More Like this

1. An expanded auxin-inducible degron toolkit for Caenorhabditis elegans

   https://doi.org/10.1093/genetics/iyab006  

   Ashley, Guinevere E; Duong, Tam; Levenson, Max T; Martinez, Michael A; Johnson, Londen C; Hibshman, Jonathan D; Saeger, Hannah N; Palmisano, Nicholas J; Doonan, Ryan; Martinez-Mendez, Raquel; et al (March 2021, Genetics)

   Greenstein, D (Ed.)

   Abstract The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion. 

   more » « less
2. Generation of an endogenous auxin inducible degron-tagged SPAS-1/spastin to investigate its targeted depletion in C. elegans neurons

   https://doi.org/10.17912/micropub.biology.001328  

   Brown, Emily; Kuszynski, Samantha; Akoachere, Faith; Feduccia, James; Malatinszky, Lili; Luth, Eric S (January 2024, microPublication biology)

   To facilitate investigations of the microtubule severing protein spastin and its specific role in neurons, we aimed to create a C. elegans strain in which the spastin homolog SPAS-1 is visible and can be degraded with spatial and temporal precision. We used CRISPR-Cas9 to fuse an auxin-inducible degron and mScarlet to the endogenous SPAS-1 protein, enabling degradation of SPAS-1 in neurons during desired life stages. DNA sequencing confirmed in-frame insertion with the SPAS-1 N-terminus and fluorescence microscopy revealed endogenous SPAS-1 throughout the CRISPR-edited worms. Auxin treatment in rgef-1::TIR1; mScarlet::AID*::3xFLAG::spas-1 animals reduced mScarlet::SPAS-1 fluorescence in neuronal ganglia. 

   more » « less
3. Rapid depletion of target proteins in plants by an inducible protein degradation system

   https://doi.org/10.1093/plcell/koae072  

   Huang, Linzhou; Rojas-Pierce, Marcela (March 2024, The Plant Cell)

   Abstract Inducible protein knockdowns are excellent tools to test the function of essential proteins in short time scales and to capture the role of proteins in dynamic events. Current approaches destroy or sequester proteins by exploiting plant biological mechanisms such as the activity of photoreceptors for optogenetics or auxin-mediated ubiquitination in auxin degrons. It follows that these are not applicable for plants as light and auxin are strong signals for plant cells. We describe here an inducible protein degradation system in plants named E3-DART for E3-targeted Degradation of Plant Proteins. The E3-DART system is based on the specific and well-characterized interaction between the Salmonella secreted protein H1 (SspH1) and its human target protein kinase N1 (PKN1). This system harnesses the E3 catalytic activity of SspH1 and the SspH1-binding activity of the Homology Region 1b (HR1b) domain from PKN1. Using Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana), we show that a chimeric protein containing the Leucine-Rich Repeat (LRR) and novel E3 ligase (NEL) domains of SspH1 efficiently targets protein fusions of varying sizes containing HR1b for degradation. Target protein degradation was induced by transcriptional control of the chimeric E3 ligase using a glucocorticoid transactivation system and target protein depletion was detected as early as 3 h after induction. This system could be used to study the loss of any plant protein with high temporal resolution and may become an important tool in plant cell biology. 

   more » « less
4. Dual Role of Auxin in Regulating Plant Defense and Bacterial Virulence Gene Expression During Pseudomonas syringae PtoDC3000 Pathogenesis

   https://doi.org/10.1094/MPMI-02-20-0047-R  

   Djami-Tchatchou, Arnaud T.; Harrison, Gregory A.; Harper, Chris P.; Wang, Renhou; Prigge, Michael J.; Estelle, Mark; Kunkel, Barbara N. (August 2020, Molecular Plant-Microbe Interactions®)

   null (Ed.)

   Modification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogen strain Pseudomonas syringae DC3000 (PtoDC3000) produces the plant hormone auxin (indole-3-acetic acid [IAA]) to promote PtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promote PtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage of Arabidopsis thaliana lines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominant axr2-1 mutation resulted in decreased bacterial growth and that this phenotype was suppressed by introducing the sid2-2 mutation, which impairs SA synthesis. Thus, host auxin signaling is required for normal susceptibility to PtoDC3000 and is involved in suppressing SA-mediated defenses. Unexpectedly, tir1 afb1 afb4 afb5 quadruple-mutant plants lacking four of the six known auxin coreceptors that exhibit decreased auxin perception, supported increased levels of bacterial growth. This mutant exhibited elevated IAA levels and reduced SA-mediated defenses, providing additional evidence that auxin promotes disease by suppressing host defense. We also investigated the hypothesis that IAA promotes PtoDC3000 virulence through a direct effect on the pathogen and found that IAA modulates expression of virulence genes, both in culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression. 

   more » « less
5. Depletion of lamins B1 and B2 promotes chromatin mobility and induces differential gene expression by a mesoscale-motion-dependent mechanism

   https://doi.org/10.1186/s13059-024-03212-y  

   Pujadas Liwag, Emily M.; Wei, Xiaolong; Acosta, Nicolas; Carter, Lucas M.; Yang, Jiekun; Almassalha, Luay M.; Jain, Surbhi; Daneshkhah, Ali; Rao, Suhas S. P.; Seker-Polat, Fidan; et al (March 2024, Genome Biology)

   Abstract BackgroundB-type lamins are critical nuclear envelope proteins that interact with the three-dimensional genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron technology. ResultsUsing live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, Stochastic Optical Reconstruction Microscopy (STORM), in situ Hi-C, CRISPR-Sirius, and fluorescence in situ hybridization (FISH), we demonstrate that lamin B1 and lamin B2 are critical structural components of the nuclear periphery that create a repressive compartment for peripheral-associated genes. Lamin B1 and lamin B2 depletion minimally alters higher-order chromatin folding but disrupts cell morphology, significantly increases chromatin mobility, redistributes both constitutive and facultative heterochromatin, and induces differential gene expression both within and near lamin-associated domain (LAD) boundaries. Critically, we demonstrate that chromatin territories expand as upregulated genes within LADs radially shift inwards. Our results indicate that the mechanism of action of B-type lamins comes from their role in constraining chromatin motion and spatial positioning of gene-specific loci, heterochromatin, and chromatin domains. ConclusionsOur findings suggest that, while B-type lamin degradation does not significantly change genome topology, it has major implications for three-dimensional chromatin conformation at the single-cell level both at the lamina-associated periphery and the non-LAD-associated nuclear interior with concomitant genome-wide transcriptional changes. This raises intriguing questions about the individual and overlapping roles of lamin B1 and lamin B2 in cellular function and disease. 

   more » « less