RESUMEN
Certain plant receptor-like cytoplasmic kinases were reported to interact with small monomeric G-proteins of the RHO of plant (ROP; also called RAC) family in planta and to be activated by this interaction in vitro. We identified a barley (Hordeum vulgare) partial cDNA of a ROP binding protein kinase (HvRBK1) in yeast (Saccharomyces cerevisiae) two-hybrid screenings with barley HvROP bait proteins. Protein interaction of the constitutively activated (CA) barley HvROPs CA HvRACB and CA HvRAC1 with full-length HvRBK1 was verified in yeast and in planta. Green fluorescent protein-tagged HvRBK1 appears in the cytoplasm and nucleoplasm, but CA HvRACB or CA HvRAC1 can recruit green fluorescent protein-HvRBK1 to the cell periphery. Barley HvRBK1 is an active kinase in vitro, and activity is enhanced by CA HvRACB or GTP-loaded HvRAC1. Hence, HvRBK1 might act downstream of active HvROPs. Transient-induced gene silencing of barley HvRBK1 supported penetration by the parasitic fungus Blumeria graminis f. sp. hordei, suggesting a function of the protein in basal disease resistance. Transient knockdown of HvRBK1 also influenced the stability of cortical microtubules in barley epidermal cells. Hence, HvRBK1 might function in basal resistance to powdery mildew by influencing microtubule organization.
Asunto(s)
Ascomicetos/patogenicidad , Resistencia a la Enfermedad , Hordeum/microbiología , Microtúbulos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , ADN Complementario/genética , ADN Complementario/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Genes de Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hordeum/enzimología , Hordeum/genética , Microtúbulos/genética , Datos de Secuencia Molecular , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Epidermis de la Planta/genética , Epidermis de la Planta/metabolismo , Inmunidad de la Planta , Proteínas de Plantas/genética , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
RHO-like GTPases of plants (ROPs, also called RACs) are involved in plant development and interaction with the environment. The barley ROP protein RACB is involved in susceptibility to the fungal pathogen Blumeria graminis f.sp. hordei (Bgh). By screening barley sequence databases for potential protein interactors of plant RHO-like proteins, we identified a ROP-interactive CRIB (CDC42/RAC interactive binding) motif containing protein of 171 amino acids (RIC171). The protein interacted with constitutively activated RACB in a targeted yeast two-hybrid assay. By use of split yellow fluorescing protein fusions, we demonstrated that RIC171 interacts with constitutively activated (CA) RACB-G15V but not with dominant negative RACB-T20N in planta. Transient overexpression of RIC171, similar to overexpression of CA RACB-G15V, rendered epidermal cells more susceptible to penetration by Bgh. In contrast, expression of a 46-amino-acid RIC171-CRIB peptide, which was sufficient to interact with CA RACB-G15V, had a dominant negative effect and reduced susceptibility to Bgh. A red fluorescing DsRED-RIC171 fusion protein colocalized with green fluorescing GFP-RACB-G15V at the cell periphery. Coexpression with CA RACB-G15V but not with RACB-T20N increased peripheral localization of DsRED-RIC171. Additionally, DsRED-RIC171 accumulated at sites of fungal attack, suggesting enhanced ROP activity at sites of attempted fungal penetration.