Plant roots encounter numerous pathogenic microbes that often cause devastating diseases. One such pathogen, Plasmodiophora brassicae (Pb), causes clubroot disease and severe yield losses on cruciferous crops worldwide. Here, we report the isolation and characterization of WeiTsing (WTS), a broad-spectrum clubroot resistance gene from Arabidopsis. WTS is transcriptionally activated in the pericycle upon Pb infection to prevent pathogen colonization in the stele. Brassica napus carrying the WTS transgene displayed strong resistance to Pb. WTS encodes a small protein localized in the endoplasmic reticulum (ER), and its expression in plants induces immune responses. The cryoelectron microscopy (cryo-EM) structure of WTS revealed a previously unknown pentameric architecture with a central pore. Electrophysiology analyses demonstrated that WTS is a calcium-permeable cation-selective channel. Structure-guided mutagenesis indicated that channel activity is strictly required for triggering defenses. The findings uncover an ion channel analogous to resistosomes that triggers immune signaling in the pericycle.
Small ubiquitin-like modifier (SUMO) conjugation is a post-translational modification associated with many human diseases. Characterization of the SUMO-modified proteome is pivotal to define the mechanistic link between SUMO conjugation and such diseases. This is particularly evident for SUMO2/3 conjugation, which is massively activated after brain ischemia/stroke, and is believed to be a protective response. The purpose of this study was to perform a comprehensive analysis of the SUMO3-modified proteome regulated by brain ischemia using a novel SUMO transgenic mouse.To enable SUMO proteomics analysis in vivo, we generated transgenic mice conditionally expressing tagged SUMO1-3 paralogues. Transgenic mice were subjected to 10 minutes forebrain ischemia and 1 hour of reperfusion. SUMO3-conjugated proteins were enriched by anti-FLAG affinity purification and analyzed by liquid chromatography-tandem mass spectrometry.Characterization of SUMO transgenic mice demonstrated that all 3 tagged SUMO paralogues were functionally active, and expression of exogenous SUMOs did not modify the endogenous SUMOylation machinery. Proteomics analysis identified 112 putative SUMO3 substrates of which 91 candidates were more abundant in the ischemia group than the sham group. Data analysis revealed processes/pathways with putative neuroprotective functions, including glucocorticoid receptor signaling, RNA processing, and SUMOylation-dependent ubiquitin conjugation.The identified proteins/pathways modulated by SUMOylation could be the key to understand the mechanisms linking SUMOylation to neuroprotection, and thus provide new promising targets for therapeutic interventions. The new transgenic mouse will be an invaluable platform for analyzing the SUMO-modified proteome in models of human disorders and thereby help to mechanistically link SUMOylation to the pathological processes.
Pattern-recognition receptors (PRRs), which consist of receptor kinases (RKs) and receptor-like proteins, sense microbe- and host-derived molecular patterns associated with pathogen infection to trigger immune responses in plants. Several kinases of the 46-member Arabidopsis (Arabidopsis thaliana) receptor-like cytoplasmic kinase (RLCK) subfamily VII play important roles in pattern-triggered immunity, but it is unclear whether different RLCK VII members act specifically or redundantly in immune signaling. Here, we constructed nine higher order mutants of this subfamily (named rlck vii-1 to rlck vii-9) and systematically characterized their immune phenotypes. The mutants rlck vii-5, -7, and -8 had compromised reactive oxygen species production in response to all patterns tested, indicating that the corresponding members are broadly required for the signaling of multiple PRRs. However, rlck vii-4 was defective specifically in chitin-induced reactive oxygen species production, suggesting that RCLK VII-4 members mediate the signaling of specific PRRs. Furthermore, RLCK VII-4 members were required for the chitin-triggered activation of MAPK, demonstrating that these kinases link a PRR to MAPK activation. Moreover, we found that RLCK VII-6 and -8 also were required for RK-mediated root growth. Together, these results show that numerous RLCK VII members are involved in pattern-triggered immune signaling and uncover both common and specific roles of these kinases in plant development and immunity mediated by various RKs.
Abstract Many Gram-negative pathogens employ the type III secretion system (T3SS) to deliver effector proteins into host cells, thereby modulating host cellular processes and suppressing host immunity to facilitate pathogenesis and colonization. In this study, we developed a straightforward, rapid, and quantitative method for detecting T3SS-mediated translocation of Pseudomonas syringae effectors using a self-assembling split Nano luciferase (Nluc)-based reporter system. It was demonstrated that this system can detect effector secretion in vitro with an exceptionally high signal-to-noise ratio and sensitivity, attributed to the strong affinity between the split domains of Nluc and the intense luminescence generated by functional Nluc. During natural infections, effectors fused to a small C-terminal fragment of Nluc were successfully translocated into plant cells and retained their virulence functions. Furthermore, upon infection of plants expressing the N-terminal fragment of Nluc with these P. syringae strains, functional Nluc proteins were spontaneously assembled and produced bright luminescence, demonstrating that this system enables the straightforward and rapid assessment of P. syringae T3SS-mediated effector translocation during natural infections. In conclusion, the self-assembling split Nluc-based reporting system developed in this study is suitable for efficient in vitro and in planta detection of effectors secreted via T3SS.
Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice) that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP+ cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express the newborn neuron marker doublecortin and PSA-NCAM. Compared to mature dentate granule cells, these newborn neurons show immature morphological features: dendritic beading, fewer dendritic branches and spines. These GFP+ newborn neurons also show immature electrophysiological properties: higher input resistance, more depolarized resting membrane potentials, small and non-typical action potentials. The bright labeling of newborn neurons with GFP makes it possible to visualize the details of dendrites, which reach the outer edge of the molecular layer, and their axon (mossy fiber) terminals, which project to the CA3 region where they form synaptic boutons. GFP expression covers the whole developmental stage of newborn neurons, beginning within the first week of cell division and disappearing as newborn neurons mature, about 4 weeks postmitotic. Thus, the GAD67-GFP transgenic mice provide a useful genetic tool for studying the development and regulation of newborn dentate granule cells.
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