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Thanks to Transgenic Research, Blight Resistant American Chestnuts Possible

Tuesday, September 10, 2019   (0 Comments)
Posted by: Dr. James Calkins, Research Information Director

Prior to 1900, the American chestnut (Castanea dentata), a member of the beech family (Fagaceae), was the dominant tree in eastern forests from Maine and southern Ontario (Canada) to Florida and west to the Ohio River valley accounting for 25% (and in some regions as much as 30-50%) of all the hardwood trees within the range of American chestnut (an estimated population of 4 billion trees). American chestnut was also commonly planted in designed landscapes both in and outside its native range as a shade tree and for its wildlife value. The stately trees grew to heights of 100 feet or more (up to 160 feet) with straight trunks that measured up to eight feet in diameter and was an important species commercially for its edible nuts and attractive, rot-resistant lumber, as a landscape species, and for its value as a reliable food source for wildlife in native ecosystems (nut crops were generally produced every year). The nuts were used as food for both people and livestock and the wood was used for just about anything that was constructed of wood including furniture, utility poles, wood flooring, musical instruments, railroad ties, and caskets, and most barns and homes east of the Mississippi that were built between 1600 and 1900 were constructed from American chestnut lumber. But that was before the effects of the accidental, and very unfortunate introduction of the pathogenic fungus that causes chestnut blight to North America – Cryphonectria parasitica (formerly Endothia parasitica), a devastating fungus that colonizes the inner bark and cambium and causes expanding, deadly cankers on the trunks, stems, and shoots of American chestnut trees of all ages, began to appear. Thus far, no inherent resistance has been found in American chestnut populations.

First detected and reported in 1904 when diseased American chestnut trees were observed by a forester in New York City in the New York Zoological Park (now the Bronx Zoo), it is believed that the fungus was introduced on Japanese chestnut trees imported as nursery stock from Asia sometime in the late 1800's. Following its introduction and initial establishment, the disease spread rapidly, and chestnut blight has subsequently devastated the American chestnut population throughout its native range and, although not yet extirpated, the species has been classified as “functionally extinct” by the United States Department of Agriculture (USDA) since 1950. In his well-known Manual of Woody Landscape Plants: Their Identification, Ornamental Characteristics, Culture, Propagation and Uses, Dr. Michael Dirr describes American chestnut as “the queen of American forest trees” that has been “reduced to a memory” by chestnut blight. Today, occasional stumps that produce sprouts are all that remain of this revered species, but the young stems are soon attacked by the fungus and never reach reproductive maturity, so seeds are never produced and the species is no longer self-sustaining. Although less susceptible, Castanea pumila (synonym Castanea alnifolia; Allegheny chinquapin/chinkapin, American chinquapin/chinkapin, or dwarf chestnut), a much smaller tree or large shrub (attaining maximum heights of only 30 feet) that is native to the eastern and southeastern United States from southern New Jersey and Pennsylvania to northern Florida and west to eastern Texas and Oklahoma, is also vulnerable to attack by the chestnut blight fungus. Breeding and selection efforts have variously focused on the development of American and hybrid chestnut trees that are resistant to chestnut blight for decades with varying degrees of success, but no final solution.

Although traditional breeding and selection efforts continue, genetic engineering has received increasing attention as a method of developing blight resistant chestnut trees in recent years. A prominent example is the American Chestnut Research and Restoration Project (https://www.esf.edu/chestnut/), a nonprofit organization housed in the College of Environmental Science and Forestry (ESF) at the State University of New York, whose goal is to reintroduce resistant American chestnut trees into forest ecosystems in New York and the rest of the eastern United States, and ultimately restore American chestnut to its native range in North America, has been pursuing the development of blight resistant trees using genetic engineering techniques. Interestingly, and although other species of chestnuts (Castanea spp.), specifically Chinese chestnut (Castanea mollissima; native to China, Taiwan, and the Korean peninsula) and Japanese chestnut (Castanea crenata; also called Korean chestnut; native to Japan and South Korea), have genetic resistance to chestnut blight and have been used in breeding efforts to develop resistant chestnut trees with characteristics that are similar to the revered American chestnut, the gene that has been used to successfully confer blight resistance to American chestnut trees by ESF researchers comes from wheat (Triticum aestivum). Cryphonectria parasitica is an oxalate-producing fungal pathogen and the oxalic acid generated by the chestnut blight fungus weakens cell walls and creates an environment that allows other fungal enzymes to degrade cell walls and membranes which results in the death of cells, the formation of girdling cankers, and the death of the portions of the tree above the cankers. The gene that has been isolated from wheat provides codes the production of an enzyme called oxalate oxidase (OxO) which, when incorporated into the genome of American chestnut trees, confers genetic resistance to chestnut blight by preventing the formation of cankers by the chestnut blight fungus in transgenic chestnut trees by converting the harmful oxalic acid to carbon dioxide and hydrogen peroxide. Although trees can still be infected by the fungus, the infections are not viable and the addition of the OxO gene to the American chestnut genome and the subsequent oxidation of oxalic acid by the oxalate oxidase enzyme has been described by some as a vaccine against the chestnut blight fungus.

A process called Agrobacterium-mediated transformation is used to facilitate the gene transfer wherein a "disarmed" (non-pathogenic) strain of Agrobacterium tumefaciens containing the resistance enhancing OxO gene is used to transform somatic American chestnut embryos. Wild type Agrobacterium tumefaciens is a pathogenic bacterium that is commonly found in the soil and is a natural genetic engineer that is commonly used in transgenic research. Using traditional tissue culture techniques, these transgenic embryos can then be multiplied and triggered to produce shoots which are then rooted and grown on to produce chestnut blight resistant trees. By crossing these transgenic trees with surviving wild American chestnut trees, the researchers hope to maintain genetic diversity and regional adaptations of America chestnut populations in future generations of American chestnuts while also protecting them from chestnut blight with the ultimate goal of producing trees that can reach reproductive maturity and produce seeds and offspring to conserve and restore the American chestnut to its native range.

Compared to traditional breeding to create inter-specific hybrids between American chestnut and resistant species like Chinese chestnut, the transfer of the single gene that is responsible for the oxalate oxidase enzyme is much more targeted and precise than the thousands of genes that are involved in the creation of inter-specific hybrids through traditional breeding and selection efforts. As a result, the introduction of undesirable characteristics from the nonnative species is generally avoided such that the inherent characteristics of American chestnut are retained. Horizontal gene transfer (HGT; the transfer of genes between organisms in a manner other than traditional reproduction) between species, including the human-mediated transfer of the OxO gene from wheat to the American chestnut, is also a natural and random occurrence in nature via a variety of pathways and can have positive or negative effects on the genetically modified species depending on the circumstances. In addition to the development of blight resistant American chestnut trees Under controlled conditions, HGT may have the potential to be used to confer a number of traits to a variety of horticultural species including abiotic stress tolerances (e.g., drought, heat, flooding, and high pH tolerance), plant habit, unique flower forms, colors and fragrances, responses to day length and flowering times, herbicide resistance, and resistance to other diseases and pests.

Needless to say, although genetic engineering may show potential for a variety of desirable outcomes, the use of biotechnology, including the use of genetic engineering and the development of genetically modified trees and other plants for agricultural, landscape, and conservation purposes is controversial and the transgenic research focused on the iconic American chestnut has become the “poster child” for both the proponents and detractors of biotechnology and genetic engineering as a means of increasing tree and forest health. In response to the growing interest in genetic engineering and the associated concerns, the National Academies of Science, Engineering and Medicine has sponsored a review of the potential of biotechnology to address forest health concerns and has subsequently published a consensus study report entitled Forest Health and Biotechnology: Possibilities and Considerations (see “Citations” section) and may be of interest to nursery and landscape professionals that have an interest in these issues. In addition, in response to concerns about the American Chestnut Research and Restoration Project’s plans to eventually release genetically engineered American chestnut trees into the wild, the Campaign to STOP GE Trees, Biofuelwatch and Global Justice Ecology Project, an international alliance of organizations formed in 2004 and dedicated to stopping the release of genetically engineered trees based on a belief that such trees would have devastating ecological and social impacts, released a white paper in April of this year (2019) describing the science and potential risks of releasing genetically engineered American chestnut trees into forests. It is important to note that the ongoing ESF American chestnut research is regulated under a permit granted by the USDA and the potential deregulation and release of transgenic American chestnut trees into an unregulated environment would be subject to stringent regulatory review by the USDA and two other federal agencies – the US Environmental Protection Agency (EPA) and the US Food and Drug Administration (FDA). It must also be noted that the activities of these regulatory agencies are not universally respected by all concerned. Regardless, if the genetically engineered American chestnuts that are currently being developed are deregulated in the next few years, they would be the first genetically modified trees to be planted in the wild; a very big and potentially consequential development.

Public angst related to genetically modified food crops, and genetically modified organisms (GMOs) in general, is certainly understandable and often justified, and public perceptions are regularly influenced by a variety of factors including a lack of clear and reliable information regarding the history and current status of traditional breeding and genetic engineering techniques, safeguards, and outcomes, media bias and a steady stream of negative stories and opinions about genetically modified organisms in the news and popular media (including social media), a general, and seemingly growing, mistrust of industry and regulatory authorities, and strong and committed opposition to GMOs by vocal activist groups. Without question, public concerns about genetically engineered plants and other organisms must be addressed if the science and desired outcomes of genetic engineering are to be understood and realized and the nursery and landscape industry needs to be educated and involved relative to the genetic engineering research and activities that have the potential to impact our industry and the plants we provide to our customers. And as the science continues to move forward, it is clear that regulatory changes will also be needed to address the many new innovations that have occurred and the future changes to come and industry will need to decide how it will be involved in this process.

Finally, it should be noted that American chestnut has also been historically threatened by Phytophthora root rot caused by the fungus Phytophthora cinnamomi in the southern reaches of its native range and the development of viable selections and restored populations of American chestnut will likely require resistance to both pathogens. Obviously, this susceptibility to another serious disease complicates efforts to return American chestnut to its former status as an important landscape and forest tree and efforts are ongoing to hopefully accomplish this goal.

Clearly, the potential benefits that might be realized through genomic research and genetic engineering are exciting, but, at the same time, these emerging technologies also present significant, and understandable concerns and research and regulatory challenges. American chestnuts that have been genetically engineered to be blight resistant could be a good test case and may set a precedent and become a model for the future. It will be interesting to see where we end up.

 Citations:

 Powell, W.A., A.E. Newhouse, and V. Coffey. 2019. Developing Blight-Tolerant American

Chestnut Trees. Cold Spring Harbor Perspectives in Biology doi:10.1101/cshperspect.a034587  https://www.esf.edu/chestnut/documents/Cold%20Spring%20Harb%20Perspect.pdf

 National Academies of Sciences, Engineering, and Medicine. 2019. Forest Health and Biotechnology: Possibilities and Considerations. The National Academies Press, Washington, DC.  This publication may be purchased, downloaded for free in pdf form, or read online at https://doi.org/10.17226/25221 or https://www.nap.edu/catalog/25221/forest-health-and-biotechnology-possibilities-and-considerations

 Smolker, R. and A. Petermann. 2019. Biotechnology for Forest Health? The Test Case of the Genetically Engineered American Chestnut. The Campaign to STOP GE Trees, Biofuelwatch and Global Justice Ecology Project. https://stopgetrees.org/wp-content/uploads/2019/04/biotechnology-for-forest-health-test-case-american-chestnut-report-WEB-1.pdf

 The following, selected resources related to the genetic engineering of plants and the regulation of genetically modified plants may also be of interest:

 Conolly, N.B. 2007 (updated January 2015). Chestnut Blight: Cryphonectria parasitica. Plant Disease Diagnostic Clinic, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY. http://plantclinic.cornell.edu/factsheets/chestnutblight.pdf

 Van Laere, K., S.C. Hokanson, R. Contreras, and J. Van Huylenbroeck. 2018. Woody Ornamentals of the Temperate Zone. In: Van Huylenbroeck J. (ed.) Ornamental Crops. Handbook of Plant Breeding, Vol. 11. Pages 803-887. Springer, Cham. https://link.springer.com/chapter/10.1007/978-3-319-90698-0_29#citeas (abstract and references only)

 Chang, S, E.L. Mahon, H.A. MacKay, W.H. Rottmann, S.H. Strauss, P.M. Pijut, W.A. Powell, V. Coffey, H. Lu, S.D. Mansfield, and T.J. Jones. 2018. In Vitro Cellular & Developmental Biology – Plant 54(4):341-376. https://link.springer.com/article/10.1007%2Fs11627-018-9914-1

 Oliver, M.J. 2014. Why We Need GMO Crops in Agriculture. Missouri Medicine 111(6):492-507. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173531/

 Leyser, O. 2014. Moving Beyond the GM Debate. PLoS Biol 12(6): e1001887. https://doi.org/10.1371/journal.pbio.1001887

 Stephen F. Chandler, S.F. and C. Sanchez. 2012. Genetic Modification; the Development of Transgenic Ornamental Plant Varieties. Plant Biotechnology Journal 10(8):891-903. https://onlinelibrary.wiley.com/doi/full/10.1111/j.1467-7652.2012.00693.x

 Lucht, J.M. 2015. Public Acceptance of Plant Biotechnology and GM Crops. Viruses 7(8):4254-4281. https://www.mdpi.com/1999-4915/7/8/2819/htm

 Herman, R.A., M. Zhuang, N.P. Storer, F. Cnudde, and B. Delaney. 2019. Risk-Only Assessment of Genetically Engineered Crops Is Risky. Trends in Plant Science 24(1):58-68. https://www.cell.com/trends/plant-science/fulltext/S1360-1385(18)30230-9

 Prakash, C.S. 2001. The Genetically Modified Crop Debate in the Context of Agricultural Evolution. Plant Physiology 126(1):8-15. http://www.plantphysiol.org/content/plantphysiol/126/1/8.full.pdf

 Westbrook, J.W., J.B. James, P.H. Sisco, J. Frampton, S. Lucas, and S.N. Jeffers. 2019. Resistance to Phytophthora cinnamomi in American Chestnut (Castanea dentata) Backcross Populations that Descended from Two Chinese Chestnut (Castanea mollissima) Sources of Resistance. Plant Disease 103(7):1631-1641. https://apsjournals.apsnet.org/doi/10.1094/PDIS-11-18-1976-RE (abstract only) https://apsjournals.apsnet.org/doi/10.1094/PDIS-11-18-1976-RE

 United States Department of Agriculture (USDA). 2014. Plant Protection Act (As Amended, December 23, 2004). USDA, Animal and Plant Health Inspection Service (APHIS), Plant Protection and Quarantine (PPQ), Professional Development Center (PDC). https://www.aphis.usda.gov/plant_health/downloads/plant-protect-act.pdf

 Executive Office of the President (EOP). 2016. National Strategy for Modernizing the Regulatory System for Biotechnology Products. https://obamawhitehouse.archives.gov/sites/default/files/microsites/ostp/biotech_national_strategy_final.pdf

 Executive Office of the President (EOP). 2017. Modernizing the Regulatory System for Biotechnology Products: An Update to the Coordinated Framework for the Regulation of Biotechnology. https://obamawhitehouse.archives.gov/sites/default/files/microsites/ostp/2017_coordinated_framework_update.pdf

 Montgomery, E. 2012. Genetically Modified Plants and Regulatory Loopholes and Weaknesses Under the Plant Protection Act. Vermont Law Review 37:351-379. https://lawreview.vermontlaw.edu/wp-content/uploads/2013/02/03-Montgomery1.pdf

  

To comment on this research update, suggest research topics of interest, or pass along a piece of research-based information that might be of interest to your industry colleagues, please email us at Research@MNLA.biz.

 

Figure 1. An American chestnut (Castanea dentata) leaf showing its characteristic shape and distinctive teeth that are responsible for the specific epithet dentata (Latin for toothed); the emerging leaves are light green and lustrous with a reddish-purple tinge and long-pointed tips, becoming dark green as they mature (Photo Credit: James Calkins).

  American Chestnut Leaf

Figure 2. American chestnut (Castanea dentata) fruits, catkin remnants, and leaves; American chestnut trees are monecious (male and female flowers produced on the same plant) and produce flowers on two types of catkins after the leaves emerge in the spring – catkins that produce only male flowers that bloom first, and catkins that produce male flowers along most of their length and several female flowers near their bases where the catkins are attached to the flowering shoots – and spine-covered fruits called burs (Photo Credit: James Calkins). 

 

American Chestnut Fruits Catkin Remnants & Leaves

Figure 3. A young American chestnut (Castanea dentata) tree infected by American chestnut blight (Cryphonectria parasitica); note the distinct, orange canker surrounding a small dead, blighted side branch in the center of the canker that will eventually girdle and kill the stem above the canker and ultimately the entire stem (Photo Credit: Linda Haugen, USDA Forest Service, Bugwood.org).


 Chestnut Blight Canker - Linda Haugen USDA


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