Methanol may function as a cross-kingdom signal

Recently, we demonstrated that leaf wounding results in the synthesis of pectin methylesterase (PME), which causes the plant to release methanol into the air. Methanol emitted by a wounded plant increases the accumulation of methanol-inducible gene mRNA and enhances antibacterial resistance as well as cell-to-cell communication, which facilitates virus spreading in neighboring plants. We concluded that methanol is a signaling molecule involved in within-plant and plant-to-plant communication. Methanol is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of methanol into toxic formaldehyde. However, recent data showed that methanol is a natural compound in normal, healthy humans. These data call into question whether human methanol is a metabolic waste product or whether methanol has specific function in humans. Here, to reveal human methanol-responsive genes (MRGs), we used suppression subtractive hybridization cDNA libraries of HeLa cells lacking ADH and exposed to methanol. This design allowed us to exclude genes involved in formaldehyde and formic acid detoxification from our analysis. We identified MRGs and revealed a correlation between increases in methanol content in the plasma and changes in human leukocyte MRG mRNA levels after fresh salad consumption by volunteers. Subsequently, we showed that the methanol generated by the pectin/PME complex in the gastrointestinal tract of mice induces the up- and downregulation of brain MRG mRNA. We used an adapted Y-maze to measure the locomotor behavior of the mice while breathing wounded plant vapors in two-choice assays. We showed that mice prefer the odor of methanol to other plant volatiles and that methanol changed MRG mRNA accumulation in the mouse brain.We hypothesize that the methanol emitted by wounded plants may have a role in plant-animal signaling. The known positive effect of plant food intake on human health suggests a role for physiological methanol in human gene regulation.     

Molecular typing of Treponema pallidum: a systematic review and meta-analysis

Background

Syphilis is resurgent in many regions of the world. Molecular typing is a robust tool for investigating strain diversity and epidemiology. This study aimed to review original research on molecular typing of Treponema pallidum (T. pallidum) with three objectives: (1) to determine specimen types most suitable for molecular typing; (2) to determine T. pallidum subtype distribution across geographic areas; and (3) to summarize available information on subtypes associated with neurosyphilis and macrolide resistance.

Methodology/Principal Findings

Two researchers independently searched five databases from 1998 through 2010, assessed for eligibility and study quality, and extracted data. Search terms included “Treponema pallidum,” or “syphilis,” combined with the subject headings “molecular,” “subtyping,” “typing,” “genotype,” and “epidemiology.” Sixteen eligible studies were included. Publication bias was not statistically significant by the Begg rank correlation test. Medians, inter-quartile ranges, and 95% confidence intervals were determined for DNA extraction and full typing efficiency. A random-effects model was used to perform subgroup analyses to reduce obvious between-study heterogeneity. Primary and secondary lesions and ear lobe blood specimens had an average higher yield of T. pallidum DNA (83.0% vs. 28.2%, χ² = 247.6, p<0.001) and an average higher efficiency of full molecular typing (80.9% vs. 43.1%, χ² = 102.3, p<0.001) compared to plasma, whole blood, and cerebrospinal fluid. A pooled analysis of subtype distribution based on country location showed that 14d was the most common subtype, and subtype distribution varied across geographic areas. Subtype data associated with macrolide resistance and neurosyphilis were limited.

Conclusions/Significance

Primary lesion was a better specimen for obtaining T. pallidum DNA than blood. There was wide geographic variation in T. pallidum subtypes. More research is needed on the relationship between clinical presentation and subtype, and further validation of ear lobe blood for obtaining T. pallidum DNA would be useful for future molecular studies of syphilis.     

Slight tissue wounding fails to induce consistent chemical defense in three willow (Salix spp.) clones

We studied the effects of natural wounding by insects and artificial wounding by clipping with scissors on the phenolic chemistry of two willows, Salix myrsinifolia and Salix pentandra. Half of the blade of a mature leaf was removed from each experimental plant either by allowing insects (chrysomellid beetles) to feed on the leaf or by clipping off half the blade of a leaf with scissors. We also examined the ability of wounded plants to warn neighboring plants of imminent wounding by an airborne signal by maintainign one set of control plants in the room containing the wounded plants and another set of control plants in a room hermetically sealed from the room containing the wounded plants. After 48 h, the experimental leaf and the fourth leaf and eighth leaf upwards in the leaf sequence from the experimental leaf were analyzed for phenols by high-pressure liquid chromatography. The same leaves in the leaf sequence from each control plant were similarly analyzed for phenols. Only one phenol, salicortin in leaves of S. myrsinifolia, increased in concentration in response to defoliation, and the observed response was small. The type of wounding affected this increase in salicortin, with natural wounding by insects causing a greater response than artificial wounding in one S. myrsinifolia clone, and artificial wounding causing a greater response than insect wounding in the other clone. This result indicates that S. myrsinifolia cannot control the effects of diffeeent types of wounding on its leaf secondary chemistry. We also found no indication of airborne warning signals between wounded and unwounded plants that trigger an elevation of leaf defenses in unwounded plants in anticipation of herbivore attack.     

Systemic movement of a tobamovirus requires host cell pectin methylesterase

Systemic movement of plant viruses through the host vasculature, one of the central events of the infection process, is essential for maximal viral accumulation and development of disease symptoms. The host plant proteins involved in this transport, however, remain unknown. Here, we examined whether or not pectin methylesterase (PME), one of the few cellular proteins known to be involved in local, cell-to-cell movement of tobacco mosaic virus (TMV), is also required for the systemic spread of viral infection through the plant vascular system. In a reverse genetics approach, PME levels were reduced in tobacco plants using antisense suppression. The resulting PME antisense plants displayed a significant degree of PME suppression in their vascular tissues but retained the wild-type pattern of phloem loading and unloading of a fluorescent solute. Systemic transport of TMV in these plants, however, was substantially delayed as compared to the wild-type tobacco, suggesting a role for PME in TMV systemic infection. Our analysis of virus distribution in the PME antisense plants suggested that TMV systemic movement may be a polar process in which the virions enter and exit the vascular system by two different mechanisms, and it is the viral exit out of the vascular system that involves PME.     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Regulation of jasmonic acid biosynthesis by silicon application during physical injury to Oryza sativa L.

We investigated the effects of silicon (Si) application on rice plants (Oryza sativa L.) and its responses in the regulation of jasmonic acid (JA) during wounding stress. Endogenous JA was significantly higher in wounded rice plants than in non-wounded. In contrast, Si treatment significantly reduced JA synthesis as compared to non-Si applications under wounding stress. mRNA expression of O. sativa genes showed down-regulation of lipoxygenase, allene oxide synthase 1, allene oxide synthase 2, 12-oxophytodienoate reductase 3, and allene oxide cyclase upon Si application and wounding stress as compared to non-Si-treated wounded rice plants. The physical injury-induced-oxidative stress was modulated by Si treatments, which resulted in higher catalase, peroxidase, and polyphenol oxidase activities as compared with non-Si-treated plants under wounding stress. The higher Si accumulation in rice plants also reduced the level of lipid peroxidation, which helped the rice plants to protect it from wounding stress. In conclusion, Si accumulation in rice plants mitigated the adverse effects of wounding through regulation of antioxidants and JA.     

The leukemia-specific fusion gene ETV6/RUNX1 perturbs distinct key biological functions primarily by gene repression

Background

ETV6/RUNX1 (E/R) (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL) and also most likely the crucial factor for disease initiation; its role in leukemia propagation and maintenance, however, remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD) of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines.

Findings

Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP) or down-regulated (KD-DOWN), the effects on biological processes and pathways differed considerably. The E/R KD-UP set was significantly enriched for genes included in the “cell activation”, “immune response”, “apoptosis”, “signal transduction” and “development and differentiation” categories, whereas in the E/R KD-DOWN set only the “PI3K/AKT/mTOR signaling” and “hematopoietic stem cells” categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories “stem cell properties”, “B-cell differentiation”, “immune response”, “cell adhesion” and “DNA damage” with RT-qPCR.

Conclusion

Our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with key regulatory functions that shape the biology of this leukemia subtype. E/R may thus indeed constitute the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets.     

The Electrophysiological Underpinnings of Processing Gender Stereotypes in Language

Despite the widely documented influence of gender stereotypes on social behaviour, little is known about the electrophysiological substrates engaged in the processing of such information when conveyed by language. Using event-related brain potentials (ERPs), we examined the brain response to third-person pronouns (lei “she” and lui “he”) that were implicitly primed by definitional (passeggera _FEM “passenger”, pensionato _MASC “pensioner”), or stereotypical antecedents (insegnante “teacher”, conducente “driver”). An N400-like effect on the pronoun emerged when it was preceded by a definitionally incongruent prime (passeggera _FEM – lui; pensionato _MASC – lei), and a stereotypically incongruent prime for masculine pronouns only (insegnante – lui). In addition, a P300-like effect was found when the pronoun was preceded by definitionally incongruent primes. However, this effect was observed for female, but not male participants. Overall, these results provide further evidence for on-line effects of stereotypical gender in language comprehension. Importantly, our results also suggest a gender stereotype asymmetry in that male and female stereotypes affected the processing of pronouns differently.     

Reduced levels of class 1 reversibly glycosylated polypeptide increase intercellular transport via plasmodesmata

Maize and Arabidopsis thaliana class 1 reversibly glycosylated polypeptides (^C1RGPs) are plasmodesmata-associated proteins. Previously, overexpression of Arabidopsis ^C1RGP AtRGP2 in Nicotiana tabacum was shown to reduce intercellular transport of photoassimilate, resulting in stunted, chlorotic plants, and inhibition of local cell-to-cell spread of tobacco mosaic virus (TMV). Here, we used virus induced gene silencing to examine the effects of reduced levels of ^C1RGPs in Nicotiana benthamiana. Silenced plants show wild-type growth and development. Intercellular transport in silenced plants was probed using fluorescently labeled TMV and its movement protein, P30. P30 shows increased cell-to-cell movement and TMV exhibited accelerated systemic spread compared with control plants. These results support the hypothesis that ^C1RGPs act to regulate intercellular transport via plasmodesmata.     

Growth and asymmetry of soil microfungal colonies from "Evolution Canyon," Lower Nahal Oren, Mount Carmel, Israel

Background

Fluctuating asymmetry is a contentious indicator of stress in populations of animals and plants. Nevertheless, it is a measure of developmental noise, typically obtained by measuring asymmetry across an individual organism''s left-right axis of symmetry. These individual, signed asymmetries are symmetrically distributed around a mean of zero. Fluctuating asymmetry, however, has rarely been studied in microorganisms, and never in fungi.

Objective and Methods

We examined colony growth and random phenotypic variation of five soil microfungal species isolated from the opposing slopes of “Evolution Canyon,” Mount Carmel, Israel. This canyon provides an opportunity to study diverse taxa inhabiting a single microsite, under different kinds and intensities of abiotic and biotic stress. The south-facing “African” slope of “Evolution Canyon” is xeric, warm, and tropical. It is only 200 m, on average, from the north-facing “European” slope, which is mesic, cool, and temperate. Five fungal species inhabiting both the south-facing “African” slope, and the north-facing “European” slope of the canyon were grown under controlled laboratory conditions, where we measured the fluctuating radial asymmetry and sizes of their colonies.

Results

Different species displayed different amounts of radial asymmetry (and colony size). Moreover, there were highly significant slope by species interactions for size, and marginally significant ones for fluctuating asymmetry. There were no universal differences (i.e., across all species) in radial asymmetry and colony size between strains from “African” and “European” slopes, but colonies of Clonostachys rosea from the “African” slope were more asymmetric than those from the “European” slope.

Conclusions and Significance

Our study suggests that fluctuating radial asymmetry has potential as an indicator of random phenotypic variation and stress in soil microfungi. Interaction of slope and species for both growth rate and asymmetry of microfungi in a common environment is evidence of genetic differences between the “African” and “European” slopes of “Evolution Canyon.”     

Non-Photochemical Quenching Capacity in Arabidopsis thaliana Affects Herbivore Behaviour

Under natural conditions, plants have to cope with numerous stresses, including light-stress and herbivory. This raises intriguing questions regarding possible trade-offs between stress defences and growth. As part of a program designed to address these questions we have compared herbivory defences and damage in wild type Arabidopsis thaliana and two “photoprotection genotypes”, npq4 and oePsbS, which respectively lack and overexpress PsbS (a protein that plays a key role in qE-type non-photochemical quenching). In dual-choice feeding experiments both a specialist (Plutella xylostella) and a generalist (Spodoptera littoralis) insect herbivore preferred plants that expressed PsbS most strongly. In contrast, although both herbivores survived equally well on each of the genotypes, for oviposition female P. xylostella adults preferred plants that expressed PsbS least strongly. However, there were no significant differences between the genotypes in levels of the 10 most prominent glucosinolates; key substances in the Arabidopsis anti-herbivore chemical defence arsenal. After transfer from a growth chamber to the field we detected significant differences in the genotypes’ metabolomic profiles at all tested time points, using GC-MS, but no consistent “metabolic signature” for the lack of PsbS. These findings suggest that the observed differences in herbivore preferences were due to differences in the primary metabolism of the plants rather than their contents of typical “defence compounds”. A potentially significant factor is that superoxide accumulated most rapidly and to the highest levels under high light conditions in npq4 mutants. This could trigger changes in planta that are sensed by herbivores either directly or indirectly, following its dismutation to H₂O₂.     

Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement

Virus-encoded movement protein (MP) mediates cell-to-cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata. The molecular pathway by which TMV MP interacts with the host cell is largely unknown. To understand this process better, a cell wall-associated protein that specifically binds the viral MP was purified from tobacco leaf cell walls and identified as pectin methylesterase (PME). In addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP showed that its domain was necessary and sufficient for association with PME. Deletion of the PME-binding region resulted in inactivation of TMV cell-to-cell movement.     

Rasnitsynala sigambrorum gen. et sp. n., a small odonatopterid ("Eomeganisoptera", "Erasipteridae") from the early Late Carboniferous of Hagen-Vorhalle (Germany)

Besides Erasipteroides valentini (Brauckmann in Brauckmann, Koch & Kemper, 1985), Zessinella siope Brauckmann, 1988, and Namurotypus sippeli Brauckmann & Zessin, 1989, Rasnitsynala sigambrorum gen. et sp. n. is the fourth species of the Odonatoptera from the early Late Carboniferous (Early Pennsylvanian: Namurian B, Marsdenian) deposits of the important Hagen-Vorhalle Konservat-Lagerstätte in Germany. With its wing-span of about 55 mm it is unusually small even for the “Eomeganisoptera”. Its venation resembles other small “Eomeganisoptera”, in particular Zessinella siope. This is why it is here assigned to the probably paraphyletic “Erasipteridae” Carpenter, 1939.     

Evidence for a senescence-associated gene induced by darkness

Tobacco (Nicotiana tabacum) plants transformed with a chimeric tobacco anionic peroxidase gene have previously been shown to synthesize high levels of peroxidase in all tissues throughout the plant. One of several distinguishable phenotypes of transformed plants is the rapid browning of pith tissue upon wounding. Pith tissue from plants expressing high levels of peroxidase browned within 24 hours of wounding, while tissue from control plants did not brown as late as 7 days after wounding. A correlation between peroxidase activity and wound-induced browning was observed, whereas no relationship between polyphenol oxidase activity and browning was found. The purified tobacco anionic peroxidase was subjected to kinetic analysis with substrates which resemble the precursors of lignin or polyphenolic acid. The purified enzyme was found to readily polymerize phenolic acids in the presence of H₂O₂ via a modified ping-pong mechanism. The percentage of lignin and lignin-related polymers in cell walls was nearly twofold greater in pith tissue isolated from peroxidase-overproducer plants compared to control plants. Lignin deposition in wounded pith tissue from control plants closely followed the induction of peroxidase activity. However, wound-induced lignification occurred 24 to 48 hours sooner in plants overexpressing the anionic peroxidase. This suggests that the availability of peroxidase rather than substrate may delay polyphenol deposition in wounded tissue.     

A new era for plant biotechnology. A combination of pharmaceutical applications, technologies and safety measures could lead to new applications for genetically modified plants

New applications and technologies, and more rigorous safety measures could herald a new era for genetically modified crops with improved traits, for use in agriculture and the pharmaceutical industry.The imminent prospect of the first approval of a plant-made pharmaceutical (PMP) for human use could herald a new era for applied plant science, after a decade of public backlash against genetically modified crops, particularly in Europe. Yet, the general resistance to genetically modified organisms might have done plant biotechnology a favour in the long run, by forcing it to adopt more-rigorous procedures for efficacy and safety in line with the pharmaceutical industry. This could, in turn, lead to renewed vigour for plant science, with the promise of developing not only food crops that deliver benefits to consumers and producers, but also a wide range of new pharmaceuticals.This is certainly the view of David Aviezer, CEO of Protalix, an Israeli company that has developed what could become the first recombinant therapeutic protein from plants to treat Gaucher disease. The protein is called taliglucerase alpha; it is a recombinant human form of the enzyme glucocerebrosidase that is produced in genetically engineered carrot cells. This enzyme has a crucial role in the breakdown of glycolipids in the cell membrane and is either used to provide energy or for cellular recognition. Deficiency of this enzyme causes accumulation of lipids with a variety of effects including premature death.“My feeling is that there is a dramatic change in this area with a shift away from the direction where a decade ago biotech companies like Monsanto and Dow went with growing transgenic plants in an open field, and instead moving this process into a more regulatory well-defined process inside a clean room,” Aviezer said. “Now the process is taking place in confined conditions and is very highly regulated as in the pharmaceutical industry.”…resistance to genetically modified organisms might have done plant biotechnology a favour […] forcing it to adopt more-rigorous procedures for efficacy and safety…He argues that this is ushering in a new era for plant biotechnology that could lead to greater public acceptance, although he denies that the move to clean-room development has been driven purely by the environmental backlash against genetically modified organisms in the late 1990s and early 2000s. “That was one aspect, but I think the move has been coming more from an appreciation that biopharmaceuticals require a more regulatory defined setting than is achieved at the moment with transgenic plants.”Interest in deriving pharmaceuticals from plants, known colloquially as ‘pharming'', first took off in the 1990s after researchers showed that monoclonal antibodies could be made in tobacco plants (Hiatt et al, 1989). This led to genetic engineering of plants to produce vaccines, antibodies and proteins for therapeutics, but none gained regulatory approval, mostly because of safety concerns. Moreover, the plants were grown in open fields, therefore attracting the same criticisms as transgenic food crops. In fact, a recent study showed that the views of the public on pharming depended on the product and the means to produce it; the researchers found increasing acceptance if the plants were used to produce therapeutics against severe diseases and grown in containment (Pardo et al, 2009).However, it was the technical challenges involved in purification and the associated regulatory issues that really delayed the PMP field, according to George Lomonossoff, project leader in biological chemistry at the John Innes Centre for plant research in Norwich in the UK, part of the Biotechnology and Biological Sciences Research Council (BBSRC). “Extraction from plants required the development of systems which are not clogged by the large amounts of fibrous material, mainly cellulose, and the development of GMP [good manufacturing practice; quality and testing guidelines for pharmaceutical manufacture] compliant methods of purification which are distinct from those required from, say, mammalian cells,” said Lomonossoff. “All this is very time consuming.”“Secondly there was no regulatory framework in place to assess the risks associated with proteins produced in plants, and determining how equivalent they are to mammalian-cell-produced material and what kind of contaminants you might have to guard against,” Lomonossoff added. “Again, attempting to address all possible concerns is a lengthy and expensive process.” Yet recent work by Protalix and a few other companies, such as Dow Agrosciences, has given grounds for optimism that purification and GMP-compliant methods of production have finally been established, Lomonossoff added.…a recent study showed that the views of the public on pharming depended on the product and the means to produce it…The first important breakthrough for PMPs came in 2006, when Dow Agrosciences gained regulatory approval from the US Department of Agriculture for a vaccine against Newcastle disease, a contagious bird infection caused by paramyxovirus PMV-1. “Though the vaccine, produced in tobacco-suspension culture cells, was never deployed commercially, it showed that regulatory approval for a plant-made pharmaceutical can be obtained, albeit for veterinary use in this case,” Lomonossoff said.As approval is imminent for taliglucerase alpha for human use, it is natural to ask why plants, as opposed to micro-organisms and animals, are worth the effort as sources of vaccines, antibiotics or hormones. There are three reasons: first, plants can manufacture some existing drugs more cheaply; second, they can do it more quickly; and third, and perhaps most significantly, they will be able to manufacture more complex proteins that cannot be produced with sufficient yield in any other way.An important example in the first category is insulin, which is being manufactured in increasing quantities to treat type 1 diabetes and some cases of type 2 diabetes. Until the arrival of recombinant DNA technology, replacement insulin was derived from the pancreases of animals in abattoirs, mostly cattle and pigs, but it is now more often produced from transgenic Escherichia coli, or sometimes yeast. Recently, there has been growing interest in using plants rather than bacteria as sources of insulin (Davidson, 2004; Molony et al, 2005). SemBioSys, a plant biotechnology company based in Calgary, Canada, is now developing systems to produce insulin and other therapeutic proteins in the seeds of safflower, an oilseed crop (Boothe et al, 2009).…plants can in principle be engineered to produce any protein, including animal ones…“We have developed technology that combines the high-capacity, low-cost production of therapeutic proteins in seeds with a novel technology that simplifies downstream purification,” said Joseph Boothe, vice president of research and development at SemBioSys. “The target proteins are engineered to associate with small, oil-containing structures within the seed known as oilbodies,” Boothe explained. “When extracted from the seed these oilbodies and associated proteins can be separated from other components by simple centrifugation. As a result, much of the heavy lifting around the initial purification is accomplished without chromatography, providing for substantial cost savings.”The second potential advantage of PMPs is their speed to market, which could prove most significant for the production of vaccines, either against emerging diseases or seasonal influenza, for which immunological changes in the virus mean that newly formulated vaccines are required each year. “In terms of a vaccine, I think influenza is very promising particularly as speed is of the essence in combating new strains,” Lomonossoff said. “Using transient expression methods, you can go from sequence to expressed protein in two weeks.” Transient gene expression involves injection of genes into a cell to produce a target protein, rather than permanently incorporating the gene into a host genome. This is emerging as a less technically difficult and faster alternative to developing stable cell lines for expressing bioengineered proteins. The process of injecting the desired gene into the target genome, known as transfection, can be effected not only by viruses, but also by non-viral agents including various lipids, polyethylenine and calcium phosphate.The last of the three advantages of plants for pharmaceutical production—the ability to manufacture proteins not available by other means—is creating perhaps the greatest excitement. The Protalix taliglucerase alpha protein falls into this category, and is likely to be followed by other candidates for treating disorders that require enzymes or complex molecules beyond the scope of bacteria, according to Aviezer. “I would say that for simpler proteins, bacteria will still be the method of choice for a while,” Aviezer said. “But for more complex proteins currently made via mammalian cells, I think we can offer a very attractive alternative using plant cells.”Indeed, plants can in principle be engineered to produce any protein, including animal ones, as Boothe pointed out. “In some cases this may require additional genetic engineering to enable the plant to perform certain types of protein modification that differ between plants and animals,” he said. “The classic example of this is glycosylation. With recent advances in the field it is now possible to engineer plants to glycosylate proteins in a manner similar to that of mammalian cells.” Glycosylation is a site-directed process that adds mono- or polysaccharides to organic molecules, and plays a vital role in folding and conferring stability on the finished molecule or macromolecule. Although plants can be engineered to perform it, bacteria generally cannot, which is a major advantage of plant systems over micro-organisms for pharmaceutical manufacture, according to Aviezer. “This enables plant systems to do complex folding and so make proteins for enzyme replacement or antibodies,” Aviezer said.Genomic-assisted breeding is being used either as a substitute for, or a complement to, genetic-modification techniques…In addition to plants themselves, their viruses also have therapeutic potential, either to display epitopes—the protein, sugar or lipid components of antigens on the surface of an infectious agent—so as to trigger an immune response or, alternatively, to deliver a drug directly into a cell. However, as Lomonossoff pointed out, regulatory authorities remain reluctant to approve any compound containing foreign nucleic acids for human use because of the risk of infection as a side effect. “I hope the empty particle technology [viruses without DNA] we have recently developed will revive this aspect,” Lomonossoff said. “The empty particles can also be used as nano-containers for targeted drug delivery and we are actively exploring this.”As pharmaceutical production is emerging as a new field for plant biology, there is a small revolution going on in plant breeding, with the emergence of genomic techniques that allow simultaneous selection across several traits. Although genetic modification can, by importing a foreign gene, provide instant expression of a desired trait, such as drought tolerance, protein content or pesticide resistance, the new field of genomics-assisted breeding has just as great potential through selection of unique variants within the existing gene pool of a plant, according to Douwe de Boer, managing director of the Netherlands biotech group Genetwister. “With this technology it will be possible to breed faster and more efficiently, especially for complex traits that involve multiple genes,” he said. “By using markers it is possible to combine many different traits in one cultivar, variety, or line in a pre-planned manner and as such breed superior crops.”“The application of genomics technologies and next generation sequencing will surely revolutionize plant breeding and will eventually allow this to be achieved with clinical precision”Genomic-assisted breeding is being used either as a substitute for, or a complement to, genetic-modification techniques, both for food crops to bolt on traits such as nutrient value or drought resistance, and for pharmaceutical products, for example to increase the yield of a desired compound or reduce unwanted side effects. Yet, there is more research required to make genomic-assisted breeding as widely used as established genetic-modification techniques. “The challenge in our research is to find markers for each trait and as such we extensively make use of bio-informatics for data storage, analysis and visualization,” de Boer said.The rewards are potentially enormous, according to Alisdair Fernie, a group leader from the Max-Planck-Institute for Molecular Plant Physiology in Potsdam, Germany. “Smart breeding will certainly have a massive impact in the future,” Fernie said. “The application of genomics technologies and next generation sequencing will surely revolutionize plant breeding and will eventually allow this to be achieved with clinical precision.” The promise of such genomic technologies in plants extends beyond food and pharmaceuticals to energy and new materials or products such as lubricants; the potential of plants is that they are not just able to produce the desired compound, but can often do so more quickly, efficiently and cheaply than competing biotechnological methods.