Translocation of Effector Proteins into Plant Cells by the Flax Rust Pathogen Melampsora lini

During infection, rust fungi secrete effector proteins into host plant cells from haustoria to aid in their colonization. How rust effectors are secreted from the haustorium and delivered into the cytoplasm of host cells remains poorly understood. We used an Agrobacterium-mediated transformation procedure to generate stable transgenic flax rust strains expressing the effectors AvrM and AvrP123 fused to yellow fluorescent protein (YFP). We showed that both AvrM-YFP and AvrP123-YFP fusion proteins were secreted by the fungus into a narrow space surrounding the haustorium, likely the extrahaustorial matrix (EHMx); however, only AvrM-YFP was delivered into host cells, triggering a typical resistance phenotype in plants carrying the corresponding resistance (R) gene M. The signal peptide of AvrM was sufficient to direct YFP secretion into the EHMx; however, delivery into the host cell required a larger 105-amino-acid N-terminal fragment of AvrM. These results indicate that translocation of this protein into the host cell from the EHMx is a separate process from secretion into the EHMx and requires a signal present in AvrM between amino acids 34 and 105. This is in contrast to previous observations of AvrM localization after transient expression in plants, highlighting the necessity for analysis in the natural infection system. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.