This is the report from a BSPP MSc/MRes Bursary.
Click here to read more/apply for one yourself.
Having kindly been provided with an MRes bursary by the BSPP, I undertook a one-year Master of Research in Molecular Plant and Microbial Sciences at Imperial College London. My MRes consisted of two rotation projects, both in the lab of Dr Ernesto Cota, each developing a different skill set within the molecular biology umbrella.
One project focussed on determining the ability of a fungal phytopathogen effector protein of interest to act on the ribosome of the Hordeum vulgare host cell. Hordeum vulgare constitutively produces a ribosome inactivating protein, which is normally rendered inactive by the presence of an inhibitor peptide loop attached to the N-terminus of the protein. On infection by a phytopathogen, defence hormone signalling triggers cleavage of this inhibitor peptide, causing activation of the ribosome inactivating protein, which then binds to a specific loop on the host cell ribosome to halt translation, ceasing cellular metabolism, and therefore initiating necrosis of the infected host plant cell, preventing spread of disease throughout the host plant. On infection by some species of obligate biotrophic fungal phytopathogens, this hypersensitive cell death response to phytopathogen attack appears to be inhibited, allowing the biotrophic pathogen to continue to live and proliferate, spreading throughout the host plant. By using a series of coupled transcription translation assays, which utilise renilla luciferase as a proxy for translation efficiency, I was able to identify effector proteins with the capacity to inhibit translation. Following these assays, a pipeline was developed to enable efficient purification of high purity samples of the main effector protein of interest, which included Ni-NTA, reverse Ni-NTA and size exclusion chromatography steps. Synthetic secondary RNA structures were synthesised in vitro, which corresponded to proposed binding sites of the effector protein of interest on the host cell ribosome. These synthetic RNAs were then utilised, alongside the effector protein of interest to undertake electrophoretic mobility shift assays (EMSA) to determine the ability of the effector protein of interest to bind specific host cell ribosome RNA sequences.
The second project focussed on increasing the solubility and stability of a notoriously difficult to purify host immune protein of interest, with the aim of developing a form of the protein amenable to purification by fast protein liquid chromatography methods such as Ni-NTA chromatography. To achieve this goal, I undertook several forms of bioinformatic analysis, including generating a JPRED consensus sequence for the protein of interest against homologues from closely related crop species, to identify potential sites where single amino acid substitutions could be implemented to increase stability and solubility of the protein without affecting surface properties. Phyre2 models were generated to predict the tertiary structure of the protein of interest, and these models were uploaded into Pymol, where cavities in the protein of interest’s hydrophobic core were identified as sited for addition of methyl groups, which should increase protein stability and solubility without changing surface properties or tertiary structure. Following structural mutagenesis, the resultant amino acid sequence was used to design a synthetic DNA sequence, which was cloned into a vector and transformed into an expression strain of E. coli, where an attempt was made to purify it in vitro. Results showed the protein of interest was present, yet remained in the insoluble fraction, therefore new and innovative approaches will be developed further to enable expression of a soluble form of this protein.
Many thanks to the BSPP for kindly supporting my MRes.
Henrietta Patterson
Imperial College London
This is the report from a BSPP MSc/MRes Bursary.
Click here to read more/apply for one yourself.
