Caracterización funcional de los genes sgb (suppressors of agb1-2 susceptibility to pathogens): Nuevos reguladores de la respuesta de inmunidad innata de Arabidopsis thaliana

Un porcentaje importante de las pérdidas de la producción agrícola se deben a las enfermedades que causan en los cultivos los hongos necrótrofos y vasculares. Para mejorar la productividad agrícola es necesario tener un conocimiento detallado de las bases genéticas y moleculares que regulan la resistencia de las plantas a este tipo de patógenos. En Arabidopsis thaliana la resistencia frente a patógenos necrótrofos, como el hongo Plectosphaerella cucumerina BMM (PcBMM), es genéticamente compleja y depende de la activación coordinada de distintas rutas de señalización, como las reguladas por las hormonas ácido salicílico (SA), ácido jasmónico (JA), etileno (ET) y ácido abscísico (ABA), así como de la síntesis de compuestos antimicrobianos derivados del Triptófano y de la integridad de la pared celular (Llorente et al., 2005, Hernández-Blanco et al., 2007; Delgado-Cerezo et al., 2012). Uno de los componentes claves en la regulación de la resistencia de las plantas a patógenos (incluidos hongos necrótrofos y biótrofos) es la proteína G heterotrimérica, un complejo proteico formado por tres subunidades (Gα, Gβ y Gγ), que también regula distintos procesos del desarrollo vegetal. En Arabidopsis hay un gen que codifica para la subunidad α (GPA1), otro para la β (AGB1), y tres genes para la subunidad γ (AGG1, AGG2 y AGG3). El complejo GPA1-AGB1-AGG (1-3) se activa y disocia tras la percepción de una señal específica, actuando el dímero AGB1-AGG1/2 como un monómero funcional que regula las respuestas de defensa (Delgado-Cerezo et al., 2012). Estudios transcriptómicos y análisis bioquímicos de la pared celular en los que se comparaban los mutantes agb1-2 y agg1 agg2, y plantas silvestres (Col-0) revelaron que la resistencia mediada por Gβ-Gγ1/2 no es dependiente de rutas de defensa previamente caracterizadas, y sugieren que la proteína G podría modular la composición/estructura (integridad) de la pared celular (Delgado-Cerezo et al., 2012). Recientemente, se ha demostrado que AGB1 es un componente fundamental de la respuesta inmune mediada por Pathogen- Associated Molecular Patterns (PTI), ya que los mutantes agb1-2 son incapaces de activar tras el tratamiento con PAMPs respuestas de inmunidad, como la producción de especies reactivas de oxígeno (ROS; Liu et al., 2013). Dada la importancia de la proteína G heterotrimérica en la regulación de la respuestas de defensa (incluida la PTI) realizamos un escrutinio de mutantes supresores de la susceptibilidad de agb1-2 al hongo necrótrofo, PcBMM, para identificar componentes adicionales de las rutas de señalización reguladas por AGB1. En este escrutinio se aislaron cuatro mutantes sgb (suppressors of agb1-2 susceptibility to pathogens), dos de los cuales, sgb10 y sgb11, se han caracterizado en la presente Tesis Doctoral. El mutante sgb10 es un segundo alelo nulo del gen MKP1 (At3g55270) que codifica la MAP quinasa-fosfatasa 1 (Bartels et al., 2009). Este mutante presenta lesiones espontáneas en plantas adultas y una activación constitutiva de las principales rutas de defensa (SA, JA y ET, y de metabolitos secundarios, como la camalexina), que explicaría su elevada resistencia a PcBMM y Pseudomonas syringae. Estudios epistáticos sugieren que la resistencia mediada por SGB10 no es dependiente, si no complementaria a la regulada por AGB1. El mutante sgb10 es capaz de restablecer en agb1-2 la producción de ROS y otras respuestas PTI (fosforilación de las MAPK6/3/4/11) tras el tratamiento con PAMPs tan diversos como flg22, elf18 y quitina, lo que demuestra el papel relevante de SGB10/MKP1 y de AGB1 en PTI. El mutante sgb11 se caracteriza por presentar un fenotipo similar a los mutantes irregular xylem (e.g. irx1) afectado en pared celular secundaria: irregularidades en las células xilemáticas, reducción en el tamaño de la roseta y altura de planta, y hojas con un mayor contenido de clorofila. La resistencia de sgb11 a PcBMM es independiente de agb1-2, ya que la susceptibilidad del doble mutante sgb11 agb1-2 es intermedia entre la de agb1-2 y sgb11. El mutante sgb11 no revierte la deficiente PTI de agb1-2 tras el tratamiento con flg22, lo que indica que está alterado en una ruta distinta de la regulada por SGB10. sgb11 presenta una sobreactivación de la ruta del ácido abscísico (ABA), lo que podría explicar su resistencia a PcBMM. La mutación sgb11 ha sido cartografiada en el cromosoma III de Arabidopsis entre los marcadores AthFUS6 (81,64cM) y nga6 (86,41cM) en un intervalo de aproximadamente 200 kb, que comprende genes, entre los que no se encuentra ninguno previamente descrito como IRX. El aislamiento y caracterización de SGB11 apoya la relevancia de la proteína G heterotrimérica en la regulación de la interconexión entre integridad de la pared celular e inmunidad. ABSTRACT A significant percentage of agricultural losses are due to diseases caused by necrotrophic and vascular fungi. To enhance crop yields is necessary to have a detailed knowledge of the genetic and molecular bases regulating plant resistance to these pathogens. Arabidopsis thaliana resistance to necrotrophic pathogens, such as Plectosphaerella cucumerina BMM (PcBMM) fungus, is genetically complex and depends on the coordinated activation of various signaling pathways. These include those regulated by salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) hormones and the synthesis of tryptophan-derived antimicrobial compounds and cell wall integrity (Llorente et al., 2005, Hernández-Blanco et al., 2007; Delgado-Cerezo et al., 2012). One key component in the regulation of plant resistance to pathogens (including biotrophic and necrotrophic fungi) is the heterotrimeric G-protein. This protein complex is formed by three subunits (Gα, Gβ and Gγ), which also regulates various plant developmental processes. In Arabidopsis only one gene encodes for subunits α (GPA1) and β (AGB1), and three genes for subunit γ (AGG1, AGG2 y AGG3). The complex GPA1- AGB1-AGG(1-3) is activated and dissociates after perception of an specific signal, AGB1- AGG1/2 acts as a functional monomer regulating defense responses (Delgado-Cerezo et al., 2012). Comparative transcriptomic studies and biochemical analyses of the cell wall of agb1-2 and agg1agg2 mutant and wild plants (Col-0), showed that Gβ-Gγ1/2-mediated resistance is not dependent on previously characterized defense pathways. In addition, it suggests that G protein may modulate the composition/structure (integrity) of the plant cell wall (Delgado-Cerezo et al., 2012). Recently, it has been shown that AGB1 is a critical component of the immune response mediated by Pathogen-Associated Molecular Patterns (PTI), as agb1-2 mutants are unable to activate immune responses such as oxygen reactive species (ROS) production after PAMPs treatment (Liu et al., 2013). Considering the importance of the heterotrimeric G protein in regulation of defense responses (including PTI), we performed a screening for suppressors of agb1-2 susceptibility to the necrotrophic fungus PcBMM. This would allow the identification of additional components of the signaling pathways regulated by AGB1. In this search four sgb mutants (suppressors of agb1-2 susceptibility to pathogens) were isolated, two of which, sgb10 and sgb11, have been characterized in this PhD thesis. sgb10 mutant is a second null allele of MKP1 gene (At3g55270), which encodes the MAP kinase-phosphatase 1 (Bartels et al., 2009). This mutant exhibits spontaneous lesions in adult plants and a constitutive activation of the main defense pathways (SA, JA and ET, and secondary metabolites, such as camalexin), which explains its high resistance to Pseudomonas syringae and PcBMM. Epistatic studies suggest that SGB10- mediated resistance is not dependent, but complementary to the regulated by AGB1. The sgb10 mutant is able to restore agb1-2 ROS production and other PTI responses (MAPK6/3/4/11 phosphorylation) upon treatment with PAMPs as diverse as, flg22, elf18 and chitin, demonstrating the relevant role of SGB10/MKP1 and AGB1 in PTI. sgb11 mutant is characterized by showing a similar phenotype to irregular xylem mutants (e.g. irx1), affected in secondary cell wall: irregular xylems cells, rosette size reduction and plant height, and higher chlorophyll content on leaves. The resistance of sgb11 to PcBMM is independent of agb1-2, as susceptibility of the double mutant agb1-2sgb11 is intermediate between agb1-2 and sgb11. The sgb11 mutant does not revert the deficient PTI response in agb1-2 after flg22 treatment, indicating that is altered in a pathway different to the one regulated by SGB10. sgb11 presents an over-activation of the abscisic acid pathway (ABA), which could explain its resistance to PcBMM. The sgb11 mutation has been mapped on chromosome III of Arabidopsis, between AthFUS6 (81.64 cM) and nga6 (86.41 cM) markers, in 200 kb interval, which does not include previously known IRX genes. The isolation and characterization of SGB11 supports the importance of heterotrimeric G protein in the regulation of the interconnection between the cell wall integrity and immunity.

Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells

Inhibitors of DNA methyltransferase, typified by 5-aza-2′-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-α. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT’s limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-α2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors.

Identification of Novel Temperature-sensitive Lethal Alleles in Essential β-Tubulin and Nonessential α2-Tubulin Genes as Fission Yeast Polarity Mutants

We have screened for temperature-sensitive (ts) fission yeast mutants with altered polarity (alp1–15). Genetic analysis indicates that alp2 is allelic to atb2 (one of two α-tubulin genes) and alp12 to nda3 (the single β-tubulin gene). atb2+ is nonessential, and the ts atb2 mutations we have isolated are dominant as expected. We sequenced two alleles of ts atb2 and one allele of ts nda3. In the ts atb2 mutants, the mutated residues (G246D and C356Y) are found at the longitudinal interface between α/β-heterodimers, whereas in ts nda3 the mutated residue (Y422H) is situated in the domain located on the outer surface of the microtubule. The ts nda3 mutant is highly sensitive to altered gene dosage of atb2+; overexpression of atb2+ lowers the restrictive temperature, and, conversely, deletion rescues ts. Phenotypic analysis shows that contrary to undergoing mitotic arrest with high viability via the spindle assembly checkpoint as expected, ts nda3 mutants execute cytokinesis and septation and lose viability. Therefore, it appears that the ts nda3 mutant becomes temperature lethal because of irreversible progression through the cell cycle in the absence of activating the spindle assembly checkpoint pathway.

Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin response element (IRE) that resembles IREs of other insulin-repressed genes

Recent data indicate that sustained elevations in plasma insulin suppress the mRNA for IRS-2, a component of the insulin signaling pathway in liver, and that this deficiency contributes to hepatic insulin resistance and inappropriate gluconeogenesis. Here, we use nuclear run-on assays to show that insulin inhibits transcription of the IRS-2 gene in the livers of intact rats. Insulin also inhibited transcription of a reporter gene driven by the human IRS-2 promoter that was transfected into freshly isolated rat hepatocytes. The human promoter contains a heptanucleotide sequence, TGTTTTG, that is identical to the insulin response element (IRE) identified previously in the promoters of insulin-repressed genes. Single base pair substitutions in this IRE decreased transcription of the IRS-2-driven reporter in the absence of insulin and abolished insulin-mediated repression. We conclude that insulin represses transcription of the IRS-2 gene by blocking the action of a positive factor that binds to the IRE. Sustained repression of IRS-2, as occurs in chronic hyperinsulinemia, contributes to hepatic insulin resistance and accelerates the development of the diabetic state.

Distinct Cyclin D Genes Show Mitotic Accumulation or Constant Levels of Transcripts in Tobacco Bright Yellow-2 Cells1

The commitment of eukaryotic cells to division normally occurs during the G1 phase of the cell cycle. In mammals D-type cyclins regulate the progression of cells through G1 and therefore are important for both proliferative and developmental controls. Plant CycDs (D-type cyclin homologs) have been identified, but their precise function during the plant cell cycle is unknown. We have isolated three tobacco (Nicotiana tabacum) CycD cyclin cDNAs: two belong to the CycD3 class (Nicta;CycD3;1 and Nicta;CycD3;2) and the third to the CycD2 class (Nicta;CycD2;1). To uncouple their cell-cycle regulation from developmental control, we have used the highly synchronizable tobacco cultivar Bright Yellow-2 in a cell-suspension culture to characterize changes in CycD transcript levels during the cell cycle. In cells re-entering the cell cycle from stationary phase, CycD3;2 was induced in G1 but subsequently remained at a constant level in synchronous cells. This expression pattern is consistent with a role for CycD3;2, similar to mammalian D-type cyclins. In contrast, CycD2;1 and CycD3;1 transcripts accumulated during mitosis in synchronous cells, a pattern of expression not normally associated with D-type cyclins. This could suggest a novel role for plant D-type cyclins during mitosis.

Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress.

Early atherosclerotic lesions develop in a topographical pattern that strongly suggests involvement of hemodynamic forces in their pathogenesis. We hypothesized that certain endothelial genes, which exhibit differential responsiveness to distinct fluid mechanical stimuli, may participate in the atherogenic process by modulating, on a local level within the arterial wall, the effects of systemic risk factors. A differential display strategy using cultured human endothelial cells has identified two genes, manganese superoxide dismutase and cyclooxygenase-2, that exhibit selective and sustained up-regulation by steady laminar shear stress (LSS). Turbulent shear stress, a nonlaminar fluid mechanical stimulus, does not induce these genes. The endothelial form of nitric oxide synthase also demonstrates a similar LSS-selective pattern of induction. Thus, three genes with potential atheroprotective (antioxidant, antithrombotic, and antiadhesive) activities manifest a differential response to distinct fluid mechanical stimuli, providing a possible mechanistic link between endothelial gene expression and early events in atherogenesis. The activities of these and other LSS-responsive genes may have important implications for the pathogenesis and prevention of atherosclerosis.

Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components.

Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers.

Genetics of ocular disease Part 2: Basic concepts: DNA, proteins and genes

This article on the basic concepts of genetics concentrates on doeoxyribose nucleic acid (DNA), the chemical constituent of the genes. First, it will cover how DNA was discovered to be the substance of the genes. Second, the structure of DNA is revealed together with how DNA molecules can make copies of themselves. Third, the nature of the genetic code contained in DNA and how this code directs the manufacture of proteins is described. Finally, the effects of mutation of the genes and how the activities of genes are regulated will be discussed together with the relevance of these concepts to ocular disease.

Expression profiling of type 2 diabetes susceptibility genes in the pancreatic islets, adipose tissue and liver of obese mice

Type 2 diabetes (T2D) is characterized by impaired beta cell function and insulin resistance. T2D susceptibility genes identified by Genome-wide association studies (GWAS) are likely to have roles in both impaired insulin secretion from the beta cell as well as insulin resistance. The aim of this study was to use gene expression profiling to assess the effect of the diabetic milieu on the expression of genes involved in both insulin secretion and insulin resistance. We measured the expression of 43 T2D susceptibility genes in the islets, adipose and liver of leptin-deficient Ob/Ob mice compared with Ob/+ littermates. The same panel of genes were also profiled in cultured rodent adipocytes, hepatocytes and beta cells in response to high glucose conditions, to distinguish expression effects due to elevated glycemia from those on the causal pathway to diabetes or induced by other factors in the diabetic microenviroment. We found widespread deregulation of these genes in tissues from Ob/Ob mice, with differential regulation of 23 genes in adipose, 18 genes in liver and one gene (Tcf7l2) in islets of diabetic animals (Ob/Ob) compared to control (Ob/+) animals. However, these expression changes were in most cases not noted in glucose-treated adipocyte, hepatocyte or beta cell lines, indicating that they may not be an effect of hyperglycemia alone. This study indicates that expression changes are apparent with diabetes in both the insulin producing beta cells, but also in peripheral tissues involved in insulin resistance. This suggests that incidence or progression of diabetic phenotypes in a mouse model of diabetes is driven by both secretory and peripheral defects. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart New York.

Early growth response genes 2 and 3 regulate the expression of Bcl6 and differentiation of T follicular helper cells

T follicular helper (Tfh) cells support differentiation of B cells to plasma cells and high affinity antibody production in germinal centers (GC) and Tfh differentiation requires the function of B cell lymphoma 6 (Bcl6). We have now discovered that early growth response gene (Egr) 2 and 3 directly regulate the expression of Bcl6 in Tfh cells which is required for their function in regulation of GC formation. In the absence of Egr2 and 3, the expression of Bcl6 in Tfh cells is defective leading to impaired differentiation of Tfh cells resulting in a failure to form GCs following virus infection and defects in production of anti-viral antibodies. Enforced expression of Bcl6 in Egr2/3 deficient CD4 T cells partially restored Tfh differentiation and GC formation in response to virus infection. Our findings demonstrate a novel function of Egr2/3 which is important for Tfh cell development and Tfh cell mediated B cell immune responses.

Ten-eleven-translocation 2 (tet2) Is Downregulated In Myelodysplastic Syndromes.

TET2, a member of the ten-eleven-translocation (TET) family genes that modify DNA by converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), is located in chromosome 4q24 and is frequently mutated in myeloid malignancies. The impact of TET2 mutation on survival outcomes is still controversial; however, functional studies have proved that it is a loss-of-function mutation that impairs myeloid cell differentiation and contributes to the phenotype of myeloid neoplasia. We, herein, aimed to investigate TET2 expression in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A significantly decreased TET2 expression was observed in bone marrow cells from AML (n = 53) and patients with MDS (n = 64), compared to normal donors (n = 22). In MDS, TET2 expression was significantly reduced in RAEB-1/RAEB-2 compared to other WHO 2008 classifications, and a lower TET2 expression was observed at the time of MDS disease progression in four of five patients. In multivariate analysis, low TET2 expression (P = 0.03), male gender (P = 0.02), and WHO 2008 classification (P < 0.0001) were independent predictors of poorer overall survival. These results suggest that defective TET2 expression plays a role in the MDS pathophysiology and predicts survival outcomes in this disease.

Selection And Validation Of Reference Genes For Functional Studies In The Calliphoridae Family.

The genera Cochliomyia and Chrysomya contain both obligate and saprophagous flies, which allows the comparison of different feeding habits between closely related species. Among the different strategies for comparing these habits is the use of qPCR to investigate the expression levels of candidate genes involved in feeding behavior. To ensure an accurate measure of the levels of gene expression, it is necessary to normalize the amount of the target gene with the amount of a reference gene having a stable expression across the compared species. Since there is no universal gene that can be used as a reference in functional studies, candidate genes for qPCR data normalization were selected and validated in three Calliphoridae (Diptera) species, Cochliomyia hominivorax Coquerel, Cochliomyia macellaria Fabricius, and Chrysomya albiceps Wiedemann . The expression stability of six genes ( Actin, Gapdh, Rp49, Rps17, α -tubulin, and GstD1) was evaluated among species within the same life stage and between life stages within each species. The expression levels of Actin, Gapdh, and Rp49 were the most stable among the selected genes. These genes can be used as reliable reference genes for functional studies in Calliphoridae using similar experimental settings.

Polymorphisms Of Cell Cycle Control Genes Influence The Development Of Sporadic Medullary Thyroid Carcinoma.

The role of key cell cycle regulation genes such as, CDKN1B, CDKN2A, CDKN2B, and CDKN2C in sporadic medullary thyroid carcinoma (s-MTC) is still largely unknown. In order to evaluate the influence of inherited polymorphisms of these genes on the pathogenesis of s-MTC, we used TaqMan SNP genotyping to examine 45 s-MTC patients carefully matched with 98 controls. A multivariate logistic regression analysis demonstrated that CDKN1B and CDKN2A genes were related to s-MTC susceptibility. The rs2066827*GT+GG CDKN1B genotype was more frequent in s-MTC patients (62.22%) than in controls (40.21%), increasing the susceptibility to s-MTC (OR=2.47; 95% CI=1.048-5.833; P=0.038). By contrast, the rs11515*CG+GG of CDKN2A gene was more frequent in the controls (32.65%) than in patients (15.56%), reducing the risk for s-MTC (OR=0.174; 95% CI=0.048-0.627; P=0.0075). A stepwise regression analysis indicated that two genotypes together could explain 11% of the total s-MTC risk. In addition, a relationship was found between disease progression and the presence of alterations in the CDKN1A (rs1801270), CDKN2C (rs12885), and CDKN2B (rs1063192) genes. WT rs1801270 CDKN1A patients presented extrathyroidal tumor extension more frequently (92%) than polymorphic CDKN1A rs1801270 patients (50%; P=0.0376). Patients with the WT CDKN2C gene (rs12885) presented larger tumors (2.9±1.8 cm) than polymorphic patients (1.5±0.7 cm; P=0.0324). On the other hand, patients with the polymorphic CDKN2B gene (rs1063192) presented distant metastases (36.3%; P=0.0261). In summary, we demonstrated that CDKN1B and CDKN2A genes are associated with susceptibility, whereas the inherited genetic profile of CDKN1A, CDKN2B, and CDKN2C is associated with aggressive features of tumors. This study suggests that profiling cell cycle genes may help define the risk and characterize s-MTC aggressiveness.