Functional Characterization of Zymoseptoria tritici Candidate Effectors Reveals Their Role in Modulating Immunity in Nicotiana benthamiana

Zymoseptoria tritici is a significant wheat pathogen responsible for Septoria tritici blotch (STB) disease and can cause up to 50% yield losses globally. Despite its economic impact, understanding of the molecular interactions between Z. tritici and its host remains limited, particularly the functions of many uncharacterized candidate effectors. To explore the roles of candidate effectors in modulating host immune responses, we selected seven Z. tritici genes with elevated expression during the early biotrophic phase and the transition to necrotrophy in a susceptible interaction. These candidates were transiently expressed in Nicotiana benthamiana, both with and without their predicted signal peptides. AlphaFold structural predictions revealed that two candidates share similarity with proteins of known function: a sterol-binding protein from Saccharomyces cerevisiae and a necrosis-inducing effector from Valsa mali. Effector activity did not always correlate with expression timing, and the presence of a signal peptide significantly influenced the activity of candidate effectors on host defense responses. Several effectors consistently attenuate the production of reactive oxygen species (ROS), while none suppress PBR1-mediated cell death, indicating they do not target this NLR or its downstream signaling. Two candidate effectors, Mycgr3107904 and Mycgr394290, induce cell death in N. benthamiana while also modulating the ROS burst, suggesting potential dual functions at different stages of infection. These findings provide new insights into how Z. tritici effectors modulate plant immunity during disease progression, either to evade host recognition or establish infection. Our results show that effector functions may extend beyond what is inferred from expression profiles alone.