We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the ‘reference’ genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates.

Summary Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes.

Cell-free transcription-translation (TXTL) is expanding as a polyvalent experimental platform to engineer biological systems outside living organisms. As the number of TXTL applications and users is rapidly growing, some aspects of this technology could be better characterized to provide a broader description of its basic working mechanisms. In particular, developing simple quantitative biophysical models that grasp the different regimes of in vitro gene expression, using relevant kinetic constants and concentrations of molecular components, remains insufficiently examined. In this work, we present an ODE (Ordinary Differential Equation)-based model of the expression of a reporter gene in an all E. coli TXTL that we apply to a set of regulatory elements spanning several orders of magnitude in strengths, far beyond the T7 standard system used in most of the TXTL platforms. Several key biochemical constants are experimentally determined through fluorescence assays. The robustness of the model is tested against the experimental parameters, and limitations of TXTL resources are described. We establish quantitative references between the performance of E. coli and synthetic promoters and ribosome binding sites. The model and the data should be useful for the TXTL community interested either in gene network engineering or in biomanufacturing beyond the conventional platforms relying on phage transcription.

Artificial cells made of molecular components and lipid membrane are emerging platforms to characterize living systems properties. Cell-free transcription−translation (TXTL) offers advantages for the bottom-up synthesis of cellular reactors. Yet, scaling up their design within welldefined geometries remains challenging. We present a microfluidic device hosting TXTL reactions of a reporter gene in thousands of microwells separated from an external buffer by a phospholipid membrane. In the presence of nutrients in the buffer, microreactors are stable beyond 24 h and yield a few mg/mL of proteins. Nutrients in the external solution feed the TXTL reaction at the picoliter scale via passive transport across the phospholipid membrane of each microfluidic well, despite the absence of pores. Replacing nutrients with an inert polymer and fatty acids at an isotonic concentration reduces microreactors efficiency, and a significant fraction yields no protein. This emphasizes the crucial role of the membrane for designing cell-free TXTL microreactors as efficient artificial cells.

Cell-free transcription–translation (TXTL) has become a highly versatile technology to construct, characterize and interrogate genetically programmed biomolecular systems implemented outside living organisms. By recapitulating gene expression in vitro, TXTL offers unparalleled flexibility to take apart, engineer and analyze quantitatively the effects of chemical, physical and genetic contexts on the function of biochemical systems, from simple regulatory elements to millimeter-scale pattern formation. Here, we review the capabilities of the current cell-free platforms for executing DNA programs in vitro. We describe the recent advances in programming using cell-free expression, a multidisciplinary playground that has enabled a myriad of novel applications in synthetic biology, biotechnology, and biological physics. Finally, we discuss the challenges and perspectives in the research area of TXTL-based constructive biology.

Abstract Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is limited by the use of channel-limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual-species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.

Recent efforts in synthetic biology have shown the possibility of engineering distributed functions in populations of living cells, which requires the development of highly orthogonal, genetically encoded communication pathways. Cell-free transcription-translation (TXTL) reactions encapsulated in microcompartments enable prototyping of molecular communication channels and their integration into engineered genetic circuits by mimicking critical cell features, such as gene expression, cell size, and cell individuality within a community. In this review, we discuss the uses of cell-free transcription–translation reactions for the development of synthetic genetic circuits, with a special focus on the use of microcompartments supporting this reaction. We highlight several studies where molecular communication between non-living microcompartments and living cells have been successfully engineered.

Detarioideae is well known for its high diversity of floral traits, including flower symmetry, number of organs, and petal size and morphology. This diversity has been characterized and studied at higher taxonomic levels, but limited analyses have been performed among closely related genera with contrasting floral traits due to the lack of fully resolved phylogenetic relationships. Here, we used four representative transcriptomes to develop an exome capture (target enrichment) bait for the entire subfamily and applied it to the Anthonotha clade using a complete data set (61 specimens) representing all extant floral diversity. Our phylogenetic analyses recovered congruent topologies using ML and Bayesian methods. Anthonotha was recovered as monophyletic contrary to the remaining three genera (Englerodendron, Isomacrolobium and Pseudomacrolobium), which form a monophyletic group sister to Anthonotha. We inferred a total of 35 transitions for the seven floral traits (pertaining to flower symmetry, petals, stamens and staminodes) that we analyzed, suggesting that at least 30% of the species in this group display transitions from the ancestral condition reconstructed for the Anthonotha clade. The main transitions were towards a reduction in the number of organs (petals, stamens and staminodes). Despite the high number of transitions, our analyses indicate that the seven characters are evolving independently in these lineages. Petal morphology is the most labile floral trait with a total of seven independent transitions in number and seven independent transitions to modification in petal types. The diverse petal morphology along the dorsoventral axis of symmetry within the flower is not associated with differences at the micromorphology of petal surface, suggesting that in this group all petals within the flower might possess the same petal identity at the molecular level. Our results provide a solid evolutionary framework for further detailed analyses of the molecular basis of petal identity.

The bottom-up assembly of synthetic cell systems capable of recapitulating biological functions has become a means to understand living matter by construction. The integration of biomolecular components into active, cell-sized, genetically programmed compartments remains, however, a major bottleneck for building synthetic cells. A primary feature of real cells is their ability to actively interact with their surroundings, particularly in stressed conditions. Here, we construct a synthetic cell equipped with an inducible genetic circuit that responds to changes in osmotic pressure through the mechanosensitive channel MscL. Liposomes loaded with an E. coli cell-free transcription-translation (TXTL) system are induced with IPTG when exposed to hypo-osmotic solution, resulting in the expression of a bacterial cytoskeletal protein (MreB). MreB associates with the membrane to generate a cortex-like structure. Our work provides the first example of molecular integration that couples mechanosensitivity, gene expression, and self-assembly at the inner membrane of synthetic cells.

Known for its remarkable biodiversity and high levels of endemism, the Brazilian Atlantic Rainforest has been characterized as one of the most threatened biomes on the planet. Despite strong interest in recent years, we still lack a comprehensive scenario to explain the origin and maintenance of diversity in this region, partially given the relatively low power of analyses involving few independent genetic loci. In this study, we examine a phylogenomic dataset of five ant species to investigate phylogeographical patterns across the Brazilian Atlantic Forest. We sequenced ultraconserved elements to generate hundreds of loci using a bait set developed specifically for hymenopterans. We analyzed the data using Bayesian and maximum likelihood approaches of phylogenetic inference. Results were then integrated with environmental niche modeling of current and past climates, including the Last Glacial Maximum and the last interglacial period. The studied species showed differentiation patterns that were consistent with the north/south division of the Atlantic Rainforest indicated in previous studies for other taxa. However, there were differences among species, both in the location of phylogeographic breaks and in the pattern of genetic variation within these areas. Samples from southern localities tended to show recent genetic structure, although a site in Tapiraí (state of São Paulo) repeatedly showed an intriguing older history of differentiation. All species experienced shifts in areas of suitability through the time. Our study suggests that distinct groups may have responded idiosyncratically to the climatic changes that took place in the Brazilian Atlantic Forest. The amount of intraspecific genetic structure was related to the inferred geographical distribution of habitat suitability according to current and past times. Also, a parallel between the amount of Quaternary climatic suitability and the level of interspecific differentiation was detected for four species. Finally, despite strong contractions at the northeastern region of the forest, the remaining areas appear to have been able to act as refugia.