Quantitative high-throughput yeast two-hybrid screen identifies proteins that contribute to immunity against Pseudomonas syringae
J. LEWIS (1), J. Hassan (2), R. Rubalcava (2), D. Ouyang (2), K. Schreiber (2) (1) USDA, UC Berkeley, U.S.A.; (2) UC Berkeley, U.S.A.

NOD-like receptors (NLRs) are critical components of effector-triggered immunity; however the pathway leading from effector recognition to effective immunity is not well-understood. The NLR ZAR1 (HopZ1-Activated Resistance 1) indirectly recognizes the Pseudomonas syringae type III effector protein HopZ1a, when HopZ1a acetylates the ZED1 pseudokinase. ZAR1 does not require any of the known immune signaling proteins for resistance, including EDS1, NDR1, salicylic acid, etc. We previously developed a high-throughput yeast two-hybrid screen that employs next-generation sequencing to quantitatively identify putative interacting proteins. To identify additional host proteins that contribute to the recognition of HopZ1a, we screened for proteins that interact with ZAR1. We identified 30 proteins that were significantly overrepresented in the ZAR1 interaction pool, compared to a luciferase control. We identified homozygous putative knockout lines for 26 of the 30 genes, and screened these lines for defects in the hypersensitive response (HR) and bacterial growth. One line displayed a delayed weaker HR, and 10 lines displayed significant differences in bacterial growth compared to Col-0. Further dissection of these candidate interacting proteins will help elucidate the molecular mechanisms of plant immunity, and the novel signaling pathway leading to HopZ1a recognition.

Abstract Number: P17-556
Session Type: Poster