Structure-guided secretome analysis of gall-forming microbes offers insights into effector diversity and evolution.

Phytopathogens secrete effector molecules to manipulate host immunity and metabolism. Recent advances in structural genomics have identified fungal effector families whose members adopt similar folds despite sequence divergence, highlighting their importance in virulence and immune evasion. To extend the scope of comparative structure-guided analysis to more evolutionarily distant phytopathogens with similar lifestyles, we used AlphaFold2 to predict the 3D structures of the secretome from selected plasmodiophorid, oomycete, and fungal gall-forming pathogens. Clustering protein folds based on structural homology revealed species-specific expansions and a low abundance of known orphan effector families. We identified novel sequence-unrelated but structurally similar (SUSS) effector clusters, rich in conserved motifs such as 'CCG' and 'RAYH'. We demonstrate that these motifs likely play a central role in maintaining the overall fold. We also identified a SUSS cluster adopting a nucleoside hydrolase-like fold conserved among various gall-forming microbes. Notably, ankyrin proteins (ANK) were significantly expanded in gall-forming plasmodiophorids, with most being highly expressed during clubroot disease, suggesting a role in pathogenicity. Subsequently, we screened ANK proteins against