Plants’ innate immunity is composed of two intricate immune networks: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is activated upon the recognition of pathogen-associated molecular patterns (PAMPs) by a pattern recognition receptor (PRRs). The outcomes of PTI are considered broad spectrum but can be easily suppressed by effectors from the adapted pathogens. Perception of the pathogen effectors by a nucleotide-binding leucine-rich (NLR) receptor activates ETI, which leads to a robust response in the form of localized programmed cell death at the site of infection. NLRs are classified into different classes based on protein structures: coiled-coil NLRs (CNLs) and Toll/interleukin-1 NLRs (TNLs). Both types deploy common and unique mechanisms to achieve immunity. CNLs can function as singletons, linked pairs or complex networks. Meanwhile, TNLs rely on the downstream immune modulator, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1). EDS1 coordinates with RPW8-like NLRs (RNLs) to attain the defense response. In this dissertation, I describe the current knowledge and perspectives about the major types of NLRs in plants, including their discovery and functions, and various mechanisms they utilize to establish immunity. I also present findings of my studies, in which the regulation of EDS1 immune signaling is elucidated by TurboID-based proximity labeling proteomics. A unique NLR, belonging to the NLR-requirement for cell death (NRC) family, is identified as a negative regulator of TNL-mediated immunity. The connection between autophagy and TNL immune network is revealed in this work from the demonstration of interactions between autophagy-related gene 6 (ATG6) and the EDS1 complexes. Overall, this dissertation contributes to a better understanding of EDS1 immune signaling in plants and highlights the interplays amongst different classes of NLRs, and also autophagy as ways to fine-tune TNL-mediated immunity.

AbstractDuring the immune response there is a significant increase in the number of protrusions of the chloroplast stroma known as stromules. These stromules were reported over 100 years ago; however, their function has remained elusive. Recently, it was shown that chloroplast stromules are induced during the plant innate immune response and serve as positive regulators of the immune response. Toward this, we wanted to identify genetic components required for stromule formation. Using a candidate approach, we found a kinesin containing a calponin homology domain, Kinesin required for Inducing Stromules 1 (KIS1) which induces constitutive stromule formation when overexpressed and is required for N-TNL mediated stromule formation and immunity to Tobacco mosaic virus (TMV). Further, we used pathogen effectors as a probe to identify proteins which are required for stromule formation and subsequent perinuclear chloroplast clustering during the N-mediated immune response. Using TurboID-based proximity labeling we identified four proteins which interact with the Type 3 secretion system (T3SS) effector SH9 and are required for immunity to TMV. Toward this, we showed that one SH9 interactor, Sh9 interactor 1 is required for perinuclear chloroplast clustering during the N-mediated immune response. Our results with KIS1 represent the first known component required for stromule formation. Further, our initial characterization of Sh9 interactor 1 suggests that perinuclear chloroplast clustering is required for immunity to TMV and is specifically inhibited by the effector Sh9.