Grapevine rust fungus Neophysopella tropicalis deploys protein effectors with plant immunity-suppressing activity during sequential stages of pathogenesis.

Germination of urediniospores marks the onset of infection by Neophysopella tropicalis, the causal agent of Asian grapevine leaf rust, yet the molecular mechanisms enabling this obligate biotrophic fungus to establish infection remain largely unknown. We combined transcriptomics, histological analyses, and functional assays to identify and characterize candidate effector proteins deployed during the earliest stages of pathogenesis. RNA sequencing of in vitro-germinated urediniospores identified 303 genes encoding predicted secreted proteins bearing hallmarks of fungal effectors, grouped into 142 sequence families and revealing a remarkably diverse repertoire of effector candidates. Fifteen highly expressed N. tropicalis effector candidate (NtEC) genes, representing thirteen distinct sequence families, were selected for expression profiling by reverse transcription quantitative PCR across infection stages. Chitin-targeted fluorescence microscopy provided a developmental framework for interpreting expression across germination, penetration, and early colonization. Although identified during germination, seven NtECs accumulated transcripts predominantly during host penetration and early biotrophic colonization, suggesting specialized roles in infection establishment. Using the Type III Secretion System of Pseudomonas fluorescens EtHAn, we demonstrated that three NtECs (NtEC-05, NtEC-09, and NtEC-10) strongly suppressed AvrB-triggered hypersensitive cell death in Nicotiana benthamiana, while three others (NtEC-11, NtEC-12, and NtEC-13) exhibited moderate immune-suppressing activity. Together, these findings provide the first comprehensive characterization of the N. tropicalis effector repertoire, demonstrating that early infection involves coordinated, sequential deployment of evolutionarily diverse effectors associated with plant immune suppression. This work establishes an integrated framework for discovery and functional prioritization of rust fungus effectors and provides a valuable molecular resource for future studies on grapevine-rust interactions.