Crystal structure of ginkbilobin‐2 with homology to the extracellular domain of plant cysteine‐rich receptor‐like kinases

Fungi are an extremely diverse group of organisms with about 250,000 species and are found in all ecosystems.1 They are also proficient in colonization and infection of plants, and often cause harm to the host organisms. Most plants have evolved a variety of potent defense mechanisms against these pathogens, such as hypersensitive responses, reinforcement of cell walls, and synthesis of phytoalexins and antifungal proteins.2 To date, hundreds of antifungal proteins have been discovered in a wide variety of plants and are classified into the following groups: cyclophilins, defensins, pathogenesis-related proteins, ribosome-inactivating proteins, and so on.1 Recently, we discovered a novel antifungal protein, ginkbilobin-2 (Gnk2), in the endosperm of Ginkgo seeds.3 Gnk2 consists of 108 amino acids as a mature protein and inhibits the growth of phytopathogenic fungi such as Fusarium oxysporum.3 This antifungal protein shows no sequence similarity to other antifungal proteins.3 On the other hand, Gnk2 has considerable homology ( 85%) to embryo-abundant proteins (EAP) from the gymnosperms Picea abies and P. glauca, which suggests that Gnk2-like proteins are widely conserved in the seeds of gymnosperms. Plant EAP are expressed in the late stage of seed maturation and are involved in protection against environmental stresses such as drought.4 However, there are no reports suggesting that these proteins help defend against fungal pathogens. The sequence of Gnk2 is also 28–31% identical to the extracellular domain of cysteine-rich receptor-like kinases (CRK) from the angiosperm Arabidopsis. CRK has been classified as a member of the plant receptor-like kinases subfamily. There are more than 40 members of CRK in Arabidopsis and they contain 1–4 copies of domain 26 of unknown function (DUF26) with a C-X8-C-X2-C motif in their extracellular regions.5,6 The CRK members are induced by pathogen infection and treatment with reactive oxygen species or salicylic acid7,8 and are involved in the hypersensitive reaction, which is a typical system of programmed cell death.9 The C-X8-C-X2-C motif is completely conserved in the sequences of both Gnk2 and gymnosperms EAP. In addition, there are at least 60 genes in Arabidopsis encoding the cysteine-rich secreted