6 Disease-Resistant Transgenic Crops
The three scourges of agricultural crops are weeds, insect predators, and infectious pathogens (namely, bacteria, viruses, and fungi). Before chemical herbicides were discovered, weeding was handled by mechanical methods. And before chemical insecticides were known, farmers learned to mitigate insect pests by planting techniques such as intercropping, crop rotation, and biological controls. Today organic farmers still use these practices in lieu of chemical pesticides.
Many plants possess natural defenses against some infectious pathogens. Although plants do not have an immune system comparable to that of animals, they do have defense mechanisms against invading microbes that exhibit some similarities in their mode of action to the immune system of animals: “Plants react to pathogen attack by activating a variety of defense mechanisms that culminate in a number of physical and biochemical changes in the host plant.”1
As in all forms of biological life, plants can detect threats to their survival and react to those threats. Plants can react to certain chemicals through receptors that are located either on their cell surface or inside the cell. After the receptors are activated by the coat proteins of a pathogen, they will issue an antiviral, antifungal, or antimicrobial response.
For example, plants naturally defend themselves against pathogens by the thickening of cell walls. If agents penetrate the walls, plants release protective proteins (pathogenesis-related proteins or phytoalexins, which are small antimicrobial chemicals that accumulate in plants as a result of infection or stress) that protect them from the immediate and future pathogen assaults. For example, scientists discovered a gene in maize that encodes an enzyme that protects it from a fungal attack by disabling the invading pathogen. In a process known as systemic acquired resistance (SAR), a plant can develop greater resistance to a pathogen after its first attack.2 SAR was first observed in 1901and has been more extensively studied over the past two decades. A leaf that is not in direct contact with a pathogen can be stimulated to protect itself from a part of the plant that has been infected—a process known as inducible microbial proteins. In some cases, farmers can spray crops with chemicals called plant activators that can artificially trigger systematic acquired resistance, essentially activating the plant’s protective mechanism. These chemicals often have less toxicity to animals and humans than traditional fungicides.
Like human pathogens, plant pathogens are constantly evolving and mutating to overcome crop resistance. In agriculture, infectious pathogens represent a significant economic loss and control cost to farmers. In the United States, it is estimated that plants are subjected to over fifty thousand different pathogens (fungi, viruses, bacteria, and nematodes). Any given agricultural region may be faced with between ten and fifteen serious plant diseases. From 1988 to 1990, there was a loss of $300 billion for eight major crops from all sources (pathogens, arthropods, and weeds), and about a third ($100 billion) of this loss was due to pathogens. Nonindigenous pathogens crossing national boundaries contribute significantly to the total crop loss:3 “Even with the extensive application of pesticides, the estimated reductions in the farm-gate value of selected vegetable crops in the United States caused by diseases range from 8 to 23%, by insects 4 to 21%, and by weeds 8 to 13%. The average losses caused by diseases, insects and weeds in Canada are 15.5, 12.5 and 10.5%, respectively. They reduced return to the vegetable industry by $172.7, $138.2 and $115.2 million, respectively, in 1990.”4 On the global scale, it is estimated that 10 to 16 percent of the total harvest is lost to plant diseases each year, costing an estimated $220 billion.5
Papaya trees can be found in the tropical and subtropical parts of the world.6 The papaya fruit (called the fruit of angels) was first introduced to the Hawaiian islands in the 1940s and is the second-largest fruit crop in the state. In the 1950s, a virus called papaya ringspot virus (PRSV) (the disease name came from the ringed spots of the fruits of infected trees) destroyed the papaya crop on Oahu island. As a result of the PRSV blight, the industry relocated to the Puna district of Hawaii island. For a while, the region was free of the virus, but in 1992, PRSV appeared in Puna, which was then Hawaii’s major papaya growing region, resulting in a decline in production by 50 percent.7 The virus is spread by aphids, which feed on the leaves of infected trees, carry the virus on their mouthparts, and transmit the virus to healthy trees.8 Young seedlings die quickly and usually do not produce healthy fruit. The virus attacks mature trees that develop mosaicism, distortion of leaves, and smaller fruit with ringspots until the trees die. Efforts to remove infected trees do not prevent the spread of the virus.
In the late 1980s, prior to the development of transgenic crops, scientists built on knowledge of systemic acquired resistance and used a method for protecting crops from pathogens that is similar to the stimulating of a human immune response to a virus. Dennis D. Gonsalves and a team at Cornell University and the University of Hawaii inoculated papaya seedlings with a mild strain of PRSV. The mild strain was developed by exposing a severe strain to nitrous oxide, which is a mutagen. Gonsalves and his colleagues found that papaya trees exposed to the mild strain of PRSV were protected from the severe strain.9 The method, which is called cross-protection strategy, was reported to have worked effectively in Taiwan from 1984 to 1993. However, it had limitations that included the nonavailability of stable attenuated strains, the requirement of a large-scale inoculation facility, and its failure to protect the trees from new strains in different countries.10
A physical method that was used to protect papaya crops against PRSV involves covering an entire orchard with netting to provide a barrier to the aphids that transmit the virus. However, this method reduces the sunlight to the trees, resulting in lower-quality fruit. Also, after the harvest, the netting is removed and burned, which results in hazardous by-products.
Modern developments in biotechnology, particularly recombinant DNA methods, led to a new approach to the PRSV virus called pathogen-derived disease resistance. It is based on a similar idea of the cross-protection strategy. Under the right circumstances, plants can be sensitized to protect themselves against invading pathogens. The plant is exposed to fragments of the invading pathogen. The transgene, which is made up from part of the pathogen’s genome, is connected to a promoter like the cauliflower mosaic virus and delivered into the plant host cells by Agrobacterium tumefaciens or a gene gun. In the gun, gold or platinum particles are laced with the transgene cassette. After the transgene cassette enters the plant cells, the cells are activated to express RNA or proteins that disable the invading pathogen. The plant response occurs after the plant attack. The chemicals that are produced by the plant are called inducible defenses. This type of generalized defense response is called RNA silencing or RNA interference. The method was deemed effective and safe according to a report published in the journal Science in the mid-1980s: “The health and environmental issues can be alleviated or totally avoided by selecting appropriate sequences from pathogens and engineering them into safer transgenes to preempt the possibility of their expression and/or persistent presence by pathogenic proteins or RNA sequences in transgenic hosts.… RNA silencing-mediated transgenic resistance is one of the strategies that does not create pathogenic nucleotide sequences or proteins in the host and is totally human-, livestock-, and environment-friendly.”11
A variant of this method was successful in protecting tobacco plants from the tobacco mosaic virus (TMV). A coat protein of TMV was inserted into the tobacco plants. The method is also termed coat protein gene-mediated transgenic resistance.12 Two genetically-modified cultivars of papaya (Rainbow and Sunup) were deregulated by the Animal and Plant Health Inspection Service (APHIS) of USDA and the EPA in May 1998. The results of the transgenic papaya cultivars resistant to PRSV in Puna, Hawaii, were deemed highly successful.
A mild Hawaiian PRSV strain was the source of the coat protein gene for the transgene cassette that was to be inserted into the papaya cells. Scientists used the cucumber mosaic virus to obtain the promoter for the transgene. In addition, they used kanamycin-resistant markers and biolistics (gene gun) in conjunction with an embryonic tissue culture technique to develop the disease-resistant papaya. The transgenic plants were tested with controls in field experiments. The transgenic papaya line had no symptoms of the pathogen, whereas the nontransgenic plants were ruined within five months. One scientific group concluded: “The transgenic resistance conferred by the viral CP [coat protein] gene has become the most effective method to prevent papaya from infection by the noxious PRSV.… Thus, the GM papaya has saved the papaya industry in Hawaii, without any significant adverse effects to environment and human health during the application for more than a decade.”13
What considerations were given to understanding the potential adverse effects of “inducible defenses”? The primary environmental risks considered by scientists and regulators were related to the transfer or recombination of transgenes in the crop, including the movement of the transgenic construct to nondomesticated papaya or wild relatives (by pollen or horizontal gene flow) and weediness of virus-resistant papaya. Among the human health effects, regulators were concerned about whether kanamycin resistance would spread and whether the essential vitamin composition or traces of toxins (such as alkaloids) of the transgenic cultivar were significantly changed.
The term heteroencapsidation refers to the process by which a virus’s nucleic acid is enclosed in a capsid by the coat proteins of another virus. For GMOs that are made disease resistant, this means that the coat proteins (CP) of the pathogenic virus inserted into the plant cells will be able to encapsulate the RNA genome of another virus that infects the plant. This is particularly relevant when a plant is infected by more than one virus. Scientists have speculated on the possibility that heteroencapsidation could result in new virus epidemics. This natural mechanism has been documented in some transgenic herbaceous plants. In reviewing the risks, Marc Fuchs and Dennis Gonsalves observe that “heteroencapsidation in transgenic plants expressing virus CP genes has been of limited significance and would be expected to be negligible in regard to adverse environmental effects.”14 Thus, these authors dismiss the risks without the need for further tests or evidence.
Another possible risk that has been raised by scientists is recombination between a genome segment from a viral genome transcript and the genome of another challenge virus entering the plant genome. Recombinations occur frequently in nature, so the issue is whether there is something unique about the recombination of an artificial gene construct. Once again, Fuchs and Gonsalves write that “So far, no recombination event has been found in CP gene-expressing transgenic plants in the field.”15
Horizontal gene flow from cultivated crops through pollen or other methods of gene transfer (transgene introgression) to compatible wild relatives was also raised as a potential risk of transgenic pathogen-derived resistance. C. Neal Stewart, Matthew D. Halfhill, and Suzanne I. Warwick have reported that “Transgenes engineered into annual crops could be unintentionally introduced into the genomes of their free-living wild relatives. The fear is that these transgenes might persist in the environment and have negative ecological consequences. Are some crops or transgenic traits of more concern than others?”16 The authors recommend that large-scale genetic modification should be avoided in high-risk crops in which evidence of introgression has occurred and that the risks and benefits of transgene introgression should be studied on a case-by-case basis.
Finally, the food safety of virus-resistant plants has been discussed regarding allergenicity. When a virus-derived transgene is inserted to create a GMO, it can have amino acid sequences that can cause new allergens or cause enhancement of intrinsic allergens. Although this is a hypothetical risk, no adverse allergenic effects have been reported for papaya transgenic disease-resistant crops with a mild strain of PRSV. Nonetheless, some scientists express caution: “it is prudent to investigate food safety aspects of virus-resistant transgenic plants.”17
From all the reviews on virus-resistant plants, there is no reliable evidence of any adverse human health effects from the consumption of such transgenic plants. One 2010 study on the compositional differences between transgenic and nontransgenic papaya for nutrients of beta-carotene and vitamin C as well as for two natural toxicants (benzyl isothiocyanate and carpaine) showed no significant differences.18 Zhe Jiao, Jianchao Deng, Li Gongke, Zhuomin Zhang, and Zongwei Cai found that the compositional variability among papayas harvested across different time periods was higher than the compositional variability between transgenic and nontransgenic varieties. Other studies recommend that precaution and continuous oversight both pre- and postrelease should be maintained in the advent of unintended consequences.
With respect to environmental effects, another paper has reported that transgenic papaya had adverse effects on soil microorganisms. Xiang-dong Wei and colleagues studied soil properties, microbial communities, and enzyme activities in soil planted with transgenic and nontransgenic papaya under field conditions.19 The transgenic papaya had a transgene consisting of a mutant gene of the papaya ringspot virus (PRSV), a neomycin antibiotic marker gene that confers antibiotic resistance to the genetically modified papaya cells, and a cauliflower mosaic virus promoter. The researchers collected soil samples before planting and after harvesting, undertook chemical and enzyme analysis, and enumerated colony-forming bacteria, actinomycetes, and fungi. They found that “transgenic papaya altered the chemical properties, enzyme activities, and microbial communities in soil.”20 Notwithstanding those effects, they observed that the transgenic papaya showed higher resistance to PRSV, had better growth, and produced more and larger fruits than the parental nontransgenic strain. Finally, the authors noted that “all these [observed effects] suggest the potential risk of field released GM plants, especially antibiotic genes like NPTII [neomycin phosphotransferase II marker genes] in GM plants, causing undesirable and unpredictable ecological effects.”21 The researchers conclude that genetically modified plants that are grown in the same soil for more than three months “could change the rhizospheric microbial metabolism; cause negative effects on soil quality, structure, and function; and affect enzyme synthesis and activity, as well as soil processes such as decomposition and mineralization of litter.”22
The use of transgenes from pathogens to stimulate the plant’s intrinsic immune system (pathogen-derived resistance) to protect itself from an invading pathogen has been approved in the United States for papayas, squash, and plums. Scientists combined the coat protein genes from three different viruses and created a squash hybrid with multiviral resistance. Similarly, genes from the plum pox virus were used to create disease resistance for the plum. Other transgenic crops in various stages of development for disease resistance include rice, wheat, apples, tomatoes, bananas, potatoes, barley, and soybeans.
Even with the widely reputed success of transgenic papaya, utilizing “pathogen-derived-disease-resistance,”23 scientists have been developing nontransgenic methods using RNA silencing. According to one report, “Compared with the transgenic method for antiviral resistance, this approach is simpler, safer, environmentally friendly, and relatively inexpensive.”24 The authors used direct mechanical inoculation of papaya plants with bacterially expressed RNA that targets the gene of the papaya ringspot virus and interferes with the virus infection.
Chapter 9 discusses whether the potential for unintended consequences for transgenic crops is any greater than it is for traditionally bred crops.
Notes
1. Richard Broglie and Karen Broglie, “Production of Disease Resistant Transgenic Plants,” Current Opinion in Biotechnology 2 (1993): 148.
2. Uwe Conrath, “Systemic Acquired Resistance,” Plant Signaling and Behavior 1, no. 4 (2006): 179–184.
3. J. Fletcher, C. Bender, B. Budowle, W. T. Cobb, S. E. Gold, et al., “Plant Pathogen Forensics: Capabilities, Needs, and Recommendations,” Microbiological and Molecular Biology Reviews 70, no. 2 (2006): 450–471.
4. Ronald J. Howard, J. Allan Garland, and W. Lloyd Seaman, “Crop Losses and Their Causes,” in Diseases and Pests of Vegetable Crops in Canada (Ottawa, CA: The Canadian Phytopathological Society and the Entomological Society of Canada, 1994), 67–78, https://
5. Society for General Microbiology, “Combatting Plant Diseases Is Key for Sustainable Crops,” Science Daily, April 13, 2011, https://
6. D. D. Jensen, “Papaya Virus Diseases with Special Reference to Papaya Ringspot,” Phytopathology 39 (1949): 191–211.
7. Jon Y. Suzuki, S. Tripathi, and D. Gonsalves, “Virus-Resistant Transgenic Papaya: Commercial Development and Regulation and Environmental Issues,” in Biotechnology and Plant Disease Management, ed. Zamir K. Punja, Solke H. De Boer, and Helene I. Sanfaçon, chap. 19 (Wallingford, UK: Centre for Agriculture and Biosciences, 2007).
8. Ronald A. Heu, Norman M. Nagata, Mach T. Fukada, and Bob Y. Yonahara, “Papaya Ringspot Virus Established on Maui,” New Pest Advisory No. 02-03, Hawaii Department of Agriculture, May 2002, https://
9. H. L. Wang, S. D. Yeh, R. J. Chiu, and D. Gonsalves, “Effectiveness of Cross Protection by Mild Mutants of Papaya Ringspot Virus for Control of Ringspot Disease of Papaya in Taiwan,” Plant Disease 71 (1987): 491–497.
10. S. D. Yeh and D. Gonsalves, “Practices and Perspectives of Control of Papaya Ringspot Virus by Cross Protection,” Advances in Disease Vector Research 10 (1994): 237–257.
11. S. D. Yeh, J. A. J. Raja, Y. J. Kung, and W. Kositratana, “Agbiotechnology: Costs and Benefits of Genetically Modified Papaya,” in Encyclopedia of Agriculture and Food Systems, ed. Neal K. Van Alfen, 35–50 (London: Elsevier, 2014).
12. P. P. Abel, R. S. Nelson, B. De, N. Hoffman, S. G. Rogers, R. T. Fraley, and R. N. Beachy, “Delay of Disease Development in Transgenic Plants That Express the Tobacco Mosaic Virus Coat Protein Gene,” Science 232 (1986): 736–743.
13. Yeh et al., “Agbiotechnology,” 47.
14. Marc Fuchs and Dennis Gonsalves, “Safety of Virus-Resistant Transgenic Plants Two Decades after Their Introduction: Lessons from the Realistic Field Assessment Studies,” Animal Review of Phytopathology 45 (2007): 183.
15. Fuchs and Gonsalves, “Safety of Virus-Resistant Transgenic Plants,” 183.
16. C. Neal Stewart Jr., Matthew D. Halfhill, and Suzanne I Warwick, “Transgence Introgression from Genetically Modified Crops to Their Wild Relatives,” Nature 4 (2003): 806.
17. Fuchs and Gonsalves, “Safety of Virus-Resistant Transgenic Plants,” 187.
18. Zhe Jiao, Jianchao Deng, Gongke Li, Zhuomin Zhang, and Zongwei Cai, “Study on the Compositional Differences between Transgenic and Non-transgenic Papaya (Carica papaya L.),” Journal of Food Composition and Analysis 23 (2010): 640–647.
19. Xiang-Dong Wei, Hui-Ling Zou, Lee-Min Chu, Bin Liao, Chang-Min Ye, and Chong-Yu Lan, “Field Released Transgenic Papaya Affects Microbial Communities and Enzyme Activities in Soil,” Plant Soil 285 (2006): 347–358.
20. Wei et al., “Field Release Transgenic Papaya,” 335.
21. Wei et al., 356.
22. Wei et al., 348.
23. Md. Abul Kalam Azad, Latifah Amin, and Nik Marzuki Sidik, “Gene Technology for Papaya Ringspot Virus Disease Management,” Scientific World Journal 2014 (2014): 1–11.
24. W. Shen, G. Yang, Y. Chen, P. Yan, D. Tuo, X. Li, and P. Zhou, “Resistance of Non-transgenic Papaya Plants to Papaya Ringspot Virus (PRSV) Mediated by Intron-Containing Hairpin dsRNAs Expressed in Bacteria,” ACTA Virologica 58 (2014): 262.