This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. One of the major plant defense mechanisms against pathogens involves the recognition of avirulence (Avr) proteins produced by pathogens. This recognition by the plant resistance (R) proteins then leads to hypersensitive response (HR) which results in programmed plant cell death at the infected site to prevent further spread of the infection.
The aim of this project is to investigate the interaction of the Avr and R proteins at the molecular level. We chose AvrL567-A, AvrL567-B and AvrL567-C from Melampsora lini flax rust and AvrL567-D from rust strain WA as they have shown to be crossed to resistant lines containing Linum usitatissimum flax R proteins (L5, L6 and L7). The sequences of the four Avr proteins are highly variable which contain 27 polymorphic sites in total of 127 amino acids. The Avr proteins were bacterially expressed and purified using affinity chromatography, SDS-PAGE and size-exclusion chromatography. The Avr proteins were characterised by MALDI-TOF and circular dichroism, and their specificities were confirmed by rust infection assay. Conditions that induce crystalisation were screened by vapor diffusion at 4oC and 16.9oC, and conditions that yielded crystals were further optimised. AvrL567-A and AvrL567-D have given rise to diffraction quality crystals and MAD phasing will be used initially to solve the structure. The structures of the AvrL567 proteins will provide the knowledge such as molecular mechanisms conferring plant disease resistance and pathogen virulence, and possibly its other functions in the rust pathogenic protein can be revealed. Therefore, the goal of this project is to perform structure determinations to examine the structural comparisons between AvrL567-A, -B, and / or D to AvrL567-C, as AvrL567-C was not recognised by any of the L5, L6, or L7 R protein, and provide a detailed analysis of its catalytic site and/or structurally conserved active site. Ultimately, by using the AvrL567 isolated from flax rust Melampsora lini, the rust defence process will be fully understood and provide a durable control to rust infection.
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