RESUMO
Pattern recognition receptors on the plant cell surface mediate the recognition of microbe/damage-associated molecular patterns (MAMPs/DAMPs) and activate downstream immune signaling. Autophosphorylation of signaling receptor-like kinases is a critical event for the activation of downstream responses but the function of each phosphorylation site in the regulation of immune signaling is not well understood. In this study, 41 Ser/Thr/Tyr and 15 Ser/Thr residues were identified as in vitro and in vivo autophosphorylation sites of Arabidopsis CERK1, which is essential for chitin signaling. Comprehensive analysis of transgenic plants expressing mutated CERK1 genes for each phosphorylation site in the cerk1-2 background indicated that the phosphorylation of T479 in the activation segment and Y428 located upstream of the catalytic loop is important for the activation of chitin-triggered defense responses. Contribution of the phosphorylation of T573 to the chitin responses was also suggested. In vitro evaluation of kinase activities of mutated kinase domains indicated that the phosphorylation of T479 and T573 is directly involved in the regulation of kinase activity of CERK1 but the phosphorylation of Y428 regulates chitin signaling independently of the regulation of kinase activity. These results indicated that the phosphorylation of specific residues in the kinase domain contributes to the regulation of downstream signaling either through the regulation of kinase activity or the different mechanisms, e.g. regulation of protein-protein interactions.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Quitina/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Treonina/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Mutação , Fosforilação/efeitos dos fármacos , Imunidade Vegetal/efeitos dos fármacos , Plantas Geneticamente Modificadas , Domínios Proteicos , Transdução de Sinais/efeitos dos fármacosRESUMO
Chitin is a representative microbe-associated molecular pattern (MAMP) molecule for various fungi and induces immune responses in many plant species. It has been clarified that the chitin signaling in rice requires a receptor kinase OsCERK1 and a receptor-like protein (Os)CEBiP, which specifically binds chitin oligosaccharides. On the other hand, Arabidopsis requires a receptor kinase (At)CERK1 for chitin signaling but it is not clear whether the plant also requires a CEBiP-like molecule for chitin perception/signaling. To clarify the similarity/difference of the chitin receptor in these two model plants, we first characterized CEBiP homologs in Arabidopsis. Only one of three CEBiP homologs, AtCEBiP (LYM2), showed a high-affinity binding for chitin oligosaccharides similar to rice CEBiP. AtCEBiP also represented the major chitin-binding protein in the Arabidopsis membrane. However, the single/triple knockout (KO) mutants of Arabidopsis CEBiP homologs and the overexpressor of AtCEBiP showed chitin-induced defense responses similar to wild-type Arabidopsis, indicating that AtCEBiP is biochemically functional as a chitin-binding protein but does not contribute to signaling. Studies of the chitin binding properties of the ectodomains of At/OsCERK1 and the chimeric receptors consisting of ecto/cytosolic domains of these molecules indicated that AtCERK1 is sufficient for chitin perception by itself.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Quitina/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Oryza/imunologia , Oryza/microbiologia , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Transdução de Sinais , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Plants have the ability to detect invading fungi through the perception of chitin fragments released from the fungal cell walls. Plant chitin receptor consists of two types of plasma membrane proteins, CEBiP and CERK1. However, the contribution of these proteins to chitin signaling is different between Arabidopsis and rice. In Arabidopsis, it seems CERK1 receptor kinase is enough for both ligand perception and signaling, whereas both CEBiP and OsCERK1 are required for chitin signaling in rice. Here we report that Arabidopsis CEBiP homolog, LYM2, is not involved in chitin signaling but contributes to resistance against a fungal pathogen, Alternaria brassicicola, indicating the presence of a novel disease resistance mechanism in Arabidopsis.