Understanding the biological functions of Puccinia striiformis f. sp. tritici (Pst) effectors is fundamental for uncovering the mechanisms of pathogenicity and variability, thereby paving the way for developing durable and effective control strategies for stripe rust. However, due to the lack of an efficient genetic transformation system in Pst, progress in effector function studies has been slow. Here, we modeled the structures of 15,201 effectors from twelve Pst races or isolates, a Puccinia striiformis isolate, and one Puccinia striiformis f. sp. hordei isolate using AlphaFold2. Of these, 8,102 folds were successfully predicted, and we performed sequence- and structure-based annotations of these effectors. These effectors were classified into 410 structure clusters and 1,005 sequence clusters. Sequence lengths varied widely, with a concentration between 101-250 amino acids, and motif analysis revealed the presence of known effector motifs such as [Y/F/W]xC and RxLR. Subcellular localization predictions indicated a predominant cytoplasmic localization, with notable chloroplast and nuclear presence. Clear annotations based on sequence and structure included superoxide dismutase and trehalose-6-phosphate phosphatase. A common feature observed was the formation of similar structures from different sequences. In our study, one of the comparative structural analyses revealed a new structure family with a core structure of four helices, including Pst27791, PstGSRE4, and PstSIE1, which target key wheat immune pathway proteins, impacting the host immune function. Further comparative structural analysis showed similarities between Pst effectors and effectors from other pathogens such as AvrSr35, AvrSr50, Zt-KP4-1, and MoHrip2, highlighting convergent evolutionary strategies. This comprehensive analysis provides novel insights into Pst effectors' structural and functional characterization, advancing our understanding of Pst pathogenicity and evolution.
