The avirulence gene AvrLm4-7 of Leptosphaeria maculans: linking crystal structure, functional and evolutionary characteristics.

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English. The avirulence gene AvrLm4-7 of Leptosphaeria maculans, the causal agent of stem canker of oilseed rape, encodes a small cysteine-rich secreted protein. AvrLm4-7 confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4-7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4-7 protein was produced in the heterologous system Pichia pastoris and its crystal structure determined. It revealed the presence of four disulfide bridges and no close structural analogs could be identified. Translocation assays in oilseed rape roots and transient expression in tobacco leaves suggested that AvrLm4-7 is translocated into plant cells in the absence of the pathogen and targeted to the cytoplasm and nucleus. Translocation of AvrLm4-7 into plant cells was found to necessitate the presence of a RAWG motif located in a loop as part of a positively charged region and also the presence of a well-conserved stretch of amino acids (R/N)(Y/F)(R/S)E(F/W) in the C-terminal part of the protein. Using bombardment assays, we determined that recognition by Rlm4 and Rlm7 occurred into the cytoplasm of plant cells. Loss of recognition of AvrLm4-7 by Rlm4 is due to mutation of a single glycine to arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutation patterns, some of which are associated with gene loss while others probably result in drastic modifications of the protein structure. Three point mutations on an a4A7 allele targeting residues either located in the (R/N)(Y/F)(R/S)E(F/W) conserved motif or close to the glycine involved in Rlm4-mediated recognition resulted in a loss of Rlm7-mediated recognition.