A mouse-made molecule is reportedly the first mammalianantibody ever to immunize a plant against a virus.
The report, "Transgenic plants expressing a functional single-chain FV antibody are specifically protected from virus attack,"appeared in last week's issue of Nature.
Molecular biologist Eugenio Benvenuto, the paper's principalauthor, directs the Biotechnology and Agriculture Departmentof Italy's New Technology, Energy and Environment Institute."Our main goal is to create plant disease resistance againstpathogens, utilizing the enormous potential of the animalimmune system," Benvenuto told BioWorld.
His strategy was to construct a gene encoding a murineantibody fragment not too big to function in a plant cell, butnot too small to fight off invading viruses. The Italian teamraised a panel of monoclonal antibodies programmed toneutralize the artichoke mottled crinkle virus virion. Then theywinnowed out the antibody with the strongest appetite for theviral coat epitope most common to various strains of theirtarget virus.
After isolating DNA clones of their candidate antibody's heavyand light chains, they amplified the variable PP active-site PPdomains, VH and VL, by PCR, for expression in Escherichia colihost cells.
What they got was a single-chain (hence very compact)recombinant antibody fragment, with both its variable domainsconnected by a linker peptide. They cloned this molecule into aplant expression vector, patented by Benvenuto's institute, andtransformed a wild-type tobacco plant, Nicotiana benthamiana.This plant model is favored for its rapid regeneration time andreceptivity to foreign DNA. Benvenuto chose it for itssensitivity to the artichoke virus.
Protection Proven in vitro and in vivo
Analysis showed that 40 percent of the regenerated transgenicplants had accumulated messenger RNA for expressing theactive single-chain antibody, amounting to 0.1 percent of totalsoluble leaf protein.
To see if this in vitro data played out in actual plant protection,the researchers infected whole transgenic plants andprotoplasts, as well as non-transgenic controls, with challengedoses of the artichoke virus particles. Specimens carrying theantibody construct accumulated smaller amounts of viral coatprotein and showed a lower frequency of infection.
Transgenic and control plants challenged with a differentpathogen PP cauliflower mosaic virus PP had equal infectionrates, which demonstrated the specificity of the artichoke-virus-primed single-chain antibody.
In a harsher test, they abraded leaves of transformed N.benthamiana with sandpaper, then rubbed on 1,000 timesmore artichoke virus than the minimum infective dose. It tookthose transformed leaves five to 14 days longer than controlsto develop necrosis (die-off) of uninoculated leaves. Thissymptomatic protection carried through to second-generationplant progeny as well.
"To our knowledge, this is the first demonstration of a plantphenotype with an attenuation of viral infection derived from aconstitutively expressed, virus-specific antibody," Benvenutostated in Nature.
Not That Others Haven't Tried
Benvenuto told BioWorld that Andrew Hiatt of the ScrippsResearch Institute made the very first attempt to put mouseantibodies into plants four years ago. He tried to smuggle anentire antibody molecule into N. benthamiana, with anti-viralintent. "We failed miserably," Hiatt told BioWorld. "We couldnever identify viral resistance with the secreted antibodies."
Hiatt explained that, unlike Benvenuto's single-chain construct,his strategy was to produce one plant expressing the antibody'sheavy chain, another the light chain. "Then we cross-pollinatedboth plants," he recalled, "and identified the offspring shoots,which were expressing both chains."
He said their lack of immune effect was due to the fact thatwhole antibodies in plant cells must be processed by theendoplasmic reticulum, which promptly secretes them out ofthe cell. "So I think it's very logical that Benvenuto's single-chain approach is probably superior because in that case theantibody is just made and sits inside the cell, where the virus isgoing to be," he concluded.
But Hiatt, who is an assistant member at Scripps, hasn't givenup. "Since that time (1989), we've been doing pretty much thebasic research to understand more about the mechanism andwhat kinds of mutagenized antibody variants can be produced.Also, to express antibodies which may have some commercialpotential," he said.
Benvenuto, too, sees vast market prospects for his "intracellularplantibodies," as he calls them. So far, the work reported inNature describes only a model system; neither tobacco plantsnor artichoke virus, he emphasizes, have any commercialsignificance.
In the near future, besides pursuing the enigmatic mechanismof antibody-conferred resistance in plants, "we will certainlytry to apply this engineered intracellular immunizationtechnology for more economically important plant-viruspathogens, such as cucumber mosaic virus and plum pox virus,"he said.
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.