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.

Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance.

Hydroperoxide lyases (HPLs) catalyze the cleavage of fatty acid hydroperoxides to aldehydes and oxoacids. These volatile aldehydes play a major role in forming the aroma of many plant fruits and flowers. In addition, they have antimicrobial activity in vitro and thus are thought to be involved in the plant defense response against pest and pathogen attack. An HPL activity present in potato leaves has been characterized and shown to cleave specifically 13-hydroperoxides of both linoleic and linolenic acids to yield hexanal and 3-hexenal, respectively, and 12-oxo-dodecenoic acid. A cDNA encoding this HPL has been isolated and used to monitor gene expression in healthy and mechanically damaged potato plants. HPL gene expression is subject to developmental control, being high in young leaves and attenuated in older ones, and it is induced weakly by wounding. HPL enzymatic activity, nevertheless, remains constant in leaves of different ages and also after wounding, suggesting that posttranscriptional mechanisms may regulate its activity levels. Antisense-mediated HPL depletion in transgenic potato plants has identified this enzyme as a major route of 13-fatty acid hydroperoxide degradation in the leaves. Although these transgenic plants have highly reduced levels of both hexanal and 3-hexenal, they show no phenotypic differences compared with wild-type ones, particularly in regard to the expression of wound-induced genes. However, aphids feeding on the HPL-depleted plants display approximately a two-fold increase in fecundity above those feeding on nontransformed plants, consistent with the hypothesis that HPL-derived products have a negative impact on aphid performance. Thus, HPL-catalyzed production of C6 aldehydes may be a key step of a built-in resistance mechanism of plants against some sucking insect pests.

Antisense-mediated depletion of a potato lipoxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pests.

De novo jasmonic acid (JA) synthesis is required for wound-induced expression of proteinase inhibitors and other defense genes in potato and tomato. The first step in JA biosynthesis involves lipoxygenase (LOX) introducing molecular oxygen at the C-13 position of linolenic acid. We previously have shown that, in potato, at least two gene families code for 13-LOX proteins. We have now produced transgenic potato plants devoid of one specific 13-LOX isoform (LOX-H3) through antisense-mediated depletion of its mRNA. LOX-H3 depletion largely abolishes accumulation of proteinase inhibitors on wounding, indicating that this specific LOX plays an instrumental role in the regulation of wound-induced gene expression. As a consequence, weight gain of Colorado potato beetles fed on antisense plants is significantly larger than those fed on wild-type plants. The poorer performance of LOX-H3-deficient plants toward herbivory is more evident with a polyphagous insect; larvae of beet armyworm reared on the antisense lines have up to 57% higher weight than those fed on nontransformed plants. LOX-H3 thus appears to regulate gene activation in response to pest attack, and this inducible response is likely to be a major determinant for reducing performance of nonspecialized herbivores. However, the regulatory role of LOX-H3 is not caused by its involvement in the wound-induced increase of JA, as wild-type and LOX-H3 deficient plants have similar jasmonate levels after wounding. LOX-H3-deficient plants have higher tuber yields. The apparent effect of suppressing the inducible defensive response on plant vigor suggests that it may pose a penalty in plant fitness under nonstress situations.

Reversible protein phosphorylation regulates jasmonic acid-dependent and -independent wound signal transduction pathways in Arabidopsis thaliana.

Plants responses to mechanical injury are complex and include the induced expression of defence-related genes. The phytohormone JA has been reported to mediate some of these responses. To elucidate further the signal transduction processes involved, the action of specific agonists and antagonists of known signalling effectors on the response of Arabidopsis thaliana plantlets to JA and wounding was investigated. The identification and characterization of a reversible protein phosphorylation step in a transduction pathway leading to JA-induced gene transcription is reported. This phosphorylation event involved the opposing activities of a staurosporine-sensitive protein kinase, negatively regulating the pathway, and a protein phosphatase, most probably of type 2 A, which activated JA-responsive gene expression. JA activation via this pathway was blocked in the A. thaliana JA-insensitive mutants jin1, jin4 and coi1, and by exogenous application of cycloheximide or auxins. Wound-induced activation of JA-responsive genes was also regulated by this protein phosphorylation step. An alternative wound signalling pathway, independent of JA, was also identified, leading to the transcriptional activation of a different set of genes. This JA-independent pathway was also regulated by a protein phosphorylation switch, in which the protein kinase positively regulated the pathway while the protein phosphatase negatively regulated it. Moreover, a labile protein apparently repressed the expression of these genes. One of the genes analysed, JR3, had a complex pattern of expression, possibly because it was regulated via both of the wound signalling pathways identified. According to the function of an homologous gene, JR3 may be involved in feedback inhibition of the JA response.

Characterization of three potato lipoxygenases with distinct enzymatic activities and different organ-specific and wound-regulated expression patterns.

Lipoxygenases are ubiquitous enzymes in eukaryotes. In plants, lipoxygenases are involved in the synthesis of the hormone jasmonic acid that regulates plant responses to wounding and, in addition, is an inducer of tuberization in potato. We have isolated potato lipoxygenase cDNA clones. From their deduced amino acid sequences, three distinct classes are defined (Lox1, Lox2, and Lox3). They are encoded in gene families that display organ-specific expression, lox1 being expressed mostly in tubers and roots, lox2 in leaves, and lox3 in leaves and roots. Consistent with their organ-specific expression pattern, Lox1 expressed in bacteria preferentially uses as substrate linoleic acid, abundant in membrane lipids of tubers, whereas linolenic acid, prevalent in leaves, is the preferred substrate for the other two classes of lipoxygenase. Analyses on reaction products of the enzymes expressed in bacteria reveal that Lox1 primarily produces 9- hydroperoxides. In contrast, the jasmonic acid precursor, 13-hydroperoxylinolenic acid, is the major product of the action of Lox2 and Lox3 on linolenic acid. Upon wounding, the levels of Lox2 and Lox3 transcripts rise markedly in leaves. While Lox3 mRNA accumulation peaks as early as 30 min after wounding, Lox2 shows a steady increase over a 24-h time course, suggesting different roles for these lipoxygenase isoforms in the synthesis of the plant hormone jasmonic acid.

LAT52 protein is essential for tomato pollen development: pollen expressing antisense LAT52 RNA hydrates and germinates abnormally and cannot achieve fertilization.

The LAT52 gene of tomato is expressed in a pollen-specific manner. It is shown that LAT52 encodes a heat-stable, glycosylated protein that traverses the secretory pathway when expressed in a baculovirus expression system. The LAT52 protein shows some similarity with Kunitz trypsin inhibitors and with pollen proteins from maize, rice and olive, but the biological function of these pollen proteins is unknown. To test whether the LAT52 protein plays an important role during pollen development, tomato plants were transformed with an antisense LAT52 gene driven by the LAT52 promoter. Because the LAT52 gene is expressed gametophytically, only 50% of the pollen of the primary transformants would be expected to express the antisense construct. Selfprogeny of 19 of the primary transformants showed the predicted 3:1 segregation for a single locus insertion of the linked kanamycin-resistance gene. However, the self-progeny of the other 32 primary transformants showed a 1:1 segregation pattern and could not transmit the linked kanamycin-resistance gene through the male. A subset of these 1:1 segregation class plants was examined in detail. The pollen showed lower levels of LAT52 mRNA and LAT52 protein when compared with wild-type. In vitro, approximately 50% of the pollen grains appear to hydrate abnormally; this anomaly is not present when the same pollen grains are incubated in a medium with higher water potential. In vivo pollination experiments showed that the growth of around 50% of the pollen tubes is arrested in the style. The 3:1 segregation class plants showed no significant differences from untransformed control plants. Taken together, the results show a direct correlation between the reduced expression of LAT52 protein and abnormal pollen function, and suggest that the LAT52 protein plays a role in pollen hydration and/or pollen germination.

Molecular analysis of gene regulation and function during male gametophyte development.

We have characterised three pollen-expressed genes (LAT52, LAT56 and LAT59) from tomato in order to determine their role in pollen development, and to determine the DNA sequences responsible for gene expression in pollen. LAT52 encodes a protein that shows amino acid sequence similarity to a protein encoded by a pollen-specific cDNA clone (pZmc13) isolated from maize, and both proteins have amino acid sequence similarity to Kunitz trypsin inhibitors of soybean and winged bean. The proteins encoded by LAT56 and LAT59 genes are 54% identical at the amino acid level, and show significant sequence similarity to bacterial pectate lyases and to a fungal pectin lyase. Additionally, regions of LAT56 and LAT59 show significant sequence similarity to tryptic peptides of ragweed and Japanese cedar pollen allergens. Preliminary results suggest that plants harboring antisense constructs of the LAT52 coding region show defects in pollen germination and fertilisation; no obvious phenotype was seen in plants harboring antisense constructs of the LAT59 coding region. Promoter fragments of these three LAT genes were fused to the reporter gene GUS and assayed using both a transient system and stably transformed plants. We have identified relatively short regions of the LAT promoters that are important for pollen expression, and are attempting to isolate trans-acting factors that interact with these cis-acting sequences, using both molecular and classical genetic approaches.

Translatability of a plant-mRNA strongly influences its accumulation in transgenic plants.

Current knowledge of parameters affecting RNA stability is very restricted in plants. Here we investigated factors which might contribute to the stability of a particular plant messenger RNA. To this end, insertion and deletion mutants were made in two different exons and an intron of the transcribed region of a well characterised patatin gene (pgT5). Mutant genes were expressed under the control of a strong leaf-stem specific promoter (ST-LS1) and analysed in vivo in transgenic tobacco plants. Northern analysis revealed the importance of the translatability of the mature messenger RNA with respect to its accumulation in transgenic plants. Enlargement of the 3' non-translated region by several hundred base-pairs reduced the steady state mRNA level slightly; the introduction of a stop codon leading to premature termination of translation of the RNA led to a dramatic decrease of the steady state mRNA level.

Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation.

Agrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to overcome this obstacle a modified prokaryotic indicator gene was constructed. The expression of this indicator gene and its use in analysing early events in Agrobacterium-mediated plant transformation are described. A portable intron, derived from a plant intron, was introduced into the beta-glucuronidase (GUS) gene. In transgenic plants containing this chimaeric gene the intron is spliced efficiently, giving rise to GUS enzymatic activity. Mapping of the splice junction indicates the exact removal of the intron. No GUS activity is detected in agrobacteria containing this construct due to the lack of a eukaryotic splicing apparatus in prokaryotes. Early phases after transformation of Arabidopsis cotyledon explants were analysed using this GUS-intron chimaeric gene showing that as early as 36 h after Agrobacterium infection significant GUS activity is detected. In vivo GUS staining of transformed cells clearly shows that quickly proliferating calli expressing GUS activity are formed, mainly at the cut surface. Minor transformation events occur however throughout the whole cotyledon. These data indicate that Agrobacterium-mediated T-DNA transfer to plants is much more efficient than has been judged from experiments where selection is applied immediately. The intron-containing GUS gene can be used as an optimised marker gene in transient and stable transformation experiments.

Expression of a Patatin-like Protein in the Anthers of Potato and Sweet Pepper Flowers.

Patatin, the major glycoprotein in potato tubers, is encoded by a multigene family. RNA and protein analyses reveal that a homologous mRNA and an immunologically cross-reacting protein can be found in potato flowers, which is similar to patatin in that it displays a lipid acyl hydrolase activity. The patatin-like protein found in flowers has a higher molecular weight than the authentic tuber patatin. Deglycosylation experiments show that this is not due to differences in the glycosylation pattern. Immunocytochemical analysis shows the patatin-like protein to be present only in the epidermal cell layer of the anther, the exothecium, and in petals of potato flowers. Furthermore, the fact that a patatin-like protein can be detected in a similar tissue in sweet pepper, another solanaceous plant, could give a clue concerning the evolutionary origin of patatin.