Identification of Haustorially Expressed Candidate Effector Proteins from <i>Gymnosporangium yamadae</i>

Biotrophic rust fungi secrete effector proteins through haustoria to promote successful colonization. Consequently, the functional analysis of haustorial effector proteins has become central to understanding rust pathogenicity. In previous work, we generated haustorial transcriptome of <i>Gymnosporangium yamadae</i>, the causal agent of apple rust disease, and predicted numerous candidate effector genes. Here, we systematically characterized the functions of 13 haustorially expressed candidate effectors of <i>G. yamadae</i> using heterologous system to assess their modulation of plant immunity and subcellular localization. Confocal microscopy revealed diverse and distinct localization patterns for both full-length and signal peptide-truncated effector constructs fused with GFP-tag at either the N- or C-terminus, including targeting to the nucleus, nuclear speck, chloroplast, and punctate cytosolic structures. Among them, eight effector genes exhibited elevated expression during early colonization, and during the spermogonia and aecia stages of fungal development. Our findings highlight the multifunctional roles of <i>G. yamadae</i> haustorial effectors in modulating plant immunity and potentially other cellular processes. This study addresses the lack of functional analyses of haustorial effectors of forest tree rust pathogens and provides new insights into rust-host interaction mechanisms.