Germination-stage transcriptomics of the grapevine rust fungus Neophysopella tropicalis reveals protein effectors with plant immunity-suppressing activity

Germination of urediniospores represents a critical early stage in the life cycle of Neophysopella tropicalis , the causal agent of Asian grapevine leaf rust in Brazil. Rust fungi deploy diverse effector proteins to manipulate host immunity and establish infection. This study identified and functionally characterized secreted proteins expressed during early fungal development using RNA-seq analysis of in-vitro-germinated urediniospores. Effectorome analysis revealed 386 putative secreted proteins with hallmarks of fungal effectors, designated as N. tropicalis effector candidates (NtECs). Fifteen highly expressed NtECs were selected for expression profiling by RT-qPCR across infection stages. Fluorescence microscopy with WGA-FITC staining defined key phases of pathogenesis. Although identified during germination, seven NtECs showed elevated transcript accumulation during the penetration and early biotrophic phases, suggesting roles in host entry and establishment. To evaluate immune-suppressing activity, nine NtECs were delivered into Nicotiana benthamiana using the Type III Secretion System of Pseudomonas fluorescens EtHAn. Three effectors (NtEC-05, -09, and -10) strongly suppressed AvrB-triggered cell death, and three others (NtEC-11, -12, and -13) showed moderate suppression. This work provides the first characterization of N. tropicalis effectors, offering insights into rust pathogenesis and supporting future effector-informed strategies for grapevine resistance.