Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Small Subunit Localization in Response to Iron Deficiency

Ribonucleotide reductase (RNR) is an essential iron-dependent enzyme that catalyzes deoxyribonucleotide synthesis in eukaryotes. Living organisms have developed multiple strategies to tightly modulate RNR function to avoid inadequate or unbalanced deoxyribonucleotide pools that cause DNA damage and genome instability. Yeast cells activate RNR in response to genotoxic stress and iron deficiency by facilitating redistribution of its small heterodimeric subunit Rnr2-Rnr4 from the nucleus to the cytoplasm, where it forms an active holoenzyme with large Rnr1 subunit. Dif1 protein inhibits RNR by promoting nuclear import of Rnr2-Rnr4. Upon DNA damage, Dif1 phosphorylation by the Dun1 checkpoint kinase and its subsequent degradation enhances RNR function. In this report, we demonstrate that Dun1 kinase triggers Rnr2-Rnr4 redistribution to the cytoplasm in response to iron deficiency. We show that Rnr2-Rnr4 relocalization by low iron requires Dun1 kinase activity and phosphorylation site Thr-380 in the Dun1 activation loop, but not the Dun1 forkhead-associated domain. By using different Dif1 mutant proteins, we uncover that Dun1 phosphorylates Dif1 Ser-104 and Thr-105 residues upon iron scarcity. We observe that the Dif1 phosphorylation pattern differs depending on the stimuli, which suggests different Dun1 activating pathways. Importantly, the Dif1-S104A/T105A mutant exhibits defects in nucleus-to-cytoplasm redistribution of Rnr2-Rnr4 by iron limitation. Taken together, these results reveal that, in response to iron starvation, Dun1 kinase phosphorylates Dif1 to stimulate Rnr2-Rnr4 relocalization to the cytoplasm and promote RNR function.This work has been supported by a predoctoral fellowship from “Conselleria d'Educació de la Generalitat Valenciana” (to N. S.), a predoctoral fellowship from the Spanish Ministry of Economy and Competitiveness (to A. M. R.), Spanish Ministry of Economy and Competitiveness Grants AGL2011-29099 and BIO2014-56298-P (to S. P.), and National Institutes of Health Grant CA125574 (to M. H.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Peer reviewe

Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis δ-endotoxin

Here we describe development of transgenic elite rice lines expressing a Bt fusion gene derived from cryIA(b) and cryIA(c) under the control of rice actinI promoter. The lines used in the study were indica CMS restorer line of Minghui 63 and its derived hybrid rice Shanyou 63. The level of Bt fusion protein CryIA(b)/CryIA(c) detected in Minghui 63 (T51-1) plants was 20 ng/mg soluble protein. The Bt Shanyou 63 was field-tested in natural and repeated heavy manual infestation of two lepidopteran insects, leaffolder and yellow stem borer. The transgenic hybrid plants showed high protection against both insect pests without reduced yield

Site-independently integrated transgenes in the elite restorer rice line Minghui 63 allow removal of a selectable marker from the gene of interest by self-segregation

In this study, we have demonstrated that two independent loci are involved in the integration of the insecticidal protein gene cryIAb/cryIAc and selectable marker gene hph in the recipient genome of the elite Chinese CMS restorer line Minghui 63. We have also documented the structural organization of these transgenes in each locus by restriction enzyme digestion and Southern blot analysis. The independent locus integration of different transgenes allowed us to remove the selectable marker gene hph from the gene of interest simply by self-segregation. Not having the selectable marker gene will enhance the commercial value of our transgenic line TT51-1, which showed a consistently high level of resistance against repeated infestations of yellow stem borers and natural outbreaks of leaf-folders, without a reduction in yield potential

QTL analysis of eating quality and cooking process of rice using a new RIL population derived from a cross between Minghui 63 and Khao Dawk Mali105

Abstract The cooking and eating quality of the rice grain is one of the most serious problems in many rice producing areas of the world. In this study, QTL analysis was performed for rice cooking and eating quality using a new recombinant inbred line (RIL) population derived from a cross between Minghui 63 (MH63), the Chinese best male sterility restorer in the hybrid rice programs, and Khao Dawk Mali105 (KDML105), the Thai jasmine rice, known as the best quality rice. The traits analyzed included amylose content (AC), gel consistency (GC), alkali spreading value (ASV), and 13 parameters from the viscosity profile. Comparison of the QTLs identified revealed 11 QTL clusters for these traits distributed on six chromosomes. The QTLs for the traits in the same class often clustered into the same chromosomal regions. A total of 29 QTLs were identified for 16 traits (or parameters) in the two years at P≤0.01 level. Our results clearly showed that the QTL cluster (six QTLs) corresponding to the Wx locus controlled six of the viscosity parameters such as BAtime-time needed from initial viscosity increase to peak viscosity (PKV), hot paste viscosity (HPV), final viscosity (FV), setback viscosity (SB) and consistency viscosity (CS), and had no effect on AC, GC, and ASV. The QTL cluster (13 QTLs) corresponding to the Alk locus played a role in ASV, GC, AC and all of the viscosity parameters except for PKV, FV and CS. In this study both AC and GC were not influenced by the Wx gene region. Our study investigated QTL analysis for the seven parameters of the viscosity profile, namely, Atemp, Atime, Btemp, Btime, BAtime, V95, and FV. Most of the QTLs previously found for these parameters on chromosome 6 in the Wx and Alk loci and on chromosome 5 and chromosome 7 were confirmed in the present study. Furthermore, new minor and major QTLs were also mapped on the chromosomes 5, 6, 7, 8, 11 and 12 for these parameters. However, we noted the instability of some of these QTLs across the environments and their small phenotypic variation value. Further investigation of these new QTLs or locus could bring more specific and comprehensive and probably complete information about them. Keywords: QTL, Recombinant inbred line, Rice quality, SSR markers, Viscosity profile. Abbreviations: AC-amylose content; Add-additive effect; Alk-alkali gene locus; Atemp-pasting temperature; Atime-pasting time; BAtime-time needed from initial viscosity increase to PKV; BD-breakdown viscosity; Btemp-peak temperature; Btime-peak time; Chrs-chromosome; CPV-cool paste viscosity; CS-consistency viscosity; FV-final viscosity at 40°C; GC-gel consistency; GTgelatinization temperature; HPV-hot paste viscosity; KDML105-Kkao Dawk Mali105; MH63-Minghui 63; PKV-peak viscosity ; QTL-quantitative trait loci; RIL-recombinant inbred lines; RVA-rapid visco analyzer; SB-setback viscosity; SD-standard deviation; SSR-simple sequence repeats; V95-viscosity at 95°C; Var%-phenotypic variation percentage; Wx-waxy gene locus

Gains in QTL Detection Using an Ultra-High Density SNP Map Based on Population Sequencing Relative to Traditional RFLP/SSR Markers

Huge efforts have been invested in the last two decades to dissect the genetic bases of complex traits including yields of many crop plants, through quantitative trait locus (QTL) analyses. However, almost all the studies were based on linkage maps constructed using low-throughput molecular markers, e.g. restriction fragment length polymorphisms (RFLPs) and simple sequence repeats (SSRs), thus are mostly of low density and not able to provide precise and complete information about the numbers and locations of the genes or QTLs controlling the traits. In this study, we constructed an ultra-high density genetic map based on high quality single nucleotide polymorphisms (SNPs) from low-coverage sequences of a recombinant inbred line (RIL) population of rice, generated using new sequencing technology. The quality of the map was assessed by validating the positions of several cloned genes including GS3 and GW5/qSW5, two major QTLs for grain length and grain width respectively, and OsC1, a qualitative trait locus for pigmentation. In all the cases the loci could be precisely resolved to the bins where the genes are located, indicating high quality and accuracy of the map. The SNP map was used to perform QTL analysis for yield and three yield-component traits, number of tillers per plant, number of grains per panicle and grain weight, using data from field trials conducted over years, in comparison to QTL mapping based on RFLPs/SSRs. The SNP map detected more QTLs especially for grain weight, with precise map locations, demonstrating advantages in detecting power and resolution relative to the RFLP/SSR map. Thus this study provided an example for ultra-high density map construction using sequencing technology. Moreover, the results obtained are helpful for understanding the genetic bases of the yield traits and for fine mapping and cloning of QTLs

A Rice Gene of De Novo Origin Negatively Regulates Pathogen-Induced Defense Response

How defense genes originated with the evolution of their specific pathogen-responsive traits remains an important problem. It is generally known that a form of duplication can generate new genes, suggesting that a new gene usually evolves from an ancestral gene. However, we show that a new defense gene in plants may evolve by de novo origination, resulting in sophisticated disease-resistant functions in rice. Analyses of gene evolution showed that this new gene, OsDR10, had homologs only in the closest relative, Leersia genus, but not other subfamilies of the grass family; therefore, it is a rice tribe-specific gene that may have originated de novo in the tribe. We further show that this gene may evolve a highly conservative rice-specific function that contributes to the regulation difference between rice and other plant species in response to pathogen infections. Biologic analyses including gene silencing, pathologic analysis, and mutant characterization by transformation showed that the OsDR10-suppressed plants enhanced resistance to a broad spectrum of Xanthomonas oryzae pv. oryzae strains, which cause bacterial blight disease. This enhanced disease resistance was accompanied by increased accumulation of endogenous salicylic acid (SA) and suppressed accumulation of endogenous jasmonic acid (JA) as well as modified expression of a subset of defense-responsive genes functioning both upstream and downstream of SA and JA. These data and analyses provide fresh insights into the new biologic and evolutionary processes of a de novo gene recruited rapidly

Altered Levels of Histone Deacetylase OsHDT1 Affect Differential Gene Expression Patterns in Hybrid Rice

Hybrids between different inbred varieties display novel patterns of gene expression resulted from parental variation in allelic nucleotide sequences. To study the function of chromatin regulators in hybrid gene expression, the histone deacetylase gene OsHDT1 whose expression displayed a circadian rhythm was over-expressed or inactivated by RNAi in an elite rice parent. Increased OsHDT1 expression did not affect plant growth in the parent but led to early flowering in the hybrid. Nonadditive up-regulation of key flowering time genes was found to be related to flowering time of the hybrid. Over-expression of OsHDT1 repressed the nonadditive expression of the key flowering repressors in the hybrid (i.e. OsGI and Hd1) inducing early flowering. Analysis of histone acetylation suggested that OsHDT1 over-expression might promote deacetylation on OsGI and Hd1 chromatin during the peak expression phase. High throughput differential gene expression analysis revealed that altered OsHDT1 levels affected nonadditive expression of many genes in the hybrid. These data demonstrate that nonadditive gene expression was involved in flowering time control in the hybrid rice and that OsHDT1 level was important for nonadditive or differential expression of many genes including the flowering time genes, suggesting that OsHDT1 may be involved in epigenetic control of parental genome interaction for differential gene expression

Conserved electron donor complex Dre2-Tah18 is required for ribonucleotide reductase metallocofactor assembly and DNA synthesis

Eukaryotic ribonucleotide reductases (RNRs) require a diferric-tyrosyl radical (Fe[III over 2]-Y•) cofactor to produce deoxynucleotides essential for DNA replication and repair. This metallocofactor is an important target of RNR-based therapeutics, although mechanisms of in vivo cofactor assembly, inactivation, and reactivation are poorly understood. Here, we demonstrate that the conserved Fe-S protein–diflavin reductase complex, Dre2–Tah18, plays a critical role in RNR cofactor biosynthesis. Depletion of Dre2 affects both RNR gene transcription and mRNA turnover through the activation of the DNA-damage checkpoint and the Aft1/Aft2-controlled iron regulon. Under conditions of comparable RNR protein levels, cells with diminishing Dre2 have significantly reduced ability to make deoxynucleotides. Furthermore, the kinetics and levels of in vivo reconstitution of the RNR cofactor are severely impaired in two conditional tah18 mutants. Together, these findings provide insight into RNR cofactor formation and reveal a shared mechanism underlying assembly of the Fe[III over 2]-Y• cofactor in RNR and the Fe-S clusters in cytosolic and nuclear proteins.National Institutes of Health (U.S.) (Grant R01GM29595)National Institutes of Health (U.S.) (Grant R01GM81393

Arabidopsis Cockayne Syndrome A-Like Proteins 1A and 1B Form a Complex with CULLIN4 and Damage DNA Binding Protein 1A and Regulate the Response to UV Irradiation[W]

This study shows that Arabidopsis CSAat1A, which encodes a Cockayne Syndrome A-like protein, and its homolog, CSAat1B, form a heterotetramer in response to DNA damage and thereby play a key role in the plant’s response to UV-B irradiation

Activation of TNF‐α/NF‐κB axis enhances CRL4BDCAF11 E3 ligase activity and regulates cell cycle progression in human osteosarcoma cells

Cullin 4B, a member of the Cullins, which serve as scaffolds to facilitate the assembly of E3 ligase complexes, is aberrantly expressed in many cancers, including osteosarcoma. Recently, we observed that CUL4B forms the CRL4BDCAF11 E3 ligase, which specifically ubiquitinates and degrades the cyclin‐dependent kinase (CDK) inhibitor p21Cip1 in human osteosarcoma cells. However, the underlying mechanisms regarding the aberrant expression of CUL4B and the upstream members of this signaling pathway are mostly unknown. In this study, we demonstrate that nuclear factor kappaB (NF‐κB) is a direct modulator of CUL4B expression. The CUL4B promoter is responsive to several NF‐κB subunits, including RelA, RelB, and c‐Rel, but not to p50 or p52. Additional studies reveal that the tumor necrosis factor alpha (TNF‐α)/NF‐κB axis pathway is activated in human osteosarcoma cells. This activation causes both CUL4B and NF‐κB subunits to become abundant in the nucleus of human osteosarcoma cells. The down‐regulation of individual genes, including TNFR1, RelA, RelB, c‐Rel, and CUL4B, or pairs of them, including TNFR1 + RelA, TNFR1 + RelB, TNFR1 + c‐Rel, and RelA+CUL4B, has similar effects on cell growth inhibition, colony formation, cell invasion, and in vivo tumor formation, whereas the overexpression of CUL4B in these knockdown cells significantly reverses their phenotypes. The inhibition of the TNF‐α/NF‐κB pathway greatly attenuates CRL4BDCAF11 E3 ligase activity and causes the accumulation of p21Cip1, thereby leading to cell cycle arrest at the S phase. Taken together, our results support a model in which the activation of the TNF‐α/NF‐κB axis contributes to an increase in CRL4BDCAF11 activity and a decrease in p21Cip1 protein levels, thereby controlling cell cycle progression in human osteosarcoma cells