Summarized findings Two candidate effectors, PM02_g378 and PM02_g2610, localize to the cell periphery while the remaining putative effectors localize to both the nucleus and cell periphery PM02_g1115, PM02_g7882 and PM02_g8240 consistently suppress chitin-dependent ROS production when transiently expressed in N. benthamiana PM02_g1115 suppresses Pseudomonas syringae DC3000-mediated cell death in maize \ Acknowledgments and Funding We thank Dr. Tesfaye Mengiste (Purdue University) for access to the microplate reader for ROS suppression assays, Dr. Rachel Hiles and Dr. Terri Cameron for their technical assistance, and the Purdue University Imaging Facility for access to the Zeiss LSM880 Axio Examiner upright confocal microscope. This research was funded by the USDA-ARS research project 5020-21220-014-00D. USDA is an equal opportunity provider and employer. Candidate effector proteins from from the maize tar spot pathogen Phyllachora maydis suppress immune responses Abigail Rogers1†,Namrata Jaiswal2 †, Emily Roggenkamp3, Hye-Seon Kim4, Joshua S. MacCready3, Martin Chilvers3, Steven R. Scofield2, Anjali Iyer-Pascuzzi1, and Matthew Helm2 * † these authors contributed equally to this work 1 Department of Botany and Plant Pathology, Purdue University West Lafayette, IN 47907, U.S.A. 2 Crop Production and Pest Control Research Unit, U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS), West Lafayette, IN 47907, U.S.A., 3 Department of Plant, Soil, and Microbial Sciences, Michigan State University, East Lansing, MI, 48824 4 Mycotoxin Prevention and Applied Microbiology Research Unit, USDA-ARS, Peoria, IL, U.S.A. * Corresponding author: M. Helm; Email: Matthew.Helm@usda.gov Significance @LabAbbie27 To better understand the molecular mechanisms underlying the P. maydis – maize interaction, we investigated how P. maydis utilizes effectors to induce disease. Leveraging the most recent genome annotation, we identified eighteen candidate virulence (effector) proteins that are expressed during disease development and assessed whether these putative effectors suppress immune responses using heterologous expression systems, including Nicotiana benthamiana and the bacterial type III secretion system from Pseudomonas syringae DC3000. Three P. maydis effector candidates suppress chitin-mediated ROS production in N. benthamiana Figure 5. Protein structures were predicted with ColabFold v1.5.2: AlphaFold2 and visualized using the ChimeraX-1.5 modeler package. Here we present the predicted structures of three PmECs which have cell surface-triggered immune suppression activities. The coloring of the models depicts the predicted local distance different test (pLDDT) which indicates the per-residue confidence score. Higher confidence scores are depicted in cool colors while low confidence scores are depicted in warmer colors. Figure 2. Transient expression assays were performed at 48 hours post infiltration. The sub-cellular localizations of PmEC-sYFP fusions were determined by laser scanning confocal microscopy (A). Expression of full-length PmEC-sYFP fusions in N. benthamiana leaves was confirmed by western blot analysis (B). Figure 3. Transient expression of candidate effector- fluorescent protein fusions suppress reactive oxygen species (ROS) burst triggered by chitin in N. benthamiana. The indicated constructs were expressed in N. benthamiana. Forty-eight hours post-agroinfiltration, N. benthamiana leaf disks were collected, challenged with chitin, and relative luminescence was monitored for 40 minutes using a luminol-based assay. Free sYFP (3xHA:sYFP) was used as a reference control P. maydis effector candidates localize primarily to the nucleus and cell periphery Predicted protein structures of the candidate effectors as determined by AlphaFold2 An introduction to Phyllachora maydis The fungal phytopathogen Phyllachora maydis is an obligate pathogen of maize and is the causal agent of tar spot disease. It was first identifie