Blight Resistance: It's in the DNA
Rebecca Hirsch | The Journal of the American Chestnut Foundation, May/June 2012
© 2012 Rebecca Hirsch
Why do American chestnut trees die from chestnut blight while Chinese chestnuts handily fight off Cryphonectria parasitica? Scientists are looking for answers to that question in the chestnut's DNA. In work supported by TACF and the Forest Health Initiative, teams of researchers from the US Forest Service, The Pennsylvania State University, University of Georgia, Clemson University, and SUNY College of Environmental Science and Forestry are working to map and sequence the chestnut's genome and identify the genes that contribute to blight resistance. What they learn will aid in the effort to restore the American chestnut to the forest ecosystem.
On the Trail of Resistance
A chestnut tree's genome—its complete set of genes—is housed in an ensemble of 12 chromosomes which together hold the instructions to make and operate the tree. Unravel one of the chromosomes and you will find long strands of DNA made of four repeating units called bases. The bases come in pairs: one strand of bases paired with another strand, forming a double helix. It is the order of the bases that determines the meaning of the genetic instructions, and physical differences between the Chinese and American chestnut trees—in traits like height, leaf shape, and the ability to fight off pathogens—can be traced back to differences in the DNA.
Studying the genome of an organism is no small task. The chestnut's genome is immense. The best estimate is that it contains, give or take, 800 million base pairs.
Genetic mapping is a way for scientists to negotiate this landscape. Researchers identify markers that act as mileposts along the chromosomes. The markers can be snippets of DNA or unique sequences of bases. Putting the markers together to create a map of the genome serves a number of useful purposes. A map gives researchers a way to compare chestnut to related species like beeches, oaks, and other forest trees. It serves as a jumping off point for sequencing the chestnut's genome, determining the exact order of those 800 million or so base pairs. And it can reveal the location of genes that control certain traits such as blight resistance.
Scientists have now pieced together a detailed map of Chinese chestnut (specifically, Castanea mollissima cv ‘Vanuxem’) and have identified genes for blight resistance in four regions on the chromosomes. These four regions, called loci, are spots where genes for blight resistance reside. Researchers are now zeroing in on the four loci, sequencing the DNA in each region in an effort to find the genes that contribute to blight resistance. The loci contains hundreds of genes—the large majority of which have nothing do with resistance—which means that scientists must use detective work to narrow down the search.
One clue researchers look for in finding a gene for blight resistance is evidence that the gene is turned on in blight cankers. Scientists have screened cankers in American and Chinese chestnut trees to determine which genes are active. They are particularly interested in genes that are turned on at high levels in the Chinese tree but are present only at low levels in the American tree. Such a pattern makes a gene a candidate for blight resistance.
Another clue researchers look for: genes that are similar to disease resistance genes from other plants. Scientists studying the blight resistance loci have noticed similarities to loci in peach that contain genes for disease resistance. The peach genes help fight powdery mildew, another fungal disease. Such similarities can greatly aid in identifying the genes that encode resistance in chestnut.
Testing Resistance
Once researchers have identified likely candidate genes for blight resistance, they can perform a direct and powerful test of each gene's function by adding the gene to an American chestnut tree and testing whether the added gene offers the tree any additional resistance to the blight. This approach allows researchers to directly answer the question; does this gene confer resistance to the blight?
To carry out this test, the gene is added to a soil bacterium known as Agrobacterium. Agrobacterium has the singular ability to attach itself to a plant and inject a small piece of DNA into a plant cell. "You can think of agrobacterium as a little shuttle," says Dr. Joe Nairn, whose lab at the University of Georgia is involved in this effort. "You put the gene in the shuttle, and the shuttle delivers it to the plant cells." Agrobacterium containing the gene of interest is mixed with American chestnut embryos, and the end result is that the injected DNA ends up spliced into the plant’s own DNA, a process known as genetic transformation.
Next the transformed embryos are moved to a growth medium—a liquid or gel-like substance filled with nutrients and hormones—and the embryos are grown into new plants. The transgenic trees are then moved to pots and later to a test-plot outdoors, where they can be tested against control plants to determine how well they can resist the blight.
Dr. Scott Merkle, a chestnut researcher at the University of Georgia, stresses that safety is a focus in working with genetically-engineered trees. Growers follow strict rules handed down by the USDA and other regulatory agencies. Nurseries are fenced, gated, locked. Inspectors visit regularly. Flowers are clipped off or bagged to prevent the spread of pollen. Every tree is labeled and monitored, and even pruned branches are tracked and discarded safely. "The major concern," said Dr. Merkle, "is that we don't allow any of the genes that we're testing to move into the wild population."
Solutions from the DNA
It will likely take years for researchers to tease apart the genetic pathways that enable Chinese chestnut to fight off the blight. Dr. Paul Sisco, retired staff geneticist with TACF, cautions that the system that emerges may be highly complex. He envisions a scenario where different genes might control resistance in different Chinese chestnut cultivars or in different Asian species such as Japanese chestnut. Researchers are already getting glimpses of complexity, with evidence that the four blight-resistant loci in Chinese chestnut all respond differently depending on the particular strain of C. parasitica.
Even though it will likely take years for scientists to unravel blight resistance, DNA studies could soon show direct benefits to the effort to restore the American chestnut. According to Dr. Sisco, the use of genetic markers that lie near the resistance genes could make the backcross breeding program more efficient. Right now trees in the breeding program must be grown for four years or more before researchers can determine their level of blight resistance. "DNA markers to identify resistance in newly-emerged seedlings could save us a lot of time, space, effort, and money," says Dr. Sisco.
Some researchers envision using genetic engineering to produce a blight-resistant American chestnut. They are experimenting with adding genes to the American chestnut in the hopes of creating a transgenic tree that can resist the blight. They are also experimenting with adding resistance genes from other species. Leading in this effort are Drs. Bill Powell and Chuck Maynard of the State University of New York College of Environmental Science and Foresty (SUNY-ESF) One promising project involves transforming American chestnut with the oxalate oxidase gene from wheat. The gene encodes an enzyme that breaks down oxalic acid, a chemical present in large amounts in blight cankers and toxic to chestnut tissues. Their hypothesis is that the enzyme will neutralize the acid, prevent the canker from growing, and enhance the tree's resistance. The first of these transgenic trees were planted in early 2011.
Genetic engineering could also be used to create a chestnut that can resist ink disease caused by Phytophthora cinnamoni. This deadly pathogen, once confined to the southeastern part of the chestnut's range, may be moving further north and to higher elevations. "It's worse than chestnut blight because there's no resprouting from the stumps," said Dr. Merkle. "Once a tree gets Phytophthora, it's dead and it's not coming back."
Researchers caution that transgenic trees would probably not be used directly for reforestation, but might be crossed to surviving American chestnut trees as a way to build in genetic diversity. Yet whether the public will welcome genetic engineering as a way to save the chestnut remains to be seen. "It's a whole other question," said Dr. Nairn. "There's a large community that will have to address that."
© 2012 Rebecca Hirsch
Why do American chestnut trees die from chestnut blight while Chinese chestnuts handily fight off Cryphonectria parasitica? Scientists are looking for answers to that question in the chestnut's DNA. In work supported by TACF and the Forest Health Initiative, teams of researchers from the US Forest Service, The Pennsylvania State University, University of Georgia, Clemson University, and SUNY College of Environmental Science and Forestry are working to map and sequence the chestnut's genome and identify the genes that contribute to blight resistance. What they learn will aid in the effort to restore the American chestnut to the forest ecosystem.
On the Trail of Resistance
A chestnut tree's genome—its complete set of genes—is housed in an ensemble of 12 chromosomes which together hold the instructions to make and operate the tree. Unravel one of the chromosomes and you will find long strands of DNA made of four repeating units called bases. The bases come in pairs: one strand of bases paired with another strand, forming a double helix. It is the order of the bases that determines the meaning of the genetic instructions, and physical differences between the Chinese and American chestnut trees—in traits like height, leaf shape, and the ability to fight off pathogens—can be traced back to differences in the DNA.
Studying the genome of an organism is no small task. The chestnut's genome is immense. The best estimate is that it contains, give or take, 800 million base pairs.
Genetic mapping is a way for scientists to negotiate this landscape. Researchers identify markers that act as mileposts along the chromosomes. The markers can be snippets of DNA or unique sequences of bases. Putting the markers together to create a map of the genome serves a number of useful purposes. A map gives researchers a way to compare chestnut to related species like beeches, oaks, and other forest trees. It serves as a jumping off point for sequencing the chestnut's genome, determining the exact order of those 800 million or so base pairs. And it can reveal the location of genes that control certain traits such as blight resistance.
Scientists have now pieced together a detailed map of Chinese chestnut (specifically, Castanea mollissima cv ‘Vanuxem’) and have identified genes for blight resistance in four regions on the chromosomes. These four regions, called loci, are spots where genes for blight resistance reside. Researchers are now zeroing in on the four loci, sequencing the DNA in each region in an effort to find the genes that contribute to blight resistance. The loci contains hundreds of genes—the large majority of which have nothing do with resistance—which means that scientists must use detective work to narrow down the search.
One clue researchers look for in finding a gene for blight resistance is evidence that the gene is turned on in blight cankers. Scientists have screened cankers in American and Chinese chestnut trees to determine which genes are active. They are particularly interested in genes that are turned on at high levels in the Chinese tree but are present only at low levels in the American tree. Such a pattern makes a gene a candidate for blight resistance.
Another clue researchers look for: genes that are similar to disease resistance genes from other plants. Scientists studying the blight resistance loci have noticed similarities to loci in peach that contain genes for disease resistance. The peach genes help fight powdery mildew, another fungal disease. Such similarities can greatly aid in identifying the genes that encode resistance in chestnut.
Testing Resistance
Once researchers have identified likely candidate genes for blight resistance, they can perform a direct and powerful test of each gene's function by adding the gene to an American chestnut tree and testing whether the added gene offers the tree any additional resistance to the blight. This approach allows researchers to directly answer the question; does this gene confer resistance to the blight?
To carry out this test, the gene is added to a soil bacterium known as Agrobacterium. Agrobacterium has the singular ability to attach itself to a plant and inject a small piece of DNA into a plant cell. "You can think of agrobacterium as a little shuttle," says Dr. Joe Nairn, whose lab at the University of Georgia is involved in this effort. "You put the gene in the shuttle, and the shuttle delivers it to the plant cells." Agrobacterium containing the gene of interest is mixed with American chestnut embryos, and the end result is that the injected DNA ends up spliced into the plant’s own DNA, a process known as genetic transformation.
Next the transformed embryos are moved to a growth medium—a liquid or gel-like substance filled with nutrients and hormones—and the embryos are grown into new plants. The transgenic trees are then moved to pots and later to a test-plot outdoors, where they can be tested against control plants to determine how well they can resist the blight.
Dr. Scott Merkle, a chestnut researcher at the University of Georgia, stresses that safety is a focus in working with genetically-engineered trees. Growers follow strict rules handed down by the USDA and other regulatory agencies. Nurseries are fenced, gated, locked. Inspectors visit regularly. Flowers are clipped off or bagged to prevent the spread of pollen. Every tree is labeled and monitored, and even pruned branches are tracked and discarded safely. "The major concern," said Dr. Merkle, "is that we don't allow any of the genes that we're testing to move into the wild population."
Solutions from the DNA
It will likely take years for researchers to tease apart the genetic pathways that enable Chinese chestnut to fight off the blight. Dr. Paul Sisco, retired staff geneticist with TACF, cautions that the system that emerges may be highly complex. He envisions a scenario where different genes might control resistance in different Chinese chestnut cultivars or in different Asian species such as Japanese chestnut. Researchers are already getting glimpses of complexity, with evidence that the four blight-resistant loci in Chinese chestnut all respond differently depending on the particular strain of C. parasitica.
Even though it will likely take years for scientists to unravel blight resistance, DNA studies could soon show direct benefits to the effort to restore the American chestnut. According to Dr. Sisco, the use of genetic markers that lie near the resistance genes could make the backcross breeding program more efficient. Right now trees in the breeding program must be grown for four years or more before researchers can determine their level of blight resistance. "DNA markers to identify resistance in newly-emerged seedlings could save us a lot of time, space, effort, and money," says Dr. Sisco.
Some researchers envision using genetic engineering to produce a blight-resistant American chestnut. They are experimenting with adding genes to the American chestnut in the hopes of creating a transgenic tree that can resist the blight. They are also experimenting with adding resistance genes from other species. Leading in this effort are Drs. Bill Powell and Chuck Maynard of the State University of New York College of Environmental Science and Foresty (SUNY-ESF) One promising project involves transforming American chestnut with the oxalate oxidase gene from wheat. The gene encodes an enzyme that breaks down oxalic acid, a chemical present in large amounts in blight cankers and toxic to chestnut tissues. Their hypothesis is that the enzyme will neutralize the acid, prevent the canker from growing, and enhance the tree's resistance. The first of these transgenic trees were planted in early 2011.
Genetic engineering could also be used to create a chestnut that can resist ink disease caused by Phytophthora cinnamoni. This deadly pathogen, once confined to the southeastern part of the chestnut's range, may be moving further north and to higher elevations. "It's worse than chestnut blight because there's no resprouting from the stumps," said Dr. Merkle. "Once a tree gets Phytophthora, it's dead and it's not coming back."
Researchers caution that transgenic trees would probably not be used directly for reforestation, but might be crossed to surviving American chestnut trees as a way to build in genetic diversity. Yet whether the public will welcome genetic engineering as a way to save the chestnut remains to be seen. "It's a whole other question," said Dr. Nairn. "There's a large community that will have to address that."
