Genomics reveals heterogeneous Plasmodium falciparum transmission and selection signals in Zambia.

BACKGROUND: Genomic surveillance is crucial for monitoring malaria transmission and understanding parasite adaptation to interventions. Zambia lacks prior nationwide efforts in malaria genomic surveillance among African countries. METHODS: We conducted genomic surveillance of Plasmodium falciparum parasites from the 2018 Malaria Indicator Survey in Zambia, a nationally representative household survey of children under five years of age. We whole-genome sequenced and analyzed 241 P. falciparum genomes from regions with varying levels of malaria transmission across Zambia and estimated genetic metrics that are informative about transmission intensity, genetic relatedness between parasites, and selection. RESULTS: We provide genomic evidence of widespread within-host polygenomic infections, regardless of epidemiological characteristics, underscoring the extensive and ongoing endemic malaria transmission in Zambia. Our analysis reveals country-level clustering of parasites from Zambia and neighboring regions, with distinct separation in West Africa. Within Zambia, identity by descent (IBD) relatedness analysis uncovers local spatial clustering and rare cases of long-distance sharing of closely related parasite pairs. Genomic regions with large shared IBD segments and strong positive selection signatures implicate genes involved in sulfadoxine-pyrimethamine and artemisinin combination therapies drug resistance, but no signature related to chloroquine resistance. Furthermore, differences in selection signatures, including drug resistance loci, are observed between eastern and western Zambian parasite populations, suggesting variable transmission intensity and ongoing drug pressure. CONCLUSIONS: Our findings enhance our understanding of nationwide P. falciparum transmission in Zambia, establishing a baseline for analyzing parasite genetic metrics as they vary over time and space. These insights highlight the urgency of strengthening malaria control programs and surveillance of antimalarial drug resistance.

Malaria is caused by a parasite that is spread to humans via mosquito bites. It is a leading cause of death in children under five years old in sub-Saharan Africa. Analysis of the malaria parasite's complete set of DNA (its genome) can help us to understand transmission of the disease and how this changes in response to different strategies to control the disease. We analyzed the genomes of malaria parasites from children across Zambia. Our study revealed that 77% of children harbored multiple parasite strains, which suggests that local transmission (transmission between people within the same local area) is high. Genetic evidence for long-distance transmission was rarer. Furthermore, our findings suggest parasites are evolving in response to antimalarial drugs. Our study enhances our understanding of malaria dynamics in Zambia and may help to inform strategies for improved surveillance and control.

Genomics reveals heterogeneous Plasmodium falciparum transmission and population differentiation in Zambia and bordering countries.

Genomic surveillance plays a critical role in monitoring malaria transmission and understanding how the parasite adapts in response to interventions. We conducted genomic surveillance of malaria by sequencing 241 Plasmodium falciparum genomes from regions with varying levels of malaria transmission across Zambia. We found genomic evidence of high levels of within-host polygenomic infections, regardless of epidemiological characteristics, underscoring the extensive and ongoing endemic malaria transmission in the country. We identified country-level clustering of parasites from Zambia and neighboring countries, and distinct clustering of parasites from West Africa. Within Zambia, our identity by descent (IBD) relatedness analysis uncovered spatial clustering of closely related parasite pairs at the local level and rare cases of long-distance sharing. Genomic regions with large shared IBD segments and strong positive selection signatures identified genes involved in sulfadoxine-pyrimethamine and artemisinin combination therapies drug resistance, but no signature related to chloroquine resistance. Together, our findings enhance our understanding of P. falciparum transmission nationwide in Zambia and highlight the urgency of strengthening malaria control programs and surveillance of antimalarial drug resistance.

Comparative Transcriptome Analysis of Tolerant and Sensitive Genotypes of Common Bean (Phaseolus vulgaris L.) in Response to Terminal Drought Stress.

We conducted a genome-wide transcriptomic analysis of three drought tolerant and sensitive genotypes of common bean to examine their transcriptional responses to terminal drought stress. We then conducted pairwise comparisons between the root and leaf transcriptomes from the resulting tissue based on combined transcriptomic data from the tolerant and sensitive genotypes. Our transcriptomic data revealed that 491 (6.4%) DEGs (differentially expressed genes) were upregulated in tolerant genotypes, whereas they were downregulated in sensitive genotypes; likewise, 396 (5.1%) DEGs upregulated in sensitive genotypes were downregulated in tolerant genotypes. Several transcription factors, heat shock proteins, and chaperones were identified in the study. Several DEGs in drought DB (data Base) overlapped between genotypes. The GO (gene ontology) terms for biological processes showed upregulation of DEGs in tolerant genotypes for sulfate and drug transmembrane transport when compared to sensitive genotypes. A GO term for cellular components enriched with upregulated DEGs for the apoplast in tolerant genotypes. These results substantiated the temporal pattern of root growth (elongation and initiation of root growth), and ABA-mediated drought response in tolerant genotypes. KEGG (kyoto encyclopedia of genes and genomes) analysis revealed an upregulation of MAPK (mitogen activated protein kinase) signaling pathways and plant hormone signaling pathways in tolerant genotypes. As a result of this study, it will be possible to uncover the molecular mechanisms of drought tolerance in response to terminal drought stress in the field. Further, genome-wide transcriptomic analysis of both tolerant and sensitive genotypes will assist us in identifying potential genes that may contribute to improving drought tolerance in the common bean.

Characterization of sow milk N-linked glycoproteome over the course of lactation.

Milk proteins serve as nutrition and affect neonate development and immunity through their bioactivity. Post-translational modifications of proteins affect their bioactivity. Glycosylation is the attachment of sugar moieties to proteins, with attachment of glycans to asparagine indicated as N-linked glycosylation. Our objective was to characterize N-linked glycosylated proteins in homogenate swine milk samples collected from sows (n = 5/6) during farrowing to represent colostrum and on days 3 and 14 post-farrowing to represent transitional and mature milk, respectively. Glycopeptides were isolated with lectin-based extraction and treated with Peptide N-glycosidase F (PNGase F) to identify N-linked glycosylation sites. Purified glycopeptides were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant software was used to align spectra to Sus scrofa Uniport database to identify proteins and measure their relative abundances. Analysis of variance and Welch's t-test analysis identified glycoproteins differentially abundant between colostrum, transitional, and mature milk (false discovery rate <0.05). Shotgun proteome analysis identified 545 N-linked and glutamine, Q, -linked, glycosylation (P > 0.75 for deamidation) sites on 220 glycoproteins in sow milk. Glycoproteins were found across all three phases of swine milk production and varied by number of glycosylation sites (1-14) and in abundance and distribution between colostrum, transitional, and mature milk. Polymeric immunoglobulin receptor was the most glycosylated protein with 14 sites identified. Also highly glycosylated were casein and mucin proteins. These data are described and the relevance of glycosylated milk proteins in neonate development, such as protection against pathogens, is discussed.

Milk is essential for healthy growth and development of neonates, with proteins in milk serving as key nutrients and regulators of these processes. Protein activity is affected by modifications made to their structure including the addition of sugar groups called glycans. Here we present the characterization of sow milk proteins modification with glycan groups on asparagine and glutamine amino acids in colostrum, transitional, and mature milk of pigs. We found 220 high confidences (found in at least two sows on one day) glycoproteins, and that the abundance of glycosylated proteins varied by stage of milk production and number of glycosylated sites.

Comparative Hessian Fly Larval Transcriptomics Provides Novel Insight into Host and Nonhost Resistance.

The Hessian fly is a destructive pest of wheat. Employing additional molecular strategies can complement wheat's native insect resistance. However, this requires functional characterization of Hessian-fly-responsive genes, which is challenging because of wheat genome complexity. The diploid Brachypodium distachyon (Bd) exhibits nonhost resistance to Hessian fly and displays phenotypic/molecular responses intermediate between resistant and susceptible host wheat, offering a surrogate genome for gene characterization. Here, we compared the transcriptomes of Biotype L larvae residing on resistant/susceptible wheat, and nonhost Bd plants. Larvae from susceptible wheat and nonhost Bd plants revealed similar molecular responses that were distinct from avirulent larval responses on resistant wheat. Secreted salivary gland proteins were strongly up-regulated in all larvae. Genes from various biological pathways and molecular processes were up-regulated in larvae from both susceptible wheat and nonhost Bd plants. However, Bd larval expression levels were intermediate between larvae from susceptible and resistant wheat. Most genes were down-regulated or unchanged in avirulent larvae, correlating with their inability to establish feeding sites and dying within 4-5 days after egg-hatch. Decreased gene expression in Bd larvae, compared to ones on susceptible wheat, potentially led to developmentally delayed 2nd-instars, followed by eventually succumbing to nonhost resistance defense mechanisms.

Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis.

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.

Phase I/II Trial of Vemurafenib in Dogs with Naturally Occurring, BRAF-mutated Urothelial Carcinoma.

BRAF-targeted therapies including vemurafenib (Zelboraf) induce dramatic cancer remission; however, drug resistance commonly emerges. The purpose was to characterize a naturally occurring canine cancer model harboring complex features of human cancer, to complement experimental models to improve BRAF-targeted therapy. A phase I/II clinical trial of vemurafenib was performed in pet dogs with naturally occurring invasive urothelial carcinoma (InvUC) harboring the canine homologue of human BRAF V600E The safety, MTD, pharmacokinetics, and antitumor activity were determined. Changes in signaling and immune gene expression were assessed by RNA sequencing and phosphoproteomic analyses of cystoscopic biopsies obtained before and during treatment, and at progression. The vemurafenib MTD was 37.5 mg/kg twice daily. Anorexia was the most common adverse event. At the MTD, partial remission occurred in 9 of 24 dogs (38%), with a median progression-free interval of 181 days (range, 53-608 days). In 18% of the dogs, new cutaneous squamous cell carcinoma and papillomas occurred, a known pharmacodynamic effect of vemurafenib in humans. Upregulation of genes in the classical and alternative MAPK-related pathways occurred in subsets of dogs at cancer progression. The most consistent transcriptomic changes were the increase in patterns of T lymphocyte infiltration during the first month of vemurafenib, and of immune failure accompanying cancer progression. In conclusion, the safety, antitumor activity, and cutaneous pharmacodynamic effects of vemurafenib, and the development of drug resistance in dogs closely mimic those reported in humans. This suggests BRAF-mutated canine InvUC offers an important complementary animal model to improve BRAF-targeted therapies in humans.

Transcriptome Responses to Defined Insecticide Selection Pressures in the German Cockroach (Blattella germanica L.).

Cockroaches are important global urban pests from aesthetic and health perspectives. Insecticides represent the most cost-effective way to control cockroaches and limit their impacts on human health. However, cockroaches readily develop insecticide resistance, which can quickly limit efficacy of even the newest and most effective insecticide products. The goal of this research was to understand whole-body physiological responses in German cockroaches, at the metatranscriptome level, to defined insecticide selection pressures. We used the insecticide indoxacarb as the selecting insecticide, which is an important bait active ingredient for cockroach control. Six generations of selection with indoxacarb bait produced a strain with substantial (>20×) resistance relative to inbred control lines originating from the same parental stock. Metatranscriptome sequencing revealed 1,123 significantly differentially expressed (DE) genes in ≥two of three statistical models (81 upregulated and 1,042 downregulated; FDR P < 0.001; log2FC of ±1). Upregulated DE genes represented many detoxification enzyme families including cytochrome-P450 oxidative enzymes, hydrolases and glutathione-S-transferases. Interestingly, the majority of downregulated DE genes were from microbial and viral origins, indicating that selection for resistance is also associated with elimination of commensal, pathogenic and/or parasitic microbes. These microbial impacts could result from: (i) direct effects of indoxacarb, (ii) indirect effects of antimicrobial preservatives included in the selecting bait matrix, or (iii) selection for general stress response mechanisms that confer both xenobiotic resistance and immunity. These results provide novel physiological insights into insecticide resistance evolution and mechanisms, as well as novel insights into parallel fitness benefits associated with selection for insecticide resistance.

Transcriptomics in Erigeron canadensis reveals rapid photosynthetic and hormonal responses to auxin herbicide application.

The perception pathway for endogenous auxin has been well described, yet the mode of action of synthetic auxin herbicides, used for >70 years, remains uncharacterized. We utilized transcriptomics and targeted physiological studies to investigate the unknown rapid response to synthetic auxin herbicides in the globally problematic weed species Erigeron canadensis. Synthetic auxin herbicide application consistently and rapidly down-regulated the photosynthetic machinery. At the same time, there was considerable perturbation to the expression of many genes related to phytohormone metabolism and perception. In particular, auxin herbicide application enhanced the expression of the key abscisic acid biosynthetic gene, 9-cis-epoxycarotenoid deoxygenase (NCED). The increase in NCED expression following auxin herbicide application led to a rapid biosynthesis of abscisic acid (ABA). This increase in ABA levels was independent of a loss of cell turgor or an increase in ethylene levels, both proposed triggers for rapid ABA biosynthesis. The levels of ABA in the leaf after auxin herbicide application continued to increase as plants approached death, up to >3-fold higher than in the leaves of plants that were drought stressed. We propose a new model in which synthetic auxin herbicides trigger plant death by the whole-scale, rapid, down-regulation of photosynthetic processes and an increase in ABA levels through up-regulation of NCED expression, independent of ethylene levels or a loss of cell turgor.

Proteomic Analysis of 3T3-L1 Adipocytes Treated with Insulin and TNF-α.

Insulin resistance is an indication of early stage Type 2 diabetes (T2D). Insulin resistant adipose tissues contain higher levels of insulin than the physiological level, as well as higher amounts of intracellular tumor necrosis factor-α (TNF-α) and other cytokines. However, the mechanism of insulin resistance remains poorly understood. To better understand the roles played by insulin and TNF-α in insulin resistance, we performed proteomic analysis of differentiated 3T3-L1 adipocytes treated with insulin (Ins), TNF-α (TNF), and both (Ins + TNF). Out of the 693 proteins identified, the abundances of 78 proteins were significantly different (p < 0.05). Carnitine parmitoyltransferase-2 (CPT2), acetyl CoA carboxylase 1 (ACCAC-1), ethylmalonyl CoA decarboxylase (ECHD1), and methylmalonyl CoA isomerase (MCEE), enzymes required for fatty acid ß-oxidation and respiratory electron transport, and ß-glucuronidase, an enzyme responsible for the breakdown of complex carbohydrates, were down-regulated in all the treatment groups, compared to the control group. In contrast, superoxide dismutase 2 (SOD2), protein disulfide isomerase (PDI), and glutathione reductase, which are the proteins responsible for cytoskeletal structure, protein folding, degradation, and oxidative stress responses, were up-regulated. This suggests higher oxidative stress in cells treated with Ins, TNF, or both. We proposed a conceptual metabolic pathway impacted by the treatments and their possible link to insulin resistance or T2D.

Genome-wide identification of histone methylation (H3K9me2) and acetylation (H4K12ac) marks in two ecotypes of switchgrass (Panicum virgatum L.).

BACKGROUND: Histone modifications play a significant role in the regulation of transcription and various biological processes, such as development and regeneration. Though a few genomic (including DNA methylation patterns) and transcriptomic studies are currently available in switchgrass, the genome-wide distribution of histone modifications has not yet been studied to help elucidate gene regulation and its application to switchgrass improvement. RESULTS: This study provides a comprehensive epigenomic analyses of two contrasting switchgrass ecotypes, lowland (AP13) and upland (VS16), by employing chromatin immunoprecipitation sequencing (ChIP-Seq) with two histone marks (suppressive- H3K9me2 and active- H4K12ac). In this study, most of the histone binding was in non-genic regions, and the highest enrichment was seen between 0 and 2 kb regions from the transcriptional start site (TSS). Considering the economic importance and potential of switchgrass as a bioenergy crop, we focused on genes, transcription factors (TFs), and pathways that were associated with C4-photosynthesis, biomass, biofuel production, biotic stresses, and abiotic stresses. Using quantitative real-time PCR (qPCR) the relative expression of five genes selected from the phenylpropanoid-monolignol pathway showed preferential binding of acetylation marks in AP13 rather than in VS16. CONCLUSIONS: The genome-wide histone modifications reported here can be utilized in understanding the regulation of genes important in the phenylpropanoid-monolignol biosynthesis pathway, which in turn, may help understand the recalcitrance associated with conversion of biomass to biofuel, a major roadblock in utilizing lignocellulosic feedstocks.

Comparative analysis of the root transcriptomes of cultivated sweetpotato (Ipomoea batatas [L.] Lam) and its wild ancestor (Ipomoea trifida [Kunth] G. Don).

BACKGROUND: The complex process of formation of storage roots (SRs) from adventitious roots affects sweetpotato yield. Identifying the genes that are uniquely expressed in the SR forming cultivated species, Ipomoea batatas (Ib), and its immediate ancestral species, Ipomoea trifida (It), which does not form SRs, may provide insights into the molecular mechanisms underlying SR formation in sweetpotato. RESULTS: Illumina paired-end sequencing generated ~208 and ~200 million reads for Ib and It, respectively. Trinity assembly of the reads resulted in 98,317 transcripts for Ib and 275,044 for It, after post-assembly removal of trans-chimeras. From these sequences, we identified 4,865 orthologous genes in both Ib and It, 60 paralogous genes in Ib and 2,286 paralogous genes in It. Among paralogous gene sets, transcripts encoding the transcription factor RKD, which may have a role in nitrogen regulation and starch formation, and rhamnogalacturonate lyase (RGL) family proteins, which produce the precursors of cell wall polysaccharides, were found only in Ib. In addition, transcripts encoding a K+ efflux antiporter (KEA5) and the ERECTA protein kinase, which function in phytohormonal regulation and root proliferation, respectively, were also found only in Ib. qRT-PCR indicated that starch and sucrose metabolism genes, such as those encoding ADP-glucose pyrophosphorylase and beta-amylase, showed lower expression in It than Ib, whereas lignin genes such as caffeoyl-CoA O-methyltransferase (CoMT) and cinnamyl alcohol dehydrogenase (CAD) showed higher expression in It than Ib. A total of 7,067 and 9,650 unique microsatellite markers, 1,037,396 and 495,931 single nucleotide polymorphisms (SNPs) and 103,439 and 69,194 InDels in Ib and It, respectively, were also identified from this study. CONCLUSION: The detection of genes involved in the biosynthesis of RGL family proteins, the transcription factor RKD, and genes encoding a K+ efflux antiporter (KEA5) and the ERECTA protein kinase only in I. batatas indicate that these genes may have important functions in SR formation in sweetpotato. Potential molecular markers (SNPs, simple sequence repeats and InDels) and sequences identified in this study may represent a valuable resource for sweetpotato gene annotation and may serve as important tools for improving SR formation in sweetpotato through breeding.

Comparative transcriptome profiling of upland (VS16) and lowland (AP13) ecotypes of switchgrass.

KEY MESSAGE: Transcriptomes of two switchgrass genotypes representing the upland and lowland ecotypes will be key tools in switchgrass genome annotation and biotic and abiotic stress functional genomics. Switchgrass (Panicum virgatum L.) is an important bioenergy feedstock for cellulosic ethanol production. We report genome-wide transcriptome profiling of two contrasting tetraploid switchgrass genotypes, VS16 and AP13, representing the upland and lowland ecotypes, respectively. A total of 268 million Illumina short reads (50 nt) were generated, of which, 133 million were obtained in AP13 and the rest 135 million in VS16. More than 90% of these reads were mapped to the switchgrass reference genome (V1.1). We identified 6619 and 5369 differentially expressed genes in VS16 and AP13, respectively. Gene ontology and KEGG pathway analysis identified key genes that regulate important pathways including C4 photosynthesis, photorespiration and phenylpropanoid metabolism. A series of genes (33) involved in photosynthetic pathway were up-regulated in AP13 but only two genes showed higher expression in VS16. We identified three dicarboxylate transporter homologs that were highly expressed in AP13. Additionally, genes that mediate drought, heat, and salinity tolerance were also identified. Vesicular transport proteins, syntaxin and signal recognition particles were seen to be up-regulated in VS16. Analyses of selected genes involved in biosynthesis of secondary metabolites, plant-pathogen interaction, membrane transporters, heat, drought and salinity stress responses confirmed significant variation in the relative expression reflected in RNA-Seq data between VS16 and AP13 genotypes. The phenylpropanoid pathway genes identified here are potential targets for biofuel conversion.

The impact of orally administered phages on host immune response and surrounding microbial communities.

Numerous studies have shown the efficacy of phage therapy in reducing foodborne pathogen carriage in food animals. Fewer studies have focused on host reactions, especially in terms of phage-mediated acute immune responses and effects on the gut microbiome. Here we administered E. coli O157:H7 phages in low (single dose of 105 PFU) or high (single dose of 107 PFU) quantities to mice. While there were time points at which cytokine levels in different treatment groups differed from one another, all cytokine levels remained within normal ranges for mice regardless of treatment. Similarly, the patterns of these differences were not dose related, indicating that phage treatment did not result in a strong acute immune response as measured here. In separate experiments, 3-week-old pigs received a diet containing an in-feed antibiotic or daily phage treatment. After two weeks, microbial DNA of ileal, cecal, and fecal contents was characterized using 16S rRNA sequencing. There were no statistical differences in performance among the different groups. Compared to control pigs (no antibiotic, no phage), antibiotic treatment significantly altered ileal microbiome composition (P < 0.05), with Bacilli being most affected (antibiotic treated: 22%; control: 76%; FDR = 0.0572). No significant differences were observed in cecal and fecal microbiome composition between antibiotic-treated and control pigs, and there were no differences in gut microbiome composition between phage treated and control pigs in any intestinal compartment. Significant abundance differences were observed at the OTU level, with OTUs belonging to genera such as Lactobacillus and Streptococcus being over- or under-represented in either antibiotic or phage treated groups compared to control pigs. Determining whether these changes are deleterious to host, however, requires further study.

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.).

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress.

Metatranscriptomic profiles of Eastern subterranean termites, Reticulitermes flavipes (Kollar) fed on second generation feedstocks.

BACKGROUND: Second generation lignocellulosic feedstocks are being considered as an alternative to first generation biofuels that are derived from grain starches and sugars. However, the current pre-treatment methods for second generation biofuel production are inefficient and expensive due to the recalcitrant nature of lignocellulose. In this study, we used the lower termite Reticulitermes flavipes (Kollar), as a model to identify potential pretreatment genes/enzymes specifically adapted for use against agricultural feedstocks. RESULTS: Metatranscriptomic profiling was performed on worker termite guts after feeding on corn stover (CS), soybean residue (SR), or 98% pure cellulose (paper) to identify (i) microbial community, (ii) pathway level and (iii) gene-level responses. Microbial community profiles after CS and SR feeding were different from the paper feeding profile, and protist symbiont abundance decreased significantly in termites feeding on SR and CS relative to paper. Functional profiles after CS feeding were similar to paper and SR; whereas paper and SR showed different profiles. Amino acid and carbohydrate metabolism pathways were downregulated in termites feeding on SR relative to paper and CS. Gene expression analyses showed more significant down regulation of genes after SR feeding relative to paper and CS. Stereotypical lignocellulase genes/enzymes were not differentially expressed, but rather were among the most abundant/constitutively-expressed genes. CONCLUSIONS: These results suggest that the effect of CS and SR feeding on termite gut lignocellulase composition is minimal and thus, the most abundantly expressed enzymes appear to encode the best candidate catalysts for use in saccharification of these and related second-generation feedstocks. Further, based on these findings we hypothesize that the most abundantly expressed lignocellulases, rather than those that are differentially expressed have the best potential as pretreatment enzymes for CS and SR feedstocks.

Transcriptomic and proteomic dynamics in the metabolism of a diazotrophic cyanobacterium, Cyanothece sp. PCC 7822 during a diurnal light-dark cycle.

BACKGROUND: Cyanothece sp. PCC 7822 is an excellent cyanobacterial model organism with great potential to be applied as a biocatalyst for the production of high value compounds. Like other unicellular diazotrophic cyanobacterial species, it has a tightly regulated metabolism synchronized to the light-dark cycle. Utilizing transcriptomic and proteomic methods, we quantified the relationships between transcription and translation underlying central and secondary metabolism in response to nitrogen free, 12 hour light and 12 hour dark conditions. RESULTS: By combining mass-spectrometry based proteomics and RNA-sequencing transcriptomics, we quantitatively measured a total of 6766 mRNAs and 1322 proteins at four time points across a 24 hour light-dark cycle. Photosynthesis, nitrogen fixation, and carbon storage relevant genes were expressed during the preceding light or dark period, concurrent with measured nitrogenase activity in the late light period. We describe many instances of disparity in peak mRNA and protein abundances, and strong correlation of light dependent expression of both antisense and CRISPR-related gene expression. The proteins for nitrogenase and the pentose phosphate pathway were highest in the dark, whereas those for glycolysis and the TCA cycle were more prominent in the light. Interestingly, one copy of the psbA gene encoding the photosystem II (PSII) reaction center protein D1 (psbA4) was highly upregulated only in the dark. This protein likely cannot catalyze O2 evolution and so may be used by the cell to keep PSII intact during N2 fixation. The CRISPR elements were found exclusively at the ends of the large plasmid and we speculate that their presence is crucial to the maintenance of this plasmid. CONCLUSIONS: This investigation of parallel transcriptional and translational activity within Cyanothece sp. PCC 7822 provided quantitative information on expression levels of metabolic pathways relevant to engineering efforts. The identification of expression patterns for both mRNA and protein affords a basis for improving biofuel production in this strain and for further genetic manipulations. Expression analysis of the genes encoded on the 6 plasmids provided insight into the possible acquisition and maintenance of some of these extra-chromosomal elements.

Draft Genome Sequence of Acetobacter aceti Strain 1023, a Vinegar Factory Isolate.

The genome sequence of Acetobacter aceti 1023, an acetic acid bacterium adapted to traditional vinegar fermentation, comprises 3.0 Mb (chromosome plus plasmids). A. aceti 1023 is closely related to the cocoa fermenter Acetobacter pasteurianus 386B but possesses many additional insertion sequence elements.

MED18 interaction with distinct transcription factors regulates multiple plant functions.

Mediator is an evolutionarily conserved transcriptional regulatory complex. Mechanisms of Mediator function are poorly understood. Here we show that Arabidopsis MED18 is a multifunctional protein regulating plant immunity, flowering time and responses to hormones through interactions with distinct transcription factors. MED18 interacts with YIN YANG1 to suppress disease susceptibility genes glutaredoxins GRX480, GRXS13 and thioredoxin TRX-h5. Consequently, yy1 and med18 mutants exhibit deregulated expression of these genes and enhanced susceptibility to fungal infection. In addition, MED18 interacts with ABA INSENSITIVE 4 and SUPPRESSOR OF FRIGIDA4 to regulate abscisic acid responses and flowering time, respectively. MED18 associates with the promoter, coding and terminator regions of target genes suggesting its function in transcription initiation, elongation and termination. Notably, RNA polymerase II occupancy and histone H3 lysine tri-methylation of target genes are affected in the med18 mutant, reinforcing MED18 function in different mechanisms of transcriptional control. Overall, MED18 conveys distinct cues to engender transcription underpinning plant responses.