Functional Characterisation of the Flax Rust AvrP/AvrP123 Avirulence Proteins

Abstract

Flax rust effector proteins AvrP and AvrP123 are recognised by the flax resistance proteins, P and P1, P2, P3, respectively. Variants of these effectors with different host-recognition specificities are found in the flax rust strains 271, 339, bs25 and WA. In this study, investigation was conducted on regions in AvrP and AvrP123 that are recognised by their cognate resistance proteins. Agrobacterium-mediated expression of AvrP and AvrP123 variants in tobacco (Nicotiana tabacum) and flax (Linum usitatissimum) was used to observe necrosis induction as an indicator of recognition. For AvrP, deletions were generated based on the structure of the protein, whereas domain swaps and mutations were generated based on amino acid polymorphisms present in the AvrP variant from strain 271, which is not recognised by P, P1, P2 or P3. Amino acid polymorphisms involved in AvrP recognition were found to be located near the C-terminus of the protein. From the structure of AvrP, it was known that AvrP and AvrP123 each bind three zinc atoms. Mutational disruption of zinc binding in AvrP resulted in a loss of protein stability and a loss of recognition by P. For AvrP123, deletions and domain swaps were generated based on amino acid polymorphisms present in AvrP123 variants from strains 339, bs25 and WA. Recognition or AvrP123 by P1 was found to be determined by the mature N-terminal region of the protein just after the signal peptide, whereas P2 and P3 were found to recognise the C-terminal region of AvrP123. In an attempt to find interactor proteins targeted by AvrP in host cells, a yeast-two hybrid assay was conducted using cDNA library derived from rust-infected flax leaves. Four candidates were identified, one candidate from the pathogen, designated SIAP (Secreted Interactor of AvrP) and three candidates from the host, phosphoglucomutase, peptidyl-prolyl cis-trans isomerase FKBP12, and DEAD-box ATP-dependent RNA helicase 56. Each of these candidates is a plausible target for AvrP effector function in the plant cells, but further analysis is required to confirm these interactions and to determine the effect of AvrP on interactor function.