cDNA Expression Library Research Articles

Screening and identification of CKI1 upstream transcription regulators in Arabidopsis

Arabidopsis CKI1 (cytokinin independent 1) is a histidine kinase protein involved in the two-component system, which can activate two-component signaling via the downstream histidine phospho-transfer proteins, playing the essential roles in central cell fate determination and development regulation in embryo sacs. However, studies on CKI1 upstream transcription regulators are still limited. In the present study, promoter activities with varying fragments were investigated, and CKI1 upstream transcription regulators were screened and identified by the yeast-one hybrid technique. Results indicated F5/R2 fragments located in the intron region showed promoter activities in embryo sacs, which is consistent with CKI1 full-length promoters. Then three tandem repeats of F5/R2 fragments were used to construct the bait expression vector, and Arabidopsis pistils were collected for cDNA library construction. Totally, 226 positive clones were screened by the yeast-one hybrid technique, 66 readable sequences were retrieved after removing sequences with low quality and redundant repeats, among which eight proteins could act as DNA-binding proteins. These results provided some important clues to study the molecular function of CKI1 in the transcription regulation network.

The 40S Ribosomal Protein S6 Response to Blue Light by Interaction with SjAUREO in Saccharina japonica.

Blue light (BL) plays an important role in regulation of the growth and development of aquatic plants and land plants. Aureochrome (AUREO), the recent BL photoreceptor identified in photosynthetic stramenopile algae, is involved in the photomorphogenesis and early development of Saccharina japonica porophytes (kelp). However the factors that interact with the SjAUREO under BL conditions specifically are not clear. Here in our study, three high quality cDNA libraries with CFU over 5 × 106 and a recombination rate of 100% were constructed respectively through white light (WL), BL and darkness (DK) treatments to the juvenile sporophytes. Based on the constructed cDNA libraries, the interactors of SjAUREO were screened and analyzed. There are eighty-four genes encoding the sixteen predicted proteins from the BL cDNA library, sixty-eight genes encoding eighteen predicted proteins from the DK cDNA library, and seventy-four genes encoding nineteen proteins from the WL cDNA library. All the predicted proteins are presumed to interact with SjAUREO when co-expressed with SjAUREO seperately. The 40S ribosomal protein S6 (RPS6), which only exists in the BL treated cDNA library except for two other libraries, and which is essential for cell proliferation and is involved in cell cycle progression, was selected for detailed analysis. We showed that its transcription was up-regulated by BL, and was highly transcribed in the basal blade (meristem region) of juvenile sporophytes but less in the distal part. Taken together, our results indicated that RPS6 was highly involved in BL-mediated kelp cellular division and photomorphogenesis by interacting with SjAUREO.

PATZ1 is required for efficient HIV-1 infection

Successful HIV-1 infection and subsequent replication deeply depend on how the virus usurps the host cell machinery. Identification and functional characterization of these host factors may represent a critical strategy for developing novel anti-HIV-1 therapy. Here, expression cloning with a cDNA expression library identified as an inhibitor of HIV-1 infection, a carboxy-terminally truncated form of human POZ/BTB and AT-hook- containing Zinc finger protein 1 (PATZ1), a transcriptional regulatory factor implicated in development and cancer. Knockdown or knockout of endogenous PATZ1 revealed a supportive role of PATZ1 in HIV-1 infection, but not in transduction with murine leukemia virus-based retroviral vector. More specifically, knockdown or knockout of PATZ1 impaired the viral cDNA synthesis but not the entry process and expression of two PATZ1 isoforms in PATZ1-KO cells restored susceptibility to HIV-1 infection. These results indicate that PATZ1 plays an important role in HIV-1 infection.

Functional characterisation of two phytochelatin synthases in rice (Oryza sativa cv. Milyang 117) that respond to cadmium stress.

Cadmium (Cd) is one of the most toxic heavy metals and a non‐essential element to all organisms, including plants; however, the genes involved in Cd resistance in plants remain poorly characterised.To identify Cd resistance genes in rice, we screened a rice cDNA expression library treated with CdCl2 using a yeast (Saccharomyces cerevisiae) mutant ycf1 strain (DTY167) and isolated two rice phytochelatin synthases (OsPCS5 and OsPCS15).The genes were strongly induced by Cd treatment and conferred increased resistance to Cd when expressed in the ycf1 mutant strain. In addition, the Cd concentration was twofold higher in yeast expressing OsPCS5 and OsPCS15 than in vector‐transformed yeast, and OsPCS5 and OsPCS15 localised in the cytoplasm. Arabidopsis thaliana plants overexpressing OsPCS5/‐15 paradoxically exhibited increased sensitivity to Cd, suggesting that overexpression of OsPCS5/‐15 resulted in toxicity due to excess phytochelatin production in A. thaliana.These data indicate that OsPCS5 and OsPCS15 are involved in Cd tolerance, which may be related to the relative abundances of phytochelatins synthesised by these phytochelatin synthases.

Evaluating and Correcting Inherent Bias of microRNA Expression in Illumina Sequencing Analysis.

microRNA (miRNA) expression profiles based on the highly powerful Illumina sequencing technology rely on the construction of cDNA libraries in which adaptor ligation is known to deeply favor some miRNAs over others. This introduces erroneous measurements of the miRNA abundances and relative miRNA quantities in biological samples. Here, by using the commercial miRXplore Universal Reference that contains an equimolar mixture of 963 animal miRNAs and TruSeq or bulged adaptors, we describe a method for correcting ligation biases in expression profiles obtained with standard protocols of cDNA library construction and provide data for quantifying the true miRNA abundances in biological samples. Ligation biases were evaluated at three ratios of miRNA to 3′-adaptor and four numbers of polymerase chain reaction amplification cycles by calculating efficiency captures/correcting factors for each miRNA. We show that ligation biases lead to over- or under-expression covering a 105 amplitude range. We also show that, at each miRNA:3′-adaptor ratio, coefficients of variation (CVs) of efficiency captures calculated over the four number of amplification cycles using sliding windows of 10 values ranged from 0.1 for the miRNAs of high expression to 0.6 for the miRNAs of low expression. Efficiency captures of miRNAs of high and low expression in profiles are therefore differently impacted by the number of amplification cycles. Importantly, we observed that at a given number of amplification cycles, CVs of efficiency captures calculated over the three miRNA:3′-adaptor ratios displayed a steady value of 0.3 +/− 0.05 STD for miRNAs of high and low expression. This allows, at a given number of amplification cycles, accurate comparison of miRNA expression between biological samples over a substantial expression range. Finally we provide tables of correcting factors that allow to measure the abundances of 963 miRNAs in biological samples from TruSeq-based expression profiles and, an example of their use by characterizing miRNAs of the let-7, miR-26, miR-29, and miR-30 families as the more abundant miRNAs of the rat adult cerebellum.

Identification of transcription factors of nitrate reductase gene promoters and NRE2 cis-element through yeast one-hybrid screening in Nicotiana tabacum

BackgroundThis study aimed to identify the transcription factors of nitrate reductase genes (NIA1 and NIA2) promoters and hypothetical cis-element of NRE2. Based on the constructed cDNA library of Nicotiana tabacum K326, a yeast one-hybrid system was established using the Matchmaker® Gold Yeast One-Hybrid Library Screening System from Clontech. The transcription factors of NIA1 andNIA2 promoters and NRE2 cis-elements were screened.ResultsAfter sequencing and bioinformatics analysis, 15 cDNA sequences were identified: 9 for NIA1 (including XP_016503563.1 and NP_001312236.1), 3 for NIA2 (including XP_016510250.1), and 3 for NRE2 (including XM_016576899.1). XP_016503563.1 was annotated in PREDICTED: CRM-domain containing factor CFM3, and NP_001312236.1chloroplastic/mitochondrial-like in Nicotiana tabacum. NP_001312236.1 was annotated in Sulfite oxidase-like of Nicotiana tabacum. XP_016510250.1 was annotated as PREDICTED: uncharacterized protein LOC107827596 in Nicotiana tabacum. XM_016576899.1 was annotated in PREDICTED: Nicotiana tabacum RING-H2 finger protein ATL16-like (LOC107759033).ConclusionA yeast one-hybrid library was successfully constructed. The identified transcription factors may provide a theoretical basis for the study of plant nitrate reductase.

Comparison of Tissue Preparation, Handling and Dissociation Methods for Total RNA

The extraction and isolation of RNA is an integral part of downstream analyses such as RT‐PCR, RT‐qPCR, Northern blotting, and cDNA library construction. The importance of using pure, intact RNA for these processes is well documented and a critical part of downstream analysis success. It is well known that RNA is sensitive to degradation due to mechanical shear, temperature, storage conditions and freeze‐thawing. Furthermore, RNA is highly susceptible to RNAse degradation following the release of nucleases during the tissue disaggregation process. Thus, proper sample handling during the tissue preparation and homogenization processes is crucial when performing any RNA based assay.Herein, we evaluate the impact of multiple methods for tissue harvesting, storage, storage buffer and mechanical dissociation processes on purified RNA yield and integrity. Multiple, freshly harvested murine tissues, including kidney, liver, heart, and lung were snap frozen in liquid nitrogen and stored without buffer or in an RNA stabilizing reagent at −80°C. Tissues were then dissociated via cryomilling or ambient bead milling and RNA was purified using a silica spin column method. The process above was repeated following one, two and three freeze‐thaw cycles. After each purification, RNA was analyzed on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano kit chip, as per the manufacturers' protocol. Gel images, electrophoretograms and RNA integrity numbers (RIN's) were visualized and analyzed on the 2100 Bioanalyzer Expert software (Agilent).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transcriptome sequencing of Himalayan Raspberry (Rubus ellipticus) and development of simple sequence repeat markers.

Rubus ellipticus is a wild crop having less number of EST-SSR markers. First attempt was made towards the transcriptomics data analysis and generation of EST-SSR markers which were used in genetic diversity and transferability studies. Rubus ellipticus is a raspberry with yellow fruits, native to tropical and subtropical India and Asia. Leaves of Rubus ellipticus 'Kumarhatti' collection were utilized for cDNA library construction. More than 15 million sequencing reads were generated using NextSeq500 Illumina RNA-seq technology. The DNASTAR software was used for de novo assembly from which 7777 unigenes with an average length of 500bp was obtained. These unigenes were annotated using public databases, including NCBI non-redundant and gene ontology. De novo assembly of R. ellipticus unigenes found the highest similarity to apple than to other members of Rosaceae. This is the first attempt to use the Illumina platform of RNA sequencing and de novo assembly for R. ellipticus without a reference genome. In this study, unigenes were used for SSR marker development. ESTs containing SSR motifs were extracted using an online Microsatellite Identification Tool (MISA). SSR primers were designed from the SSR containing 704 EST sequences using the Websat software. Total 304 EST-SSRs primers were successfully designed, out of which 68 randomly selected primer pairs were custom synthesized and used for validation. Real-time PCR was also performed to analyze the relationship of transcriptional factors with fruit ripening. Out of 68 primer pairs, 61 were found to be informative in R. ellipticus, whereas 65 primer pairs were informative in the five tested genera of Rosaceae, i.e., pear, peach, apple, rose, and strawberry with 95.3% and 93.5% polymorphism, leading to the conclusion that these marker systems are very efficient to carryout diversity and cross transferability study in Rosaceae genera.

De novo characterization of the Goji berry (Lycium barbarium L.) fruit transcriptome and analysis of candidate genes involved in sugar metabolism under different CO2 concentrations.

Goji berry (Lycium barbarum L.) is one of the important economic crops due to its exceptional nutritional value and medicinal benefits. Although reduced sugar levels in goji berry exposed to long-term elevated carbon dioxide (CO2) have been documented, the underlying molecular mechanisms remain unknown. The objective of this study was to explore the transcriptome of goji berry fruit under ambient and elevated CO2 concentrations and further to screen the differentially expressed genes (DEGs) for functions related to sugar metabolism. Fruit samples from goji berry exposed to ambient (400 μmol mol-1) and elevated (700 μmol mol-1) levels of CO2 for 120 days were analyzed for total sugar, carotenoid and flavone analysis. In this study, a reduction in total sugar and carotenoid levels in the fruits grown under elevated CO2 levels were observed. Fruit samples were also used to construct cDNA libraries using a HiSeqTM2500 platform. Consequently, 81,100 unigenes were assembled, of which 35,111 (43.3%) were annotated using various databases. Through DEGs analysis, it was found that 55 genes were upregulated and 18 were down-regulated in response to elevated CO2 treatment. Genes involved in the sugar metabolism and the related pathways were identified by Gene Ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Furthermore, three genes, LBGAE (Lycium barbarum UDP-glucuronate 4-epimerase), LBGALA (Lycium barbarum alpha-galactosidase) and LBMS (Lycium barbarum malate synthase), associated with sugar metabolism were identified and discussed with respect to the reduction in the total sugar levels along with the enzymes acid invertase (AI), sucrose synthase (SS) and sucrose phosphate synthase (SPS) of the sucrose metabolism. This study can provide gene sources for elucidating the molecular mechanisms of sugar metabolism in the fruit of goji berry under elevated CO2.

The Construction of Full-Length cDNA Library for Otodectes cynotis.

Otodectes cynotis (Hering, 1838) is the pathogen of otodectic mange distributed worldwide. The mite mainly infests carnivores and, sometimes, humans. However, due to the lack of cDNA library, research on its pathogenesis has been challenging. To solve this problem, the present study first sampled O. cynotis mites from an infested cat from Xi'an, China, for RNA extraction. Then, the full-length cDNA library was constructed using the SMART technique. Finally, positive clones > 500 bp and Hsc70-5 gene fragment specifically amplified from the cDNA library were sequenced and analyzed to verify the library's reliability. Results showed that RNA extracted from 300 mites had good quality with a concentration of 149 ng/μl and OD260/OD280 of 1.99. The library satisfied the quality standard of a good library with a titer of 5.02 × 105 PFU/ml and a combination rate of 97.61%. In addition, clone 4 and Hsc70-5 showed 98.38% and 99.72% identity with Ef1-α and Hsc70-5 gene sequences of O. cynotis in GenBank, respectively. The cDNA library of O. cynotis constructed here was successful and reliable, creating the basis for research on RNA sequencing and functional genes of O. cynotis.

Constructing and Screening Beta-Glucanase Activity of Metagenomic cDNA Expression Library of Digestive Gland of Achatina fulica

Constructing and Screening Beta-Glucanase Activity of Metagenomic cDNA Expression Library of Digestive Gland of Achatina fulica

Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments.

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT50, temperature that results in 50% injury of maximum, approximately −7°C) and cold-acclimated (CA; leaf LT50 approximately −50°C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-à-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F2S2Kn type; and RcDhn 2 as F3SKn type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

Small RNA-seq: The RNA 5'-end adapter ligation problem and how to circumvent it.

The preparation of small RNA cDNA sequencing libraries depends on the unbiased ligation of adapters to the RNA ends. Small RNA with 5’ recessed ends are poor substrates for enzymatic adapter ligation, but this 5’ adapter ligation problem can go undetected if the library preparation steps are not monitored. Here we illustrate the severity of the 5’ RNA end ligation problem using several pre-miRNA-like hairpins that allow us to expand the definition of the problem to include 5’ ends close to a hairpin stem, whether recessed or in a short extension. The ribosome profiling method can avoid a difficult 5’ adapter ligation, but the enzyme typically used to circularize the cDNA has been reported to be biased, calling into question the benefit of this workaround. Using the TS2126 RNA ligase 1 (a.k.a. CircLigase) as the circularizing enzyme, we devised a bias test for the circularization of first strand cDNA. All possible dinucleotides were circle-ligated with similar efficiency. To re-linearize the first strand cDNA in the ribosome profiling approach, we introduce an improved method wherein a single ribonucleotide is placed between the sequencing primer binding sites in the reverse transcriptase primer, which later serves as the point of re-linearization by RNase A. We incorporate this step into the ribosomal profiling method and describe a complete improved library preparation method, Coligo-seq, for the sequencing of small RNA with secondary structure close to the 5’ end. This method accepts a variety of 5’ modified RNA, including 5’ monophosphorylated RNA, as demonstrated by the construction of a HeLa cell microRNA cDNA library.

First Report of Rose Rosette Virus Associated with Rose Rosette Disease Affecting Roses in California

Rose rosette disease (RRD) affecting rose (Rosa spp.) was first described in the western United States and Canada in the early 1940s (Conners 1941) and is now widely distributed in the eastern United States. Typical RRD symptoms include excessive lateral shoot growth and thorniness, witches’ broom, leaf proliferation and malformation, mosaic, red pigmentation, and eventual plant decline and death. RRD is caused by the negative-sense RNA virus, rose rosette virus (RRV), which is vectored by the eriophyid mite (Phyllocoptes fructiphilus Keifer) (Di Bello et al. 2015; Laney et al. 2011). California is one of the main producers of roses in the United States, with a wholesale value of US$17.6 million in 2016 (NASS 2016). In August 2017, symptoms suggestive of RRD were observed in a commercial nursery in Wasco, CA. Leaf tissues were collected from a total of 415 plants, representing many rose cultivars, throughout the entire nursey. Two plants (cvs. Veterans’ Honor and Brilliant Pink Iceberg) showed RRD symptoms, whereas the rest of the samples were asymptomatic. Total RNA was extracted from leaf petioles using the MagMA-96 viral RNA isolation kit (Thermo Fisher Scientific, Waltham, MA) and tested for RRV using a modified quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay developed by Dobhal et al. (2016). Only the two symptomatic plants were positive for RRV. Plants were also microscopically examined, and the P. fructiphilus was observed on several plants throughout the nursery. In June 2018, three garden roses (unknown cultivars) planted in two neighboring homes in Bakersfield, CA, approximately 50 km from the 2017 finds, were identified with RRD symptoms. Leaves from all three plants tested positive for RRV by RT-qPCR and were also infested with P. fructiphilus. To confirm these results, total nucleic acid was extracted from stored (–80°C) leaves from each of the five symptomatic plants and used for cDNA library construction after a ribosomal RNA depletion step (TruSeq Stranded Total RNA with RiboZero Plant kit, Illumina, San Diego, CA). High-throughput sequencing (HTS) was performed on the Illumina NextSeq 500 platform, generating between 15 million and 41 million 75-nucleotide (nt) single-end raw data reads per sample. Analysis of the HTS reads identified nearly complete sequences (>97%) of the seven RRV genome segments from all five samples. The RRV genomes from the two nursery plants sampled in 2017 shared 100% nt identity to one another. The RRV genomes from the three homeowner plants sampled in 2018 shared 100% nt identity to one another but not to the nursery samples (nursery versus homeowner showed 98% nt identity). Sequences from the two locations shared 98% nt identity to the NCBI reference RRV RNA3 (NC_015300) and RNA7 (NC_034981) and 99% nt identity to the NCBI reference RRV RNA1-2 (NC_015298-99) and RNA4-6 (NC_015301, NC_034979, and NC_034980). These results suggest that the samples from these two locations are likely two separate introductions of RRV into California. Two genomic sequences, one from each location, were submitted to GenBank under the accession numbers MH581213 to MH581226. To our knowledge, this is the first report of RRV associated with RRD in California. Further studies will be necessary to assess the relevance and potential threat of this virus to the garden rose nursery industry in California.

Isolation of Bacterial RNA from Foods Inoculated with Pathogens.

One strategy bacteria use to acclimate to changing environmental conditions is modulation of gene expression. Alterations in gene expression are indicative of activation or repression of certain physiological responses. In order to understand which genetic responses are utilized to cope with various environmental conditions by analyzing transcriptomes, obtaining RNA of high quality, yield, and integrity is paramount. Here, we describe an acid phenol-chloroform method employed to extract RNA from laboratory grown cell cultures, as well as cultures inoculated onto complex matrices such as lettuce and cold-smoked salmon. The method results in high-quality RNA, which can be used for various downstream processes such as cDNA library construction, RNA sequencing, real-time quantitative PCR, and northern analysis. Extraction of RNA from bacterial foodborne pathogens in conjunction with transcriptome sequencing is a useful technique to elucidate pathogens' transcriptional responses.

Isolation of living apical and basal cell lineages of early proembryos for transcriptome analysis

Plant zygotes usually undergo asymmetrical cell division, giving rise to the formation of two daughter cells with distinct developmental cell fate. The small apical cell will develop into the major part of embryo proper, whereas the larger basal cell will divide to form a transient suspensor. Thus, the apical and basal cell lineages are an excellent model to study cell fate determination in relation to zygote polarity. However, the molecular mechanism underlying the differentiation of two distinct cell lineages is not yet understood, possibly due to the technique limitations. Previously, we have established a protocol for isolating apical cell and basal cell for cDNA library construction in tobacco. However, the method for isolating tiny Arabidopsis embryos has long been considered much more difficult. Here, we present a detailed protocol for isolating early Arabidopsis proembryos and separating apical and basal cell lineages of proembryos, which allow us to establish cell lineage-specific transcriptomes of early proembryos.

Construction of a full-length cDNA library from Typha laxmanni Lepech. and T. angustifolia L. from an EST dataset

Construction of a full-length cDNA library from Typha laxmanni Lepech. and T. angustifolia L. from an EST dataset

Downstream components of the calmodulin signaling pathway in the rice salt stress response revealed by transcriptome profiling and target identification

BackgroundCalmodulin (CaM) is an important calcium sensor protein that transduces Ca2+ signals in plant stress signaling pathways. A previous study has revealed that transgenic rice over-expressing the calmodulin gene OsCam1–1 (LOC_Os03g20370) is more tolerant to salt stress than wild type. To elucidate the role of OsCam1–1 in the salt stress response mechanism, downstream components of the OsCam1–1-mediated response were identified and investigated by transcriptome profiling and target identification.ResultsTranscriptome profiling of transgenic ‘Khao Dawk Mali 105’ rice over-expressing OsCam1–1 and wild type rice showed that overexpression of OsCam1–1 widely affected the expression of genes involved in several cellular processes under salt stress, including signaling, hormone-mediated regulation, transcription, lipid metabolism, carbohydrate metabolism, secondary metabolism, photosynthesis, glycolysis, tricarboxylic acid (TCA) cycle and glyoxylate cycle. Under salt stress, the photosynthesis rate in the transgenic rice was slightly lower than in wild type, while sucrose and starch contents were higher, suggesting that energy and carbon metabolism were affected by OsCam1–1 overexpression. Additionally, four known and six novel CaM-interacting proteins were identified by cDNA expression library screening with the recombinant OsCaM1. GO terms enriched in their associated proteins that matched those of the differentially expressed genes affected by OsCam1–1 overexpression revealed various downstream cellular processes that could potentially be regulated by OsCaM1 through their actions.ConclusionsThe diverse cellular processes affected by OsCam1–1 overexpression and possessed by the identified CaM1-interacting proteins corroborate the notion that CaM signal transduction pathways compose a complex network of downstream components involved in several cellular processes. These findings suggest that under salt stress, CaM activity elevates metabolic enzymes involved in central energy pathways, which promote or at least maintain the production of energy under the limitation of photosynthesis.

Contribution of the cold shock protein CspA to virulence in Xanthomonas oryzae pv. oryzae.

Summary Xanthomonas oryzae pv. oryzae (Xoo) causes a damaging bacterial leaf blight disease in rice. Cold shock proteins (Csps) are highly conserved nucleic acid‐binding proteins present in various bacterial genera, but relatively little is known about their functions in Xanthomonas. Herein, we identified four Csps (CspA–CspD) in the Xoo PXO99A strain. Deletion of cspA decreased cold adaptation and a few known pathogenic factors, including bacterial pathogenicity, biofilm formation and polysaccharide production. Furthermore, we performed transcriptomic and chromosome immunoprecipitation (ChIP) experiments to identify direct targets of CspA and to determine its DNA‐binding sequence. Integrative data analysis revealed that CspA directly regulates two genes, PXO_RS11830 and PXO_RS01060, by binding to a conserved CCAAT sequence in the promoter region. We generated single‐deletion mutants of each gene and the results indicate that both are responsible for Xanthomonas pathogenicity. In addition, quantitative real‐time polymerase chain reaction and western blotting showed that CspA suppressed the expression of its direct targets. In summary, our study clarifies the characteristics of Csps in Xanthomonas and greatly advances our understanding of the mechanisms underlying the contribution of CspA to bacterial virulence.

Coding/non-coding cross-talk in intestinal epithelium transcriptome gives insights on how fish respond to stocking density

Fish respond to increasing stocking density as a stressor, adjusting physiological functions to increase energy supply for coping with deleterious effects and adapting. These responses are complex and systemic, and the molecular mechanisms involved remain undetermined. One of the most sensitive organs to environmental and homeostatic disruptions is the intestine, and since it plays several vital functions, understanding the molecular underpinnings of this organ under deleterious conditions is imperative for health improvement in aquaculture systems. This study aimed to understand how different stocking densities of rainbow trout (Oncorhynchus mykiss) modulates the coding and non-coding RNAs profiling, that in turn, play key roles to maintain the fish homeostasis. For this, the intestine tissue of juvenile trout stocked for 30 days either at low (LD: 3 kg m−3) or high density (HD: 40 kg m−3) were sampled to isolate total RNA and then construct cDNA libraries for an illumina sequencing platform. Differential gene expression analysis revealed a generalized downregulation of transcripts, including coding and long non-coding RNAs (lncRNAs). Notably, significant differences in transcripts involved in metabolic pathways, as well as immune and epithelium integrity and stability related pathways were found. A high number of downregulated transcripts enriched these pathways, and a strong correlation was observed between the most differentially expressed transcripts and the highly expressed lncRNAs. This study suggests a cross-talk between coding and non-coding RNAs in the intestine of fish exposed to suboptimal conditions, providing new insights into the regulatory role of the lncRNAs on fish response to stressors.