ExploitingPseudomonas syringaeType 3 secretion to study effector contribution to disease in spinach

Abstract

Intensive cultivation practices of spinach create favourable conditions for the emergence and rapid evolution of pathogens, causing substantial economic damage. Research on host-pathogen interactions and host immunity in various leafy greens benefits from advanced biotechnological tools. The absence of specialised tools for spinach, however, constrains our understanding of spinach immunity. Here, we explored the potential of Type III Secretion System (T3SS)-mediated delivery to study the activity of pathogen effectors in spinach. We identified thePseudomonas syringaepv. tomatoDC3000 (DC3000) polymutant D36E, which lacks 36 known T3SS effectors (T3Es), as a promising T3SS-dependent effector delivery system in spinach. Unlike DC3000, which causes visual disease symptoms on spinach, D36E did not induce visible disease symptoms. Using D36E effector delivery, we screened 28 known DC3000 T3Es individually on spinach for effects on disease symptom development, bacterial proliferation reflecting bacterial virulence, and ROS bursts as a proxy for early immune responses. All three assays identified T3Es AvrE1 and HopM1 as crucial determinants of DC3000-like infection on spinach. Additionally, we observed that the T3E HopAD1 strongly suppressed ROS production in spinach. We present the first experimental evidence of plant pathogen effector activities in spinach. By establishing the D36E-effector delivery system in spinach, we pave the way for high-throughput effector studies on spinach. This system provides a critical link between genomics-based effector predictions in spinach pathogens and experimental validation, which is a crucial step for knowledge-driven resistance breeding in non-model crops like spinach.