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Aspalathin, the main polyphenol of rooibos (Aspalathus linearis), is associated with diverse health promoting properties of the tea. During fermentation, aspalathin is oxidized and concentrations are significantly reduced. Standardized methods for quality control of rooibos products do not investigate aspalathin, since current techniques of aspalathin detection require expensive equipment and expertise. Here, we describe a simple and fast thin-layer chromatography (TLC) method that can reproducibly visualize aspalathin in rooibos herbal tea and plant extracts at a limit of detection (LOD) equal to 178.7 ng and a limit of quantification (LOQ) equal to 541.6 ng. Aspalathin is a rare compound, so far only found in A. linearis and its (rare) sister species A. pendula. Therefore, aspalathin could serve as a marker compound for authentication and quality control of rooibos products, and the described TLC method represents a cost-effective approach for high-throughput screening of plant and herbal tea extracts.

Symbiotic associations between plants and fungi are a dominant feature of many terrestrial ecosystems, yet relatively little is known about the signaling, and associated transcriptome profiles, that define the symbiotic metabolic state. Using the Epichloë festucae-perennial ryegrass (Lolium perenne) association as a model symbiotic experimental system, we show an essential role for the fungal stress-activated mitogen-activated protein kinase (sakA) in the establishment and maintenance of this mutualistic interaction. Deletion of sakA switches the fungal interaction with the host from mutualistic to pathogenic. Infected plants exhibit loss of apical dominance, premature senescence, and dramatic changes in development, including the formation of bulb-like structures at the base of tillers that lack anthocyanin pigmentation. A comparison of the transcriptome of wild-type and sakA associations using high-throughput mRNA sequencing reveals dramatic changes in fungal gene expression consistent with the transition from restricted to proliferative growth, including a down-regulation of several clusters of secondary metabolite genes and up-regulation of a large set of genes that encode hydrolytic enzymes and transporters. Analysis of the plant transcriptome reveals up-regulation of host genes involved in pathogen defense and transposon activation as well as dramatic changes in anthocyanin and hormone biosynthetic/responsive gene expression. These results highlight the fine balance between mutualism and antagonism in a plant-fungal interaction and the power of deep mRNA sequencing to identify candidate sets of genes underlying the symbiosis.

Plant genomes provide information on biosynthetic pathways involved in the production of industrially relevant compounds. Genome size estimates are essential for the initiation of genome projects. The genome size of rooibos (Aspalathus linearis species complex) was estimated using DAPI flow cytometry and k-mer analyses. For flow cytometry, a suitable nuclei isolation buffer, plant tissue and a transport medium for rooibos ecotype samples collected from distant locations were identified. When using radicles from commercial rooibos seedlings, Woody Plant Buffer and Vicia faba as an internal standard, the flow cytometry-estimated genome size of rooibos was 1.24 ± 0.01 Gbp. The estimates for eight wild rooibos growth types did not deviate significantly from this value. K-mer analysis was performed using Illumina paired-end sequencing data from one commercial rooibos genotype. For biocomputational estimation of the genome size, four k-mer analysis methods were investigated: A standard formula and three popular programs (BBNorm, GenomeScope, and FindGSE). GenomeScope estimates were strongly affected by parameter settings, specifically CovMax.

While plant genome analysis is gaining speed worldwide, few plant genomes have been sequenced and analyzed on the African continent. Yet, this information holds the potential to transform diverse industries as it unlocks medicinally and industrially relevant biosynthesis pathways for bioprospecting. Considering that South Africa is home to the highly diverse Cape Floristic Region, local establishment of methods for plant genome analysis is essential. Long-read sequencing is becoming standard procedure for plant genome research, as these reads can span repetitive regions of the DNA, substantially facilitating reassembly of a contiguous genome. With the MinION, Oxford Nanopore offers a cost-efficient sequencing method to generate long reads; however, DNA purification protocols must be adapted for each plant species to generate ultra-pure DNA, essential for these analyses.

In western North America, the current outbreak of the mountain pine beetle (MPB) and its microbial associates has destroyed wide areas of lodgepole pine forest, including more than 16 million hectares in British Columbia. Grosmannia clavigera (Gc), a critical component of the outbreak, is a symbiont of the MPB and a pathogen of pine trees. To better understand the interactions between Gc, MPB, and lodgepole pine hosts, we sequenced the ∼30-Mb Gc genome and assembled it into 18 supercontigs. We predict 8,314 protein-coding genes, and support the gene models with proteome, expressed sequence tag, and RNA-seq data. We establish that Gc is heterothallic, and report evidence for repeat-induced point mutation. We report insights, from genome and transcriptome analyses, into how Gc tolerates conifer-defense chemicals, including oleoresin terpenoids, as they colonize a host tree. RNA-seq data indicate that terpenoids induce a substantial antimicrobial stress in Gc, and suggest that the fungus may detoxify these chemicals by using them as a carbon source. Terpenoid treatment strongly activated a ∼100-kb region of the Gc genome that contains a set of genes that may be important for detoxification of these host-defense chemicals. This work is a major step toward understanding the biological interactions between the tripartite MPB/fungus/forest system.

Background Subtilisin-like proteases (SLPs) form a superfamily of enzymes that act to degrade protein substrates. In fungi, SLPs can play either a general nutritive role, or may play specific roles in cell metabolism, or as pathogenicity or virulence factors. Results Fifteen different genes encoding SLPs were identified in the genome of the grass endophytic fungus Epichloë festucae . Phylogenetic analysis indicated that these SLPs belong to four different subtilisin families: proteinase K, kexin, pyrolysin and subtilisin. The pattern of intron loss and gain is consistent with this phylogeny. E. festucae is exceptional in that it contains two kexin-like genes. Phylogenetic analysis in Hypocreales fungi revealed an extensive history of gene loss and duplication. Conclusion This study provides new insights into the evolution of the SLP superfamily in filamentous fungi.

Background De novo transcriptome assembly of short transcribed fragments (transfrags) produced from sequencing-by-synthesis technologies often results in redundant datasets with differing levels of unassembled, partially assembled or mis-assembled transcripts. Post-assembly processing intended to reduce redundancy typically involves reassembly or clustering of assembled sequences. However, these approaches are mostly based on common word heuristics and often create clusters of biologically unrelated sequences, resulting in loss of unique transfrags annotations and propagation of mis-assemblies. Results Here, we propose a structured framework that consists of a few steps in pipeline architecture for I nferring F unctionally R elevant A ssembly-derived T ranscripts (IFRAT). IFRAT combines 1) removal of identical subsequences, 2) error tolerant CDS prediction, 3) identification of coding potential, and 4) complements BLAST with a multiple domain architecture annotation that reduces non-specific domain annotation. We demonstrate that independent of the assembler, IFRAT selects bona fide transfrags (with CDS and coding potential) from the transcriptome assembly of a model organism without relying on post-assembly clustering or reassembly. The robustness of IFRAT is inferred on RNA-Seq data of Neurospora crassa assembled using de Bruijn graph-based assemblers, in single (Trinity and Oases-25) and multiple (Oases-Merge and additive or pooled) k -mer modes. Single k -mer assemblies contained fewer transfrags compared to the multiple k -mer assemblies. However, Trinity identified a comparable number of predicted coding sequence and gene loci to Oases pooled assembly. IFRAT selects bona fide transfrags representing over 94% of cumulative BLAST-derived functional annotations of the unfiltered assemblies. Between 4-6% are lost when orphan transfrags are excluded and this represents only a tiny fraction of annotation derived from functional transference by sequence similarity. The median length of bona fide transfrags ranged from 1.5kb (Trinity) to 2kb (Oases), which is consistent with the average coding sequence length in fungi. The fraction of transfrags that could be associated with gene ontology terms ranged from 33-50%, which is also high for domain based annotation. We showed that unselected transfrags were mostly truncated and represent sequences from intronic, untranslated (5′ and 3′) regions and non-coding gene loci. Conclusions IFRAT simplifies post-assembly processing providing a reference transcriptome enriched with functionally relevant assembly-derived transcripts for non-model organism.

Background Grosmannia clavigera is a bark beetle-vectored fungal pathogen of pines that causes wood discoloration and may kill trees by disrupting nutrient and water transport. Trees respond to attacks from beetles and associated fungi by releasing terpenoid and phenolic defense compounds. It is unclear which genes are important for G. clavigera 's ability to overcome antifungal pine terpenoids and phenolics. Results We constructed seven cDNA libraries from eight G. clavigera isolates grown under various culture conditions, and Sanger sequenced the 5' and 3' ends of 25,000 cDNA clones, resulting in 44,288 high quality ESTs. The assembled dataset of unique transcripts (unigenes) consists of 6,265 contigs and 2,459 singletons that mapped to 6,467 locations on the G. clavigera reference genome, representing ~70% of the predicted G. clavigera genes. Although only 54% of the unigenes matched characterized proteins at the NCBI database, this dataset extensively covers major metabolic pathways, cellular processes, and genes necessary for response to environmental stimuli and genetic information processing. Furthermore, we identified genes expressed in spores prior to germination, and genes involved in response to treatment with lodgepole pine phloem extract (LPPE). Conclusions We provide a comprehensively annotated EST dataset for G. clavigera that represents a rich resource for gene characterization in this and other ophiostomatoid fungi. Genes expressed in response to LPPE treatment are indicative of fungal oxidative stress response. We identified two clusters of potentially functionally related genes responsive to LPPE treatment. Furthermore, we report a simple method for identifying contig misassemblies in de novo assembled EST collections caused by gene overlap on the genome.

Rooibos (Aspalathus linearis), widely known as a herbal tea, is endemic to the Cape Floristic Region of South Africa (SA). It produces a wide range of phenolic compounds that have been associated with diverse health promoting properties of the plant. The species comprises several growth forms that differ in their morphology and biochemical composition, only one of which is cultivated and used commercially. Here, we established methodologies for non-invasive transcriptome research of wild-growing South African plant species, including (1) harvesting and transport of plant material suitable for RNA sequencing; (2) inexpensive, high-throughput biochemical sample screening; (3) extraction of high-quality RNA from recalcitrant, polysaccharide- and polyphenol rich plant material; and (4) biocomputational analysis of Illumina sequencing data, together with the evaluation of programs for transcriptome assembly (Trinity, IDBA-Trans, SOAPdenovo-Trans, CLC), protein prediction, as well as functional and taxonomic transcript annotation. In the process, we established a biochemically characterized sample pool from 44 distinct rooibos ecotypes (1–5 harvests) and generated four in-depth annotated transcriptomes (each comprising on average ≈86,000 transcripts) from rooibos plants that represent distinct growth forms and differ in their biochemical profiles. These resources will serve future rooibos research and plant breeding endeavours.