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April 21, 2020

A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana.

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.Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

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April 21, 2020

PacBio sequencing reveals bacterial community diversity in cheeses collected from different regions.

Cheese is a fermented dairy product that is popular for its unique flavor and nutritional value. Recent studies have shown that microorganisms in cheese play an important role in the fermentation process and determine the quality of the cheese. We collected 12 cheese samples from different regions and studied the composition of their bacterial communities using PacBio small-molecule real-time sequencing (Pacific Biosciences, Menlo Park, CA). Our data revealed 144 bacterial genera (including Lactobacillus, Streptococcus, Lactococcus, and Staphylococcus) and 217 bacterial species (including Lactococcus lactis, Streptococcus thermophilus, Staphylococcus equorum, and Streptococcus uberis). We investigated the flavor quality of the cheese samples using an electronic nose system and we found differences in flavor-quality indices among samples from different regions. We found a clustering tendency based on flavor quality using principal component analysis. We found correlations between lactic acid bacteria and the flavor quality of the cheese samples. Biodegradation and metabolism of xenobiotics, and lipid-metabolism-related pathways, were predicted to contribute to differences in cheese flavor using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). This preliminary study explored the bacterial communities in cheeses collected from different regions and their potential genome functions from the perspective of flavor quality.Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Comparative genome analysis provides novel insight into the interaction of Aquimarina sp. AD1, BL5 and AD10 with their macroalgal host.

The Aquimarina genus is widely distributed throughout the marine environment, however little is understood regarding its ecological role, particularly when in association with eukaryotic hosts. Here, we examine the genomes of two opportunistic pathogens, Aquimarina sp. AD1 and BL5, and a non-pathogenic strain Aquimarina sp. AD10, that were isolated from diseased individuals of the red alga Delisea pulchra. Each strain encodes multiple genes for the degradation of marine carbohydrates and vitamin biosynthesis. These traits are hypothesised to promote nutrient exchange between the Aquimarina strains and their algal host, facilitating a close symbiotic relationship. Moreover, each strain harbours the necessary genes for the assembly of a Type 9 Secretion System (T9SS) and the associated gliding motility apparatus. In addition to these common features, pathogenic strains AD1 and BL5, encode genes for the production of flexirubin type pigments and a number of unique non-ribosomal peptide synthesis (NRPS) gene clusters, suggesting a role for these uncharacterised traits in virulence. This study provides valuable insight into the potential ecological role of Aquimarina in the marine environment and the complex factors driving pathogenesis and symbiosis in this genus.Copyright © 2019 Elsevier B.V. All rights reserved.

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April 21, 2020

The genome sequence of Streptomyces rochei 7434AN4, which carries a linear chromosome and three characteristic linear plasmids.

Streptomyces rochei 7434AN4 produces two structurally unrelated polyketide antibiotics, lankacidin and lankamycin, and carries three linear plasmids, pSLA2-L (211?kb), -M (113?kb), and -S (18?kb), whose nucleotide sequences were previously reported. The complete nucleotide sequence of the S. rochei chromosome has now been determined using the long-read PacBio RS-II sequencing together with short-read Illumina Genome Analyzer IIx sequencing and Roche 454 pyrosequencing techniques. The assembled sequence revealed an 8,364,802-bp linear chromosome with a high G?+?C content of 71.7% and 7,568 protein-coding ORFs. Thus, the gross genome size of S. rochei 7434AN4 was confirmed to be 8,706,406?bp including the three linear plasmids. Consistent with our previous study, a tap-tpg gene pair, which is essential for the maintenance of a linear topology of Streptomyces genomes, was not found on the chromosome. Remarkably, the S. rochei chromosome contains seven ribosomal RNA (rrn) operons (16S-23S-5S), although Streptomyces species generally contain six rrn operons. Based on 2ndFind and antiSMASH platforms, the S. rochei chromosome harbors at least 35 secondary metabolite biosynthetic gene clusters, including those for the 28-membered polyene macrolide pentamycin and the azoxyalkene compound KA57-A.

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April 21, 2020

Metatranscriptomic evidence for classical and RuBisCO-mediated CO2 reduction to methane facilitated by direct interspecies electron transfer in a methanogenic system.

In a staged anaerobic fluidized-bed ceramic membrane bioreactor, metagenomic and metatranscriptomic analyses were performed to decipher the microbial interactions on the granular activated carbon. Metagenome bins, representing the predominating microbes in the bioreactor: syntrophic propionate-oxidizing bacteria (SPOB), acetoclastic Methanothrix concilii, and exoelectrogenic Geobacter lovleyi, were successfully recovered for the reconstruction and analysis of metabolic pathways involved in the transformation of fatty acids to methane. In particular, SPOB degraded propionate into acetate, which was further converted into methane and CO2 by M. concilii via the acetoclastic methanogenesis. Concurrently, G. lovleyi oxidized acetate into CO2, releasing electrons into the extracellular environment. By accepting these electrons through direct interspecies electron transfer (DIET), M. concilii was capable of performing CO2 reduction for further methane formation. Most notably, an alternative RuBisCO-mediated CO2 reduction (the reductive hexulose-phosphate (RHP) pathway) is transcriptionally-active in M. concilii. This RHP pathway enables M. concilii dominance and energy gain by carbon fixation and methanogenesis, respectively via a methyl-H4MPT intermediate, constituting the third methanogenesis route. The complete acetate reduction (2 mole methane formation/1 mole acetate consumption), coupling of acetoclastic methanogenesis and two CO2 reduction pathways, are thermodynamically favorable even under very low substrate condition (down to to 10-5?M level). Such tight interactions via both mediated and direct interspecies electron transfer (MIET and DIET), induced by the conductive GAC promote the overall efficiency of bioenergy processes.

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April 21, 2020

Modulation of metabolome and bacterial community in whole crop corn silage by inoculating homofermentative Lactobacillus plantarum and heterofermentative Lactobacillus buchneri.

The present study investigated the species level based microbial community and metabolome in corn silage inoculated with or without homofermentative Lactobacillus plantarum and heterofermentative Lactobacillus buchneri using the PacBio SMRT Sequencing and time-of-flight mass spectrometry (GC-TOF/MS). Chopped whole crop corn was treated with (1) deionized water (control), (2) Lactobacillus plantarum, or (3) Lactobacillus buchneri. The chopped whole crop corn was ensiled in vacuum-sealed polyethylene bags containing 300 g of fresh forge for 90 days, with three replicates for each treatment. The results showed that a total of 979 substances were detected, and 316 different metabolites were identified. Some metabolites with antimicrobial activity were detected in whole crop corn silage, such as catechol, 3-phenyllactic acid, 4-hydroxybenzoic acid, azelaic acid, 3,4-dihydroxybenzoic acid and 4-hydroxycinnamic acid. Catechol, pyrogallol and ferulic acid with antioxidant property, 4-hydroxybutyrate with nervine activity, and linoleic acid with cholesterol lowering effects, were detected in present study. In addition, a flavoring agent of myristic acid and a depression mitigation substance of phenylethylamine were also found in this study. Samples treated with inoculants presented more biofunctional metabolites of organic acids, amino acids and phenolic acids than untreated samples. The Lactobacillus species covered over 98% after ensiling, and were mainly comprised by the L. acetotolerans, L. silagei, L. parafarraginis, L. buchneri and L. odoratitofui. As compared to the control silage, inoculation of L. plantarum increased the relative abundances of L. acetotolerans, L. buchneri and L. parafarraginis, and a considerable decline in the proportion of L. silagei was observed; whereas an obvious decrease in L. acetotolerans and increases in L. odoratitofui and L. farciminis were observed in the L. buchneri inoculated silage. Therefore, inoculation of L. plantarum and L. buchneri regulated the microbial composition and metabolome of the corn silage with different behaviors. The present results indicated that profiling of silage microbiome and metabolome might improve our current understanding of the biological process underlying silage formation.

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April 21, 2020

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea’s unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762?bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.

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April 21, 2020

Complete Genome Sequence of Sequevar 14M Ralstonia solanacearum Strain HA4-1 Reveals Novel Type III Effectors Acquired Through Horizontal Gene Transfer.

Ralstonia solanacearum, which causes bacterial wilt in a broad range of plants, is considered a “species complex” due to its significant genetic diversity. Recently, we have isolated a new R. solanacearum strain HA4-1 from Hong’an county in Hubei province of China and identified it being phylotype I, sequevar 14M (phylotype I-14M). Interestingly, we found that it can cause various disease symptoms among different potato genotypes and display different pathogenic behavior compared to a phylogenetically related strain, GMI1000. To dissect the pathogenic mechanisms of HA4-1, we sequenced its whole genome by combined sequencing technologies including Illumina HiSeq2000, PacBio RS II, and BAC-end sequencing. Genome assembly results revealed the presence of a conventional chromosome, a megaplasmid as well as a 143 kb plasmid in HA4-1. Comparative genome analysis between HA4-1 and GMI1000 shows high conservation of the general virulence factors such as secretion systems, motility, exopolysaccharides (EPS), and key regulatory factors, but significant variation in the repertoire and structure of type III effectors, which could be the determinants of their differential pathogenesis in certain potato species or genotypes. We have identified two novel type III effectors that were probably acquired through horizontal gene transfer (HGT). These novel R. solanacearum effectors display homology to several YopJ and XopAC family members. We named them as RipBR and RipBS. Notably, the copy of RipBR on the plasmid is a pseudogene, while the other on the megaplasmid is normal. For RipBS, there are three copies located in the megaplasmid and plasmid, respectively. Our results have not only enriched the genome information on R. solanacearum species complex by sequencing the first sequevar 14M strain and the largest plasmid reported in R. solanacearum to date but also revealed the variation in the repertoire of type III effectors. This will greatly contribute to the future studies on the pathogenic evolution, host adaptation, and interaction between R. solanacearum and potato.

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April 21, 2020

Comparative Genomics of Marine Sponge-Derived Streptomyces spp. Isolates SM17 and SM18 With Their Closest Terrestrial Relatives Provides Novel Insights Into Environmental Niche Adaptations and Secondary Metabolite Biosynthesis Potential.

The emergence of antibiotic resistant microorganisms has led to an increased need for the discovery and development of novel antimicrobial compounds. Frequent rediscovery of the same natural products (NPs) continues to decrease the likelihood of the discovery of new compounds from soil bacteria. Thus, efforts have shifted toward investigating microorganisms and their secondary metabolite biosynthesis potential, from diverse niche environments, such as those isolated from marine sponges. Here we investigated at the genomic level two Streptomyces spp. strains, namely SM17 and SM18, isolated from the marine sponge Haliclona simulans, with previously reported antimicrobial activity against clinically relevant pathogens; using single molecule real-time (SMRT) sequencing. We performed a series of comparative genomic analyses on SM17 and SM18 with their closest terrestrial relatives, namely S. albus J1074 and S. pratensis ATCC 33331 respectively; in an effort to provide further insights into potential environmental niche adaptations (ENAs) of marine sponge-associated Streptomyces, and on how these adaptations might be linked to their secondary metabolite biosynthesis potential. Prediction of secondary metabolite biosynthetic gene clusters (smBGCs) indicated that, even though the marine isolates are closely related to their terrestrial counterparts at a genomic level; they potentially produce different compounds. SM17 and SM18 displayed a better ability to grow in high salinity medium when compared to their terrestrial counterparts, and further analysis of their genomes indicated that they possess a pool of 29 potential ENA genes that are absent in S. albus J1074 and S. pratensis ATCC 33331. This ENA gene pool included functional categories of genes that are likely to be related to niche adaptations and which could be grouped based on potential biological functions such as osmotic stress, defense; transcriptional regulation; symbiotic interactions; antimicrobial compound production and resistance; ABC transporters; together with horizontal gene transfer and defense-related features.

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April 21, 2020

Hybrid sequencing of the Gynostemma pentaphyllum transcriptome provides new insights into gypenoside biosynthesis.

Gypenosides are a group of triterpene saponins from Gynostemma pentaphyllum that are the same as or very similar to ginsenosides from the Panax species. Several enzymes involved in ginsenoside biosynthesis have been characterized, which provide important clues for elucidating the gypenoside biosynthetic pathway. We suppose that gypenosides and ginsenosides may have a similar biosynthetic mechanism and that the corresponding enzymes in the two pathways may have considerable similarity in their sequences. To further understand gypenoside biosynthesis, we sequenced the G. pentaphyllum transcriptome with a hybrid sequencing-based strategy and then determined the candidate genes involved in this pathway using phylogenetic tree construction and gene expression analysis.Following the PacBio standard analysis pipeline, 66,046 polished consensus sequences were obtained, while Illumina data were assembled into 140,601 unigenes with Trinity software. Then, these output sequences from the two analytical routes were merged. After removing redundant data with CD-HIT software, a total of 140,157 final unigenes were obtained. After functional annotation, five 2,3-oxidosqualene cyclase genes, 145 cytochrome P450 genes and 254 UDP-glycosyltransferase genes were selected for the screening of genes involved in gypenoside biosynthesis. Using phylogenetic analysis, several genes were divided into the same subfamilies or closely related evolutionary branches with characterized enzymes involved in ginsenoside biosynthesis. Using real-time PCR technology, their expression patterns were investigated in different tissues and at different times after methyl jasmonate induction. Since the genes in the same biosynthetic pathway are generally coexpressed, we speculated that GpOSC1, GpCYP89, and GpUGT35 were the leading candidates for gypenoside biosynthesis. In addition, six GpWRKYs and one GpbHLH might play a possible role in regulating gypenoside biosynthesis.We developed a hybrid sequencing strategy to obtain longer length transcriptomes with increased accuracy, which will greatly contribute to downstream gene screening and characterization, thus improving our ability to elucidate secondary metabolite biosynthetic pathways. With this strategy, we found several candidate genes that may be involved in gypenoside biosynthesis, which laid an important foundation for the elucidation of this biosynthetic pathway, thus greatly contributing to further research in metabolic regulation, synthetic biology and molecular breeding in this species.

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April 21, 2020

Prediction of Host-Specific Genes by Pan-Genome Analyses of the Korean Ralstonia solanacearum Species Complex.

The soil-borne pathogenic Ralstonia solanacearum species complex (RSSC) is a group of plant pathogens that is economically destructive worldwide and has a broad host range, including various solanaceae plants, banana, ginger, sesame, and clove. Previously, Korean RSSC strains isolated from samples of potato bacterial wilt were grouped into four pathotypes based on virulence tests against potato, tomato, eggplant, and pepper. In this study, we sequenced the genomes of 25 Korean RSSC strains selected based on these pathotypes. The newly sequenced genomes were analyzed to determine the phylogenetic relationships between the strains with average nucleotide identity values, and structurally compared via multiple genome alignment using Mauve software. To identify candidate genes responsible for the host specificity of the pathotypes, functional genome comparisons were conducted by analyzing pan-genome orthologous group (POG) and type III secretion system effectors (T3es). POG analyses revealed that a total of 128 genes were shared only in tomato-non-pathogenic strains, 8 genes in tomato-pathogenic strains, 5 genes in eggplant-non-pathogenic strains, 7 genes in eggplant-pathogenic strains, 1 gene in pepper-non-pathogenic strains, and 34 genes in pepper-pathogenic strains. When we analyzed T3es, three host-specific effectors were predicted: RipS3 (SKWP3) and RipH3 (HLK3) were found only in tomato-pathogenic strains, and RipAC (PopC) were found only in eggplant-pathogenic strains. Overall, we identified host-specific genes and effectors that may be responsible for virulence functions in RSSC in silico. The expected characters of those genes suggest that the host range of RSSC is determined by the comprehensive actions of various virulence factors, including effectors, secretion systems, and metabolic enzymes.

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April 21, 2020

Closing the Yield Gap for Cannabis: A Meta-Analysis of Factors Determining Cannabis Yield.

Until recently, the commercial production of Cannabis sativa was restricted to varieties that yielded high-quality fiber while producing low levels of the psychoactive cannabinoid tetrahydrocannabinol (THC). In the last few years, a number of jurisdictions have legalized the production of medical and/or recreational cannabis with higher levels of THC, and other jurisdictions seem poised to follow suit. Consequently, demand for industrial-scale production of high yield cannabis with consistent cannabinoid profiles is expected to increase. In this paper we highlight that currently, projected annual production of cannabis is based largely on facility size, not yield per square meter. This meta-analysis of cannabis yields reported in scientific literature aimed to identify the main factors contributing to cannabis yield per plant, per square meter, and per W of lighting electricity. In line with previous research we found that variety, plant density, light intensity and fertilization influence cannabis yield and cannabinoid content; we also identified pot size, light type and duration of the flowering period as predictors of yield and THC accumulation. We provide insight into the critical role of light intensity, quality, and photoperiod in determining cannabis yields, with particular focus on the potential for light-emitting diodes (LEDs) to improve growth and reduce energy requirements. We propose that the vast amount of genomics data currently available for cannabis can be used to better understand the effect of genotype on yield. Finally, we describe diversification that is likely to emerge in cannabis growing systems and examine the potential role of plant-growth promoting rhizobacteria (PGPR) for growth promotion, regulation of cannabinoid biosynthesis, and biocontrol.

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April 21, 2020

Genomics-driven discovery of a biosynthetic gene cluster required for the synthesis of BII-Rafflesfungin from the fungus Phoma sp. F3723.

Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis.Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined.We describe the NRPS-t1PKS cluster ‘BIIRfg’ compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.

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April 21, 2020

Hybrid-Transcriptome Sequencing and Associated Metabolite Analysis Reveal Putative Genes Involved in Flower Color Difference in Rose Mutants.

Gene mutation is a common phenomenon in nature that often leads to phenotype differences, such as the variations in flower color that frequently occur in roses. With the aim of revealing the genomic information and inner mechanisms, the differences in the levels of both transcription and secondary metabolism between a pair of natural rose mutants were investigated by using hybrid RNA-sequencing and metabolite analysis. Metabolite analysis showed that glycosylated derivatives of pelargonidin, e.g., pelargonidin 3,5 diglucoside and pelargonidin 3-glucoside, which were not detected in white flowers (Rosa ‘Whilte Mrago Koster’), constituted the major pigments in pink flowers. Conversely, the flavonol contents of petal, such as kaempferol-3-glucoside, quercetin 3-glucoside, and rutin, were higher in white flowers. Hybrid RNA-sequencing obtained a total of 107,280 full-length transcripts in rose petal which were annotated in major databases. Differentially expressed gene (DEG) analysis showed that the expression of genes involved in the flavonoid biosynthesis pathway was significantly different, e.g., CHS, FLS, DFR, LDOX, which was verified by qRT-PCR during flowering. Additionally, two MYB transcription factors were found and named RmMYBAN2 and RmMYBPA1, and their expression patterns during flowering were also analyzed. These findings indicate that these genes may be involved in the flower color difference in the rose mutants, and competition between anthocyanin and flavonol biosynthesis is a primary cause of flower color variation, with its regulation reflected by transcriptional and secondary metabolite levels.

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