Project Summary Every living organism possesses a defense system crucial for distinguishing self from potentially harmful foreign invaders. This defense system is essential for survival and is finely tuned to protect against threats that may compromise the organism's integrity. The long-term goal of this proposal is to unravel the immune surveillance mechanisms employed by a new class of immune GTPases, the 65~120kDa Guanylate binding protein-like (GBPL) proteins, in the model plant species Arabidopsis thaliana. Preliminary evidence suggests that this new three-member GBPL protein family bridges both sensing and sterilizing activities in plants via mechanisms distinct from well-established plant defense pathways. We are focused in part on how GBPL1, a pseudoGTPase, fine-tunes immune homeostasis as a negative regulator of defense. Here a combination of targeted mutagenesis, structural modeling and organismal genetics will be used to determine the sequence- enzyme relationships governing GBPL1's functions and, in turn, understand its implications in host defense. We are also testing the importance of GBPL2 to act as a broad-spectrum danger sensor under physiological conditions to elicit sterilizing activities. This will be examined using powerful host-pathogen genetics, biochemistry and advanced imaging strategies to unveil the intricate molecular processes that enable GBPL2 to sense external cues and trigger effective immune responses. Lastly, we are exploring the role of GBPL3 in enhancing defense responses and influencing gene expression through the realm of biological phase separation. We are employing facile protein organelle biochemistry, proteomics, imaging and reconstitution techniques to probe how GBPL3 condensates are regulated in vivo in the context of infection. Collectively, this research addresses fundamental questions in plant host defense and offers insights into immune mechanisms applicable to diverse organisms including humans.