Insights into chloroplast genome structure and phylogenetic relationships within the Sesamum species complex (Pedaliaceae).

Background: In the Sesamum species complex, the lack of wild species genomic resources hinders the evolutionary comprehension of phylogenetic relationships. Results: In the present study, we generated complete chloroplast genomes of six wild relatives (Sesamum alatum, Sesamum angolense, Sesamum pedaloides, Ceratotheca sesamoides (syn. Sesamum sesamoides), Ceratotheca triloba (syn. Sesamum trilobum), and Sesamum radiatum) and a Korean cultivar, Sesamum indicum cv. Goenbaek. A typical quadripartite chloroplast structure, including two inverted repeats (IR), a large single copy (LSC), and a small single copy (SSC), was observed. A total of 114 unique genes encompassing 80 coding genes, four ribosomal RNAs, and 30 transfer RNAs were counted. The chloroplast genomes (152, 863-153, 338 bp) exhibited the IR contraction/expansion phenomenon and were quite conserved in both coding and non-coding regions. However, high values of the nucleotide diversity index were found in several genes, including ndhA, ndhE, ndhF, ycf1, and psaC-ndhD. Concordant tree topologies suggest ndhF as a useful marker for taxon discrimination. The phylogenetic inference and time divergence dating indicate that S. radiatum (2n = 64) occurred concomitantly with the sister species C. sesamoides (2n = 32) approximately 0.05 million years ago (Mya). In addition, S. alatum was clearly discriminated by forming a single clade, showing its long genetic distance and potential early speciation event in regards to the others. Conclusion: Altogether, we propose to rename C. sesamoides and C. triloba as S. sesamoides and S. trilobum, respectively, as suggested previously based on the morphological description. This study provides the first insight into the phylogenetic relationships among the cultivated and wild African native relatives. The chloroplast genome data lay a foundation for speciation genomics in the Sesamum species complex.

Non-targeted metabolomic analysis for the comparative evaluation of volatile organic compounds in 20 globally representative cucumber lines.

Volatile organic compounds (VOCs) are one of the main fruit-quality determinants in cucumber. Here, we investigated the differences in the VOC and primary metabolite composition among 20 representative cucumber lines. Results of non-targeted metabolomics revealed that the cucumber breeding line of the Korean group showed a unique VOC composition in the fruit peel compared to the other groups. Fruit-flesh VOCs significantly differed among Korean, European, and Thai fruits. The main cucumber flavor components, 2-hexenal, hexanal, 6-nonenal, 2,4-nonadienal, and 2,6-nonadienal, were lower in the Korean cucumber lines than in the others. Conversely, linoleic acid derivatives and α-linolenic acid, which are precursors of these VOCs, were abundant in Korean cucumber line. This suggests that the metabolism related to the characteristic flavor of cucumber are downregulated in Korean cucumber line. This study provides novel insights into the fruit flavor-associated metabolome in various cucumber lines.

Development of SNP Markers for White Immature Fruit Skin Color in Cucumber (Cucumis sativus L.) Using QTL-seq and Marker Analyses.

Despite various efforts in identifying the genes governing the white immature fruit skin color in cucumber, the genetic basis of the white immature fruit skin color is not well known. In the present study, genetic analysis showed that a recessive gene confers the white immature fruit skin-color phenotype over the light-green color of a Korean slicer cucumber. High-throughput QTL-seq combined with bulked segregation analysis of two pools with the extreme phenotypes (white and light-green fruit skin color) in an F2 population identified two significant genomic regions harboring QTLs for white fruit skin color within the genomic region between 34.1 and 41.67 Mb on chromosome 3, and the genomic region between 12.2 and 12.7 Mb on chromosome 5. Further, nonsynonymous SNPs were identified with a significance of p < 0.05 within the QTL regions, resulting in eight homozygous variants within the QTL region on chromosome 3. SNP marker analysis uncovered the novel missense mutations in Chr3CG52930 and Chr3CG53640 genes and showed consistent results with the phenotype of light-green and white fruit skin-colored F2 plants. These two genes were located 0.5 Mb apart on chromosome 3, which are considered strong candidate genes. Altogether, this study laid a solid foundation for understanding the genetic basis and marker-assisted breeding of immature fruit skin color in cucumber.

Metabolite Profiling of Dioscorea (Yam) Leaves to Identify Bioactive Compounds Reveals Their Potential as Renewable Resources.

Yams (Dioscorea spp.) are cultivated and consumed as edible tubers, while their leaves are discarded as waste or burned with negative environmental impact. Herein, the metabolites of two yam species (Danma, DAN; Dunggeunma, DUN), harvested in June, July, and August, were profiled using GC-TOF-MS and UHPLC-LTQ-Orbitrap-MS/MS and the antioxidant activity of the extracts was evaluated to stimulate the utilization of yam leaves as a by-product. We observed that the relative levels of amino acids, organic acids, sugars, and saponins decreased linearly with prolonged harvest time, while fatty acid, phenanthrene, and flavonoid levels gradually increased. Furthermore, the leaf extracts obtained in August exhibited the highest antioxidant activity. To determine the antioxidant-contributing metabolites, OPLS-DA was performed for the leaf metabolites of DAN and DUN leaves harvested in August. Hydroxytyrosol-glucoside, apigenin-rhamnoside, and rutin were more abundant in DUN, while luteolin, phenanthrene derivatives, epicatechin, and kaempferide were relatively higher in DAN and their respective metabolites were positively correlated with the antioxidant activity. Moreover, secondary metabolites were more abundant in the leaves than in the roots, and consequently, the antioxidant activity of the former was also higher. Overall, the potential value of yam leaves as a renewable source of bioactive compounds is proposed.

Combined Analysis of the Time-Resolved Transcriptome and Proteome of Plant Pathogen Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a plant pathogen responsible for causing bacterial blight in rice. The immediate alterations in Xoo upon initial contact with rice are essential for pathogenesis. We studied time-resolved genome-wide gene expression in pathogenicity-activated Xoo cells at the transcriptome and proteome levels. The early response genes of Xoo include genes related to cell motility, inorganic ion transport, and effectors. The alteration of gene expression is initiated as early as few minutes after the initial interaction and changes with time. The time-resolved comparison of the transcriptome and proteome shows the differences between transcriptional and translational expression peaks in many genes, although the overall expression pattern of mRNAs and proteins is conserved. The discrepancy suggests an important role of translational regulation in Xoo at the early stages of pathogenesis. The gene expression analysis using time-resolved transcriptome and proteome provides unprecedented valuable information regarding Xoo pathogenesis.

Identification of an Allelic Variant of the CsOr Gene Controlling Fruit Endocarp Color in Cucumber (Cucumis sativus L.) Using Genotyping-By-Sequencing (GBS) and Whole-Genome Sequencing.

The cucumber is a major vegetable crop around the world. Fruit flesh color is an important quality trait in cucumber and flesh color mainly depends on the relative content of ß-carotene in the fruits. The ß-carotene serves as a precursor of vitamin A, which has dietary benefits for human health. Cucumbers with orange flesh contain a higher amount of ß-carotene than white fruit flesh. Therefore, development of orange-fleshed cucumber varieties is gaining attention for improved nutritional benefits. In this study, we performed genotyping-by-sequencing (GBS) based on genetic mapping and whole-genome sequencing to identify the orange endocarp color gene in the cucumber breeding line, CS-B. Genetic mapping, genetic sequencing, and genetic segregation analyses showed that a single recessive gene (CsaV3_6G040750) encodes a chaperone DnaJ protein (DnaJ) protein at the Cucumis sativus(CsOr) locus was responsible for the orange endocarp phenotype in the CS-B line. The Or gene harbored point mutations T13G and T17C in the first exon of the coding region, resulting in serine to alanine at position 13 and isoleucine to threonine at position 17, respectively. CS-B line displayed increased ß-carotene content in the endocarp tissue, corresponding to elevated expression of CsOr gene at fruit developmental stages. Identifying novel missense mutations in the CsOr gene could provide new insights into the role of Or mechanism of action for orange fruit flesh in cucumber and serve as a valuable resource for developing ß-carotene-rich cucumbers varieties with increased nutritional benefits.

Genome Resource of Podosphaera xanthii, the Host-Specific Fungal Pathogen That Causes Cucurbit Powdery Mildew.

Approximately 33 types of commonly consumed fruits and vegetables are members of the family Cucurbitaceae, making it an important crop family worldwide. However, pathogen resistance to pesticides and fungicides has become a growing problem in cultivation practices. The identification of the effector proteins in each unique fungus-host pair would help toward the development of strategies for preventing the infection of important crops. In this study, we characterized the genome of Podosphaera xanthii, the fungal pathogen that causes powdery mildew disease in cucurbitaceous plants. A first-draft genome of 209.08 MB was assembled and compared with those of 25 other fungal pathogens, particularly for identifying candidate secreted effector proteins. This draft genome can serve as a valuable resource for future genomic and proteomic studies of P. xanthii and its host-specific pathogenesis.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Integrated Metabolomics and Transcriptomics Unravel the Metabolic Pathway Variations for Different Sized Beech Mushrooms.

Beech mushrooms (Hypsizygus marmoreus) are largely relished for their characteristic earthy flavor, chewy-texture, and gustatory and nutritional properties in East Asian societies. Intriguingly, the aforementioned properties of beech mushroom can be subsumed under its elusive metabolome and subtle transcriptome regulating its various stages of growth and development. Herein, we carried out an integrated metabolomic and transcriptomic profiling for different sized beech mushrooms across spatial components (cap and stipe) to delineate their signature pathways. We observed that metabolite profiles and differentially expressed gene (DEGs) displayed marked synergy for specific signature pathways according to mushroom sizes. Notably, the amino acid, nucleotide, and terpenoid metabolism-related metabolites and genes were more abundant in small-sized mushrooms. On the other hand, the relative levels of carbohydrates and TCA intermediate metabolites as well as corresponding genes were linearly increased with mushroom size. However, the composition of flavor-related metabolites was varying in different sized beech mushrooms. Our study explores the signature pathways associated with growth, development, nutritional, functional and organoleptic properties of different sized beech mushrooms.

Efficacy of an antiadhesive agent for the prevention of intra-abdominal adhesions after radical gastrectomy: A prospective randomized, multicenter trial.

BACKGROUND: Guardix-SG is a poloxamer-based antiadhesive agent. The aim of this study was to investigate its efficacy in preventing abdominal adhesions in gastric cancer patients undergoing gastrectomy. Few clinical studies have reported that antiadhesive agent reduces the incidence of adhesion after gastrectomy. METHODS: We conducted a multicenter trial from June 2013 and August 2015 in patients with gastric adenocarcinoma undergoing radical gastrectomy. Patients were randomly assigned to the Guardix treatment or control group. Postoperative adhesions were diagnosed based on postoperative symptoms, plain x-ray films, and computed tomography. The primary endpoint of the study was the incidence of small bowel obstruction in the first postoperative year. The secondary end-point was the safety of Guardix-SG. RESULTS: The study included 109 patients in the Guardix group and 105 patients in the control group. The groups were similarly matched with pathological stage, operation type, anastomosis method, midline incision length, and the extent of lymph node dissection. Eight in the Guardix group and 21 in the control group experienced intestinal obstruction during the 1-year follow-up period. The cumulative incidence of small bowel obstruction was significantly lower in the Guardix group compared to that seen in the control group (4.7% vs 8.6% at 6 months and 7.3% vs 20% at 1 year; P = .007, log-rank test). There were no differences in postoperative complications and adverse events. CONCLUSION: Guardix-SG significantly decreased the incidence of intestinal obstruction without affecting the incidence of postoperative complications.

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.

Mitochondrial genome of the Podosphaera xanthii: a plant pathogen causes powdery mildew in cucurbits.

In this study, we sequenced the complete mitochondrial genome of the Podosphaera xanthii, which is the powdery mildew diseases causative pathogen for cucurbits. The total size of the mitochondrial genome is 26,052 bp, which includes 15 coding genes, 25 tRNAs, and 2 rRNAs. The cytochrome c oxidase subunit I (COXI) used for the phylogenetic construction, which grouped this species into Hypocreomycetidae taxonomy family, which could aid the researchers to place the fungal in an appropriate taxonomy clade.

Genomic discovery of the hypsin gene and biosynthetic pathways for terpenoids in Hypsizygus marmoreus.

BACKGROUND: Hypsizygus marmoreus (Beech mushroom) is a popular ingredient in Asian cuisine. The medicinal effects of its bioactive compounds such as hypsin and hypsiziprenol have been reported, but the genetic basis or biosynthesis of these components is unknown. RESULTS: In this study, we sequenced a reference strain of H. marmoreus (Haemi 51,987-8). We evaluated various assembly strategies, and as a result the Allpaths and PBJelly produced the best assembly. The resulting genome was 42.7 Mbp in length and annotated with 16,627 gene models. A putative gene (Hypma_04324) encoding the antifungal and antiproliferative hypsin protein with 75% sequence identity with the previously known N-terminal sequence was identified. Carbohydrate active enzyme analysis displayed the typical feature of white-rot fungi where auxiliary activity and carbohydrate-binding modules were enriched. The genome annotation revealed four terpene synthase genes responsible for terpenoid biosynthesis. From the gene tree analysis, we identified that terpene synthase genes can be classified into six clades. Four terpene synthase genes of H. marmoreus belonged to four different groups that implies they may be involved in the synthesis of different structures of terpenes. A terpene synthase gene cluster was well-conserved in Agaricomycetes genomes, which contained known biosynthesis and regulatory genes. CONCLUSIONS: Genome sequence analysis of this mushroom led to the discovery of the hypsin gene. Comparative genome analysis revealed the conserved gene cluster for terpenoid biosynthesis in the genome. These discoveries will further our understanding of the biosynthesis of medicinal bioactive molecules in this edible mushroom.

Analysis of Genetic and Pathogenic Diversity of Ralstonia solanacearum Causing Potato Bacterial Wilt in Korea.

The Ralstonia solanacearum species complex (RSSC) can be divided into four phylotypes, and includes phenotypically diverse bacterial strains that cause bacterial wilt on various host plants. This study used 93 RSSC isolates responsible for potato bacterial wilt in Korea, and investigated their phylogenetic relatedness based on the analysis of phylotype, biovar, and host range. Of the 93 isolates, twenty-two were identified as biovar 2, eight as biovar 3, and sixty-three as biovar 4. Applied to the phylotype scheme, biovar 3 and 4 isolates belonged to phylotype I, and biovar 2 isolates belonged to phylotype IV. This classification was consistent with phylogenetic trees based on 16S rRNA and egl gene sequences, in which biovar 3 and 4 isolates clustered to phylotype I, and biovar 2 isolates clustered to phylotype IV. Korean biovar 2 isolates were distinct from biovar 3 and 4 isolates pathologically as well as genetically - all biovar 2 isolates were nonpathogenic to peppers. Additionally, in host-determining assays, we found uncommon strains among biovar 2 of phylotype IV, which were the tomato-nonpathogenic strains. Since tomatoes are known to be highly susceptible to RSSC, to the best of our knowledge this is the first report of tomato-nonpathogenic potato strains. These results imply the potential prevalence of greater RSSC diversity in terms of host range than would be predicted based on phylogenetic analysis.

Spatial (cap & stipe) metabolomic variations affect functional components between brown and white beech mushrooms.

The beech mushrooms have customarily been revered by oriental societies for their nutritional and health benefits. We explored the mass spectrometry (MS) based spatial metabolomic variations between parts (cap and stipe) of two beech mushroom strains (brown and white). The principal component analysis (PCA) revealed their distinct primary (cap and stipe: PC1, 25.5%; strains: PC2, 12.5%) and secondary (cap and stipe: PC1, 10.3%; strains: PC2, 7.6%) metabolite patterns. The caps were rich in amino acids, fatty acids, and N-acetylglucosamine with higher protein and nitrogen contents. The stipes had abundant ß-glucans, malic acid, and fructose. The discriminant secondary metabolites, especially, hypsiziprenols were higher in caps from brown strains. A fatty acid derivative, azelaic acid, was abundant in white strains (cap>stipe). We established a positive correlation for the cytotoxic activities of hypsiziprenols against ACHN cells. These spatial inter-strain metabolomic distinctions are potentially helpful for mushroom selection and improvement.

Crystal Structures of Peptide Deformylase from Rice Pathogen Xanthomonas oryzae pv. oryzae in Complex with Substrate Peptides, Actinonin, and Fragment Chemical Compounds.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight on rice; this species is one of the most destructive pathogenic bacteria in rice cultivation worldwide. Peptide deformylase (PDF) catalyzes the removal of the N-formyl group from the N-terminus of newly synthesized polypeptides in bacterial cells and is an important target to develop antibacterial agents. We determined crystal structures of Xoo PDF (XoPDF) at up to 1.9 Å resolution, which include apo, two substrate-bound (methionine-alanine or methionine-alanine-serine), an inhibitor-bound (actinonin), and six fragment chemical-bound structures. Six fragment chemical compounds were bound in the substrate-binding pocket. The fragment chemical-bound structures were compared to the natural PDF inhibitor actinonin-bound structure. The fragment chemical molecules will be useful to design an inhibitor specific to XoPDF and a potential pesticide against Xoo.

Time-resolved pathogenic gene expression analysis of the plant pathogen Xanthomonas oryzae pv. oryzae.

BACKGROUND: Plant-pathogen interactions at early stages of infection are important to the fate of interaction. Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, which is a devastating disease in rice. Although in vivo and in vitro systems have been developed to study rice-Xoo interactions, both systems have limitations. The resistance mechanisms in rice can be better studied by the in vivo approach, whereas the in vitro systems are suitable for pathogenicity studies on Xoo. The current in vitro system uses minimal medium to activate the pathogenic signal (expression of pathogenicity-related genes) of Xoo, but lacks rice-derived factors needed for Xoo activation. This fact emphasizes the need of developing a new in vitro system that allow for an easy control of both pathogenic activation and for the experiment itself. RESULTS: We employed an in vitro system that can activate pathogenicity-related genes in Xoo using rice leaf extract (RLX) and combined the in vitro assay with RNA-Seq to analyze the time-resolved genome-wide gene expression of Xoo. RNA-Seq was performed with samples from seven different time points within 1 h post-RLX treatment and the expression of up- or downregulated genes in RNA-Seq was validated by qRT-PCR. Global analysis of gene expression and regulation revealed the most dramatic changes in functional categories of genes related to inorganic ion transport and metabolism, and cell motility. Expression of many pathogenicity-related genes was induced within 15 min upon contact with RLX. hrpG and hrpX expression reached the maximum level within 10 and 15 min, respectively. Chemotaxis and flagella biosynthesis-related genes and cyclic-di-GMP controlling genes were downregulated for 10 min and were then upregulated. Genes related to inorganic ion uptake were upregulated within 5 min. We introduced a non-linear regression fit to generate continuous time-resolved gene expression levels and tested the essentiality of the transcriptionally upregulated genes by a pathogenicity assay of lesion length using single-gene knock-out Xoo strains. CONCLUSIONS: The in vitro system combined with RNA-Seq generated a genome-wide time-resolved pathogenic gene expression profile within 1 h of initial rice-Xoo interactions, demonstrating the expression order and interaction dependency of pathogenic genes. This combined system can be used as a novel tool to study the initial interactions between rice and Xoo during bacterial blight progression.

Metabolomics-Based Chemotaxonomic Classification of Streptomyces spp. and Its Correlation with Antibacterial Activity.

Secondary metabolite-based chemotaxonomic classification of Streptomyces (8 species, 14 strains) was performed using ultraperformance liquid chromatography-quadrupole-time-offlight- mass spectrometry with multivariate statistical analysis. Most strains were generally well separated by grouping under each species. In particular, S. rimosus was discriminated from t he r emaining s ev en s pecies ( S. coelicolor, S. griseus, S. indigoferus, S. peucetius, S. rubrolavendulae, S. scabiei, and S. virginiae) in partial least squares discriminant analysis, and oxytetracycline and rimocidin were identified as S. rimosus-specific metabolites. S. rimosus also showed high antibacterial activity against Xanthomonas oryzae pv. oryzae , the pathogen responsible for rice bacterial blight. This study demonstrated that metabolite-based chemotaxonomic classification is an effective tool for distinguishing Streptomyces spp. and for determining their species-specific metabolites.

Crystallization and preliminary X-ray crystallographic analysis of the XoGroEL chaperonin from Xanthomonas oryzae pv. oryzae.

Along with the co-chaperonin GroES, the chaperonin GroEL plays an essential role in enhancing protein folding or refolding and in protecting proteins against misfolding and aggregation in the cellular environment. The XoGroEL gene (XOO_4288) from Xanthomonas oryzae pv. oryzae was cloned and the protein was expressed, purified and crystallized. The purified XoGroEL protein was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to a resolution of 3.4 Å. The crystal belonged to the orthorhombic space group P212121 with 14 monomers in the asymmetric unit, with a corresponding VM of 2.7 Å(3) Da(-1) and a solvent content of 54.5%.

Crystal structure of XoLAP, a leucine aminopeptidase, from Xanthomonas oryzae pv. oryzae.

Aminopeptidases are metalloproteinases that degrade N-terminal residues from protein and play important roles in cell growth and development by controlling cell homeostasis and protein maturation. We determined the crystal structure of XoLAP, a leucyl aminopeptidase, at 2.6 Å resolution from Xanthomonas oryzae pv. oryzae, causing the destructive rice disease of bacterial blight. It is the first crystal structure of aminopeptidase from phytopathogens as a drug target. XoLAP existed as a hexamer and the monomer structure consisted of an N-terminal cap domain and a C-terminal peptidase domain with two divalent zinc ions. XoLAP structure was compared with BlLAP and EcLAP (EcPepA) structures. Based on the structural comparison, the molecular model of XoLAP in complex with the natural aminopeptidase inhibitor of microginin FR1 was proposed. The model structure will be useful to develop a novel antibacterial drug against Xoo.