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.

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.

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.

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.

Expression, crystallization and preliminary X-ray crystallographic analysis of cystathionine γ-synthase (XometB) from Xanthomonas oryzae pv. oryzae.

Cystathionine γ-synthase (CGS) catalyzes the first step in the transsulfuration pathway leading to the formation of cystathionine from O-succinylhomoserine and L-cysteine through a γ-replacement reaction. As an antibacterial drug target against Xanthomonas oryzae pv. oryzae (Xoo), CGS from Xoo (XometB) was cloned, expressed, purified and crystallized. The XometB crystal diffracted to 2.4 Šresolution and belonged to the tetragonal space group I4(1), with unit-cell parameters a=b=165.4, c=241.7 Å. There were four protomers in the asymmetric unit, with a corresponding solvent content of 73.9%.

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.

Crystallization and preliminary X-ray crystallographic analysis of ß-ketoacyl-ACP synthase I (XoFabB) from Xanthomonas oryzae pv. oryzae.

The proteins in the fatty-acid synthesis pathway in bacteria have significant potential as targets for the development of antibacterial agents. An essential elongation step in fatty-acid synthesis is performed by ß-ketoacyl-acyl carrier protein synthase I (FabB). The organism Xanthomonas oryzae pv. oryzae (Xoo) causes a destructive bacterial blight disease of rice. The XoFabB protein from Xoo was expressed, purified and crystallized for the three-dimensional structure determination that is essential for the development of specific inhibitors of the enzyme. An XoFabB crystal diffracted to 3.0 Å resolution and belonged to the tetragonal space group P4(1), with unit-cell parameters a = b = 82.2, c = 233.2 Å. Assuming that the crystallographic structure contains four molecules in the asymmetric unit, the corresponding V(M) would be 2.18 Å(3) Da(-1) and the solvent content would be 43.5%. The initial structure was determined by the MOLREP program with an R factor of 44.0% and does contain four monomers in the asymmetric unit.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of the co-chaperonin XoGroES from Xanthomonas oryzae pv. oryzae.

Bacterial blight (BB), a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo), causes serious production losses of rice in Asian countries. Protein misfolding may interfere with the function of proteins in all living cells and must be prevented to avoid cellular disaster. All cells naturally contain molecular chaperones that assist the unfolded proteins in folding into the native structure. One of the well characterized chaperone complexes is GroEL-GroES. GroEL, which consists of two chambers, captures misfolded proteins and refolds them. GroES is a co-chaperonin protein that assists the GroEL protein as a lid that temporarily closes the chamber during the folding process. Xoo4289, the GroES gene from Xoo, was cloned and expressed for X-ray crystallographic study. The purified protein (XoGroES) was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to 2.0 Šresolution. The crystal belonged to the hexagonal space group P6(1), with unit-cell parameters a=64.4, c=36.5 Å. The crystal contains a single molecule in the asymmetric unit, with a corresponding VM of 2.05 Å3 Da(-1) and a solvent content of 39.9%.

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.

Identification, fermentation, and bioactivity against Xanthomonas oryzae of antimicrobial metabolites isolated from Phomopsis longicolla S1B4.

Bacterial blight, an important and potentially destructive bacterial disease in rice, is caused by Xanthomonas oryzae. Recently, this organism has developed resistance to available antibiotics, prompting scientists to find a suitable alternative. This study focused on secondary metabolites of Phomopsis longicolla to target X. oryzae. Five bioactive compounds were isolated by activity-guided fractionation from ethyl acetate extracts of mycelia and were identified by LC/MS and NMR spectroscopy as dicerandrol A, dicerandrol B, dicerandrol C, deacetylphomoxanthone B, and fusaristatin A. This is the first time fusaristatin A has been isolated from Phomopsis sp. Deacetylphomoxanthone B showed a higher antibacterial effect against X. oryzae KACC 10331 than the positive control (2,4-diacetyphloroglucinol). Dicerandrol A also showed high antimicrobial activity against Grampositive bacteria (Staphylococcus aureus, Bacillus subtilis) and yeast (Candida albicans). In addition, high production yields of these compounds were obtained at the stationary and death phases.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of glutamyl-tRNA synthetase (Xoo1504) from Xanthomonas oryzae pv. oryzae.

The gltX gene from Xanthomonas oryzae pv. oryzae (Xoo1504) encodes glutamyl-tRNA synthetase (GluRS), one of the most important enzymes involved in bacterial blight (BB), which causes huge production losses of rice worldwide. GluRS is a class I-type aminoacyl-tRNA synthetase (aaRS) that is primarily responsible for the glutamylation of tRNA(Glu). It plays an essential role in protein synthesis, as well as the regulation of cells, in all organisms. As it represents an important target for the development of new antibacterial drugs against BB, determination of the three-dimensional structure of GluRS is essential in order to understand its catalytic mechanism. In order to analyze its structure and function, the gltX gene was cloned and the GluRS enzyme was expressed, purified and then crystallized. A GluRS crystal belonging to the monoclinic space group C2 diffracted to 2.8 A resolution and had unit-cell parameters a = 186.8, b = 108.4, c = 166.1 A, beta = 96.3 degrees . The unit-cell volume of the crystal allowed the presence of six to eight monomers in the asymmetric unit, with a corresponding Matthews coefficient (V(M)) range of 2.70-2.02 A(3) Da(-1) and a solvent-content range of 54.5-39.3%.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of beta-ketoacyl-ACP synthase III (FabH) from Xanthomonas oryzae pv. oryzae.

The bacterial beta-ketoacyl-ACP synthase III (KASIII) encoded by the gene fabH (Xoo4209) from Xanthomonas oryzae pv. oryzae, a plant pathogen, is an important enzyme in the elongation steps of fatty-acid biosynthesis. It is expected to be one of the enzymes responsible for bacterial blight (BB), a serious disease that results in huge production losses of rice. As it represents an important target for the development of new antibacterial drugs against BB, determination of the crystal structure of the KAS III enzyme is essential in order to understand its reaction mechanism. In order to analyze the structure and function of KAS III, the fabH (Xoo4209) gene was cloned and the enzyme was expressed and purified. The KASIII crystal diffracted to 2.05 A resolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 69.8, b = 79.5, c = 62.3 A. The unit-cell volume of the crystal is compatible with the presence of a single monomer in the asymmetric unit, with a corresponding Matthews coefficient V(M) of 2.27 A(3) Da(-1) and a solvent content of 45.8%.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of leucine aminopeptidase (LAP) from the pepA gene of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes the serious disease bacterial blight in rice. The pepA (Xoo0834) gene from Xoo is one of around 100 genes that have been selected for the design of antibacterial drugs. The pepA gene encodes leucine aminopeptidase (LAP), an exopeptidase that catalyzes the hydrolysis of leucine residues from the N-terminus of a protein or peptide. This enzyme was expressed in Escherichia coli, purified and crystallized, and preliminary X-ray structural studies have been carried out. The LAP crystal diffracted to 2.6 A resolution and belonged to the cubic space group P2(1)3. The unit-cell volume of the crystal was compatible with the presence of two monomers in the asymmetric unit.

Mutational analysis of the gum gene cluster required for xanthan biosynthesis in Xanthomonas oryzae pv oryzae.

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae.

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.

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.