This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
It is estimated that 20% of agricultural yield is lost to pathogen interference. With the constant demand for food due to an ever-growing population, it is crucial to address this issue and reduce these losses. In plants, Resistance proteins recognise pathogen effectors and trigger effector-triggered immunity (ETI). Previous research has discovered that the proteins TFT3, NRC3, and MAPKKKa are part of the tomato Prf/Pto Resistance complex. Our aim is to confirm these interactions and observe how protein-protein interactions within the Prf/Pto complex change following recognition of the effector protein AvrPtoB. We have shown that the dissociation of the TFT3 and NRC3 proteins from the Prf/Pto complex occurs at different times, with the TFT3 pathway being activated first. As we deepen our understanding of how ETI is activated, we come closer to reducing the agricultural loss caused by pathogens.
Plants detect pathogen-associated molecules (PAMPs) using pattern recognition receptors (PRRs), leading to MAMP-triggered immunity (MTI). However, pathogens can counteract MTI with effector protein, resulting in susceptibility. To counter this, plant resistance complexes recognise the effector proteins and activate effector-triggered immunity (ETI). The tomato Prf/Pto Resistance complex recognises the effector AvrPtoB from the bacterium Pseudomonas syringae. The Prf/Pto complex is well-studied, making it a suitable model for studying and understanding the protein-protein interactions between R proteins and their effectors.
To characterise protein-protein interactions within the Prf/Pto complex we utilised split-luciferase complementation imaging. Constructs were cloned into the pDEST-NLUCGW and pDEST-CLUCGW binary destination vectors and then transformed into Agrobacterium tumefaciens. Bacteria was cultured in LB broth for 24 hours with antibiotic selections. The cultures were resuspended in infiltration buffer at OD600 0.8. Mixed constructs were prepared in equal ratios and infiltrated in Nicotiana benthamiana leaves. Leaves were collected 2 days later, and luciferase signal was detected in a Photek HRPCS single photon counting system. Image32 software was used to analyse the luminescence images.
The first set of experiments that were carried out investigated the interactions within the Prf/Pto resistance complex. We examined four key interactions: Prf-Prf; Prf-NRC3; Prf-Pto and Pto-TFT3. Three control spots were infiltrated, using mCherry as an empty vector. Then, we infiltrated one spot with both proteins of interest. For all four interactions, there was only signal in the spot with both proteins.
The second set of experiments investigated the effect that the effector protein AvrPtoB had on the complex. We studied what happened to the Prf-NRC3 and Pto-TFT3 interaction following AvrPtoB recognition. On the leaf, two spots contained the proteins of interest, one had an empty vector and the other AvrPtoB. The spot with the empty vector had a stronger signal compared to the signal seen on the spot with AvrPtoB.
The final set of experiments investigated the timing of the Prf-NRC3 and Pto-TFT3 dissociation following AvrPtoB recognition. Proteins of interest were infiltrated. 24 hours later AvrptoB was infiltrated, overlapping both spots. The percentage decrease in signal was then measured 2 days after AvrPtoB was infiltrated. The Pto-TFT3 signal had a greater decrease compared to the Prf-NRC3 interaction.
In the first set of experiments, signal was only seen where both proteins of interest were present. This suggests that the proteins have interacted, forming a complete luciferase enzyme. The second set of experiments showed a weaker signal when AvrPtoB is present for both the Prf-NRC3 and Pto-TFT3 interactions. This suggests that the proteins are dissociating due to AvrPtoB presence, resulting in the observed weaker signal.
The final set of experiment showed a larger percentage decrease in signal in the Pto-TFT3 reaction 2 days after the AvrPtoB infiltration. This indicates that TFT3 dissociates prior to NRC3 from the Prf/Pto complex.
This project has been a great experience that has improved my confidence in a lab environment and has allowed me to learn techniques unavailable on my undergraduate course. I would like to thank the University of Warwick for allowing me to use their facilities and Professor Vardis Ntoukakis for supervising me during the project. I would also like to thank the BSPP for granting me this opportunity.
Kyle Willis
University of Warwick
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
