Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, UK.
Department of Molecular Microbiology, Faculty of Life Sciences, Tokyo University of Agriculture, Tokyo, Japan.
J Biol Chem. 2021 Jan-Jun;296:100371. doi: 10.1016/j.jbc.2021.100371. Epub 2021 Feb 4.
Microbial plant pathogens secrete effector proteins, which manipulate the host to promote infection. Effectors can be recognized by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik effector from the rice blast fungus Magnaporthe oryzae is recognized by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a noncanonical integrated heavy-metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defenses. The host targets of AVR-Pik are also HMA-domain-containing proteins, namely heavy-metal-associated isoprenylated plant proteins (HIPPs) and heavy-metal-associated plant proteins (HPPs). Here, we demonstrate that one of these targets interacts with a wider set of AVR-Pik variants compared with the Pik-1 HMA domains. We define the biochemical and structural basis of the interaction between AVR-Pik and OsHIPP19 and compare the interaction to that formed with the HMA domain of Pik-1. Using analytical gel filtration and surface plasmon resonance, we show that multiple AVR-Pik variants, including the stealthy variants AVR-PikC and AVR-PikF, which do not interact with any characterized Pik-1 alleles, bind to OsHIPP19 with nanomolar affinity. The crystal structure of OsHIPP19 in complex with AVR-PikF reveals differences at the interface that underpin high-affinity binding of OsHIPP19-HMA to a wider set of AVR-Pik variants than achieved by the integrated HMA domain of Pik-1. Our results provide a foundation for engineering the HMA domain of Pik-1 to extend binding to currently unrecognized AVR-Pik variants and expand disease resistance in rice to divergent pathogen strains.
微生物植物病原体分泌效应蛋白，这些蛋白操纵宿主以促进感染。效应蛋白可以被植物细胞内的核苷酸结合富含亮氨酸重复（NLR）受体识别，从而引发免疫反应。来自稻瘟病菌 Magnaporthe oryzae 的 AVR-Pik 效应子被一对水稻 NLR 受体 Pik-1 和 Pik-2 识别。Pik-1 含有一个非典型的整合重金属相关（HMA）结构域，该结构域直接结合 AVR-Pik 以激活植物防御。AVR-Pik 的宿主靶标也是含有 HMA 结构域的蛋白质，即重金属相关异戊烯基化植物蛋白（HIPPs）和重金属相关植物蛋白（HPPs）。在这里，我们证明这些靶标之一与更广泛的 AVR-Pik 变体相互作用，而不是与 Pik-1 HMA 结构域相互作用。我们定义了 AVR-Pik 与 OsHIPP19 之间相互作用的生化和结构基础，并将该相互作用与 Pik-1 的 HMA 结构域形成的相互作用进行了比较。使用分析凝胶过滤和表面等离子体共振，我们表明，多个 AVR-Pik 变体，包括不与任何鉴定的 Pik-1 等位基因相互作用的隐匿变体 AVR-PikC 和 AVR-PikF，都以纳摩尔亲和力结合到 OsHIPP19 上。OsHIPP19 与 AVR-PikF 复合物的晶体结构揭示了界面上的差异，这些差异为 OsHIPP19-HMA 与更广泛的 AVR-Pik 变体形成高亲和力结合提供了基础，而这是 Pik-1 的整合 HMA 结构域所无法实现的。我们的研究结果为工程 Pik-1 的 HMA 结构域提供了基础，以扩展对当前未识别的 AVR-Pik 变体的结合，并扩大水稻对不同病原体菌株的抗病性。
