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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Autoregulation of the LIM kinases by their PDZ domain

LIM domain kinases (LIMK) are important regulators of actin cytoskeletal remodeling. These protein kinases phosphorylate the actin depolymerizing factor cofilin to suppress filament severing, and are key nodes between Rho GTPase cascades and actin. The two mammalian LIMKs, LIMK1 and LIMK2, contain consecutive LIM domains and a PDZ domain upstream of the C-terminal kinase domain. The roles of the N-terminal regions are not fully understood, and the function of the PDZ domain remains elusive. Here, we determine the 2.0 Å crystal structure of the PDZ domain of LIMK2 and reveal features not previously observed in PDZ domains including a core-facing arginine residue located at the second position of the ‘x-Φ-G-Φ’ motif, and that the expected peptide binding cleft is shallow and poorly conserved. We find a distal extended surface to be highly conserved, and when LIMK1 was ectopically expressed in yeast we find targeted mutagenesis of this surface decreases growth, implying increased LIMK activity. PDZ domain LIMK1 mutants expressed in yeast are hyperphosphorylated and show elevated activity in vitro. This surface in both LIMK1 and LIMK2 is critical for autoregulation independent of activation loop phosphorylation. Overall, our study demonstrates the functional importance of the PDZ domain to autoregulation of LIMKs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Structural basis for the subtype-selectivity of K Ca 2.2 channel activators

Small-conductance (K Ca 2.2) and intermediate-conductance (K Ca 3.1) Ca 2+ -activated K + channels are gated by a Ca 2+ -calmodulin dependent mechanism. NS309 potentiates the activity of both K Ca 2.2 and K Ca 3.1, while rimtuzalcap selectively activates K Ca 2.2. Rimtuzalcap has been used in clinical trials for the treatment of spinocerebellar ataxia and essential tremor. We report cryo-electron microscopy structures of NS309-bound K Ca 2.2 and K Ca 3.1, in addition to structures of rimtuzalcap-bound K Ca 2.2 and mutant K Ca 3.1_R355K. The different conformations of calmodulin and the cytoplasmic HC helices in the two channels underlie the subtype-selectivity of rimtuzalcap for K Ca 2.2. NS309 binds to pre-existing pockets in both channels, while the bulkier rimtuzalcap binds in an induced-fit pocket in K Ca 2.2 requiring conformational changes. In K Ca 2.2, calmodulin’s N-lobes are sufficiently far apart to enable conformational changes to accommodate either NS309 or rimtuzalcap. In K Ca 3.1, calmodulin’s N-lobes are closer to each other and constrained by K Ca 3.1’s HC helices, which allows binding of NS309 but not rimtuzalcap. Replacement of arginine-355 in K Ca 3.1’s HB helix with lysine (K Ca 3.1_R355K) allows the binding of rimtuzalcap and renders the mutant channel sensitive to rimtuzalcap. These structures provide a framework for structure-based drug design targeting K Ca 2.2 channels.

Nam, Young-Woo [Chapman Univ., Irvine, CA (United ↗
DOE OSTI2020

Dynamic resource allocation drives growth under nitrogen starvation in eukaryotes

Cells can sense changes in their extracellular environment and subsequently adapt their biomass composition. Nutrient abundance defines the capability of the cell to produce biomass components. Under nutrient-limited conditions, resource allocation dramatically shifts to carbon-rich molecules. Here, we used dynamic biomass composition data to predict changes in growth and reaction flux distributions using the available genome-scale metabolic models of five eukaryotic organisms (three heterotrophs and two phototrophs). We identified temporal profiles of metabolic fluxes that indicate long-term trends in pathway and organelle function in response to nitrogen depletion. Surprisingly, our calculations of model sensitivity and biosynthetic cost showed that free energy of biomass metabolites is the main driver of biosynthetic cost and not molecular weight, thus explaining the high costs of arginine and histidine. We demonstrated how metabolic models can accurately predict the complexity of interwoven mechanisms in response to stress over the course of growth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Bioinformatics of cyanophycin metabolism genes and characterization of promiscuous isoaspartyl dipeptidases that catalyze the final step of cyanophycin degradation

Cyanophycin is a bacterial biopolymer used for storage of fixed nitrogen. It is composed of a backbone of L-aspartate residues with L-arginines attached to each of their side chains. Cyanophycin is produced by cyanophycin synthetase 1 (CphA1) using Arg, Asp and ATP, and is degraded in two steps. First, cyanophycinase breaks down the backbone peptide bonds, releasing β-Asp-Arg dipeptides. Then, these dipeptides are broken down into free Asp and Arg by enzymes with isoaspartyl dipeptidase activity. Two bacterial enzymes are known to possess promiscuous isoaspartyl dipeptidase activity: isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA). We performed a bioinformatic analysis to investigate whether genes for cyanophycin metabolism enzymes cluster together or are spread around the microbial genomes. Many genomes showed incomplete contingents of known cyanophycin metabolizing genes, with different patterns in various bacterial clades. Cyanophycin synthetase and cyanophycinase are usually clustered together when recognizable genes for each are found within a genome. Cyanophycinase and isoaspartyl dipeptidase genes typically cluster within genomes lacking cphA1. About one-third of genomes with genes for CphA1, cyanophycinase and IaaA show these genes clustered together, while the proportion is around one-sixth for CphA1, cyanophycinase and IadA. We used X-ray crystallography and biochemical studies to characterize an IadA and an IaaA from two such clusters, in Leucothrix mucor and Roseivivax halodurans, respectively. The enzymes retained their promiscuous nature, showing that being associated with cyanophycin-related genes did not make them specific for β-Asp-Arg dipeptides derived from cyanophycin degradation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus

The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of the T = 4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry relaxation followed by asymmetric reconstruction reveal that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the ε stem-loop in the gRNA, where polymerase binds in vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors.

99 GENERAL AND MISCELLANEOUS↗
DOE OSTI2022

A structure-based mechanism for displacement of the HEXIM adapter from 7SK small nuclear RNA

Productive transcriptional elongation of many cellular and viral mRNAs requires transcriptional factors to extract pTEFb from the 7SK snRNP by modulating the association between HEXIM and 7SK snRNA. In HIV-1, Tat binds to 7SK by displacing HEXIM. However, without the structure of the 7SK-HEXIM complex, the constraints that must be overcome for displacement remain unknown. Furthermore, while structure details of the Tat NL4-3 -7SK complex have been elucidated, it is unclear how subtypes with more HEXIM-like Tat sequences accomplish displacement. Here we report the structures of HEXIM, Tat G , and Tat Fin arginine rich motifs in complex with the apical stemloop-1 of 7SK. While most interactions between 7SK with HEXIM and Tat are similar, critical differences exist that guide function. First, the conformational plasticity of 7SK enables the formation of three different base pair configurations at a critical remodeling site, which allows for the modulation required for HEXIM binding and its subsequent displacement by Tat. Furthermore, the specific sequence variations observed in various Tat subtypes all converge on remodeling 7SK at this region. Second, we show that HEXIM primes its own displacement by causing specific local destabilization upon binding — a feature that is then exploited by Tat to bind 7SK more efficiently.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural and functional insights into δ-poly-L-ornithine polymer biosynthesis from Acinetobacter baumannii

Cationic homo-polyamino acid (CHPA) peptides containing isopeptide bonds of diamino acids have been identified from Actinomycetes strains. However, none has been reported from other bacteria. Here, we report a δ-poly-L-ornithine synthetase from Acinetobacter baumannii, which we name PosA. Surprisingly, structural analysis of the adenylation domain and biochemical assay shows L-ornithine as the substrate for PosA. The product from the enzymatic reaction was purified and identified as poly-L-ornithine composed of 7-12 amino acid units. Chemical labeling of the polymer confirmed the isopeptide linkage of δ-poly-L-ornithine. We examine the biological activity of chemically synthesized 12-mer δ-poly-L-ornithine, illustrating that the polymer may act as an anti-fungal agent. Structures of the isolated adenylation domain from PosA are presented with several diamino acids and biochemical assays identify important substrate binding residues. Structurally-guided genome-mining led to the identification of homologs with different substrate binding residues that could activate additional substrates. A homolog from Bdellovibrionales sp. shows modest activity with L-arginine but not with any diamino acids observed to be substrates for previously examined CHPA synthetases. Our study indicates the possibility that additional CHPAs may be produced by various microbes, supporting the further exploration of uncharacterized natural products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Genetic control of morphological transitions in a coacervating protein template

Nature routinely exploits liquid–liquid phase separation (LLPS) of proteins to control the assembly and mineralization of hybrid materials. Here, we show that fusion of the Car9 silica-binding peptide to an elastin-like polypeptide (ELP) yields temperature- and sequence-programmable soft matter templates for the synthesis of silicified architectures ranging in size from nanometers to micrometers. Specifically, we demonstrate unprecedented control over the diameter of silica nanoparticles (SiNP) in the 30–60 nm range with 4 nm precision, show that a single arginine residue (R4) in the Car9 sequence underpins the transition from micelles to proteinosomes, and find that substitutions in other basic residues modulate electrostatic repulsion and solvation to enable access to kinetically trapped species. These structures, which include interconnected micelles, small (∼200 nm) and large (>5 µm) vesicles, are readily visualized by SEM imaging following silicification. Molecular dynamics (MD) simulations and AlphaFold predictions reveal that mutations in positively charged residues alter interfacial packing, hydration, and conformational freedom of the silica-binding segments. Overall, our results establish sequence and thermal energy as synergistic levers for morphological control across length scales using solid-binding ELPs and establish mineralization as a powerful tool to visualize the structure of dynamic soft matter assemblies.

hierarchy↗
DOE OSTI2023

Cancer-associated somatic mutations in human phosphofructokinase-1 reveal a critical electrostatic interaction for allosteric regulation of enzyme activity

Metabolic reprogramming, including increased glucose uptake and lactic acid excretion, is a hallmark of cancer. The glycolytic ‘gatekeeper’ enzyme phosphofructokinase-1 (PFK1), which catalyzes the step committing glucose to breakdown, is dysregulated in cancers. While altered PFK1 activity and expression in tumors have been demonstrated, little is known about the effects of cancer-associated somatic mutations. Somatic mutations in PFK1 inform our understanding of allosteric regulation by identifying key amino acid residues involved in the regulation of enzyme activity. Here, we characterized mutations disrupting an evolutionarily conserved salt bridge between aspartic acid and arginine in human platelet (PFKP) and liver (PFKL) isoforms. Using purified recombinant proteins, we showed that disruption of the Asp–Arg pair in two PFK1 isoforms decreased enzyme activity and altered allosteric regulation. We determined the crystal structure of PFK1 to 3.6 Å resolution and used molecular dynamic simulations to understand molecular mechanisms of altered allosteric regulation. We showed that PFKP-D564N had a decreased total system energy and changes in the electrostatic surface potential of the effector site. Cells expressing PFKP-D564N demonstrated a decreased rate of glycolysis, while their ability to induce glycolytic flux under conditions of low cellular energy was enhanced compared with cells expressing wild-type PFKP. Taken together, these results suggest that mutations in Arg–Asp pair at the interface of the catalytic-regulatory domains stabilizes the t-state and presents novel mechanistic insight for therapeutic development in cancer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Structure and assembly of the diiron cofactor in the heme-oxygenase–like domain of the N-nitrosourea–producing enzyme SznF

In biosynthesis of the pancreatic cancer drug streptozotocin, the tridomain nonheme-iron oxygenase SznF hydroxylates N δ and N ω ' of N ω -methyl- L -arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the monoiron cofactor in the enzyme’s C-terminal cupin domain, which promotes the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase–like (HO-like) central domain. We leveraged our recent observation that the N-oxygenating µ-peroxodiiron(III/III) intermediate can form in the HO-like domain after the apo protein self-assembles its diiron(II/II) cofactor to solve structures of SznF with both of its iron cofactors bound. These structures of a biochemically validated member of the emerging heme-oxygenase–like diiron oxidase and oxygenase (HDO) superfamily with intact diiron cofactor reveal both the large-scale conformational change required to assemble the O 2 -reactive Fe 2 (II/II) complex and the structural basis for cofactor instability—a trait shared by the other validated HDOs. During cofactor (dis)assembly, a ligand-harboring core helix dynamically (un)folds. The diiron cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulations. The additional carboxylate ligand is conserved in another N-oxygenating HDO but not in two HDOs that cleave carbon–hydrogen and carbon–carbon bonds to install olefins. Among ~9,600 sequences identified bioinformatically as members of the emerging HDO superfamily, ~25% conserve this additional carboxylate residue and are thus tentatively assigned as N-oxygenases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Discovery of cyanophycin dipeptide hydrolase enzymes suggests widespread utility of the natural biopolymer cyanophycin

Cyanophycin is a bacterial polymer mainly used for nitrogen storage. It is composed of a peptide backbone of L-aspartate residues with L-arginines attached to their side chains through isopeptide bonds. Cyanophycin is degraded in two steps: Cyanophycinase cleaves the polymer into β-Asp-Arg dipeptides, which are hydrolyzed into free Asp and Arg by enzymes possessing isoaspartyl dipeptide hydrolase activity. Two unrelated enzymes with this activity, isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA) have been shown to degrade β-Asp-Arg dipeptides, but bacteria which encode cyanophycin-metabolizing genes can lack iaaA and iadA genes. In this study, we investigate a previously uncharacterized enzyme whose gene can cluster with cyanophycin-metabolizing genes. This enzyme, which we name cyanophycin dipeptide hydrolase (CphZ), is specific for dipeptides derived from cyanophycin degradation. Accordingly, a co-complex structure of CphZ and β-Asp-Arg shows that CphZ, unlike IadA or IaaA, recognizes all portions of its β-Asp-Arg substrate. Bioinformatic analyses showed that CphZ is found in very many proteobacteria and is homologous to an uncharacterized protein encoded in the “arginine/ornithine transport” (aot) operon of many pseudomonas species, including Pseudomonas aeruginosa . In vitro assays show that AotO is indeed a CphZ, and in cellulo growth experiments show that this enzyme and the aot operon allow P. aeruginosa to take up and use β-Asp-Arg as a sole carbon and nitrogen source. Together the results establish the novel, highly specific enzyme subfamily of CphZs, suggesting that cyanophycin is potentially used by a much wider range of bacteria than previously appreciated.

Science & Technology - Other Topics↗
DOE OSTI2024

Homologous mutations in human β, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in β-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman–Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human β, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in β myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in β myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not β, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin’s highly allosteric nature.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Characterization of SETD3 methyltransferase–mediated protein methionine methylation

Most characterized protein methylation events encompass arginine and lysine N-methylation, and only a few cases of protein methionine thiomethylation have been reported. Newly discovered oncohistone mutations include lysine-to-methionine substitutions at positions 27 and 36 of histone H3.3. In these cases, the methionine substitution localizes to the active-site pocket of the corresponding histone lysine methyltransferase, thereby inhibiting the respective transmethylation activity. SET domain–containing 3 (SETD3) is a protein (i.e. actin) histidine methyltransferase. Here, we generated an actin variant in which the histidine target of SETD3 was substituted with methionine. As for previously characterized histone SET domain proteins, the methionine substitution substantially (76-fold) increased binding affinity for SETD3 and inhibited SETD3 activity on histidine. Unexpectedly, SETD3 was active on the substituted methionine, generating S-methylmethionine in the context of actin peptide. The ternary structure of SETD3 in complex with the methionine-containing actin peptide at 1.9 Å resolution revealed that the hydrophobic thioether side chain is packed by the aromatic rings of Tyr 312 and Trp 273 , as well as the hydrocarbon side chain of Ile 310 . Our results suggest that placing methionine properly in the active site—within close proximity to and in line with the incoming methyl group of SAM—would allow some SET domain proteins to selectively methylate methionine in proteins.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

High-quality genome of the basidiomycete yeast Dioszegia hungarica PDD-24b-2 isolated from cloud water

The genome of the basidiomycete yeast Dioszegia hungarica strain PDD-24b-2 isolated from cloud water at the summit of puy de $D\hat{o}me$ (France) was sequenced using a hybrid PacBio and Illumina sequencing strategy. The obtained assembled genome of 20.98 Mb and a GC content of 57% is structured in 16 large-scale contigs ranging from 90 kb to 5.56Mb, and another 27.2 kb contig representing the complete circular mitochondrial genome. In total, 8,234 proteins were predicted from the genome sequence. The mitochondrial genome shows 16.2% cgu codon usage for arginine but has no canonical cognate tRNA to translate this codon. Detected transposable element (TE)-related sequences account for about 0.63% of the assembled genome. A dataset of 2,068 hand-picked public environmental metagenomes, representing over 20 Tbp of raw reads, was probed for D. hungarica related ITS sequences, and revealed worldwide distribution of this species, particularly in aerial habitats. Growth experiments suggested a psychrophilic phenotype and the ability to disperse by producing ballistospores. The high-quality assembled genome obtained for this D. hungarica strain will help investigate the behavior and ecological functions of this species in the environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structures of CTCF–DNA complexes including all 11 zinc fingers

The CCCTC-binding factor (CTCF) binds tens of thousands of enhancers and promoters on mammalian chromosomes by means of its 11 tandem zinc finger (ZF) DNA-binding domain. In addition to the 12–15-bp CORE sequence, some of the CTCF binding sites contain 5' upstream and/or 3' downstream motifs. Here, we describe two structures for overlapping portions of human CTCF, respectively, including ZF1–ZF7 and ZF3–ZF11 in complex with DNA that incorporates the CORE sequence together with either 3' downstream or 5' upstream motifs. Like conventional tandem ZF array proteins, ZF1–ZF7 follow the right-handed twist of the DNA, with each finger occupying and recognizing one triplet of three base pairs in the DNA major groove. ZF8 plays a unique role, acting as a spacer across the DNA minor groove and positioning ZF9–ZF11 to make cross-strand contacts with DNA. We ascribe the difference between the two subgroups of ZF1–ZF7 and ZF8–ZF11 to residues at the two positions -6 and -5 within each finger, with small residues for ZF1–ZF7 and bulkier and polar/charged residues for ZF8–ZF11. ZF8 is also uniquely rich in basic amino acids, which allows salt bridges to DNA phosphates in the minor groove. Highly specific arginine–guanine and glutamine–adenine interactions, used to recognize G:C or A:T base pairs at conventional base-interacting positions of ZFs, also apply to the cross-strand interactions adopted by ZF9–ZF11. The differences between ZF1–ZF7 and ZF8–ZF11 can be rationalized structurally and may contribute to recognition of high-affinity CTCF binding sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

‘ Candidatus Liberibacter asiaticus’-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress In Planta

The lipopolysaccharides (LPS) of gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic ‘Candidatus Liberibacter’ spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on nonhost tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host orange (Citrus sinensis), confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione pool, callose deposition, and activation of the salicylic acid and azelaic acid (AzA) signaling networks. Transient expression of ‘Ca. L. asiaticus’ bacterioferritin comigratory protein (BCP) peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that ‘Ca. L. asiaticus’ BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. ‘Ca. L. asiaticus’ BCP peroxiredoxin (i) attenuates NO-mediated SAR signaling and (ii) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of ‘Ca. L. asiaticus’ acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The essential effector SCRE1 in Ustilaginoidea virens suppresses rice immunity via a small peptide region

Summary The biotrophic fungal pathogen Ustilaginoidea virens causes rice false smut, a newly emerging plant disease that has become epidemic worldwide in recent years. The U. virens genome encodes many putative effector proteins that, based on the study of other pathosystems, could play an essential role in fungal virulence. However, few studies have been reported on virulence functions of individual U. virens effectors. Here, we report our identification and characterization of the secreted cysteine‐rich protein SCRE1, which is an essential virulence effector in U. virens . When SCRE1 was heterologously expressed in Magnaporthe oryzae , the protein was secreted and translocated into plant cells during infection. SCRE1 suppresses the immunity‐associated hypersensitive response in the nonhost plant Nicotiana benthamiana . Induced expression of SCRE1 in rice also inhibits pattern‐triggered immunity and enhances disease susceptibility to rice bacterial and fungal pathogens. The immunosuppressive activity is localized to a small peptide region that contains an important ‘cysteine‐proline‐alanine‐arginine‐serine’ motif. Furthermore, the scre1 knockout mutant generated using the CRISPR/Cas9 system is attenuated in U. virens virulence to rice, which is greatly complemented by the full‐length SCRE1 gene. Collectively, this study indicates that the effector SCRE1 is able to inhibit host immunity and is required for full virulence of U. virens .

Zhang, Nan↗