RESUMEN
Plant cellulose is synthesized by rosette-structured cellulose synthase (CESA) complexes (CSCs). Each CSC is composed of multiple subunits of CESAs representing three different isoforms. Individual CESA proteins contain conserved catalytic domains for catalyzing cellulose synthesis, other domains such as plant-conserved sequences, and class-specific regions that are thought to facilitate complex assembly and CSC trafficking. Because of the current lack of atomic-resolution structures for plant CSCs or CESAs, the molecular mechanism through which CESA catalyzes cellulose synthesis and whether its catalytic activity influences efficient CSC transport at the subcellular level remain unknown. Here, by performing chemical genetic analyses, biochemical assays, structural modeling, and molecular docking, we demonstrate that Endosidin20 (ES20) targets the catalytic site of CESA6 in Arabidopsis (Arabidopsis thaliana). Chemical genetic analysis revealed important amino acids that potentially participate in the catalytic activity of plant CESA6, in addition to previously identified conserved motifs across kingdoms. Using high spatiotemporal resolution live cell imaging, we found that inhibiting the catalytic activity of CESA6 by ES20 treatment reduced the efficiency of CSC transport to the plasma membrane. Our results demonstrate that ES20 is a chemical inhibitor of CESA activity and trafficking that represents a powerful tool for studying cellulose synthesis in plants.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efectos de los fármacos , Celulosa/biosíntesis , Glucosiltransferasas/antagonistas & inhibidores , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Recuperación de Fluorescencia tras Fotoblanqueo , Glucosiltransferasas/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Simulación del Acoplamiento Molecular , Mutación , Plantas Modificadas Genéticamente , Conformación ProteicaRESUMEN
Somatic embryogenesis receptor kinases (SERKs) are transmembrane receptors involved in plant immunity. Tomato (Solanum lycopersicum) carries three SERK members. One of these, SlSERK1, is required for Mi-1.2-mediated resistance to potato aphids (Macrosiphum euphorbiae). Mi-1.2 encodes a coiled-coil nucleotide-binding leucine-rich repeat protein that in addition to potato aphids confers resistance to two additional phloem-feeding insects and to root-knot nematodes (Meloidogyne spp.). How SlSERK1 participates in Mi-1.2-mediated resistance is unknown, and no Mi-1.2 cognate pest effectors have been identified. Here, we study the mechanistic involvement of SlSERK1 in Mi-1.2-mediated resistance. We show that potato aphid saliva and protein extracts induce the Mi-1.2 defense marker gene SlWRKY72b, indicating that both saliva and extracts contain a Mi-1.2 recognized effector. Resistant tomato cultivar Motelle (Mi-1.2/Mi-1.2) plants overexpressing SlSERK1 were found to display enhanced resistance to potato aphids. Confocal microscopy revealed that Mi-1.2 localizes at three distinct subcellular compartments: the plasma membrane, cytoplasm, and nucleus. Coimmunoprecipitation experiments in these tomato plants and in Nicotiana benthamiana transiently expressing Mi-1.2 and SlSERK1 showed that Mi-1.2 and SlSERK1 colocalize only in a microsomal complex. Interestingly, bimolecular fluorescence complementation analysis showed that the interaction of Mi-1.2 and SlSERK1 at the plasma membrane distinctively changes in the presence of potato aphid saliva, suggesting a model in which a constitutive complex at the plasma membrane participates in defense signaling upon effector binding.