Stem cells and cancer cells have enough molecular similarities that the former can be used to trigger immunity against the latter.
Plants are locked in an ancient arms race with hostile viruses, but genome editing is giving crops the upper hand.
February 1, 2018|
© BILL BARKSDALE/DESIGN PICS/GETTY IMAGES
In 2011, Noah Phiri was working with local farmers in Kenya to combat the fungal pathogen that causes coffee leaf rust when another virulent plant disease began wiping out maize in the country’s southwest corner. Infected plants developed pale streaks on their leaves, then wilted and died. Some farmers lost as much as 90 percent of their crop that year. Phiri, a plant pathologist at the U.K.-based Centre for Agriculture and Biosciences International (CABI), raced to identify the culprit. He and his colleagues collected samples of sick plants and sent them off to the plant clinic at the Food and Environment Research Agency (now Fera Science) in York, U.K. There, researchers sequenced RNA molecules expressed in the infected corn and identified two viruses that were at the root of the epidemic.1
The viruses were already familiar to the researchers—in the second half of the 20th century, corn crops in Kansas suffered a similar fate. Known as maize lethal necrosis, the disease is caused by a combination of sugarcane mosaic virus (SMV), a common virus that is not usually harmful to maize, and a strain of maize chlorotic mottle virus (MCMV). MCMV is damaging to maize crops on its own, but in combination with SMV, the effect is exacerbated. While there hasn’t been a major outbreak of maize lethal necrosis in Kansas since 1988—thanks to a rotation of disease-tolerant corn varieties—when the viruses struck Kenya in 2011, the local maize had no defense. By the following year, the disease had infected 77,000 hectares of Kenyan farmland, costing an estimated USD $52 million. It has now spread to most east African countries and threatens food security for millions of people.
Unfortunately, maize lethal necrosis is hardly unique; in general, plants are just as susceptible to viral infections as humans and other animals are. And viruses are particularly dangerous because, unlike bacteria and other pathogens, they cannot be killed with antibiotics or pesticides. “At the moment, there’s not much to be done with plants that are infected,” says Jean-François Laliberté, a virologist at the National Institute of Scientific Research (INRS) in Quebec, Canada. So when viruses strike, farmers are often forced to destroy crops, clean tools and machinery, and then replant with seeds from elsewhere.
In recent years, however, scientists have turned to inventive new ways to protect crops. Genetic modification techniques developed over the last 30 years, for example, can arm plants with defenses against viral invasion, while leaving crop yields and food quality unaffected. Some of these modified plants are now in the food chain. More-recent gene editing techniques are refining this approach, allowing researchers to make precise changes in plants’ DNA to engineer a more resistant generation of crops. Several such varieties are now being tested in lab and field trials, while a handful await safety approval from national regulatory bodies.
“With climate change, there will be more new insects appearing, and those insects will be carrying new viruses and new strains,” says Laliberté. To secure crop production around the world, “we have to find those means of genome editing.”
The study of plant viruses has a long history. In fact, it was in plants that viruses were first discovered. In the late 1850s, a devastating disease began spreading across tobacco plantations in the Netherlands. Scientists at the time found that injecting sap from infected plants into healthy ones spread the symptoms—mottling and discoloration of the plants’ leaves—leading researchers to assume that the affliction must be caused by a bacterium. However, additional experiments in the 1890s showed that the infectious agent spreading the disease could pass through the tiny pores of a porcelain water filter—far too small to allow the passage of any known bacterium. In 1898, Dutch microbiologist and botanist Martinus Beijerinck coined the term “virus” to describe the mystery contagion, though it would be another few decades before researchers characterized exactly what it was.
STEPHEN AUSMUS, USDA/ARS
Even after scientists identified viruses as protein-encapsulated nucleic acids in the first half of the 20th century, many questions remained about how these particles acted within host cells to cause disease. Again, the study of plants fueled the young field of virology. In the 1950s, scientists began using electron microscopy to view plant-virus interactions in detail, revealing huge cellular rearrangements in infected cells. “[Researchers] noticed that there were a lot of structures that resembled vesicles,” says Laliberté. “In [healthy] plant cells we have chloroplasts, a nucleus, and mitochondria, but in infected cells we have novel organelles.”
With climate change, there will be more new insects appearing, and those insects will be carrying new viruses and new strains.—Jean-François Laliberté,
Institut National de la Recherche Scientifique
More than 30 years later, researchers discovered that those strange vesicles, ranging in diameter from around 50 to 350 nanometers, are the powerhouses of viral infection. Now known as viroplasms or viral factories, the membrane-bound compartments collect resources from the plant to replicate the viral genome and produce RNAs that will direct the production of proteins and the construction of new viral particles poised to infect new hosts. (See illustration below.) The close proximity and high concentrations of the biomolecules made in these factories make for a highly efficient production line, notes Peter Nagy, a virologist at the University of Kentucky. For example, “tomato bushy stunt virus can produce close to one million progeny per cell in 24 hours,” he says. “This is an unbelievably powerful process.”
By cordoning off viral replication into membrane-bound compartments, the factories also serve to protect the pathogen against the plant immune system. In replicating their genomes, which are commonly single-stranded RNA, plant viruses typically generate a complementary copy to temporarily produce double-stranded RNA, an extremely unusual sight in a plant cell. “This double-stranded RNA does not exist in plant cells,” says Nagy, so if not for the protective membrane around the viral factory, “the plant cell would right away know that this was an invading virus.”
New viral genomes, sometimes packaged into a new protein capsid, are then carried away to neighboring cells through small channels in cell walls called plasmodesmata. But it takes a little coaxing, as these passageways typically allow the transit of small molecules, but not of proteins and RNA. So viral factories produce what are called movement proteins, which trigger the channels to widen. Some viral particles also manage to make their way into the phloem, where they have a chance of being sucked up by a sap-feeding insect like an aphid and carried away to infect other plants, often decimating entire fields of crops.
Of course, plants are not passive victims in this relationship, and many have evolved genetic resistance to viral infections. (See “Holding Their Ground,” The Scientist, February 2016.) Understanding how plants defend themselves against attack has given scientists a head start in the race to protect crops, allowing them to engineer new, resistant varieties.
PEGGY GREB, USDA/ARSOne of the first lines of plants’ natural defense against viruses, deployed when the cell detects double-stranded RNA, is RNA silencing. Plant enzymes called Dicer-like proteins take viral RNAs and turn them into small interfering RNAs (siRNA). These siRNAs bind to a family of proteins called Argonaute as part of the RNA-induced silencing complex (RISC), which tracks down viral RNAs based on their similarity to the siRNA sequence and chops them into tiny fragments. “We now know that this is the major mechanism by which plants defend themselves against viruses,” says Hanu Pappu, a plant virologist at Washington State University in Pullman.
Researchers have been boosting plants’ ability to use this mechanism to fight off viruses for more than 20 years. In Hawaii in the 1990s, the $11 million papaya industry was nearly decimated by papaya ringspot virus (PRSV), which yellows the leaves of the fruit trees and slowly kills them. A team at Cornell University inserted the ringspot virus coat protein gene into a bacterial plasmid, and fired gold particles carrying the plasmid at papaya cell cultures. They then germinated plant embryos that were expressing the foreign RNA, which would trigger RNA silencing against the virus. This new papaya variety, named Rainbow, is primed for PRSV, ready to silence its RNA as soon as it invades a cell. Rainbow papaya has been hugely successful and has come to dominate the Hawaiian papaya market since its commercial release in 1998.
Over the past few decades, plant geneticists have employed similar techniques to combat other damaging crop viruses. For example, regulators in Canada and the U.S. approved a transgenic variety of squash engineered using a bacterial plasmid to carry genes coding for the coat proteins of cucumber mosaic virus, watermelon mosaic virus, and zucchini yellow mosaic virus, in the late 1990s. And in 2001, researchers used a bacterial plasmid to insert the coat protein of the soybean mosaic virus into soybean plants to confer resistance to the virus, although a commercial variety was not developed.2
More recently, scientists have been taking advantage of this natural plant defense system to protect cassava, a starchy root vegetable that’s a staple in the diets of hundreds of millions of people in Africa, Asia, the Pacific, and South America. Cassava brown streak disease is caused by two viruses, Ugandan cassava brown streak virus (UCBSV) and cassava brown streak virus (CBSV), which have been infecting cassava crops since the 1980s. The disease only began causing serious problems for farmers in 2004, when the viruses spread out from coastal regions and across Tanzania, Uganda, Rwanda, and the Democratic Republic of Congo. Cassava plants had no natural resistance to the disease.
In 2011, researchers used a bacterial plasmid to insert the full gene sequence for the coat protein of UCBSV into the genomes of cultured cassava cells, which were then regenerated into whole plants, successfully priming cassava’s natural RNA silencing machinery against the virus.3 Cassava engineered to express the UCBSV coat protein gene showed 100 percent resistance to the virus when infected cuttings were grafted onto them in greenhouse experiments. And researchers at the Virus Resistant Cassava for Africa (VIRCA) initiative found that the best performing transgenic cassava line was more than 98 percent resistant to CBSV in confined field trials.4
Meanwhile, to produce a crop with resistance to a greater variety of CBSV strains, VIRCA researchers have combined the full coat protein gene sequences from UCBSV and CBSV into a plasmid and inserted it into the genome of an East African cassava variety that is preferred by farmers.6 This new variety, part of a project dubbed VIRCA Plus, performed well in confined field trials in Kenya and Uganda, with 16 out of 25 transgenic lines remaining symptom-free after 12 months.6 Field trials with these resistant lines continue, as the team works with national government regulators in Uganda and Kenya to seek approval for the new variety to be released for use by farmers.
© PENS AND BEETLES STUDIOS
“The biggest challenge is still trying to negotiate this regulatory infrastructure,” says Becky Bart, a plant geneticist at the Donald Danforth Plant Science Center in St. Louis.
Another way plants can defend themselves against viral infection is through the accumulation of mutations in proteins targeted by viral pathogens. For example, research in the 1990s showed that the viral protein VPg interacts with plant proteins in the eIF4E family of translation initiation factors to produce other proteins critical for viral replication. In 2002, a research team in France showed that naturally occurring resistance to several viruses in peppers (Capsicum annum) was caused by a mutation that gave one eIF4E protein a slightly different molecular structure.7 At the same time, an overlapping group of researchers identified a mutant line of the plant model organism Arabidopsis thaliana in which the gene for an isoform of eIF4E was disabled, leaving normal plant growth unaffected but hampering viral replication.8 More recently, researchers at the University of Tokyo in Japan identified variants of the nCBP protein, part of the eIF4E family, in Arabidopsis that prevent the accumulation of certain movement proteins, trapping the Plantago asiatica mosaic virus in a single plant cell and saving the whole plant from infection.9
JUANE WANPlant breeders have long been making use of such naturally occurring genetic resistance, selectively crossing wild varieties to produce more-resistant crops. For example, in the 1980s, work led by scientists at the International Institute of Tropical Agriculture succeeded in breeding partial resistance to the geminivirus-caused cassava mosaic disease—resistance that’s found in closely related wild species of the root vegetable—into cultivated varieties across Africa.10 By cross-breeding cultivated cassava (Manihot esculenta) with its wild relative, tree cassava (M. glaziovii), the team was able to introduce naturally occurring resistance to the disease, controlled by multiple genes.
Such traditional breeding approaches can take decades, however—a cumbersome prospect when new resistance genes must be introduced for each new viral strain that evolves. More recently, scientists have used genetic engineering techniques to more swiftly and precisely arm crops with such resistance. “Genome editing has just completely revolutionized every part of biology,” says Bart.
Last year, doing screens in yeast, Bart and her collaborators identified two eIF4E proteins from cassava that interact with CBSV and UCBSV VPg proteins. Then, using the CRISPR-Cas9 system, they edited the sequence of those genes to prevent their expression, resulting in a cassava variety that showed improved resistance to the viruses in greenhouse trials.11 The CRISPRed cassava plants weren’t fully resistant, however, suggesting that the viruses may also be able to interact with the three remaining unedited eIF4E proteins. The team hopes to fine-tune the system to engineer a fully resistant cassava plant.
Recent studies have revealed other tricks used by plants to fight off viral infections. For instance, the process designed to recycle damaged or unwanted objects in the cell—autophagy—has been coopted to remove viral particles, too. Working with tomato plants (Solanum lycopersicum), Yakupjan Haxim at Tsinghua University in Beijing, China, and colleagues found that the plant’s autophagy protein ATG8 binds the viral ßC1 gene, which encodes an essential protein for infection by geminiviruses, transporting it to an autophagosome for degradation.12 Viruses carrying a mutated version of ßC1, which cannot be bound by ATG8, cause more-severe symptoms and replicate more rapidly. Conversely, when the researchers promoted autophagy—by preventing the expression of enzymes that inhibit the process—plants of the model organism Nicotiana benthamiana, a close relative of tobacco, showed more resistance to three geminiviruses: cotton leaf curl Multan virus, tomato yellow leaf curl virus, and tomato yellow leaf curl China virus.
We have a long way to go to develop sustainable and environmentally sound approaches to really control virus diseases.—Bryce Falk, University of California, Davis
As researchers continue to learn more about the natural defenses plants use to protect themselves against viral pathogens, and as they enlist rapidly advancing genetic engineering techniques to equip plants with such weaponry, the field is on its way to having the tools it needs to develop a new generation of resistant crops. But it will be an uphill battle. Viruses are constantly evolving, many times faster than the plants they infect, and it is only a matter of time before each virus develops a countermeasure to such resistance mechanisms. For example, many viruses manufacture a protein that can mop up siRNAs, binding them before Dicer-like enzymes can form a RISC complex; and potyviruses such as tobacco vein mottling virus have mutated their VPg protein, allowing them to bind to the modified eIF4E proteins that previously offered the plant resistance.
“I think we have a long way to go to develop sustainable and environmentally sound approaches to really control virus diseases,” says Bryce Falk, a plant pathologist at the University of California, Davis.
While genetic editing may be paving the way to more-resistant crops, the approach’s application to agriculture is still in its infancy. Each new variety requires extensive testing for safety before the engineered plants can be deployed in the field. For now, quick identification of new viral threats and strict hygiene and quarantine regulations remain critical, by containing outbreaks before they can spread and cause widespread crop losses, particularly in developing nations.
The Plantwise project is a global program led by CABI that aims to do just that. Launched in 2011, the project works with farmers in Africa to help diagnose and treat plant health problems. One of their key innovations has been Plant Clinics, where local farmers can meet with trained plant health experts to identify pests and pathogens. These meetings “have been pivotal in the identification of some of these new pests that are coming to different countries,” says Phiri. It was at one of the clinics that maize lethal necrosis was first detected in Africa six years ago. “Early detection is crucial, and plant health clinics are playing that role,” he says.
But new technology can also help. For example, MinION portable DNA sequencers being used by the Cassava Virus Action Project are helping farmers in developing nations identify new viruses, allowing them to quickly take action to minimize transmission.
Although many challenges remain in the fight against plant viruses, such technological advances are giving researchers the upper hand, says Falk. For now, he adds, it is a fight we are winning. “We’re winning it because we’re feeding people.”
Claire Asher is a freelance science writer living in London, U.K.
The good viruses
For example, Roossinck’s research group has found that brome mosaic virus and cucumber mosaic virus (latter shown in background image) both help some plants cope with drought stress, possibly as a result of the changes to plant cell metabolism caused by viral infection (New Phytol, 180:911-21, 2008). In both Arabidopsis and tobacco plants, for instance, researchers at the Centro de Investigaciones Biológicas in Madrid, Spain, found last year that simultaneous infection with two different viruses increased levels of salicylic acid, a plant hormone linked to stress and drought tolerance (Plant Cell Environ, 40:2909-30, 2017).
“If the conditions are normal, then the virus may be harmful,” Roossinck explains. “But when you have a drought, then the virus becomes beneficial.” While the precise mechanisms by which viruses make their hosts more drought resistant are still poorly understood, it’s possible that one day the molecular mechanisms underpinning such viral infections could be deployed in an agricultural setting to help crops deal with dry conditions, she adds.
In addition to such acute viral infections, plants harbor an array of persistent viruses, which reside permanently within healthy organisms and are transmitted from one generation to the next via seeds. “In wild plants we find about 60 percent of the viruses fall into this persistent category,” she says. Many of these viruses, too, may benefit their hosts. For instance, white clover cryptic virus inhibits the formation of nitrogen-fixing nodules in legumes such as lotus when nitrogen levels are high, helping the plants use resources more efficiently.
More research is needed to understand the huge variety of viruses in wild plants and how they coexist with—and even benefit—their plant partners, says Roossinck. “Plant virus disease . . . is almost certainly not the norm for a virus.”
February 8, 2018
Like many basic scientists, this article takes the perspective that "traditional breeding" is slow and outdated and that genetic engineering is the only way to go. That perspective ignores the relevant data on which technique is actually producing advances in disease resistance in crops. Genetic engineering techniques cause great enthusiasm among biologists, but they remain far more expensive, fail more in real-world testing and are rarely faster to market than breeding by other techniques.
I enourage my fellow plant scientists not be be sucked in by the hype and look at the data.