ABSTRACTRust fungi are plant pathogens that secrete an arsenal of effector proteins interfering with plant functions and promoting parasitic infection. Effectors are often species-specific, evolve rapidly, and display low sequence similarities with known proteins or domains. How rust fungal effectors function in host cells remains elusive, and biochemical and structural approaches have been scarcely used to tackle this question. In this study, we used a strategy based on recombinant protein production in Escherichia coli to study eleven candidate effectors of the leaf rust fungus Melampsora larici-populina. We successfully purified and solved the three-dimensional structure of two proteins, MLP124266 and MLP124017, using NMR spectroscopy. Although both proteins show no sequence similarity with known proteins, they exhibit structural similarities to knottin and nuclear transport factor 2-like proteins, respectively. Altogether, our findings show that sequence-unrelated effectors can adopt folds similar to known proteins, and encourage the use of biochemical and structural approaches to functionally characterize rust effector candidates.