Dataset Information

Application of ALFA-Tagging in the Nematode Model Organisms Caenorhabditis elegans and Pristionchus pacificus.

ABSTRACT: The detection, manipulation and purification of proteins is key in modern life sciences studies. To achieve this goal, a plethora of epitope tags have been employed in model organisms from bacteria to humans. Recently, the introduction of the rationally designed ALFA-tag resulted in a highly versatile tool with a very broad spectrum of potential applications. ALFA-tagged proteins can be detected by nanobodies, the single-domain antibodies of camelids, allowing for super-resolution microscopy and immunoprecipitation in biochemical applications. Here, we introduce ALFA-tagging into the two nematode model organisms Caenorhabditis elegans and Pristionchus pacificus. We show that the introduction of the DNA sequence, corresponding to the 13 amino acid sequence of the ALFA-tag, can easily be accommodated by CRISPR engineering. We provide examples of high-resolution protein expression in both nematodes. Finally, we use the GW182 ortholog Ppa-ain-1 to show successful pulldowns in P. pacificus. Thus, the ALFA-tag represents a novel epitope tag for nematode research with a broad spectrum of applications.

SUBMITTER: Igreja C

PROVIDER: S-EPMC9740511 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Publications

Application of ALFA-Tagging in the Nematode Model Organisms <i>Caenorhabditis elegans</i> and <i>Pristionchus pacificus</i>.

Igreja Catia C Loschko Tobias T Schäfer Alejandra A Sharma Radhika R Quiobe Shiela Pearl SP Aloshy Elbin E Witte Hanh H Sommer Ralf J RJ

Cells 20221201 23

The detection, manipulation and purification of proteins is key in modern life sciences studies. To achieve this goal, a plethora of epitope tags have been employed in model organisms from bacteria to humans. Recently, the introduction of the rationally designed ALFA-tag resulted in a highly versatile tool with a very broad spectrum of potential applications. ALFA-tagged proteins can be detected by nanobodies, the single-domain antibodies of camelids, allowing for super-resolution microscopy and ...[more]

PMID: 36497133

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Project description:The evolution of genetic mechanisms used to combat bacterial infections is critical for the survival of animals and plants, yet how these genes evolved to produce a robust defense system is poorly understood. Studies of the nematode Caenorhabditis elegans have uncovered a plethora of genetic regulators and effectors responsible for surviving pathogens. However, comparative studies utilizing other free-living nematodes and therefore providing an insight into the evolution of innate immunity have been lacking. Here, we take a systems biology approach and use whole genome microarrays to profile the transcriptional response of C. elegans and the necromenic nematode Pristionchus pacificus after exposure to the four different pathogens Serratia marcescens, Xenorhabdus nematophila, Staphylococcus aureus and Bacillus thuringiensis DB27. C. elegans is susceptible to all four pathogens whilst P. pacificus is only susceptible to S. marcescens and X. nematophila. We show an unexpected level of specificity in host responses to distinct pathogens within and across species, revealing an enormous complexity of effectors of innate immunity. Functional domains enriched in the transcriptomes on different pathogens are similar within a nematode species but different across them, suggesting differences in pathogen sensing and response networks. We find translation inhibition to be a potentially conserved response to gram-negative pathogens in both the nematodes. Further computational analysis indicates that both nematodes when fed on pathogens up-regulate genes known to be involved in other stress responses like heat shock, oxidative and osmotic stress, and genes regulated by DAF-16/FOXO and TGF-beta pathways. This study presents a platform for comparative systems analysis of two nematode model species, and a catalog of genes involved in the evolution of nematode immunity and identifies both pathogen specific and pan-pathogen responses. We discuss the potential effects of ecology on evolution of downstream effectors and upstream regulators on evolution of nematode innate immunity.

Project description:BackgroundAn organism can respond to changing environmental conditions by adjusting gene regulation and by forming alternative phenotypes. In nematodes, these mechanisms are coupled because many species will form dauer larvae, a stress-resistant and non-aging developmental stage, when exposed to unfavorable environmental conditions, and execute gene expression programs that have been selected for the survival of the animal in the wild. These dauer larvae represent an environmentally induced, homologous developmental stage across many nematode species, sharing conserved morphological and physiological properties. Hence it can be expected that some core components of the associated transcriptional program would be conserved across species, while others might diverge over the course of evolution. However, transcriptional and metabolic analysis of dauer development has been largely restricted to Caenorhabditis elegans. Here, we use a transcriptomic approach to compare the dauer stage in the evolutionary model system Pristionchus pacificus with the dauer stage in C. elegans.ResultsWe have employed Agilent microarrays, which represent 20,446 P. pacificus and 20,143 C. elegans genes to show an unexpected divergence in the expression profiles of these two nematodes in dauer and dauer exit samples. P. pacificus and C. elegans differ in the dynamics and function of genes that are differentially expressed. We find that only a small number of orthologous gene pairs show similar expression pattern in the dauers of the two species, while the non-orthologous fraction of genes is a major contributor to the active transcriptome in dauers. Interestingly, many of the genes acquired by horizontal gene transfer and orphan genes in P. pacificus, are differentially expressed suggesting that these genes are of evolutionary and functional importance.ConclusionOur data set provides a catalog for future functional investigations and indicates novel insight into evolutionary mechanisms. We discuss the limited conservation of core developmental and transcriptional programs as a common aspect of animal evolution.

Dataset Information

Application of ALFA-Tagging in the Nematode Model Organisms Caenorhabditis elegans and Pristionchus pacificus.

Publications

Application of ALFA-Tagging in the Nematode Model Organisms <i>Caenorhabditis elegans</i> and <i>Pristionchus pacificus</i>.

Similar Datasets

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