Plant-pathogen interactions are primarily constituted of the cyclic release of pathogenic effectors, intended to suppress plant immunity, followed by plant recognition of these effectors, resulting in enhanced plant immunity. The Banfield lab at the John Innes Centre, Norwich, investigates the structure and function of plant receptors and pathogenic effectors with the intention of elucidating novel approaches for the improvement of crop disease tolerance. The Magnaporthe oryzae fungus causes sufficient crop loss of wheat and rice to feed well over 60 million people annually. PWT3 and PWT4 are two effectors of M. oryzae recognised by immune receptors in wheat; PWL2 is an effector that acts as a host range determinant. My work was focused on the expression, purification and characterisation of PWT3, PWT4 and PWL2.
The expression of each effector required amplification and cloning of the effector genes into appropriate vectors (various alternatives were investigated to attain the optimal level of expression), with which to transform E. coli. The transformed E. coli were grown in ample quantity so when induced to express the protein of interest, gave sufficient protein for purification. Despite the satisfactory expression of each effector, each presented significant challenges during purification: purification of PWT4 showed a significant level of degradation, resulting in contamination; PWT3 appeared to be prone to aggregation/conformational change; and PWL2 showed a high affinity to the purification columns, leading to a high level of contamination or loss of protein. From a student perspective, these challenges provided an ideal opportunity to improve one’s approach to troubleshooting, deepening knowledge and understanding of the research area and applicable techniques. In many cases, alteration or repetition of relevant protocols permitted the challenges to be overcome. Such amendments resulted in adequately pure protein for the characterisation of PWL2, PWT3 and PWT4 by differential scanning fluorimetry and circular dichroism. The data indicated the proteins’ folded state and provided information regarding their stability. Sufficient PWT3 was also purified to begin crystallisation trials.
The knowledge and understanding gathered during this short project has formed the foundation on which to build a greater comprehension of the effectors’ roles in the M. oryzae infection of staple crops. The results and conclusions of the project will be built on by a member of the Banfield lab.
The personal experienced gained by undertaking a vacation project greatly assists in providing the student with a unique multitude of skills and erudition of a niche scientific area, but more importantly an invaluable insight into the world of research. This allows the student to determine their suitability to a research career: was the student overwhelmed or were they sparked into pledging themselves to scientific discovery? The latter was undoubtedly the case here.
John Innes Centre