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

植物-病原体界面的军备竞赛是生物学中一个引人入胜的领域，它可以为作物保护提供重要的新的细胞外策略。我们使用尖端的人工智能来预测植物-病原体界面上新的蛋白质-蛋白质相互作用，使用Alphafold2 multitimer （A2M）。利用A2M筛选番茄分泌的P69B免疫蛋白酶的新型病原体来源抑制剂，我们发现P69B被（至少）四种不同的番茄病原体产生的五种不相关的抑制剂靶向：来自卵霉菌晚疫病病原体疫霉的Epi1；P3来自细菌性斑点病原体穿孔黄单胞菌；Ecp36来自真菌叶霉病原菌黄枝霉，TIL和Six15来自真菌枯萎病病原菌尖孢镰刀菌。P69B是番茄病原菌的主要靶点，这与以下事实是一致的：1)P69B是诱导的，在受感染植株的外质体中含量很高；ii) P69B在野生番茄中可能与抑制剂相互作用的残基处于正选择状态；iii) P69B从一个快速进化的基因簇中编码了9个类似物，它们的区别主要在于底物结合槽周围的残基，而底物结合槽是抑制剂相互作用的地方。在本建议中，我们的目标是阐明这种军备竞赛，并利用这些知识来设计细胞外免疫。我们将首先解决抑制机制，并确定与P69B同源物和同源物相互作用的特异性，也来自非寄主植物。其次，我们将利用番茄和病原菌的反向遗传学来确定P69s在免疫中的作用以及抑制剂在病原菌毒力中的作用。第三，我们将阐明茄科植物P69基因家族的进化，并设计对抑制剂不敏感的P69，以建立持久的细胞外抗性策略。

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.