Peter R. Sternes

Design rules for efficient transgene expression in plants.

Sustained expression of transgenes in specified developmental patterns is commonly needed in plant biotechnology, but obstructed by transgene silencing. Here, we present a set of gene design rules, tested on the silencing-susceptible beetle luc and bacterial ims genes, expressed in sugarcane. Designs tested independently or in combination included removal of rare codons, removal of RNA instability sequences, blocking of likely endogenous sRNA binding sites and randomization of non-rare codons. Stable transgene expression analyses, on multiple independent lines per construct, showed greatest improvement from the removal of RNA instability sequences, accompanied by greatly reduced transcript degradation evident in northern blot analysis. We provide a set of motifs that readily can be eliminated concurrently with rare codons and undesired structural features such as repeat sequences, using Gene Designer 2.0 software. These design rules yielded 935- and 5-fold increased expression in transgenic callus, relative to the native luc and ims sequences; and gave sustained expression under the control of sugarcane and heterologous promoters over several years in greenhouse and field trials. The rules can be applied easily with codon usage tables from any plant species, providing a simple and effective means to achieve sustained expression of otherwise silencing-prone transgenes in plants.

Deep Sequencing Reveals Divergent Expression Patterns Within the Small RNA Transcriptomes of Cultured and Vegetative Tissues of Sugarcane

Deep sequencing has advanced the discovery and analysis of the small RNA component of transcriptomes and has revealed developmentally-regulated populations of small RNAs consistent with key roles in plant development. To study small RNA transcriptome complexity and explore their roles in sugarcane development, we obtained almost 50 million small RNA reads from suspension cells, embryogenic calli, leaf, apex and a developmental series of stem internodes. The complexity of the small RNA component of the transcriptome varied between tissues. The undifferentiated and young tissue type libraries had lower redundancy levels than libraries generated from maturing and mature tissues. The ratio of 21:24 nt small RNAs also varied widely between different tissue types, as did the proportion of abundant small RNAs derived from each putative origin of small RNA biogenesis. Cluster analysis indicates many abundant small RNAs display developmental expression patterns. There was substantial variation in isomiR composition, abundance and expression patterns within sugarcane microRNA (miRNA) families. Two hundred and fifty-six isomiRs from 36 miRNA families were identified by homology to known miRNA families from a range of plant species. Many isomiRs and miRNA families appear to be developmentally regulated, including a subset of miRNAs that are progressively upregulated during stem internode maturation. Transcribed sequences putatively targeted by abundant sugarcane small RNAs were predicted and miRNA directed cleavage of 18 predicted sugarcane targets were validated by 5′ RACE.

A combined meta-barcoding and shotgun metagenomic analysis of spontaneous wine fermentation

Abstract Wine is a complex beverage, comprising hundreds of metabolites produced through the action of yeasts and bacteria in fermenting grape must. Commercially, there is now a growing trend away from using wine yeast (Saccharomyces) starter cultures, toward the historic practice of uninoculated or “wild” fermentation, where the yeasts and bacteria associated with the grapes and/or winery perform the fermentation. It is the varied metabolic contributions of these numerous non-Saccharomyces species that are thought to impart complexity and desirable taste and aroma attributes to wild ferments in comparison to their inoculated counterparts. To map the microflora of spontaneous fermentation, metagenomic techniques were employed to characterize and monitor the progression of fungal species in 5 different wild fermentations. Both amplicon-based ribosomal DNA internal transcribed spacer (ITS) phylotyping and shotgun metagenomics were used to assess community structure across different stages of fermentation. While providing a sensitive and highly accurate means of characterizing the wine microbiome, the shotgun metagenomic data also uncovered a significant overabundance bias in the ITS phylotyping abundance estimations for the common non-Saccharomyces wine yeast genus Metschnikowia. By identifying biases such as that observed for Metschnikowia, abundance measurements from future ITS phylotyping datasets can be corrected to provide more accurate species representation. Ultimately, as more shotgun metagenomic and single-strain de novo assemblies for key wine species become available, the accuracy of both ITS-amplicon and shotgun studies will greatly increase, providing a powerful methodology for deciphering the influence of the microbial community on the wine flavor and aroma.

Genome Sequences of Three Species of Hanseniaspora Isolated from Spontaneous Wine Fermentations

ABSTRACT Members of the genus Hanseniaspora represent a significant proportion of the normal flora of grape berries and play a significant role in wine fermentation. Here, we present genome sequences for three species of Hanseniaspora, H. opuntiae, H. osmophila, and H. uvarum, which were isolated from spontaneous Chardonnay wine fermentation.

Whole transcriptome RNAseq analysis of Oenococcus oeni reveals distinct intra-specific expression patterns during malolactic fermentation, including genes involved in diacetyl metabolism

We report the first whole transcriptome RNAseq analysis of the wine-associated lactic acid bacterium Oenococcus oeni using a combination of reference-based mapping and de novo transcript assembly in three distinct strains during malolactic fermentation in Cabernet Sauvignon wine. Two of the strains (AWRIB551 and AWRIB552) exhibited similar transcriptomes relative to the third strain (AWRIB419) which was dissimilar by comparison. Significant intra-specific variation for genes related to glycolysis/gluconeogenesis, purine metabolism, aminoacyl-tRNA biosynthesis, ABC transporters and phosphotransferase systems was observed. Importantly, thirteen genes associated with the production of diacetyl, a commercially valuable aroma and flavour compound, were also found to be differentially expressed between the strains in a manner that correlated positively with total diacetyl production. This included a key strain-specific gene that is predicted to encode a l-lactate dehydrogenase that may enable l-lactic acid to be utilised as a precursor for the production of diacetyl. In conjunction with previous comparative genomic studies of O. oeni, this study progresses the understanding of genetic variations which contribute to the phenotypes of this industrially-important bacterium.

Erratum to: ‘Consensus pan-genome assembly of the specialised wine bacterium Oenococcus oeni’

Unfortunately, the version of this article as originally published [1] contained an error. The quality of Figs. ​Figs.1,1, ​,2,2, ​,3,3, ​,4,4, ​,5,5, ​,66 and ​and77 were affected due to an error in sampling the original images and an incorrect version of the article being used during the production process. For the sake of clarity, the legible Figs. ​Figs.1,1, ​,2,2, ​,3,3, ​,4,4, ​,5,5, ​,66 and ​and77 have been included below.