The chitin connection: developing plants' own self-defenses through biotechnology.
What could a fungus, a crab, a lobster, a shrimp, and a beetle possibly have in common?They all contain forms of chitin, a substance found in the outer shell of crustaceans and insects and in the cell walls of many fungi. It is chitin that makes that shell, or exoskeleton, hard; it serves as protection against predators.
Plants, too, have many built-in defense mechanisms, but chitin, as such, is not one of them. However, ARS scientists at the U.S. Horticultural Research Laboratory in Orlando, Florida, have found large numbers of related enzymatic proteins in some citrus.
"These are enzymes that break down chitin," explains lab director Richard T. Mayer. "We're hoping to put the genes for these enzymes, called chitinases and chitosanases, into the citrus and other plants that don't have them--or to manipulate the genes already present. This would build into otherwise vulnerable plants natural protection against pathogens, insects, and harmful nematodes."
Mayer hopes that these plant defensive proteins could be exploited as an alternative to methyl bromide, a protectant chemical used extensively in agriculture.
The U.S. Environmental Protection Agency has scheduled methyl bromide to be taken out of production and off the market by the year 2001 because it has been identified as being harmful to the atmosphere.
Of the 64 million pounds used in the United States in 1990, more than 80 percent were used in agriculture. After soil fumigation, commodity fumigation and quarantine are the next largest use for this chemical.
About $1.5 billion in agricultural losses might occur annually as a result of this ban, according to a report by the National Agricultural Pesticide Assessment Program. The estimate does not include the impact on nonquarantine postharvest uses, quarantine treatment of nonfood imports, and some soil fumigation.
"This adds urgency to our research on plant defensive proteins like chitinases," Mayer says. "Our ultimate goal from this research is to put genes into plants that will enable the plants to protect themselves."
Mayer says that plant chitinases and chitosanases can work independently of, or in concert with, each other or with other types of enzymes to degrade invading fungal pathogens.
This is possible, he says, because the pathogens, insects, and nematodes contain chitin and chitosan as structural components in their cell walls and exoskeletons.
Chitin also forms a membrane that lines the digestive tract of insects. This protective membrane serves as a barrier against internal penetration of the insect by bacteria or pathogens. Chitinases would break down or dissolve this membrane, leaving the insect open to infection.
If citrus plants could be engineered to produce more of the defensive proteins, then the enzymes would counterattack a pathogen or repel insects and harmful nematodes.
Pie in the sky?
"No," says Mayer. "We discovered 20 or more different forms of chitinases and chitosanases in oranges and grapefruit. We are now describing the qualities of and purifying these proteins and are looking for the genes responsible for producing them."
Mayer and colleagues are also studying what activates the genes. "We've found that we can raise the levels of the defensive proteins by applications of simple sugar compounds that are environmentally safe. These elicitors could be incorporated into a grower's routine crop-management scheme," he says. "We also plan to test this approach as a control measure for pests of citrus and other fruit and vegetable crops."
A Defense Against Whitefly?
Work on one such pest, the silverleaf whitefly, Bemisia argentifolii (formerly called the sweetpotato whitefly, Bemisia tabaci), is well under way at Orlando.
Entomologist Jeff Shapiro says that when this insect feeds on pumpkin and squash plants, the feeding activity triggers production of a few select proteins in the plant. He is looking for the gene promoters that cause this response. "Once we discover and isolate these DNA promoters, they could be linked to the chitinase genes," he says.
The next step would be incorporating both the promoters and the genes into plants such as citrus, for built-in protection against whitefly.
Finding Where the Genes Are
Plant physiologist Greg McCollum and molecular biologists Hamed Doostdar and Joseph Nairn are trying to identify the chitinase genes. They are preparing antibodies that are important as a selection tool in identifying the responsible genes.
"We've seen varied responses from the forms of enzymes we've found in citrus," McCollum says. "From the responses, we think these enzymes may be players in defending citrus against pests."
It's the multifunctional aspect of the proteins that fascinates Doostdar.
"We saw that different enzymes are expressed at different times," he says. "Once we have the enzymes purified and the genes identified that produce them, the next step will be cloning the genes and inserting them back into citrus plants to see if the genes can produce the enzymes at high enough rates to give protection."
Since the genes must be inserted in the earliest stage of plant growth, Randall P. Niedz, Orlando plant geneticist, has a citrus cell culture ready to go.
"We know that chitinases and chitosanases are in callus [a cell mass that forms during tissue culturing] and that the enzymes have many forms, so we have a lot to work with. We plan to treat callus cells with different substances to see if we can elicit the enzymes," Niedz says.
He plans to challenge the tissue-cultured citrus callus with several different types of elicitors to see what responses are triggered.
"Maybe we won't need a transgenic plant. We could find an elicitor that might be sprayed on a citrus tree to change its normal enzyme levels," says Niedz. "There are lots of possibilities."
In other research at Orlando, scientists found that the levels of chitinase in grapefruit decrease dramatically as the fruit matures. By applying plant hormones, like gibberellic acid, this trend was reversed.
"This is intriguing, especially since the presence of these enzymes appears to correlate with greater resistance to fungal infections and Caribbean fruit fly attacks," Mayer says.
Additional experiments at Orlando show other proteins that also make plants and their fruits undesirable hosts for pests. ARS horticulturist Roy McDonald is working with peroxidases--proteins that limit penetration of disease pathogens and, possibly, insects by increasing the cross-linkage of plant cell wall components.
"We've found that several elicitors cause these protective proteins to show up in citrus fruit, trees, and cell cultures," McDonald says. "We plan to use these elicitors in citrus, other fruits, and vegetables."
Any proteins elicited by these compounds will need to be characterized to determine which are plant defensive. Plant breeders will need to know which proteins to select to produce new, improved citrus varieties that can defend themselves.
Genetically engineered plants that do not require a foreign gene are generally regarded as safe by the U.S. Food and Drug Administration. Therefore, Mayer expects that FDA review would not be necessary, since these defensive proteins are already present in plants and nothing foreign is being introduced.
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| Author: | Stanley, Doris |
|---|---|
| Publication: | Agricultural Research |
| Date: | Jul 1, 1994 |
| Words: | 1150 |
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