Potency, immunogenicity, and efficacy of rabies vaccine: In vitro and in vivo approach.

BACKGROUND: Rabies, a potentially lethal virus, affects more than 150 countries. Although the rabies vaccine and immunoglobulin have been available since 1908, Bangladesh is new to vaccine manufacturing. We checked the quality of the local manufacturing rabies vaccine for substandard. METHODS: The potency and immunogenicity of 20 vaccines were analyzed by three in vivo and in vitro methods from March 2020 to May 2023. Single radial immunodiffusion, fluorescent antibody virus neutralization, and national institutes of health tests were carried out to evaluate the vaccine's efficacy to provide sufficient protection against the rabies virus. RESULTS: The potency of the rabies vaccine was determined by the in vitro SRID method by measuring glycoprotein content. An average of 16 articles from each batch was calculated. The minimum and maximum average mean values of the 20 batches were 5.058 and 5.346, respectively. The variance was calculated at 0.00566. We found a coefficient of variation (CV) between 9.36% and 14.80%. The 100% sample was satisfactory, as these samples had a potency of over 2.5 IU/mL. To observe immunogenicity, we applied the FAVN method for determining antibody titers. An average of 16 articles from every batch were counted to quantify antibody titers. The mean quantity of antibody titers ranged from 2.389 to 3.3875. The CV was slightly lower because of the dispersion of the data. At last, we performed an in vivo method, the NIH test method, to determine potency based on mortality rate. We found a mean value of 4.777 IU/SHD with a standard deviation of 1.13 IU/SHD. All 20 batches were found 100% satisfactory in the NIH test. CONCLUSION: The study implies that the rabies human vaccines manufactured in Bangladesh are potent enough to provide sufficient immunogenicity. Our research is warranted testimony for healthcare providers who work to extirpate rabies.

Mutation and sequencing-based cloning and functional studies of a rust resistance gene in sunflower (Helianthus annuus).

Rust, caused by the fungus Puccinia helianthi Schwein., is one of the most devastating diseases of sunflower (Helianthus annuus L.), affecting global production. The rust R gene R11 in sunflower line HA-R9 shows broad-spectrum resistance to P. helianthi virulent races and was previously mapped to an interval on sunflower chromosome 13 encompassing three candidate genes annotated in the XRQr1.0 reference genome assembly. In the current study, we combined ethyl methane sulfonate (EMS) mutagenesis with targeted region capture and PacBio long-read sequencing to clone the R11 gene. Sequencing of a 60-kb region spanning the R11 locus from the R11 -HA-R9 rust-resistant line and three EMS-induced susceptible mutants facilitated the identification of R11 and definition of induced mutations. The R11 gene is predicted to have a single 3996-bp open reading frame and encodes a protein of 1331 amino acids with CC-NBS-LRR domains typical of genes conferring plant resistance to biotrophic pathogens. Point mutations identified in the R11 rust-susceptible mutants resulted in premature stop codons, consistent with loss of function leading to rust susceptibility. Additional functional studies using comparative RNA sequencing of the resistant line R11 -HA-R9 and R11 -susceptible mutants revealed substantial differences in gene expression patterns associated with R11 -mediated resistance at 7 days post-inoculation with rust, and uncovered the potential roles of terpenoid biosynthesis and metabolism in sunflower rust resistance.

The tomato yellow leaf curl virus C4 protein alters the expression of plant developmental genes correlating to leaf upward cupping phenotype in tomato.

Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus in the family Geminiviridae, is efficiently transmitted by the whitefly, Bemisia tabaci, and causes serious economic losses to tomato crops around the world. TYLCV-infected tomato plants develop distinctive symptoms of yellowing and leaf upward cupping. In recent years, excellent progress has been made in the characterization of TYLCV C4 protein function as a pathogenicity determinant in experimental plants, including Nicotiana benthamiana and Arabidopsis thaliana. However, the molecular mechanism leading to disease symptom development in the natural host plant, tomato, has yet to be characterized. The aim of the current study was to generate transgenic tomato plants expressing the TYLCV C4 gene and evaluate differential gene expression through comparative transcriptome analysis between the transgenic C4 plants and the transgenic green fluorescent protein (Gfp) gene control plants. Transgenic tomato plants expressing TYLCV C4 developed phenotypes, including leaf upward cupping and yellowing, that are similar to the disease symptoms expressed on tomato plants infected with TYLCV. In a total of 241 differentially expressed genes identified in the transcriptome analysis, a series of plant development-related genes, including transcription factors, glutaredoxins, protein kinases, R-genes and microRNA target genes, were significantly altered. These results provide further evidence to support the important function of the C4 protein in begomovirus pathogenicity. These transgenic tomato plants could serve as basic genetic materials for further characterization of plant receptors that are interacting with the TYLCV C4.

Hymenoptera Genome Database: new genomes and annotation datasets for improved go enrichment and orthologue analyses.

We report an update of the Hymenoptera Genome Database (HGD; http://HymenopteraGenome.org), a genomic database of hymenopteran insect species. The number of species represented in HGD has nearly tripled, with fifty-eight hymenopteran species, including twenty bees, twenty-three ants, eleven wasps and four sawflies. With a reorganized website, HGD continues to provide the HymenopteraMine genomic data mining warehouse and JBrowse/Apollo genome browsers integrated with BLAST. We have computed Gene Ontology (GO) annotations for all species, greatly enhancing the GO annotation data gathered from UniProt with more than a ten-fold increase in the number of GO-annotated genes. We have also generated orthology datasets that encompass all HGD species and provide orthologue clusters for fourteen taxonomic groups. The new GO annotation and orthology data are available for searching in HymenopteraMine, and as bulk file downloads.

Effectiveness of disinfectants against the spread of tobamoviruses: Tomato brown rugose fruit virus and Cucumber green mottle mosaic virus.

BACKGROUND: Tobamoviruses, including tomato brown rugose fruit virus (ToBRFV) on tomato and pepper, and cucumber green mottle mosaic virus (CGMMV) on cucumber and watermelon, have caused many disease outbreaks around the world in recent years. With seed-borne, mechanical transmission and resistant breaking traits, tobamoviruses pose serious threat to vegetable production worldwide. With the absence of a commercial resistant cultivar, growers are encouraged to take preventative measures to manage those highly contagious viral diseases. However, there is no information available on which disinfectants are effective to deactivate the virus infectivity on contaminated hands, tools and equipment for these emerging tobamoviruses. The purpose of this study was to evaluate a collection of 16 chemical disinfectants for their effectiveness against mechanical transmission of two emerging tobamoviruses, ToBRFV and CGMMV. METHODS: Bioassay was used to evaluate the efficacy of each disinfectant based on virus infectivity remaining in a prepared virus inoculum after three short exposure times (10 s, 30 s and 60 s) to the disinfectant and inoculated mechanically on three respective test plants (ToBRFV on tomato and CGMMV on watermelon). Percent infection of plants was measured through symptom observation on the test plants and the presence of the virus was confirmed through an enzyme-linked immunosorbent assay with appropriate antibodies. Statistical analysis was performed using one-way ANOVA based on data collected from three independent experiments. RESULTS: Through comparative analysis of percent infection of test plants, a similar trend of efficacy among 16 disinfectants was observed between the two pathosystems. Four common disinfectants with broad spectrum activities against two different tobamoviruses were identified. Those effective disinfectants with 90-100% efficacy against both tobamoviruses were 0.5% Lactoferrin, 2% Virocid, and 10% Clorox, plus 2% Virkon against CGMMV and 3% Virkon against ToBRFV. In addition, SP2700 generated a significant effect against CGMMV, but poorly against ToBRFV. CONCLUSION: Identification of common disinfectants against ToBRFV and CGMMV, two emerging tobamoviruses in two different pathosystems suggest their potential broader effects against other tobamoviruses or even other viruses.

Map and sequence-based chromosome walking towards cloning of the male fertility restoration gene Rf5 linked to R11 in sunflower.

The nuclear fertility restorer gene Rf5 in HA-R9, originating from the wild sunflower species Helianthus annuus, is able to restore the widely used PET1 cytoplasmic male sterility in sunflowers. Previous mapping placed Rf5 at an interval of 5.8 cM on sunflower chromosome 13, distal to a rust resistance gene R11 at a 1.6 cM genetic distance in an SSR map. In the present study, publicly available SNP markers were further mapped around Rf5 and R11 using 192 F2 individuals, reducing the Rf5 interval from 5.8 to 0.8 cM. Additional SNP markers were developed in the target region of the two genes from the whole-genome resequencing of HA-R9, a donor line carrying Rf5 and R11. Fine mapping using 3517 F3 individuals placed Rf5 at a 0.00071 cM interval and the gene co-segregated with SNP marker S13_216392091. Similarly, fine mapping performed using 8795 F3 individuals mapped R11 at an interval of 0.00210 cM, co-segregating with two SNP markers, S13_225290789 and C13_181790141. Sequence analysis identified Rf5 as a pentatricopeptide repeat-encoding gene. The high-density map and diagnostic SNP markers developed in this study will accelerate the use of Rf5 and R11 in sunflower breeding.

MaizeMine: A Data Mining Warehouse for the Maize Genetics and Genomics Database.

MaizeMine is the data mining resource of the Maize Genetics and Genome Database (MaizeGDB; http://maizemine.maizegdb.org). It enables researchers to create and export customized annotation datasets that can be merged with their own research data for use in downstream analyses. MaizeMine uses the InterMine data warehousing system to integrate genomic sequences and gene annotations from the Zea mays B73 RefGen_v3 and B73 RefGen_v4 genome assemblies, Gene Ontology annotations, single nucleotide polymorphisms, protein annotations, homologs, pathways, and precomputed gene expression levels based on RNA-seq data from the Z. mays B73 Gene Expression Atlas. MaizeMine also provides database cross references between genes of alternative gene sets from Gramene and NCBI RefSeq. MaizeMine includes several search tools, including a keyword search, built-in template queries with intuitive search menus, and a QueryBuilder tool for creating custom queries. The Genomic Regions search tool executes queries based on lists of genome coordinates, and supports both the B73 RefGen_v3 and B73 RefGen_v4 assemblies. The List tool allows you to upload identifiers to create custom lists, perform set operations such as unions and intersections, and execute template queries with lists. When used with gene identifiers, the List tool automatically provides gene set enrichment for Gene Ontology (GO) and pathways, with a choice of statistical parameters and background gene sets. With the ability to save query outputs as lists that can be input to new queries, MaizeMine provides limitless possibilities for data integration and meta-analysis.

Deep Sequencing of Small RNAs in the Whitefly Bemisia tabaci Reveals Novel MicroRNAs Potentially Associated with Begomovirus Acquisition and Transmission.

The whitefly Bemisia tabaci (Gennadius) is a notorious insect vector that transmits hundreds of plant viruses, affecting food and fiber crops worldwide, and results in the equivalent of billions of U.S. dollars in crop loss annually. To gain a better understanding of the mechanism in virus transmission, we conducted deep sequencing of small RNAs on the whitefly B. tabaci MEAM1 (Middle East-Asia Minor 1) that fed on tomato plants infected with tomato yellow leaf curl virus (TYLCV). Overall, 160 miRNAs were identified, 66 of which were conserved and 94 were B. tabaci-specific. Among the B. tabaci-specific miRNAs, 67 were newly described in the present study. Two miRNAs, with predicted targets encoding a nuclear receptor (Bta05482) and a very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase 2 (Bta10702), respectively, were differentially expressed in whiteflies that fed on TYLCV-infected versus uninfected plants. To better understand the regulatory effects of identified miRNAs and their target genes, we correlated expression profiles of miRNAs and their target transcripts and found that, interestingly, miRNA expression was inversely correlated with the expression of ~50% of the predicted target genes. These analyses could serve as a model to study gene regulation in other systems involving arthropod transmission of viruses to plants and animals.

The regulatory landscape of early maize inflorescence development.

BACKGROUND: The functional genome of agronomically important plant species remains largely unexplored, yet presents a virtually untapped resource for targeted crop improvement. Functional elements of regulatory DNA revealed through profiles of chromatin accessibility can be harnessed for fine-tuning gene expression to optimal phenotypes in specific environments. RESULT: Here, we investigate the non-coding regulatory space in the maize (Zea mays) genome during early reproductive development of pollen- and grain-bearing inflorescences. Using an assay for differential sensitivity of chromatin to micrococcal nuclease (MNase) digestion, we profile accessible chromatin and nucleosome occupancy in these largely undifferentiated tissues and classify at least 1.6% of the genome as accessible, with the majority of MNase hypersensitive sites marking proximal promoters, but also 3' ends of maize genes. This approach maps regulatory elements to footprint-level resolution. Integration of complementary transcriptome profiles and transcription factor occupancy data are used to annotate regulatory factors, such as combinatorial transcription factor binding motifs and long non-coding RNAs, that potentially contribute to organogenesis, including tissue-specific regulation between male and female inflorescence structures. Finally, genome-wide association studies for inflorescence architecture traits based solely on functional regions delineated by MNase hypersensitivity reveals new SNP-trait associations in known regulators of inflorescence development as well as new candidates. CONCLUSIONS: These analyses provide a comprehensive look into the cis-regulatory landscape during inflorescence differentiation in a major cereal crop, which ultimately shapes architecture and influences yield potential.

Bovine Genome Database: new annotation tools for a new reference genome.

The Bovine Genome Database (BGD) (http://bovinegenome.org) has been the key community bovine genomics database for more than a decade. To accommodate the increasing amount and complexity of bovine genomics data, BGD continues to advance its practices in data acquisition, curation, integration and efficient data retrieval. BGD provides tools for genome browsing (JBrowse), genome annotation (Apollo), data mining (BovineMine) and sequence database searching (BLAST). To augment the BGD genome annotation capabilities, we have developed a new Apollo plug-in, called the Locus-Specific Alternate Assembly (LSAA) tool, which enables users to identify and report potential genome assembly errors and structural variants. BGD now hosts both the newest bovine reference genome assembly, ARS-UCD1.2, as well as the previous reference genome, UMD3.1.1, with cross-genome navigation and queries supported in JBrowse and BovineMine, respectively. Other notable enhancements to BovineMine include the incorporation of genomes and gene annotation datasets for non-bovine ruminant species (goat and sheep), support for multiple assemblies per organism in the Regions Search tool, integration of additional ontologies and development of many new template queries. To better serve the research community, we continue to focus on improving existing tools, developing new tools, adding new datasets and encouraging researchers to use these resources.

Understanding the Transmissibility of Cucumber Green Mottle Mosaic Virus in Watermelon Seeds and Seed Health Assays.

Cucumber green mottle mosaic virus (CGMMV), an emerging tobamovirus, has caused serious disease outbreaks to cucurbit crops in several countries, including the United States. Although CGMMV is seed-borne, the mechanism of its transmission from a contaminated seed to germinating seedling is still not fully understood, and the most suitable seed health assay method has not been well established. To evaluate the mechanism of seed transmissibility, using highly contaminated watermelon seeds collected from CGMMV-infected experimental plants, bioassays were conducted in a greenhouse through seedling grow-out and by mechanical inoculation. Through natural seedling grow-out, we did not observe seed transmission of CGMMV to germinating seedlings. However, efficient transmission of CGMMV was observed using bioassays on melon plants through mechanical inoculation of seed extract prepared from CGMMV-contaminated seeds. Understanding the seed-borne property and the ease of mechanical transmission of CGMMV from a contaminated seed to seedling is an important finding. In comparative evaluation of various laboratory techniques for seed health assays, we found that enzyme-linked immunosorbent assay and loop-mediated isothermal amplification were the most sensitive and reliable methods to detect CGMMV on cucurbit seeds. Because CGMMV is a seed-borne and highly contagious virus, a new infection might not result in a natural seedling grow-out; it could occur through mechanical transmission from contaminated seeds. Therefore, a sensitive seed health test is necessary to ensure CGMMV-free seed lots are used for planting.

Genome-wide profiling of piRNAs in the whitefly Bemisia tabaci reveals cluster distribution and association with begomovirus transmission.

The whitefly Bemisia tabaci MEAM1 is a notorious vector capable of transmitting many plant viruses, resulting in serious crop loss and food shortage around the world. To investigate potential sRNA-mediated regulatory mechanisms in whiteflies that are affected by virus acquisition and transmission, we conducted small RNA (sRNA) deep sequencing and performed genome-wide profiling of piwi-interacting RNAs (piRNAs) in whiteflies that were fed on tomato yellow leaf curl virus (TYLCV)-infected or non-infected tomato plants for 24, 48, and 72 h. In the present study, piRNA reads ranging from 564,395 to 1,715,652 per library were identified and shown to distribute unevenly in clusters (57 to 96 per library) on the whitefly (B. tabaci MEAM1) genome. Among them, 53 piRNA clusters were common for all treatments. Comparative analysis between libraries generated from viruliferous and non-viruliferous whiteflies identified five TYLCV-induced and 24 TYLCV-suppressed piRNA clusters. Approximately 62% of piRNAs were derived from non-coding sequences including intergenic regions, introns, and untranslated regions (UTRs). The remaining 38% were derived from coding sequences (CDS) or repeat elements. Interestingly, six protein coding genes were targeted by the TYLCV-induced piRNAs. We identified a large number of piRNAs that were distributed in clusters across the whitefly genome, with 60% being derived from non-coding regions. Comparative analysis revealed that feeding on a virus-infected host caused induction and suppression of only a small number of piRNA clusters in whiteflies. Although piRNAs primarily regulate the activity of transposable elements, our results suggest that they may have additional functions in regulating protein coding genes and in insect-virus interactions.

Ribosome profiling reveals changes in translational status of soybean transcripts during immature cotyledon development.

To understand translational capacity on a genome-wide scale across three developmental stages of immature soybean seed cotyledons, ribosome profiling was performed in combination with RNA sequencing and cluster analysis. Transcripts representing 216 unique genes demonstrated a higher level of translational activity in at least one stage by exhibiting higher translational efficiencies (TEs) in which there were relatively more ribosome footprint sequence reads mapping to the transcript than were present in the control total RNA sample. The majority of these transcripts were more translationally active at the early stage of seed development and included 12 unique serine or cysteine proteases and 16 2S albumin and low molecular weight cysteine-rich proteins that may serve as substrates for turnover and mobilization early in seed development. It would appear that the serine proteases and 2S albumins play a vital role in the early stages. In contrast, our investigation of profiles of 19 genes encoding high abundance seed storage proteins, such as glycinins, beta-conglycinins, lectin, and Kunitz trypsin inhibitors, showed that they all had similar patterns in which the TE values started at low levels and increased approximately 2 to 6-fold during development. The highest levels of these seed protein transcripts were found at the mid-developmental stage, whereas the highest ribosome footprint levels of only up to 1.6 TE were found at the late developmental stage. These experimental findings suggest that the major seed storage protein coding genes are primarily regulated at the transcriptional level during normal soybean cotyledon development. Finally, our analyses also identified a total of 370 unique gene models that showed very low TE values including over 48 genes encoding ribosomal family proteins and 95 gene models that are related to energy and photosynthetic functions, many of which have homology to the chloroplast genome. Additionally, we showed that genes of the chloroplast were relatively translationally inactive during seed development.

The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ring-spot virus resistance locus.

Bottle gourd (Lagenaria siceraria) is an important vegetable crop as well as a rootstock for other cucurbit crops. In this study, we report a high-quality 313.4-Mb genome sequence of a bottle gourd inbred line, USVL1VR-Ls, with a scaffold N50 of 8.7 Mb and the longest of 19.0 Mb. About 98.3% of the assembled scaffolds are anchored to the 11 pseudomolecules. Our comparative genomic analysis identifies chromosome-level syntenic relationships between bottle gourd and other cucurbits, as well as lineage-specific gene family expansions in bottle gourd. We reconstructed the genome of the most recent common ancestor of Cucurbitaceae, which revealed that the ancestral Cucurbitaceae karyotypes consisted of 12 protochromosomes with 18 534 protogenes. The 12 protochromosomes are largely retained in the modern melon genome, while have undergone different degrees of shuffling events in other investigated cucurbit genomes. The 11 bottle gourd chromosomes derive from the ancestral Cucurbitaceae karyotypes followed by 19 chromosomal fissions and 20 fusions. The bottle gourd genome sequence has facilitated the mapping of a dominant monogenic locus, Prs, conferring Papaya ring-spot virus (PRSV) resistance in bottle gourd, to a 317.8-kb region on chromosome 1. We have developed a cleaved amplified polymorphic sequence (CAPS) marker tightly linked to the Prs locus and demonstrated its potential application in marker-assisted selection of PRSV resistance in bottle gourd. This study provides insights into the paleohistory of Cucurbitaceae genome evolution, and the high-quality genome sequence of bottle gourd provides a useful resource for plant comparative genomics studies and cucurbit improvement.

Direct detection of transcription factors in cotyledons during seedling development using sensitive silicon-substrate photonic crystal protein arrays.

Transcription factors control important gene networks, altering the expression of a wide variety of genes, including those of agronomic importance, despite often being expressed at low levels. Detecting transcription factor proteins is difficult, because current high-throughput methods may not be sensitive enough. One-dimensional, silicon-substrate photonic crystal (PC) arrays provide an alternative substrate for printing multiplexed protein microarrays that have greater sensitivity through an increased signal-to-noise ratio of the fluorescent signal compared with performing the same assay upon a traditional aminosilanized glass surface. As a model system to test proof of concept of the silicon-substrate PC arrays to directly detect rare proteins in crude plant extracts, we selected representatives of four different transcription factor families (zinc finger GATA, basic helix-loop-helix, BTF3/NAC [for basic transcription factor of the NAC family], and YABBY) that have increasing transcript levels during the stages of seedling cotyledon development. Antibodies to synthetic peptides representing the transcription factors were printed on both glass slides and silicon-substrate PC slides along with antibodies to abundant cotyledon proteins, seed lectin, and Kunitz trypsin inhibitor. The silicon-substrate PC arrays proved more sensitive than those performed on glass slides, detecting rare proteins that were below background on the glass slides. The zinc finger transcription factor was detected on the PC arrays in crude extracts of all stages of the seedling cotyledons, whereas YABBY seemed to be at the lower limit of their sensitivity. Interestingly, the basic helix-loop-helix and NAC proteins showed developmental profiles consistent with their transcript patterns, indicating proof of concept for detecting these low-abundance proteins in crude extracts.

Transcription factors and glyoxylate cycle genes prominent in the transition of soybean cotyledons to the first functional leaves of the seedling.

During early seedling growth, the cotyledons transition from a storage tissue to become effectively the first leaf-like structures of the plant. In this programmed developmental process, they likely undergo a massive change in gene expression to redirect their metabolism and physiological processes. To define the developmental shifts in gene expression and begin to understand the gene regulatory networks that set this transition in motion, we carried out high-throughput RNA sequencing of cotyledons from seven developmental stages of soybean seedlings. We identified 154 gene models with high expression exclusively in the early seedling stages. A significant number (about 25 %) of those genes with known annotations were involved in carbohydrate metabolism. A detailed examination of glyoxylate cycle genes revealed the upregulation of their expression in the early stages of development. A total of approximately 50 % of the highly expressed genes whose expression peaked in the mid-developmental stages encoded ribosomal family proteins. Our analysis also identified 219 gene models with high expression at late developmental stages. The majority of these genes are involved in photosynthesis, including photosystem I- and II-associated genes. Additionally, the advantage of RNA-Seq to detect genes expressed at low levels revealed approximately 460 transcription factors with notable expression in at least one stage of the developing soybean seedling. Relatively over-represented transcription factor genes encode AP2, zinc finger, NAC, WRKY, and MYB families. These transcription factor genes may lead to the transcriptional reprogramming during the transition of seedling cotyledons from storage tissue to metabolically active organs that serve as the first functional leaves of the plant.

Genome-wide identification of binding sites for NAC and YABBY transcription factors and co-regulated genes during soybean seedling development by ChIP-Seq and RNA-Seq.

BACKGROUND: Two plant-specific transcription factors, NAC and YABBY, are involved in important plant developmental processes. However their molecular mechanisms, especially DNA binding sites and co-regulated genes, are largely unknown during soybean seedling development. RESULTS: In order to identify genome-wide binding sites of specific members of the NAC and YABBY transcription factors and co-regulated genes, we performed Chromatin Immunoprecipitation Sequencing (ChIP-Seq) and RNA Sequencing (RNA-Seq) using cotyledons from soybean seedling developmental stages. Our RNA-Seq data revealed that these particular NAC and YABBY transcription factors showed a clear pattern in their expression during soybean seedling development. The highest level of their expression was found in seedling developmental stage 4 when cotyledons undergo a physiological transition from non-photosynthetic storage tissue to a metabolically active photosynthetic tissue. Our ChIP-Seq data identified 72 genes potentially regulated by the NAC and 96 genes by the YABBY transcription factors examined. Our RNA-Seq data revealed highly differentially expressed candidate genes regulated by the NAC transcription factor include lipoxygense, pectin methyl esterase inhibitor, DEAD/DEAH box helicase and homeobox associated proteins. YABBY-regulated genes include AP2 transcription factor, fatty acid desaturase and WRKY transcription factor. Additionally, we have identified DNA binding motifs for the NAC and YABBY transcription factors. CONCLUSIONS: Genome-wide determination of binding sites for NAC and YABBY transcription factors and identification of candidate genes regulated by these transcription factors will advance the understanding of complex gene regulatory networks during soybean seedling development. Our data imply that there is transcriptional reprogramming during the functional transition of cotyledons from non-photosynthetic storage tissue to metabolically active photosynthetic tissue.

Identification of soybean seed developmental stage-specific and tissue-specific miRNA targets by degradome sequencing.

BACKGROUND: MicroRNAs (miRNAs) regulate the expression of target genes by mediating gene silencing in both plants and animals. The miRNA targets have been extensively investigated in Arabidopsis and rice using computational prediction, experimental validation by overexpression in transgenic plants, and by degradome or PARE (parallel analysis of RNA ends) sequencing. However, miRNA targets mostly remain unknown in soybean (Glycine max). More specifically miRNA mediated gene regulation at different seed developmental stages in soybean is largely unexplored. In order to dissect miRNA guided gene regulation in soybean developing seeds, we performed a transcriptome-wide experimental method using degradome sequencing to directly detect cleaved miRNA targets. RESULTS: In this study, degradome libraries were separately prepared from immature soybean cotyledons representing three stages of development and from seed coats of two stages. Sequencing and analysis of 10 to 40 million reads from each library resulted in identification of 183 different targets for 53 known soybean miRNAs. Among these, some were found only in the cotyledons representing cleavage by 25 miRNAs and others were found only in the seed coats reflecting cleavage by 12 miRNAs. A large number of targets for 16 miRNAs families were identified in both tissues irrespective of the stage. Interestingly, we identified more miRNA targets in the desiccating cotyledons of late seed maturation than in immature seed. We validated four different auxin response factor genes as targets for gma-miR160 via RNA ligase mediated 5' rapid amplification of cDNA ends (RLM-5'RACE). Gene Ontology (GO) analysis indicated the involvement of miRNA target genes in various cellular processes during seed development. CONCLUSIONS: The miRNA targets in both the cotyledons and seed coats of several stages of soybean seed development have been elucidated by experimental evidence from comprehensive, high throughput sequencing of the enriched fragments resulting from miRNA-guided cleavage of messenger RNAs. Nearly 50% of the miRNA targets were transcription factors in pathways that are likely important in setting or maintaining the developmental program leading to high quality soybean seeds that are one of the dominant sources of protein and oil in world markets.

De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks.

Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.