Investigating NLR-mediated immunity and Pseudomonas syringae effector interactions with two MACPF domain proteins in Arabidopsis thaliana.
To disarm plant defenses, successful pathogens secrete an arsenal of diverse virulence factors, termed effectors, into the host cell to shut down key components of the plant immune signaling pathway. To protect against effector sabotage, plants have evolved intracellular NUCLEOTIDE BINDING AND LEUCINE RICH REPEAT RECEPTORS (NLRs) that monitor the integrity of critical immune signaling components likely to be effector targets. NLR activation upregulates plant immune responses and triggers a form of localized cell death known as the hypersensitive response (HR). To prevent damaging levels of cell death NLR activation is under tight control. NLRs are maintained in an inactive “OFF” state under stable conditions, and only turn ‘ON’ when perturbations of monitored proteins are detected. MEMBRANE ATTACK COMPLEX/PERFORIN (MACPF) proteins are well known agents of defense in the mammalian immune system, though the exact role of this protein family in plants has not been established. Mutations in two of the four MACPF domain proteins encoded in Arabidopsis thaliana cause salicylic acid - dependent autoimmunity, a phenotype that has been linked to the constitutive activation of NLR receptors. In constitutively activated defense 1 (cad1) mutants this autoimmunity is dependent on ENHANCED DISEASE SUSCEPTIBILITY 1, a component of the signaling pathway mediated by a subset of NLR receptors. The first major objective of this thesis was to utilize a recently published library of dominant negative NLR mutants to identify the receptor(s) involved in cad1 autoimmunity. Through screening 67 unique dominant negative transgenic lines for the suppression of cad1-5 rosette stunting and lesion formation phenotypes I did not identify the NLR activated in cad1-5. Additionally, as my second objective I conducted a yeast two hybrid screen to test whether CAD1 or its close relative NECROTIC SPOTTED LESION 1 (NSL1) are targeted by effectors of the Arabidopsis pathogen Pseudomonas syringae pv tomato. From this screen I identified one effector that may interact with NSL1. Collectively, this work provides novel insight into the molecular aspects of host-pathogen interactions.