Leishmania genetic exchange is mediated by IgM natural antibodies.

Host factors that mediate Leishmania genetic exchange are not well defined. Here we demonstrate that natural IgM (IgMn)1-4 antibodies mediate parasite genetic exchange by inducing the transient formation of a spherical parasite clump that promotes parasite fusion and hybrid formation. We establish that IgMn from Leishmania-free animals binds to the surface of Leishmania parasites to induce significant changes in the expression of parasite transcripts and proteins. Leishmania binding to IgMn is partially lost after glycosidase treatment, although parasite surface phosphoglycans, including lipophosphoglycan, are not required for IgMn-induced parasite clumping. Notably, the transient formation of parasite clumps is essential for Leishmania hybridization in vitro. In vivo, we observed a 12-fold increase in hybrid formation in sand flies provided a second blood meal containing IgMn compared with controls. Furthermore, the generation of recombinant progeny from mating hybrids and parental lines were only observed in sand flies provided with IgMn. Both in vitro and in vivo IgM-induced Leishmania crosses resulted in full genome hybrids that show equal patterns of biparental contribution. Leishmania co-option of a host natural antibody to facilitate mating in the insect vector establishes a new paradigm of parasite-host-vector interdependence that contributes to parasite diversity and fitness by promoting genetic exchange.

A novel pectin methylesterase inhibitor, PMEI3, in common bean suggests a key role of pectin methylesterification in Pseudomonas resistance.

The mechanisms underlying susceptibility to and defense against Pseudomonas syringae (Pph) of the common bean (Phaseolus vulgaris) have not yet been clarified. To investigate these, 15-day-old plants of the variety Riñón were infected with Pph and the transcriptomic changes at 2 h and 9 h post-infection were analysed. RNA-seq analysis showed an up-regulation of genes involved in defense/signaling at 2 h, most of them being down-regulated at 9 h, suggesting that Pph inhibits the transcriptomic reprogramming of the plant. This trend was also observed in the modulation of 101 cell wall-related genes. Cell wall composition changes at early stages of Pph infection were associated with homogalacturonan methylation and the formation of egg boxes. Among the cell wall genes modulated, a pectin methylesterase inhibitor 3 (PvPMEI3) gene, closely related to AtPMEI3, was detected. PvPMEI3 protein was located in the apoplast and its pectin methylesterase inhibitory activity was demonstrated. PvPMEI3 seems to be a good candidate to play a key role in Pph infection, which was supported by analysis of an Arabidopsis pmei3 mutant, which showed susceptibility to Pph, in contrast to resistant Arabidopsis Col-0 plants. These results indicate a key role of the degree of pectin methylesterification in host resistance to Pph during the first steps of the attack.

Drosophila DAxud1 Has a Repressive Transcription Activity on Hsp70 and Other Heat Shock Genes.

Drosophila melanogaster DAxud1 is a transcription factor that belongs to the Cysteine Serine Rich Nuclear Protein (CSRNP) family, conserved in metazoans, with a transcriptional transactivation activity. According to previous studies, this protein promotes apoptosis and Wnt signaling-mediated neural crest differentiation in vertebrates. However, no analysis has been conducted to determine what other genes it might control, especially in connection with cell survival and apoptosis. To partly answer this question, this work analyzes the role of Drosophila DAxud1 using Targeted-DamID-seq (TaDa-seq), which allows whole genome screening to determine in which regions it is most frequently found. This analysis confirmed the presence of DAxud1 in groups of pro-apoptotic and Wnt pathway genes, as previously described; furthermore, stress resistance genes that coding heat shock protein (HSP) family genes were found as hsp70, hsp67, and hsp26. The enrichment of DAxud1 also identified a DNA-binding motif (AYATACATAYATA) that is frequently found in the promoters of these genes. Surprisingly, the following analyses demonstrated that DAxud1 exerts a repressive role on these genes, which are necessary for cell survival. This is coupled with the pro-apoptotic and cell cycle arrest roles of DAxud1, in which repression of hsp70 complements the maintenance of tissue homeostasis through cell survival modulation.

Proteomic and Low-Polar Metabolite Profiling Reveal Unique Dynamics in Fatty Acid Metabolism during Flower and Berry Development of Table Grapes.

Grapevine development and ripening are complex processes that involve several biochemical pathways, including fatty acid and lipid metabolism. Fatty acids are essential components of lipids, which play crucial roles in fruit maturation and flavor development. However, the dynamics of fatty acid metabolism in grape flowers and berries are poorly understood. In this study, we present those dynamics and investigate the mechanisms of fatty acid homeostasis on 'Thompson Seedless' berries using metabolomic and proteomic analyses. Low-polar metabolite profiling indicated a higher abundance of fatty acids at the pre-flowering and pre-veraison stages. Proteomic analyses revealed that grape flowers and berries display unique profiles of proteins involved in fatty acid biosynthesis, triacylglycerol assembly, fatty acid ß-oxidation, and lipid signaling. These findings show, for the first time, that fatty acid metabolism also plays an important role in the development of non-oil-rich tissues, opening new perspectives about lipid function and its relation to berry quality.

Development of Inapparent Dengue Associated With Increased Antibody Levels to Aedes aegypti Salivary Proteins: A Longitudinal Dengue Cohort in Cambodia.

BACKGROUND: We established the first prospective cohort to understand how infection with dengue virus is influenced by vector-specific determinants such as humoral immunity to Aedes aegypti salivary proteins. METHODS: Children aged 2-9 years were enrolled in the PAGODAS (Pediatric Assessment Group of Dengue and Aedes Saliva) cohort with informed consent by their guardians. Children were followed semi-annually for antibodies to dengue and to proteins in Ae. aegypti salivary gland homogenate using enzyme-linked immunosorbent assays and dengue-specific neutralization titers. Children presented with fever at any time for dengue testing. RESULTS: From 13 July to 30 August 2018, we enrolled 771 children. At baseline, 22% (173/770) had evidence of neutralizing antibodies to 1 or more dengue serotypes. By April 2020, 51 children had symptomatic dengue while 148 dengue-naive children had inapparent dengue defined by neutralization assays. In a multivariate model, individuals with higher antibodies to Ae. aegypti salivary proteins were 1.5 times more likely to have dengue infection (hazard ratio [HR], 1.47 [95% confidence interval {CI}, 1.05-2.06]; P = .02), particularly individuals with inapparent dengue (HR, 1.64 [95% CI, 1.12-2.41]; P = .01). CONCLUSIONS: High levels of seropositivity to Ae. aegypti salivary proteins are associated with future development of dengue infection, primarily inapparent, in dengue-naive Cambodian children. CLINICAL TRIALS REGISTRATION: NCT03534245.

Transcriptome analysis of Echinococcus granulosus sensu stricto protoscoleces reveals differences in immune modulation gene expression between cysts found in cattle and sheep.

Cystic Echinococcosis (CE), a zoonotic parasitic disease, is caused by the cestode Echinococcus granulosus sensu lato. CE inflicts severe damage in cattle, sheep, and human hosts worldwide. Fertile CE cysts are characterized by the presence of viable protoscoleces. These parasite forms are studied with minimal contamination with host molecules. Hosts, cattle and sheep, show differences in their CE cyst fertility. The effect of the host in protoscolex transcriptome is not known. We genotyped and performed transcriptomic analysis on sheep protoscoleces obtained from liver and lung CE cysts. The transcriptomic data of Echinococcus granulosus sensu stricto protoscoleces from 6 lung CE cysts and 6 liver CE cysts were Collected. For host comparison analysis, 4 raw data files belonging to Echinococcus granulosus sensu stricto protoscoleces from cattle liver CE cysts were obtained from the NCBI SRA database. Principal component and differential expression analysis did not reveal any statistical differences between protoscoleces obtained from liver or lung cysts, either within the same sheep or different sheep hosts. Conversely, there are significant differences between cattle and sheep protoscolex samples. We found differential expression of immune-related genes. In cattle, 7 genes were upregulated in protoscoleces from liver cysts. In sheep, 3 genes were upregulated in protoscoleces from liver and lung CE cysts. Noteworthy, are the differential expression of antigen B, tegument antigen, and arginase-2 in samples obtained from sheep CE cysts, and basigin in samples from cattle CE cysts. These findings suggest that the host species is an important factor involved in the differential expression of immune related genes, which in turn is possibly related to the fertility of Echinococcus granulosus sensu stricto cysts.

Safety and immunogenicity of a mosquito saliva peptide-based vaccine: a randomised, placebo-controlled, double-blind, phase 1 trial.

BACKGROUND: In animal models, immunity to mosquito salivary proteins protects animals against mosquito-borne disease. These findings provide a rationale to vaccinate against mosquito saliva instead of the pathogen itself. To our knowledge, no vector salivary protein-based vaccine has been tested for safety and immunogenicity in humans. We aimed to assess the safety and immunogenicity of Anopheles gambiae saliva vaccine (AGS-v), a peptide-based vaccine derived from four A gambiae salivary proteins, in humans. METHODS: In this randomised, placebo-controlled, double-blind, phase 1 trial, participants were enrolled at the National Institutes of Health Clinical Center in Bethesda, MD, USA. Participants were eligible if they were healthy adults, aged 18-50 years with no history of severe allergic reactions to mosquito bites. Participants were randomly assigned (1:1:1), using block randomisation and a computer-generated randomisation sequence, to treatment with either 200 nmol of AGS-v vaccine alone, 200 nmol of AGS-v with adjuvant (Montanide ISA 51), or sterile water as placebo. Participants and clinicians were masked to treatment assignment. Participants were given a subcutaneous injection of their allocated treatment at day 0 and day 21, followed by exposure to feeding by an uninfected Aedes aegypti mosquito at day 42 to assess subsequent risk to mosquito bites in a controlled setting. The primary endpoints were safety and immunogenicity at day 42 after the first immunisation. Participants who were given at least one dose of assigned treatment were assessed for the primary endpoints and analysis was by intention to treat. The trial was registered with ClinicalTrials.gov, NCT03055000, and is closed for accrual. FINDINGS: Between Feb 15 and Sept 10, 2017, we enrolled and randomly assigned 49 healthy adult participants to the adjuvanted vaccine (n=17), vaccine alone (n=16), or placebo group (n=16). Five participants did not complete the two-injection regimen with mosquito feeding at day 42, but were included in the safety analyses. No systemic safety concerns were identified; however, one participant in the adjuvanted vaccine group developed a grade 3 erythematous rash at the injection site. Pain, swelling, erythema, and itching were the most commonly reported local symptoms and were significantly increased in the adjuvanted vaccine group compared with both other treatment groups (nine [53%] of 17 participants in the adjuvanted vaccine group, two [13%] of 16 in the vaccine only group, and one [6%] of 16 in the placebo group; p=0·004). By day 42, participants who were given the adjuvanted vaccine had a significant increase in vaccine-specific total IgG antibodies compared with at baseline than did participants who were give vaccine only (absolute difference of log10-fold change of 0·64 [95% CI 0·39 to 0·89]; p=0·0002) and who were given placebo (0·62 [0·34 to 0·91]; p=0·0001). We saw a significant increase in IFN-γ production by peripheral blood mononuclear cells at day 42 in the adjuvanted vaccine group compared with in the placebo group (absolute difference of log10 ratio of vaccine peptide-stimulated vs negative control 0·17 [95% CI 0·061 to 0·27]; p=0·009) but we saw no difference between the IFN-γ production in the vaccine only group compared with the placebo group (0·022 [-0·072 to 0·116]; p=0·63). INTERPRETATION: AGS-v was well tolerated, and, when adjuvanted, immunogenic. These findings suggest that vector-targeted vaccine administration in humans is safe and could be a viable option for the increasing burden of vector-borne disease. FUNDING: Office of the Director and the Division of Intramural Research at the National Institute of Allergy and Infectious Diseases, and National Institutes of Health.

Leishmania infection induces a limited differential gene expression in the sand fly midgut.

BACKGROUND: Sand flies are the vectors of Leishmania parasites. To develop in the sand fly midgut, Leishmania multiplies and undergoes various stage differentiations giving rise to the infective form, the metacyclic promastigotes. To determine the changes in sand fly midgut gene expression caused by the presence of Leishmania, we performed RNA-Seq of uninfected and Leishmania infantum-infected Lutzomyia longipalpis midguts from seven different libraries corresponding to time points which cover the various Leishmania developmental stages. RESULTS: The combined transcriptomes resulted in the de novo assembly of 13,841 sand fly midgut transcripts. Importantly, only 113 sand fly transcripts, about 1%, were differentially expressed in the presence of Leishmania parasites. Further, we observed distinct differentially expressed sand fly midgut transcripts corresponding to the presence of each of the various Leishmania stages suggesting that each parasite stage influences midgut gene expression in a specific manner. Two main patterns of sand fly gene expression modulation were noted. At early time points (days 1-4), more transcripts were down-regulated by Leishmania infection at large fold changes (> 32 fold). Among the down-regulated genes, the transcription factor Forkhead/HNF-3 and hormone degradation enzymes were differentially regulated on day 2 and appear to be the upstream regulators of nutrient transport, digestive enzymes, and peritrophic matrix proteins. Conversely, at later time points (days 6 onwards), most of the differentially expressed transcripts were up-regulated by Leishmania infection with small fold changes (< 32 fold). The molecular functions of these genes have been associated with the metabolism of lipids and detoxification of xenobiotics. CONCLUSION: Overall, our data suggest that the presence of Leishmania produces a limited change in the midgut transcript expression profile in sand flies. Further, Leishmania modulates sand fly gene expression early on in the developmental cycle in order to overcome the barriers imposed by the midgut, yet it behaves like a commensal at later time points where a massive number of parasites in the anterior midgut results only in modest changes in midgut gene expression.

Identification of SNPs and InDels associated with berry size in table grapes integrating genetic and transcriptomic approaches.

BACKGROUND: Berry size is considered as one of the main selection criteria in table grapes breeding programs, due to the consumer preferences. However, berry size is a complex quantitive trait under polygenic control, and its genetic determination of berry weight is not yet fully understood. The aim of this work was to perform marker discovery using a transcriptomic approach, in order to identify and characterize SNP and InDel markers associated with berry size in table grapes. We used an integrative analysis based on RNA-Seq, SNP/InDel search and validation on table grape segregants and varieties with different genetic backgrounds. RESULTS: Thirty SNPs and eight InDels were identified using a transcriptomic approach (RNA-Seq). These markers were selected from SNP/InDel found among segregants from a Ruby x Sultanina population with contrasting phenotypes for berry size. The set of 38 SNP and InDel markers was distributed in eight chromosomes. Genotype-phenotype association analyses were performed using a set of 13 RxS segregants and 41 table grapes varieties with different genetic backgrounds during three seasons. The results showed several degrees of association of these markers with berry size (10.2 to 30.7%) as other berry-related traits such as length and width. The co-localization of SNP and /or InDel markers and previously reported QTLs and candidate genes associated with berry size were analysed. CONCLUSIONS: We identified a set of informative and transferable SNP and InDel markers associated with berry size. Our results suggest the suitability of SNPs and InDels as candidate markers for berry weight in seedless table grape breeding. The identification of genomic regions associated with berry weight in chromosomes 8, 15 and 17 was achieved with supporting evidence derived from a transcriptome experiment focused on SNP/InDel search, as well as from a QTL-linkage mapping approach. New regions possibly associated with berry weight in chromosomes 3, 6, 9 and 14 were identified.

Transcriptomic study of pedicels from GA3-treated table grape genotypes with different susceptibility to berry drop reveals responses elicited in cell wall yield, primary growth and phenylpropanoids synthesis.

BACKGROUND: Gibberellins (GA3) are the most sprayed growth regulator for table grape production worldwide, increasing berry size of seedless varieties through pericarp cell expansion. However, these treatments also exacerbate berry drop, which has a detrimental effect on the postharvest quality of commercialized clusters. Several studies have suggested that pedicel stiffening caused by GA3 would have a role in this disorder. Nevertheless, transcriptional and phenotypic information regarding pedicel responses to GA3 is minimal. RESULTS: Characterization of responses to GA3 treatments using the lines L23 and Thompson Seedless showed that the former was up to six times more susceptible to berry drop than the latter. GA3 also increased the diameter and dry matter percentage of the pedicel on both genotypes. Induction of lignin biosynthesis-related genes by GA3 has been reported, so the quantity of this polymer was measured. The acetyl bromide method detected a decreased concentration of lignin 7 days after GA3 treatment, due to a higher cell wall yield of the isolated fractions of GA3-treated pedicel samples which caused a dilution effect. Thus, an initial enrichment of primary cell wall components in response to GA3 was suggested, particularly in the L23 background. A transcriptomic profiling was performed to identify which genes were associated with these phenotypic changes. This analysis identified 1281 and 1787 genes differentially upregulated by GA3 in L23 and cv. Thompson Seedless, respectively. Concomitantly, 1202 and 1317 downregulated genes were detected in L23 and cv. Thompson Seedless (FDR < 0.05). Gene ontology analysis of upregulated genes showed enrichment in pathways including phenylpropanoids, cell wall metabolism, xylem development, photosynthesis and the cell cycle at 7 days post GA3 application. Twelve genes were characterized by qPCR and striking differences were observed between genotypes, mainly in genes related to cell wall synthesis. CONCLUSIONS: High levels of berry drop are related to an early strong response of primary cell wall synthesis in the pedicel promoted by GA3 treatment. Genetic backgrounds can produce similar phenotypic responses to GA3, although there is considerable variation in the regulation of genes in terms of which are expressed, and the extent of transcript levels achieved within the same time frame.

Rapid sequence modification in the highly polymorphic region (HPR) of the hemagglutinin gene of the infectious salmon anaemia virus (ISAV) suggests intra-segmental template switching recombination.

The ISAV has a genome composed of eight segments of (-)ssRNA, segment 6 codes for the hemagglutinin-esterase protein, and has the most variable region of the genome, the highly polymorphic region (HPR), which is unique among orthomyxoviruses. The HPR has been associated with virulence, infectivity and pathogenicity. The full length of the HPR is called HPR0 and the strain with this HPR is avirulent, in contrast to strains with deleted HPR that are virulent to varying degrees. The molecular mechanism that gives rise to the different HPRs remains unclear. Here, we studied in vitro the evolution of reassortant recombinant ISAV (rISAV) in Atlantic salmon head kidney (ASK) cells. To this end, we rescued and cultivated a set of rISAV with different segment 6-HPR genotypes using a reverse genetics system and then sequencing HPR regions of the viruses. Our results show rapid multiple recombination events in ISAV, with sequence insertions and deletions in the HPR, indicating a dynamic process. Inserted sequences can be found in four segments of the ISAV genome (segments 1, 5, 6, and 8). The results suggest intra-segmental heterologous recombination, probably by class I and class II template switching, similar to the proposed segment 5 recombination mechanism.

Early transcriptomic responses associated with the membrane-initiated action of cortisol in the skeletal muscle of rainbow trout (Oncorhynchus mykiss).

Cortisol is a critical neuroendocrine regulator of the stress response in fish. Cortisol practically affects all tissues by interacting with an intracellular receptor and modulating target gene expression. However, cortisol also interacts with components of the plasma membrane in a nongenomic process that activates rapid signaling. Until now, the implication of this novel cortisol signaling for the global transcriptional response has not been explored. In the present work, we evaluated the effects of the membrane-initiated actions of cortisol on the in vivo transcriptome of rainbow trout (Oncorhynchus mykiss) skeletal muscle. RNA-Seq analyses were performed to examine the transcriptomic changes in rainbow trout stimulated by physiological concentrations of cortisol and cortisol coupled with bovine serum albumin (cortisol-BSA), a membrane-impermeable analog of cortisol. A total of 660 million paired-ends reads were generated. Reads mapped onto the reference genome revealed that 1,737; 897; and 1,012 transcripts were differentially expressed after 1, 3, and 9 h of cortisol-BSA treatment, respectively. Gene Ontology analysis showed that this novel action of cortisol modulates several biological processes, such as mRNA processing, ubiquitin-dependent protein catabolic processes, and transcription regulation. In addition, a KEGG analysis revealed that focal adhesion was the main signaling pathway that was upregulated at all the times tested. Taking these results together, we propose that the membrane-initiated cortisol action contributes significantly in the regulation of stress-mediated gene expression.

De novo assembly of Persea americana cv. 'Hass' transcriptome during fruit development.

BACKGROUND: Avocado (Persea americana Mill.) is a basal angiosperm from the Lauraceae family. This species has a diploid genome with an approximated size of ~ 920 Mbp and produces a climacteric, fleshy and oily fruit. The flowering and fruit set are particularly prolonged processes, lasting between one to three months, generating important differences in physiological ages of the fruit within the same tree. So far there is no detailed genomic information regarding this species, being the cultivar 'Hass' especially important for avocado growers worldwide. With the aim to explore the fruit avocado transcriptome and to identify candidate biomarkers to monitore fruit development, we carried out an RNA-Seq approach during 4 stages of 'Hass' fruit development: 150 days after fruit set (DAFS), 240 DAFS, 300 DAFS (harvest) and 390 DAFS (late-harvest). RESULTS: The 'Hass' de novo transcriptome contains 62,203 contigs (x̅=988 bp, N50 = 1050 bp). We found approximately an 85 and 99% of complete ultra-conserved genes in eukaryote and plantae database using BUSCO (Benchmarking Universal Single-Copy Orthologs) and CEGMA (Core Eukaryotic Gene Mapping Approach), respectively. Annotation was performed with BLASTx, resulting in a 58% of annotated contigs (90% of differentially expressed genes were annotated). Differentially expressed genes analysis (DEG; with False Discovery Rate ≤ 0.01) found 8672 genes considering all developmental stages. From this analysis, genes were clustered according to their expression pattern and 1209 genes show correlation with the four developmental stages. CONCLUSIONS: Candidate genes are proposed as possible biomarkers for monitoring the development of the 'Hass' avocado fruit associated with lipid metabolism, ethylene signaling pathway, auxin signaling pathway, and components of the cell wall.

New steps in mucilage biosynthesis revealed by analysis of the transcriptome of the UDP-rhamnose/UDP-galactose transporter 2 mutant.

Upon imbibition, epidermal cells of Arabidopsis thaliana seeds release a mucilage formed mostly by pectic polysaccharides. The Arabidopsis mucilage is composed mainly of unbranched rhamnogalacturonan-I (RG-I), with low amounts of cellulose, homogalacturonan, and traces of xylan, xyloglucan, galactoglucomannan, and galactan. The pectin-rich composition of the mucilage and their simple extractability makes this structure a good candidate to study the biosynthesis of pectic polysaccharides and their modification. Here, we characterize the mucilage phenotype of a mutant in the UDP-rhamnose/galactose transporter 2 (URGT2), which exhibits a reduction in RG-I and also shows pleiotropic changes, suggesting the existence of compensation mechanisms triggered by the lack of URGT2. To gain an insight into the possible compensation mechanisms activated in the mutant, we performed a transcriptome analysis of developing seeds using RNA sequencing (RNA-seq). The results showed a significant misregulation of 3149 genes, 37 of them (out of the 75 genes described to date) encoding genes proposed to be involved in mucilage biosynthesis and/or its modification. The changes observed in urgt2 included the up-regulation of UAFT2, a UDP-arabinofuranose transporter, and UUAT3, a paralog of the UDP-uronic acid transporter UUAT1, suggesting that they play a role in mucilage biosynthesis. Mutants in both genes showed changes in mucilage composition and structure, confirming their participation in mucilage biosynthesis. Our results suggest that plants lacking a UDP-rhamnose/galactose transporter undergo important changes in gene expression, probably to compensate modifications in the plant cell wall due to the lack of a gene involved in its biosynthesis.

A Prunus persica genome-wide RNA-seq approach uncovers major differences in the transcriptome among chilling injury sensitive and non-sensitive varieties.

Chilling injury represents a major constrain for crops productivity. Prunus persica, one of the most relevant rosacea crops, have early season varieties that are resistant to chilling injury, in contrast to late season varieties, which display chilling symptoms such as mealiness (dry, sandy fruit mesocarp) after prolonged storage at chilling temperatures. To uncover the molecular processes related to the ability of early varieties to withstand mealiness, postharvest and genome-wide RNA-seq assessments were performed in two early and two late varieties. Differences in juice content and ethylene biosynthesis were detected among early and late season fruits that became mealy after exposed to prolonged chilling. Principal component and data distribution analysis revealed that cold-stored late variety fruit displayed an exacerbated and unique transcriptome profile when compared to any other postharvest condition. A differential expression analysis performed using an empirical Bayes mixture modeling approach followed by co-expression and functional enrichment analysis uncover processes related to ethylene, lipids, cell wall, carotenoids and DNA metabolism, light response, and plastid homeostasis associated to the susceptibility or resistance of P. persica varieties to chilling stress. Several of the genes related to these processes are in quantitative trait loci (QTL) associated to mealiness in P. persica. Together, these analyses exemplify how P. persica can be used as a model for studying chilling stress in plants.

Evaluating the genetic structure of wild and commercial red cusk-eel (Genypterus chilensis) populations through the development of novel microsatellite markers from a reference transcriptome.

The red cusk-eel (Genypterus chilensis) is a native Chilean species with a high-value market, with the potential to diversify Chilean aquaculture. The objective of this study was to develop a set of microsatellite markers, estimate genetic parameters, determine population differentiation, and identify the population structure of wild and commercial populations of G. chilensis. We discovered 6427 microsatellites markers from RNA-seq data, of which 54.9%, 20.2% and 16.8% were di-, tri-, and tetranucleotides, respectively. We used 12 of these markers to genotype two sets of broodstock, one group from commercial fish, and one group from wild fish from the Coquimbo Region of G. chilensis. We estimate the genetic parameters of the markers, selecting ten polymorphic markers (PIC > 0.5). We observed differences in the inbreeding coefficient among populations, with high values of inbreeding in one broodstock set and lower values in the other groups. The evaluation of population differentiation using Fst showed small (0.0195) to large (0.1888) genetic differentiation between the groups. The structure analysis showed that commercial and wild groups were formed by three clusters, without relevant evidence of admixture process, suggesting that groups evaluated in this study are formed of at least three subpopulations of G. chilensis, which could be explained by the low or lack of migration suggested for this species. This is the first study that identifies a high number of molecular markers in G. chilensis, providing relevant information of the genetic structure of commercial and wild population of this species.

Copper-induced increased expression of genes involved in photosynthesis, carotenoid synthesis and C assimilation in the marine alga Ulva compressa.

BACKGROUND: The marine alga Ulva compressa is the dominant species in coastal areas receiving effluents from copper mines. The alga can accumulate high amounts of copper and possesses a strong antioxidant system. Here, we performed short-term transcriptomic analyses using total RNA of the alga cultivated with 10 µM of copper for 0, 3, 6, 12 and 24 h by RNA-seq. RESULTS: De novo transcriptomes were assembled using the Trinity software, putative proteins were annotated and classified using Blast2GO. Differentially expressed transcripts were identified using edgeR. Transcript levels were compared by paired times 0 vs 3, 0 vs 6, 0 vs 12 and 0 vs 24 h at an FDR < 0.01 and Log2 Fold Change > 2. Up-regulated transcripts encode proteins belonging to photosystem II (PSII), Light Harvesting II Complex (LHCII), PSI and LHCI, proteins involved in assembly and repair of PSII, and assembly and protection of PSI. In addition, transcripts encoding enzymes leading to ß-carotene synthesis and enzymes belonging to the Calvin-Benson cycle were also increased. We further analyzed photosynthesis and carotenoid levels in the alga cultivated with 10 µM of copper for 0 to 24 h. Photosynthesis was increased from 3 to 24 h as well as the level of total carotenoids. The increase in transcripts encoding enzymes of the Calvin-Benson cycle suggests that C assimilation may also be increased. CONCLUSIONS: Thus, U. compressa displays a short-term response to copper stress enhancing the expression of genes encoding proteins involved in photosynthesis, enzymes involved carotenoids synthesis, as well as those belonging to the Calvin-Benson cycle, which may result in an increase in C assimilation.

Identification of a novel long noncoding RNA that promotes osteoblast differentiation.

Long noncoding RNAs (lncRNAs) are a heterogeneous class of transcripts, longer than 200 nucleotides, 5'-capped, polyadenylated, and poorly conserved among mammalian species. Several studies have shown the contribution of lncRNAs to different cellular processes, including regulation of the chromatin structure, control of messenger RNA translation, regulation of gene transcription, regulation of embryonic pluripotency, and differentiation. Although limited numbers of functional lncRNAs have been identified so far, the immense regulatory potential of these RNAs is already evident, indicating that a functional characterization of lncRNAs is needed. In this study, mouse preosteoblastic cells were induced to differentiate into osteoblasts. At 3 sequential differentiation stages, total RNA was isolated and libraries were constructed for Illumina sequencing. The resulting sequences were aligned and transcript abundances were determined. New lncRNA candidates that displayed differential expression patterns during osteoblast differentiation were identified by combining bioinformatics and reverse transcription polymerase chain reaction analyses. Among these, lncRNA-1 that exhibited increased expression during osteogenesis and was downregulated during myogenesis. Importantly, knockdown of lncRNA-1 expression in primary mouse preosteoblasts was found to inhibit osteogenic differentiation, reflected by a reduced transcription of the Runx2/p57 and Sp7 bone master genes. Together, our results indicate that lncRNA-1 represents a new regulatory RNA that plays a relevant role during the early stages of osteogenesis.

bZIP17 regulates the expression of genes related to seed storage and germination, reducing seed susceptibility to osmotic stress.

Low temperatures, salinity, and drought cause significant crop losses. These conditions involve osmotic stress, triggering transcriptional remodeling, and consequently, the restitution of cellular homeostasis and growth recovery. Protein transcription factors regulate target genes, thereby mediating plant responses to stress. bZIP17 is a transcription factor involved in cellular responses to salinity and the unfolded protein response. Because salinity can also produce osmotic stress, the role of bZIP17 in response to osmotic stress was assessed. Mannitol treatments induced the transcript accumulation and protein processing of bZIP17. Transcriptomic analyses showed that several genes associated with seed storage and germination showed lower expression in bzip17 mutants than in wild-type plants. Interestingly, bZIP17 transcript was more abundant in seeds, and germination analyses revealed that wild-type plants germinated later than bzip17 mutants in the presence of mannitol, but no effects were observed when the seeds were exposed to ABA. Finally, the transcript levels of bZIP17 target genes that control seed storage and germination were assessed in seeds exposed to mannitol treatments, which showed lower expression levels in bzip17 mutants compared to the wild-type seeds. These results suggest that bZIP17 plays a role in osmotic stress, acting as a negative regulator of germination through the regulation of genes involved in seed storage and germination.

Draft Whole Genome Sequence Analyses on Pseudomonas syringae pv. actinidiae Hypersensitive Response Negative Strains Detected from Kiwifruit Bleeding Sap Samples.

Kiwifruit bleeding sap samples, collected in Italian and Chilean orchards from symptomatic and asymptomatic plants, were evaluated for the presence of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker. The saps were sampled during the spring in both hemispheres, before the bud sprouting, during the optimal time window for the collection of an adequate volume of sample for the early detection of the pathogen, preliminarily by molecular assays, and then through its direct isolation and identification. The results of molecular analyses showed more effectiveness in the P. syringae pv. actinidiae detection when compared with those of microbiological analyses through the pathogen isolation on the nutritive and semiselective media selected. The bleeding sap analyses allowed the isolation and identification of two hypersensitive response (HR) negative and hypovirulent P. syringae pv. actinidiae strains from different regions in Italy. Moreover, multilocus sequence analysis (MLSA) and whole genome sequence (WGS) were carried out on selected Italian and Chilean P. syringae pv. actinidiae virulent strains to verify the presence of genetic variability compared with the HR negative strains and to compare the variability of selected gene clusters between strains isolated in both countries. All the strains showed the lack of argK and coronatine gene clusters as reported for the biovar 3 P. syringae pv. actinidiae strains. Despite the biologic differences obtained in the tobacco bioassays and in pathogenicity assays, the MLSA and WGS analyses did not show significant differences between the WGS of the HR negative and HR positive strains; the difference, on the other hand, between PAC_ICE sequences of Italian and Chilean P. syringae pv. actinidiae strains was confirmed. The inability of the hypovirulent strains IPV-BO 8893 and IPV-BO 9286 to provoke HR in tobacco and the low virulence shown in this host could not be associated with mutations or recombinations in T3SS island.