An improved Nicotiana benthamiana bioproduction chassis provides novel insights into nicotine biosynthesis.

The model plant Nicotiana benthamiana is an increasingly attractive organism for the production of high-value, biologically active molecules. However, N. benthamiana accumulates high levels of pyridine alkaloids, in particular nicotine, which complicates the downstream purification processes. Here, we report a new assembly of the N. benthamiana genome as well as the generation of low-nicotine lines by CRISPR/Cas9-based inactivation of berberine bridge enzyme-like proteins (BBLs). Triple as well as quintuple mutants accumulated three to four times less nicotine than the respective control lines. The availability of lines without functional BBLs allowed us to probe their catalytic role in nicotine biosynthesis, which has remained obscure. Notably, chiral analysis revealed that the enantiomeric purity of nicotine was fully lost in the quintuple mutants. In addition, precursor feeding experiments showed that these mutants cannot facilitate the specific loss of C6 hydrogen that characterizes natural nicotine biosynthesis. Our work delivers an improved N. benthamiana chassis for bioproduction and uncovers the crucial role of BBLs in the stereoselectivity of nicotine biosynthesis.

Long read sequencing reveals novel isoforms and insights into splicing regulation during cell state changes.

BACKGROUND: Alternative splicing is a key mechanism underlying cellular differentiation and a driver of complexity in mammalian neuronal tissues. However, understanding of which isoforms are differentially used or expressed and how this affects cellular differentiation remains unclear. Long read sequencing allows full-length transcript recovery and quantification, enabling transcript-level analysis of alternative splicing processes and how these change with cell state. Here, we utilise Oxford Nanopore Technologies sequencing to produce a custom annotation of a well-studied human neuroblastoma cell line SH-SY5Y, and to characterise isoform expression and usage across differentiation. RESULTS: We identify many previously unannotated features, including a novel transcript of the voltage-gated calcium channel subunit gene, CACNA2D2. We show differential expression and usage of transcripts during differentiation identifying candidates for future research into state change regulation. CONCLUSIONS: Our work highlights the potential of long read sequencing to uncover previously unknown transcript diversity and mechanisms influencing alternative splicing.

Genome-Wide Transcription During Early Wheat Meiosis Is Independent of Synapsis, Ploidy Level, and the Ph1 Locus.

Polyploidization is a fundamental process in plant evolution. One of the biggest challenges faced by a new polyploid is meiosis, particularly discriminating between multiple related chromosomes so that only homologous chromosomes synapse and recombine to ensure regular chromosome segregation and balanced gametes. Despite its large genome size, high DNA repetitive content and similarity between homoeologous chromosomes, hexaploid wheat completes meiosis in a shorter period than diploid species with a much smaller genome. Therefore, during wheat meiosis, mechanisms additional to the classical model based on DNA sequence homology, must facilitate more efficient homologous recognition. One such mechanism could involve exploitation of differences in chromosome structure between homologs and homoeologs at the onset of meiosis. In turn, these chromatin changes, can be expected to be linked to transcriptional gene activity. In this study, we present an extensive analysis of a large RNA-seq data derived from six different genotypes: wheat, wheat-rye hybrids and newly synthesized octoploid triticale, both in the presence and absence of the Ph1 locus. Plant material was collected at early prophase, at the transition leptotene-zygotene, when the telomere bouquet is forming and synapsis between homologs is beginning. The six genotypes exhibit different levels of synapsis and chromatin structure at this stage; therefore, recombination and consequently segregation, are also different. Unexpectedly, our study reveals that neither synapsis, whole genome duplication nor the absence of the Ph1 locus are associated with major changes in gene expression levels during early meiotic prophase. Overall wheat transcription at this meiotic stage is therefore highly resilient to such alterations, even in the presence of major chromatin structural changes. Further studies in wheat and other polyploid species will be required to reveal whether these observations are specific to wheat meiosis.

An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations.

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

Heterarchy of transcription factors driving basal and luminal cell phenotypes in human urothelium.

Cell differentiation is affected by complex networks of transcription factors that co-ordinate re-organisation of the chromatin landscape. The hierarchies of these relationships can be difficult to dissect. During in vitro differentiation of normal human uro-epithelial cells, formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) and RNA-seq was used to identify alterations in chromatin accessibility and gene expression changes following activation of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) as a differentiation-initiating event. Regions of chromatin identified by FAIRE-seq, as having altered accessibility during differentiation, were found to be enriched with sequence-specific binding motifs for transcription factors predicted to be involved in driving basal and differentiated urothelial cell phenotypes, including forkhead box A1 (FOXA1), P63, GRHL2, CTCF and GATA-binding protein 3 (GATA3). In addition, co-occurrence of GATA3 motifs was observed within subsets of differentiation-specific peaks containing P63 or FOXA1. Changes in abundance of GRHL2, GATA3 and P63 were observed in immunoblots of chromatin-enriched extracts. Transient siRNA knockdown of P63 revealed that P63 favoured a basal-like phenotype by inhibiting differentiation and promoting expression of basal marker genes. GATA3 siRNA prevented differentiation-associated downregulation of P63 protein and transcript, and demonstrated positive feedback of GATA3 on PPARG transcript, but showed no effect on FOXA1 transcript or protein expression. This approach indicates that as a transcriptionally regulated programme, urothelial differentiation operates as a heterarchy, wherein GATA3 is able to co-operate with FOXA1 to drive expression of luminal marker genes, but that P63 has potential to transrepress expression of the same genes.

Microbiota and metabolite profiling reveal specific alterations in bacterial community structure and environment in the cystic fibrosis airway during exacerbation.

Chronic polymicrobial infections of the lung are the foremost cause of morbidity and mortality in cystic fibrosis (CF) patients. The composition of the microbial flora of the airway alters considerably during infection, particularly during patient exacerbation. An understanding of which organisms are growing, their environment and their behaviour in the airway is of importance for designing antibiotic treatment regimes and for patient prognosis. To this end, we have analysed sputum samples taken from separate cohorts of CF and non-CF subjects for metabolites and in parallel, and we have examined both isolated DNA and RNA for the presence of 16S rRNA genes and transcripts by high-throughput sequencing of amplicon or cDNA libraries. This analysis revealed that although the population size of all dominant orders of bacteria as measured by DNA- and RNA- based methods are similar, greater discrepancies are seen with less prevalent organisms, some of which we associated with CF for the first time. Additionally, we identified a strong relationship between the abundance of specific anaerobes and fluctuations in several metabolites including lactate and putrescine during patient exacerbation. This study has hence identified organisms whose occurrence within the CF microbiome has been hitherto unreported and has revealed potential metabolic biomarkers for exacerbation.