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.
In sexually reproducing metazoans, spermatogenesis is the process by which uncommitted germ cells give rise to haploid sperm. Work in model systems has revealed mechanisms controlling commitment to the sperm fate, but how this fate is subsequently executed remains less clear. While studying the well-established role of the conserved nuclear hormone receptor transcription factor, NHR-23/NR1F1, in regulating C. elegans molting, we discovered NHR-23/NR1F1 is also constitutively expressed in developing 1° spermatocytes and is a critical regulator of spermatogenesis. In this novel role, NHR-23/NR1F1 functions downstream of the canonical sex determination pathway. Degron-mediated depletion of NHR-23/NR1F1 within hermaphrodite or male germlines causes sterility due to an absence of functional sperm as depleted animals produce arrested primary spermatocytes rather than haploid sperm. These spermatocytes arrest in prometaphase I and fail to either progress to anaphase or attempt spermatid-residual body partitioning. They make sperm-specific membranous organelles (MOs) but fail to assemble their major sperm protein into fibrous bodies. NHR-23/NR1F1 appears to function independently of the known SPE-44 gene regulatory network, revealing the existence of an NHR-23/NR1F1-mediated module that regulates the spermatogenesis program.
Vibrio parahaemolyticus cells transit from free swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP are low or when the phosphodiesterase is catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called Trigger phosphodiesterases, hence we named the product of VP1881 TpdA, for Trigger phosphodiesterase A. The absence of tpdA showed a negative effect on swimming motility while its overexpression from an IPTG inducible promoter showed a positive effect on both swimming and swarming motility, and a negative effect on biofilm formation. Changes in TpdA abundance altered the expression of representative polar and lateral flagellar genes as well as the biofilm related gene cpsA. Our results also revealed that autoactivation of the native PtpdA promoter is sufficient to alter c-di-GMP signaling responses such as swarming and biofilm formation in V. parahaemolyticus, an observation that could have important implications in the dynamics of these social behaviors. IMPORTANCE C-di-GMP trigger phosphodiesterases (PDEs) could play a key role in controlling the heterogeneity of biofilm-matrix composition, a property that endows characteristics that are potentially relevant for sustaining integrity and functionality of biofilms in a variety of natural environments. Trigger PDEs are not always easy to identify based on their sequence, hence not many examples of these type of signaling proteins have been reported in the literature. Here we report on the identification of a novel trigger PDE in V. parahaemolyticus and provide evidence suggesting that its autoactivation could play an important role in the progression of swarming motility and biofilm formation, multicellular behaviors that are important for the survival and dissemination of this environmental pathogen.
1The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally 2 deplete proteins in a range of organisms and cell-types. Here, we describe a toolkit to 3 augment the use of the AID system in Caenorhabditis elegans. We have generated a set 4 of single-copy, tissue-specific (germline, intestine, neuron, muscle, hypodermis, seam 5 cell, anchor cell) and pan-somatic TIR1-expressing strains carrying an equimolar co-6 expressed blue fluorescent reporter to enable use of both red and green channels in 7 experiments. We have also constructed a set of plasmids to generate fluorescent 8 protein::AID fusions through CRISPR/Cas9-mediated genome editing. These templates 9can be produced through frequently used cloning systems (Gibson assembly or SapTrap) 10 or through ribonucleoprotein complex-mediated insertion of PCR-derived, linear repair 11 templates. We have generated a set of sgRNA plasmids carrying modifications shown to 12 boost editing efficiency, targeting standardized transgene insertion sites on 13 chromosomes I and II. Together these reagents should complement existing TIR1 strains 14 and facilitate rapid and high-throughput fluorescent protein::AID* tagging of factors of 15 interest. This battery of new TIR1-expressing strains and modular, efficient cloning 16 vectors serves as a platform for facile assembly of CRISPR/Cas9 repair templates for 17 conditional protein depletion. 18 19
The second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) plays a central role in controlling decision making processes of vital importance for the environmental survival of the human pathogenVibrio parahaemolyticus. The mechanisms by which c-di-GMP levels are dynamically controlled inV. parahaemolyticusare poorly understood. Here we report our findings regarding the involvement of OpaR in controlling c-di-GMP metabolism in planktonic and surface-attached cells through controlling the expression of the trigger phosphodiesterase (PDE) TpdA and other PDEs such as ScrC. Our results revealed that OpaR negatively modulates the expression oftpdAby maintaining a baseline level of c-di-GMP. The OpaR-regulated PDEs ScrC, ScrG and VP0117 enable the upregulation oftpdA, to a different degree, in the absence of OpaR. We also found that TpdA plays the dominant role in c-di-GMP degradation under planktonic conditions compared to the other OpaR-regulated PDEs. In cells growing over solid media the dominant c-di-GMP degrader role is played by ScrC for 72 hours and passes to TpdA after 96 hours of growth. We also report negative and positive effects of the absence of OpaR oncpsAexpression in cells growing over solid media or forming biofilms over glass, respectively. These results suggest that OpaR can act as a double-edged sword to control c-di-GMP accumulation andcpsAexpression positively or negatively in response to poorly understood environmental factors. Finally, through an in-silico analysis we point out outlets of the OpaR regulatory module that can impact decision making during the motile to sessile transition inV. parahaemolyticus.
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.