Asim Esen, biology professor in Virginia Tech's College of Science, and David R. Bevan, biochemistry professor in the College of Agriculture and Life Sciences, received a four-year, $711,000 grant from the National Science Foundation to study the specific interaction between an enzyme and another protein, both of which are believed to be involved in helping plants defend themselves against pests. "If we can understand how the plant defense system works, we can optimize it in such a way that plants can defend themselves without using pesticides," Esen said.
"Plants have been around for millions of years and defended themselves before chemical pesticides," Esen said. However, because of selection of plant traits by humans, some plants can't even propagate themselves now. Eight thousand years ago, maize, or corn, could both defend itself and drop seeds to grow a new generation. But as humans selected for the cob and the ear, they made it impossible for the seed to get out and disperse itself; so maize now can't sustain itself. "We mutilated it," Esen said. "It can't survive on its own."
However, maize can still defend itself. Esen and Bevan are looking at the way its defense mechanism works. Young maize--the young seedlings and any growing tissues and organs--has two enzymes that help protect against insect attacks. Beta-glucosidases reside in the plastid of the maize cells, and their substrate DIMBOA-glucoside resides in the vacuole part of the cell. Usually, the two do not meet each other in an intact cell. However, when an insect starts gnawing at the young maize, it breaks the cell compartments. The enzyme beta-glucosidase breaks the DIMBOA-glucoside down into glucose and DIMBOA, and the DIMBOA is toxic to insects. However, 14 of 463 inbred lines of maize tested in a study seemed to lack the enzyme. They are called NULL.
Using spectrophotometric detection, Esen and Bevan found that all the NULL lines actually did have active beta-glucosidase, but the enzyme became aggregated and could not be extracted efficiently. From this discovery, the scientists knew the enzyme was there, but something was keeping it in the large aggregate.
Using a procedure called gel filtration that separates proteins according to size, the researchers then found that the cause of aggregation was another protein, the beta-glucosidase aggregating factor (BGAF), which NULL lines produced in excess. They isolated BGAF and proved its aggregating activity.
After further study, the scientists found that BGAF was a hybrid protein with two distinct regions or domains, a disease-response region and a carbohydrate-binding region (lectin). In nature, the two occur as separate proteins, but in all the grass species studied so far, they were fused, probably millions of years ago in the ancestors of the grasses. Such things usually happen as accidents (mutations), and, if advantageous, they get selected and passed to future generations.
Surfaces of cells have glycoproteins that lectins recognize by their carbohydrate portion and bind to. The BGAF's lectin region is similar to lectins that recognize mannose sugar. Esen and Bevan hypothesized that one of the functions of BGAF is in defense when foreign cells, such as bacteria, fungus, or viruses, try to enter the cell. BGAF probably binds foreign cells, marks them, and recruits other components of the defense system to eventually arrest the development of the foreign elements and kill them. So the beta-glucosidase-BGAF aggregate is involved in defense, Esen said, and behaves much like a football team that surrounds the ball carrier and keeps him from moving.
The researchers' project is to understand the interaction between beta-glucosidase and BGAF--how they recognize each other and bind so tightly. Thus far, they have evidence of three genes that make BGAF, but they need to find out which one, which part of the molecule, is recognized. They will do that through genetic engineering--changing the gene for BGAF, producing the protein in bacteria and yeast, and then testing it with the enzyme.
The ultimate goal is to provide evidence of the biological function of the binding and aggregation, understand the defense system, and produce plants that can once again defend themelves--to reengineer the plants in an artificial setting to enable them to do what they could originally do: survive on their own.
The NSF grant is the third major grant to Esen and Bevan for this type of study.