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The Giovanni Armenise-Harvard Foundation Session 4: Plant Defense/Pathogenesis Overview The major challenge in modern plant biology is to understand plant resistance at the molecular level. This knowledge would help biologists devise rational strategies for protecting crops against pests, which could lead to a new agricultural revolution. These strategies will probably involve manipulating and potentiating natural plant defenses, predicted Dr. Giulia De Lorenzo as she introduced this session of the symposium. When plants are attacked by any virus, fungus, or other pathogen, they almost always respond by initiating rapid programmed cell death at the invasion site and by synthesizing antimicrobial compounds that rush to the scene. The more that researchers learn about the specific pathways within this general response, the more complex and the more familiar this story appears. “The emerging picture is that the plant’s cellular defense is analogous to the defense response in vertebrates,” which suggests that self-protection is an ancient evolutionary mechanism , said Dr. De Lorenzo, of the Universita’ di Roma La Sapienza. Plant biologists now know that resistance occurs only when certain complementary genes are present in the plant and its attacker. It is easy to see how plants would benefit from a resistance gene, but less obvious why pathogens would carry avirulence genes that alert the watchful plant to their presence. These avirulence molecules probably have a separate function that confers a survival advantage, although that has yet to be demonstrated. The four papers in this session homed in on the details of plant-pathogen interactions, shedding light on the character of both the attacker and the attacked.
Pseudomonas aeruginosa, a multi-host pathogen of mice, insects,
nematodes and plants In order to explore bacterial pathogenesis from an evolutionary point of view, Dr. Ausubel’s lab developed a novel strain of the opportunistic human bacterial pathogen Pseudomonas aeruginosa. This versatile model, called PA14, is infectious in several mouse models and causes disease in the plant Arabidopsis thaliana and in the insects Drosophilia melanogaster and Galleria mellonella. Moreover, PA14 also kills the nematode Caenorhabditis elegans. The researchers created an array of selective mutations, and screened them for virulence in the Arabidopsis and C. elegans models. This process identified 16 PA14 mutants that are not only less pathogenic in the Arabidopsis and/or C. elegans models, but also are generally less pathogenic in the insect and mouse models as well. While many of these had mutations in known virulence genes, 8 out of 10 identified in the Arabidopsis screen exhibited alterations to previously unknown genes. On the host side of the equation, Dr. Ausubel sought genes that are important for fighting off the bacteria. He found that some C. elegans mutants, which experience high oxidative stress when exposed to a small molecule secreted by PA14, are especially likely to be killed quickly by the bacterium. A different set of C. elegans mutants, which appear less vulnerable to oxidative stress, are more resistant to the bacteria. These studies of PA14 have identified a cluster of novel pathogenesis genes that could be targets for anti-infective compounds, Dr. Ausubel said.
Recognition of non-self molecules in plants: the role of polygalacturonase-inhibiting
proteins (PGIPs) Like an animal’s skin, the cell wall of a leaf is the first battleground between pathogen and host. Here it may be decided whether the plant is going to resist disease or fall prey to it. And just as the human immune system is activated by recognition of a pathogenic antigen, plants resistance mechanisms are turned on when they recognize pathogenicity factors produced by enemy microbes. Fungi secrete endopolygalacturonases (PG), enzymes that not only macerate the plant cell wall so that fungi can enter but also elicit a defensive response from the plant. The plant’s protective response is triggered by fragments of its own cell wall, called oligogalacturonides, that are released when PG interacts with polygalacturonase-inhibiting proteins (PGIPs), which are common in the cell walls of plants. PGIP recognizes fungal PG and modulates its enzymatic activity so that oligogalacturonides accumulate, thus sounding a loud alarm. Like most plant resistance genes, PGIP belongs to the superfamily of leucine-rich repeat (LRR) proteins. These proteins specialize in the recognition of non-self molecules, which is a key step in immunologic functioning.
Molecular basis of specificity in polygalacturonase-inhibiting proteins
(PGIPs) Like the previous speaker, Dr. De Lorenzo studies polygalacturonase-inhibiting proteins (PGIPs). Her work centers on the role of leucine-rich repeats (LRR) in recognition and regulation of specific polygalacturonases (PG) produced by pathogenic fungi. Almost one hundred proteins with LRR have been identified in animals and plants. These are needed for molecular recognition in processes as diverse as cell adhesion, signal perception in cell development, resistance to pathogens, DNA repair, and RNA processing. Structural biologists are especially intrigued with the functional versatility of the LRR protein. Just as the humoral immune system is able to form antibodies against myriad attackers, plants must recognize and resist a wide array of pathogens. It is the functional flexibility of the LRR proteins that enables them to protect against a range of phytopathogenic fungi, Dr. De Lorenzo hypothesized. In her laboratory, a series of experiments has explored the structure-function relationships of PGIP1 and PGIP2, proteins that differ only by a few amino acids yet can recognize and regulate the activity of polygalacturonases (PGs) from a wide variety of fungi. Site-directed mutagenesis has been used to improve or disable recognition function and to change ligand binding. These experiments suggest that in the future it may be possible to create custom-made receptors on plant surfaces that could make them highly resistant to specific pests.
Signal transduction pathways specifying bacterial disease resistance
in Arabidopsis thaliana This presentation illustrates one of the concepts introduced by Dr. De Lorenzo when she opened this session: the right genes must be present in both pathogen and host for resistance to occur. Dr. Staskawicz has been studying RPS2, a gene that enables Arabidopsis thaliana to fight off strains of Pseudomonas syringae containing the corresponding avirulence gene, avrRpt2. In order to understand specific molecular events in pathogen recognition and subsequent signal transduction in this plant, his laboratory constructed an epitope-tagged avrRpt2 avirulence gene and produced polyclonal antisera that detect the AvrRpt2 protein. With these tools, they have been able to identify AvrRpt2 protein in induced bacteria, inoculated plants and stable transgenic plants. These results suggest that this protein, which sets in motion a signal transduction cascade that leads to a protective response, is processed as the pathogen enters the plant cell or just after entry. Dr. Staskawicz and his colleagues are now trying to pin down the exact location of AvrRpt2 processing and to determine the sub-cellular location of the protein once it has penetrated the cell. They are also developing a screening technique, using transgenic Arabidopsis plants, that will enable them to screen for mutations in resistance genes that make plants especially vulnerable to pathogen attack.
Symposium Topics
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