Dissection of Regulatory Networks Mediating Resistance and Susceptibility of Arabidopsis thaliana to the Downy Mildew Pathogen Hyaloperonospora parasitica
Plants and pathogenic microorganisms are in constant conflict with each other. Understanding the molecular networks that trigger resistance, along with the molecular networks that pathogens might co-opt to infect susceptible plants, is important for developing the integrated, holistic perspective that is necessary for innovative development of engineered resistance to current and emerging pathogens.
The first objective of the dissertation was to increase the understanding of mechanisms by which plants recognize pathogen attack and mount an appropriate defense response. These experiments focused on resistance triggered by the Arabidopsis thaliana R gene, RPP7, which encodes a coiled-coil nucleotide binding-leucine-rich repeat (CC-NB-LRR) protein that activates race-specific resistance to the downy mildew pathogen, Hyaloperonospora parasitica (Hpa). Previously-published genetic epistasis tests have established that RPP7 activates defense responses through a signaling mechanism that does not require accumulation of salicylic acid (SA), or components of the ethylene and jasmonate response pathways. Furthermore, RPP7 is not strongly compromised by mutations in genes associated with defense signal transduction (PAD4, NDR1, NPR1, RAR1). Double mutant combinations of these signal transduction components were analyzed to detect additive or functionally-redundant contributions to RPP7-dependent resistance. Most of the double mutants support an enhanced level of asexual sporulation compared to the single mutant parental lines. Time-course experiments with histochemical stains revealed that these double mutants delay, but do not suppress, the oxidative burst and the hypersensitive response. These results suggest that RPP7 activates multiple signaling pathways, each of which makes incremental contributions to the timing of defense activation.
The second objective of the dissertation was to investigate the role that auxin plays in enabling virulent H. parasitica to colonize Arabidopsis. Transcript profiling revealed induction of auxin-associated genes in response to infection of Arabidopsis thaliana by virulent strains of the oÃ¶mycete pathogen, H. parasitica. Experiments with the DR5::GUS reporter gene demonstrated that auxin responses are activated at interaction sites following hyphal penetration. A strong reduction-of-susceptibility phenotype was observed in the cyp79B2/cyp79B3 double mutant that lacks cytochrome p450 monoxygenases that catalyze for a key step in biosynthesis of indole acetic acid. Cytological and molecular experiments demonstrated that uninfected cyp79B2/cyp79B3 double mutants do not exhibit constitutive defense activation. However, penetration by an otherwise virulent H. parasitica isolate induces cell death at infection sites and transcription of defense-marker genes, suggesting that reduction of susceptibility in cyp79B2/cyp79B3 is due to derepression of defenses. Similar phenotypes were observed in the twisted dwarf (TWD1) auxin transport mutant, twd1-1, and in response to application of auxin export inhibitors. Together, these data indicate that auxin is a susceptibility factor for successful infection by H. parasitica, most likely because it retards defense responses at early stages in the interaction. Manipulation of auxin biosynthesis and transport may therefore play an important role in colonization of susceptible hosts by H. parasitica.
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Advisor:Dr. James Westwood; Dr. Brenda Winkel; Dr. Brett Tyler; Dr. John McDowell; Dr. David Beven
School:Virginia Polytechnic Institute and State University
School Location:USA - Virginia
Source Type:Master's Thesis
Keywords:plant pathology physiology and weed science
Date of Publication:01/22/2009