A2M: Exploring in-silico predicted arms-races at the plant-pathogen interface

The arms race at the plant-pathogen interface is a fascinating field of biology that can deliver important new, extracellular strategies for crop protection. We have used cutting edge artificial intelligence to predict novel protein-protein interactions at the plant-pathogen interface using Alphafold2 Multimer (A2M). Using an A2M screen for novel, pathogen-derived inhibitors of the secreted P69B immune protease of tomato, we discovered that P69B is targeted by (at least) five unrelated inhibitors produced by four different tomato pathogens: Epi1 from the oomycete late blight pathogen Phytophthora infestans; P3 from the bacterial spot pathogen Xanthomonas perforans; Ecp36 from the fungal leaf mould pathogen Cladosporium fulvum and TIL and Six15 from the fungal Fusarium wilt pathogen Fusarium oxysporum. That P69B is a major target for tomato pathogens is consistent with the facts that: i) P69B is induced and highly abundant in the apoplast of infected plants; ii) P69B is under positive selection in wild tomato at residues that probably interact with inhibitors; iii) P69B has nine paralogs encoded from a fast evolving gene cluster, that differ mostly in residues surrounding the substrate binding groove, where inhibitors interact. In this proposal, we aim to elucidate this arms-race and use this knowledge to engineer extracellular immunity. We will first resolve the inhibition mechanisms and determine the specificities of interactions with P69B paralogs and homologs, also from non-host plants. Second, we will determine the role of P69s in immunity and the role of inhibitors in pathogen virulence using reverse genetics on tomato and the pathogens. Third, we will elucidate the evolution of the P69 gene family in solanaceous plants and engineer inhibitor-insensitive P69s to build a strategy for durable extracellular resistance to apoplastic pathogens.

The arms race at the plant-pathogen interface is a fascinating field of biology that can deliver important new, extracellular strategies for crop protection. We have used Alphafold2 Multimer (A2M) to screen 1,879 small secreted proteins from seven tomato pathogens for proteins that bind to immune protease P69B with an intrinsic fold that obstructs the active site. This way we identified 10 putative P69B inhibitors, four of which could be produced and purified as soluble proteins from E. coli and at least three proteins inhibit labelling of the active site of P69B with fluorescent activity-based probes. Together with Epi1 from Phytophthora infestans, we have confirmed P69B inhibition by P3 from Xanthomonas perforans; P5(Ecp36) from Cladosporium fulvum and P8(TIL) from Fusarium oxysporum. We suspect P69B inhibition also by P9(Six15) from Fusarium oxysporum. These 3-4 new P69B inhibitors are present in related plant pathogens and these homologs contain conserved structural features and signatures for adaptation to target different P69s. P69a, on the other hand, are encoded by gene clusters that seem to evolve fast and encodes P69 proteins that differ mostly at positions that are likely interact with the different inhibitors. In this proposal, we aim to explore this new, in-silico predicted arms race at the plant-pathogen interface and use this knowledge to engineer extracellular immunity. We will first resolve the molecular mechanism of inhibition and determine the specificities of interactions with P69B paralogs and homologs, also from non-host plants. Second, we will determine the role of P69s in immunity and the role of inhibitors in pathogen virulence using reverse genetics on tomato and the pathogens by genome editing. Third, we will elucidate the evolution of the P69 gene family in solanaceous plants and produce inhibitor-insensitive P69s and combine these mutations to increase extracellular, durable resistance to apoplastic pathogens.