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Strategy Expands Screening for Bacterial Effectors Key to Plant Diseases

Arabidopsis thaliana, a small annual weed which is proving to be valuable for rapid genetic and molecular analysis, including identifying genes that encode potential effector proteins in pathogens. (Scanning electron micrograph courtesy of Thomas Laux and Jurgen Berger, Max-Planck-Institute/Science Photo Library.)

Researchers at the University of Toronto (UT) and University of Chicago (UC) recently developed a means for identifying genes whose products enable certain bacteria to infect specific types of plants. This functional screening method, which is based on monitoring responses of Arabidopsis thaliana to putative effector proteins from plant pathogens, could yield insights that would enable researchers to control bacterial infections in crops and other economically important plants, and might also prove applicable to infectious diseases in humans.

David Guttman and his colleagues at the UT in Toronto, Ontario, Canada, and their collaborators at UC in Chicago, Ill., are studying several proteins in Pseudomonas syringae, a gram-negative plant pathogen, that make up the type III secretion system of that bacterium. These type III secretion proteins, whose close relatives are found among many other gram-negative bacteria, enable other proteins, known as effectors, to enter cells of host plants—as if "the type III secretion apparatus is the syringe while the type III effectors are the drugs," he says.

Highly varied effector proteins are found in other bacteria, including P. syringae and other plant pathogens such as Xanthomonas campestris, as well as among other pathogens that infect animals, including humans. The effector proteins from plant pathogens are responsible for damaging infected plant hosts, explaining why researchers are seeking to identify such effectors and find means to block their sometimes devastating effects.

However, Guttman says, "Up until now, there hasn't been a direct system for looking at the genes used in the natural interaction between a bacterium and its host." Moreover, identifying genes that encode potential effector proteins is difficult with traditional mutational analyses. Typically, a phenotype resulting from mutations in a single effector gene can be so slight as to be indistinguishable from the wild type, according to Jim Alfano of the University of Nebraska, Lincoln, Neb., who also is studying plant pathogens.

The functional screen devised by Guttman is based on measuring responses by a relatively simple plant, Arabidopsis thaliana, to potential effector proteins. The technique relies on hooking a region of a gene that codes for the functional portion of a known effector to a region in a candidate effector that is suspected of being a type III secretion signal. "The secretion signal from the unknown effector could then move the effector domain of the known effector into the plant cell," says Guttman, in which case the plant will mount a hypersensitive response against the combined material.

To test this screening procedure, a portion of gene of a known effector gene minus its type III secretion signal was inserted randomly into many different sites of the genome of P. syringae, including into genes encoding unidentified effectors, which themselves contain secretion signals. When the effector gene attaches to these secretion gene signals, the fused genes become detectable when bacteria containing them are injected into A. thaliana plants. The detected candidate effectors are then traced within the genome, and their genes identified. Additional details of the screening method are described in a report published recently in Science (295:1722-1726, March 1, 2002).

In one series of experiments, 25 hypersensitive plants led Guttman to identify 13 new effectors. Computer analysis conducted with colleagues at the University of Chicago expanded the number of possible type III effectors in P. syringae to 38, more than double the number of previously known bacterial plant effectors.

"[Guttman and colleagues] have designed a very clever screen to identify just the proteins that are transferred from a pathogen to its host via the type III secretion system[which] will be very useful to researchers using the P. syringae bacterial genome sequence," says Sarah Grant of the University of North Carolina at Chapel Hill, who is also studying P. syringae pathogenicity factors. "This paper increases the inventory of possible effectors, which is quite important," Alfano says, noting that some of the proteins identified through such screens may not be bona fide effectors but could have supporting roles in pathogenesis.

"When we understand how these natural interactions work, we can control the bacteria either by traditional plant breeding methods or by engineering plants to resist the bacteria," Guttman says. Grant agrees with this outlook, noting that "the effectors will lead us to a variety of host proteins with which they interact to cause disease. The plant proteins that are the targets of conserved effectors might be logical starting points to mutate or genetically engineer in order to get disease-resistant plants."

Guttman hopes to use the functional screen to learn how certain bacterial strains survive on some hosts but not others. "In the long term, we may be able to extend this research to identify elements of bacterial pathogens of animals such as Salmonella or E. coli," he says. "The new screening process has opened up a tremendous pool of resources to study and understand the whole process of pathogenesis."

Brian Hoyle
Brian Hoyle is President of Square Rainbow Limited, a science writing and editing company located in Bedford, Nova Scotia, Canada.