Exploiting Plants' Protective Proteins
ARS chemist Richard Mayer (left) and George Butler, vice president of Morse
Enterprises, examine tomatoes that have been treated with KeyPlex 350-DP, a
cooperatively developed product that deters whiteflies.
Like our own bodies that valiantly fight off infections, plants also have
built-in protective mechanisms.
In plants, this system can be triggered by pathogenesis-related (PR)
proteins. These proteins are induced and start working when an outside
enemy--an insect or a disease-causing pathogen, for example--invades the plant.
As it feeds on a plant, an insect can release substances in saliva that can
turn on defensive proteins. Or the action of plant cells being disrupted by
insect feeding can trigger the proteins. And proteins and carbohydrates
secreted by pathogenic fungi can also initiate protective measures in plants.
"For several years, we've been studying plant responses to disease and
insect pests," says Agricultural
Research Service chemist Richard T. Mayer. "A plant may have
resistance mechanisms, but the damage from pathogens or pests often overwhelms
the plant before it can marshal its defenses."
Mayer heads research at the U.S. Horticultural Research Laboratory, the ARS
research facility that will soon move from Orlando to Fort Pierce, Florida. At
Orlando, Mayer and colleagues Hamed Doostdar, Moshe Inbar, Gregory T. McCollum,
and Roy E. McDonald have identified more than 20 PR proteins in citrus.
According to Mayer, scientists have known that plants contain protective
proteins but not that they exist in citrus. He and colleagues found two classes
of enzymes--chitinases and glucanases--in citrus.
"We found these enzymes in citrus roots, leaves, blossoms, and
fruit," Mayer reports. "Once we knew that the compounds were present
in citrus, our next goal was to find a way to elicit them, or make them become
To help learn more about plant defenses, the Florida Citrus Production
Research Advisory Council supports the ARS research with funding from a
self-imposed grower tax. If plants can better defend themselves, growers will
spend less money on fungicides and insecticides and greatly benefit the
Three New Defense Products
The plant defense research has already led to one industry partnership, a
cooperative research and development agreement (CRADA) with Morse Enterprises
Limited, Inc., in Miami, Florida. From this, three products--KeyPlex 250-DP,
350-DP, and 445-DP--are being marketed in the United States, and the company is
expanding the market to Central America and the Caribbean Basin. These products
are as effective as fungicides against two diseases that plague citrus: greasy
spot, Mycosphaerella citri, and postbloom fruit drop, Colletotrichum
Morse Enterprises markets fertilizer supplements for foliar or soil
application. Because Florida's well-drained soil often contains lots of calcium
and very little organic matter and gets ample rain, growers usually add
nutrients to the soil or the foliage to boost productivity.
Grapefruit leaves on the right have been treated with KeyPlex 445-DP, which
reduces the incidence of greasy spot, Mycosphaerella citri, shown on
George C. Butler, Jr., vice president of Morse Enterprises, explains,
"We brought three products to Dr. Mayer's lab for evaluation. Our original
products were micronutrients that could be used to make plants healthier. ARS
scientists added other naturally occurring compounds that increase plant
resistance by causing them to produce more pathogenesis-related proteins."
These jointly developed products can be sprayed on plants or incorporated
into the soil to protect citrus and tomato crops. Butler says that in field
tests, KeyPlex 350-DP reduced the incidence of postbloom fruit drop of navel
oranges by 80 percent.
KeyPlex is also effective against whiteflies. Their feeding on crops can
introduce a geminivirus that significantly affects tomato plants.
"Treatment of tomato plants with KeyPlex 350-DP reduced the numbers of
whitefly adults, pupae, and eggs. Whiteflies seem to prefer untreated
plants," says Butler.
KeyPlex products are being used on citrus, bush beans, and tomatoes and
tested on limes, bananas, bell peppers, and cotton. Future tests will include
squash and other cucurbits. The label list is being extended to include most
vegetable and fruit crops.
"We've tested KeyPlex 350-DP on bananas in Florida and Central America
against yellow and black Sigatoka," says Butler. "We have had
significant reduction of both diseases, which are significant problems for
growers in Central America and the Caribbean Basin. Three major banana growers
there are interested in testing KeyPlex 350-DP because treating Sigatoka
diseases with expensive fungicides is not totally effective."
Made from naturally occurring compounds, KeyPlex products don't require
registration by the Environmental Protection Agency. "KeyPlex products are
no more toxic to humans, animals, plants, or insects than common
fertilizer," Butler says. "They work by causing the plant to produce
more of the compounds that resist pathogens and repel insects."
Boosting Plants' Protective Proteins
For 6 years, ARS chemist Hamed Doostdar has been working on identifying and
purifying plant-protective proteins in citrus.
Chemist Richard Mayer analyzes chlorophyll fluorescence in Valencia oranges as
a measure of the tree's stress.
"Purifying the proteins enables us to characterize them--that is, to
determine their composition and activity. We must know how these proteins work
and where they're located within the plant's structure," he says. "If
we expect to manipulate these proteins in plants, then we must first understand
how and why they occur."
Along with McCollum, Doostdar has isolated genes that produce these
protective proteins. The newly isolated genes are now in gene banks for public
"Once you have the gene, it can be inserted into a plant that doesn't
have that gene, if it does something you want that plant to do," Doostdar
says. "Even if a plant has a desirable gene, it might be useful to have a
transgenic plant that expresses the protein in larger quantities or at a
different place in the plant or at a different time.
"The next step for us has been to see if we can manipulate the levels
of the compounds in existing plants without having to produce transgenic
plants," says Doostdar. "It takes years to get transgenic plants
approved and released, and in tree crops like citrus, you get no fruit for the
first 4 or 5," he says. This means that it would not be economically
feasible for growers to get rid of existing groves and replant with transgenic
plants, even if plants were available. Growers could possibly replace diseased
or nonproductive trees with transgenic ones, but most could not start over.
Because of this, Doostdar and colleagues have begun looking at ways to evoke
protective compounds in existing plants.
"We started looking at chemicals that would start the protective
mechanism without the plant being actually attacked by a pest," says ARS
Inbar got his best results with BTH (benzothiadiazole), a nontoxic chemical
developed by Novartis, formerly Ciba Geigy. The compound does not harm humans,
livestock, wild or domestic animals, or plants. When sprayed on crops such as
tomatoes, it starts an internal chemical chain reaction that reduces the number
A secondary pest of Florida's vegetable crops, leafminers are now treated
with chemicals and biocontrol strategies.
Tomato fields treated with BTH had 30 percent less leafminers than untreated
fields. A tomato crop takes about 2 to 3 months to grow, and treatment should
begin 1 week before young seedlings are transplanted to the field. After
transplanting, plants should be sprayed about every third week.
Because of this research, Novartis has amended its patents on BTH to include
insect pests. The patents originally covered protection against pathogenic
fungi and bacteria.
Postharvest Use of Elicitors
Harvested fruits and vegetables also have built-in protective mechanisms,
according to ARS horticulturist Roy McDonald.
"When we found the enzyme chitinase in citrus fruit, we knew it must be
a protective compound because its purpose is to break down chitin and there is
none in citrus," he explains. "But insects' exoskeletons are made of
chitin--as are the walls of microbial pathogens. So we presume chitinase is
there to protect the fruit, since it becomes active when the fruit is attacked
by a pathogen."
Made from naturally occurring compounds, KeyPlex products stimulate plants to
increase production of their own defensive compounds. Already used on several
crops, they don't require registration by the U.S. Environmental Protection
Plant physiologist McCollum has been working on glucanases. These enzymes
break down glucans, which are anti-fungal compounds found in citrus. He has
also purified a polygalacturonase inhibitor protein (PGIP) from grapefruit
"This protein may play a role in fungal resistance," says
McCollum. "Fungi release pectinases during the infection process. PGIP
proteins inhibit pectinase production, thereby preventing the fungus from
Lab director Mayer says that PGIP proteins may also prove valuable in
increasing resistance to insect pests.
"Insects secrete pectinase when they feed. If we can get transgenic
citrus plants to express PGIP, then we have an excellent chance of producing
Dual Protection for Sweet Oranges
The West Indies sugarcane rootstalk borer weevil, Diaprepes
abbreviatus, and the fungal pathogen Phytophthora parasitica are two
interrelated problems for citrus growers.
Diaprepes larvae feed on citrus roots, causing plant decline and
death. Injured roots are more susceptible to infection by the fungi that cause
foot rot. Most of the best citrus varieties are sweet oranges whose roots are
highly susceptible to foot rot, so they must be grafted on more resistant
rootstocks. Mayer and colleagues are using the pathogenesis-related protein
approach on these problems.
"We've identified substances that stop the larvae from feeding, and
we've isolated a gene from citrus varieties resistant to foot rot that may
confer Phytophthora resistance," Mayer reports.
"What we're hoping for is to put this gene into a sweet orange like
Valencia to see if it can be grown on its own roots and still resist
Phytophthora." -- ByDoris
Stanley, Agricultural Research Service Information staff, 128 Smallwood
Village Center, Waldorf, MD 20602, phone (301) 893-6727.
Richard T. Mayer and other
scientists in this article can be reached at the USDA-ARS
Horticultural Research Laboratory, 2120 Camden Road, Orlando, FL 32803;
phone 407-897-7300, fax 407-897-7309.
"Exploiting Plants' Protective Proteins" was published in
the May 1998 issue of Agricultural Research magazine. Click here to see this
issue's table of contents.