Subtractive Library Research Articles

Extracellular Matrix Peptides of Artemia Cyst Shell Participate in Protecting Encysted Embryos from Extreme Environments

BackgroundMany species of the brine shrimp Artemia are found in various severe environments in many parts of the world where extreme salinity, high UV radiation levels, high pH, anoxia, large temperature fluctuations, and intermittent dry conditions are often recorded. To withstand adverse environments, Artemia undergoes an oviparous developmental pathway to release cysts whereas, under favorable conditions, swimming nauplius larvae are formed directly via an ovoviviparous pathway. In the former case these cysts have an extraordinary ability to keep the embryos protected from the harsh environment for long periods. This is achieved through the protection by a complex out-wrapping cyst shell. However, the formation and function of the cyst shell is complex; the details remain largely unclear.Principal FindingA shell gland-specific gene (SGEG2) was cloned and identified from a suppression subtractive hybridization library. Western blot analysis showed that SGEG2 presumably requires post-translational proteolysis in order to be processed into two mature peptides (SGEG2a and 2b). The three matrix peptides (SGEG1 reported previously, 2a, and 2b) were found to distribute throughout the cyst shell. The results of gene knockdown by RNAi and subsequent resistance to environmental stresses assays indicated that these matrix peptides are required for cyst shell formation and are involved in protecting the encysted embryos from environmental stress.Conclusions/SignificanceThis study revealed that extracellular matrix peptides participate in protecting embryos from extreme salinity, UV radiation, large temperature fluctuations and dry environments, thereby facilitating their survival. The cyst shell provides an excellent opportunity to link the ecological setting of an organism to the underlying physiological and biochemical processes enabling its survival. The cyst shell material has also a high potential to become an excellent new biomaterial with a high number of prospective uses due, specifically, to such biological characteristics.

Screening of the target genes transactivated by PS1TP1 protein with suppression subtractive hybridization technique

To construct a subtractive cDNA library of genes transactivated by PS1TP1 protein with suppression subtractive hybridization (SSH) technique. Suppression subtractive hybridization technique and bioinformatics technique were used, the mRNA from HepG2 cells transfected with pcDNA3.1 (-) -PS1TP1 and pcDNA3. 1 (-) empty vector was isolated, respectively; cDNA underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. The subtractive library of genes transactivated by PS1TP1 was constructed successfully. Sequence analysis was performed in 43 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 12 coding sequences were gotten, which consisted of 10 known and 2 unknown ones. The obtained sequences may be target genes transactivated by PS1TP1 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity and cell apoptosis. This finding brought some clues for studying the biological functions of PS1T1.

Identification and expression profile of multiple genes in Nile tilapia in response to formalin killed Streptococcus iniae vaccination

Twenty-eight expressed sequence tags (ESTs) were isolated from a Nile tilapia ( Oreochromis niloticus) vaccinated vs non-vaccinated subtractive library at 12-h post injection of a formalin killed Streptococcus iniae ARS-98-60 vaccine. The 28 ESTs were classified in terms of their putative functions. Half of the ESTs identified were unknown proteins. Of the remaining half ESTs, 17% have putative functions in protein biosynthesis and 11% have putative functions in immunity, energy production, and signal transduction, respectively. Immunity-related ESTs identified included high density lipoprotein-binding protein vigilin, immunoglobulin heavy chain, and QM-like protein. Quantitative PCR revealed that one EST (cytochrome c oxidase subunit II) was highly upregulated (1825 ± 336 fold) in vaccinated fish compared to that in non-vaccinated fish. Of the remaining 27 ESTs, nine were significantly ( P < 0.05) upregulated (<20 fold) in vaccinated fish. The nine significantly upregulated genes included five unknown or hypothetical proteins and four known proteins (high density lipoprotein-binding protein vigilin, QM-like protein, ribosomal protein S13, and ribosomal protein L5). The upregulation of these genes induced by killed S. iniae vaccines suggest that they might play important role in Nile tilapia defense against S. iniae infection.

Construction of cDNA subtractive library from pearl oyster (Pinctada fucata Gould) with red color shell by SSH

The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization (SSH) to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively. Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6, shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COI. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and COI so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.

PR-10, defensin and cold dehydrin genes are among those over expressed in Oxytropis (Fabaceae) species adapted to the arctic

In many studied plants, typical responses to cold treatment include up-regulating the hydrophilic COR/LEA genes and down-regulating photosynthesis-related genes, carbohydrate metabolism, GDSL-motif lipase, hormone metabolism and oxidative regulation genes. However, next to nothing is known about gene expression in arctic plants, which are actually adapted to a harsh, cold environment. The molecular mechanisms behind the many specific adaptations of arctic plants, such as slow growth, well-developed root systems and short stature, are not well understood. In this study, we examine whole plantlet transcriptome differences between two arctic and two temperate Oxytropis (Fabaceae) species, grown under their respective controlled environmental conditions. Gene expression differences are analyzed using cDNA library subtraction followed by expressed sequence tags sequencing and annotation. Sequences from a total of nearly 2,000 clones cluster into 121 and 368 unique genes from the arctic and from the temperate plants, respectively. The predominant biological process for genes from the arctic-enriched library is “response to stimulus”. A concurrent overexpression of pathogenesis-related class 10 proteins (PR-10), plant defensin and cold dehydrin genes is a novel feature for species adapted to stressful growth environment. The temperate-enriched genes are involved in photosynthesis, translation and nucleosome assembly. Interestingly, both arctic and temperate-enriched libraries also contain genes involved in ribosome biogenesis and assembly, however of different types. Real-time reverse transcription PCR of cold dehydrin and two PR-10 genes, as well as the light harvesting complex b1 genes demonstrates that the gene expression is dependent on species and growth conditions.Electronic supplementary materialThe online version of this article (doi:10.1007/s10142-011-0223-6) contains supplementary material, which is available to authorized users.

Identification of unique DNA sequences present in highly virulent 2009 Alabama isolates of Aeromonas hydrophila

In 2009, a disease outbreak caused by Aeromonas hydrophila occurred in 48 catfish farms in West Alabama, causing an estimated loss of more than 3 million pounds of food size channel catfish. Virulence studies have revealed that the 2009 isolates of A. hydrophila are at least 200-fold more virulent than a 1998 Alabama isolate AL98-C1B. However, up to now, no molecular markers have been identified to differentiate the highly virulent 2009 isolates from other isolates of A. hydrophila. To understand the genetic differences between the highly virulent 2009 isolates and the less virulent AL98-C1B at molecular level, PCR-select bacterial genome subtractive hybridization was used in this study. A total of 96 clones were selected from the subtractive genomic DNA library. Sequencing results revealed that the 96 clones represented 64 unique A. hydrophila sequences. Of the 64 sequences, three (hypothetical protein XAUC_13870, structural toxin protein RtxA, and putative methyltransferase) were confirmed to be present in the three virulent 2009 Alabama isolates but absent in the less virulent AL98-C1B. Using genomic DNAs from nine field isolates of A. hydrophila with different virulence as templates, two sequences (hypothetical protein XAUC_13870 and putative methyltransferase) were found to be only present in highly virulent A. hydrophila isolates, but absent in avirulent isolates.

Abstract 3860: Differential gene expression and HPV in penile carcinoma

Abstract Penile cancer (PC) is an invasive epithelium tumor, representing more than 10% of the malignancies in men in some developing countries, mainly in Brazil. Studies on the aetiopathogenesis of this cancer have focused on its association with the HPV. Patients with PC without treatment usually die within 2 years after diagnosis, because of uncontrollable loco regional disease or from distant metastasis. The aim of this study is evaluating the possible role of HPV in the development of PC and the use of the RaSH technique to analyze gene expression in normal tissues and penile tumors. HPV detection was done by PCR with generic primers GP5+/GP6+ and HPV typing was done by direct sequencing. RaSH methodology identified differentially expressed genes, generating subtractive cDNA libraries. The presence of HPV was analyzed in 58 samples of patients with PC. It has been observed high prevalence (85.12%) of HPV. Of the 16 samples sequenced, HPV-16 was found in 14 (87.5%) samples, HPV-11 in 1 (6.3%) and HPV-35 in 1 (6.3%). The RaSH subtractive libraries identified the presence of 57 clones differentially expressed between samples, 30 in tumor samples and 27 in the normal tissue. The genes PBEF1, ANXA1, RPL6 and KIAA1033 were overexpressed in the tumors. On the other hand, the gene p16 was overexpressed in the normal tissue. Finally, the expression of the selected genes was confirmed by qPCR. Only ANXA1 was validated with overexpression in 80% of all samples. It was also analyzed ANXA1 protein expression by immunohistochemistry in 25 samples of penile cancer. It was observed overexpression of ANXA1 protein 100% of PC samples compared to normal tissue. The results obtained are capable of revealing differences in patterns of gene expression between normal and tumor tissues. Moreover, it was observed high prevalence of HPV, suggesting an important role of this virus in penile carcinogenesis. Besides, overexpression of ANXA1 was observed in most of the penile cancer samples and such information will contribute to develop a possible marker for penis tumor diagnostic and prognostic, improving the development of directed therapies against this new putative marker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3860. doi:10.1158/1538-7445.AM2011-3860

MARCH-I expression in cord blood CD34+KDR+ cells

Hematopoietic stem cells transplantation has been successfully used in the treatment of patients with hematological malignances. A better knowledge of the mechanisms beyond their ability to completely repopulate the entire hematopoietic system would help in the treatment of hematological diseases. For this reason we focused our studies on a cell population that has been demonstrated to have some peculiar characteristics among the stem cells: CD34+KDR+ cells. These cells, an extremely rare population among the CD34 (0.1%–0.5%) cells, have been demonstrated from different groups to have the potential to give rise to the hematopoietic and endothelial lineage. By a subtraction library approach we found different sequences more expressed in CD34+KDR+ than their CD34+KDR− counterpart. In particular, we found an open reading frame correspondent to a newly characterized E3 ligase, MARCH-I. This gene is part of a recently described family involved in immune response modulation through the proteosomal mediated degradation. MARCH-I expression in stem cells could be important for their intrinsic immune properties.

Construction and analysis of suppression subtractive cDNA libraries of continuous monoculture Rehmannia glutinosa

To explore the molecular mechanism of continuous monoculture problem by constructing the cDNA libraries of continuous monoculture Rehmannia glutinosa. To use the suppression subtractive hybridization (SSH) technique to construct the forward and reverse subtractive cDNA libraries of continuous monoculture R. glutinosa to adopt blue-white colony screening and PCR to detect the positive clones which would be sequenced and analyzed by bioinformatics. The subtracted cDNA libraries of continuous monoculture R. glutinosa. were successfully constructed, and the result showed that the forward and reverse subtracted libraries obtained 300 positive clones, respectively. The forward and reverse libraries got different ESTs, and produced 232 (forward library) and 214 (reverse library) unique ESTs by sequencing. Based on homology search of BLASTX and BLASTN in NCBI, 200 and 195 of unique ESTs were homologous to known genes in the forward and reverse libraries, respectively. Categories of orthologous group (COG) showed that the forward and reverse libraries got 60 and 61 ESTs with the corresponding gene annotation, involving 21 metabolic pathways. The information of differential expression genes in continuous monoculture R. glutinosa, and their functional annotation of differentially expressed genes indicate that continuous monoculture has a profound effect on expression of the genes in R. glutinosa. Furthermore, the research analyzed several key genes in response to replant problem, which provided a foundation for revealing the molecular mechanism of continuous monoculture R. glutinosa.

Construction and Primary Analysis of Subtractive cDNA Library of Capsicum annuum L. Under Phytophthora capsici Stress

To identify the Phytophthora capsici stress induced genes of pepper,a high P.capsici resistant pepper cultivar was used as main material in this study.With cDNA from pepper seedlings treated with P.capsici as the tester and cDNA from this plant in normal growth as the driver,we constructed subtracted library using suppression subtractive hybridization(SSH).Thirty positive clones were picked out randomly from SSH library,plasmid PCR suggested that the inserts were between 200-1 000 bp and library was suitable for the following work.Forty clones were randomly selected and sequenced,35 differential gene fragments were obtained.Blastx analysis showed that 30 ESTs have high homology with known genes in GenBank,5 ESTs was unknown function genes.The obtained known function ESTs code NAC transcription factor protein,Serine/threonine protein kinase,cytochrome P450 monooxygenase,chlorophyll a b-binding protein,glutathione s-transferase,chitinase,respectively.These expressed genes are involved in disease-resistant signal transduction,antioxidative stress,transcriptional regulation,photosynthesis,physiological processes.The research established a basis for cloning stress resistance genes and further studying genes expression in Capsicum annuum L.seedlings under P.capsici stress.

A potato skin SSH library yields new candidate genes for suberin biosynthesis and periderm formation

Potato (Solanum tuberosum) tubers are underground storage organs covered by the skin or periderm, a suberized layer that protects inner flesh from dehydration and pathogens. Understanding the molecular processes associated with periderm formation is of great importance for a better knowledge of this protective tissue and for improving the storage life of tubers. Here, to isolate new candidate genes for potato periderm, a suppression subtractive hybridization library from potato skin was performed. This library yielded a comprehensive list of 108 candidate genes that were manually sorted in functional categories according to the main cellular and metabolic processes in periderm. As expected, the list contains Suberin and wax genes, including some genes with a demonstrated role in the biosynthesis of these cell wall aliphatic compounds. Moreover, Regulation and Stress and defence genes are highly abundant in the library in general agreement with previous potato skin proteomic studies. The putative function of the genes in periderm is discussed.

Analysis of gene expression by ESTs from suppression subtractive hybridization library in Chenopodium album L. under salt stress

To identify genes expression in Chenopodium album exposed to NaCl stress and screen ESTs related to salt stress, a subtractive suppression hybridization (SSH) library of C. album under salt stress was constructed in the present study. Random EST sequencing produced 825 high-quality ESTs with GenBank ID GE746311-GE747007, which had 301 bp of average size and were clustered into 88 contigs and 550 singletons. They were classified into 12 categories according to their function annotations. 635 ESTs (76.97%) showed similarities to gene sequences in the non-redundancy database, while 190 ESTs (23.03%) showed low or no similarities. The transcriptional profiles of 56 ESTs randomly selected from 347 unknown or novel ESTs of SSH library under varying NaCl concentration and at different time points were analyzed. The results indicated that a high proportion of tested ESTs were activated by salt stress. Four in 56 ESTs responded to NaCl were also enhanced in expression level when exposed to ABA and PEG stresses. The above four ESTs were validated by northern blotting which was consistent with the results of RT-PCR. The results suggested that genes corresponded to these ESTs might be involved in stress response or regulation. The complete sequences and detailed function of these ESTs need to be further studied.

Enhancement of GDP-Dissociation Inhibitor Gene Expression in Osteoblasts by Low-Level Laser Irradiation

Low-level laser irradiation (LLLI) stimulates bone regeneration and may be useful for bone fracture and oral implant therapy. However, the molecular mechanism of LLLI in accelerating bone formation is not well known. In order to understand the mechanism of LLLI in accelerating bone formation, we attempted to identify gene expressions enhanced by LLLI by a subtractive gene cloning and DNA microarray analysis. Osteoblastic cell MC3T3-E1 was treated with 830 nm Ga-Al-As diode laser irradiation and total mRNA was recovered and used to construct a subtractive gene library of genes with transcription enhanced by LLLI. The DNA sequences of subtracted gene was subjected to a homology search using BLASTN program in the National Center for Biotechnology Information (NCBI). Genes with mRNA levels altered by LLLI were also analysed using Affymetrix GeneChip. The mRNA levels were confirmed by RT-PCR and real-time PCR. Subtracted gene clones MCL101 indicated high homology with GDP-dissociation inhibitor beta (GDPIB) genes. Affymetrix GeneChipTM analysis was further analyzed and found that GDI alpha gene expression was also enhanced by LLLI. The increased mRNA level of GDIA and GDIB by LLLI was successfully confirmed by RT-PCR and real-time PCR. Since the up-regulation of GDIA is one of the mechanisms underlying the synergistic boneforming effect of parathyroid hormone and oestrogen. Moreover, GDIA and GDIB involve in the regulation of vesicle-mediated cellular transport and anti-apoptosis, the enhancement of GDIA and GDIB gene expressions by LLLI may be a part of mechanisms in the acceleration of bone formation by LLLI.

A Sexual Shift Induced by Silencing of a Single Insulin-Like Gene in Crayfish: Ovarian Upregulation and Testicular Degeneration

In sequential hermaphrodites, intersexuality occurs naturally, usually as a transition state during sexual re-differentiation processes. In crustaceans, male sexual differentiation is controlled by the male-specific androgenic gland (AG). An AG-specific insulin-like gene, previously identified in the red-claw crayfish Cherax quadricarinatus (designated Cq-IAG), was found in this study to be the prominent transcript in an AG cDNA subtractive library. In C. quadricarinatus, sexual plasticity is exhibited by intersex individuals in the form of an active male reproductive system and male secondary sex characters, along with a constantly arrested ovary. This intersexuality was exploited to follow changes caused by single gene silencing, accomplished via dsRNA injection. Cq-IAG silencing induced dramatic sex-related alterations, including male feature feminization, a reduction in sperm production, extensive testicular degeneration, expression of the vitellogenin gene, and accumulation of yolk proteins in the developing oocytes. Upon silencing of the gene, AG cells hypertrophied, possibly to compensate for low hormone levels, as reflected in the poor production of the insulin-like hormone (and revealed by immunohistochemistry). These results demonstrate both the functionality of Cq-IAG as an androgenic hormone-encoding gene and the dependence of male gonad viability on the Cq-IAG product. This study is the first to provide evidence that silencing an insulin-like gene in intersex C. quadricarinatus feminizes male-related phenotypes. These findings, moreover, contribute to the understanding of the regulation of sexual shifts, whether naturally occurring in sequential hermaphrodites or abnormally induced by endocrine disruptors found in the environment, and offer insight into an unusual gender-related link to the evolution of insulins.

Identification of Differentially Expressed Genes in Lung Tissues of Nickel-Exposed Rats Using Suppression Subtractive Hybridization

Occupational exposure to nickel compound, such as nickel refining, electroplating, and in conjunction with other metals, is harmful to the health, causing respiratory distress, and lung and nasal cancer. In this work, the different gene expression patterns of lung tissues from nickel-exposed rats and controls were investigated. The suppression subtractive hybridization (SSH) method was used to generate two subtracted cDNA libraries with gene transcripts differentially expressed after nickel inducing. Dot-blot hybridizations were used to confirm differential ratios of expression of obtained SSH clones. Out of 768 unique SSH clones, which were chosen randomly from the two subtraction libraries (384 of each), 319 could be verified as differentially expressed. According to blast screening and functional annotation, 28% genes in nickel-induced cDNA library were related to cell differentiation, whereas 21% in driver library were related to oxygen transport. Two novel expressed sequence tags (ESTs; NCBI Accession No. FC809414 and No. FC809411) in nickel-induced cDNA library were obtained. The genes detected in the present study are probably important genes associated with nickel-induced lung cancer.

Roles for Stress-inducible Lambda Glutathione Transferases in Flavonoid Metabolism in Plants as Identified by Ligand Fishing

The glutathione transferases (GSTs) of plants are a superfamily of abundant enzymes whose roles in endogenous metabolism are largely unknown. For example, the lambda class of GSTs (GSTLs) have members that are selectively induced by chemical stress treatments and based on their enzyme chemistry are predicted to have roles in redox homeostasis. However, using conventional approaches these functions have yet to be determined. To address this, recombinant GSTLs from wheat and Arabidopsis were tagged with a Strep tag and after affinity-immobilization, incubated with extracts from Arabidopsis, tobacco, and wheat. Bound ligands were then recovered by solvent extraction and identified by mass spectrometry (MS). With the wheat enzyme TaGSTL1, the ligand profiles obtained with in vitro extracts from tobacco closely matched those observed after the protein had been expressed in planta, demonstrating that these associations were physiologically representative. The stress-inducible TaGSTL1 was found to selectively recognize flavonols (e.g. taxifolin; K(d) = 25 nM), with this binding being dependent upon S-glutathionylation of an active site cysteine. In the case of the wheat extracts, this selectivity in ligand recognitions lead to the detection of flavonols that had not been previously described in this cereal. Subsequent in vitro assays showed that the co-binding of flavonols, such as quercetin, to the thiolated TaGSTL1 represented an intermediate step in the reduction of the respective S-glutathionylated quinone derivatives to yield free flavonols. These results suggest a novel role for GSTLs in maintaining the flavonoid pool under stress conditions.

Induction of transposase and polyprotein RNA levels in disseminated neoplastic hemocytes of soft-shell clams: Mya arenaria

In Prince Edward Island, a high mortality of soft-shell clams Mya arenaria was found to be related to the disease known as disseminated neoplasia (DN). However, the molecular mechanisms by which hemocytes of clams are transformed in the course of DN remain by far unknown. This study aims at identifying the transcripts involved in the development of the disease. Four subtractive cDNA sequence libraries were generated and more than 200,000 reads were obtained. Following similarity searches in genome databases, the transcripts were assigned to cellular functions including mitochondrial respiration, structural proteins, cytoskeleton, nucleic acid regulation, general metabolism, signal transduction, apoptosis, cell cycle regulation, as well as virus transcripts. The expression levels of transposase and polyprotein genes were evaluated in clams with various percentages of tetraploid hemocytes. Data have shown that expression levels were significantly higher in clams with a high percentage of tetraploid hemocytes. These results reinforce the hypothesis of endogenous retrotransposon involvement in the etiology of the disease. Further investigations are needed, however, to elucidate the role of transposase and polyprotein in the disease development.

Massively parallel sequencing and analysis of expressed sequence tags in a successful invasive plant

Invasive species pose a significant threat to global economies, agriculture and biodiversity. Despite progress towards understanding the ecological factors associated with plant invasions, limited genomic resources have made it difficult to elucidate the evolutionary and genetic factors responsible for invasiveness. This study presents the first expressed sequence tag (EST) collection for Senecio madagascariensis, a globally invasive plant species. We used pyrosequencing of one normalized and two subtractive libraries, derived from one native and one invasive population, to generate an EST collection. ESTs were assembled into contigs, annotated by BLAST comparison with the NCBI non-redundant protein database and assigned gene ontology (GO) terms from the Plant GO Slim ontologies. Assembly of the 221,746 sequence reads resulted in 12,442 contigs. Over 50 % (6183) of 12,442 contigs showed significant homology to proteins in the NCBI database, representing approx. 4800 independent transcripts. The molecular transducer GO term was significantly over-represented in the native (South African) subtractive library compared with the invasive (Australian) library. Based on NCBI BLAST hits and literature searches, 40 % of the molecular transducer genes identified in the South African subtractive library are likely to be involved in response to biotic stimuli, such as fungal, bacterial and viral pathogens. This EST collection is the first representation of the S. madagascariensis transcriptome and provides an important resource for the discovery of candidate genes associated with plant invasiveness. The over-representation of molecular transducer genes associated with defence responses in the native subtractive library provides preliminary support for aspects of the enemy release and evolution of increased competitive ability hypotheses in this successful invasive. This study highlights the contribution of next-generation sequencing to better understanding the molecular mechanisms underlying ecological hypotheses that are important in successful plant invasions.

The promoters of 3 celery salt-induced phloem-specific genes as new tools for monitoring salt stress responses

Genes induced by a progressive 3 week salt stress (final NaCl concentration 300 mM) were identified in the phloem of celery (Apium graveolens L., cv Vert d'Elne). A subtractive library was constructed and screened for salt-induced, phloem-specific genes. Work was focused on phloem due to its central role in inter-organ exchanges. Three genes were studied in more details, 2 coding for metallothioneins (AgMT2 and AgMT3) and one for a new mannitol transporter (AgMaT3). Expression of a reporter gene in transgenic Arabidopsis under control of promoter of each gene was located in the phloem. pAgMT2 has a typical phloem pattern with slight induction by salt stress. pAgMT3 and pAgMaT3 expression was induced by salt stress, except in minor veins. pAgMaT3 was highly active in stressed roots. The promoters described here could be regarded as new tools for engineering salt-resistant plants.

Upregulation in response to infection and antibacterial activity of oyster histone H4

Several histones and histone-derived peptides have been shown to have antimicrobial activity and a potential role in innate immune defenses. A histone H4 sequence was identified in a subtractive suppression library containing genes upregulated in American cupped oysters, Crassostrea virginica, in response to challenge with the protozoan parasite Perkinsus marinus. Oyster histone H4 protein levels significantly increased in hemocyte lysates and cell free hemolymph of oysters experimentally challenged with P. marinus. The complete histone H4 coding sequence of C. virginica was cloned into a Saccharomyces cerevisiae yeast expression system and recombinant expression was confirmed using SDS-PAGE analysis and western blot. Delivery of yeast cells expressing recombinant oyster histone H4 into the gut of brine shrimp, Artemia salinas, challenged with a streptomycin resistant strain of Vibrio anguillarum resulted in a significant and dose-dependent decrease in the load of V. anguillarum. Purified recombinant histone H4 showed antimicrobial activity against V. anguillarum and Escherichia coli at micromolar concentrations, but did not affect the viability of P. marinus in culture. These results support the role of histone H4 in the defense of oysters against bacterial infection and validate the use of a novel oyster antimicrobial H4 in a yeast feed-based delivery system for the treatment of bacterial infections in aquaculture applications.