This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
Plant diseases are a major threat to global agriculture, with up to 40% of crops being lost to plant diseases and pests annually. Meanwhile, the growing human population means 50% more food will be needed by 2050. The creation of disease-resistant crop varieties to increase crop yields has thus become a priority. The initial ‘battleground’ between plants and pathogens is the extracellular space known as the apoplast. Plants have evolved to secrete hydrolytic enzymes to resist attacks by pathogens. To survive, pathogens in turn produce proteins that form inhibitory complexes with these defensive enzymes, neutralising them. The Plant Chemetics lab utilises the new AI-based protein-structure prediction technology AlphaFold-Multimer to understand these apoplastic interactions and ultimately produce more resilient crops. My project aimed to validate and characterise AI-predicted interactions between the model plant Nicotiana benthamiana and the model pathogen Pseudomonas syringae.
Pathogens are not the only producers of hydrolase inhibitors; plants have been shown to secrete inhibitors of their own, although their purpose remains largely elusive. These proteins have been hypothesised to inhibit the plant’s immune response to propagate immune signalling or inhibit pathogen-derived proteases. An Alphafold-Multimer screen identified a number of N. benthamiana-derived proteins that form predicted complexes with host cysteine and serine hydrolases which were previously shown to be suppressed upon infection with Pseudomonas syringae. For example, the protein NbE44070139 (annotated as non-specific lipid-transfer protein 1-like) was highly scored to form a complex with Cathepsin B, a cysteine protease. Alphafold-Multimer also predicted that several P. syringae-derived proteins form complexes with the suppressed hydrolases.
Predicted interactions between endogenous inhibitors and hydrolases were investigated in planta, where N. benthamiana was transformed to co-express a candidate inhibitor and the hydrolase it was predicted to inhibit. Transformation was achieved using Agrobacterium tumefaciens. Agrobacterium transfers DNA (T-DNA) into the host genome. This DNA naturally encodes genes for the production of opines; the host then expresses these opines which the bacterium uses for energy. Here, the T-DNA was modified to encode the inhibitors and hydrolases under investigation. Agrobacterium was infiltrated into the leaves of N. benthamiana, after which the plant secretes the enzymes and inhibitors into the apoplast. After 4-6 days, the apoplastic fluid was extracted using the infiltration-centrifugation method.
Predicted P. syringae-derived inhibitors were produced in Escherichia coli using constructs containing an IPTG-inducible promoter. A His tag was included on the protein, allowing the protein to be purified though a nickel affinity column. Apoplastic fluid containing the overexpressed hydrolase was obtained as above.
Most validations were performed using a technique called Activity Based Protein Profiling (ABPP) (see figure). ABPP can be used to selectively label the active site a particular class of hydrolase. This allows us to analyse the effect of an inhibitor candidate on hydrolase activity in the apoplast. For example, ABPP was used to investigate interactions between NbE44070139 and Cathepsin B. The apoplastic fluid containing these proteins was incubated with a fluorescent probe. The proteins were then separated out using SDS-PAGE, and the gel was scanned for fluorescent signals using a Typhoon scanner.
ABPP can only be conducted when there is a bespoke probe available for the suppressed hydrolase being investigated. If no probe is available, an in vitro pull-down experiment is an alternative. Such was the case for pathogenesis-related protein 3 (PR3), which was predicted to form a complex with the candidate P. syringae-derived inhibitors CIP1 and PSPTO4974. The pull-down experiment made use of the His tag present on CIP1 and PSPTO4974 (termed the ‘bait’), which can bind to His beads. The bait-bound beads were then incubated with the apoplastic fluid containing the PR3 (termed the ‘prey’). Anti-His western blots were used to confirm that the bait proteins successfully bound to the beads, and then an anti-PR3 western blot was used to see whether PR3 bound to the inhibitor candidates.
Contributing to research at the University of Oxford has been a brilliant opportunity to both develop my practical skills and learn about cutting-edge plant pathology research going on at the Department of Biology. I will certainly look to do research in plant pathology in the future. Finally, I would like to thank the Plant Chemetics lab for hosting me, and especially Dr Brian Mooney, who has been a fantastic supervisor and has taught me invaluable skills over the past six weeks.
Fletcher Tomkins
University of York
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
