A multi-omic Nicotiana benthamiana resource for fundamental research and biotechnology.

Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

Assessment of root-specific promoters in banana and tobacco and identification of a banana TIP2 promoter with strong root activity.

Genetic modification is one possible strategy to generate bananas (Musa spp.) with resistance to the soil-borne pathogen causing Fusarium wilt. The availability of banana root-specific promoters to target transgene expression to the sites of infection would be beneficial. We have assessed 18 promoter sequences derived from a range of plant species for their expression profiles in banana tissues to identify those with root-specific activity. Promoter sequences were isolated and fused to the ß-glucuronidase (GUS) gene to assess their expression levels and tissue specificity in both banana and the model plant tobacco. Two heterologous promoters conferring high root expression levels in banana were identified, including a ß-glucosidase 1 (GLU1) promoter from maize and the RB7-type tonoplast intrinsic protein (TIP)-2 promoter from strawberry. Further, a novel Musa TIP2-2 promoter sequence was isolated and characterized which, when fused to the GUS gene, conferred very high GUS expression levels in banana roots. These promoters will expand the options for the control of gene expression in genetically modified bananas, providing a tool to develop plants with resistance not only to soil-borne diseases such as Fusarium wilt, but also for the improvement of other traits, such as nematode resistance, nutrition or abiotic stress resistance.

Triple action of FeCl3-assisted hydrothermal treatment of digested sludge for deep dewatering.

In this study, a FeCl3-assisted hydrothermal treatment (HTT) process under mild conditions (90 °C-130 °C) was developed for deep dewatering of anaerobically digested sludge. HTT of sludge at 90 °C-130 °C with 4%-6% Fe3+ ions loading based on total sludge solids followed by mechanical dewatering reduced sludge water content from 82% to 38%-53% and sludge weight by 62%-72%. The treatment increased the flowability of sludge through reduction of apparent viscosity and disintegration of colloidal forces between sludge particles. This study unveiled that FeCl3-assisted HTT process had three mechanisms for improving sludge dewaterability and flowability. The treatment hydrolysed sludge flocs in the presence of Lewis acid FeCl3 and high temperature (90-130 °C). Fe3+ ions also improved dewaterability through the formation of double electric layers and neutralisation of surface negative charges, leading to flocculation of sludge flocs. More importantly, the hydrolysed sludge components produced during HTT process acted as reducing agents and led to in-situ generation of iron oxyhydroxide nanoparticles through reduction-oxidation reactions, further enhancing flocculation/co-precipitation of sludge flocs. The treatment reduced EPS content and changed conformational structures of EPS proteins by breaking down hydrogen bond-maintaining α-helix which led to a loose EPS protein structure and enhanced hydrophobicity and flocculability. Furthermore, the FeCl3-assisted treatment promoted immobilisation of the majority of heavy metals in the sludge matrix through co-precipitation/complexation reactions with iron species and organic/inorganic matters. This indicates that the FeCl3-assisted treatment reduced direct toxicity/bioavailability of the majority of heavy metals and the treated sludge may be suitable for land application. Overall, this study provides new insights into mechanism of FeCl3-assisted HTT process for dewaterability of anaerobically digested sludge and immobilisation of heavy metals.

Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species.

Next-generation sequencing technologies rely on high quality DNA that is suitable for library preparation followed by sequencing. Some plant species store large amounts of phenolics and polysaccharides within their leaf tissue making genomic DNA extraction difficult. While many DNA extraction methods exist that contend with the presence of phenolics and polysaccharides, these methods rely on long incubations, multiple precipitations or commercially available kits to produce high molecular weight and contaminant-free DNA. In this protocol, we describe simple modifications to the established CTAB- based extraction method that allows for reliable isolation of high molecular weight genomic DNA from difficult to isolate plant species Corymbia (a eucalypt) and Coffea (coffee). The simplified protocol does not require multiple clean up steps or commercial based kits, and the isolated DNA passed stringent quality control standards for whole genome sequencing on Illumina HiSeq and TruSeq sequencing platforms.