C9orf82ko/ko mice were born at a Mendelian ratio and did not display any overt macroscopic or histological abnormalities. DSBs repair dependent processes like lymphocyte development and class switch recombination (CSR) appeared normal, arguing against a link between the C9orf82 encoded protein and V(D)J recombination or CSR. Most relevant, primary pre-B cell cultures and Tp53 transformed mouse embryo fibroblasts (MEFs) derived from C9orf82ko/ko E14.5 and wild type embryos displayed comparable sensitivity to a number of DNA lesions, including DSBs breaks induced by the topoisomerase II inhibitors, etoposide and doxorubicin. Likewise, the kinetics of γH2AX formation and resolution in response to etoposide of C9orf82 protein proficient, deficient and overexpressing MEFs were indistinguishable. These data argue against a direct role of C9orf82 protein in delaying repair of Topo II generated DSBs and regulating apoptosis. The genetically defined systems generated in this study will be of value to determine the actual function of C9orf82 protein.C9orf82 protein, or conserved anti-apoptotic protein 1 or caspase activity and apoptosis inhibitor 1 (CAAP1) has been implicated as a negative regulator of the intrinsic apoptosis pathway by modulating caspase expression and activity. In contrast, an independent genome wide screen for factors capable of driving drug resistance to the topoisomerase II (Topo II) poisons doxorubicin and etoposide, implicated a role for the nuclear protein C9orf82 in delaying DSBs repair downstream of Topo II, hereby sensitizing cells to DSB induced apoptosis. To determine its function in a genetically defined setting  DNA double-strand breaks (DSBs), that arise for example upon exposure to ionizing irradiation, are very dangerous lesions. If not repaired correctly DSBs can lead to DNA rearrangements and generate gain or loss of function mutations involving oncogenes and tumor suppressor genes, respectively , 2. ThesC9orf82 was found to augment subsequent DSBs repair in HAP1 cells and its overexpression delayed DSB repair in MelJuSo melanoma cells [In an independent, unbiased genome-wide gene knockout approach, we previously searched for factors capable of driving drug resistance to the topoisomerase II (Topo II) poisons doxorubicin and etoposide, two established longstanding cornerstones of chemotherapy. Keap1, the SWI/SNF complex, and C9orf82 protein were found to drive drug resistance through diverse molecular mechanisms, all converging at the level of DSBs formation and repair. Loss of Keap1 or the SWI/SNF complex was found to inhibit the generation of DSBs by attenuating the expression and activity of topoisomerase IIα, respectively, whereas deletion of ma cells .C9orf82 expression was found to increase Caspase-10 expression and activation and be required for Bid fragmentation and Caspase-9 activation. This study in human A-549 lung and MCF7/casp3-10b breast carcinoma cell lines, which made use of siRNA, suggested an anti-apoptotic function, where CAAP1 was proposed to modulate a Caspase-10 dependent mitochondrial Caspase-3/9 feedback amplification loop [C9orf82 protein, also known as conserved anti-apoptotic protein 1 (CAAP1), or caspase activity and apoptosis inhibitor 1, was first related to the regulation of apoptosis . Knock dion loop .C9orf82 knock down would accelerate DSBs repair and decrease DSBs induced apoptosis, positioning C9orf82 protein not as a direct negative regulator of apoptosis [In summary, while C9orf82 protein was initially identified as a negative regulator of the intrinsic apoptosis pathway , a subsepoptosis  but rathpoptosis . Clearlypoptosis .C9orf82ko/ko mouse model, and (re)investigated the function of C9orf82 protein in regulating apoptosis and DSBs repair using a genetically defined setting. Our studies failed to confirm previous conclusions on a role of C9orf82 protein in regulating apoptosis and DNA DSBs repair.In this study, we applied the CRISPR/Cas9 technology to generate a C9orf82 knock out (ko) mouse model was generated by co-injecting zygotes isolated from C57BL/6N mice with in vitro transcribed CRISPR/Cas9 encoding mRNA along with guide RNAs (gRNAs) from pX330 plasmid. To inactivate the C9orf82 locus in the mouse germline (ENSMUSG00000028578), gRNAs flanking exon 2–4 were designed using the crispr.mit.edu tool (A edu tool . To distC9orf82wt/ko) mice were used to isolate primary mouse embryonic fibroblasts (MEFs) and fetal livers. Pre-B cells were generated from single cell suspensions of fetal livers and subsequently grown on irradiated stromal cell line, ST2 feeder cells in complete IMDM medium , 50 μM 2-mercapthoethanol, penicillin/streptomycin) containing IL-7. Primary MEFs (2 per genotype) were isolated using Trypsin and cell strainers according to [E14.5 embryos resulting from the intercrosses of heterozygous  using gene specific primers (RNeasy mini (Qiagen) was used to isolate total RNA from wild type MEF for cloning of  primers . The PCRC9orf82 and pcl-Eco, according to [HEK293 T cells were transfected with pMX-IRES-GFP-rding to , to prodC9orf82 and expression of C9orf82 was normalized with that of GAPDH.qRT-PCR was performed on LightCycler 4800II (Roche) using Fast SYBR Green Master Mix (Thermo Scientific). Specific primers  were useC9orf82ko/ko mice and their wild-type littermates. Following erythrocytes lysis, naïve splenic B cells were enriched by the depletion of CD43 expressing cell using biotinylated anti-CD43 antibody , BD IMag Streptavidin Particles Plus and the IMag system (BD Biosciences), as described by the manufacturer. To measure their proliferative capacity, naïve B cells were labelled for 10 min at 37°C with 5 μM Carboxyfluorescein succinimidyl ester  in IMDM medium containing 2% FCS. After washing, cells were cultured in complete IMDM medium at a density of 105 cells/well in 24 well plates. CSR to IgG3 and IgG1 was induced by exposure to LPS  or LPS+rIL-4 (rIL4 20 ng/ml). Four days later, the cells were harvested and stained with CD19-PercpCy5.5 (BD), IgM-APC, and IgG3-PE (LPS cultures) or IgG1-PE (LPS/rIL4 cultures) to determine CSR frequency along with CFSE dilution as an indicator of cell multiplication. IgM-APC, IgG1-PE and IgG3-PE were purchased from Southern Biotech. Data were acquired by flow cytometry  and analyzed using FlowJo software (Version: 10.0.8r1).Single cells suspensions were prepared from the spleen of 8 to 10-week-old 5 fetal liver derived pre-B cells from wild type and C9orf82ko/ko mice were seeded on irradiated ST2 feeder layers in 0.5 ml of complete IMDM medium containing IL-7. For UV  or γ- irradiation (from 137Cs source) treatment, 15 min after seeding, the cells were irradiated with different doses and cultured in 1 ml of complete IMDM medium supplemented with IL-7. For Doxorubicin, Cisplatin, Etoposide and MMS, the pre-B cells seeded on irradiated ST2 feeder layers were continuously exposed to different doses of DNA damage agents in 1.0 ml of complete IMDM medium containing IL-7. For determining the survival, pre-B cells were harvested after three days of culture and live (PI negative) cells were counted using FACSArray (Becton Dickinson). Data were analyzed using FlowJo software (Version: 10.0.8r1).1*102O. After 1.5 h, the staining solution was removed, the dishes were washed with H2O and dried overnight. Colonies were counted and the survival of drug-treated cells were normalized for the plating efficiency of untreated cells. Data points represent the mean survival relative to the untreated control cells.MEFs were seeded in 10 cm dishes at a density of 200 cells/dish in complete medium with varying concentrations of Doxorubicin or Etoposide (Pharmachemie). One day later, the medium was removed and replaced with complete medium containing the indicated concentrations of respective drugs. After eight days, the medium containing drug was removed and the cells were washed with PBS and fixed in 5 mL 3:1 v/v Methanol: Acetic acid for 1 h. Following the fixation, colonies were stained by adding 3 ml of 0.3% Coomassie brilliant blue (Merck) solution prepared in HMEFs proficient or deficient for C9orf82 protein were seeded and next day treated with the indicated amount of etoposide. After an hour, etoposide was washed out and cells were lysed directly in lysis buffer . Samples were analyzed by SDS-PAGE and Western blotting. Blocking of the filter and antibody incubations (mouse anti-γH2AX (Millipore), mouse anti-actin (Sigma)) were done in PBS supplemented with 0.1% (v/v) Tween and 5% (w/v) milk powder. Blots were imaged using the Odyssey Imaging System (LI-COR).All the data were acquired by flow cytometry  unless mentioned otherwise and analyzed using FlowJo software (Version: 10.0.8r1). All the antibodies were purchased from BD Pharmingen unless mentioned otherwise.Lymphoid cells from bone marrow, thymus and spleen were isolated from 8–10 weeks old mice. To identify different B and T cell progenitor subsets and mature lymphocytes the cell suspension from bone marrow, thymus and spleen were stained with different mixture of conjugated antibodies .Primary pre-B cells were exposed to different concentrations of DNA damage agents like mentioned in pre-B cells survival assay. After three days of culture, the cells were harvested and stained with Annexin-V according to the manufacturer  instructions. DAPI was added at the time of measurement to identify different subsets of apoptotic cells. For caspase-3 activation assay, the cells were stained intracellularly with anti-active caspase-3 antibody according to the manufacturer  instructions.5) proficient or deficient for C9orf82 protein were exposed to different concentrations of Doxorubicin for 24 hrs. After the treatment, cells were fixed in 70% ethanol and kept at -4°C. Following fixation, cells were treated with RNAse A  for 20 min and then resuspended in PBS containing 5 μg/ml PI. Cells were measured on a FACSCalibur (Becton Dickinson) and the data were analyzed using Flow Jo software (10.0.8r1).Fetal liver derived pre-B cells .C9orf82 gene is encoded on the reverse strand of chromosome 4 and is expressed as a single protein-coding mRNA of 2107nt containing an open reading frame 1068nt, i.e. 356aa of an expected molecular weight of 37.83kD. The spliced transcript comprises 6 exons. To functionally characterize C9orf82 protein in mice, C9orf82 was inactivated by CRISPR/Cas9 genome editing. To avoid potential linkage mutations associated with long-term embryonic stem cell (ES) culturing, we chose for zygote injection. C57BL/6 zygotes were injected with in vitro transcribed Cas9 mRNA and two gRNAs flanking exon 2 and 4 of C9orf82 at chromosomal position 94555956 to 94555978 (gRNA1) and 94548569 to 94548591(gRNA2) . The absence of exon 2–4 was validated by Sanger sequencing. Deletion of exon 2–4 was independently confirmed at the cDNA level  . The nex . The neant mice . Four puNA level . The CRIed decay . This wa ko mice . C9orf82malities . These dC9orf82ko/ko mice were found indistinguishable from wild-type littermates , pre-B cells , immature B cells , and mature B cells , by staining with a cocktail of IgM, CD19, CD25, CD45R and CD117 specific antibodies each conjugated with a differential fluorophore. Within immature B cells, the developmental subsets were further distinguished as pro B (CD117+CD25-) and pre-B (CD117-CD25+). Dead cells were identified as propidium iodide+ and excluded from the analysis. B-lineage precursor subsets were found normal in number and frequencies between the genotypes , immature single positive (CD4-/CD8+/ TCRßlow), double positive (DP), i.e. CD4+/CD8+, and mature single positive (SP), i.e. CD4+/CD8-/TCRßhigh or CD4-/CD8+/TCRßhigh were analyzed. Within the DN compartment the developmental subsets were further distinguished as DN1 (CD44+CD25-), DN2 (CD44+CD25+), DN3 (CD44-CD25+) and DN4 (CD44-CD25-). Like for B lineage precursors in the bone marrow, the absolute and relative number of T cell progenitor subsets in the thymus did not differ between the genotypes , CD8+ (cytotoxic T cells), CD19+ (B cells), as well as CD3-/CD19- (non-B/T cells). In addition, the proportional size of different subsets of mature B cells as defined by IgM and IgD surface expression  were also included in the analyses. Like in primary lymphatic organs, the cellularity and composition of mature lymphocyte subsets in the spleen were found similar between the C9orf82ko/ko and wild type mice  from C9orf82ko/ko and wild type littermates were compared. Antigen inexperienced B cells from C9orf82ko/ko and wild type mice showed similar percentage of switching to IgG3 and IgG1 after being exposed to LPS alone or LPS and IL4, respectively  transformed by a TP53kd were also tested for their sensitivity against the Topo II poisons, Doxorubicin and Etoposide. This also included retrovirally transduced MEFs overexpressing C9orf82 protein. The colony survival rate of MEFs from wild type was indistinguishable from that of C9orf82 protein deficient and over expressing MEFs . AnalysiAs mentioned earlier, C9orf82 protein has been associated with delaying the repair of Topo II poisons induced DSBs, measured by the kinetics of γH2AX resolution, a surrogate marker for DNA damage . If so, C9orf82ko/ko cells repair DNA damage faster than wild type and over expressing cells [Apparently, two independent systems measuring the survival of primary cells as well as primary transformed cells upon DNA damage contradict previous findings in cell lines, suggesting that ng cells , which sTo allow DNA synthesis to proceed, the positive supercoils must be relaxed. In eukaryotes this is accomplished by type I and type II topoisomerases . This leC9orf82ko/ko fetal liver pre-B cells were exposed to different concentrations of Doxorubicin for 72 hrs and then co-stained with DAPI and Annexin V for subsequent FACS measurement. Early and late apoptotic cells were distinguished as Annexin V+ DAPI- and Annexin V+ DAPI+, respectively. This analysis revealed that C9orf82 protein deficient pre-B cells were as sensitive as the wild type controls  in mature B cells excluded a critical role of C9orf82 protein in this DSB-repair dependent process.Given the previous insights on the role of C9orf82 protein in delaying DSB repair, and the dependence of early lymphocytes on Rag-induced DSB repair during V(D)J recombination, we first investigated the impact of the As we identified C9orf82 protein as a factor conferring resistance to Topo II inhibitors, we first wanted to validate our previous findings in tumor cell lines to two independent, well-established primary and secondary cellular systems, i.e. primary pre-B cell cultures and immortalized MEFs (TP53kd), respectively. Testing the DNA damage sensitivity of C9orf82 protein proficient and deficient cells, including MEFs that overexpress C9orf82 protein against various DNA damaging agents we observed, that loss of C9orf82 protein does not confer resistance to DNA damaging agents, including Topo II inhibitors.As Topo I and II play a crucial role in DNA unwinding during replication and thereby influences the cell cycle , we reasTo test anti-apoptotic potential of C9orf82 protein as suggested by , C9orf82C9orf82 and the lack of any known related protein family members argue against transspecies differences and compensatory back-up mechanisms in the absence of C9orf82 protein, respectively. The discrepancies between our primary study in human HAP1 and MelJuSo cell lines and mouse knock out system likely relate to differences in the cell type rather the strategy to knock out C9orf82. We like to note, that our initial functional analysis of C9orf82 inactivation was limited to the characterization of the HAP1 C9orf82 ko clone whereas our analysis of triple negative breast cancer patients failed to reveal any correlation between C9orf82 protein expression and the response to the treatment with doxorubicin-containing regimen [The evolutionary conserved nature of  regimen . It remaC9orf82 should be omitted as it is misleading and be reconsidered once the exact function has been identified.Future attempts that aim to determine the actual function of C9orf82 protein may benefit from an unbiased hypothesis generating approach, like combined co-immunoprecipitation/mass spectroscopy studies. The identification of specific binding partners of C9orf82 protein may pinpoint the key biological pathway(s) controlled by C9orf82 protein. We propose that the term CAAP1 for the protein encoded by in vivo as well as in vitro settings, where confounding issue scan be excluded.In conclusion, although our data failed to confirm previous findings, this study has generated a valuable, genetically well-defined mouse model and cell lines thereof. These will enable the scientific community to explore the actual role of C9orf82 protein at the systemic, organ, cellular, and molecular level


Stem Cell Reports, Morales-Hernández et al. identify one particular difference—disruption of Nicotinamide Nucleotide Transhydrogenase (Nnt)—that increases reactive oxygen exposure and impairs hematopoietic progenitor cell function in C57BL/6J, as compared to C57BL/6N, mice.C57BL/6J and C57BL/6N inbred mice are widely, and often interchangeably, used for stem cell research; yet, these substrains harbor discrete genetic differences that can cause phenotypic disparities. In this issue of  Stem Cell Reports, Morales-Hernández et al. identify one particular difference—disruption of Nicotinamide Nucleotide Transhydrogenase (Nnt)—that increases reactive oxygen exposure and impairs hematopoietic progenitor cell function in C57BL/6J, as compared to C57BL/6N, mice.C57BL/6J and C57BL/6N inbred mice are widely, and often interchangeably, used for stem cell research; yet, these substrains harbor discrete genetic differences that can cause phenotypic disparities. In this issue of  It was there that Lathrop established and maintained several colonies of mice, some of which she later sold to the Harvard geneticist William Castle. In 1921, Castle’s student, C.C. Little (who later went on to found the Jackson Laboratory), crossed female mouse 57 to male mouse 52 from Lathrop’s stocks to generate progeny termed C57 Black (C57BL). Little continued propagating these mice until he established the C57BL/6J substrain at the Jackson Laboratory in 1948. In 1951, a colony of C57BL/6J mice was transferred to the National Institutes of Health (NIH), establishing the C57BL/6N substrain, which was later distributed to several companies including Charles River Laboratories (C57BL/6NCrl), Harlan Sprague Dawley (C57BL/6NHsd), and Taconic Farms (C57BL/6NTac).The laboratory mouse has been a highly valuable tool for biological and biomedical research, and the inbred C57BL/6 strain is one of the most widely used across the globe, thanks in part to its fecundity, lifespan, and relatively low predisposition to spontaneous tumors. The origins of the C57BL/6 strain date back to the early 20Crb1 and Cyfip2, resulting in photoreceptor degeneration and a lowered cocaine response, respectively  significantly alters hematopoietic stem and progenitor cell (HSPC) frequency and function in C57BL/6N versus C57BL6/J animals.Now, nearly a century after that fateful 57 × 52 cross, both the C57BL/6J and C57BL/6N substrains serve frequently as “wild-type” controls for scientific inquiry and discovery in individual laboratories and global consortia. The Mouse Genome Sequencing Consortium and the Allen Brain Atlas, for example, chose C57BL/6J as their reference substrain, while the International Knockout Mouse Consortium  has disrupted tens of thousands of genes in the C57BL/6N background. In the minds of many investigators, these physically indistinguishable substrains A are conectively . In this biology , revealiNnt results in complete absence of Nnt protein  cells, which boosted production of mature T and B lymphocytes in recipients of C57BL/6N HSPCs. Interestingly, these substrain-specific differences in hematopoietic reconstitution were no longer statistically significant at 16 weeks after transplantation, indicating that the C57BL/6J repopulation defect affects primarily short-term reconstituting, rather than long-term reconstituting, HSPCs.Nnt is an enzyme that localizes to the inner mitochondrial membrane and functions to eliminate reactive oxygen species (ROS). In the C57BL/6J substrain, a deletion involving exons 7–11 of  protein  and redu protein . As prio protein , MoralesTo clarify the cellular mechanism underlying impaired short-term reconstitution by C57BL/6J HSPCs, Morales-Hernández and colleagues next isolated and transplanted into sub-lethally irradiated recipients 5 different subsets of HSPCs from C57BL/6N or C57BL/6J mice: Long-Term (LT)-HSCs, Short-Term (ST)-HSCs, and three different lineage-biased populations of multipotent progenitor cells . Analysis of hematopoietic reconstitution via peripheral blood cell chimerism revealed that C57BL/6N-derived MPP3 and MPP4 cells consistently showed greater frequencies of donor cell reconstitution relative to their C57BL/6J counterparts, suggesting that increased functional activity of the lymphoid-biased MPP4s, which are also relatively more abundant than other MPP populations , likely Nnt in the C57BL/6J substrain and prior work demonstrating functional deficits in HSPCs from mice with overabundant ROS, the authors next tested whether the defects in short-term hematopoietic reconstitution they observed in the C57BL/6J substrain might be due to impaired ROS clearance in Nnt mutant HSPCs. Using a fluorescent probe to monitor ROS levels, the authors found that C57BL/6J-derived CLPs indeed exhibited higher ROS levels than C57BL/6N-derived CLPs following transplantation. Furthermore, experiments using polyinosinic:polycytidylic acid (pI:pC) to increase oxidative stress revealed that C57BL/6J HSPCs exhibited higher ROS levels in in vivo transplantation assays and ex vivo colony formation assays. Finally, the authors linked their findings of increased ROS back to the Nnt deficiency within the C57BL/6J substrain. Using shRNAs to suppress Nnt in C57BL/6N HSPCs (thereby mimicking the Nnt deficiency of the C57BL/6J substrain), the authors showed that Nnt knockdown in C57BL/6N HSPCs recapitulated the impaired short-term hematopoietic reconstitution activity and increased ROS levels observed with C57BL/6J HSPCs. Taken together, these findings support the conclusion that Nnt disruption in C57BL/6J mice leads to impaired ROS clearance and compromised short-term reconstituting HPSC function, particularly within the lymphoid compartment, after transplantation  of published scientific reports using genetically modified mice fail to specify which C57BL/6 substrain was used . Issues /6N mice  could alIn conclusion, as unknown or uncontrolled substrain differences have the potential to impact any experiment where mixed genetic compositions of “control” and “experimental” genotypes could unintentionally confound research results, the results of Morales-Hernández et al. mark an important “call to action” for both researchers and scientific journals to increase their efforts to ensure proper selection and reporting of mouse strains and substrains in scientific publications and to review the detailed genetic compositions of their own “C57BL/6” mice.


Three-dimensional modeling of the HMA4 protein in Arabidopsis reveals the zinc permeation pathway across the plasma membrane, and mutations in the pathway alter zinc and cadmium transport differently in plants. 1B ATPase heavy metal ATPase 4 (HMA4) is responsible for zinc and cadmium translocation from roots to shoots in Arabidopsis thaliana. It couples ATP hydrolysis to cytosolic domain movements, enabling metal transport across the membrane. The detailed mechanism of metal permeation by HMA4 through the membrane remains elusive. Here, homology modeling of the HMA4 transmembrane region was conducted based on the crystal structure of a ZntA bacterial homolog. The analysis highlighted amino acids forming a metal permeation pathway, whose importance was subsequently investigated functionally through mutagenesis and complementation experiments in plants. Although the zinc pathway displayed overall conservation among the two proteins, significant differences were observed, especially in the entrance area with altered electronegativity and the presence of a ionic interaction/hydrogen bond network. The analysis also newly identified amino acids whose mutation results in total or partial loss of the protein function. In addition, comparison of zinc and cadmium accumulation in shoots of A. thaliana complemented lines revealed a number of HMA4 mutants exhibiting different abilities in zinc and cadmium translocation. These observations could be instrumental to design low cadmium-accumulating crops, hence decreasing human cadmium exposure.The P IB ATPases, a subfamily of P-type ATPases, are metal cation transporters essential for the survival of organisms ranging from bacteria to eukaryotes such as plants and animals  transporters. This subgroup is found in all domains of life and includes the well-characterized CopA bacterial transporter, as well as the human ATP7A and ATP7B proteins, whose mutations lead, respectively, to Menkes syndrome and Wilson’s disease  and are only present in bacteria and plants, with, for instance, the bacterial pumps ZntA and CadA and the Arabidopsis thaliana heavy metal ATPases 2–4 (HMA2–HMA4)  forming the TM domain, two cytoplasmic catalytic domains, the actuator domain (A domain), and the ATP-binding domain (ATP domain) divided into a nucleotide-binding domain (N domain) and a phosphorylation domain (P domain), and for most of them N- and occasionally C-terminal cytosolic extensions. As members of the P-type ATPase superfamily, these proteins use the energy of ATP to transport their substrate following the E1/E2 Post–Albers cycle  with a typical βαββαβ-fold structure containing a consensus CxxC motif binding one Cu+ or one Zn2+ ion, respectively  preceded by two additional helices in many of them (TMA and TMB). A characteristic sequence of three amino acids (CPx motif) in TM4 together with amino acids in TM5 and TM6 may determine ion selectivity 7K and DxG(x)7N on TM5 and TM6, respectively (i) of the Shigella sonnei ZntA protein, a homolog of HMA4, has recently been determined  that directs free Zn2+ ions to the intramembrane Zn2+-binding site. This site consists of C392 and C394 of the conserved CPx motif and D714 of the DxG(x)7N PIB-2 signature sequence. Their involvement in Zn2+ binding had already been characterized biochemically 7K motif] located near D714 forms a salt bridge with the latter, preventing Zn2+ reflux to the binding site  (4 in a short-day growth chamber (22 °C and 8 h d–1 photoperiod) during 7 weeks. They were then transferred in long days (16 h d–1 photoperiod) where they were supplied with 3 mM ZnSO4 for 5 weeks to allow flowering. The plants were transformed using Agrobacterium tumefaciens by floral dipping ( dipping .1) heterozygous transgenic seeds  were germinated on hygromycin B (20 µg ml–1) 1/2 MS (Murashige and Skoog) agar medium containing 1% sucrose in short days. After 14 d, seedlings were transferred to soil , watered with tap water, and grown for 5 weeks in long days prior to imaging and sample harvesting.For phenotyping on soil and metal accumulation analysis, first-generation  fragment was cloned into the pAhHMA4-2::AhHMA4::GFP pBluescript II KS+ vector . Site-directed mutagenesis, using the QuickChange Site-Directed Mutagenesis method (Agilent Technologies) and mutagenic primers (see JXB online), was then performed on the newly constructed vector to create the TM variants  on a DigiPrep Graphite Block Digestion System (SCP Science) as follows: 15 min at 45 °C, 15 min at 65 °C, and 90 min at 105 °C. After cooling, sample volumes were adjusted to 10 ml with milliQ water, and 200 µl of ≥65% HNO3 was added. Metal concentrations were determined using inductively coupled plasma atomic emission spectroscopy (ICP-AES) with a Vista-AX instrument  as described (Shoot tissues were cleaned with milliQ water and dried at 60 °C for 3 d. Shoot samples (10–50 mg of tissues) were then acid-digested in DigiPrep tubes with 3 ml of ≥65% HNO1 seeds of A. thaliana hma2hma4 plants expressing the WT and variant AtHMA4 proteins fused to green fluorescent protein (GFP) as described above were germinated on hygromycin B (20 µg ml–1) 1/2 MS agar medium containing 1% sucrose in short days. After 14 d, seedlings were transferred on the same medium without antibiotic. After 3 d, roots of 2–4 independent lines per construct from two independent experiments were analyzed. Images were collected at a 1024 × 1024 pixel resolution using a TCS SP5 inverted confocal laser microscope (Leica Microsystems) with a ×63 water immersion PlanApochromat 1.20 objective (Leica Microsystems) as previously described (https://imagej.nih.gov/ij/) and plot profile analysis. Briefly, in pericycle cells expressing HMA4 fused to GFP, 10 optical sections were drawn across the transversal plasma membranes. GFP fluorescence intensity values (n=20) were then used to calculate a mean fluorescence intensity for each independent mutant line.Thttp://www.uniprot.org). This protein has an N-terminal MBD (residues 1–96) that has been resolved by NMR (PDB ID: 2KKH) (https://blast.ncbi.nlm.nih.gov) (http://www.rcsb.org/pdb)  (https://swissmodel.expasy.org) through the QMEAN6 Z-score (The Uniprot code of the AtHMA4 protein is O64474 (sn-glycero-3-phosphocholine) bilayer of 347 lipids with the insane tool (XY and Z. Non-bonded interactions were computed up to 1.2 nm with the shift method. Electrostatics were treated with dielectric permittivity constant ε=15. The compressibility was 10–5 (1/bar). The system was then transformed to an atomistic resolution with backwards (–5 (1/bar) and τP=5 ps. Non-bonded interactions were evaluated using a twin-range cut-off scheme. Interactions within the shorter range cut-off (0.8 nm) were calculated every step, whereas interactions within the longer cut-off (1.4 nm) were updated every five steps, together with the pair list. In all the simulations, a reaction ﬁeld correction was applied to the electrostatic interactions beyond the long-range cut-off of 35 using a relative dielectric permittivity constant εRF of 62 (http://www.PyMOL.org) and VMD software  in HMA4 instead of one in ZntA (G391) . Additioin planta the functional importance of the amino acids depicted in the model, HMA4 transmembrane variants were generated by directed mutagenesis (HMA4 (WT) and mutated genes were fused to the GFP gene (HMA4::GFP) and expressed under the control of the endogenous A. halleri HMA4 promoter 2 (pAhHMA4-2) in the loss-of-function hma2hma4 A. thaliana mutant  accumulated ~5-fold higher zinc in shoots than the hma2hma4 mutant, reflecting the inability of the latter to translocate zinc from root to shoot   bordering the motif  was significantly decreased, suggesting that HMA4 might be more Zn efficient than many of its plant homologs  and Oryza sativa HMA2 (OsHMA2) and HMA3 (OsHMA3) proteins  for molecular dynamic simulations.Fig. S1. Amino acid sequence alignment of Shigella sonnei ZntA and Arabidopsis thaliana HMA4.Fig. S2. Complementation of the A. thaliana hma2hma4 zinc deficiency phenotype.Fig. S3. HMA4 TM variant localization in A. thaliana.Fig. S4. HMA4 TM variant expression level in A. thaliana.Fig. S5. Sequence conservation of the TM region among plant PIB-2 ATPases.Fig. S6. Distribution of residue types at the surface of the N-terminal MBDs of HMA4 and ZntA.Fig. S7. View of the transmembrane domain of HMA4 in the E2P and E2-Pi states in a membrane environment during molecular dynamic simulations.Fig. S8. Amino acid sequence alignment of Arabidopsis thaliana HMA4, Nicotiana tabacum HMA4, Oryza sativa HMA2, and Oryza sativa HMA3.Supplementary Figures S1-S8 and Tables S1-S2Click here for additional data file.


The prognostic value and the best method of testing of topoisomerase II alpha (TOP2A) status have not been established in modern tailored therapy based on breast cancer subtype.TOP2A amplified were 55.8% and 9.5%, respectively. TOP2A overexpression correlated strongly with non-luminal A subtype . TOP2A overexpression was significantly associated with relapse-free survival in luminal B breast cancer  but not in other breast cancer subtypes. Cox regression analysis showed that TOP2A overexpression is a significant prognostic factor in luminal B breast cancer (hazard ratio (HR) 4.00, 95% confidence interval (CI) 1.65–9.54, p = 0.002). TOP2A amplified was recognized in HER2 positive breast cancer (p < 0.001). In HER2 positive breast cancer, TOP2A amplified  appeared to be a better prognostic factor.The frequencies of TOP2A overexpression and TOP2A amplified could be a better prognostic factor in HER2 positive breast cancer.In modern tailored therapy, TOP2A overexpression can be a poor prognostic factor in luminal B breast cancer. In contrast, TOP2A amplified using fluorescence in situ hybridization analysis (FISH) and for TOP2A overexpression using the immunohistochemistry assay. FISH ratios of 2 or higher were designated as TOP2A amplified, and TOP2A staining >10% was defined as TOP2A overexpression. The prognostic values of TOP2A amplified and TOP2A overexpression were retrospectively evaluated.Between May 2005 and April 2015, a total of 643 consecutive non-metastatic invasive breast cancers were evaluated for  TOP2A amplified or deleted are predictive markers of anthracycline-containing adjuvant chemotherapy regimens for early breast cancer [Topoisomerase IIA TOP2A) is an essential nuclear enzyme that cleaves and recombines double-stranded DNA in processes such as replication, transcription, condensation, and segregation . TOP2A iA is an et cancer –13.Although TOP2A status has several important implications in breast cancer, the standard tools and cutoff values for estimating TOP2A status have not been established . AdditioTOP2A amplified had been consecutively evaluated using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in patients with breast cancer between May 2005 and April 2015. During this period, adjuvant therapies, including taxane and HER2 targeting therapy, were administered based on breast cancer subtype and the risk for recurrence. We retrospectively evaluated the prognostic value of TOP2A status in these patients with non-metastatic breast cancer.At our institution, both TOP2A overexpression and TOP2A amplified, normal, and deleted were 61 (9.5%), 577 (89.7%), and 5 (0.8%), respectively. The associations of the clinicopathological factors with TOP2A overexpression and TOP2A amplified are shown in Tables p < 0.001), node positivity (p < 0.001), high nuclear grade (p < 0.001), ER negativity (p < 0.001), PgR negativity (p < 0.001), HER2 positivity (p < 0.001), and high Ki67 index (p < 0.001) using the χ2-test. TOP2A overexpression was recognized in non-luminal A subtype . Although the associations between TOP2A amplified and several clinicopathological factors, including larger T, high nuclear grade, and high Ki67 index, were significant, almost TOP2A amplified was observed in HER2 positive breast cancers; specifically, the rates of TOP2A amplified were 42.2% (57/135) and 0.8% (4/508) in HER2-positive and -negative cancers, respectively. Thus, the characteristics of TOP2A amplified breast cancer reflected those of HER2 positive breast cancer.The median and mean of TOP2A expression were 11.3% and 18.4%, respectively. Histogram of continuous IHC proportion scores of TOP2A is shown in . The frep < 0.001, log-rank test)  Figure .p < 0.001, log-rank test); however, no such significant association was observed in other subtypes  . A continuous value of TOP2A expression was also shown to be the significant prognostic factor in luminal B breast cancer in Cox regression analysis .In the subgroup analysis of breast cancer subtypes, TOP2A overexpression was a significant prognostic factor in luminal B breast cancer . As exploratory analysis, the prognostic value of adjuvant anthracycline therapy was examined with respect to the presence of TOP2A amplified; however, adjuvant anthracycline therapy was not associated with survival advantages for patients with or without TOP2A amplified  [TOP2A status  [TOP2A amplified compared anthracycline-based chemotherapy regimens with CMF [TOP2A amplified may lack clinical meaning.TOP2A is known as a molecular target of anthracycline, and previous meta-analysis has shown that regimens . A meta- studies , 28. Unlotherapy , 33. In vs. 83%) . Second,with CMF , which iwith CMF . ConsideTOP2A amplified in anthracycline treatment.This study has some limitations. First, this retrospective analysis from a single institution could have biases and a multiple-institutional prospective study is needed to confirm our results. Second, PgR expression and Ki67 index were substituted for a multigene expression classifier, such as Oncotype Dx or Mammaprint, to distinguish luminal B from luminal A breast cancer in this study. Some discrepancies between the multi-gene assay and analysis of conventional pathological markers in determining intrinsic subtype may exist. Third, a small number of cases and events in HER2 positive breast cancer is underpowered to make conclusion about the predictive value of TOP2A amplified is limited in HER2 positive breast cancer, and the presence of the TOP2A amplified does not influence the survival advantage of adjuvant anthracycline therapy. To tailor the adjuvant therapy based on breast cancer subtype, the assessment of TOP2A overexpression may be considered for risk assessment in luminal B breast cancer.In conclusion, our study indicates that TOP2A overexpression is a marker for poor prognosis, especially in cases of luminal B breast cancer. In contrast, the TOP2A amplified. The prognostic value of TOP2A status was retrospectively evaluated in this study. The Kure Medical Center review board approved this study (29–23). The requirement for informed consents from individual patients was waived because this was a retrospective review of a prospectively maintained patient database.Between May 2005 and Apr 2015, a total of 643 consecutive non-metastatic invasive breast cancers were evaluated at the Kure Medical Center and the Chugoku Cancer Center, Kure, Japan, for their TOP2A status using IHC to analyze TOP2A overexpression and FISH analysis for The clinicopathological factors in this study, derived from a prospectively maintained database at our institute, included patient age, histological type of cancer, prescribed neoadjuvant therapy, T and N stage according to the TNM classification, nuclear grade, estrogen receptor (ER) status, progesterone receptor (PgR) status, human epidermal growth factor receptor 2 (HER2) status, Ki67 index, breast cancer subtype, prescription of anthracycline and taxane, and recurrence Table . ER and Luminal A: ER positive, HER2 negative, Ki67 <14%, and PgR positive ≥20%Luminal B: ER positive, HER2 negative, and Ki67 ≥14%, or PgR positive <20%HER2: HER2 positive, and any one of the following: ER, PgR and Ki67Triple negative breast cancer (TNBC): ER, PgR, and HER2 negativeAdjuvant therapy was essentially determined in accordance with clinical guidelines. Patients with luminal A or B breast cancers were administered adjuvant endocrine therapy for 5 years. The determination of adjuvant chemotherapy for luminal type breast cancer was based on breast cancer recurrence risks and the preference of the patients. One-year adjuvant trastuzumab therapy was administered in cases of HER2 positive breast cancer. Patients with TNBC received adjuvant chemotherapy. Adjuvant chemotherapy essentially consisted of anthracycline- and taxane-based regimens administered individually or in combination. Postoperative follow-up was performed in accordance with the guidelines for medical checks and annual mammography. In cases with signs of recurrence, image tests and biopsies were performed to confirm the status. Recurrence-free survival (RFS) was defined as the elapsed time from the date of surgery until the date of the first event (relapse or death from any cause) or of last follow-up.TOP2A amplified was evaluated using FISH analysis, as described previously [TOP2A gene and a spectrum green-labeled centromere control for chromosome 17 were evaluated (DAKO). FISH ratios of 2 or higher, 0.8 to 1.9, and <0.8 were designated as TOP2A amplified, normal, and deleted, respectively. Representative microscopic findings of TOP2A/CEP17 for TOP2A amplified and normal breast cancer are shown in . A p-value of < 0.05 was considered as statistically significant.The association between the clinicopathological factors and TOP2A status was assessed using the χ


Candida albicans is an opportunistic pathogen that normally lives on the human body without causing any harm. However, C. albicans is also a dangerous pathogen responsible for millions of infections annually. C. albicans is such a successful pathogen because it can adapt to and thrive in different environments. Chemical modifications of chromatin, the structure that packages DNA into cells, can allow environmental adaptation by regulating gene expression and genome organization. Surprisingly, the contribution of chromatin modification to C. albicans biology is still largely unknown. For the first time, we analyzed C. albicans chromatin modifications on a genome-wide basis. We demonstrate that specific chromatin states are associated with distinct regions of the C. albicans genome and identify the roles of the chromatin modifiers Sir2 and Set1 in shaping C. albicans chromatin and gene expression.The fungus  Candida albicans, a prevalent fungal pathogen in humans, can rapidly adapt and thrive in diverse host niches. The contribution of chromatin to C. albicans biology is largely unexplored. Here, we generated the first comprehensive chromatin profile of histone modifications  across the C. albicans genome and investigated its relationship to gene expression by harnessing genome-wide sequencing approaches. We demonstrated that gene-rich nonrepetitive regions are packaged into canonical euchromatin in association with histone modifications that mirror their transcriptional activity. In contrast, repetitive regions are assembled into distinct chromatin states; subtelomeric regions and the ribosomal DNA (rDNA) locus are assembled into heterochromatin, while major repeat sequences and transposons are packaged in chromatin that bears features of euchromatin and heterochromatin. Genome-wide mapping of γH2A, a marker of genome instability, identified potential recombination-prone genomic loci. Finally, we present the first quantitative chromatin profiling in C. albicans to delineate the role of the chromatin modifiers Sir2 and Set1 in controlling chromatin structure and gene expression. This report presents the first genome-wide chromatin profiling of histone modifications associated with the C. albicans genome. These epigenomic maps provide an invaluable resource to understand the contribution of chromatin to C. albicans biology and identify aspects of C. albicans chromatin organization that differ from that of other yeasts.Eukaryotic genomes are packaged into chromatin structures that play pivotal roles in regulating all DNA-associated processes. Histone posttranslational modifications modulate chromatin structure and function, leading to rapid regulation of gene expression and genome stability, key steps in environmental adaptation.  Packaging of genomes into chromatin is the key determinant of nuclear organization , 2. The 3) and acetylated on lysine 9 (H3K9Ac), while coding sequences are enriched in a different set of histone modifications, such as acetylated lysine 16 on histone H4 (H4K16Ac)  and is enriched in repressive histone marks such as methylation of lysine 9 on histone H3 (H3K9me) and methylation of lysine 27 on histone H3 (H3K27me)  –10. In c4K16Ac) –.These re4K16Ac) –, 11, 12.Saccharomyces cerevisiae, heterochromatin is devoid of H3K9me and H3K27me marks but contains hypomethylated H3K4 and hypoacetylated H3K9 and H4K16  depend on the chromatin state of the genomic region undergoing repair. Furthermore, extensive chromatin changes, including recruitment of γH2A, are linked to repair of DNA breaks . ConsequChromatin modifications are reversible, and specific histone modifiers maintain or erase the histone modification state associated with different chromatin regions. Among these, histone acetyltransferases (HATs) and histone deacetylases (HDACs) maintain and erase histone acetylation, respectively, while histone methyltransferases (HMTs) and demethylases (HDMs) are responsible for the methylation state of histones , 3. ThesCandida albicans. C. albicans is a benign commensal organism that colonizes the mouth, the skin, and the urointestinal and reproductive tracts of most individuals. However, C. albicans is also the most common causal agent of invasive fungal infections, and systemic infections are associated with high mortality rates (up to 50%)  . C. albiaptation .C. albicans genome has 8 diploid chromosomes that contain 6,408 annotated protein-coding genes, as well as a large number of noncoding RNAs (ncRNAs) (–TLO genes (The (ncRNAs) –28. The ncRNAs) –. TelomerLO genes , 30.The rDNA locus consists of a tandem array of ∼12-kb units repeated 50 to 200 times. Each unit contains the two highly conserved 35S and 5S rRNA genes that are separated by nontranscribed spacer region 1 (NTS1) and NTS2, whose sequences are not conserved with other eukaryotes , 31.C. albicans chromosomes  units flanked by nonrepetitive HOK and RBP-2 elements. Each RBP-2 element contains a protein-coding gene, FGR6, important for morphological switching (MRS loci are long (10-to-100-kb) tracts of nested DNA repeats found on 7 of the 8 omosomes , 32. Thewitching .C. albicans genome, including 16 classes of LTR retrotransposons (Tca1 to Tca16) and Zorro non-LTR retrotransposons that are present in 5 to 10 copies per cell, dispersed along the chromosomes. Among those, Tca2, Tca4, Tca5, Zorro-2, and Zorro-3 are capable of transposition  events, chromosome rearrangements, and aneuploidy , marking euchromatic and heterochromatic regions and potential recombination-prone unstable sites. Genome-wide mapping of RNA polymerase II (RNAPII) and transcriptome expression profiling unveiled the link between histone modification states and transcriptional activity. We demonstrate that specific chromatin states are associated with the repetitive and nonrepetitive C. albicans genomes. While gene-rich regions are associated with active chromatin marks mirroring their transcriptional state, different types of repetitive elements are assembled into distinct chromatin types. Finally, we present the first C. albicans quantitative ChIP-seq (q-ChIP-seq) methodology, which has permitted us to elucidate the roles of HDAC Sir2 and HMT Set1 in shaping the chromatin state of C. albicans genome and regulating gene expression.In this study, we used chromatin immunoprecipitation with massively parallel deep sequencing (ChIP-seq) technology to establish the first comprehensive genome-wide map of C. albicans genome contains two homologous pairs of divergently transcribed histone H2A and H2B genes as well as histone H3 and H4 genes in addition to a single histone H3 gene  Histone gene names and locations, according to assembly 22. (B) Alignment of C. albicans H2A, H3, and H4 protein sequences against S. cerevisiae and S. pombe homologues. Residues highlighted in red were investigated in this study and are conserved across all species. Download FIG S1, TIF file, 0.9 MB.(A) (Top) Diagram of genome organization of Copyright © 2019 Price et al.2019Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans repetitive and nonrepetitive regions, we globally mapped the genomic locations of H3K4me3, H3K9Ac, H4K16Ac, and γH2A by performing chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Since nucleosomes are not equally distributed across genomes, we accounted for nucleosome occupancy by performing genome-wide profiling of unmodified histone H3 and histone H4. To correlate specific histone modification profiling with transcriptional activity, we performed genome-wide mapping of RNA polymerase II (RNAPII) occupancy. We also performed transcriptome analysis by strand-specific RNA sequencing (RNA-seq) to profile gene expression levels.To explore the chromatin signature of C. albicans wild-type (WT) cells grown under standard laboratory growth conditions  using antibodies specific for modified or unmodified histones. Input (I) samples and immunoprecipitated (Ip) samples were sequenced using an Illumina HiSeq2000 platform , with the exception of centromeric regions, where the histone H3 variant Cse4CENP-A replaced histone H3 (r = 0.72) (ChIP-seq was performed using s genome . Unmodifstone H3 . Further = 0.72) .10.1128/mBio.01376-19.2FIG S22) of histone H3 relative to unmodified H4 across the centromeric and pericentromeric regions of all 8 chromosomes in C. albicans. The CenpA enrichment profile is shown for comparison. The blue bars indicate statistically significantly depleted regions for histone H3. (B) Histogram of RNA-seq counts (log2) in WT cells across all protein-coding genes, according to assembly 22. Genes were grouped into four sets organized by expression level as follows: no expression , low expression , medium expression  and high expression . Download FIG S2, TIF file, 0.9 MB.(A) Fold enrichment  and 5′ regions of genes, and H4K16Ac was enriched across gene bodies  as revealed by RNA-seq analysis . Enrichmed genes . TherefoC. albicans coding genes, we analyzed the chromatin state of the C. albicans repetitive genome, focusing on the major classes of DNA repeats: subtelomeric regions, the rDNA locus, MRSs, and transposable elements (LTR and non-LTR retrotransposons). Sequence analysis of these elements can be problematic because of incomplete sequencing and because of their repetitive nature  of H3K4me3, H3K9Ac, H4K16Ac, γH2A, and H3 relative to unmodified H4 across the 20-kb left and right terminal regions of each of the 8 C. albicans chromosomes. (Middle) Diagrams of coding genes found at these regions (grey: TLO), according to assembly 22. (Bottom) Diagram depicting statistically significantly enriched (red) or depleted (blue) domains for each histone modification. Download FIG S3, TIF file, 1.1 MB.(Top) Fold enrichment  are associated with chromatin, which is largely hypomethylated on H3K4  and D. IC. albicans genome are marked by different chromatin modifications depending on their transcriptional state  than the 50-kb γH2A domains surrounding DSBs  Regions of enriched/depleted histone modifications compared to unmodified histone H4 in WT cells, as determined by DESeq2. (F – H) Regions of enriched/depleted histone modifications in mutant strains compared to the WT cells, as determined by two sample t-test.  RNA-seq analysis in mutant strains compared to the WT cells, as determined by DESeq2. Download Copyright © 2019 Price et al.2019Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Analysis of γ-sites indicates that they are present with three classes of genomic loci: (i) subtelomeric regions, (ii) longer genes, which are often convergent; and (iii) origins of replication  Fig. 4A.We detected statistically significant γH2A enrichment at 13/16 subtelomeres . We suspr = 0.062), γ-sites are more likely to mark genomic regions that are acetylated on H4K16 and H3K9 . We also detected a weak negative correlation between γH2A occupancy and H3K4me3 occupancy  for each histone modification normalized to unmodified histone H4 is displayed within a region spanning ±2 kb around the γH2A peak summits. The blue-to-red color gradient indicates high to low levels of enrichment in the corresponding region. MIN (minimum), −1.5 log2; MAX (maximum), +1.5 log2. Download FIG S4, TIF file, 1.5 MB.Chromatin signature of γ-sites throughout the Copyright © 2019 Price et al.2019Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the +URA3 marker gene integrated at different genomic locations (URA3 loss was more frequent when the gene is integrated in proximity to γ-sites (rDNA locus and subtelomeres and less than 15 kb away from a γ-site[s]) than when integrated far away (>30 Kb) from γ-sites  of H3K9Ac and H4K16Ac relative to unmodified H4 in WT cells, and relative to the WT in sir2 Δ/Δ cells, across the 20 kb left and right terminal regions of each of the 8 C. albicans chromosomes. (Bottom) Diagrams of coding genes found at these regions (grey: TLO), according to assembly 22. Download FIG S5, TIF file, 0.9 MB.(Top) Fold enrichment  or downregulated  in strain set1Δ/Δ compared to the WT  of H3K4me3 relative to unmodified H4 in WT cells, and relative to the WT in set1 Δ/Δ cells, across the 20 kb left and right terminal regions of each of the 8 C. albicans chromosomes. (Bottom) Diagrams of coding genes found at these regions (grey: TLO), according to assembly 22. Download FIG S6, TIF file, 0.8 MB.(Top) Fold enrichment  the use of antibodies that recognize modified histones with high specificity and (ii) the use of appropriate biological controls. The specificity of antibodies used in this study has been tested using S. pombe or S. cerevisiae histone mutants lacking the modifiable amino acid  (Our first objective was to obtain “proof-of-concept” epigenomic maps of chromatin modifications associated with gene-rich regions of the H2AS129) , 59. To C. albicans active chromatin conforms to the histone modification pattern reported in other organisms: active genes, associated with high levels of RNA Pol II, are assembled into canonical euchromatin, with H3K4me3 and H3K9Ac associated with promoters and H4K16Ac enriched at gene bodies. Thus, the specific histone modification pattern predictive of active transcription in other organisms is conserved in C. albicans.The results confirm that the experimental approach is valid and that C. albicans genome that are more likely to have properties of heterochromatin. The chromatin in NTS regions of the rDNA locus and in the subtelomeric regions resembles the heterochromatic structure of the budding yeast S. cerevisiae, which lacks H3K9me/H3K27me systems  Sir2 is specifically targeted to subtelomeres and the rDNA locus or (ii) other histone deacetylases act redundantly with Sir2 in regulating hypoacetylation and gene expression at other genomic locations. In support of the second hypothesis, note that the C. albicans genome encodes 9 histone deacetylases and that functional redundancy among these enzymes has been reported in C. albicans and other organisms , anti-H3 , anti-H4 , anti-H3K4me3 , anti-H3K9ac , anti-H4K16ac , and anti-RNA polymerase II .600] = 0.6 to 0.8). Cells (OD600 = 20) were fixed with 1% formaldehyde (Sigma) for 15 min at room temperature. Reactions were quenched by the addition of glycine to reach a final concentration of 125 mM. Cells were lysed using acid-washed glass beads (Sigma) and a DisruptorGenie (Scientific Industries) for four cycles of 30 min at 4°C with 5 min on ice between cycles. Chromatin was sheared to 200 to 500 bp using a BioRuptor sonicator (Diagenode) for a total of 20 min  at 4°C. Immunoprecipitation was performed overnight at 4°C using 2 μl of the appropriate antibody and 25 μl of protein G magnetic Dynabeads (Invitrogen). ChIP DNA was eluted, and cross-links were reversed at 65°C in the presence of 1% SDS. All samples were then treated with RNase A and proteinase K before being purified by phenol/chloroform extraction and ethanol precipitation. Libraries were prepared and sequenced as 50-bp single-end reads on an Illumina HiSeq2000 platform by the Genomics Core Facility at EMBL . All ChIP-seq experiments were carried out in biological duplicate.Chromatin immunoprecipitation with massively parallel deep sequencing (ChIP-seq) was performed as follows. A 5-ml volume of an overnight culture grown in YPAD was diluted into fresh YPAD and grown until the exponential phase was reached . C. albicans cells were combined with S. cerevisiae cells (OD600 = 10) and then fixed with 1% formaldehyde (Sigma) for 15 min at room temperature. After the cells had been fixed, the q-ChIP-seq sample was processed as a single ChIP-seq sample throughout the experiment until completion of DNA sequencing. All q-ChIP-seq experiments were carried out in biological duplicate.Quantitative ChIP-seq (q-ChIP-seq) was performed similarly to the ChIP-seq method, except 5 ml of an overnight culture of 600 = 0.6 to 0.8) using a yeast RNA extraction kit  following the manufacturer’s instructions. RNA quality was checked by electrophoresis under denaturing conditions in 1% agarose–1× HEPES–6% formaldehyde (Sigma). RNA concentrations were measured using a NanoDrop ND-1000 spectrophotometer. Strand-specific cDNA Illumina barcoded libraries were generated from 1 μg of total RNA and sequenced as 50-bp single-end reads using an Illumina HiSeq2000 sequencer by the Genomics Core Facility at EMBL . All RNA-seq experiments were carried out in biological duplicate.RNA was extracted from exponential cultures  were pregrown overnight from independent single colonies. Each culture was diluted to a concentration of 100 cells/μl and grown for nine generations (18 h). Cells were plated on synthetic complete (SC) plates containing 1 mg/ml 5-FOA  and on nonselective SC plates and grown at 30°C. Colonies were counted after 2 days of growth, and the frequency of 5‐FOA‐resistant (5‐FOAr) colony appearance  was calculated using the formula F = m/M, where m represents the median number of colonies obtained on 5‐FOA medium corrected by the dilution factor used and the fraction of culture plated and M the average number of colonies obtained on YPD corrected by the dilution factor used and the fraction of culture plated . For each coverage track, reads per million (RPM) were calculated. The histone modification coverage tracks were normalized to unmodified histone H4, and the RNAPII track was normalized to the corresponding input sample. All coverage tracks were visualized using IGV (Illumina reads were mapped using Bowtie2  to a cuss genome . Reads ts genome  and the s genome . For eac package  69). Ge. GeC. al package , and datsing IGV . Metaplo10.1128/mBio.01376-19.7FIG S73, H3 and H4 IPs in WT strains; (B) H3K9Ac, H4K16Ac IP in WT and sir2 Δ/Δ strains; (C) H3K4me3 IP in WT and set1 Δ/Δ strains; (D) RNA-seq WT and sir2 Δ/Δ strains; (E) RNA-seq in WT and set1 Δ/Δ strains. Download FIG S7, TIF file, 1.7 MB.Heat maps of pairwise correlations between different ChIP-seq and RNA-seq samples. The Pearson correlation coefficients are shown. The gradient red-to-blue color indicates the strength of correlation between the corresponding samples. (A) H3K9Ac, H4K16Ac, H2AS129P, H3K4meCopyright © 2019 Price et al.2019Price et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (ii) q-ChIP-seq analysis. To isolate the reads that uniquely aligned to the C. albicans genome, the full data sets were first aligned to the S. cerevisiae genome (sacCer3). The unaligned reads were outputted as separate fastq files, and these reads were then aligned to a custom haploid version of assembly 22 of the C. albicans genome , according to the method described previously by Orlando et al. , and the mutant strain track coverage data were normalized to the WT coverage. All tracks were visualized using IGV . Ge. Ge(ii)  package , and RRPsing IGV . Metaplo(iii) RNA-seq analysis. Reads were aligned to a custom haploid version of assembly 22 of the C. albicans genome using HISAT2 , an, an(iii)g HISAT2 . Differekage see  69). Sc. Sc(iii)https://www.ncbi.nlm.nih.gov/bioproject) under BioProject identifier (ID) PRJNA503946. Coverage tracks and genome files used in our analyses are available at https://www.kentfungalgroup.com/price-2019-data.Raw data sets generated and analyzed during this study are available in the BioProject NCBI repository (


Here, we used an ethylene (ET) receptor mutant Nr and its wild-type to determine the function of the ET signaling pathway in O3-induced leaf senescence, and bottom-up effects on the performance of Bemisia tabaci in field open-top chambers (OTCs). Our results showed that elevated O3 reduced photosynthetic efficiency and chlorophyll content and induced leaf senescence of plant regardless of plant genotype. Leaf senescence in Nr plants was alleviated relative to wild-type under elevated O3. Further analyses of foliar quality showed that elevated O3 had little effect on phytohormone-mediated defenses, but significantly increased the concentration of amino acids in two plant genotypes. Furthermore, Nr plants had lower amino acid content relative to wild-type under elevated O3. These results provided an explanation of O3-induced increase in abundance of B. tabaci. We concluded that O3-induced senescence of plant was ET signal-dependent, and positive effects of O3-induced leaf senescence on the performance of B. tabaci largely resulted from changes of nutritional quality of host plants.Elevated ozone (O Our specific goals were to determine the differences in these two plant genotypes in (i) leaf senescence under elevated O3; (ii) nitrogenous nutrition and resistance; and (iii) population abundance of B. tabaci.smission . Underst3 treatments were set up as: current tropospheric O3 levels (40 nL L-1) and elevated O3 levels (90 nL L-1). The O3 treatment was performed in four paired OTCs. Each OTC with elevated O3 was matched with one OTC with ambient O3. The OTCs were ventilated with air daily from 8:00 a.m. to 6:00 p.m. In the elevated O3 treatment, the method of O3 generation was offered by 3 concentrations were monitored  four times per day throughout the studies to keep relatively stable O3 levels within the OTCs. The O3 levels throughout the research were 42 ± 3.8 nL L-1 in the ambient O3 OTCs and 89 ± 5.3 nL L-1 in the elevated O3 OTCs. Air temperatures were measured and there was not obviously difference between the two treatments (22.7 ± 1.9°C in ambient O3 chambers vs 24.2 ± 2.0°C in elevated O3 chambers).The field experiments were carried out in eight 2.1 m diameter and 2 m height octagonal, open-top chambers (OTCs) at the Observation Station of the Global Change Biology Group, Institute of Zoology, Chinese Academy of Sciences in Xiaotangshan County, Beijing, China . The ONever ripe (Nr) tomato plants were kindly supplied by Professor Chuanyou Li . The N-terminal coding region of an ET receptor (Le-ETR3/NR) was associated with a single base substitution in ET-insensitive mutation Nr. Nr plants showed defects in the ET-induced triple response in etiolated hypocotyls and also exhibited the lack of fruit ripening (Wild-type Ailsa Craig (AC) and ET-insensitive mutation ripening .Nr plants. The insecticides were not used throughout the research. The plants were irrigated every 2 days.The germinated seeds were individually sown into approximate 1.5-L small pots. Tomato plants were maintained in the OTCs for 43 days from seedling with two to three leaves to the end of the experiment (19 May to 30 June 2015). The position of pot within each chamber was re-randomized once every week. There were 88 tomato plants within each OTC , which contained 54 AC plants and 34 B. tabaci Mediterranean genetic group, also called Q biotype, was kindly provided by Professor Youjun Zhang . The B. tabaci population was maintained on the cotton plants in separated cages in a greenhouse at 25 ± 2°C and 75 ± 10% relative humidity, with a photoperiod of 14 h light: 10 h dark. The 30 adults were sampled to determine the B. tabaci biotypes of colony by sequencing a molecular marker mtCO I  gene (The  I) gene .3 exposure, 320 plants in total in eight OTCs, which contained 40 tomato plants (30 AC plants and 10 Nr plants) with uniform size in per OTC, were randomly selected. Ten AC plants were sprayed with ACC  and 10 AC plants were sprayed with 1-MCP . Ten AC plants and 10 Nr plants were sprayed with H2O (AC/H2O and Nr/H2O), which was regarded as control treatment. Both sides of leaves were sprayed once every two days at 8:00 a.m. along the 43 days of the experiment. Treatment of samples with ACC was conducted by dissolving 30 mg of ACC in 1 L of distilled water with 100 μL L-77 at a final concentration of 50 ppm. The final concentration of 1-MCP was 1 ppm by dissolving 0.005 g 1-MCP into distilled water (1 L) with 100 μL L-77 (3 fumigation (19 May to 25 June 2015), the leaves from 40 tomato plants of each OTC  were collected and immediately frozen in liquid nitrogen to analyze ROS accumulation, ET emission, and the expression of ET synthase genes.During O μL L-77 . After 3B. tabaci. After 21-day O3 fumigation (19 May to 9 June 2015), 128 tomato plants in total in eight OTCs, which contained 16 tomato plants (eight AC plants and eight Nr plants) with uniform size in per OTC, were randomly selected for 21-day B. tabaci infestation experiment. Each plant was inoculated with five pairs of newly emerging B. tabaci. The B. tabaci was maintained in a clip-cage to develop and produce offspring on tomato plants for 3 weeks. All B. tabaci stages  on per plant were counted as the B. tabaci abundance in 30 June 2015.Plants were arranged for two different treatments with Nr plants) with uniform size in per OTC, were randomly selected after 41-day O3 fumigation (19 May to 28 June 2015). Each plant was damaged with ten pairs of newly emerging B. tabaci. The B. tabaci, which was maintained within a clip-cage, infested freely for 24 h. Another 128 tomato plants in total in eight OTCs, which included 16 tomato plants (eight AC plants and eight Nr plants) with uniform size in per OTC, were also randomly selected after 41-day O3 fumigation (19 May to 28 June 2015). These plants were not infested with B. tabaci, which served as un-infested control. The un-infested and 24 h-infested leaves of each tomato plants were harvested in 29 June 2015 and immediately frozen in liquid nitrogen for amino acid, N concentration, hormone, ET emission, and the expression of hormone-signal related genes analysis.In the second part of this experiment, 128 tomato plants in total in eight OTCs, which 16 tomato plants . Leaf chlorophyll content of tomato plant was measured with a Minolta SPAD-502 plus .2DCFDA , which was dissolved in DMSO, and incubated for 10 min in darkness at room temperature. Fluorescence absorbance was determined by a SpectraMax i3 . The quantification of total protein was measured by Bradford dye. The ROS production was expressed as relative fluorescence units (RFUs) per milligram of protein.Frozen powder, which was hand ground in liquid nitrogen, was weighed and immediately homogenized with 10 mM Tris-HCl buffer (pH 7.3). The homogenized extract was centrifuged twice at 15,000 rpm for 5 min. The quantification of ROS was determined by 10 mM Ho-phthaldialdehyde (OPA) and 9-fluorenylmethyloxycarbonyl (FMOC). The quantification of amino acids was calculated with reference to the standard curves of AA-S-17  amino acid mixture, supplemented with asparagine, glutamine, and tryptophan . Free amino acid concentrations of the five standard solutions were 250, 100, 50, 25, and 10 pmol μL-1. The mixed sample with 10 μL amino acid sample, 20 μL sodium borate buffer , 10 μL OPA, 10 μL FMOC, and 50 μL water was injected to the HPLC . The HPLC analysis was performed using a method provided by TM instruments, Model 2100, Hillerød, Denmark).Approximately 0.2 g leaf samples were homogenized in liquid nitrogen, and then were extracted within 2.5 mL of cold 5% acetic acid. The extraction was agitated for 1 h on a shaker  at room temperature. Homogenates were centrifuged at 4000 rpm for 15 min, and the supernatants were used for leaf individual amino acid analyses. The leaf amino acids were measured by reverse-phase HPLC with precolumn derivatization using -1 to 250°C and held for 10.8 min. The flow rate of hydrogen carrier gas was 40 mL min-1 and was detected by flame ionization detector (FID). The ET production rate was estimated by comparison of sample peak areas of known ET standards  and corrected for tissue fresh weight and the duration of incubation to determine ET emission rate.The ET emission from leaves was determined according to 2Cl2, and then agitated for 30 min on a shaker at 4°C. The homogenized sample was centrifuged at 13,000 g for 10 min. After centrifugation, the lower layer was collected, and then was concentrated in a dry machine. The concentrated sample was re-solubilized in 200 μL of MeOH. Next, 1 μL of the sample was injected into an Agilent ZORBAX SB-Aq column  for hormone analysis. The hormone contents were calculated with reference to standard curves.The foliar hormone was measured according to 3) containing 100 μL 0.6% (w/v) azocasein , and the mixture was incubated at 37°C for 3 h. The reaction was terminated by adding 400 μL 10% TCA . The mixture was maintained at 4°C for 30 min and then centrifuged at 4°C, 10,000 rpm for 10 min. The absorbance of the filtrate at 366 nm was determined by fluorescence spectrophotometry  at 4°C for 30 min. The homogenized extract was centrifuged at 4°C, 12,000 rpm for 40 min. After centrifugation, the upper layer was collected for protease activity analysis. The supernatants (100 μL) was mixed with 600 μL 50 mM pH 8.0 Na-Pi buffer  to determine the mRNA levels. Specific primers for each gene were designed from The EST sequences was used to design specific primers for target genes using PRIMER5 software (Table 1). The qPCR reactions were performed in a 20 μL total reaction volume that included 10 μL of 2× SYBR Premix EX TaqTM (Qiagen) master mix, 5 mM of each gene-specific primer, and 1 μL of pure cDNA template. Reactions were carried out using the Mx 3000P detection system (Stratagene), with the parameters [the elongation temperature (68°C)] as described in ACTIN, EF-1, TIPL-41, GADPH, and TUB, to get the best reference gene, which is expressed at a relative constant level among different experimental treatments, in my experimental conditions. We used TIP41 and Actin as the internal qPCR standard. The expression level of each target gene was standardized to the tomato TIP41 gene and Actin gene  was used to isolate total RNA from the leaves. The cDNA was generated from 1 tin gene .3 and block (a pair of OTCs with elevated and ambient OTCs), the subplot factor was tomato genotypes, and the sub-subplot factor was B. tabaci infestation. The main effects of O3 concentrations, tomato genotype, and B. tabaci infestation on plant were tested according to the following model:The statistical package IBM SPSS Statistics 21.0 was used for statistical analyses. A split–split plot design was applied to analyze the hormone content and gene expression of defense signaling, which the main factor was OO is the O3 treatment (i = 2), B is the block (j = 4), G is the tomato genotype (k = 2), and W is the B. tabaci infestation (l = 2). Xijklm represents the error because of the smaller scale differences between samples and variability within blocks . Effects were considered significant if P < 0.05. Tukey’s multiple range tests were used to separate means when ANOVAs were significant (P < 0.05). The ET emission, biomass, photosynthetic rate, chlorophyll content, ROS, curl leaves, O3-damaged stippled leaves, deciduous leaves of plants, population abundance, individual amino acids, and total amino acid under two O3 concentrations were analyzed by a split-plot design, which O3 and block as the main effects and tomato genotype as the subplot effect.where 3 significantly increased the ET production by 37% in AC plants spraying with H2O (AC/H2O plants), by 24% in AC plant spraying with ACC (AC/ACC plants), and by 38% in Nr plants spraying with H2O (Nr/H2O plants). However, ET production was not affected by elevated O3 in AC plants spraying with 1-MCP (AC/1-MCP plants). Under both O3 concentrations, the ET production was the highest in AC/ACC plants and the lowest in AC/1-MCP plants. The level of ET emission was significantly higher in AC/H2O plants than in Nr/H2O plants . We also analyzed ACS and ACO genes, which were two important ET synthesis genes in tomato plants. The expression of foliar ACO and ACS genes were consistent with the level of ET emission, which were upregulated under elevated O3 in AC/H2O plants, AC/ACC plants, and Nr/H2O plants .Elevated O3 decreased the plant biomass by 37%, the photosynthetic rate by 62%, and chlorophyll content by 17% in AC/H2O plants. The plant biomass, photosynthetic rate, and chlorophyll content were not affected by elevated O3 in AC/1-MCP plants. Elevated O3 had the most detrimental effects on AC/ACC plants, reducing plant biomass by 66%, photosynthetic rate by 67%, and chlorophyll content by 14%. Compared with AC/H2O plant, elevated O3 had marginal effects on Nr/H2O plants, with decreased biomass of 27%, photosynthetic rate by 34%, and chlorophyll content by 15% .Elevated O3 also increased ROS accumulation and leaf injury, including numbers of O3-damaged stippled leaves, deciduous leaves, and curl leaves in AC/H2O plants, AC/ACC plants, and Nr/H2O plants. ROS accumulation and leaf injury in AC/H2O plants were significantly lower than these in AC/ACC plants, and higher than these in Nr/H2O plants. In AC/1-MCP plants, ROS accumulation and leaf injury were similar under both O3 concentrations .Elevated OB. tabaci. Relative to ambient O3, the number of B. tabaci was increased 3-fold on AC plants and 0.5-fold on Nr plants under elevated O3 .Ozone concentration and plant genotype had significant effects on the population abundance of 3-induced leaf senescence activated leaf salicylic acid (SA) and ET signaling pathway, but had no effects on jasmonic acid (JA) signaling pathway.OB. tabaci infestation had significant effects on the foliar SA content. Regardless of B. tabaci infestation, elevated O3 significantly increased foliar SA content by 54% in AC plants and by 51% in Nr plants . Under both O3 levels, B. tabaci infestation increased the foliar SA content in AC and Nr plants. Regardless of B. tabaci infestation and O3 concentration, foliar SA content was equivalent in AC and Nr plants. The expression of β-1,3-glucanase (GLU) and pathogenesis-related protein (PR) genes, two downstream genes of SA signaling pathway, was consistent with foliar SA content, which was increased under elevated O3 and B. tabaci infestation, and was not affected by plant genotype .Ozone concentration and 3 did not increase the foliar JA accumulation and the relative expression level of lipoxygenase (LOX) and proteinase inhibitor (PI), which were two marker genes of JA signaling pathway, in AC and Nr plants with and without B. tabaci infestation. Under both O3 concentrations, B. tabaci infestation significantly reduced the foliar JA concentration and the relative expression level of LOX and PI. Regardless of B. tabaci infestation and O3 concentration, the foliar JA concentration and the expression of LOX and PI were markedly higher in AC plants than in Nr plants .Elevated O3 significantly increased the emission of ET without B. tabaci infestation in AC plants, increasing from 33 to 44 nL g-1FW h-1. When infested by B. tabaci, elevated O3 had no effects on the emission of ET. B. tabaci infestation significantly decreased the ET emission by 38 % under ambient O3, and by 49% under elevated O3 in AC plants. Compared with AC plants, the ET emission was significantly lower in Nr plants regardless of B. tabaci infestation and O3 concentrations . We also analyzed the ethylene-response factor 1 (ERF1) and ethylene-response factor 2 (ERF2), two down-stream genes of ET signaling pathway, and found that their expression was increased by elevated O3, and decreased by B. tabaci infestation in both tomato genotypes .Elevated O3 significantly increased foliar nitrogen content, total amino acid content, and protease activity in AC and Nr plants, but the response was greater in AC plants  compared with Nr plants .Elevated O3 markedly increased the concentrations of 13 individual amino acids, including nine essential amino acids  and four non-essential amino acids  in AC/H2O plants, and 11 individual amino acids including six essential amino acids  and four non-essential amino acids  in Nr/H2O plants. Furthermore, the increase in individual amino acids was greater in AC plants than in Nr plants .A total of 17 individual amino acids in leaves were analyzed including essential and non-essential amino acids. Elevated O2 or O3) and insect infestation . It is likely that different fumigation regimes of O3 exposure may be important for the different function of NR receptor in regulating O3 sensitivity. Acute O3 exposure means that plants are fumigated with O3 concentration exceeding 120 ppb within a few hours, while chronic O3 exposure is daily peak concentration in the range of 40–120 ppb within several days  under acute O3 exposure . ET signal also involves secondary symplastic ROS accumulation in O3-exposure tomato plants, in which plant spraying with ET inhibitors accumulates less H2O2 under elevated O3 (Arabidopsis A-Fifteen (AAF) gene, the A. thaliana ortholog of sweet potato senescence-associated gene-SPA15, is involved in balancing the ROS homeostasis to regulate the age and dark-induced leaf senescence, in which leaf senescence is suppressed in aaf T-DNA insertion mutant and promoted in AAF over-expression plants . Thus, these suggest that ET signaling pathway is required in ROS accumulation and leaf senescence under elevated O3.A direct role of ET signaling pathway in regulating abiotic stress-induced leaf premature senescence has been demonstrated. Experiments in maize show that a deficiency in the ET synthase inhibits drought-induced senescence, and the delayed drought-induced senescence in ET synthase mutants is complemented by spraying with ET precursor ACC . Similarch trees . We alsovated O3 . ROS canvated O3 . For exan plants . The reg 2 EIN2; , which i 2 EIN2; . A recen 2 EIN2; . It is aB. tabaci infestation  plants , which increased the population abundance of B. tabaci . Compared with wild-type plants, the lower amino acid content sustained lower population number of B. tabaci on Nr plants under elevated O3. Thus, these results indicate that the rise in leaf amino acid concentrations, which is caused by leaf senescence, is an important aspect of the improved population fitness of B. tabaci under elevated O3.Leaf senescence, which changes plant nutrition and defense metabolisms, could be utilized by plant to regulate insect growth. There are two hypotheses to explain the effects of senescing leaves with the sign of yellowing on herbivores: (i) handicap signal hypothesis and (ii) nutrient re-translocation hypothesis. estation . However defense . When in1 plants . In the estation . For exa) plants . Thus, i) plants , explain pendula . It is w pendula . Total aus padus . In Arabnescence . For sapnescence . Plants  feeding . O3-induB. tabaci was increased when fed foliage grown under elevated O3 in the current research. This finding coincides with early results showing that elevated O3 increases the population fitness of aphids  plants. This is in agreement with previous reports of multiple variations in nutrient responses to O3 exposure existing among plant genotypes and/or species (B. tabaci to elevated O3 is dependent on the biotypes. Previous studies also support that Q biotype exhibits higher population fitness than B biotype, such as better feeding efficiency, greater reproductive ability, shorter development time, and greater tolerance to heat stress (3 may exacerbate intra-species competitions between different biotypes of B. tabaci.The population abundance of Q biotypes of f aphids . Howevervated O3 . One pos species . Anothert stress . Thus, o3 activates ET signaling pathway, which accelerates leaf senescence associated with decrease in biomass, photosynthesis, and increase in numbers of yellow leaves; however, the performance of B. tabaci on tomato plants is improved by increasing nitrogenous nutrition of O3-induced senescing leaves. This study has generated several significant findings. First, oxidative stress can accelerate leaf senescence via regulating endogenous ET signals. Second, our results support the “nutrient re-translocation hypothesis” that O3-induced senescing leaves with higher amino acid contents enhance the population fitness of B. tabaci. Finally, such changes suggest that tomato plants may suffer greater damage due to the interacting stress of direct O3-damage and additive infested by B. tabaci if tropospheric O3 levels increase continuously.Elevated OHG, YS, and FG planned and designed the research. HG performed the experiments, conducted the fieldwork, and analyzed the data. CL provided tomato seeds. HY provided field support. HG wrote the first draft of the manuscript. YS and FG contributed to the subsequent manuscript development.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sorghum bicolor) is cultivated worldwide for food, bioethanol, and fodder production. Although nitrogen fixation in sorghum has been studied since the 1970s, N2-fixing bacteria have not been widely examined in field-grown sorghum plants because the identification of functional diazotrophs depends on the culture method used. The aim of this study was to identify functional N2-fixing bacteria associated with field-grown sorghum by using “omics” approaches. Four lines of sorghum  were grown in a field in Fukushima, Japan. The nitrogen-fixing activities of the roots, leaves, and stems were evaluated by acetylene reduction and 15N2-feeding assays. The highest nitrogen-fixing activities were detected in the roots of lines KM1 and KM2 at the late growth stage. Bacterial cells extracted from KM1 and KM2 roots were analyzed by metagenome, proteome, and isolation approaches and their DNA was isolated and sequenced. Nitrogenase structural gene sequences in the metagenome sequences were retrieved using two nitrogenase databases. Most sequences were assigned to nifHDK of Bradyrhizobium species, including non-nodulating Bradyrhizobium sp. S23321 and photosynthetic B. oligotrophicum S58T. Amplicon sequence and metagenome analysis revealed a relatively higher abundance (2.9–3.6%) of Bradyrhizobium in the roots. Proteome analysis indicated that three NifHDK proteins of Bradyrhizobium species were consistently detected across sample replicates. By using oligotrophic media, we purified eight bradyrhizobial isolates. Among them, two bradyrhizobial isolates possessed 16S rRNA and nif genes similar to those in S23321 and S58T which were predicted as functional diazotrophs by omics approaches. Both free-living cells of the isolates expressed N2-fixing activity in a semi-solid medium according to an acetylene reduction assay. These results suggest that major functional N2-fixing bacteria in sorghum roots are unique bradyrhizobia that resemble photosynthetic B. oligotrophicum S58T and non-nodulating Bradyrhizobium sp. S23321. Based on our findings, we discuss the N2-fixing activity level of sorghum plants, phylogenetic and genomic comparison with diazotrophic bacteria in other crops, and Bradyrhizobium diversity in N2 fixation and nodulation.Sorghum ( Sorghum has little breeding history compared to sugarcane and maize but has the potential for broad agro-ecological adaptation  in the rhizosphere soils of field-grown sorghum in Brazil based on nifH PCR of soil DNA extracts. However, N2-fixing bacteria associated with sorghum plant tissues have not been fully explored.For nitrogen fixation in sorghum plants, 2-fixing bacteria .We used four lines  of sorghum developed by Earthnote Co., Ltd. . KM1 is a late-ripening line with vigorous leaf growth. KM2 is an early-ripening line with lodging resistance and salt tolerance. KM4 and KM5 were pre-selected because of their high (KM4) and low (KM5) N′′140°34′13.7′′) on June 6, 2016. The soil had the following chemical properties: pH (H2O), 5.9; total C, 13.9 g kg-1 dry soil; total N, 0.8 g kg-1 dry soil; available phosphorus, 560.4 mg P kg-1 dry soil (Truog method). Before transplanting the seedlings, the field was treated with 85 kg N as urea, 84 kg N as controlled-release coated-urea fertilizer , which releases 80% of its total N over 100 days, and 85 kg K2O as potassium sulfate per hectare. This is the standard fertilization regime used for sorghum cropping under the local conditions. Each line was planted in three plots, with 50 plants per plot .Seeds were sown in 200-cell plug trays on May 10, 2016. The seedlings were transplanted into a field owned by Earthnote   were performed based on three biological replicates.At least three plants were sampled individually from three different field plots or from the seedling trays for transplanting. When the sorghum seedlings were transplanted into the field, six seedlings were randomly selected from the plug trays [0 days after transplanting (DAT)], which were subjected to the acetylene reduction assay  as descrolation) . Thus, tNitrogen-fixing activity was examined by the acetylene reduction assay  at four At 0 DAT, seedling roots were washed in tap water to remove adhering soil and placed in a 100-mL vial with a butyl rubber septum  . At 27, Acetylene gas  was injected to provide a concentration of 10% (vol/vol), and the vial or jar was incubated for 1 day at ambient temperature. We drew 20 mL of headspace gas in a sampling syringe and stored it in a vacuumed gas vial  until analysis. Gas samples (0.5 mL) were analyzed to determine their ethylene concentrations with a Shimadzu GC-18A gas chromatograph equipped with a flame ionization detector and Porapak R column as described previously .15N2 /20% O2/48% Ar. The negative control included air gas containing atmospheric N2 [natural abundance of 15N (0.366 atom %)]. After static incubation in the chamber at 25°C for 23 h in the dark, the root samples were dried at 80°C for 3 days and then powdered in a Multi-Beads Shocker . To determine the level of 15N2 fixation in the roots, we analyzed 15N and total N contents of the powdered root tissues with an elemental analyzer/isotope ratio mass spectrometer .At 102 DAT, the divided roots from the same plant used for ARA were introduced into a gas chamber with a sampling port . The gas15N2-feeding experiments were stored at -80°C for metagenome analysis . The size and quality of the libraries were assessed using a BioAnalyzer High Sensitivity Chip, and the average fragment size was around 700 base pairs (bp). Next, 250-bp paired-end libraries were sequenced on an Illumina MiSeq sequencer. The quality of the sequence data was checked by using the FastQC v. 0.10.1 softwaree 0.20.4  with thenifH, we first assigned the metagenomic reads in each sample according to the results of BLAST analysis against the nifH database  NCBI databases matched with respective keywords  and (ii) nifD and nifK encoded on genome sequences of nifH best-hits. The metagenomic reads in each sample were assigned according to the BLAST analysis against the nifD and nifK database with a threshold identity of 80% in-house. Next, the candidate reads of nifD and nifK from the metagenomic data were subjected to BLAST searches against the NCBI NR public database with an E-value threshold of 10-10, and false nifD or nifK reads were removed by manual inspection of the BLAST results. The resulting DNA sequences of nifD and nifK were taxonomically assigned based on the best BLAST hit, translated into amino acid sequences, and incorporated in our nitrogenase database for proteome analysis with taxonomic information.To extract Amplicon sequencing of 16S rRNA and metagenomics analysis targeting 31 AMPHORA genes , which aA partial bacterial 16S rRNA gene fragment was amplified by two-step PCR. First, a partial sequence of the V4 variable region was amplified with simultaneous addition of sequencing priming sites. The target region was amplified using primers 515F (5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT-3′) . In the A universal marker set of 31 protein-encoding phylogenetic marker genes was extracted from the metagenome data of KM1 and KM2 using AMPHORA2 with default settings .Proteins were extracted from the EBCs from the roots, and ∼50 μg of sample was separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and stained with Coomassie blue. Gel strips containing proteins ranging from 21.5 to 66.2 kDa (NifHDK protein sizes) were excised . Proteom-1 polymixin B or 1/100-strength HM salt medium containing 1.5% agar and 10 mg L-1 polymixin B. This isolation strategy was used because Bradyrhizobium species are oligotrophic slow-growing bacteria  and sequenced by direct PCR using universal forward (27f) and reverse (1492r) primers . The PCRGenomic DNA of bradyrhizobial isolates TM122 or TM124 was extracted with an Illustra Bacteria GenomicPrep Mini Spin Kit . DNA libraries were prepared with a Nextera DNA Sample Preparation Kit and sequenced on an MiSeq system using a MiSeq Reagent Nano Kit v. 2 (500 cycles) (both Illumina). Raw reads were trimmed in CLC Genomics Workbench v. 7.5.1 software  with the following parameters: ambiguous limit, 2; quality limit, 0.05; number of 5′ terminal nucleotides, 20; number of 3′ terminal nucleotides, 5; minimum number of nucleotides in reads, 70.B. diazoefficiens USDA110T .Shotgun sequence reads of the isolates were mapped to a reference genome of USDA110T . MappingBradyrhizobium sp. TM122, and TM124 were pre-cultured in HM medium  and washed twice with sterilized water. The cell density was adjusted to 107 cells/mL, and 1 mL of the cell suspension was inoculated into 7 mL of Rennie semi-solid medium  (M medium  with shar per L)  in a 28-4NO3 under aseptic conditions  under a daily cycle of 16 h of light and 8 h of dark at 25°C. After 7 days, the seedlings were thinned out to one plant. At 28 days after inoculation, the roots were carefully harvested and then transferred into a 100-mL vial with a butyl rubber septum . Acetylene (10 mL) was introduced into the bottles, which were incubated for 24 h at 25°C in the dark.The plants were grown in a growth cabinet  that provided 65 mol photons mDRA006465, DRA006466, DRA006492, and DRA006493 in the DDBJ Sequence Read Archive, respectively. The sequences of 16S rRNA genes of bacterial isolates were deposited in DDBJ/EMBL/GenBank under the accession numbers LC367220–LC367221 and LC433572–LC433596, respectively.DNA sequences obtained by the metagenomic analysis and 16S rRNA gene amplicon, TM122 genome, and TM124 genome were deposited under the accession numbers 2-fixing activities were monitored at different growth stages for four sorghum lines  by an acetylene reduction assay in closed bottles  at 71 DAT in the washed roots (-1 h-1), followed by line KM2 (332.5 nmol plant-1 h-1). The ARA of KM5 roots (6.7 nmol plant-1 h-1) was significantly lower than that of KM1  and total N amount (biomass × N content) , which was significantly higher than the values in the other lines (-1 day-1).The above results suggest that N 102 DAT . When thfixed N2 . We calccontent) . Althouger lines . KM2 sho15N2-feeding experiments  in both methods. Thus, we focused on the root microbiomes of KM1 and KM2 at 102 DAT for subsequent metagenome, proteome, and isolation analyses , we retrieved genes encoding nitrogenase reductase (NifH) and dinitrogenase (NifDK) from the six datasets. Most nitrogenase genes were proteobacterial nifHDK genes  of Bradyrhizobium were consistently detected among the six datasets including sorghum lines KM1 and KM2  (B. oligotrophicum S58T (AP012603)  and S58 (11–29%) among Bradyrhizobium nifDK reads of sorghum lines KM1 and KM2   and photP012603)  (SupplemP012603) , S7. Amoectively , S7. Abu and KM2 , S7. Thef energy . Thus, wnifHDK genes from our metagenome data (Data Set 1). Next, amino acid sequences of the peptide generated by proteome analysis (Data Set 2) were assigned based on 100% identity (Data Set 2) in the NifHDK database (Data Set 1). The results were summarized at the genus level  generated by dada2 (Bradyrhizobium (ASV 0004) in KM1 and KM2 root microbiomes were 2.9 and 3.0%, respectively (Bradyrhizobium of KM1 and KM2 (3.6%)  . These rnif genes among several linages of bacteria  containing polymixin B. Twenty-eight slow-growing white colonies were isolated, eight of which were identified as Bradyrhizobium based on 16S rRNA gene sequencing  were close to B. japonicum. Both species are typical soybean endosymbionts  required for legume nodulation  in the roots inoculated with TM122 compared to that in uninoculated roots. In contrast, TM124-inoculated roots showed no ARA under the two different treatments of N supply under our experimental conditions , the usage of nifDK genes encoding NifDK facilitated the reliable identification of functional diazotrophs in sorghum roots  were determined by both the ARA and 15N2-feeding methods  was abundant in sugarcane stems, sweet potato stems, and pineapple leaves, which are rich in sugars and organic acids (2-fixing bacteria exclusively reside in the roots of field-grown sorghum.Active expression of the dinitrogenase reductase-encoded gene (ic acids . The leaic acids . In contic acids . These rBradyrhizobium are versatile bacteria inhabiting soils and plants (B. diazoefficiens and B. japonicum) because of their prominent roles in nodule formation and their agricultural significance , at least in sorghum seedlings inoculated with TM122 (28 days after sowing) and in the free-living state for TM122 and TM124  of cultivated and wild rice plants  in the metagenome sequences were assigned to Bradyrhizobium species by metagenome analysis. Proteome analysis indicated that three NifHDK proteins of Bradyrhizobium species were consistently detected across sample replicates. Most of these proteins were assigned to photosynthetic B. oligotrophicum S58T and non-nodulating Bradyrhizobium sp. S23321. The polyphasic approach suggested that major functional N2-fixing bacteria in the sorghum roots are unique bradyrhizobia that resemble non-nodulating Bradyrhizobium sp. S23321 and photosynthetic B. oligotrophicum S58T.The highest nitrogen-fixing activities were detected in the roots of sorghum lines KM1 and KM2 in the late growth stage (102 DAT). Nitrogenase structural genes (SH, TK, KY, TF, TT, and KM designed the research. SH, TM, SW, and YK analyzed the data. SH and TM analyzed the DNA sequences of PCR products and metagenome. YK performed the proteome analysis. TK, SH, and SW characterized isolated diazotrophs. SH and KM wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.