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The Giovanni Armenise-Harvard Foundation Session 3: Plant Defense/Pathogenesis Overview The more that scientists learn about how plants protect themselves against disease, the more parallels they see between plant and mammalian defense systems. Much of what is known about how plants respond to pathogen attack has come from studies of Arabadopsis thaliana or rice. This type of research will get a boost in 2000, when the genomes of these model systems are expected to be fully sequenced, Dr. Brian Staskawicz predicted in his introduction to this session. Plants have many enemies, including bacteria, fungi, viruses, and nematodes. Agriculturists have been developing disease-resistant plants since the turn of the century, relying almost entirely on classical breeding, hybridization, and recurrent selection for desirable traits. Long before genes could be isolated, it was obvious that a single resistance gene could make the difference between a bountiful harvest and a failed crop, Dr. Staskawicz noted. After genetic mapping and map-based cloning became available, scientists began cloning specific genes for disease resistance. Many plant resistance genes have leucine-rich repeat(LRR) domains, which are important for recognizing non-self proteins in the environment and setting in motion a signal transduction pathway that ultimately leads to defensive action. The machinery for recognition and response closely resembles the mammalian immune system. In recent years, studies of bacteria that prey on plants have revealed that they share many characteristics with human pathogens, including similarities in effector proteins known as virulence factors. Now that both host plants and their pathogens can be genetically manipulated, scientists are going to find better ways to give plants an edge over their enemies. "The field of plant pathogenesis and defense is exploding right now," Dr. Staskawicz said.
Plants can be resistant to disease only when there is a match between a plant resistance gene and an avirulence (Avr) gene in the pathogen. Resistance genes are thought to code for receptors that recognize specific Avr products. With one exception, all the plant resistance genes that have been identified have leucine-rich repeats (LRR), which encode specific receptors for a wide variety of Avr proteins. Dr. De Lorenzo suspects that recognition of specific pathogens hinges on a hypervariable region in resistance genes that codes for variations in a specific region within LRR proteins. This is theoretical at present, however, because her team has not yet observed a direct interaction between LRR and Avr proteins. Polygalacturonase-inhibiting proteins (PGIPs), present in the cell wall of many plants, belong to the large family of LRR proteins and are structurally similar to other known products of resistance genes. PGIP recognizes endopolygalacturonases (PG), enzymes that disease-causing fungi use to breach the cell walls of plants. PGIPs and PGs offer a unique opportunity to analyze how LRR proteins recognize specific attackers. Dr. De Lorenzo's laboratory has been using site-directed mutagenesis to explore how the recognition capacity of PGIPs can be manipulated. In one experiment, alteration of a single amino acid residue caused a PGIP to lose function, she reported. Now her team is working with mutations that may increase recognition, seeking to create chimeric proteins that will enable plants to identify and resist a wider range of pathogens.
Dr. Cervone's laboratory focuses on events in the plant cell wall, which is the organism's first line of defense against pathogenic invaders. For some years, they have pursued genetic mutations in the cell wall that may be important in both defense and normal development. When these proved elusive, the researchers decided to see if cell-wall mutations could be detected if they used well-characterized bacterial enzymes to manipulate extracellular matrix architecture. Dr. Cervone's team used Agrobacterium-mediated transformation to produce plants that overexpress polygalacturonase (PG) and polygalacturonase-inhibiting proteins (PGIPs). They were able to generate two kinds of transgenic plants: Arabidopsis and tobacco plants expressing PG from Aspergillus niger, and Arabidopsis, tobacco and tomato plants expressing PGIP from Phaseolus vulgaris. These alterations changed the pectins in the cells walls. Although they expected that the morphogenesis of transformed plants would not be the same as normal ones, they were surprised when the altered plants grew much larger and more vigorously than the wild-type, a kind of plant version of super mouse. Another unexpected finding was that pectins from tomatoes with the pgip transgene exhibited a higher degree of methylation and acetylation than those isolated from non-transformed plants. This is consistent with earlier findings that PGIP in vitro interacts with methylated pectins better than with non-methylated homogalacturonans, a perference that probably protects pectins from demethylation. Although much more research is needed, even at this early stage it is clear that plant transformation with PGs and PGIPs is a valuable tool for exploring how changes in pectin structure affect plant development, physiology, and defense.
Dr. Rahme's laboratory studies the molecular mechanisms underlying Pseudomonas aeruginosa pathogenesis in mammals. This is a medically important opportunistic bacterium that infects burn and trauma patients, as well as other immunocompromised individuals, and it is the leading cause of death in people with cystic fibrosis. She and her collaborators have shown that a novel strain of P. aeruginosa uses the same subset of virulence factors to cause disease both in plants and in a wide range of animals, including humans. This important finding gave them the opportunity to use plants as a screening system for bacterial virulence factors, which once found could point the way to the identification of new antibiotics. This screening algorithm markedly decreases the use of laboratory animals, yet it generates data relevant to pathogenesis in mammals. In plants, Dr. Rahme's group has identified several novel P. aeruginosa virulence-related factors, the majority of them affecting the persistence and severity of infection. When these factors were tested in a mouse model that involves infection after a non-lethal burn, they made infections worse and accelerated sepsis development. More recently, Dr. Rahme has been using C. elegans to identify avirulence mutations that enable the worm to feed on pathogenic P. aeruginosa and survive. She has found several mutations that appear to increase the host's ability to limit disease development, not just in worms but in mammals as well.
Scientists have long anticipated a time when transgenic plants will be resistant to diseases that farmers now combat with chemical pesticides. Dr. Staskawicz and his coworkers will soon discover whether this moment has arrived for a type of bacterial spot disease caused by Xanthomonas campestris pv vesicatorio (XCV), which afflicts both peppers and tomatoes. A gene that produces durable resistance in peppers is being experimentally introduced into tomato plants, which have no natural resistance to this pathogen. It took Dr. Staskawicz' lab more than 8 years to nail down the function of this gene, isolate it, and prepare it for use as a transgene. This work grew out of a broader inquiry into the molecular genetics of disease resistance, which showed that plants are protected against XCV-caused spot disease when the bacterium carries the avirulence gene (actually an effector protein) avrBs2 and the host plant has the resistance gene Bs2. A series of genetic and biochemical experiments demonstrated that the Bs2 gene product recognizes the business end of XCV-an effector protein that the pathogen needs to be at its most virulent. Having decided that Bs2 was a good candidate for insertion into the tomato genome, Dr. Staskawicz and his colleagues set out to map and clone the gene-no trivial task in a genome four times as large as the human genetic endowment. The next step is to show protection in field trials, which are expected to begin this year.
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Exploiting polygalacturonase-inhibiting proteins (PGIPs) to engineer
novel plant receptors
