Assessing Zymoseptoria tritici candidate effectors in the non-host Nicotiana benthamiana reveals immune recognition and potential host-independent activities

Authors: GOMEZ-GUTIERREZ, SANDRA - PURDUE UNIVERSITY; RODRIGUEZ-DIAZ, CATALINA - PURDUE UNIVERSITY; JAISWAL, NAMRATA; GRIBSKOV, MICHAEL - PURDUE UNIVERSITY; Helm, Matthew; GOODWIN, STEPHEN - Steve

Submitted to: Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2026
Publication Date: N/A
Citation: N/A

Interpretive Summary: Zymoseptoria tritici is a fungus that causes Septoria tritici blotch (STB) disease on wheat, leading to crop yield losses of up to 50% during bad epidemics. This fungus uses small proteins called effectors to cause disease, but exactly how or where they function in the host are not known. To better understand the role of these effectors in causing disease, results from our previous biotechnology experiments in gene expression identified effectors that were expressed during early and middle phases of disease development. Artificial intelligence analyses identified how they might function and helped develop hypotheses. To test these hypotheses, predicted effector genes were expressed in tobacco plants with and without the peptides that signal their location in the cells. The results showed that activity of the effector proteins often differed from the timing of their expression and that presence of the signal affected their activity on host defense responses. Several effector proteins reduced plant defense responses and two appeared to have dual functions at different stages of infection. These results will be useful to plant pathologists trying to control Septoria tritici blotch disease in wheat and demonstrated how combining artificial intelligence and biotechnology can improve our understanding of host-pathogen interactions to make plants more resistant in the future.

Technical Abstract: Zymoseptoria tritici is a significant wheat pathogen responsible for Septoria tritici blotch (STB) disease and for causing up to 50% yield losses globally. Despite its economic impact, only a few effectors have been characterized functionally. 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 expressed transiently in Nicotiana benthamiana, both with and without their predicted signal peptides. AlphaFold structural predictions revealed that two candidates share structural similarity with proteins of known function, offering insights into their potential activities. Our results demonstrate that effector activity did not always correlate with expression timing, and that the presence of a signal peptide significantly influenced their activity on host defense responses. Several effectors consistently attenuate the production of reactive oxygen species (ROS), while none of them suppress PBR1-mediated cell death, indicating they do not target this NLR or its downstream signaling. Notably, 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. Collectively, these findings provide new insights into how Z. tritici effectors modulate plant immunity at distinct stages of disease progression, either to evade host recognition or to establish infection. Our results demonstrate that the functional roles of candidate effectors may extend beyond what is inferred from expression profiles alone.