Sulfenylated proteins in the Medicago truncatula-Sinorhizobium meliloti symbiosis

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)), play a crucial role as signaling molecules in the establishment and functioning of the nitrogen-fixing legume-Rhizobium symbiosis. The regulation of protein function through oxidative modification has emerged as an important molecular mechanism modulating various biological processes. Protein cysteine residues are known to be sensitive targets of H(2)O(2), in a posttranslational modification called sulfenylation. We trapped and identified sulfenylated proteins in the Medicago truncatula-Sinorhizobium meliloti symbiosis, by combining the use of chemical and genetic probes with mass spectrometry analysis. We identified 44 M. truncatula proteins sulfenylated in inoculated roots (two days post infection, 2dpi) and 65 such proteins in the functioning symbiotic organ, the nodule (four weeks post infection, 4wpi); 18 proteins were identified at both time points. However, the largest functional groups at 2dpi and 4wpi were different: redox state-linked proteins early in the interaction and proteins involved in amino-acid and carbohydrate metabolism in the nodule. Twenty proteins from S. meliloti, including some directly involved in nitrogen fixation, were also identified as sulfenylated. These results suggest that sulfenylation may regulate the activity of proteins playing major roles in the development and functioning of the symbiotic interaction.     

Nitric oxide is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiosis

Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure. Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia. Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process. Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula-S. meliloti symbiotic interaction.     

Phosphate concentration alters the effective bacterial quorum in the symbiosis of Medicago truncatula-Sinorhizobium meliloti

The symbiosis of Medicago truncatula-Sinorhizobium meliloti is affected by phosphate (P) deficiency in the environment. Quorum sensing (QS) is a regulatory pathway in S. meliloti that controls various functions of free-living and symbiotic bacteria in response to phosphate availability and regulation is mediated by a periplasmic protein PstS, and also bacterial density. The quorum sensing pathway of S. meliloti, involves three genes named sinI, sinR and expR and also some bacterial auto-inducers such as N-acyl homoserine lactones (AHLs). In the current study, the expression of the different genes of quorum sensing and pstS were evaluated under 0.1, 0.5 and 2 mM P. The qRT-PCR results showed an increased expression of pstS and also the quorum sensing genes sinI and sinR but not expR, following phosphate starvation. Indeed, the enhanced level of sinR induces the expression of sinI that is responsible for the N-acyl homoserine lactones (AHL) production in S. meliloti. The different response of expR may be due to its negative control on sinR expression. In the symbiosis of M. truncatula-S. meliloti, it was shown that the concentration of phosphate in the medium alters the effective inoculating bacterial quorum (density). By increasing the phosphate concentration in the medium from 0.1 to 0.5 and 2 mM, considering the optimal plant growth and pink nodule (nitrogen-fixing) formation, the effective inoculating bacterial densities were 10⁵, 10⁷ and 10⁹ CFU ml^?1, respectively. Therefore, low phosphate concentrations can compensate for a low bacterial density by inducing the quorum sensing pathway and establishing a symbiosis. Conversely, bacterial density plays the main role in the formation of symbiosis at high phosphate concentrations.     

Identification of genes associated with fruit ripening in Ziziphus jujuba using suppression subtractive hybridization approach

Owing to the high nutritional value and extensive medicinal use of its products, Chinese jujube (Ziziphus jujuba Mill) is one of the most important fruit crops in China. However, jujube fruits are highly perishable and thus have a short shelf life, which is a serious hindrance to the industry. Better understanding of the molecular mechanisms underlying jujube fruit softening is fundamental to overcome the problem. Thus, both forward and reverse suppression subtractive hybridization (SSH) cDNA libraries were constructed to identify differentially expressed genes for fruit at half-red ripening stage and complete red stage. As a result of dot blot confirmation, a total of 154 differentially expressed genes were identified. After removed low-quality regions and screened for vector contamination, blasted with the non-redundant NCBI databases, 78.6 % of sequences exhibited high homology to previously identified or putative proteins. All the ESTs were annotated and classified according to the terms of the three main Gene Ontology vocabularies using the Blast2GO software. Furthermore, the quantitative real-time PCR was carried out for 17 genes to validate the genes differentially expressed from the SSH libraries. And the full-length sequences of galactose oxidase and aldehyde dehydrogenase genes were obtained. It is the first step to explore the functional genomics and regulatory networks during the storage period of jujube fruit. The identification of the genes differentially expressed is helpful to understand the ripening and softening of the jujube fruit at the molecular level.     

Identification of a pheA gene associated with Streptococcus mitis by using suppression subtractive hybridization

We performed suppression subtractive hybridization to identify genomic differences between Streptococcus mitis and Streptococcus pneumoniae. Based on the pheA gene, a primer set specific to S. mitis detection was found in 18 out of 103 S. mitis-specific clones. Our findings would be useful for discrimination of S. mitis from other closely related cocci in the oral environment.     

Identification of immunostimulatory DNA-induced genes by suppression subtractive hybridization

Bacterial DNA and related synthetic immunostimulatory oligodeoxyribo-nucleotides (ISS-ODN) have stimulatory effects on mammalian immune cells through a Toll-like receptor, TLR9. Genes upregulated in ISS-ODN-stimulated immune cells are obviously significant to delineate the mechanism of the induced innate immunity. Employing suppression subtractive hybridization (SSH), we have generated a profile of genes induced by ISS-ODN in spleen cells. Sequencing of 87 clones isolated by the SSH showed 39 clones corresponding to known mouse genes in the public database. Eleven clones appeared to possess 80-90% homology with known mouse genes and the remaining 37 clones showed no significant homology with any known mouse genes. A series of known genes which have not previously been reported to be induced with ISS-ODN were confirmed to be induced in ISS-ODN-stimulated bone marrow-derived macrophages: NF-kappaB p105, IRF-1, PA28beta, IRG2, and MyD88. These genes were suggested to be involved in the molecular process of innate host defense mechanisms.     

Stolbur phytoplasma genome survey achieved using a suppression subtractive hybridization approach with high specificity

Phytoplasmas are unculturable bacterial plant pathogens transmitted by phloem-feeding hemipteran insects. DNA of phytoplasmas is difficult to purify because of their exclusive phloem location and low abundance in plants. To overcome this constraint, suppression subtractive hybridization (SSH) was modified and used to selectively amplify DNA of the stolbur phytoplasma infecting a periwinkle plant. Plasmid libraries were constructed, and the origins of the DNA inserts were verified by hybridization and PCR screenings. After a single round of SSH, there was still a significant level of contamination with plant DNA (around 50%). However, the modified SSH, which included a second round of subtraction (double SSH), resulted in an increased phytoplasma DNA purity (97%). Results validated double SSH as an efficient way to produce a genome survey for microbial agents unavailable in culture. Assembly of 266 insert sequences revealed 181 phytoplasma genetic loci which were annotated. Comparative analysis of 113 kbp indicated that among 217 protein coding sequences, 83% were homologous to "Candidatus Phytoplasma asteris" (OY-M strain) genes, with hits widely distributed along the chromosome. Most of the stolbur-specific SSH sequences were orphan genes, with the exception of two partial coding sequences encoding proteins homologous to a mycoplasma surface protein and riboflavin kinase.     

Identification of differentially expressed genes in a porcine in vivo model of adipogenesis using suppression subtractive hybridization

Although they provide valuable information, in vitro models of adipocyte development often require high doses of hormones and growth factors, which may influence gene expression and adipocyte differentiation patterns. To overcome these problems, a novel in vivo model of adipose tissue development was used to characterize genes involved in adipogenesis. The suppression subtractive hybridization technique was used to identify genes showing differential expression between the adipose tissue of a day 90 gestating sow, which is enriched in adipocytes, and day 90 fetal adipose tissue, which is enriched in preadipocytes. A total of 149 expressed sequence tags corresponding to identified genes and tentative consensus sequences emerged. Thirty-seven clones matched expressed sequence tags or genomic DNA sequences and six novel sequences were also identified. Adipogenesis-related genes were identified, many of which have never been reported to be expressed in mammalian adipose tissue, and may play a role in regulation of adipose tissue differentiation. Validation of differentially expressed genes was confirmed for perilipin, monocyte to macrophage differentiation-associated, myocilin, paraoxonase 3, stearoyl-CoA desaturase, angiotensinogen and adiponectin genes using real-time RT-PCR.     

Identification of genes preferentially expressed in goat hair follicle anagen-catagen transition using suppression subtractive hybridization

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes in goat (Capra hircus) hair follicle anagen-catagen transition. The cDNA fragments, derived from SSH positive subtractive library (tester: anagen-catagen transition, driver: later anagen), were cloned into pEGM-T vector. Two hundred cDNA fragments screened from this library were subjected to identify forty-five unregulated isolates. Sequence analysis revealed that these fragments represented twenty-three genes. Blasting analysis with database in GenBank showed that twenty genes were previously clearly annotated, two were homologous to un-annotated expressed sequence tag (ESTs), and one might be novel. To identify characters of gene expression, seven genes in later anagen and anagen-catagen transition skin tissues were chosen for quantitative real-time PCR. Results indicated that expression of these seven genes varied much, reaching threefold among them, furthering indicating that expression of those genes was up-regulation in the anagen-catagen transition. We characterized expression levels of this potential novel gene and the goat ectodysplasin A during differential stages of hair cycle. These profiles suggested that these two genes might play a role in the goat secondary hair follicle cycle.     

Identification of differentially regulated genes of Plasmodium by suppression subtractive hybridization

Plasmodium, the causative agent of malaria, has many morphologically and functionally distinct developmental stages. In the mosquito host alone, there are five transitions during the development of a gametocyte into a sporozoite. Determining which genes are expressed at the different developmental stages is vital to our understanding of the parasite. There are a growing number of techniques designed to study gene expression, including microarray. Here, Johannes Dessens, Gabrielle Margos, Maria del Carmen Rodriguez and Robert Sinden describe a novel method: suppression subtractive hybridization (SSH) and its successful application in obtaining mosquito midgut stage-specific genes of Plasmodium.     

Identification of a novel gene, HCR2, in aorta of hypercoagulable rats using suppression subtractive hybridization

Screening and identification of novel genes involved in hypercoagulable state (HCS) is important in understanding the underlying mechanisms of development of atheroslerosis, which implicated in critical vascular diseases such as coronary heart disease and stroke. HCS was induced in rats with high carbohydrate diet. Subtractive hybridization experiments between aorta tissues from hypercoagulable rats and controls showed that a novel cDNA (GenBank Accession No. AY234417), designated as hypercoagulability related gene-2 (HCR2), was highly expressed in aorta tissue. The predicted protein encoded by HCR2 contains 78 amino acids, which has a theoretical isoelectric point (pI) of 8.59 and molecular weight (MW) of 8841.7 based on sequence analysis. Our data suggest that HCR2 may be involved in HCS and identification of the gene may contribute to our understanding of the mechanisms for the production of a hypercoagulable state of several critical clinical disorders.     

SSHSuite: an integrated software package for analysis of large-scale suppression subtractive hybridization data

Suppression subtractive hybridization (SSH) is a widely used technique for the identification of differentially expressed genes. SSH as well as other types of sequencing projects generate large amounts of anonymous sequences. SSHSuite automates the handling and storage of these sequences and enables identification through similarity searches. SSHSuite also offers analysis tools for the retrieval and comparison of the resulting similarity data. SSHSuite consists of four programs: SSHHandler, SSHOverview, SSHAnalysis, and SSHCompare.     

Identification and characterization of hypoxia-induced genes in Carassius auratus blastulae embryonic cells using suppression subtractive hybridization

Organisms living in water are inevitably exposed to periods of hypoxia. Environmental hypoxia has been an important stressor having manifold effects on aquatic life. Many fish species have evolved behavioral, physiological, biochemical and molecular adaptations that enable them to cope with hypoxia. However, the molecular mechanisms of hypoxia tolerance in fish, remain unknown. In this study, we used suppression subtractive hybridization to examine the differential gene expression in CAB cells (Carassius auratus blastulae embryonic cells) exposed to hypoxia for 24 h. We isolated 2100 clones and identified 211 differentially expressed genes (e-value ≤ 5e? 3; Identity > 45%). Among the genes whose expression is modified in cells, a vast majority involved in metabolism, signal transduction, cell defense, angiogenesis, cell growth and proliferation. Twelve genes encoding for ERO1-L, p53, CPO, HO-1, MKP2, PFK-2, cystatin B, GLUT1, BTG1, TGFβ1, PGAM1, hypothetical protein F1508, were selected and identified to be hypoxia-induced using semi-quantitive RT-PCR and real-time PCR. Among the identified genes, two open reading frames (ORFs) encoding for CaBTG1 and Cacystatin B were obtained. The deduced amino acid sequence of CaBTG1 had 94.1%, 72.8%, 72.8%, 72.8%, 68.6% identity with that of DrBTG1, HsBTG1, BtBTG1, MmBTG1 and XlBTG1. Comparison of Cacystatin B with known cystatin B, the molecules exhibited 49.5 to 76.0% identity overall. These results may provide significant information for further understanding of the adaptive mechanism by which C. auratus responds to hypoxia.     

Identification of up-regulated genes in human uterine leiomyoma by suppression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.     

Identification of differentially expressed genes in human heart with ventricular septal defect using suppression subtractive hybridization

Ventricular septal defect (VSD) accounts for the largest number of birth congenital heart defects in human, but the genetic programs that control ventricular septation are poorly understood. To identify differentially expressed genes between ventricular septal defect and normal ventricular septum myocardium, we have undertaken suppression subtractive hybridization (SSH) and generated reciprocal cDNA collections of representative mRNAs specific to human heart with ventricular septal defect versus normal control. Following SSH, 1378 clones were sequenced and found to derive from 551 different genes. These predominately expressed genes included genes involved in energy metabolism, cell cycle and growth, cytoskeleton and cell adhesion, LIM protein, zinc finger protein, and development. It is anticipated that further study of genes identified will provide insights into their specific roles in the etiology of VSD, even in cardiac development, aging, and disease.     

Identification of up-regulated genes in human uterine leiomyoma by suppression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression subtractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88 genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes were firstly associated with UL. Three genes with notable difference were selected for Northern confirmation. Our results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showed up-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obvious expression in prostate, testis, liver, heart and skeletal muscle.