Comparative transcriptomics analysis reveals defense mechanisms of Manihot esculenta Crantz against Sri Lanka Cassava MosaicVirus.

BACKGROUND: Cassava mosaic disease (CMD), caused by Sri Lankan cassava mosaic virus (SLCMV) infection, has been identified as a major pernicious disease in Manihot esculenta Crantz (cassava) plantations. It is widespread in Southeast Asia, especially in Thailand, which is one of the main cassava supplier countries. With the aim of restricting the spread of SLCMV, we explored the gene expression of a tolerant cassava cultivar vs. a susceptible cassava cultivar from the perspective of transcriptional regulation and the mechanisms underlying plant immunity and adaptation. RESULTS: Transcriptomic analysis of SLCMV-infected tolerant (Kasetsart 50 [KU 50]) and susceptible (Rayong 11 [R 11]) cultivars at three infection stages-that is, at 21 days post-inoculation (dpi) (early/asymptomatic), 32 dpi (middle/recovery), and 67 dpi (late infection/late recovery)-identified 55,699 expressed genes. Differentially expressed genes (DEGs) between SLCMV-infected KU 50 and R 11 cultivars at (i) 21 dpi to 32 dpi (the early to middle stage), and (ii) 32 dpi to 67 dpi (the middle stage to late stage) were then identified and validated by real-time quantitative PCR (RT-qPCR). DEGs among different infection stages represent genes that respond to and regulate the viral infection during specific stages. The transcriptomic comparison between the tolerant and susceptible cultivars highlighted the role of gene expression regulation in tolerant and susceptible phenotypes. CONCLUSIONS: This study identified genes involved in epigenetic modification, transcription and transcription factor activities, plant defense and oxidative stress response, gene expression, hormone- and metabolite-related pathways, and translation and translational initiation activities, particularly in KU 50 which represented the tolerant cultivar in this study.

Mutation mapping of a variegated EMS tomato reveals an FtsH-like protein precursor potentially causing patches of four phenotype classes in the leaves with distinctive internal morphology.

BACKGROUND: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F2 population to reveal candidate genes responsible for the variegation. RESULTS: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation. CONCLUSION: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation.

Comparative Transcriptomic Analysis of Genes in the 20-Hydroxyecdysone Biosynthesis in the Fern Microsorum scolopendria towards Challenges with Foliar Application of Chitosan.

Microsorum scolopendria is an important medicinal plant that belongs to the Polypodiaceae family. In this study, we analyzed the effects of foliar spraying of chitosan on growth promotion and 20-hydroxyecdysone (20E) production in M. scolopendria. Treatment with chitosan at a concentration of 50 mg/L in both young and mature sterile fronds induced the highest increase in the amount of accumulated 20E. Using RNA sequencing, we identified 3552 differentially expressed genes (DEGs) in response to chitosan treatment. The identified DEGs were associated with 236 metabolic pathways. We identified several DEGs involved in the terpenoid and steroid biosynthetic pathways that might be associated with secondary metabolite 20E biosynthesis. Eight upregulated genes involved in cholesterol and phytosterol biosynthetic pathway, five upregulated genes related to the methylerythritol 4-phosphate (MEP) and mevalonate (MVA) pathways, and several DEGs that are members of cytochrome P450s and ABC transporters were identified. Quantitative real-time RT-PCR confirmed the results of RNA-sequencing. Taken together, we showed that chitosan treatment increased plant dry weight and 20E accumulation in M. scolopendria. RNA-sequencing and DEG analyses revealed key enzymes that might be related to the production of the secondary metabolite 20E in M. scolopendria.

Preliminary Study on Comparative Efficacy of Four Light Sources for Trapping Culicoides spp. (Diptera: Ceratopogonidae) in Prachuap Khiri Khan Province, Thailand.

The light trap is an important tool to determine the presence and abundance of vectors in the field. However, no one has studied the efficiency of light traps for collecting Culicoides in Thailand. In the present study, the efficacy of four light sources was evaluated in Prachuap Khiri Khan province, Thailand. Incandescent (INCND) light, white fluorescent (WHT-FLR) light, ultraviolet fluorescent (UV-FLR) light, and UV light-emitting diode (UV-LED) light were tested using commercial traps. In total, 30,866 individuals of Culicoides species were collected from November 2020 to June 2021, of which 21,016 were trapped on site 1 and 6,731 were trapped on site 2. The two most abundant Culicoides species were C. imicola (54%) and C. oxystoma (31.2%). UV-FLR was highly effective, followed by UV-LED light, WHT-FLR light, and INCND light, respectively, for Culicoides collection. Significantly, more Culicoides species were collected in those traps baited with UV-FLR light, UV-LED light, or WHT-FLR light than for INCND light traps. Traps equipped with UV-FLR lights can be recommended to trap Culcoides biting midges for monitoring purposes.

Genome-wide association mapping and genomic prediction of yield-related traits and starch pasting properties in cassava.

KEY MESSAGE: GWAS identified eight yield-related, peak starch type of waxy and wild-type starch and 21 starch pasting property-related traits (QTLs). Prediction ability of eight GS models resulted in low to high predictability, depending on trait, heritability, and genetic architecture. Cassava is both a food and an industrial crop in Africa, South America, and Asia, but knowledge of the genes that control yield and starch pasting properties remains limited. We carried out a genome-wide association study to clarify the molecular mechanisms underlying these traits and to explore marker-based breeding approaches. We estimated the predictive ability of genomic selection (GS) using parametric, semi-parametric, and nonparametric GS models with a panel of 276 cassava genotypes from Thai Tapioca Development Institute, International Center for Tropical Agriculture, International Institute of Tropical Agriculture, and other breeding programs. The cassava panel was genotyped via genotyping-by-sequencing, and 89,934 single-nucleotide polymorphism (SNP) markers were identified. A total of 31 SNPs associated with yield, starch type, and starch properties traits were detected by the fixed and random model circulating probability unification (FarmCPU), Bayesian-information and linkage-disequilibrium iteratively nested keyway and compressed mixed linear model, respectively. GS models were developed, and forward predictabilities using all the prediction methods resulted in values of - 0.001-0.71 for the four yield-related traits and 0.33-0.82 for the seven starch pasting property traits. This study provides additional insight into the genetic architecture of these important traits for the development of markers that could be used in cassava breeding programs.

Genetic diversity and population structure of ridge gourd (Luffa acutangula) accessions in a Thailand collection using SNP markers.

This study explored a germplasm collection consisting of 112 Luffa acutangula (ridge gourd) accessions, mainly from Thailand. A total of 2834 SNPs were used to establish population structure and underlying genetic diversity while exploring the fruit characteristics together with genetic information which would help in the selection of parental lines for a breeding program. The study found that the average polymorphism information content value of 0.288 which indicates a moderate genetic diversity for this L. acutangula germplasm. STRUCTURE analysis (ΔK at K = 6) allowed us to group the accessions into six subpopulations that corresponded well with the unrooted phylogenetic tree and principal coordinate analyses. When plotted, the STRUCTURE bars to the area of collection, we observed an admixed genotype from surrounding accessions and a geneflow confirmed by the value of FST = 0.137. AMOVA based on STRUCTURE clustering showed a low 12.83% variation between subpopulations that correspond well with the negative inbreeding coefficient value (FIS = - 0.092) and low total fixation index (FIT = 0.057). There were distinguishing fruit shapes and length characteristics in specific accessions for each subpopulation. The genetic diversity and different fruit shapes in the L. acutangula germplasm could benefit the ridge gourd breeding programs to meet the demands and needs of consumers, farmers, and vegetable exporters such as increasing the yield of fruit by the fruit width but not by the fruit length to solve the problem of fruit breakage during exportation.

EnHERV: Enrichment analysis of specific human endogenous retrovirus patterns and their neighboring genes.

Human endogenous retroviruses (HERVs) are flanked by long terminal repeats (LTRs), which contain the regulation part of the retrovirus. Remaining HERVs constitute 7% to 8% of the present day human genome, and most have been identified as solo LTRs. The HERV sequences have been associated with several molecular functions as well as certain diseases in human, but their roles in human diseases are yet to be established. We designed EnHERV to make accessible the identified endogenous retrovirus repetitive sequences from Repbase Update (a database of eukaryotic repetitive elements) that are present in the human genome. Defragmentation process was done to improve the RepeatMasker annotation output. The defragmented elements were used as core database in EnHERV. EnHERV is available at http://sysbio.chula.ac.th/enherv and can be searched using either gene lists of user interest or HERV characteristics. Besides the search function, EnHERV also provides an enrichment analysis function that allows users to perform enrichment analysis between selected HERV characteristics and user-input gene lists, especially genes with the expression profile of a certain disease. EnHERV will facilitate exploratory studies of specific HERV characteristics that control gene expression patterns related to various disease conditions. Here we analyzed 25 selected HERV groups/names from all four HERV superfamilies, using the sense and anti-sense directions of the HERV and gene expression profiles from 49 specific tissue and disease conditions. We found that intragenic HERVs were associated with down-regulated genes in most cancer conditions and in psoriatic skin tissues and associated with up-regulated genes in immune cells particularly from systemic lupus erythematosus (SLE) patients. EnHERV allowed the analysis of how different types of LTRs were differentially associated with specific gene expression profiles in particular disease conditions for further studies into their mechanisms and functions.

Snake Venom Metalloproteinases and Their Peptide Inhibitors from Myanmar Russell's Viper Venom.

Russell's viper bites are potentially fatal from severe bleeding, renal failure and capillary leakage. Snake venom metalloproteinases (SVMPs) are attributed to these effects. In addition to specific antivenom therapy, endogenous inhibitors from snakes are of interest in studies of new treatment modalities for neutralization of the effect of toxins. Two major snake venom metalloproteinases (SVMPs): RVV-X and Daborhagin were purified from Myanmar Russell's viper venom using a new purification strategy. Using the Next Generation Sequencing (NGS) approach to explore the Myanmar RV venom gland transcriptome, mRNAs of novel tripeptide SVMP inhibitors (SVMPIs) were discovered. Two novel endogenous tripeptides, pERW and pEKW were identified and isolated from the crude venom. Both purified SVMPs showed caseinolytic activity. Additionally, RVV-X displayed specific proteolytic activity towards gelatin and Daborhagin showed potent fibrinogenolytic activity. These activities were inhibited by metal chelators. Notably, the synthetic peptide inhibitors, pERW and pEKW, completely inhibit the gelatinolytic and fibrinogenolytic activities of respective SVMPs at 5 mM concentration. These complete inhibitory effects suggest that these tripeptides deserve further study for development of a therapeutic candidate for Russell's viper envenomation.

Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions.

The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out "deep" proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, and HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 gigabytes of spectral data. As a result, we identified 6,766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over eight orders of magnitude of protein abundance. The data are provided to users as a public data base (https://helixweb.nih.gov/ESBL/Database/mpkFractions/). The mass spectrometry data were mapped back to their gel slices to generate "virtual Western blots" for each protein. For most of the 6,766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, posttranslational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular mass <20 kDa.

Biomarkers for Refractory Lupus Nephritis: A Microarray Study of Kidney Tissue.

The prognosis of severe lupus nephritis (LN) is very different among individual patients. None of the current biomarkers can be used to predict the development of refractory LN. Because kidney histology is the gold standard for diagnosing LN, the authors hypothesize that molecular signatures detected in kidney biopsy tissue may have predictive value in determining the therapeutic response. Sixty-seven patients with biopsy-proven severely active LN by International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification III/IV were recruited. Twenty-three kidney tissue samples were used for RNA microarray analysis, while the remaining 44 samples were used for validation by real-time polymerase chain reaction (PCR) gene expression analysis. From hundreds of differential gene expressions in refractory LN, 12 candidates were selected for validation based on gene expression levels as well as relevant functions. The candidate biomarkers were members of the innate immune response molecules, adhesion molecules, calcium-binding receptors, and paracellular tight junction proteins. S100A8, ANXA13, CLDN19 and FAM46B were identified as the best kidney biomarkers for refractory LN, and COL8A1 was identified as the best marker for early loss of kidney function. These new molecular markers can be used to predict refractory LN and may eventually lead to novel molecular targets for therapy.

Analysis of methylation microarray for tissue specific detection.

The role of human DNA methylation has been extensively studied in genomic imprinting, X-inactivation, and disease. However, studies of tissue-specific methylation remain limited. In this study, we use bioinformatics methods to analyze methylation data and reveal loci that are exclusively methylated or unmethylated in individual tissues. We collect 39 previously published DNA methylation profiles using an Illumina® HumanMethylation 27 BeadChip Kit containing 22 common tissues and involving 27,578 CpG loci across the human genome. We found 86 positions of tissue specific methylation CpG (TSM) that encompass 34 hypermethylated TSMs (31 genes) and 52 hypomethylated TSMs (47 genes). Tissues were found to contain 1 to 25 TSM loci, with the majority in the liver (25), testis (18), and brain (16). Fewer TSM loci were found in the muscle (8), ovary (7), adrenal gland (3), pancreas (2-4), kidney, spleen, and stomach (1 each). TSMs are predominantly located 0-300 base pairs in the 3' direction after the transcription start site. Similar to known promoters of methylation, hypermethylated TSM genes suppress transcription, while hypomethylated TSMs allow gene transcription. The majority of hypermethylated TSM genes encode membrane proteins and receptors, while hypomethylated TSM genes primarily encode signal peptides and tissue-specific proteins. In summary, the database of TSM loci produced herein is useful for the selection of tissue-specific DNA markers as diagnostic tools, as well as for the further study of the mechanisms and roles of TSM.

Detecting novel genetic variants associated with isoniazid-resistant Mycobacterium tuberculosis.

BACKGROUND: Isoniazid (INH) is a highly effective antibiotic central for the treatment of Mycobacterium tuberculosis (MTB). INH-resistant MTB clinical isolates are frequently mutated in the katG gene and the inhA promoter region, but 10 to 37% of INH-resistant clinical isolates have no detectable alterations in currently known gene targets associated with INH-resistance. We aimed to identify novel genes associated with INH-resistance in these latter isolates. METHODOLOGY/PRINCIPAL FINDINGS: INH-resistant clinical isolates of MTB were pre-screened for mutations in the katG, inhA, kasA and ndh genes and the regulatory regions of inhA and ahpC. Twelve INH-resistant isolates with no mutations, and 17 INH-susceptible MTB isolates were subjected to whole genome sequencing. Phylogenetically related variants and synonymous mutations were excluded and further analysis revealed mutations in 60 genes and 4 intergenic regions associated with INH-resistance. Sanger sequencing verification of 45 genes confirmed that mutations in 40 genes were observed only in INH-resistant isolates and not in INH-susceptible isolates. The ratios of non-synonymous to synonymous mutations (dN/dS ratio) for the INH-resistance associated mutations identified in this study were 1.234 for INH-resistant and 0.654 for INH-susceptible isolates, strongly suggesting that these mutations are indeed associated with INH-resistance. CONCLUSION: The discovery of novel targets associated with INH-resistance described in this study may potentially be important for the development of improved molecular detection strategies.