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Abstract Transcription profiling assays have revealed substantial changes in gene expression during plant-microbe interactions, but it is often time- and labor-consuming to define the causative roles of the differentially expressed genes that finetune the plant responses to diverse pathogens. We improved the duplex-specific nuclease-mediated transcriptome subtraction method and integrated it with SMART cDNA library construction technology to generate normalized libraries consisting of full-length cDNAs derived from transcripts upregulated in the rice-Magnaporthe oryzae interaction. By adding BP recombination sites to the sequence of primers used for the synthesis of full-length cDNAs, we were able to transfer the full-length cDNAs of the library to an expression binary vector with CaMV 35S promoter, which allowed a subsequent screening for candidate genes potentially involved in the disease process by Agrobacterium-mediated transient assay. Results showed that the newly established approach preferentially suppressed the cDNA abundance of most constitutive genes and enriched or normalized that of the upregulated ones. Subsequent screening with transient expression in planta isolated 61 clones, which carry full-length cDNAs of 32 distinct genes, were capable of causing cell death on Nicotiana benthamiana. When transiently expressed in barley leaf, five of the identified genes were able to induce chlorosis, and additional 11 genes were found to promote disease symptoms caused by infection with the blast pathogen while the others did not enhance the symptoms of pathogen infection. These observations demonstrate that the subtractive hybridization-assisted functional screening is an efficient approach that provides initial leads to the role of candidate genes in the complex process of plant-microbe interactions.

Detection of microbial antigens in clinical samples can lead to rapid diagnosis of an infection and administration of appropriate therapeutics. A major barrier in diagnostics development is determining which of the potentially hundreds or thousands of antigens produced by a microbe are actually present in patient samples in detectable amounts against a background of innumerable host proteins. In this report, we describe a strategy, termed in vivo microbial antigen discovery (InMAD), that we used to identify circulating bacterial antigens. This technique starts with "InMAD serum," which is filtered serum that has been harvested from BALB/c mice infected with a bacterial pathogen. The InMAD serum, which is free of whole bacterial cells, is used to immunize syngeneic BALB/c mice. The resulting " InMAD immune serum" contains antibodies specific for the soluble microbial antigens present in sera from the infected mice. The InMAD immune serum is then used to probe blots of bacterial lysates or bacterial proteome arrays. Bacterial antigens that are reactive with the InMAD immune serum are precisely the antigens to target in an antigen immunoassay. By employing InMAD, we identified multiple circulating antigens that are secreted or shed during infection using Burkholderia pseudomallei and Francisella tularensis as model organisms. Potential diagnostic targets identified by the InMAD approach included bacterial proteins, capsular polysaccharide, and lipopolysaccharide. The InMAD technique makes no assumptions other than immunogenicity and has the potential to be a broad discovery platform to identify diagnostic targets from microbial pathogens. IMPORTANCE Effective treatment of microbial infection is critically dependent on early diagnosis and identification of the etiological agent. One means for rapid diagnosis is immunoassay for antigens that are shed into body fluids during infection. Immunoassays can be inexpensive, rapid, and adaptable to a point-of-care format. A major impediment to immunoassay for diagnosis of infectious disease is identification of appropriate antigen targets. This report describes a strategy that can be used for identification of microbial antigens that are shed into serum during infection by the biothreats Burkholderia pseudomallei and Francisella tularensis. Termed InMAD (in vivo microbial antigen discovery), the strategy has the potential for application to a broad spectrum of microbial pathogens.

ABSTRACT The profiling of gene expression by RNA sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is rRNA, it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued abruptly and for an extended period of time. Here, we report the development of a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin-coated beads then depletes rRNA from a complex, total RNA sample such that ∼75 to 80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold change measurements in RNA-seq experiments between our method and the Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or community of interest. IMPORTANCE The ability to examine global patterns of gene expression in microbes through RNA sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of rRNA from total RNA samples. Otherwise, rRNA would comprise upward of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from mRNA or requiring high total read depth. A commonly used kit for rRNA subtraction from Illumina was recently unavailable for an extended period of time, disrupting routine rRNA depletion. Here, we report the development of a “do-it-yourself” kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligonucleotides enable sufficient rRNA depletion to produce RNA-seq data with 75 to 80% of reads coming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

Background: Tuberculosis remains a worldwide public health emergency. To better understand M. tuberculosis and to identify genomic variations characteristic to the Indian clinical isolates by a low-cost method, a genomic subtractive hybridization between M. tuberculosis H37Rv and a clinical isolate from South India was performed. Results: This revealed a novel 0.4-kb subtractive fragment which was used as a handle to pull out a 4.5-kb genomic region characteristic to the clinical isolate and was absent in H37Rv. On further studies, this 4.5-kb region was found to be present in 91% of the M. tuberculosis clinical isolates screened from Kerala, a state in South India. Interestingly, this novel region has 99% identity (with 100% query coverage) with genomic regions of M. canettii. Discussion: The present study hypothesizes that this locus was present in the recent common environmental ancestor of mycobacteria, retained to the maximum extent in M. canettii and ancestral isolates of M. tuberculosis, and later deleted in other modern lineages of M. tuberculosis. Thus, this region may serve as one of the links between the pathogenic mycobacteria and the environmental species. We also propose that the Indian isolates of M. tuberculosis might be closely related to the putative progenitor M. prototuberculosis with respect to this locus. More studies on other genomic loci from different strains of M. tuberculosis are required to establish more links in this direction. [ABSTRACT FROM AUTHOR]

On December 10, 2018, I was sitting among the big crowd of audience, as one of the invited guests to the ceremony, in the Stockholm Concert Hall. When King of Sweden Carl XVI Gustaf bestowed the diploma and medal of Nobel Prize of Physiology or Medicine 2018 on Dr. Tasuku Honjo and shook his hand for a while, surrounded by the thunderous applause and energetically blessing orchestral music, I thought that it had been a long journey for the molecule that we had first isolated in the early 1990s. Although it was truly a commemorable moment in the history of the programmed death-1 (PD-1) research, I believe we still have a long way to go. In this review article, I will explain why I think so, particularly by focusing on the potential role(s) that PD-1 appears to play in self-nonself discrimination by the immune system.

Molecular processes that either regulate ovarian atresia or are consequences of atresia are poorly understood in teleost fishes. We hypothesized that feed restriction that perturbs normal ovarian growth and induces follicular atresia would alter ovarian gene expression patterns. Previtellogenic, two-year old coho salmon ( Oncorhynchus kisutch ) were subjected to prolonged fasting to induce atresia or maintained on a normal feeding schedule that would promote continued ovarian development. To identify genes that were specifically up- or down-regulated during oocyte growth in healthy, growing fish compared to fasted fish, reciprocal suppression subtractive hybridization (SSH) cDNA libraries were generated using ovaries from fed and fasted animals. Differential expression of genes identified by SSH was confirmed with quantitative PCR. The SSH library representing genes elevated in ovaries of fed fish relative to those of fasted fish contained steroidogenesis-related genes (e.g., hydroxy-delta-5-steroid dehydrogenase ), Tgf-beta superfamily members (e.g., anti-Mullerian hormone ) and cytoskeletal intermediate filament proteins (e.g., type I keratin s8 ). Overall, these genes were associated with steroid production, cell proliferation and differentiation, and ovarian epithelialization. The library representing genes elevated in ovaries of fasted fish relative to fed fish contained genes associated with apoptosis (e.g., programmed cell death protein 4 ), cortical alveoli (e.g., alveolin ), the zona pellucida (e.g., zona pellucida protein c ), and microtubules (e.g., microtubule associated protein tau ). Elevated expression of this suite of genes was likely associated with the initiation of atresia and/or a reduced rate of follicle development in response to fasting. This study revealed ovarian genes involved in normal early secondary oocyte growth and potential early markers of atresia. [ABSTRACT FROM AUTHOR]

Background: Some plants had been used in the treatment of cancer and one of these has attracted scientific interest, the Euphorbia tirucalli (E. tirucalli), used in the treatment of asthma, ulcers, warts has active components with activities scientifically proven as antimutagenic, anti-inflammatory and anticancer. Methods: We evaluate the influence of the antitumoral fraction of the E. tirucalli latex in the larynx squamous cell carcinoma (Hep-2), on the morphology, cell proliferation and gene expression. The Hep-2 cells were cultivated in complete medium (MEM 10 %) and treated with E. tirucalli latex for 1, 3, 5 and 7 days. After statistically analyzing the proliferation of the tested cells, the cells were cultivated again for RNA extraction and the Rapid Subtractive Hybridization (RaSH) technique was used to identify genes with altered expression. The genes found using the RaSH technique were analyzed by Gene Ontology (GO) using Ingenuity Systems. Results: The five genes found to have differential expression were validated by real-time quantitative PCR. Though treatment with E. tirucalli latex did not change the cell morphology in comparison to control samples, but the cell growth was significantly decreased. The RaSH showed change in the expression of some genes, including ANXA1, TCEA1, NGFRAP1, ITPR1 and CD55, which are associated with inflammatory response, transcriptional regulation, apoptosis, calcium ion transport regulation and complement system, respectively. The E. tirucalli latex treatment down-regulated ITPR1 and up-regulated ANXA1 and CD55 genes, and was validated by real-time quantitative PCR. Conclusions: The data indicate the involvement of E. tirucalli latex in the altered expression of genes involved in tumorigenic processes, which could potentially be applied as a therapeutic indicator of larynx cancer. [ABSTRACT FROM AUTHOR]

Introduction: Current prognostic markers have improved survival prediction, however, it has not advanced treatment strategies. Gene expression profiling may identify biological markers suitable as therapeutic targets. Leukaemia stem cell is associated with adverse outcome, however, its biological characteristics are still being investigated. We observed higher in vitro cell viability in acute myeloid leukaemia (AML) samples with poor prognosis, which may be stem cell related. Objective: The objective of this study was to profile highly expressed genes in an AML sample of poor prognosis/high viability and compare with a sample of good prognosis/low viability. Method: Subtractive hybridization was performed on two AML samples with high blast counts (>80%), a poor prognosis, PP (disease free survival, DFS<12 months) and a good prognosis, GP (DFS>12 months) sample. The PP sample had higher CD34+ counts (73% vs 46%) and higher cell viability than the GP sample. cDNA libraries were subsequently cloned and sequenced. Results: cDNA subtracted from the PP samples was identified as genes active during fetal/embryonic development (LCOR, CNOT1, ORMDL1), HOX-related genes (HOXA3, PBX3, SF3B1), hematopoiesis (SELL, IL-3RA) and aerobic glycolysis/hypoxia (PGK1, HIGD1A)-associated genes. Majority of GP clones isolated contained genes involved in oxidative phosphorylation, OXPHOS (COXs, ATPs, MTND4 and MTRNR2), protein synthesis (including ribosomal proteins, initiating and elongation factors), chromatin remodeling (H2AFZ, PTMA), cell motility (MALAT1, CALM2, TMSB4X), and mitochondria (HSPA9, MPO) genes. Conclusion: Thus, the PP sample exhibited stem cell-like features while the GP sample showed cells at a high level of cell activity. These genes are potential prognostic markers and targets for therapy. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphoma (DLBCL) is more prevalent and more often fatal in HIV-infected patients and SIV-infected monkeys compared to immune-competent individuals. Molecular, biological, and immunological data indicate that virus-associated lymphomagenesis is similar in both infected hosts. To find genes specifically overexpressed in HIV/SIV-associated and non-HIV/SIV-associated DLBCL we compared gene expression profiles of HIV/SIV-related and non-HIV-related lymphomas using subtractive hybridization and Northern blot analysis. Our experimental approach allowed us to detect two genes (a-myb and pub) upregulated solely in HIV/SIV-associated DLBCLs potentially involved in virus-specific lymphomagenesis in human and monkey. Downregulation of the pub gene was observed in all non-HIV-associated lymphomas investigated. In addition, we have found genes upregulated in both non-HIV- and HIV-associated lymphomas. Among those were genes both with known (set, ND4, SMG-1) and unknown functions. In summary, we have demonstrated that simultaneous transcriptional upregulation of at least two genes (a-myb and pub) was specific for AIDS-associated lymphomas.

Cervical cancer is the second most frequent cancer in women worldwide and is associated with genetic alterations, infection with human papilloma virus (HPV), angiogenesis and inflammatory processes. The idea that inflammation is involved in tumorigenesis is supported by the frequent appearance of cancer in areas of chronic inflammation. On the other hand, the inflammatory response is controlled by the action of anti-inflammatory mediators, among these mediators, annexin A1 (ANXA1), a 37 kDa protein was detected as a modulator of inflammatory processes and is expressed by tumor cells. The study was carried out on the epithelial cancer cell line (SiHa) treated with the peptide of annexin A1 (ANXA1 Ac2-26 ). We combined subtraction hybridization approach, Ingenuity Systems software and quantitative PCR, in order to evaluate gene expression influenced by ANXA1. We observed that ANXA1 Ac2-26 inhibited proliferation in SiHa cells after 72 h. In these cells, 55 genes exhibited changes in expression levels in response to peptide treatment. Six genes were selected and the expression results of 5 up-regulated genes ( TPT1 , LDHA , NCOA3 , HIF1A , RAB13 ) and one down-regulated gene ( ID1 ) were research by real time quantitative PCR. Four more genes ( BMP4 , BMPR1B , SMAD1 and SMAD4 ) of the ID1 pathway were investigated and only one ( BMPR1B ) shows the same down regulation. The data indicate the involvement of ANXA1 Ac2-26 in the altered expression of genes involved in tumorigenic processes, which could potentially be applied as a therapeutic indicator of cervical cancer. [ABSTRACT FROM AUTHOR]

Garden asparagus ( Asparagus officinalis L.) is a dioecious species whose male and female flowers are found in separate unisexual individuals. A region called the M-locus, located on a pair of homomorphic sex chromosomes, controls sexual dimorphism in asparagus. To date, no sex determining gene has been isolated from asparagus. To identify more genes involved in flower development in asparagus, subtractive hybridization library of male flowers in asparagus was constructed by suppression subtraction hybridization. A total of 107 expressed sequence tags (ESTs) were identified. BLASTX analysis showed that the library contained several genes that could be related to flower development. The expression patterns of seven selected genes believed to be involved in the development of asparagus male flower were further analyzed by semi-quantitative or real-time reverse-transcription polymerase chain reaction (RT-PCR). Results showed that AOEST, AOEST, and AOEST were strongly expressed in the male flower stage, whereas no transcript level of AOEST was detected in the female flower stage. The expression levels of AOEST, AOEST, AOEST, and AOEST in the male flower stage were also higher than those in the female flower stage, although these transcripts were also expressed in other tissues. The identified genes can provide a strong starting point for further studies on the underlying molecular differences between the male and female flowers of asparagus. [ABSTRACT FROM AUTHOR]

The Serum- and Glucocorticoid-induced Kinase 1, SGK1, exhibits a broad range of cellular functions that include regulation of the number of ion channels in plasma membrane and modulation of signaling pathways of cell survival. This diversity of functions is made possible by various regulatory processes acting upon the SGK1 gene, giving rise to various isoforms: SGK1_v1-5, each with distinct properties and distinct aminotermini that serve to target proteins to different subcellular compartments. Among cellular effects of SGK1 expression is to indirectly modulate gene transcription by phosphorylating transcriptional factors of the FOXO family. Here we examined if SGK1.1 (SGK1_v2; NM_001143676), which associates primarily to the plasma membrane, is also able to regulate gene expression. Using a differential gene expression approach we identified six genes upregulated by SGK1.1 in HeLa cells. Further analysis of transcript and protein levels validated two genes: BCL2-associated athanogene 4 (BAG-4) and Brox. The results indicate that SGK1.1 regulates gene transcription upon a different set of genes some of which participate in cell survival pathways (BAG-4) and others in intracellular vesicular traffic (Brox). [ABSTRACT FROM AUTHOR]

Background: Salt stress is a major challenge for growth and development of plants. The mangrove tree Avicennia officinalis has evolved salt tolerance mechanisms such as salt secretion through specialized glands on its leaves. Although a number of structural studies on salt glands have been done, the molecular mechanism of salt secretion is not clearly understood. Also, studies to identify salt gland-specific genes in mangroves have been scarce. Results: By subtractive hybridization (SH) of cDNA from salt gland-rich cell layers (tester) with mesophyll tissues as the driver, several Expressed Sequence Tags (ESTs) were identified. The major classes of ESTs identified include those known to be involved in regulating metabolic processes (37%), stress response (17%), transcription (17%), signal transduction (17%) and transport functions (12%). A visual interactive map generated based on predicted functional gene interactions of the identified ESTs suggested altered activities of hydrolase, transmembrane transport and kinases. Quantitative Real-Time PCR (qRT-PCR) was carried out to validate the expression specificity of the ESTs identified by SH. A Dehydrin gene was chosen for further experimental analysis, because it is significantly highly expressed in salt gland cells, and dehydrins are known to be involved in stress remediation in other plants. Full-length Avicennia officinalis Dehydrin1 (AoDHN1) cDNA was obtained by Rapid Amplification of cDNA Ends. Phylogenetic analysis and further characterization of this gene suggested that AoDHN1 belongs to group II Late Embryogenesis Abundant proteins. qRT-PCR analysis of Avicennia showed up-regulation of AoDHN1 in response to salt and drought treatments. Furthermore, some functional insights were obtained by growing E. coli cells expressing AoDHN1. Growth of E. coli cells expressing AoDHN1 was significantly higher than that of the control cells without AoDHN1 under salinity and drought stresses, suggesting that the mangrove dehydrin protein helps to mitigate the abiotic stresses. Conclusions: Thirty-four ESTs were identified to be enriched in salt gland-rich tissues of A. officinalis leaves. qRT-PCR analysis showed that 10 of these were specifically enriched in the salt gland-rich tissues. Our data suggest that one of the selected genes, namely, AoDHN1 plays an important role to mitigate salt and drought stress responses. [ABSTRACT FROM AUTHOR]

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Papaver somniferum produces therapeutically useful benzylisoquinoline alkaloids (BIAs) like papaverine, thebaine, codeine, and morphine that accumulate in its capsular latex. Morphine is a potent analgesic but is also abused as a narcotic, which has increased the demand for non-narcotic thebaine that can be converted into various analgesics. To curtail the narcotic menace, many distinct genotypes of the plant have been developed that are deficient in morphine and/or latex. Sujata is one such latex-less low alkaloid-producing variety developed from the alkaloid-rich gum harvest variety Sampada. Its utility for gene prospecting and studying differential gene regulation responsible for its low alkaloid, nutritive seed oil, and latex-less phenotype has been exploited in this study. BIA profiling of Sujata and Sampada capsules at the early and late stages indicated that except for thebaine, Sujata had a depressed alkaloid phenotype as compared to Sampada. Comparative transcript-based analysis of the two genotypes was carried out in the early stage capsule (higher thebaine) using subtractive hybridization and microarray. Interrogation of a P. somniferum array yielded many differentially expressing transcripts. Their homology-based annotation classified them into categories-latex related, oil/lipid related, alkaloid related, cell wall related, and others. These leads will be useful to characterize the highly sought after Sujata phenotype. [ABSTRACT FROM AUTHOR]

In Ayurveda, a healthy body is defined by a balance among the three doshas ( Vata, Pitta, Kapha) and ailments result due to imbalances among them. It prescribes specific plant parts/tissues collected in a season-specific manner for curing dosha-related imbalances but the plants prescribed for treating a particular dosha imbalance belong to taxonomically diverse families and often contain similar classes of phytomolecules, making it difficult to provide a phytochemical validation for any similarity that might exist among them. This exploratory study hypothesised that plants of the same dosha-curing group may have similarity at the transcript level. For proving/disproving the hypothesis, cDNA-AFLP and specific expression subset analysis (SESA) were carried out on the Ayurveda-defined active tissues of four representative plants each of the three dosha-balancing groups. cDNA-AFLP analyses indicated that even though the plants belonging to a particular dosha-group may widely differ at the transcript level, there is a small fraction of transcripts that is monomorphic among their active tissues. SESA (Tester-active tissue cDNA; Driver-cDNA from other major tissue[s]) generated 803 subtractive ESTs from the twelve plants that yielded 150 unigenes upon assembly (of ESTs from each plant separately). Cross-plant EST assembly for plants in the same dosha group also corroborated the results. Although a distinct pattern of transcripts was not observed across all the plants in a particular dosha group, some commonalities were obtained that need further characterization towards searching for the hitherto elusive similarity among plants of the same group. [ABSTRACT FROM AUTHOR]

A locally isolated co-culture of two photosynthetic species [ Chlorella vulgaris ( Chlorophyta) and Leptolyngbya sp. ( Cyanobacteria)] displayed enhanced growth when compared to a Chlorella monoculture; however, the biological mechanisms driving such improvement are currently not well understood. To investigate these mechanisms, this study examined the differential gene expression in the Chlorella between the co-culture and the monoculture. Suppression subtractive hybridization was performed between m RNA from Chlorella in the co-culture and in a monoculture, and 105 genes were identified as being putatively differentially expressed. Nine of these genes, corresponding to the key functional categories of energy, metabolism, and protein synthesis, were further examined using quantitative real-time PCR and showed differential regulation of photosystem I and photosystem II and upregulation of stress-response genes and a gene encoding an oil-globule-associated gene in the co-culture Chlorella. This differential gene expression study of a Chlorella/cyanobacteria co-culture will aid in the development of culture strategies capable of taking advantage of these differences for the production of biomass and bioproducts of interest. Knowledge of the underlying genetic causes of the changes in growth and productivity of the species in co-culture provides insights on possible target genes for optimization of the culture. [ABSTRACT FROM AUTHOR]

Abstract: Follicle-stimulating hormone (Fsh) function in fishes is poorly understood. This study aimed to reveal Fsh-regulated genes in coho salmon previtellogenic ovarian follicles in vitro. Four suppression subtractive hybridization libraries were generated with RNA isolated from Fsh-treated and control follicles or follicle cell-enriched tissue fractions. Fsh induced steroidogenesis and dynamically upregulated several genes predominantly expressed in follicle cells, including WAP domain-containing protease, connexin 34.3, clusterin (clu1, clu2), fibronectin, wilms tumor 2-like, and influenza virus NS1A-binding protein a. Genes downregulated by Fsh included connective tissue growth factor, alcohol dehydrogenase 8-like, and serine/threonine-protein kinase pim-1. This study demonstrates for the first time in fishes that Fsh influences the expression of a unique suite of ovarian genes involved in processes like cell communication, survival and differentiation, and extracellular matrix remodeling. Collectively, these findings suggest that Fsh and/or steroids induce differentiation of granulosa cells and remodeling of the follicle in preparation for onset of vitellogenesis. [Copyright &y& Elsevier]

Abstract: Citrus is one of the most economically important fruit crops growing in subtropical and tropical regions. Most commercially important Citrus varieties are susceptible to cold; therefore, low and freezing temperatures are the main limiting factors for citrus production in subtropical areas. Since Poncirus trifoliata (L.) Raf. is a cold-hardy, interfertile Citrus relative, it serves as a genetic resource for improving cold tolerance in cold sensitive commercial Citrus species. While gene induced in response to long-term cold acclimation was previously identified in Poncirus, early response of Poncirus to cold has not been explored in detail. To identify early cold-responsive genes, a subtractive cDNA library was constructed using 4-h cold-treated and untreated control Poncirus seedlings in this study. A total of 210 randomly picked clones from the subtracted library with cold-induced genes were sequenced. The sequences obtained from the majority of these clones shared homology with previously identified cold-induced and/or environmental stress-regulated genes in other plants. Reverse northern blot analysis of the expression of these cDNAs with cold-treated and untreated control probes revealed that expression of 64 cDNAs was increased two to 11 fold in response to 4-h cold treatment. While the majority of these genes were related with cell rescue, defense, cell death and aging, transcription, metabolism, protein fate, energy, cellular communication and signal transduction, transport facilitation and development, some of them did not show homology with genes with known functions. Individual expression analysis of nine selected genes by semi-quantitative RT-PCR using mRNA from cold-treated and untreated control plants confirmed that the expression of selected cDNAs was all induced in response to cold. The results demonstrated that although a few genes were commonly induced in response to both short and long-term cold acclimation in Poncirus, majority of early cold-responsive genes were different from previously identified late cold-responsive genes in Poncirus. [Copyright &y& Elsevier]

Drought is the most crucial environmental factor that limits productivity of many crop plants. Exploring novel genes and gene combinations is of primary importance in plant drought tolerance research. Stress tolerant genotypes/species are known to express novel stress responsive genes with unique functional significance. Hence, identification and characterization of stress responsive genes from these tolerant species might be a reliable option to engineer the drought tolerance. Safflower has been found to be a relatively drought tolerant crop and thus, it has been the choice of study to characterize the genes expressed under drought stress. In the present study, we have evaluated differential drought tolerance of two cultivars of safflower namely, A1 and Nira using selective physiological marker traits and we have identified cultivar A1 as relatively drought tolerant. To identify the drought responsive genes, we have constructed a stress subtracted cDNA library from cultivar A1 following subtractive hybridization. Analysis of ~1,300 cDNA clones resulted in the identification of 667 unique drought responsive ESTs. Protein homology search revealed that 521 (78 %) out of 667 ESTs showed significant similarity to known sequences in the database and majority of them previously identified as drought stress-related genes and were found to be involved in a variety of cellular functions ranging from stress perception to cellular protection. Remaining 146 (22 %) ESTs were not homologous to known sequences in the database and therefore, they were considered to be unique and novel drought responsive genes of safflower. Since safflower is a stress-adapted oil-seed crop this observation has great relevance. In addition, to validate the differential expression of the identified genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), and northern blot analysis. We showed that these clones were differentially expressed under different abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance are discussed in our study. [ABSTRACT FROM AUTHOR]

The isolation of unknown DNA sequences is an important task in molecular biology research. The subtractive hybridization by magnetic beads technique reduces time and cost when the aim of the study is to isolate new genes. We have developed an alternative strategy that combines the subtractive hybridization method with an enrichment microsatellite strategy to isolate a de novo DNA sequence of the phaC gene involved in the polymerization of polyhydroxyalkanoates. A 113-bp fragment was developed as a probe and, based on its melting temperature calculated using the nearest-neighbor model, used to isolate the full-length phaC gene (GenBank Accession number HM026759) of the marine bacteria Paracoccus homiensis strain E33, which was isolated from polluted microbial mats. [ABSTRACT FROM AUTHOR]

Pichia fermentans (strain DISAABA 726) is an effective biocontrol agent against Monilinia fructicola and Botrytis cinerea when inoculated in artificially wounded apple fruit but is an aggressive pathogen when inoculated on wounded peach fruit, causing severe fruit decay. Pichia fermentans grows as budding yeast on apple tissue and exhibits pseudohyphal growth on peach tissue, suggesting that dimorphism may be associated with pathogenicity. Two complementary suppressive subtractive hybridization ( SSH) strategies, that is, rapid subtraction hybridization ( Ra SH) and PCR-based subtraction, were performed to identify genes differentially expressed by P. fermentans after 24-h growth on apple vs. peach fruit. Gene products that were more highly expressed on peach than on apple tissue, or vice versa, were sequenced and compared with available yeast genome sequence databases. Several of the genes more highly expressed, when P. fermentans was grown on peach, were related to stress response, glycolysis, amino acid metabolism, and alcoholic fermentation but surprisingly not to cell wall degrading enzymes such as pectinases or cellulases. The dual activity of P. fermentans as both a biocontrol agent and a pathogen emphasizes the need for a thorough risk analysis of potential antagonists to avoid unpredictable results that could negatively impact the safe use of postharvest biocontrol strategies. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is the causative agent of melioidosis. We initiated this investigation with a virulent and an attenuated strain of B. pseudomollei. Pulsed-field gel electrophoresis was carried out initially for macrogenomic com- parison of both strains of B. pseudomal!ei. However, the pulsotypes obtained were identical and therefore we applied a subtractive hybridization technique to distinguish and determine the possible differences between the two strains. Six virulence strain-specific DNA fragments were obtained and the encoding homolog proteins were identified as a xenobiotic-responsive element family of transcriptional regulator, a hypothetical protein, an unknown protein, a plasmid recombination enzyme, a regulatory protein and a putative hemolysin activator protein. A combination of at least three of these determinants was identified in 45 clinical isolates when screening was carried out with self-designed multiplex PCR targeting the six putative virulent determinants. Our data demonstrated that different combinations of the six putative virulence genes were present in the clinical isolates indicating their probable role in the pathogenesis of B. pseudomallel infections. [ABSTRACT FROM AUTHOR]

Electric organs (EOs) have evolved independently in vertebrates six times from skeletal muscle (SM). The transcriptional changes accompanying this developmental transformation are not presently well understood. Mormyrids and gymnotiforms are two highly convergent groups of weakly electric fish that have independently evolved EOs: while much is known about development and gene expression in gymnotiforms, very little is known about development and gene expression in mormyrids. This lack of data limits prospects for comparative work. We report here on the characterization of 28 differentially expressed genes between SM and EO tissues in the mormyrid Brienomyrus brachyistius, which were Identified using suppressive subtractive hybridization (SSH). Forward and reverse SSH was performed on tissue samples of EO and SM resulting in one cDNA library enriched with mRNAs expressed in EO, and a second library representing mRNAs unique to SM. Nineteen expressed sequence tags (ESTs) were identified in EO and nine were identified in SM using BLAST searching of Danio rerio sequences available in NCBI databases. We confirmed differential expression of all 28 ESTs using RT-PCR. In EO, these ESTs represent four classes of proteins: (1) ion pumps, including the &agr;- and &bgr;-subunits of Na+/K+-ATPase, and a plasma membrane Ca2+-ATPase; (2) Ca2+-binding protein S100, several parvalbumin paralogs, calcyclin-binding protein and neurogranin; (3) sarcomeric proteins troponin I, myosin heavy chain and actin-related protein complex subunit 3 (Arcp3); and (4) the transcription factors enhancer of rudimentary homolog (ERH) and myocyte enhancer factor 2A (MEF2A). Immunohistochemistry and western blotting were used to demonstrate the translation of seven proteins (myosin heavy chain, Na+/K+-ATPase, plasma membrane Ca2+-ATPase, MEF2, troponin and parvalbumin) and their cellular localization in EO and SM. Our findings suggest that mormyrids express several paralogs of muscle-specific genes and the proteins they encode in EOs, unlike gymnotiforms, which may post-transcriptionally repress several sarcomeric proteins. In spite of the similarity in the physiology and function of EOs in mormyrids and gymnotiforms, this study indicates that the mechanisms of development in the two groups may be considerably different. [ABSTRACT FROM AUTHOR]

Penicillium purpurogenum is a filamentous ascomycete which grows on a variety of natural carbon sources, among them sugar beet pulp, and secretes a large variety of cellulolytic and hemicellulolytic enzymes into the culture medium. The purpose of this work was to analyse the difference in the expression of genes when those transcribed in a medium with glucose as carbon source (a highly repressive substrate) are subtracted from those expressed when it is grown on sugar beet pulp. A subtractive cDNA library was constructed by means of suppression subtractive hybridization. In all, 101 clones were selected and 80 were found to have inserts of cDNA differentially expressed in sugar beet pulp. These cDNAs were sequenced and were queried for similarities with BLAST. Sixty-two unique expressed sequence tags (ESTs) of interest were obtained. Of them, 58% are unidentified and 5% are unclassified. The remaining sequences correspond to: 18% metabolism, 5% gene/protein expression, 5% cell/organism defence, 6% cell signalling/cell communication and 3% cell division. The complete sequences of the genes and cDNAs of two of the ESTs was performed; one of them (EST 19) codes for a putative transcriptional regulator and the other (EST 182) for a putative DNA repair protein. [ABSTRACT FROM AUTHOR]

Abstract: The potential impact of commercial salmon aquaculture along the coast of British Columbia on the health of non-target marine wildlife is of growing concern. In the current initiative, the biological effects on gene expression within spot prawn (Pandalus platyceros) exposed to the sea lice controlling agent, emamectin benzoate (EB; 0.1–4.8mg/kg sediment), were investigated. A mean sediment/water partitioning coefficient (K p) was determined to be 21.81 and significant levels of EB were detected in the tail muscle tissue in all exposed animals. Animals selected for the experiment did not have eggs and were of similar weight. Significant mortality was observed within 8 days of EB treatment at concentrations between 0.1 and 0.8mg/kg and there was no effect of EB on molting. Twelve spot prawn cDNA sequences were isolated from the tail muscle either by directed cloning or subtractive hybridization of control versus EB exposed tissues. Three of the transcripts most affected by EB exposure matched sequences encoding the 60S ribosomal protein L22, spliceosome RNA helicase WM6/UAP56, and the intracellular signal mediator histidine triad nucleotide binding protein 1 suggesting that translation, transcription regulation, and apoptosis pathways were impacted. The mRNA encoding the molting enzyme, β-N-acetylglucosaminidase, was not affected by EB treatment. However, the expression of this transcript was extremely variable making it unsuitable for effects assessment. The results suggest that short-term exposure to EB can impact biological processes within this non-target crustacean. [Copyright &y& Elsevier]

Abstract: The potential of using a QCDCR (quilA:cholesterol:dimethyl dioctadecyl ammonium bromide:carbopol:R1005 glycolipid) formulated CpG oligodeoxynucleotide (ODN), ODN 2007, to confer protection in Nile tilapia against Streptococcus iniae infection was evaluated in this study. At two days post treatment, QCDCR formulated ODN 2007 elicited significant (P <0.05) protection to Nile tilapia, with relative percent survival of 63% compared to fish treated by QCDCR alone. To understand the molecular mechanisms involved in the protective immunity elicited by ODN 2007, suppression subtractive cDNA hybridization technique was used to identify upregulated genes induced by ODN 2007. A total of 69 expressed sequence tags (ESTs) were identified from the subtractive cDNA library. Quantitative PCR revealed that 44 ESTs were significantly (P <0.05) upregulated by ODN 2007, including 29 highly (>10-fold) and 15 moderately (<10-fold) upregulated ESTs. Of all ESTs, putative peroxisomal sarcosine oxidase was upregulated the highest. The 69 ESTs only included six genes that had putative functions related to immunity, of which only two (putative glutaredoxin-1 and carboxypeptidase N catalytic chain) were confirmed to be significantly upregulated. Our results suggest that the protection elicited by ODN 2007 is mainly through innate immune responses directly or indirectly related to immunity. [Copyright &y& Elsevier]

Drought is one of the major abiotic stresses limiting crop productivity in arid and semi-arid regions and influences many aspects of plant development. Groundnut ( Arachis hypogaea L.) is an important oil yielding crop and considered as relatively drought tolerant. In this study, two groundnut cultivars were first tested for their drought tolerance based on physiological marker attributes such as relative water content, total chlorophyll content, cell membrane stability and free proline content and identified cultivar K-134 as a drought tolerant and cultivar JL-24 as drought susceptible. To gain a better understanding of the drought stress responses at molecular level, we carried out a genomic analysis of stress-responsive genes/transcripts in drought-tolerant cultivar K-134. As a first step toward characterization of stress-responsive genes, construction and analysis of subtracted cDNA library from drought-tolerant cultivar (K-134) is reported here. Using this strategy a total of 200 ESTs were isolated, sequenced, of which 120 high-quality ESTs were obtained and clustered. Further, our analysis revealed that 31% sequences were unique and no homology to known proteins in the database. This observation has great relevance since groundnut is a stress-adapted legume crop. Further, to validate the identified differentially expressed genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), northern blot analysis. We showed that these clones are differentially expressed under different abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance were discussed. [ABSTRACT FROM AUTHOR]

Abstract: To understand the molecular mechanisms involved in response of Nile tilapia (Oreochromis niloticus) to bacterial infection, suppression subtractive cDNA hybridization technique was used to identify upregulated genes in the posterior kidney of Nile tilapia at 6h post infection with Aeromonas hydrophila. A total of 31 unique expressed sequence tags (ESTs) were identified from 192 clones of the subtractive cDNA library. Quantitative PCR revealed that nine of the 31 ESTs were significantly (p <0.05) upregulated in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 105 CFU per fish (∼20% mortality). Of the nine upregulated genes, four were also significantly (p <0.05) induced in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 106 CFU per fish (∼60% mortality). Of the four genes induced by A. hydrophila at both injection doses, three were also significantly (p <0.05) upregulated in Nile tilapia at 6h post infection with Streptococcus iniae at doses of 106 and at 105 CFU per fish (∼70% and ∼30% mortality, respectively). The three genes induced by both bacteria included EST 2A05 (similar to adenylate kinase domain containing protein 1), EST 2G11 (unknown protein, shared similarity with Salmo salar IgH locus B genomic sequence with e value of 0.02), and EST 2H04 (unknown protein). Significant upregulation of these genes in Nile tilapia following bacterial infections suggested that they might play important roles in host response to infections of A. hydrophila and S. iniae. [Copyright &y& Elsevier]

A high content of noncoding RNA in the total bacterial RNA significantly impedes analysis of their transcriptome using standard approaches, i.e., full-scale sequencing, analysis of gene expression profiles, and subtractive hybridization. A method of preparation of bacterial cDNA for transcriptome analysis is proposed that includes a depletion of the excess of rRNA and tRNA copies with the preservation of the relative abundance of transcripts. The method is based on the kinetics of DNA hybridization and the unique features of duplex-specific nuclease from the Kamchatka crab. The efficiency of the method was confirmed in a number of model experiments. [ABSTRACT FROM AUTHOR]

Abstract: Ovarian cancer cells are usually initially sensitive to platinum-based chemotherapy, such as cisplatin (CDDP), but typically become resistant over time. Such drug resistance is a serious impediment to successful disease treatment, and the molecular mechanisms responsible for resistance are not fully understood. In search of novel mechanisms that may lead to the development of CDDP chemoresistance, we used subtractive hybridization to identify differentially expressed genes in CDDP resistant CP70 andC200 cells vs. CDDP sensitive A2780 human ovarian adenocarcinoma cells. We analyzed 256 randomly selected clones. Subtraction efficiency was determined by dot blot and DNA sequencing. Confirmation of differentially expressed cDNAs was done by virtual northern blot analysis, and 17 genes that were differentially expressed in CDDP resistant cell lines vs. CDDP sensitive A2780 cells were identified. The expression of 10 of these genes was low or undetectable in sensitive A2780 cells in comparison to resistant cells and an additional seven genes were more highly expressed in resistant CP70 and C200 vs. A2780 cells. Our identified genes are involved in numerous and diverse cellular processes, such as inhibition of apoptosis (ARHGDIB), stress response (HSPCA, TRA1), chromatin condensation (CNAP1, RanBP2), invasiveness of cells (MMP10), alteration of Ca2+ homeostasis (ASPH, ATP2B1) and others. Further characterization of these genes and gene products should yield important insights into the biology of CDDP resistance in ovarian carcinoma. [Copyright &y& Elsevier]

Abstract: Tomato leaf curl viruses (ToLCV) infect tomato plants and eventually cause several phenotypic defects, notably in the leaves in the form of upward curling. The entry of virus triggers plants’ basal defense responses which eventually introduce temporal changes in the transcriptome to evade the pathogen attack. In this study, we have identified about 20 tomato ESTs using subtractive hybridization that were induced in tomato leaves upon agro-infection with the constructs bearing the dimers of Tomato leaf curl New Delhi virus (ToLCNDV) DNA-A and DNA-B components. The induced ESTs belonged to the class of genes that play crucial roles in innate immunity, plants metabolism and ethylene signaling. The expression of few of these ESTs was validated by northern blot analysis and two out of six selected genes expressed exclusively in the infected leaf tissues. Besides leaves, the expression status of selected genes was checked in a wide variety of tissues (flower, fruit, stem and root) of both healthy and infected plants by RT-PCR. These results suggest that the flower and fruit tissues, similar to leaves, exhibited induction of most of the genes while the stem and root tissues suffered from down-regulation. Overall, these results indicate that the hosts’ transcriptome undergoes considerable changes in response to viral infection. [Copyright &y& Elsevier]

Abstract: Verticillium dahliae is the main pathogen causing Verticillium wilt in potato. Management of this vascular disease is very challenging due to the soilborne nature of the pathogen. A better understanding of the molecular host–pathogen interactions is important for the development of novel strategies to control Verticillium wilt. In this pathosystem, the disease cycle starts with stimulation and germination of the V. dahliae microsclerotia through host root exudates. The present study reports on the use of potato root extracts derived from a susceptible (Kennebec) and a moderately resistant (Ranger Russet) cultivar to elicit pathogenicity-related genes in highly and weakly aggressive isolates of V. dahliae. Using a combined approach of subtractive hybridization and cDNA-AFLP, 573 transcripts differentially accumulated in one or the other isolate in response to root extracts were detected. Sixteen primer combinations representing EcoRI/MseI AFLP primers+A, T, C, or G were used to provide a complete coverage of the subtractive hybridization products. The detected differentially expressed transcripts in the highly and weakly aggressive isolates were 301 and 272, respectively. Among the amplified transcripts, 185 were recovered from the PAGE gel then re-amplified by PCR and further sequenced. BLAST search against the NCBI, the Broad Institute V. dahliae genome, and V. dahliae ESTs collection COGEME databases showed that some of the differentially expressed transcripts matched with known sequences, with assigned functions in V. dahliae such as polygalacturonases or with conserved hypothetical proteins. The remaining sequences had no match in these databases. The results are discussed based on the potential involvement of these genes in V. dahliae''s pathogenesis. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Mulberry, the backbone of sericulture industry, is a rainfed crop, and its biomass production is affected adversely under drought conditions. In this study, genes expressed differentially during drought stress response have been examined by PCR-Select subtractive hybridization. The sensitive and tolerant genotypes were identified based on physiological evaluation by determination of proline content, electrolyte leakage, and measurement of relative water content. In total, 1,920 clones were sequenced, representing 208 contigs and 151 singletons. The expressed sequence tags generated from this subtracted cDNA library comprises a broad repertoire of stress-responsive genes, which contribute to the process of drought tolerance in mulberry. Additionally, 23% of the cDNA library is represented by transcripts of unknown function. The expression of a select number of these drought-inducible genes was studied based on cDNA macroarray and Northern blot analyses. In order to unravel the crosstalk with other abiotic stresses, expression profile of Arabidopsis homologs of selected genes in response to a wide range of different stresses was studied using Genevestigator as a reference expression database. The results of this study show that subtractive hybridization coupled with validation steps for differential screening is an effective method for identification of stress/drought-induced genes in plants with limited sequence information available. [ABSTRACT FROM AUTHOR]

The brine shrimp, Artemia, reproduces either oviparously, producing encysted embryos (diapause cysts), or ovoviviparously, producing free-swimming nauplii. Environmental factors, such as photoperiod, have been applied to control the reproduction mode of Artemia, but when the determination of a reproductive mode occurs remains unknown. We analysed the differential gene expression between oocytes from oviparous and ovoviviparous Artemia reared under different photoperiods. A total of 692 qualified cDNA clones were obtained by subtractive hybridization, 327 of which matched GenBank® Nucleotide Sequence Database entries. Gene expressions of 44 cDNAs (representing 56 clones) were analysed in oocytes using real-time PCR. Among these genes, 11 (21 clones) were significantly (P<0.05) upregulated and 7 (9 clones) down-regulated in Artemia oocytes that subsequently enter diapause. Remarkably, known diapause-related proteins such as ArHsp22 (Artemia heat-shock protein 22) and chitin-binding proteins are found to be already differentially expressed. Furthermore, RNAi (RNA interference) knockdown of a differentially expressed gene, polo-like kinase 1, in oocyte of ovoviviparous Artemia led to the production of white embryos rather than free-swimming nauplii. In summary, our results provide evidence at the molecular level that the reproductive mode of Artemia is already determined at the oocyte stage of their life cycle. [ABSTRACT FROM AUTHOR]

We report identification of putative innate immune related genes from a cell line of the mosquito Aedes albopictus challenged with heat-killed bacteria. Using a subtractive hybridization and sequencing approach, we analyzed a total 309 expressed sequence tags (ESTs) which clustered in 40 contigs. Thirty-five percent of genes yielded homology to known immune genes corresponding to antimicrobial peptides (AMPs), pathogen-associated molecular patterns, protease and immune signaling cascades. Interestingly, most of the genes have not been previously described from this mosquito and thus represent a class of novel immune genes. Further, 25% sequences did not match to any known species in the non-redundant databases, appear to be specific to the mosquito A. albopictus and merit further study. [ABSTRACT FROM AUTHOR]

The harvesting and storing of alfalfa is a routine practice in the agricultural industry worldwide. To investigate gene expression in harvested alfalfa, cDNA from non-harvested and harvested plants in the field was subjected to subtractive hybridization to identify, in particular, those genes that are induced by the harvesting treatment. One cDNA clone, named hi11, was isolated and analysed. The full length cDNA of the hi11 gene was cloned by RACE amplification. The hi11 gene, which has high homology to a putative protein of unknown function in Arabidopsis, was induced in alfalfa following harvesting, a 38°C heat shock and a wounding treatment. Northern blot analysis confirmed that the expression patterns of hi11 in alfalfa in response to harvesting, heat shock, and wounding. In addition, genomic walking was performed to isolate the 5′ flanking region of the hi11 gene. The promoter of the hi11 gene was fused to the GUS reporter gene and transferred to Medicago truncatula and tobacco. In all transgenic plants of M. truncatula and tobacco, GUS gene expression was observed in harvested tissue, especially in the transgenic tobacco plants, but not in the non-harvested control tissue. [ABSTRACT FROM AUTHOR]