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All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Distant-acting Enhancers in Development and Disease

Multiple converging lines of evidence from experimental and human genetic studies indicate that both common and rare sequence variants involving enhancers play major roles in Mendelian and complex human disease phenotypes. We use a combination of sequence-based molecular approaches including chromatin mapping and synthesis-enabled modification of enhancer sequences, large-scale transgenic mouse studies (http://enhancer.lbl.gov), and CRISPR genome editing in the mouse model to study the in vivofunction of enhancers in developmental, evolutionary, and disease-related processes. Using examples from our ongoing work, I will illustrate how these methods provide insight into the function and evolution of distant-acting regulatory sequences and offer a starting point for understanding their role in human disease. I will discuss how improved in vivo mouse assays can be used to study candidate pathogenic human mutations in enhancers at scale, revealing the extent to which human genetic studies alone can be insufficient to identify truly causal mutations in enhancers. I will also show how such assays can be used to explore the sensitivity of enhancers that are extremely conserved ("ultraconserved") in evolution to mutations, revealing a remarkable functional robustness to sequence changes that contrasts with apparent evolutionary constraints.

Visel, A↗
DOE OSTI2014

Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix. Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C{sup α} atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗
DOE OSTI2016

Crystal structures of apo and inhibitor-bound TGFβR2 kinase domain: insights into TGFβR isoform selectivity

The cytokine TGF-β modulates a number of cellular activities and plays a critical role in development, hemostasis and physiology, as well as in diseases including cancer and fibrosis. TGF-β signals through two transmembrane serine/threonine kinase receptors: TGFβR1 and TGFβR2. Multiple structures of the TGFβR1 kinase domain are known, but the structure of TGFβR2 remains unreported. Wild-type TGFβR2 kinase domain was refractory to crystallization, leading to the design of two mutated constructs: firstly, a TGFβR1 chimeric protein with seven ATP-site residues mutated to their counterparts in TGFβR2, and secondly, a reduction of surface entropy through mutation of six charged residues on the surface of the TGFβR2 kinase domain to alanines. These yielded apo and inhibitor-bound crystals that diffracted to high resolution (<2 Å). Comparison of these structures with those of TGFβR1 reveal shared ligand contacts as well as differences in the ATP-binding sites, suggesting strategies for the design of pan and selective TGFβR inhibitors.

Tebben, Andrew J.↗
DOE OSTI2024

The expression of genes encoding novel Sesame oleosin variants facilitates enhanced triacylglycerol accumulation in Arabidopsis leaves and seeds

Summary Triacylglycerols (TAG), accumulate within lipid droplets (LD), predominantly surrounded by OLEOSINs (OLE), that protect TAG from hydrolysis. We tested the hypothesis that identifying and removing degradation signals from OLE would promote its abundance, preventing TAG degradation and enhancing TAG accumulation. We tested whether mutating potential ubiquitin‐conjugation sites in a previously reported improved Sesamum indicum OLE ( SiO ) variant, o3‐3 Cys‐OLE ( SiCO herein), would stabilize it and increase its lipogenic potential. SiCOv1 was created by replacing all five lysines in SiCO with arginines. Separately, six cysteine residues within SiCO were deleted to create SiCOv2. SiCOv1 and SiCOv2 mutations were combined to create SiCOv3. Transient expression of SiCOv3 in Nicotiana benthamiana increased TAG by two‐fold relative to SiCO. Constitutive expression of SiCOv3 or SiCOv5 , containing the five predominant TAG‐increasing mutations from SiCOv3 , in Arabidopsis along with mouse DGAT2 ( mD ) increased TAG accumulation by 54% in leaves and 13% in seeds compared with control lines coexpressing SiCO and mD. Lipid synthesis rates increased, consistent with an increase in lipid sink strength that sequesters newly synthesized TAG, thereby relieving the constitutive BADC‐dependent inhibition of ACCase reported for WT Arabidopsis. These OLE variants represent novel factors for potentially increasing TAG accumulation in a variety of oil crops.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The molecular mechanism of sialic acid transport mediated by Sialin

Malfunction of the sialic acid transporter caused by various genetic mutations in the SLC17A5 gene encoding Sialin leads to a spectrum of neurodegenerative conditions called free sialic acid storage disorders. Unfortunately, how Sialin transports sialic acid/proton (H + ) and how pathogenic mutations impair its function are poorly defined. Here, we present the structure of human Sialin in an inward-facing partially open conformation determined by cryo–electron microscopy, representing the first high-resolution structure of any human SLC17 member. Our analysis reveals two unique features in Sialin: (i) The H + coupling/sensing requires two highly conserved Glu residues (E171 and E175) instead of one (E175) as implied in previous studies; and (ii) the normal function of Sialin requires the stabilization of a cytosolic helix, which has not been noticed in the literature. By mapping known pathogenic mutations, we provide mechanistic explanations for corresponding functional defects. We propose a structure-based mechanism for sialic acid transport mediated by Sialin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Structure-function analysis of the FCRL5–IgG1 Fc complex reveals an unappreciated pathway for B cell modulation by Fc-attenuated IgG

The Fc region of therapeutic IgG antibodies is often engineered to remove or “silence” Fc effector functions, but it remains unclear whether these mutations eliminate all Fc-mediated effector activity. Human Fc receptor-like 5 (FCRL5/FcRH5) is a low-affinity IgG Fc receptor expressed on B cells and is an actively pursued antibody target in multiple myeloma. Here, we show that common Fc function-silencing mutations do not impair FCRL5-mediated activity and therefore attenuate, rather than eliminate, Fc effector function. The crystal structure of the FCRL5-IgG1 Fc complex, solved at 3.4 Å resolution, revealed that FCRL5 binds IgG1 Fc in a 1:1 complex through a binding mode distinct from that of classical Fcγ receptors, explaining why mutations that attenuate Fc effector function spare FCRL5 binding. Fc-engineered antibodies that selectively engage FCRL5 inhibited B cell receptor-induced Ca 2+ flux in FCRL5-expressing B cells. These findings demonstrate that Fc-attenuated therapeutic IgG retains the ability to engage FCRL5, identifying an unappreciated pathway for B cell modulation.

Herpers, Bart M. [Department of Biomedical Enginee↗
DOE OSTI2019

How C-terminal additions to insulin B-chain fragments create superagonists for T cells in mouse and human type 1 diabetes

In type 1 diabetes (T1D), proinsulin is a major autoantigen and the insulin B:9-23 peptide contains epitopes for CD4 + T cells in both mice and humans. This peptide requires carboxyl-terminal mutations for uniform binding in the proper position within the mouse IA g7 or human DQ8 major histocompatibility complex (MHC) class II (MHCII) peptide grooves and for strong CD4 + T cell stimulation. In this work, we present crystal structures showing how these mutations control CD4 + T cell receptor (TCR) binding to these MHCII-peptide complexes. Our data reveal stricking similarities between mouse and human CD4 + TCRs in their interactions with these ligands. We also show how fusions between fragments of B:9-23 and of proinsulin C-peptide create chimeric peptides with activities as strong or stronger than the mutated insulin peptides. We propose transpeptidation in the lysosome as a mechanism that could accomplish these fusions in vivo, similar to the creation of fused peptide epitopes for MHCI presentation shown to occur by transpeptidation in the proteasome. Were this mechanism limited to the pancreas and absent in the thymus, it could provide an explanation for how diabetogenic T cells escape negative selection during development but find their modified target antigens in the pancreas to cause T1D.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858)

AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two β-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer. Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal β-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Combination Attenuation Offers Strategy for Live Attenuated Coronavirus Vaccines

With an ongoing threat posed by circulating zoonotic strains, new strategies are required to prepare for the next emergent coronavirus (CoV). Previously, groups had targeted conserved coronavirus proteins as a strategy to generate live attenuated vaccine strains against current and future CoVs. With this in mind, we explored whether manipulation of CoV NSP16, a conserved 2'O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the severe acute respiratory syndrome-CoV mouse model, an NSP16 mutant vaccine was evaluated for protection from heterologous challenge, efficacy in the aging host, and potential for reversion to pathogenesis. Despite some success, concerns for virulence in the aged and potential for reversion makes targeting NSP16 alone an untenable approach. However, combining a 2'O MTase mutation with a previously described CoV fidelity mutant produced a vaccine strain capable of protection from heterologous virus challenge, efficacy in aged mice, and no evidence for reversion. Together, the results indicate that targeting the CoV 2'O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live attenuated coronavirus vaccines. IMPORTANCE Emergent coronaviruses remain a significant threat to global public health and rapid response vaccine platforms are needed to stem future outbreaks. However, failure of many previous CoV vaccine formulations has clearly highlighted the need to test efficacy under different conditions and especially in vulnerable populations such as the aged and immunocompromised. This study illustrates that despite success in young models, the 2'O methyltransferase mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the 2'O methyltransferase mutation can be paired with other attenuating approaches to provide robust protection from heterologous challenge and in vulnerable populations. Coupled with increased safety and reduced pathogenesis, the study highlights the potential for 2'O methyltransferase attenuation as a major component of future live attenuated coronavirus vaccines.

Evolview, evolgenius, SARS-CoV, NSP14 exonuclease↗
DOE OSTI2017

The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations in rctB that reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in a dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Physiological Effect of XoxG(4) on Lanthanide-Dependent Methanotrophy

ABSTRACT A recent surprising discovery of the activity of rare earth metals (lanthanides) as enzyme cofactors as well as transcriptional regulators has overturned the traditional assumption of biological inertia of these metals. However, so far, examples of such activities have been limited to alcohol dehydrogenases. Here we describe the physiological effects of a mutation in xoxG , a gene encoding a novel cytochrome, XoxG(4), and compare these to the effects of mutation in XoxF, a lanthanide-dependent methanol dehydrogenase, at the enzyme activity level and also at the community function level, using Methylomonas sp. strain LW13 as a model organism. Through comparative phenotypic characterization, we establish XoxG as the second protein directly involved in lanthanide-dependent metabolism, likely as a dedicated electron acceptor from XoxF. However, mutation in XoxG caused a phenotype that was dramatically different from the phenotype of the mutant in XoxF, suggesting a secondary function for this cytochrome, in metabolism of methane. We also purify XoxG(4) and demonstrate that this protein is a true cytochrome c , based on the typical absorption spectra, and we demonstrate that XoxG can be directly reduced by a purified XoxF, supporting one of its proposed physiological functions. Overall, our data continue to suggest the complex nature of the interplay between the calcium-dependent and lanthanide-dependent alcohol oxidation systems, while they also suggest that addressing the roles of these alternative systems is essential at the enzyme and community function level, in addition to the gene transcription level. IMPORTANCE The lanthanide-dependent biochemistry of living organisms remains a barely tapped area of knowledge. So far, only a handful of lanthanide-dependent alcohol dehydrogenases have been described, and their regulation by lanthanides has been demonstrated at the transcription level. Little information is available regarding the concentrations of lanthanides that could support sufficient enzymatic activities to support specific metabolisms, and so far, no other redox proteins involved in lanthanide-dependent methanotrophy have been demonstrated. The research presented here provides enzyme activity-level data on lanthanide-dependent methanotrophy in a model methanotroph. Additionally, we identify a second protein important for lanthanide-dependent metabolism in this organism, XoxG(4), a novel cytochrome. XoxG(4) appears to have multiple functions in methanotrophy, one function as an electron acceptor from XoxF and another function remaining unknown. On the basis of the dramatic phenotype of the XoxG(4) mutant, this function must be crucial for methanotrophy.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Structural basis for varying drug resistance of SARS-CoV-2 M pro E166 variants

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M pro ) has an essential role in the virus lifecycle and, accordingly, it is a target for antiviral drugs. Multiple studies have identified an M pro mutation (E166V) that confers strong resistance to clinically relevant inhibitors, including nirmatrelvir, but the underlying mechanism is not fully understood. Here, we report on crystal structures of SARS-CoV-2 M pro E166V in complex with nirmatrelvir, ensitrelvir, and bofutrelvir. The structures suggest that resistance is caused in part by the loss of a direct hydrogen bond and also, especially for nirmatrelvir, by a steric clash with the substituted valine residue. In comparison, the binding of bofutrelvir shows greater flexibility, which may help alleviate this steric effect and allow bofutrelvir to fit the mutant active site despite the loss of a direct polar contact. Thermal stability analyses also corroborate E166V most severely affecting the binding of nirmatrelvir and, to lesser and different extents, ensitrelvir and bofutrelvir. We further show that E166I causes even more severe nirmatrelvir resistance, whereas E166A and E166L have much milder effects. These studies shed light on the molecular mechanisms of a key M pro drug resistance mutation and may help inform the design of next-generation inhibitors. IMPORTANCE Using a combination of high-resolution X-ray crystallographic and biochemical analyses, we reveal the molecular mechanisms by which a mutation in the severe acute respiratory syndrome coronavirus 2 main protease (M pro ) confers strong resistance against clinically relevant antiviral drugs that inhibit M pro activity. The results presented here may help inform the design of next-generation inhibitors to combat the problem of therapy resistance.

Microbiology↗
DOE OSTI2023

Comparison of phage-derived recombinases for genetic manipulation of Pseudomonas species

ABSTRACT Several strains in the Pseudomonas genus are categorized as plant growth-promoting rhizobacteria (PGPR). Although several of these strains are strong candidates for applications as biofertilizers or biopesticides, genome editing approaches are generally limited and require further development. Editing genomes in PGPR could enable more robust agricultural applications, persistence, and biosafety measures. In this study, we investigate the use of five phage-encoded recombinases to develop a recombineering workflow in three PGPR strains: Pseudomonas protegens Pf-5, Pseudomonas protegens CHA0, and Pseudomonas putida KT2440. Using point mutations in the rpoB gene, we reach maximum recombineering efficiencies of 1.5 × 10 −4 , 3 × 10 −4 , and 5 × 10 −5 , respectively, in these strains using λ-Red Beta recombinase from Escherichia coli . We further examine recombineering efficiencies across these strains as a function of selected mutation, editing template concentration, and phosphorothiolate bond protection. This work validates the use of these tools across several environmentally and biotechnologically relevant strains to expand the possibilities of genetic manipulation in the Pseudomonas genus. IMPORTANCE The Pseudomonas genus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol, and synthetic biology. Though several strains have been identified with beneficial properties, chromosomal manipulations to further improve these strains for commercial applications have been limited due to the lack of efficient genetic tools that have been tested across this genus. Here, we test the recombineering efficiencies of five phage-derived recombinases across three biotechnologically relevant Pseudomonas strains: P. putida KT2440, P. protegens Pf-5, and P. protegens CHA0. These results demonstrate a method to generate targeted mutations quickly and efficiently across these strains, ideally introducing a method that can be implemented across the Pseudomonas genus and a strategy that may be applied to develop analogous systems in other nonmodel bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu , Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Precancer Atlas to Drive Precision Prevention Trials

Cancer development is a complex process driven by inherited and acquired molecular and cellular alterations. Prevention is the holy grail of cancer elimination, but making this a reality will take a fundamental rethinking and deep understanding of premalignant biology. Here, we propose a national concerted effort to create a Precancer Atlas (PCA), integrating multi-omics and immunity – basic tenets of the neoplastic process. The biology of neoplasia caused by germline mutations has led to paradigm-changing precision prevention efforts, including: tumor testing for mismatch repair (MMR) deficiency in Lynch syndrome establishing a new paradigm, combinatorial chemoprevention efficacy in familial adenomatous polyposis (FAP), signal of benefit from imaging-based early detection research in high-germline risk for pancreatic neoplasia, elucidating early ontogeny in BRCA1-mutation carriers leading to an international breast cancer prevention trial, and insights into the intricate germline-somatic-immunity interaction landscape. Emerging genetic and pharmacologic (metformin) disruption of mitochondrial (mt) respiration increased autophagy to prevent cancer in a Li-Fraumeni mouse model (biology reproduced in clinical pilot) and revealed profound influences of subtle changes in mt DNA background variation on obesity, aging, and cancer risk. The elaborate communication between the immune system and neoplasia includes an increasingly complex cellular microenvironment and dynamic interactions between host genetics, environmental factors, and microbes in shaping the immune response. Cancer vaccines are in early murine and clinical precancer studies, building on the recent successes of immunotherapy and HPV vaccine immune prevention. Molecular monitoring in Barrett's esophagus to avoid overdiagnosis/treatment highlights an important PCA theme. Next generation sequencing (NGS) discovered age-related clonal hematopoiesis of indeterminate potential (CHIP). Ultra-deep NGS reports over the past year have redefined the premalignant landscape remarkably identifying tiny clones in the blood of up to 95% of women in their 50s, suggesting that potentially premalignant clones are ubiquitous. Similar data from eyelid skin and peritoneal and uterine lavage fluid provide unprecedented opportunities to dissect the earliest phases of stem/progenitor clonal (and microenvironment) evolution/diversity with new single-cell and liquid biopsy technologies. Cancer mutational signatures reflect exogenous or endogenous processes imprinted over time in precursors. In conclusion, accelerating the prevention of cancer will require a large-scale, longitudinal effort, leveraging diverse disciplines (from genetics, biochemistry, and immunology to mathematics, computational biology, and engineering), initiatives, technologies, and models in developing an integrated multi-omics and immunity PCA – an immense national resource to interrogate, target, and intercept events that drive oncogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Whole-Genome Sequencing Identifies a Rice Grain Shape Mutant, gs9–1

Breeding for genes controlling key agronomic traits is an important goal of rice genetic improvement. To gain insight into genes controlling grain morphology, we screened M 3 plants derived from 1,000 whole-genome sequenced (WGS) M 2 Kitaake mutants to identify lines with altered grain size. RESULTS:In this study, we isolated a mutant, named fast-neutron (FN) 60-4, which exhibits a significant reduction in grain size. We crossed FN60-4 with the parental line Kitaake and analyzed the resulting backcross population. Segregation analysis of 113 lines from the BC 2 F 2 population revealed that the mutant phenotype is controlled by a single semi-dominant locus. Mutant FN60-4 is reduced 20% in plant height and 8.8% in 1000-grain weight compared with Kitaake. FN60-4 also exhibits an 8% reduction in cell number and a 9% reduction in cell length along the vertical axis of the glume. We carried out whole-genome sequencing of DNA pools extracted from segregants with long grains or short grains, and revealed that one gene, LOC_Os09g02650, cosegregated with the grain size phenotype in the BC 1 F 2 and BC 2 F 2 populations. This mutant allele was named grain shape 9-1 (gs9-1). gs9-1 carries a 3-bp deletion that affects two amino acids. This locus is a new allele of the BC12/GDD1/MTD1 gene that encodes a kinesin-like protein involved in cell-cycle progression, cellulose microfibril deposition and gibberellic acid (GA) biosynthesis. The GA biosynthesis-related gene KO2 is down-regulated in gs9-1 . The dwarf phenotype of gs9-1 can be rescued by adding exogenous GA3. In contrast to the phenotypes for the other alleles, the gs9-1 is less severe, consistent with the nature of the mutation, which does not disrupt the open reading frame as observed for the other alleles. In this study, we isolated a mutant, which exhibits altered grain shape and identified the mutated gene, gs9-1 . Our study reveals that gs9-1 is a semi-dominant gene that carries a two-amino acid mutation. gs9-1 is allelic to the BC12/GDD1/MTD1 gene involved in GA biosynthesis. These results demonstrate the efficiency and convenience of cloning genes from the whole-genome sequenced Kitaake mutant population to advance investigations into genes controlling key agronomic traits in rice.

59 BASIC BIOLOGICAL SCIENCES↗