Cyanovirin-N produced in rice endosperm offers effective pre-exposure prophylaxis against HIV-1BaL infection in vitro.

KEY MESSAGE: Cyanovirin-N produced in rice endosperm provides efficient pre-exposure prophylaxis against HIV-1 BaL infection in vitro. Cyanovirin-N (CV-N) is a lectin with potent antiviral activity that has been proposed as a component of microbicides for the prevention of infection with Human immunodeficiency virus (HIV). The production of protein-based microbicide components requires a platform that is sufficiently economical and scalable to meet the demands of the large at-risk population, particularly in resource poor developing countries. We, therefore, expressed CV-N in rice endosperm, because the dried seed is ideal for storage and transport and crude extracts could be prepared locally and used as a microbicide component without further purification. We found that crude extracts from rice seeds expressing up to 10 µg CV-N per gram dry seed weight showed dose-dependent gp120 binding activity, confirming that the protein was soluble, correctly folded and active. The recombinant lectin ((OS)CV-N) reduced the infectivity of HIV-1BaL (an R5 virus strain representing the majority of transmitted infections) by ~90 % but showed only weak neutralization activity against HIV-1RF (representative of X4 virus, rarely associated with transmission), suggesting it would be highly effective for pre-exposure prophylaxis against the vast majority of transmitted strains. Crude extracts expressing (OS)CV-N showed no toxicity towards human cells at working dilutions indicating that microbicide components produced in rice endosperm are safe for direct application as topical microbicides in humans.

Can plant biotechnology help break the HIV-malaria link?

The population of sub-Saharan Africa is at risk from multiple, poverty-related endemic diseases. HIV and malaria are the most prevalent, but they disproportionately affect different groups of people, i.e. HIV predominantly affects sexually-active adults whereas malaria has a greater impact on children and pregnant women. Nevertheless, there is a significant geographical and epidemiological overlap which results in bidirectional and synergistic interactions with important consequences for public health. The immunosuppressive effects of HIV increase the risk of infection when individuals are exposed to malaria parasites and also the severity of malaria symptoms. Similarly, acute malaria can induce a temporary increase in the HIV viral load. HIV is associated with a wide range of opportunistic infections that can be misdiagnosed as malaria, resulting in the wasteful misuse of antimalarial drugs and a failure to address the genuine cause of the disease. There is also a cumulative risk of toxicity when antiretroviral and antimalarial drugs are given to the same patients. Synergistic approaches involving the control of malaria as a strategy to fight HIV/AIDS and vice versa are therefore needed in co-endemic areas. Plant biotechnology has emerged as a promising approach to tackle poverty-related diseases because plant-derived drugs and vaccines can be produced inexpensively in developing countries and may be distributed using agricultural infrastructure without the need for a cold chain. Here we explore some of the potential contributions of plant biotechnology and its integration into broader multidisciplinary public health programs to combat the two diseases in developing countries.

Functional characterization of the recombinant HIV-neutralizing monoclonal antibody 2F5 produced in maize seeds.

Monoclonal antibodies (mAbs) that neutralize human immunodeficiency virus (HIV) can be used as microbicides to help prevent the spread of HIV in human populations. As an industry standard, HIV-neutralizing mAbs are produced as recombinant proteins in mammalian cells, but the high manufacturing costs and limited capacity reduce the ability of target populations in developing countries to gain access to these potentially life-saving medicines. Plants offer a more cost-effective and deployable production platform because they can be grown inexpensively and on a large scale in the region where the products are required. Here we show that the maize-derived HIV-neutralizing mAb 2F5 is assembled correctly in planta and binds to its antigen with the same affinity as 2F5 produced in mammalian cells. Although 2F5 has been produced at high levels in non-plant platforms, the yield in maize seeds is lower than previously achieved with another HIV-neutralizing mAb, 2G12. This suggests that the intrinsic properties of the antibody (e.g. sensitivity to specific proteases) and the environment provided by the production host (e.g. the relative abundance of different proteases, potential transgene silencing) may combine to limit the accumulation of some antibodies on a case-by-case basis.

Transgenic plants for insect pest control: a forward looking scientific perspective.

One of the first successes of plant biotechnology has been the creation and commercialisation of transgenic crops exhibiting resistance to major insect pests. First generation products encompassed plants with single insecticidal Bt genes with resistance against major pests of corn and cotton. Modelling studies predicted that usefulness of these resistant plants would be short-lived, as a result of the ability of insects to develop resistance against single insecticidal gene products. However, despite such dire predictions no such collapse has taken place and the acreage of transgenic insect resistance crops has been increasing at a steady rate over the 9 years since the deployment of the first transgenic insect resistant plant. However, in order to assure durability and sustainability of resistance, novel strategies have been contemplated and are being developed. This perspective addresses a number of potentially useful strategies to assure the longevity of second and third generation insect resistant plants.

Producing transglutaminases by molecular farming in plants: minireview article.

Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities, both in terms of biology and biotechnology, is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases in research, medicine and industry has increased the demand for an inexpensive, efficient and safe source of recombinant enzymes. We describe initial results concerning the production of a mammalian transglutaminase in transgenic rice plants as a first step towards the large-scale molecular farming of this enzyme.

Transgenic rice as a vehicle for the production of the industrial enzyme transglutaminase.

Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases has increased the demand for an inexpensive, efficient and safe source of recombinant enzyme. We explored the use of plant-based systems for the production of this important industrial enzyme. Transgenic rice plants engineered with a rat prostate transglutaminase (rTGp), driven by the strong constitutive maize-1 ubiquitin promoter and its first intron, were shown to express the recombinant enzyme at the mRNA and protein levels. The Ca2+ dependence of the recombinant enzyme was confirmed by the biotin-labelled cadaverine-incorporation assay. In this communication we report the molecular and biochemical characterisation of transgenic plants expressing rTGp and this sets the stage for establishing a bioreactor system for the production of transglutaminases in plants.

Endogenous enzyme activities and polyamine levels in diverse rice cultivars depend on the genetic background and are not affected by the presence of the hygromycin phosphotransferase selectable marker.

We used the polyamine biosynthetic pathway and rice as a relevant model to understand the genetic basis of variation in endogenous levels of metabolites and key enzymes involved in the pathway. Wild-type tissues and also tissues containing a commonly used selectable marker gene were employed. We detected a wide variation in levels of arginine decarboxylase activity and in the three polyamines, putrescine, spermidine and spermine, in different tissues and varieties, but this was not dependent on the presence of the selectable marker. A more-extensive profile of enzyme activities (ADC, ODC, SAMDC, DAO and PAO) and polyamine levels in different tissues was generated in two different varieties. Our results indicate that genetic background is important in terms of the basal levels of metabolites and enzyme activity, particularly in situations in which we aim to engineer metabolic pathways that are also encoded by homologous endogenous genes. We did not find any evidence that the presence of a selectable marker in any way influences enzyme activity or metabolite levels.

Over-expression of a cDNA for human ornithine decarboxylase in transgenic rice plants alters the polyamine pool in a tissue-specific manner.

We investigated how over-expression of a cDNA for human ornithine decarboxylase (odc) affects the polyamine pools in transgenic rice. We further investigated tissue-specific expression patterns and product accumulation levels of the transgene driven by either constitutive or seed-specific promoters. Our results indicate that: (1) whereas the expression of a heterologous arginine decarboxylase (adc) cDNA in rice resulted in increased putrescine and spermine levels only in seeds, plants engineered to express odc cDNA exhibited significant changes in the levels of all three major polyamines in seeds and also in vegetative tissues (leaves and roots); (2) there was no linear correlation between odc mRNA levels, ODC enzyme activity and polyamine accumulation, suggesting that control of the polyamine pathway in plants is more complex than in mammalian systems; (3) ODC activity and polyamine changes varied in different tissues, indicating that the pathway is regulated in a tissue-specific manner. Our results suggest that ODC rather than ADC is responsible for the regulation of putrescine synthesis in plants.

Simultaneous reduction of the activity of two related enzymes, involved in early steps of the polyamine biosynthetic pathway, by a single antisense cDNA in transgenic rice.

Transgenic rice cell lines transformed with a heterologous cDNA derived from the arginine decarboxylase gene of oat, in an antisense orientation, exhibited significant (P < 0.05) down-regulation of the activity of the endogenous arginine and ornithine decarboxylases, compared to wild type and controls transformed only with the selectable marker (hpt). Changes in enzyme activity were reflected in a marked decrease in the level of putrescine (P < 0.001) and spermidine (P < 0.01) but not spermine (P > 0.05) in the majority of cell lines analyzed. In agreement with previous results, we confirmed that cell lines with low levels of polyamines exhibited normal morphogenic responses. In vegetative tissue at the whole plant level no significant variation (P > 0.05) in polyamine levels was observed. However, we measured significant reductions (P < 0.001) in putrescine levels in seeds derived from three out of five plants analyzed in detail. Thus, simultaneous reduction of the activity of the two alternative enzymes in the early steps of the polyamine pathway results in significant reduction in end-product accumulation in the seeds of transgenic plants.

A transgenic rice cell lineage expressing the oat arginine decarboxylase (adc) cDNA constitutively accumulates putrescine in callus and seeds but not in vegetative tissues.

We introduced the oat adc cDNA into rice under the control of the constitutive maize ubiquitin 1 promoter. We studied molecularly and biochemically sixteen independent transgenic plant lines. Significant increases in mRNA levels, ADC enzyme activity and polyamines were measured in transgenic callus. These increases were not maintained in vegetative tissue or seeds in regenerated plants, with the exception of one lineage. This particular lineage showed very significant increases in putrescine preferentially in seeds (up to 10 times compared to wild type and controls transformed with the hpt selectable marker alone). We have demonstrated that in cereals such as rice, over-expression of the oat adc cDNA results in increased accumulation of polyamines at different stages of development. We have also demonstrated that strong constitutive promoters, such as the maize ubiquitin 1 promoter, are sufficient to facilitate heritable high-level polyamine accumulation in seed. Our results demonstrate that by screening adequate numbers of independently derived transgenic plants, it is possible to identify those individuals which express a desired phenotype or genotype.

Promoter strength influences polyamine metabolism and morphogenic capacity in transgenic rice tissues expressing the oat adc cDNA constitutively.

We analyzed molecularly and biochemically a series of transgenic rice lines expressing the oat adc (arginine decarboxylase) cDNA under the control of the constitutive maize ubiquitin 1 promoter. We established baseline biochemical parameters to elucidate the role of polyamines (PAs) during morphogenesis. We measured mRNA levels, ADC enzyme activity and cellular PAs in dedifferentiated callus. Polyamine levels were also quantified in two subsequent developmental stages--regenerating tissue and differentiated shoots. We observed significant (P < 0.05) differences in the levels of individual PAs at the three developmental stages. The amounts of putrescine (Put) and spermidine (Spd) in dedifferentiated transgenic callus were lower than those in the wild type or in hpt (hygromycin resistant)-controls, whereas the amount of spermine (Spm) was increased up to two-fold. In regenerating tissue, this trend was reversed, with significantly higher levels of Put and Spd (P < 0.05), and lower levels of Spm (P < 0.05) compared to non-transformed or hpt-control tissues at the same developmental stage. In differentiated shoots, there was a general increase in PA levels, with significant increases in Put, Spd, and Spm (P < 0.05); on occasion reaching six times the level observed in wild type and hpt-control tissues. These results contrast those we reported previously using the weaker CaMV 35S promoter driving adc expression. mRNA measurements and ADC enzyme activity were consistently higher (P < 0.01) in all tissues expressing pUbiadcs compared to equivalent tissues engineered with 35Sadc. Our findings are consistent with a threshold model which postulates that high adc expression leading to production of Put above a basal level is necessary to generate a big enough metabolic pool to trigger PA flux through the pathway leading to an increase in the concentration of Spd and Spm. This can be best accomplished by a strong constitutive promoter driving adc. We discuss our results in the context of flux through the PA pathway and its impact on morphogenesis.

Rapid high-performance liquid chromatographic method for the quantitation of polyamines as their dansyl derivatives: application to plant and animal tissues.

A rapid high-performance liquid chromatographic method for the separation of polyamines as their dansyl derivative has been developed. The chromatographic system used consisted of a reversed-phase column and a mobile phase of acetonitrile and water. The separation of 1,3-diaminopropane, putrescine, cadaverine, spermidine and spermine takes only 9 min. This method provides a good resolution between 1,3-diaminopropane and putrescine. It has been applied to quantify polyamines from seeds of wheat, petals of Phalaenopsis hybrids and various rat tissues.