My BSPP-funded summer project was undertaken in Sophien Kamoun’s group, at ‘The Sainsbury Laboratory’, which focuses upon interactions between plants and filamentous pathogens, particularly the oomycete Phytophthora infestans. Previous work in the lab revealed that Solanaceous plants possess a network of interconnected intracellular nucleotide-binding leucine-rich (NLR) receptors that function as key mediators of plant immunity. Pathogen secreted virulence proteins, termed effectors, are recognised by ‘Sensor NLR’ proteins, which rely on downstream ‘Helper NLRs’ called NRCs (NLRs required for Cell death) for immune signalling. This is usually accompanied by a form of localized cell-death known as the hypersensitive response. However, the pathway which connects effector recognition by the NRC network and the hypersensitive response is almost completely unmapped.
As this network involves an array of sensor NLRs dependent on a reduced number of downstream helpers, these NRCs represent key nodes in plant immunity, and one would therefore expect pathogens to deploy effectors targeting them. Previously in the Kamoun lab, Lida Derevnina performed a screen to identify these hypothesised effectors. This screen identified AVRcap1b, produced by Phytophthora infestans, which acts downstream of the NRC’s to abrogate NLR-triggered cell death. My project was based on the idea that by identifying AVRcap1b’s target, we could identify a novel protein required for the hypersensitive response pathway. Prior to my arrival, IP-MS and yeast two-hybrid assays were used to identify potential AVRcap1b host targets. One potential target identified was a member of the ESCRT complex involved in multivesicular body formation which we named target of AVRcap1b (Toa). Toa is present in all examined multicellular organism genomes, but interestingly has more homologues in plant species suggesting a functional diversification (such as this proposed immunity role). For example, the key solanaceous model species Nicotiana benthamiana has five identified homologues, which we named T1-T5.
The hypothesis that we investigated during my summer project was that Toa is genetically required for the NRC-mediated hypersensitive response. Therefore, my project’s main focus was to employ virus-induced gene silencing (VIGS) to silence Toa and test whether this compromised the hypersensitive response mediated by the NRCs. Starting with a synthetic silencing fragment targeting all five N. benthamiana Toa homologues, I designed primers for Phusion PCR to generate individual silencing fragments for each homologue along with combinations such as T1+2+5 and T3+4. These fragments were then inserted into the TRV2 VIGS vector via Golden Gate assembly and transformed into E. coli. After culturing up this E. coli, the silencing fragment-containing plasmid was isolated using MiniPrep kits and agrotransformation was performed using electroporation. The resulting agrobacterium stocks for each of the silencing combinations can be used in the future by other members of the lab. We then infiltrated N. benthamiana plants with suspensions of these agrobacterium transformants to deliver the silencing constructs into the plant. We intended to perform hypersensitive response assays, however as we did not have enough time to achieve this it will be continued by my supervisor in the future.
A second strand to my project was to test the association between AvrCap1b and Toa in planta using co-immunoprecipitation. We co-infiltrated HA-tagged Toa homologues and GFP-tagged AvrCap1b into N. benthamiana plants. After harvesting leaf tissue and grinding, we performed both GFP and HA pulldowns. In the GFP pulldown we found AvrCap1b to associate with T1, T2 and T5 but not with T3 or T4. This suggests a differential association between AvrCap1b and the different Toa homologues, and as we have performed 3 replicates, we can be fairly confident of the results. In the HA pulldown, we only found an association between T1 and AvrCap1b. However, fewer replicates were performed so it may be an issue with the level of infiltrated GFP-tagged AVRcap1b expression. In order to check whether this association between AvrCap1b and T1 is specific, or random due to T1 associating non-specifically, we also co-infiltrated plants with HA-tagged T1 and GFP-tagged RD54 as a negative control. No association was found, therefore T1 is specifically associating with AvrCap1b.
I have thoroughly enjoyed my summer undertaking this work, and it has given me the confidence that a PhD, specifically in plant-pathogen interactions, is the route for me. I would like to thank my supervisor Mauricio Contreras for invaluable help and advice over the summer, along with Professor Sophien Kamoun for hosting me. I would also like to thank the entire Kamounity for making me feel so welcome, and the BSPP for providing the funds that made this all possible.
University of York