scholarly journals Several downy mildew resistance genes in Arabidopsis require signaling via a homologue of yeast SGT1

2017 ◽  
Vol 38 (SI 2 - 6th Conf EFPP 2002) ◽  
pp. 510-512
Author(s):  
M. Tör ◽  
P. Gordon ◽  
A. Cuzick ◽  
A. Yemm ◽  
E.B. Holub
Keyword(s):  
Downy Mildew ◽  
Crop Improvement ◽  
Regulatory Protein ◽  
Carboxyl Terminus ◽  
Peronospora Parasitica ◽  
Wild Type ◽  
Altered Expression ◽  
Centromere Function ◽  
N Terminus ◽  
Potential Use

A fast neutron mutant in Arabidopsis (Columbia) was identified that exhibits enhanced downy mildew (edm1) susceptibility to several Peronospora parasitica isolates, including the RPP7-diagnostic isolate Hiks1. The mutation was mapped to chr.4 and physically characterised as a 35kb deletion spanning seven genes. One of these genes restored wild-type resistance to all of the P. parasitica isolates. This gene (AtSGT1b) encodes a predicted protein that is orthologous to yeast SGT1, originally described as a key regulatory protein in centromere function and ubiquitin-mediated proteolysis. AtSGT1b contains three tatratrico-peptide repeats at the N terminus followed by a bipartite “CS” (CHORD containing Sgt) domain and an SGT specific (SGTS) domain at the carboxyl terminus. Altered expression of this gene is being investigated in Arabidopsis and Brassica olarecea to determine its potential use for crop improvement.

scholarly journals GAL4 mutations that separate the transcriptional activation and GAL80-interactive functions of the yeast GAL4 protein.

Genetics ◽  
1990 ◽  
Vol 125 (1) ◽  
pp. 21-27 ◽  
Author(s):  
J M Salmeron ◽  
K K Leuther ◽  
S A Johnston
Keyword(s):  
Amino Acid ◽  
Transcriptional Activation ◽  
Regulatory Protein ◽  
Activation Function ◽  
Single Amino Acid ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Wild Type Phenotype ◽  
Transcriptional Activator Protein ◽  
Carboxy Terminal

Abstract The carboxy-terminal 28 amino acids of the Saccharomyces cerevisiae transcriptional activator protein GAL4 execute two functions--transcriptional activation and interaction with the negative regulatory protein, GAL80. Here we demonstrate that these two functions are separable by single amino acid changes within this region. We determined the sequences of four GAL4C-mutations, and characterized the abilities of the encoded GAL4C proteins to activate transcription of the galactose/melibiose regulon in the presence of GAL80 and superrepressible GAL80S alleles. One of the GAL4C mutations can be compensated by a specific GAL80S mutation, resulting in a wild-type phenotype. These results support the idea that while the GAL4 activation function tolerates at least minor alterations in the GAL4 carboxyl terminus, the GAL80-interactive function is highly sequence-specific and sensitive even to single amino acid alterations. They also argue that the GAL80S mutations affect the affinity of GAL80 for GAL4, and not the ability of GAL80 to bind inducer.

scholarly journals Mutants of downy mildew resistance in Lactuca sativa (lettuce).

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 867-874
Author(s):  
P A Okubara ◽  
P A Anderson ◽  
O E Ochoa ◽  
R W Michelmore
Keyword(s):  
Molecular Marker ◽  
Downy Mildew ◽  
Genomic Dna ◽  
Spontaneous Mutation ◽  
Multigene Families ◽  
Downy Mildew Resistance ◽  
Bremia Lactucae ◽  
Wild Type ◽  
Mildew Resistance ◽  
Recessive Mutations

Abstract As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm 1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from gamma- or fast neutron-irradiated seed. In two separate Dm 1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.

scholarly journals Glia maturation factor-γ regulates murine macrophage iron metabolism and M2 polarization through mitochondrial ROS

2019 ◽  
Vol 3 (8) ◽  
pp. 1211-1225 ◽  
Author(s):  
Wulin Aerbajinai ◽  
Manik C. Ghosh ◽  
Jie Liu ◽  
Chutima Kumkhaek ◽  
Jianqing Zhu ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Mitochondrial Function ◽  
Regulatory Protein ◽  
M2 Macrophage ◽  
Macrophage Phenotype ◽  
Glia Maturation Factor ◽  
Altered Expression ◽  
Murine Macrophages ◽  
Cellular Iron ◽  
Maturation Factor

Abstract In macrophages, cellular iron metabolism status is tightly integrated with macrophage phenotype and associated with mitochondrial function. However, how molecular events regulate mitochondrial activity to integrate regulation of iron metabolism and macrophage phenotype remains unclear. Here, we explored the important role of the actin-regulatory protein glia maturation factor-γ (GMFG) in the regulation of cellular iron metabolism and macrophage phenotype. We found that GMFG was downregulated in murine macrophages by exposure to iron and hydrogen peroxide. GMFG knockdown altered the expression of iron metabolism proteins and increased iron levels in murine macrophages and concomitantly promoted their polarization toward an anti-inflammatory M2 phenotype. GMFG-knockdown macrophages exhibited moderately increased levels of mitochondrial reactive oxygen species (mtROS), which were accompanied by decreased expression of some mitochondrial respiration chain components, including the iron-sulfur cluster assembly scaffold protein ISCU as well as the antioxidant enzymes SOD1 and SOD2. Importantly, treatment of GMFG-knockdown macrophages with the antioxidant N-acetylcysteine reversed the altered expression of iron metabolism proteins and significantly inhibited the enhanced gene expression of M2 macrophage markers, suggesting that mtROS is mechanistically linked to cellular iron metabolism and macrophage phenotype. Finally, GMFG interacted with the mitochondrial membrane ATPase ATAD3A, suggesting that GMFG knockdown–induced mtROS production might be attributed to alteration of mitochondrial function in macrophages. Our findings suggest that GMFG is an important regulator in cellular iron metabolism and macrophage phenotype and could be a novel therapeutic target for modulating macrophage function in immune and metabolic disorders.

scholarly journals The Constitutive Centromere Component CENP-50 Is Required for Recovery from Spindle Damage

2005 ◽  
Vol 25 (23) ◽  
pp. 10315-10328 ◽  
Author(s):  
Yukinori Minoshima ◽  
Tetsuya Hori ◽  
Masahiro Okada ◽  
Hiroshi Kimura ◽  
Tokuko Haraguchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Mitotic Checkpoint ◽  
Loss Of Function ◽  
Wild Type ◽  
Centromere Function ◽  
Dt40 Cells ◽  
Precise Role ◽  
Chicken Dt40 Cell

ABSTRACT We identified CENP-50 as a novel kinetochore component. We found that CENP-50 is a constitutive component of the centromere that colocalizes with CENP-A and CENP-H throughout the cell cycle in vertebrate cells. To determine the precise role of CENP-50, we examined its role in centromere function by generating a loss-of-function mutant in the chicken DT40 cell line. The CENP-50 knockout was not lethal; however, the growth rate of cells with this mutation was slower than that of wild-type cells. We observed that the time for CENP-50-deficient cells to complete mitosis was longer than that for wild-type cells. Centromeric localization of CENP-50 was abolished in both CENP-H- and CENP-I-deficient cells. Coimmunoprecipitation experiments revealed that CENP-50 interacted with the CENP-H/CENP-I complex in chicken DT40 cells. We also observed severe mitotic defects in CENP-50-deficient cells with apparent premature sister chromatid separation when the mitotic checkpoint was activated, indicating that CENP-50 is required for recovery from spindle damage.

scholarly journals Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions

2016 ◽  
Vol 91 (3) ◽  
Author(s):  
Jolene Ramsey ◽  
Emily C. Renzi ◽  
Randy J. Arnold ◽  
Jonathan C. Trinidad ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Plasma Membrane ◽  
Sindbis Virus ◽  
Molecular Mechanisms ◽  
Wild Type Virus ◽  
Membrane Localization ◽  
Clear Understanding ◽  
Wild Type ◽  
Plasma Membrane Localization ◽  
C Terminus ◽  
N Terminus

ABSTRACT Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a −1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. IMPORTANCE Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo. Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans.

scholarly journals Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80.

1989 ◽  
Vol 9 (7) ◽  
pp. 2950-2956 ◽  
Author(s):  
J M Salmeron ◽  
S D Langdon ◽  
S A Johnston
Keyword(s):  
Transcriptional Activation ◽  
Kluyveromyces Lactis ◽  
Regulatory Protein ◽  
Wild Type ◽  
Galactose Metabolism ◽  
Metabolism Regulation ◽  
Transcriptional Activator Protein ◽  
The Difference ◽  
Carboxy Terminal

In Saccharomyces cerevisiae, transcriptional activation mediated by the GAL4 regulatory protein is repressed in the absence of galactose by the binding of the GAL80 protein, an interaction that requires the carboxy-terminal 28 amino acids of GAL4. The homolog of GAL4 from Kluyveromyces lactis, LAC9, activates transcription in S. cerevisiae and is highly similar to GAL4 in its carboxyl terminus but is not repressed by wild-type levels of GAL80 protein. Here we show that GAL80 does repress LAC9-activated transcription in S. cerevisiae if overproduced. We sought to determine the molecular basis for the difference in the responses of the LAC9 and GAL4 proteins to GAL80. Our results indicate that this difference is due primarily to the fact that under wild-type conditions, the level of LAC9 protein in S. cerevisiae is much higher than that of GAL4, which suggests that LAC9 escapes GAL80-mediated repression by titration of GAL80 protein in vivo. The difference in response to GAL80 is not due to amino acid sequence differences between the LAC9 and GAL4 carboxyl termini. We discuss the implications of these results for the mechanism of galactose metabolism regulation in S. cerevisiae and K. lactis.

scholarly journals Rice NIN-LIKE PROTEIN 4 is a master regulator of nitrogen use efficiency

2020 ◽  
Author(s):  
Jie Wu ◽  
Zi-Sheng Zhang ◽  
Jing-Qiu Xia ◽  
Alamin Alfatih ◽  
Ying Song ◽  
...  
Keyword(s):  
N Uptake ◽  
Crop Improvement ◽  
Field Trials ◽  
Growth And Yield ◽  
Wild Type ◽  
Inorganic N ◽  
Master Regulator ◽  
Nitrogen Use ◽  
Use Efficiency ◽  
N Use

AbstractNitrogen (N) is one of the key essential macronutrients that affects rice growth and yield. Inorganic N fertilizers are excessively used to boost yield and generate serious collateral environmental pollution. Therefore, improving crop N use efficiency (NUE) is highly desirable and has been a major endeavor in crop improvement. However, only a few regulators have been identified that can be used to improve NUE in rice to date. Here we show that the NIN-like protein OsNLP4 significantly improves the rice NUE and yield. Field trials consistently showed that loss-of-OsNLP4 dramatically reduced yield and NUE compared with wild type under different N regimes. In contrast, the OsNLP4 overexpression lines remarkably increased yield by 30% and NUE by 47% under moderate N level compared with wild type. Transcriptomic analyses revealed that OsNLP4 orchestrates the expression of a majority of known N uptake, assimilation and signaling genes by directly binding to the nitrate-responsive cis-element in their promoters to regulate their expression. Moreover, overexpression of OsNLP4 can recover the phenotype of Arabidopsis nlp7 mutant and enhance its biomass. Our results demonstrate that OsNLP4 is a master regulator of NUE in rice and sheds light on crop NUE improvement.

scholarly journals Identification of TAZ as a Binding Partner of the Polyomavirus T Antigens

2004 ◽  
Vol 78 (22) ◽  
pp. 12657-12664 ◽  
Author(s):  
Yu Tian ◽  
Dawei Li ◽  
Jean Dahl ◽  
John You ◽  
Thomas Benjamin
Keyword(s):  
Dna Replication ◽  
Host Range ◽  
T Antigen ◽  
Binding Motif ◽  
Wild Type ◽  
T Antigens ◽  
Ww Domain ◽  
Binding Partner ◽  
N Terminus ◽  
The Common

ABSTRACT A polyomavirus mutant isolated by the tumor host range selection procedure (19) has a three-amino-acid deletion (Δ2-4) in the common N terminus of the T antigens. To search for a cellular protein bound by wild-type but not the mutant T antigen(s), a yeast two-hybrid screen of a mouse embryo cDNA library was carried out with a bait of wild-type small T antigen (sT) fused N terminally to the DNA-binding domain of Gal4. TAZ, a transcriptional coactivator with a WW domain and PDZ-binding motif (17), was identified as a binding partner. TAZ bound in vivo to all three T antigens with different apparent affinities estimated as 1:7:100 (large T antigen [lT]:middle T antigen [mT]:sT). The Δ2-4 mutant T antigens showed no detectable binding. The sT and mT of the host range transformation-defective (hr-t) mutant NG59 with an alteration in the common sT/mT region (179 D→NI) and a normal N terminus also failed to bind TAZ, while the unaltered lT bound but with reduced affinity compared to that seen in a wild-type virus infection. The WW domain but not the PDZ-binding motif of TAZ was essential for T antigen binding. The Δ2-4 mutant was defective in viral DNA replication. Forced overexpression of TAZ blocked wild-type DNA replication in a manner dependent on the binding site for the polyomavirus enhancer-binding protein 2α. Wild-type polyomavirus T antigens effectively block transactivation by TAZ. The functional significance of TAZ interactions with polyomavirus T antigens is discussed.

scholarly journals Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions

2007 ◽  
Vol 81 (15) ◽  
pp. 7833-7843 ◽  
Author(s):  
Joshua C. Grieger ◽  
Jarrod S. Johnson ◽  
Brittney Gurda-Whitaker ◽  
Mavis Agbandje-McKenna ◽  
R. Jude Samulski
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Wild Type ◽  
Dot Blot ◽  
Adeno Associated Virus ◽  
N Terminus ◽  
Localization Signal ◽  
Significant Effort

ABSTRACT Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.

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