to PCR analysis, and that proved to be much faster and more reliable than published protocols.