RESUMO
This protocol combines fluorescent in situ hybridization and immunostaining to simultaneously detect, in histological sections from the same animal, subpopulations of neurons activated after two episodes of sensory stimulation. It allows the identification of groups of cells singly activated by either stimulus or co-activated by both stimuli. Our method results in nuclear staining for c-Fos mRNA and c-Fos protein, allowing better spatial and temporal resolution than previously published protocols, although it requires quick brain fixation. For complete details on the use and execution of this protocol, please refer to Carvalho et al. (2015, 2020).
Assuntos
Neurônios/citologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , RNA/metabolismo , Coloração e Rotulagem , Fatores de TempoRESUMO
Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation.