Colorimetric gene detection based on gold nanoparticles (AuNPs) is an attractive detection format due to its simplicity. Here, we report a new design for a colorimetric gene-sensing platform based on the CRISPR/Cas system that has improved specificity, sensitivity, and universality. CRISPR/Cas12a and CRISPR/Cas13a have two distinct catalytic activities and are used for specific target gene recognition. Programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary sequence activates the nonspecific trans-ssDNA or -RNA cleavage, respectively, thus degrading the ssDNA or RNA linkers which are designed as a hybridization template for the AuNP-DNA probe pair. Target-induced trans -ssDNA or RNA cleavage leads to a distance-dependent color change for the AuNP-DNA probe pair. In this platform, naked eye detection of transgenic rice, African swine fever virus (ASFV), and a miRNA can be completed within 1 hour. Our colorimetric gene-sensing method shows superior characteristics, such as probe universality, isothermal reaction conditions, on-site detection capability, and sensitivity that is comparable to that of the fluorescent detection; thus, this method represents a robust next generation gene detection platform.
Development requires the proper execution and regulation of the cell cycle via precise, conserved mechanisms. Critically, the E2F/DP complex controls the expression of essential genes during cell cycle transitions. Here, we discovered the molecular function of the SUMO E3 ligase METHYL METHANESULFONATE SENSITIVITY GENE21 (AtMMS21) in regulating the cell cycle via the E2Fa/DPa pathway. DPa was identified as an AtMMS21-interacting protein and AtMMS21 competes with E2Fa for interaction with DPa. Moreover, DPa is a substrate for SUMOylation mediated by AtMMS21, and this SUMOylation enhances the dissociation of the E2Fa/DPa complex. AtMMS21 also affects the subcellular localization of E2Fa/DPa. The E2Fa/DPa target genes are upregulated in the root of and mutants showed increased endoreplication. Overexpression of affected the root development of , and overexpression of completely recovered the abnormal phenotypes of plants. Our results suggest that AtMMS21 dissociates the E2Fa/DPa complex via competition and SUMOylation in the regulation of plant cell cycle.
The nucleo-mitochondrial dual-localized proteins can act as gene expression regulators; however, few instances of these proteins have been described in plants. Arabidopsis (Arabidopsis thaliana) PROHIBITIN 3 (PHB3) is involved in stress responses and developmental processes, but it is unknown how these roles are achieved at the molecular level in the nucleus. In this study, we show that nucleo-mitochondrial PHB3 plays an essential role in regulating genome stability and cell proliferation. PHB3 is upregulated by DNA damage agents, and the stress-induced PHB3 proteins accumulate in the nucleus. Loss of function of PHB3 results in DNA damage and defective maintenance of the root stem cell niche. Subsequently, the expression patterns and levels of the root stem cell regulators are altered and down-regulated, respectively. In addition, the phb3 mutant shows aberrant cell division and altered expression of cell cycle-related genes, such as CycB1 and Cyclin dependent kinase 1. Moreover, the minichromosome maintenance (MCM) genes, e.g. MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7, are up-regulated in the phb3 mutant. Reducing the MCM2 expression level substantially recovers the DNA damage in the phb3 mutant and partially rescues the altered cell proliferation and root deficiency of phb3 seedlings. PHB3 acts as a transcriptional coregulator that represses MCM2 expression by competitively binding to the promoter E2F-cis-acting elements with E2Fa so as to modulate primary root growth. Collectively, these findings indicate that nuclear-localized PHB3 acts as a transcriptional coregulator that suppresses MCM2 expression to sustain genome integrity and cell proliferation for stem cell niche maintenance in Arabidopsis.
Chromatin remodeling is essential for gene expression regulation in plant development and response to stresses. Brahma (BRM) is a conserved ATPase in the SWI/SNF chromatin remodeling complex and is involved in various biological processes in plant cells, but the regulation mechanism on BRM protein remains unclear. Here, we report that BRM interacts with AtMMS21, a SUMO ligase in Arabidopsis (). The interaction was confirmed in different approaches in vivo and in vitro. The mutants of and displayed a similar defect in root development. In the mutant, the protein level of BRM-GFP was significantly lower than that in wild type, but the RNA level of did not change. Biochemical evidence indicated that BRM was modified by SUMO3, and the reaction was enhanced by AtMMS21. Furthermore, overexpression of wild-type AtMMS21 but not the mutated AtMMS21 without SUMO ligase activity was able to recover the stability of BRM in Overexpression of in partially rescued the developmental defect of roots. Taken together, these results supported that AtMMS21 regulates the protein stability of BRM in root development.
DNA damage occurs in all cells and can hinder chromosome stability and cell viability. Structural Maintenance of Chromosomes5/6 (SMC5/6) is a protein complex that functions as an evolutionarily conserved chromosomal ATPase critical for repairing DNA double-strand breaks (DSBs). However, the mechanisms regulating this complex in plants are poorly understood. Here, we identified the transcriptional coactivator ALTERATION/DEFICIENCY IN ACTIVATION2B (ADA2b) as an interactor of SMC5 in Arabidopsis (). ADA2b is a conserved component of the Spt-Ada-Gcn5 acetyltransferase complex, which functions in transcriptional regulation. Characterization of mutant and knockdown Arabidopsis lines showed that disruption of either or resulted in enhanced DNA damage. Both SMC5 and ADA2b were associated with γ-H2AX, a marker of DSBs, and the recruitment of SMC5 onto DSBs was dependent on ADA2b. In addition, overexpression of in the mutant background stimulated cell death. Collectively, our results show that the interaction between ADA2b and SMC5 mediates DNA repair in plant cells, suggesting a functional association between these conserved proteins and further elucidating mechanisms of DNA damage repair in plants.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.