Periodic Reporting for period 2 - ExtraImmune (Unlocking extracellular immunity for new crop protection strategies.)
Période du rapport: 2023-07-01 au 2024-12-31
The extracellular space within plant tissues (the apoplast) is defended by plants against microbial colonization by depositing toxic and harmful molecules into the apoplast during defense responses. Plant pathogens that have adapted to this environment cause disease that can have serious implications on our food security. This project aims at elucidating how plant pathogens manipulate plant-secreted proteins using state-of-the art chemical proteomic technologies. Using activity-based protein profiling (ABPP) we will identify manipulated hydrolytic enzymes such as proteases, lipases and glycosidases. Using reactivity-based protein profiling (RBPP) we will monitor the differential availability of reactive lysine residues on the surface of secreted plant proteins, beyond the active site of hydrolases. And with crosslinking mass spectrometry (CLMS) we will monitor the formation of protein complexes and conformational changes in secreted plant proteins. These three chemical proteomic experiments will be performed to study the impact of infection of the model plant Nicotiana benthamiana upon infection with three diverse strains of the bacterial model pathogen Pseudomonas syringae. Suspect manipulations will be studied further to determine the molecular mechanism underpinning the manipulation (how), and the effect this has on the interaction (why). Ultimately, the identification of manipulated plant proteins will enable us to engineer these plant proteins such that they cannot be manipulated by plant pathogens and this will deliver a novel way of making crops ‘ExtraImmune’ against plant pathogens.
To provide proof-of-concept, we have established RBPP in plant science and used this to demonstrate that this can display differential labeling in the presence/absence of ATP (Mooney et al., manuscript in prep). We have also established CLMS in plant science and identified hundreds of crosslinked peptides and are using AlphaFold Multimer to confirm the detected close proximities in silico (Mahadevan et al., manuscript in prep). We have established robust infection assays for the three bacterial strains on Nicotiana benthamiana and have isolated large quantities of apoplastic fluids from the three infections and the mock control at the start of infection (t=0) in n=4 replicates and two days after infection (2dpi) in n=6 replicates. We performed chemical proteomics using ABPP, RBPP and CLMS on the t=0 samples and analysed the results. The chemical proteomics experiments on the t=0 samples did not yield the large number of labeling that we anticipated. ABPP displayed only 12 active hydrolases but only one protease activity was suppressed in the presence of the pathogen. Likewise, RBPP displayed only 1718 unique labeling sites in the plant proteome. Interestingly, one site in a glycosidase is consistently more reactive in the presence of the pathogen, indicating that this glycosidase is manipulated at t=0. Furthermore, CLMS revealed 32 interlinks between two different protein, including six plant-pathogen interactions. These experiments showed that, although the between-sample variation is low for the t=0 samples, the protein concentration is relatively low and we did not monitor the hundreds of proteins that we aimed at. Protein concentrations are much higher in the 2dpi samples and we are currently labeling these samples for proteomic analysis with sufficient replicates (n=6). Meanwhile, we have used AlphaFold Multimer (AFM) to predict interactions between 48 hydrolases that are suppressed upon infection with one of the three strains (pv tomato, PtoDC3000, Sueldo et al., 2024) with 211 PtoDC3000-produced proteins that were detected in the apoplast of infected plants. This screen identified >500 robust protein complexes, of which 30 complexes seem to involve a PtoDC3000 protein that might inhibit the plant hydrolase. We have produced some of these candidate inhibitors from PtoDC3000 in E. coli and expressed their presumed target hydrolases by agroinfiltration and tested if the PtoDC3000 proteins are indeed hydrolase inhibitors. Preliminary data indicate that at least some of these PtoDC3000 proteins are indeed novel inhibitors, for instance of glycosidases BGAL2 and PR3.
This project will continue with the discovery and validation of extracellular pathogen-manipulated plant proteins. We expect to identify more differential protein interactions in the 2dpi samples and will use AFM to identify candidate pathogen proteins that might be responsible for host manipulation. We will establish co-immunoprecipitation (CoIP) experiments to confirm the physical interactions between AFM-predicted complexes and use chemical probes to demonstrate that these interactions are responsible for the manipulations observed by ABPP/RBPP/CLMS. The structural models will be used to design mutant proteins that should no longer interact and test these in the CoIP and labeling experiments. We will perform disease assays with plants depleted for the host proteins using virus-induced gene silencing and overexpressing the WT host protein or the non-interacting mutant host protein to determine if these conditions alter bacterial growth. We have established robust assays to detect the impact of deletion and overexpression of antibacterial plant proteins, illustrated with PR3 (Sueldo et al., 2024) and PRp27 (Morimoto et al., 2022). We recently demonstrated the concept that we can engineer host proteins such that they are not manipulated by pathogen-secreted proteins and enhance plant immunity by engineering immune protease Pip1 (Schuster et al., 2024). We will use this same experimentation to demonstrate the importance of extracellular manipulation and show that its avoidance can lead to a novel type of extracellular immunity.