Plant biologists are genetically transforming a wide array ofcommercial crops PP from alfalfa to zucchini -- to resist virusesthat savage farm plantings every year.

Extensive field trials in the U.S. and Europe show that thispathogen-derived cross-protection works, and companies areeagerly awaiting regulatory permission to bring their virus-proof fruits, vegetables and field crops to market.

"One of the things the government wants to take a very closelook at is the risk of their environmental release," said plantvirologist Richard Allison. "Specifically, the U.S. Department ofAgriculture (USDA) is asking whether the small segment of theviral genome inserted into the plant is available to recombinewith any other virus that might challenge the plant.

Allison, of Michigan State University's botany and plantpathology department, is the principal author of a paper intoday's issue of Science that answers this question. Its title is"Recombination Between Viral RNA and Transgenic PlantTranscripts."

"It turns out," Allison explained to BioWorld, "that if you take avery mild strain of a virus and inoculate a plant with it, it thenbecomes resistant to more severe strains. This process isactually economically used; it's called cross-protection.

"We wanted to establish whether that could occur in transgenicplants," he continued. "Our sole hypothesis to test was, 'Is thatRNA available to a replicating virus for recombination?' Andthe conclusion that we have arrived at is, 'Yes it is.' "

He hastened to add that far from evoking the specter of anAndromeda Strain viral blight down on the farm, thisconclusion "should not hinder the release of virus-resistanttransgenic plants." The positive aspect, he explained, is thatsince submitting his paper to Science last October, ongoingexperiments in his laboratory indicate that "modifying theportion of the viral RNA that we express in the plant appears toreduce the rather high rate of recombination.

"Finding that recombination does occur between two parts ofthe same virus is no big deal," Allison added. "We've justestablished that it can happen. As for recombination betweentwo distinctly different viruses, chances for its survival wouldbe infinitesimally small," he said.

In their experiments so far, Allison and his co-workers havestarted with the gene that encodes the capsid, or coat protein,of the cowpea chlorotic mottle virus (CCMV). This, he said, "is alab rat rather than a serious crop pathogen."

They have ligated a small stretch of this gene into the genomeof Nicotiana benthamiana, a lab-model plant that, despite itsname, has little in common with tobacco. Another portion of thecapsid gene has gone back into the virus.

For a virus to function, its individual virions have to be able tomove physically from the leaf they initially infect out into thevascular system, which spreads their replicating progenythroughout the plant. But to move, their capsids must be intact.

By thus separating the original capsid gene into a plantcomponent and a defective virus segment, Allison knew thatthe pathogen could replicate but would remain grounded on itsleaf of entry. If it moved after all -- as evidenced by outlyingleaves becoming infected -- this meant it had restored an intactcapsid, which could only be the result of recombination.

In fact, this is what happened. Four of 125 transgenic plantschallenged with intact virus became systemically infected.

For proof positive, Allison and his team then altered a couple ofnucleotides in the plant's capsid sequence to mark it as distinctfrom the initial wild-type virus. When they took out andsequenced the RNA from infected leaves, these mutantfragments showed up in a complete gene. "Therefore," Allisonsaid, "we knew that that piece of RNA expressed in the planthad united the transgenic messenger RNA and the challengervirus because the joining of these two pieces together wouldoccur only during recombination events."

The molecular mechanism that confers pathogen-derived viralresistance on a plant is not understood, Allison said. "But Iassure you that there are many labs in the U.S. and elsewherethat are working on this."

One of these is the laboratory of plant molecular biologist RogerBeechy, who discovered this phenomenon in 1986. Beechy, whowas then at Washington University in St. Louis, now heads thedepartment of plant biology science at the Scripps Institute."The idea came about because based on the whole concept ofcross-protection, we were simply testing which of severalhypotheses was correct for that kind of protection," he toldBioWorld.

In cooperation with nearby Monsanto Corp., Beechy and hisassociates engineered some tobacco and tomato plants with thetobacco mosaic virus coat protein, some with antisense RNAand other constructs. "The coat protein turned out to becorrect," he said.

Beechy recalled that "back in 1986, very few people weredoing plant transformation. Monsanto did the transformationfor us; we analyzed the resistance." (Monsanto, jointly with theconsortium for plant biotechnology research, also supportsAllison's risk-assessment work jointly with the MichiganAgricultural Experiment Service.)

Beechy is now trying to "do some definitive experiments thisyear to figure out the mechanism." So far, his evidence suggeststhat "the coat protein in the cell interferes with thedisencapsidation of the challenge virus, so the infection doesn'ttake off."

Beechy said the commercial impact of transgenically conferredviral resistance will be different for different crops. "Manyfield crops in the U.S. -- corn and soybeans, for example PPsuffer annual losses of between 2 and 10 percent depending onthe number of aphids, which carry the virus, in a given year,"he said.

The aphid, a small winged insect that is also called the "plantlouse," transports the virus to its crop target. Farmers normallyspray incoming aphids -- a costly and largely ineffectivepractice. "Losses can be as severe as 80 percent in some areasof New Jersey and the Northeast where there are severeviruses on plants such as squash and cucumber," Beechyobserved.

Such crops, along with canteloupe, are called cucurbits, and theUpJohn Co. of Kalamazoo, Mich., through its subsidiary, theAsgrow Seed Co., is busy virus-proofing them. "We are in theprocess of producing virus-resistant transgenic cucurbits usingthe coat proteins of other viruses," Asgrow's co-director ofvegetable biotechnology, Hector Quemada, told BioWorld. He isworking with cucumber and watermelon mosaic viruses,zucchini yellow and papaya ringspot viruses.

Asgrow has had marketing applications pending with USDA forseveral of its transgenic vegetables since July 1992. Quemadadoubts that Allison's paper in today's Science will influence theagency's decision. "His work demonstrates that what one wouldexpect to happen, does. It's known that such recombinationoccurs in nature anyway."

Quemada added that Monsanto has developed transgenic potatolines resistant to the two main potato viruses and that Pioneer-Hybrid is working on protecting alfalfa from the alfalfa mosaicvirus.

"Most of the major and many of the minor seed companiesaround the world are using it," said Beechy. "For example, theFrench champagne makers Mot-Hennessey and Mot-Chandonhave reported virus-resistant transgenic grapevines."

As for the recombination risk, the Scripps researcher observed,"This is going to be a virus-by-virus case; some may recombine,others may not." He predicted that "the environmentalists andothers will say, 'Gee, this will lead to unleashing all sorts of newviruses that nature never saw before.' " But he emphasized that"it's important to say it's what happens in nature naturally."

Two plant pathologists, Bryce Falk and George Bruening of theUniversity of California, Davis, wrote an editorial in Scienceaccompanying Allison's paper. It concludes: "The virus-resistant cultivars developed by traditional plant breedinghave fostered the emergence of virulent virus strains.However, abandoning plant breeding does not seem to be asensible alternative.

"Equally, abandoning virus-derived genes for resistance againstviruses because of a vanishingly small risk of creating new andharmful viruses (which would occur naturally anyway) seemsto us to be a foolish approach," they wrote.

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.