Genetic engineering takes the poison out of cassava

Cassava is one of the most important staple food crops in the developing world but is also one of the most poisonous, its tissues releasing large quantities of cyanide when they are macerated during eating. Although the cyanide can be effectively removed by thorough cooking or by fermentation, chronic cynaide poisoning due to consumption of poorly processed cassava is common throughout tropical Africa and leads to serious neurological conditions. To solve this problem, a team of researchers led by Richard Sayre at the Danforth Plant Science Center, St Louis, Missouri have created genetically engineered cassava plants that contain an enzyme that improves the processing efficiency of the crop. Reporting their findings in Plos One, they show that not only do the transgenic cassava roots contain less cyanide after processing, but that the high amount of the added enzyme also significantly raised the total protein content of the root. This is a major added benefit because cassava contains the lowest protein to energy ratio of any crop leading to protein energy malnutrition if it is the primary food source.

Plants that produce cyanide in their tissues do so as a protective mechanism to discourage their consumption by herbivorous animals and insects. However, cyanide is as toxic to plants as it is to animals and so to prevent self-poisoning, cyanide is only release when then cells of the plants are disrupted, as would occur when an animal or insect chews the tissue. This relies on the accumulation of non-toxic cyanide-containing compounds called cyanogenic glucosides in one part of the cell (the vacuole) being kept separate from an enzyme that can act on these compounds. When the cell is disrupted by chewing, the two are mixed and the enzyme causes the release of the cyanide. It is this enzyme, hydroxynitrile lyase, that the Danforth team have increased. The processing of cassava is based on allowing cyanide, which is volatile, to evaporate. The increased amount of hydroxynitrile lyase ensures a rapid conversion of cyanogenic glucosides to cyanide and reduces the risk that cyanogenic glucosides could remain in the tissue after processing such that cyanide is still produced.

The beauty of this approach is that it does not interfere with the natural cyanogenic process, meaning that the pest-resistance of the plants is not affected. This is in contrast to previous attempts to solve the cyanide problem by preventing the synthesis of the cyanogenic glucosides in the first place.

Take your partner by the hand: a map of protein interactions in plants

A global consortium of scientists have established the first large-scale map of protein-protein interactions in the model plant Arabidopsis thaliana. The findings are reported in two papers in the 29th of July issue of Science. Using the yeast-two-hybrid technique, The Arabidopsis Interactome Consortium, exhaustively catalogued binary interactions between 8000 proteins (about 30% of the proteins encoded in the Arabidopsis genome). It was established that there were 6,200 interactions between 2,700 proteins.

Protein-protein ‘interactomes’ of bacteria, yeast and a few animals have revealed that proteins do not work alone. Almost all proteins physically interact with other proteins and this is thought to be important for controlling and modulating their function. The production of an interactome map for a plant is an important step in understanding the function of proteins that may have relevance for agriculture. A large fraction of the Arabidopsis genome encodes proteins of unknown function and working out what these proteins do is critical for a full understanding of the biology of the model plant. It is also an extreme challenge. Most of these proteins contain no clues in their sequence or predicted structure that might tell us what they do. Researchers are faced with a blank canvas of endless possibilities and such proteins tend not to get worked on because they are too ‘hard’ or the problem is too risky. An interactome map can help assign function to unknown proteins by using the ‘guilt by association’ principle. In the Arabidopsis interactome, it was found that proteins of similar function are more likely to interact with each other than those of different function. If proteins of unknown function are included in a particular interacting ‘community’ this makes it likely that they are part of the same biological process and helps guide research into their function.

An interaction maps also provides a topology that hypotheses can be tested against; the degree of interaction amongst proteins is not uniform - some proteins interact with more partners than others. These constitute ‘hubs’ in the functional interaction network and may be considered to more important for maintaining function. In a companion paper by Mukhtar et al this topological information was used to understand basic features of plant immunity against pathogens. Both bacterial and fungal pathogens secrete effector proteins into host plant cells that act to disable the plant defences. It was hypothesised that the effectors are more likely to disable hub proteins than those that are only weakly connected and this was confirmed by the interactome map that included pathogen effectors. Moreover, it was found that, despite being separated by 2 billion years of evolution, both the bacterial and fungal pathogens targeted the same hubs, providing important information that could be used to make broad-spectrum treatments for crop diseases.

Protein interactome maps take us a step beyond the genome, revealing aspects of biological function that are not accessible from gene sequence alone. "This starts to give us a big, systems-level picture of howArabidopsis works, and much of that systems-level picture is going to be relevant to--and guide further research on--other plant species, including those used in human agriculture and even pharmaceuticals," says Salk Institute biologist Joseph Ecker, a senior member of the Consortium.

Much remains to be done though, with the consortium estimating that the current interaction map only covers 2% of the possible 300,000 interactions that occur in Arabidopsis.

Why is the sequencing of the potato genome such big news?

The genome sequence of the humble potato was published in Nature last week (Nature 475, 189–195 (14 July 2011) and the story was taken up by the world’s media. As ever, hyperbole ruled, with claims that the new genome information would solve the problem of serious diseases of the crop and prevent starvation in the developing world. But given the fact that next generation sequencing technologies mean that whole genome sequencing is now both cheap and quick, why was this particular genome paper afforded the accolade of a place in Nature? Part of the answer is undoubtedly the ‘newsworthy’ nature of the work. Potato is the world’s fouth most important food crop and is especially important in the developing world. But there are two particular parts of the science presented in the paper that represent genuine breakthroughs.

The first is related to the difficulty of sequencing the genome. Like many of our crops potato is polyploid – meaning it has more than two copies of each chromosome – and is highly heterozygous with considerable variation between the same gene present on each of the 4 copies of each chromosome. This is a result of the fact that commercial tetraploid potato varieties are infertile and are propagated exclusively clonally through the tubers or stem cuttings. Without the homologous recombination that occurs between chromosomes during sexual reproduction, mutations in an individual copy of a gene are not redistributed to the other copies, permitting substantial differences between the four gene copies to accumulate. For technical reasons, this makes it much harder to reassemble the genome from the short fragment sequences that are obtained using current sequencing methods. The international consortium behind the potato genome sequencing solved this problem by creating an artificial ‘double monoploid’ clone (called ‘DM’) in which there were only two copies of each chromosome and more importantly, each copy was identical. This allowed them to completely sequence this simplified genome which then formed a framework for comparison and assembly of a more complex diploid genome (from a variety called ‘RH’ which closely resembles varieties that we consume).

The second breakthrough was the comparison of the DM and RH genomes revealed possible explanations for two major problems of potato: inbreeding depression and susceptibility to disease. Inbreeding depression in the phenomenon whereby a lack of genetic diversity in a population leads to reduced fitness of individuals. Although there is wide genetic diversity amongst the many thousands of species in South America, only a limited number of species were introduced from Europe and these formed the genetic base from which all modern farmed potatoes were derived.  Inbreeding depression is apparent in the two sequenced varieties: DM is much less vigourous than the RH variety. By examining the sequences, the consortium were able to show that there was a greater prevalence of genome mutations that disable gene function in the DM variety suggesting a possible reason for the reduced vigour. The sequencing also revealed a possible explanation for the susceptibility of modern potato to devastating pests such as  potato cyst nematode and diseases such as potato blight. More than 800 disease resistance genes were identified in the sequenced genome and importantly, many appeared to be broken due to mutation. While a second potato famine is unlikely to afflict the modern world, blight still is a cause of major losses of the crop every year and the genome sequencing now provides a possible route to a genetic fix.

Breeders create wheat ‘immune’ to a super-blight threatening world's crops

Scientists from the Mexico-based International Maize and Wheat Improvement Center (CIMMYT) will announce next week that they have developed new varieties of wheat that are resistant to UG99, a virulent new form of black stem rust that is spreading rapidly from East Africa and threatening the world’s wheat supply. The research will be presented at a ‘technical workshop’ of the Borlaug Global Rust Initiative, an international consortium to tackle the threat of new crop pathogens set up in 2005 by Norman Borlaug, the father of the green revolution. Borlaug had already taken on stem rust in the past, succeeding in breeding high-yielding, rust-resistant wheat in the 1950s and 1960s, after the pathogen had claimed 40 percent of the wheat crop in the US and Canada. It is estimated that 90% of the world’s wheat now uses the Sr31 gene introduced by Borlaug to confer resistance to stem rust and is now susceptible to UG99, the new race of Puccinia graminis tritici, the fungus responsible for black stem rust. First identified in Uganda, UG99 spores are rapidly dispersed in the wind and, according to David Hodson of the Global Cereal Rust Monitoring System at the Food and Agriculture Organization of the United Nations (FAO), “Future spread of these variants outside of Africa is inevitable". The new CIMMYT varieties are based on the introduction of several ‘minor’ resistance genes into CIMMYT’s wheat stocks, an outcome of a breeding experiment on a grand scale involving about 2 million wheat plants grown in field sites in Mexico and Kenya. The hard graft of the plant breeding done, Ravi Singh who led the CIMMYT research is now calling for political support for the introduction of the new varieties: “We need to see national governments making the investments in seed systems development, including seed production and distribution. In many areas there will need to be support and leadership from wealthy countries and international institutions to carry these innovations into farmers' fields."

BIOEFFECT EGF serum: a scientific revolution that only benefits its makers

I have steered away from poking at the cosmetics industry in this blog; it is simply too obvious, a sitting duck of marketing flim-flam atop nonsensical pseudoscience. But the latest skincare 'revolution' that has been sweeping the beauty world deserves a comment. It comes from Sif Cosmetics based in Iceland and is called BIOEFFECT EGF serum. The media is awash with the news of this miracle cream whose bioactive component is 'grown inside bio-engineered barley plants that thrive in bacteria-free ­volcanic ash' www.dailymail.co.uk. And there is plenty of chatter on the beauty blogs, not all of it as gushing as you might imagine (see www.juliegabriel.com)

The key ingredient in the magical cream is EGF or Epidermal Growth Factor. EGF is one of many proteins in our bodies that tells cells when to divide by binding to a receptor on their surface and triggering an internal signalling pathway. Human EGF has been used in a number of skin creams and it is claimed to improve your skin  / prevent ageing by speeding up the rate at which skin cells are replaced. As it is impossible to go around extracting EGF from 'donor' humans just so that other richer, vainer humans can spread it on their faces, EGF for face creams has to be produced in genetically modified organisms. Usually these are bacteria, yeast, or animal cell cultures engineered to express the human gene that encodes the EGF protein. The EGF protein is then extracted from these GM organisms or cells, purified and added to the usual concoction of glycerine and water to make a face cream.

The BIOEFFECT serum is different in that the EGF is produced in genetically modified barley plants. I can just about see why people might be more comfortable smearing a protein on their face that has been isolated from barley than bacteria, but is there really any benefit to making the protein in plants? Not to you there isn't. The protein is pretty much exactly the same whichever organism it is produced in and if it is properly isolated, then background contaminants are not an issue. In fact, almost all the benefits are in favour of the company making the product, Sif Cosmetics and their parent biotechnology company ORF genetics. The latter website helpfully lists all these benefits.

Most of them relate to the purity of the EGF. From the consumer point of view, pure ingredients are obviously best, but there is relatively little evidence that EGF from barley is any purer than that from bacteria or animal cells. However, it is almost certainly cheaper to produce pure EGF from barley than from bacteria. When any recombinant protein is produced, either for pharmaceutical or cosmetic purposes, it has to be separated from all the other proteins and molecules in the host organism. Usually this is done by altering the gene sequence slightly so that the recombinant protein is made with what is known as an 'affinity tag' at one end - a string of extra amino acids that has a high affinity for another molecule that in turn can be immobilised onto a solid chromatography matrix. This allows the recombinant protein to be purified by affinity chromatography. In the ORF genetics system, the recombinant EGF protein is expressed only in the seeds of the genetically modified barley plants. In comparison to bacteria or animal cells, the seed endosperm is a very simple system with only a few storage proteins present. This means that the protein profile (the number of different types of protein present) is very simple and cheaper, less sophisticated protein fractionation techniques can be used to isolate the EGF protein.

ORF genetics also claims that their EGF preparation from barley has lower endotoxin content (nonsense: endotoxins are small metabolites easily separated from proteins in all protein purification approaches), lists 'lower endoprotease activity' as an advantage (this just means that the recombinant protein is less likely to be degraded by host protein-digesting enzymes making it easier for ORF genetics to recover more intact recombinant protein) and says that there is no risk of transmission of 'infective agents' (ie disease causing bacteria or viruses). This last one is probably true, but rather overlooks other known dangers of plant materials such as allergenicity and the presence of bacteria such as Bacillus cereus that cause food poisoning (the latter is well known in connection with rice, but in fact is present in most cereals including barley!).

The other benefit that the ORF genetics website fails to mention is that it is substantially cheaper in general to produce recombinant proteins in plants than in bacteria or animal cell cultures. This is because the latter are grown in large vats known as chemostats which are expensive to run, requiring a constant supply of nutrient solution to support the growth of the bacteria or cells. Plants by contrast, can be grown relatively cheaply in a greenhouse (in the Icelandic case, the greenhouse is heated by geothermal energy). This, and the fact that production of recombinant proteins in plants can be massively scaled up (think fields of GM crops) has lead to an increasing uptake of plants as 'biofactories' for production of useful molecules. And not all of this sophisticated biotechnology is wasted on cosmetics. Sometimes, it is put to better use. For example, insulin is now being produced in a thistle-like plant called safflower (www.sembiosys.com).