Exploiting Pseudomonas syringae Type 3 Secretion to Study Effector Contribution to Disease in Spinach.

Intensive spinach cultivation creates favorable conditions for the emergence and rapid evolution of pathogens, leading to substantial economic losses. Research on host–pathogen interactions in leafy greens would benefit from advanced biotechnological tools; however, the absence of such tools in spinach hampers our understanding of spinach immunity. Here, we explored the potential of type III secretion system (T3SS)mediated effector delivery to study pathogen effector activity in spinach. We identified the Pseudomonas syringae pv. tomato DC3000 (DC3000) polymutant D36E, which lacks 36 known T3SS effectors (T3Es), as a promising T3SS-dependent effector delivery system for spinach. Unlike DC3000, which causes necrotic symptoms on spinach and reaches high bacterial titers, D36E did not proliferate and caused no visible symptoms. Using D36E, we screened 28 DC3000 T3Es in spinach, assessing symptom development, bacterial proliferation, and reactive oxygen species (ROS) bursts as a proxy for early immune responses. AvrE1 and HopM1 emerged as key determinants of DC3000-like infection, inducing water-soaked lesions, whereas HopAD1 strongly suppressed ROS production. Our findings establish the D36E-based effector delivery system as a powerful tool for high-throughput effector studies in spinach. It bridges the gap between genomics-based effector predictions and experimental validation, paving the way for knowledge-driven resistance breeding in non-model crops such as spinach.