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The Giovanni Armenise-Harvard Foundation Session 4: Pathogens and Defense Overview Since its inception, the Armenise-Harvard Foundation has sponsored basic research on plants as well as animals, believing that advances in agriculture as well as medicine hold tremendous benefits for future generations. Thus, each year's symposium has featured lectures on pathogenesis and defense mechanisms in the plant world. This year, the synergy between plant and animal science was more apparent than ever before. Mammalian biologist Tomas Kirchhausen, who chaired the session, admitted that he knew little about plants before he began preparing for the symposium. And the Harvard researcher was astonished to learn that plant and animal defense systems not only resemble one another, but sometimes deploy the same genetic and molecular elements when battling their enemies. The first presentation in this session, for example, concerned a plant pathogen that injects harmful proteins into its leafy victims with the same syringe-like structure that salmonella uses to invade the lining of the human gut. In the second talk, the emphasis shifted to experimental methods that can be applied to either kingdom. Here it became clear that X-ray crystallography, which has provided remarkable insights into human dramas such as the binding of HIV to T cells, can also illuminate pathogen-host interactions in plants. The third paper focused on a class of proteins that act as both sentinels and warriors, recognizing certain invaders and doing battle with them. Pathogen strategies for evading host defenses were the focus of the closing talk, which made the transition from plants to animals by concentrating on how peptide antigens are presented on cell surfaces.
Presentations
The first task of any plant-attacking microbe is to batter its way through the ramparts of the cell wall. A high resolution structural analysis of a fungal battering ram, done by Drs. Mattei and Federici, reveals that the best form of this weapon is also the most recognizable to plant defenses. Fusarium moniliforme, like other fungi, uses endopolygalacturonases (PGs) to break cell-wall proteins into pieces and dissolve them. These investigators used X-ray crystallography to determine the structure of PG from F. moniliforme at 1.73 Å resolution. Like other pectinolytic enzymes, this PG resembles a squared-off coil of spring, with 10 coils each made up of three or four parallel beta-helical strands, in a coil-coiled helical organization. The researchers prepared an assortment of site-directed mutants of PG and used these to sort out which PG residues are involved in catalysis and which interact with defensive polygalacturonase inhibiting protein (PGIP). It appeared that three aspartic acids and one histidine may be involved in catalysis, but play no role in recognition by PGIP. The researchers found that amino acid substitutions at two residues (Lys269 and His188) interfered dramatically with the binding of PG and its inhibitor. These and other results led the investigators to hypothesize that inhibition mechanisms involve both competition between substrate and inhibitor and the covering of an active site cleft.
Their data suggest that X. campestris delivers the AvrBs2 avirulence protein via a syringe-like structure called the Hrp Type III secretion system. This mechanism is familiar to eukaryotic biologists because salmonella, shigella, and other animal pathogens use it to inject proteins into host cells. It's remarkable that plant and animal pathogens have both come up with this machinery, Dr. Staskawicz observed, and many researchers are now analyzing its evolution and genetic underpinnings. In the meantime, Dr. Staskawicz' findings about effector proteins made by avirulence genes and the receptors that recognize them are being put to use in the field. Years ago, agriculturists found a Bs2 resistance gene in wild peppers and successfully bred it into commercial pepper strains. As a result, many farms grow Xcv-resistant peppers. There is no indigenous Bs2 gene in tomatoes, however, so Dr. Staskawicz and his collaborators have used the pepper resistance gene to create transgenic tomato plants. These are now being field tested for resistance to Xcv. The environmental benefits of these plants could be substantial, as bacterial spot disease is presently controlled by spraying fields with massive amounts of copper and other toxic chemicals.
Although polygalacturonase-inhibiting proteins (PGIPs) were named for their ability to defend plant cells against fungal endopolygalacturonases (PGs), it turns out that they wear other hats as well. This is to be expected, Dr. De Lorenzo said, because PGIPs are made by genes in the leucine-rich repeat (LRR) family, some of which play key roles in development while others confer resistance to pathogens. In addition to PGs, PGIPs interact with macromolecules including methylated pectins and membrane-associated lipoxygenases. Dr. De Lorenzo, in collaboration with Dr. Fred Ausubel of Harvard, has been exploring the physiologic significance of these interactions in Phaseolus vulgaris and Arabidopsis. They have found significant redundancy in pgip gene families, with several genes encoding the same or related products. Using knock-out and over-expression mutants, they have begun to identify differences in recognition specificity, regulation, and function for pgip genes. One of the most exciting findings is that a pgip transgene generates a more heavily methylated pectin than the type found in normal cell walls. This may be useful in helping toughen plants against pests. The researchers have also used pgip transgenes to grow arabidopsis plants that are bushier than usual.
Experiments in Dr. Ploegh's lab recently showed how these two proteins disrupt antigen presentation. US2 and US11 appear to grab newly synthesized MHC Class I products by their tails, which protrude from the endoplasmic reticulum (ER), and drag them into the cytosol. Being ripped from the ER in an untimely fashion, these complexes are viewed by the cell as mis-folded proteins; ubiquitin marks them for destruction and they are whisked off to the proteasome and shredded. There are some other actors in this plot, such as unknown proteins that strip off ubiquitin immediately before proteolysis, and the researchers are still looking for them. CMV's wiles may help explain why this virus can infect such a wide range of cell types, especially in patients with AIDS or other forms of immune suppression. In the long term, experiments such as these may contribute to the design of superior gene therapy vectors, which might be able to evade the immune system en route to their targets.
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Structural
studies on a fungal pathogenicity factor
