Nucleotide Binding Site Research Articles

Large-scale gene gains and losses molded the NLR defense arsenal during the Cucurbita evolution

Main conclusionGenome-wide annotation reveals that the gene birth–death process of the Cucurbita R family is associated with a species-specific diversification of TNL and CNL protein classes.The Cucurbitaceae family includes nearly 1000 plant species known universally as cucurbits. Cucurbita genus includes many economically important worldwide crops vulnerable to more than 200 pathogens. Therefore, the identification of pathogen-recognition genes is of utmost importance for this genus. The major class of plant-resistance (R) genes encodes nucleotide-binding site and leucine-rich repeat (NLR) proteins, and is divided into three sub-classes namely, TIR-NB-LRR (TNL), CC-NB-LRR (CNL) and RPW8-NB-LRR (RNL). Although the characterization of the NLR gene family has been carried out in important Cucurbita species, this information is still linked to the availability of sequenced genomes. In this study, we analyzed 40 de novo transcriptomes and 5 genome assemblies, which were explored to investigate the Cucurbita expressed-NLR (eNLR) and NLR repertoires using an ad hoc gene annotation approach. Over 1850 NLR-encoding genes were identified, finely characterized and compared to 96 well-characterized plant R-genes. The maximum likelihood analyses revealed an unusual diversification of CNL/TNL genes and a strong RNL conservation. Indeed, several gene gain and loss events have shaped the Cucurbita NLR family. Finally, to provide a first validation step Cucurbita, eNLRs were explored by real-time PCR analysis. The NLR repertories of the 12 Cucurbita species presented in this paper will be useful to discover novel R-genes.

The RING-type protein BOI negatively regulates the protein level of a CC-NBS-LRR in Arabidopsis

Nucleotide-binding site and leucine-rich repeat receptors (NLRs) play pivotal roles in plant immunity. The regulation of NLR stability is essential to ensure effective immunity, whereas the exact mechanism is largely unclear. The Arabidopsis CC-NBS-LRR protein L5 (At1g12290) can induce cell death in Nicotiana benthamiana, but not in Arabidopsis thaliana. We screened the interactors of L5 by yeast two-hybrid, and found that the BOI can interact with the CC domain of L5. Transiently expressed BOI reduced the protein level of L5, and suppressed the auoactivity of L5 in N. benthamiana. BOI can interact and ubiquitinate L5 in vitro, and mediate the proteasomal degradation of L5 in N. benthamiana and Arabidopsis. The Lys425 in the NBS domain of L5 is the critical unbiquitin site for the degradation. In conclusion, our results reveal a mechanism for the control of the stability of L5 protein and for the suppressed of L5-triggered cell death by a RING-type E3 ligase through the ubiquitin proteasome system.

Inheritance of Black Rot Resistance and Development of Molecular Marker Linked to Xcc Races 6 and 7 Resistance in Cabbage.

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), produces V-shaped chlorotic lesions on the leaves of cabbage (Brassica oleracea var. capitata L.), causing darkened veins and drastically reducing yield and quality. Of the 11 Xcc races identified, races 1, 4, and 6 are predominant globally. In the present study, we aimed to develop a molecular marker linked to black rot resistance against Xcc races 6 and 7. Crossed between black rot-resistant (‘SCNU-C-3470’) and -susceptible (‘SCNU-C-3328’) lines obtained 186 F2 plants. Resistance to Xcc race 6 segregated in a 3:1 (susceptible:resistant) ratio in the F2 population, which is consistent with a monogenic recessive trait. Nucleotide-binding site (NBS) leucine rich repeat (LRR)-encoding resistance (R) genes play a crucial role in plant defenses to various pathogens. The candidate R gene (Bol031422) located on chromosome C08, previously reported by our research group, was cloned and sequenced in resistant and susceptible cabbage lines. The R gene Bol031422 consisted of a single exon with a 3 bp insertion/deletions (InDels), a 292 bp polymorphism (an insertion in the exon of the resistant line relative to the susceptible line) and several single nucleotide polymorphisms (SNPs). Here, we developed the InDel marker BR6-InDel to assess linkage between variation at Bol031422 and resistance to Xcc races 6 and 7. This marker will help cabbage breeders develop cabbage cultivars resistant to Xcc races 6 and 7.

High-resolution mapping of the quantitative trait locus (QTLs) conferring resistance to false smut disease in rice

Rice false smut (RFS), an emerging major fungal disease worldwide caused by Ustilaginoidea virens, affects rice grain quality and yield. RFS cause 2.8-49% global yield loss depending upon disease severity and cultivars. In India, the yield loss due to RFS ranged from 2 to 75%. Identification of the genes or quantitative trait loci (QTLs) governing disease resistance would be of utmost importance towards mitigating the economic losses incurred due to RFS. Here, we report mapping of RFS resistance QTLs from a resistant breeding line RYT2668. The mapping population was evaluated for RFS resistance under the field condition in three cropping seasons 2013, 2015, and 2016. A positive correlation among infected panicle/plant, total smut ball/panicle, and disease score was observed in the years 2013, 2015, and the mean data. A total of seven QTLs were mapped on rice chromosomes 2, 4, 5, 7, and 9 using 2326 single nucleotide polymorphism markers. Of these, two QTLs, qRFSr5.3 and qRFSr7.1a, were associated with the infected panicle per plant, one QTL qRFsr9.1 with total smut ball per panicle, and four QTLs qRFSr2.2, qRFSr4.3, qRFSr5.4, and qRFSr7.1b with disease score. Among them, a novel QTL qRFSr9.1 on chromosome 9 exhibits the largest phenotypic effect. The prediction of putative candidate genes within the qRFSr9.1 revealed four nucleotide-binding sites-leucine-rich repeat (NBS-LRR) domain-containing disease resistance proteins. In summary, our findings mark the hotspot region of rice chromosomes carrying genes/QTLs for resistance to the RFS disease.

Electrical polarity-dependent gating and a unique subconductance of RyR2 induced by S-adenosyl methionine via the ATP binding site.

S-Adenosyl-L-methionine (SAM) was used to probe the functional effects exerted via the cardiac RyR isoform (RyR2) adenine nucleotide binding site. Single channel experiments revealed that SAM applied to the cytoplasmic face of RyR2 had complex voltage dependent effects on channel gating and conductance. At positive transmembrane holding potentials, SAM caused a striking reduction in channel openings and a reduced channel conductance. In contrast, at negative potentials, SAM promoted a clearly resolved subconductance state. At membrane potentials between -75 and -25mV, the open probability of the subconductance state was independent of voltage. ATP, but not the non-adenosine-based ryanodine receptor (RyR) activator 4-chloro-m-cresol, interfered with the effects of SAM at both negative and positive potentials. This suggests that ATP and SAM interact with a common binding site. Molecular docking showed SAM bound to the adenine nucleotide binding site and formed a hydrogen bond to Glu4886 in the C-terminal end of the S6 alpha helix. In this configuration, SAM may alter the conformation of the RyR2 ion conduction pathway. This work provides novel insightGraphical Abstract into potential functional outcomes of ligand binding to the RyR adenine nucleotide binding site.

Aegilops tauschii genome assembly Aet v5.0 features greater sequence contiguity and improved annotation.

Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and an important genetic resource. The reference-quality genome sequence Aet v4.0 for Ae. tauschii acc. AL8/78 was therefore an important milestone for wheat biology and breeding. Further advances in sequencing acc. AL8/78 and release of the Aet v5.0 sequence assembly are reported here. Two new optical maps were constructed and used in the revision of pseudomolecules. Gaps were closed with Pacific Biosciences long-read contigs, decreasing the gap number by 38,899. Transposable elements and protein-coding genes were reannotated. The number of annotated high-confidence genes was reduced from 39,635 in Aet v4.0 to 32,885 in Aet v5.0. A total of 2245 biologically important genes, including those affecting plant phenology, grain quality, and tolerance of abiotic stresses in wheat, was manually annotated and disease-resistance genes were annotated by a dedicated pipeline. Disease-resistance genes encoding nucleotide-binding site domains, receptor-like protein kinases, and receptor-like proteins were preferentially located in distal chromosome regions, whereas those encoding transmembrane coiled-coil proteins were dispersed more evenly along the chromosomes. Discovery, annotation, and expression analyses of microRNA (miRNA) precursors, mature miRNAs, and phasiRNAs are reported, including miRNA target genes. Other small RNAs, such as hc-siRNAs and tRFs, were characterized. These advances enhance the utility of the Ae. tauschii genome sequence for wheat genetics, biotechnology, and breeding.

Recent insights into the relative timing of myosin's powerstroke and release of phosphate.

Myosin is a motor enzyme that converts the chemical energy in ATP into mechanical work to drive a myriad of intracellular processes, from muscle contraction to vesicular transport. Key steps in the transduction of energy are the force-generating powerstroke, and the release of phosphate (Pi ) from the nucleotide-binding site. Both events occur rapidly after binding to actin, making it difficult to determine which event occurs first. Early efforts suggested that these events occur simultaneously; however, recent findings indicate that they are separate and distinct events that occur at different rates. High-resolution crystal structures of myosin captured in intermediate states of the ATPase cycle suggest that when Pi is in the active site it prevents the powerstroke from occurring, leading to the hypothesis that Pi -release precedes the powerstroke. However, advances in functional assays, enabling sub-millisecond temporal and nanometer spatial resolution, are challenging this hypothesis. For example, Föster Resonance Energy Transfer (FRET) based assays, as well as single molecule laser trap assays, suggest the opposite; that the powerstroke occurs prior to the release of Pi from myosin's active site. This review provides some historical context and then highlights recent reports that reveal exciting new insight into this fundamental mechanism of energy transduction by this prototypical motor enzyme.

Melatonin enhances the resistance of ginger rhizomes to postharvest fungal decay

Significant losses in harvested ginger can be directly attributed to decay fungi, including Fusarium oxysporum and Penicillium brevicompactum. Eco-friendly treatments, utilizing non-conventional chemical methods to manage postharvest decay on ginger rhizome are being actively investigated. In the current study, the application of melatonin (N-acetyl-5-methoxytryptamine) was evaluated for controlling postharvest decay of ginger rhizomes, artificially-inoculated with F. oxysporum or P. brevicompactum. Results showed that the melatonin treatment (0.1 mM, 15-min immersion) reduced F. oxysporum and P. brevicompactum rots on rhizomes. Melatonin induced the expression of defense-related genes, including β-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), and coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR). GLU and PAL enzyme activity was also induced in ginger rhizomes, and the level of total phenols in rhizomes was elevated. Importantly, melatonin did not have a negative impact on rhizome quality. The ability of melatonin to enhance disease resistance may be partially attributed to the induction of defense response in ginger rhizomes. The findings of the present study have practical implications for the use of melatonin to reduce postharvest decays in ginger rhizomes.

Discovery and Genetic Mapping of PM1, a Powdery Mildew Resistance Gene in Cannabis sativa L.

Powdery mildew is among the most common diseases of both hemp- and marijuana-type cultivated Cannabis sativa. Despite its prevalence, no documented studies have characterized sources of natural genetic resistance in this pathosystem. Here we provide evidence for the first resistance (R) gene in C. sativa, represented by a single dominant locus that confers complete resistance to an isolate of the powdery mildew pathogen Golovinomyces ambrosiae, found in the Pacific Northwest of the United States. Linkage mapping with nearly 10,000 single nucleotide polymorphism (SNP) markers revealed that this R gene (designated PM1) is located on the distal end of the long arm of one of the largest chromosomes in the C. sativa genome. According to reference whole genome sequences and Sanger sequencing, the marker was tentatively placed in a cluster of R genes of the nucleotide-binding site (NBS) and leucine-rich repeat (LRR) protein type. PM1's dominant behavior, qualitative penetrance, and a co-segregating qPCR marker to track its inheritance were confirmed in two separate genetic backgrounds totaling 185 recombinant F1 plants. The goal of this study is to provide a foundation for the discovery and characterization of additional sources of genetic resistance to pathogens that infect C. sativa.

Search for putative heme binding sites in the integrated membrane protein of human erythrocytes SLC4A1 and protein kinases SYK and LYN

SLC4A1 protein or band 3 protein (band 3) is one of the most common erythrocyte membrane proteins. Along with the function of the anion exchanger, it contributes to the control of cell shape and lifespan through the formation of various complexes with cytoskeletal components and enzymes. Under oxidative stress, the protein oxidative modifications occur, in particular, due to the binding of hemoglobin aggregates, but the direct effect of heme as the major product of hemoglobin degradation on the band 3 protein activity has not been described in the literature. It is known that hemolytic conditions are accompanied by phosphorylation of the SLC4A1 protein, non-receptor tyrosine protein kinases LYN (by residue Y359), and SYK (by residues Y8 and Y21), while SYK kinase inhibitors have a stabilizing effect on erythrocytes. The regulatory effect of heme on Src kinases, which include SYK and LIN kinases, has been clarified, but the sites of their interaction with heme have not been investigated. Therefore, the aim of this study was to predict putative heme binding sites in the integral protein of the erythrocyte membrane SLC4A1, as well as in the protein kinases SYK and LYN and their complexes with SLC4A1, modeled in silico. Sequence analysis of proteins with HeMoQuest revealed several nonapeptides with potential heme binding sites in the SLC4A1 protein, including the cytosolic His98 and Tyr553 and Tyr555 residues in the region between the TM5 and TM6 transmembrane segments. These residues, as well as the amino acids Tyr216, His303, and His192, were also predicted as heme-binding sites by HemeBind tool. The largest number of putative heme binding sites was found for SYK protein kinase, including the two cytosolic residues Tyr216 and His303. Molecular docking of the SLC4A1 protein revealed a heme binding site in the cavity between His192 and region 173–176 in the cytosolic domain structure (PDB ID 4KY9 and 1HYN), also as part of a simulated complex with SYK or LYN kinases. It should be noted that site 175–185 is known as the ankyrin binding site. Docking heme to the membrane domain (PDB ID 4YZF) revealed a putative heme binding site near Lys539 in TM5, which, according to the literature, belongs to one of the reactive centers sensitive to the anionic transport inhibitor DIDS. Molecular docking to SYK protein kinase with ATP in the active site (PDB ID 4FL2) revealed two putative heme binding sites – near Tyr64 and near His243, but when ATP was removed from pdb-file, the heme occupied a nucleotide binding site in the cavity near Lys402 and His531. LYN protein kinase (PDB ID 5XY1) had a heme binding site near Tyr321 in the structure with inhibitor molecule (piperazine derivative). After removing the inhibitor, the heme occupied its area near Glu290 and Ala371. In most model complexes, the band 3 protein was revealed to be a more likely heme binding site than LYN and SYK protein kinases, but kinases with free active sites can apparently bind a heme instead of substrate, which will interfere phosphorylation. Disruption of band 3 protein under heme accumulation can inhibit an anion transport or complicate the formation of SLC4A1 complexes with cytoskeletal proteins that together with the effects on phosphorylation could be a mechanism for reducing erythrocyte stability.

Molecular basis of cholesterol efflux via ABCG subfamily transporters

The ABCG1 homodimer (G1) and ABCG5-ABCG8 heterodimer (G5G8), two members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter G family, are required for maintenance of cellular cholesterol levels. G5G8 mediates secretion of neutral sterols into bile and the gut lumen, whereas G1 transports cholesterol from macrophages to high-density lipoproteins (HDLs). The mechanisms used by G5G8 and G1 to recognize and export sterols remain unclear. Here, we report cryoelectron microscopy (cryo-EM) structures of human G5G8 in sterol-bound and human G1 in cholesterol- and ATP-bound states. Both transporters have a sterol-binding site that is accessible from the cytosolic leaflet. A second site is present midway through the transmembrane domains of G5G8. The Walker A motif of G8 adopts a unique conformation that accounts for the marked asymmetry in ATPase activities between the two nucleotide-binding sites of G5G8. These structures, along with functional validation studies, provide a mechanistic framework for understanding cholesterol efflux via ABC transporters.

Systematic dissection of disease resistance to southern corn rust by bulked-segregant and transcriptome analysis

Southern corn rust (SCR) is a destructive maize disease caused by Puccinia polysora Underw. To investigate the mechanism of SCR resistance in maize, a highly resistant inbred line, L119A, and a highly susceptible line, Lx9801, were subjected to gene mapping and transcriptome analysis. Bulked-segregant analysis coupled with whole-genome sequencing revealed several quantitative trait loci (QTL) on chromosomes 1, 6, 8, and 10. A set of 25 genes, including two coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) genes, were identified as candidate genes for a major-effect QTL on chromosome 10. To investigate the mechanism of SCR resistance in L119A, RNA-seq of P. polysora-inoculated and non-inoculated plants of L119A and Lx9801 was performed. Unexpectedly, the number of differentially expressed genes in inoculated versus non-inoculated L119A plants was about 10 times that of Lx9801, with only 29 common genes identified in both lines, suggesting extensive gene expression changes in the highly resistant but not in the susceptible line. Based on the transcriptome analysis, one of the CC-NBS-LRR candidate genes was confirmed to be upregulated in L119A relative to Lx9801 independently of P. polysora inoculation. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that transcription factors, as well as genes involved in defense responses and metabolic processes, were dominantly enriched, with the phenylpropanoid biosynthesis pathway most specifically activated. Consistently, accumulation of phenylpropanoid-derived lignin, especially S lignin, was drastically increased in L119A after P. polysora inoculation, but remained unchanged in Lx9801, suggesting a critical role of lignin in SCR resistance. A regulatory network of defense activation and metabolic change in SCR-resistant maize upon P. polysora infection is described.

Structural and Functional Genomics of the Resistance of Cacao to Phytophthora palmivora.

Black pod disease, caused by Phytophthora spp., is one of the main diseases that attack cocoa plantations. This study validated, by association mapping, 29 SSR molecular markers flanking to QTL (Quantitative Trait Loci) associated with Phytophthora palmivora Butler (Butler) (PP) resistance, in three local ancient varieties of the Bahia (Comum, Pará, and Maranhão), varieties that have a high potential in the production of gourmet chocolate. Four SSR loci associated with resistance to PP were detected, two on chromosome 8, explaining 7.43% and 3.72% of the Phenotypic Variation (%PV), one on chromosome 2 explaining 2.71%PV and one on chromosome 3 explaining 1.93%PV. A functional domains-based annotation was carried out, in two Theobroma cacao (CRIOLLO and MATINA) reference genomes, of 20 QTL regions associated with cocoa resistance to the pathogen. It was identified 164 (genome CRIOLLO) and 160 (genome MATINA) candidate genes, hypothetically involved in the recognition and activation of responses in the interaction with the pathogen. Genomic regions rich in genes with Coiled-coils (CC), nucleotide binding sites (NBS) and Leucine-rich repeat (LRR) domains were identified on chromosomes 1, 3, 6, 8, and 10, likewise, regions rich in Receptor-like Kinase domain (RLK) and Ginkbilobin2 (GNK2) domains were identified in chromosomes 4 and 6.

Characterization of Nucleotide Binding ­Site-Encoding Genes in Sweetpotato, Ipomoea batatas(L.) Lam., and Their Response to Biotic and Abiotic Stresses

Sweetpotato, Ipomoea batatas (L.) Lam., is an important and widely grown crop, yet its production is affected severely by biotic and abiotic stresses. The nucleotide binding site (NBS)-encoding genes have been shown to improve stress tolerance in several plant species. However, the characterization of NBS-encoding genes in sweetpotato is not well-documented to date. In this study, a comprehensive analysis of NBS-encoding genes has been conducted on this species by using bioinformatics and molecular biology methods. A total of 315 NBS-encoding genes were identified, and 260 of them contained all essential conserved domains while 55 genes were truncated. Based on domain architectures, the 260 NBS-encoding genes were grouped into 6 distinct categories. Phylogenetic analysis grouped these genes into 3 classes: TIR, CC (I), and CC (II). Chromosome location analysis revealed that the distribution of NBS-encoding genes in chromosomes was uneven, with a number ranging from 1 to 34. Multiple stress-related regulatory elements were detected in the promoters, and the NBS-encoding genes’ expression profiles under biotic and abiotic stresses were obtained. According to the bioinformatics analysis, 9 genes were selected for RT-qPCR analysis. The results revealed that IbNBS75, IbNBS219, and IbNBS256 respond to stem nematode infection; Ib­NBS240, IbNBS90, and IbNBS80 respond to cold stress, while IbNBS208, IbNBS71, and IbNBS159 respond to 30% PEG treatment. We hope these results will provide new insights into the evolution of NBS-encoding genes in the sweetpotato genome and contribute to the molecular breeding of sweetpotato in the future.

Fine dissection of limber pine resistance to Cronartium ribicola using targeted sequencing of the NLR family

BackgroundProteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains (NLR) make up one of most important resistance (R) families for plants to resist attacks from various pathogens and pests. The available transcriptomes of limber pine (Pinus flexilis) allow us to characterize NLR genes and related resistance gene analogs (RGAs) in host resistance against Cronartium ribicola, the causal fungal pathogen of white pine blister rust (WPBR) on five-needle pines throughout the world. We previously mapped a limber pine major gene locus (Cr4) that confers complete resistance to C. ribicola on the Pinus consensus linkage group 8 (LG-8). However, genetic distribution of NLR genes as well as their divergence between resistant and susceptible alleles are still unknown.ResultsTo identify NLR genes at the Cr4 locus, the present study re-sequenced a total of 480 RGAs using targeted sequencing in a Cr4-segregated seed family. Following a call of single nucleotide polymorphisms (SNPs) and genetic mapping, a total of 541 SNPs from 155 genes were mapped across 12 LGs. Three putative NLR genes were newly mapped in the Cr4 region, including one that co-segregated with Cr4. The tight linkage of NLRs with Cr4-controlled phenotypes was further confirmed by bulked segregation analysis (BSA) using extreme-phenotype genome-wide association study (XP-GWAS) for significance test. Local tandem duplication in the Cr4 region was further supported by syntenic analysis using the sugar pine genome sequence. Significant gene divergences have been observed in the NLR family, revealing that diversifying selection pressures are relatively higher in local duplicated genes. Most genes showed similar expression patterns at low levels, but some were affected by genetic background related to disease resistance. Evidence from fine genetic dissection, evolutionary analysis, and expression profiling suggests that two NLR genes are the most promising candidates for Cr4 against WPBR.ConclusionThis study provides fundamental insights into genetic architecture of the Cr4 locus as well as a set of NLR variants for marker-assisted selection in limber pine breeding. Novel NLR genes were identified at the Cr4 locus and the Cr4 candidates will aid deployment of this R gene in combination with other major/minor genes in the limber pine breeding program.

Kolaflavanone, a biflavonoid derived from medicinal plant Garcinia, is an inhibitor of mitotic kinesin Eg5.

Mitotic kinesin Eg5 remains a validated target in antimitotic therapy because of its essential role in the formation and maintenance of bipolar mitotic spindles. Although numerous Eg5 inhibitors of synthetic origin are known, only a few inhibitors derived from natural products have been reported. In our study, we focused on identifying novel Eg5 inhibitors from medicinal plants, particularly Garcinia species. Herein, we report the inhibitory effect of kolaflavanone (KLF), a Garcinia biflavonoid, on the ATPase and microtubule-gliding activities of mitotic kinesin Eg5. Additionally, we showed the interaction mechanism between Eg5 and KLF via in vitro and in silico analyses. The results revealed that KLF inhibited both the basal and microtubule-activated ATPase activities of Eg5. The inhibitory mechanism is allosteric, without a direct competition with adenosine-5'-diphosphate for the nucleotide-binding site. KLF also suppressed the microtubule gliding of Eg5 in vitro. The Eg5-KLF model obtained from molecular docking showed that the biflavonoid exists within the α2/α3/L5 (α2: Lys111-Glu116 and Ile135-Asp149, α3: Asn206-Thr226; L5: Gly117-Gly134) pocket, with a binding pose comparable to known Eg5 inhibitors. Overall, our data suggest that KLF is a novel allosteric inhibitor of mitotic kinesin Eg5.

Abstract 225: Computational analysis of 5-fluorouracil antitumor activity in colon cancer using a mechanistic pharmacokinetic/pharmacodynamic model

Abstract The purpose of this study is to develop a mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model to quantitatively analyze the treatment outcome of 5-fluorouracil (5-FU) in colon cancer. Predicting the outcome of chemotherapeutic agents plays an essential role in improving dose strategies. To that end, we have developed an integrated mechanistic PK/PD model to analyze the kinetics of tumor response to 5-FU treatment. Compared to the existing PK/PD models, our model is novel in the way it incorporates the 5-FU metabolism pathway and downstream antiproliferative events to provide a comprehensive model of treatment outcome. 5-FU has been widely used to treat cancers, including colon cancer. 5-FU metabolites can be incorporated into RNA, DNA and also interfere with thymidylate synthase (TS) catalytic activity, which is the main mechanism of 5-FU cytotoxicity. TS activity has been shown to be the predictor for treatment efficacy. The conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate catalyzed by TS is the sole intracellular de novo source of thymidylate. The formation of a ternary complex between fluorodeoxyuridylate (FdUMP) and TS obstructs the access of dUMP to the TS nucleotide-binding site. Resultant thymidine deprivation and dUMP accumulation give rise to the deoxynucleotide triphosphate (dNTP) pool imbalance, which could trigger S-phase specific DNA fragmentation, cell cycle arrest and apoptosis. In our PK model, compartmental analysis is developed to track the distribution of 5-FU from its entry into body to interstitial fluid outside the tumor cells and to its cellular uptake. In the cell model, the connection of TS inhibition and dNTP pool imbalance is implemented. In addition to the central plasma and peripheral tissue compartments, our model includes compartments for: 5-FU concentration in the extracellular fluids; intracellular 5-FU; 5-FU anabolites; genomic 5-FU in RNA, and in DNA; and, TS - FdUMP complex. Posttreatment free TS level is used to compute the perturbed TS catalytic activity. Using the model outcome on TS inhibition, we modeled the kinetics of dNTP pool perturbation. The dNTP pool imbalance leads to double strand break (DSB) formation that initiates DNA damage repair response, kinetics of which are also modeled. Our model uses the law of mass action and Michaelian kinetics and each molecular reaction is expressed as a nonlinear differential equation. Time course data from the literature are used in parameter estimation. Our simulation results successfully captured the kinetics of treatment outcome from the administration of the 5-FU to downstream effects in the tumor. The next step in our effort is to expand the model to include the tumor growth kinetics. Citation Format: Chenhui Ma, Alexandru Almasan, Evren Gurkan-Cavusoglu. Computational analysis of 5-fluorouracil antitumor activity in colon cancer using a mechanistic pharmacokinetic/pharmacodynamic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 225.

Gapless indica rice genome reveals synergistic contributions of active transposable elements and segmental duplications to rice genome evolution

The ultimate goal of genome assembly is a high-accuracy gapless genome. Here, we report a new assembly pipeline that is used to produce a gapless genome for the indica rice cultivar Minghui 63. The resulting 397.71-Mb final assembly is composed of 12 contigs with a contig N50 size of 31.93 Mb. Each chromosome is represented by a single contig and the genomic sequences of all chromosomes are gapless. Quality evaluation of this gapless genome assembly showed that gene regions in our assembly have the highest completeness compared with the other 15 reported high-quality rice genomes. Further comparison with the japonica rice genome revealed that the gapless indica genome assembly contains more transposable elements (TEs) and segmental duplications (SDs), the latter of which produce many duplicated genes that can affect agronomic traits through dose effect or sub-/neo-functionalization. The insertion of TEs can also affect the expression of duplicated genes, which may drive the evolution of these genes. Furthermore, we found the expansion of nucleotide-binding site with leucine-rich repeat disease-resistance genes and cis-zeatin-O-glucosyltransferase growth-related genes in SDs in the gapless indica genome assembly, suggesting that SDs contribute to the adaptive evolution of rice disease resistance and developmental processes. Collectively, our findings suggest that active TEs and SDs synergistically contribute to rice genome evolution.

A spinach genome assembly with remarkable completeness, and its use for rapid identification of candidate genes for agronomic traits.

Spinach (Spinacia oleracea) is grown as a nutritious leafy vegetable worldwide. To accelerate spinach breeding efficiency, a high-quality reference genome sequence with great completeness and continuity is needed as a basic infrastructure. Here, we used long-read and linked-read technologies to construct a de novo spinach genome assembly, designated SOL_r1.1, which was comprised of 287 scaffolds (total size: 935.7 Mb; N50 = 11.3 Mb) with a low proportion of undetermined nucleotides (Ns = 0.34%) and with high gene completeness (BUSCO complete 96.9%). A genome-wide survey of resistance gene analogues identified 695 genes encoding nucleotide-binding site domains, receptor-like protein kinases, receptor-like proteins and transmembrane-coiled coil domains. Based on a high-density double-digest restriction-site associated DNA sequencing-based linkage map, the genome assembly was anchored to six pseudomolecules representing ∼73.5% of the whole genome assembly. In addition, we used SOL_r1.1 to identify quantitative trait loci for bolting timing and fruit/seed shape, which harbour biologically plausible candidate genes, such as homologues of the FLOWERING LOCUS T and EPIDERMAL PATTERNING FACTOR-LIKE genes. The new genome assembly, SOL_r1.1, will serve as a useful resource for identifying loci associated with important agronomic traits and for developing molecular markers for spinach breeding/selection programs.

A recombined Sr26 and Sr61 disease resistance gene stack in wheat encodes unrelated NLR genes

The re-emergence of stem rust on wheat in Europe and Africa is reinforcing the ongoing need for durable resistance gene deployment. Here, we isolate from wheat, Sr26 and Sr61, with both genes independently introduced as alien chromosome introgressions from tall wheat grass (Thinopyrum ponticum). Mutational genomics and targeted exome capture identify Sr26 and Sr61 as separate single genes that encode unrelated (34.8%) nucleotide binding site leucine rich repeat proteins. Sr26 and Sr61 are each validated by transgenic complementation using endogenous and/or heterologous promoter sequences. Sr61 orthologs are absent from current Thinopyrum elongatum and wheat pan genome sequences, contrasting with Sr26 where homologues are present. Using gene-specific markers, we validate the presence of both genes on a single recombinant alien segment developed in wheat. The co-location of these genes on a small non-recombinogenic segment simplifies their deployment as a gene stack and potentially enhances their resistance durability.