Septuple Mutant Research Articles

Plasmodium falciparum drug resistance-associated mutations in isolates from children living in endemic areas of Burkina Faso

BackgroundArtemisinin-based combinations therapy (ACT) is the current frontline curative therapy for uncomplicated malaria in Burkina Faso. Sulfadoxine-pyrimethamine (SP) is used for the preventive treatment of pregnant women (IPTp), while SP plus amodiaquine (SP-AQ) is recommended for children under five in seasonal malaria chemoprevention (SMC). This study aimed to assess the proportions of mutations in the P. falciparum multidrug-resistance 1 (Pfmdr1), P. falciparum chloroquine resistance transporter (Pfcrt), P. falciparum dihydrofolate reductase (pfdhfr), and P. falciparum dihydropteroate synthase (pfdhps), genes from isolates collected during household surveys in Burkina Faso.MethodsDried blood spots from Plasmodium falciparum-positive cases at three sites (Orodara, Gaoua, and Banfora) collected during the peak of transmission were analysed for mutations in Pfcrt (codons 72–76, 93, 97, 145, 218, 343, 350 and 353), Pfmdr-1 (codons 86, 184, 1034, 1042 and 1246) dhfr (codons 51, 59, 108, 164) and dhps (at codons 431, 436, 437, 540, 581, 613) genes using deep sequencing of multiplexed Polymerase chaine reaction (PCR) amplicons.ResultsOf the 377 samples analysed, 346 (91.7%), 369 (97.9%), 368 (97.6%), and 374 (99.2%) were successfully sequenced for Pfcrt, Pfmdr-1, dhfr, and dhps, respectively. Most of the samples had a Pfcrt wild-type allele (89.3%). The 76T mutation was below 10%. The most frequent Pfmdr-1 mutation was detected at codon 184 (Y > F, 30.9%). The single mutant genotype (NFSND) predominated (66.7%), followed by the wild-type genotype (NYSND, 30.4%). The highest dhfr mutations were observed at codon 59R (69.8%), followed by codons 51I (66.6%) and 108 N (14.7%). The double mutant genotype (ACIRSI) predominated (52.4%). For mutation in the dhps gene, the highest frequency was observed at codon 437 K (89.3%), followed by codons 436 A (61.2%), and 613 S (14.4%). The double mutant genotype (IAKKAA) and the single mutant genotype (ISKKAA) were predominant (37.7% and 37.2%, respectively). The most frequent dhfr/dhps haplotypes were the triple mutant ACIRSI/IAKKAA (23%), the wild-type ACNCSI/ISKKAA (19%) and the double mutant ACIRSI/ISKKAA (14%). A septuple mutant ACIRNI/VAKKGA was observed in 2 isolates from Gaoua (0.5%).ConclusionThe efficacy of ACT partner drugs and drugs used in IPTp and SMC does not appear to be affected by the low proportion of highly resistant mutants observed in this study. Continued monitoring, including molecular surveillance, is critical for decision-making on effective treatment policy in Burkina Faso.

Combining multiplex gene editing and doubled haploid technology in maize.

A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.

Systematic Large Fragment Deletions in the Genome of Synechococcus elongatus and the Consequent Changes in Transcriptomic Profiles.

Genome streamlining, as a natural process in the evolution of microbes, has become a common approach for generating ideal chassis cells for synthetic biology studies and industrial applications. However, systematic genome reduction remains a bottleneck in the generation of such chassis cells with cyanobacteria, due to very time-consuming genetic manipulations. Synechococcus elongatus PCC 7942, a unicellular cyanobacterium, is a candidate for systematic genome reduction, as its essential and nonessential genes have been experimentally identified. Here, we report that at least 20 of the 23 over 10 kb nonessential gene regions could be deleted and that stepwise deletions of these regions could be achieved. A septuple-deletion mutant (genome reduced by 3.8%) was generated, and the effects of genome reduction on the growth and genome-wide transcription were investigated. In the ancestral triple to sextuple mutants (b, c, d, e1), an increasingly large number of genes (up to 998) were upregulated relative to the wild type, while slightly fewer genes (831) were upregulated in the septuple mutant (f). In a different sextuple mutant (e2) derived from the quintuple mutant d, much fewer genes (232) were upregulated. Under the standard conditions in this study, the mutant e2 showed a higher growth rate than the wild type, e1 and f. Our results indicate that it is feasible to extensively reduce the genomes of cyanobacteria for generation of chassis cells and for experimental evolutionary studies.

CLE3 and its homologs share overlapping functions in the modulation of lateral root formation through CLV1 and BAM1 in Arabidopsis thaliana.

Lateral roots are important for a wide range of processes, including uptake of water and nutrients. The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-RELATED (CLE) 1 ~ 7 peptide family and their cognate receptor CLV1 have been shown to negatively regulate lateral root formation under low-nitrate conditions. However, little is known about how CLE signaling regulates lateral root formation. A persistent obstacle in CLE peptide research is their functional redundancies, which makes functional analyses difficult. To address this problem, we generate the cle1 ~ 7 septuple mutant (cle1 ~ 7-cr1, cr stands for mutant allele generated with CRISPR/Cas9). cle1 ~ 7-cr1 exhibits longer lateral roots under normal conditions. Specifically, in cle1 ~ 7-cr1, the lateral root density is increased, and lateral root primordia initiation is found to be accelerated. Further analysis shows that cle3 single mutant exhibits slightly longer lateral roots. On the other hand, plants that overexpress CLE2 and CLE3 exhibit decreased lateral root lengths. To explore cognate receptor(s) of CLE2 and CLE3, we analyze lateral root lengths in clv1 barely any meristem 1(bam1) double mutant. Mutating both the CLV1 and BAM1 causes longer lateral roots, but not in each single mutant. In addition, genetic analysis reveals that CLV1 and BAM1 are epistatic to CLE2 and CLE3. Furthermore, gene expression analysis shows that the LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes, which promote lateral root formation, are upregulated in cle1 ~ 7-cr1 and clv1 bam1. We therefore propose that CLE2 and CLE3 peptides are perceived by CLV1 and BAM1 to mediate lateral root formation through LBDs regulation.

Coffee silverskin as a substrate for biobased production of biomass and hydrogen by Escherichia coli

Currently, coffee consumption reached 9.92 million tons, being responsible for the generation of huge amounts of waste associated with coffee bean processing steps and further operations. Coffee silverskin (CS) and spent coffee grounds having high organic content are generated as a result of roasting and brewing processes. Escherichia coli wild-type and septuple mutant (ΔhyaB ΔhybC ΔhycA ΔfdoG ΔldhA ΔfrdC ΔaceE) growth and hydrogen production were investigated in mediums containing 20 to 200 g L−1 CS with dilutions, hydrolyzed for 25 or 45 min. During utilization of CS, hydrolysate phenolic and flavonoid content does affect bacterial growth, moreover, shorter hydrolyzing was more optimal for biomass generation. Highest specific growth rate of 0.64 ± 0.02 h−1 was observed in wild-type cells when 5 times diluted 200 g L−1 CS was applied to yield 0.495 ± 0.015 g L−1 biomass. Prolonged hydrogen (H2) production determined as an oxidation reduction potential drop was observed during utilization of 20% CS containing twice diluted medium started from 3 h until 72 h. Highest H2 yield was observed in the twice diluted medium after 24 h growth containing 65 g L−1 CS. Particularly it was 1.66 mL (g CS)−1 and 2.15 mL (g CS)−1 in wild-type and septuple mutant, respectively. It can be concluded that CS also can be considered as perspective, cost-effective efficient carbon source for the development of biomass and bio-H2 production technologies.

Anti-folate quintuple mutations in Plasmodium falciparum asymptomatic infections in Yaoundé, Cameroon

A major challenge in the fight to effectively control malaria is the emergence of resistant parasite to drugs used in therapy as well as for chemoprevention. In this study, single nucleotide polymorphisms (SNPs) associated with Plasmodium falciparum resistance to sulfadoxine-pyrimethamine (SP), one of the partner drugs in artemisinin-based therapies (ACTs) were studied in asymptomatic P. falciparum isolates from Cameroon. Dried Blood spots were collected from children with asymptomatic malaria enrolled during a household survey. The P. falciparum dihydrofolate reductase (Pfdhfr), dihydropteroate synthase (Pfdhps) and Kelch 13 genes were amplified and point mutations in these gene sequences were analyzed by sequencing. Among a total of 234 samples collected, 51 showed parasitaemia after microscopic examination of which 47 were P. falciparum mono-infections. Molecular analysis revealed 97.3% of mutant alleles at codons 51I, 59R and 108 N in Pfdhfr gene. In Pfdhps gene the most common mutation was 437G (83.3%); followed by 436A (47.6%) and 436F (28.6%). The association of mutations in the two genes (dhfr + dhps) showed 11 different haplotypes including three sextuple mutants (IRNI + AGKGA, IRNI + AAKGS, IRNI + AGKAS) and one septuple mutant (IRNI + AGKGS). For K13 gene no SNPs were seen in the studied asymptomatic malaria samples. The findings revealed presence of SP-resistant alleles in asymptomatic infected individuals with presence of sextuples and septuple SNPs. This emphasizes that regular profiling of antimalarial drugs resistance markers in such population is essential for malaria control and elimination programmes.

Type one protein phosphatase regulates fixed-carbon starvation-induced autophagy in Arabidopsis.

Autophagy, a conserved pathway that carries out the bulk degradation of cytoplasmic material in eukaryotic cells, is critical for plant physiology and development. This process is tightly regulated by ATG13, a core component of the ATG1 kinase complex, which initiates autophagy. Although ATG13 is known to be dephosphorylated immediately after nutrient starvation, the phosphatase regulating this process is poorly understood. Here, we determined that the Arabidopsis (Arabidopsis thaliana) septuple mutant (topp-7m) and octuple mutant (topp-8m) of TYPE ONE PROTEIN PHOSPHATASE (TOPP) exhibited significantly reduced tolerance to fixed-carbon (C) starvation due to compromised autophagy activity. Genetic analysis placed TOPP upstream of autophagy. Interestingly, ATG13a was found to be an interactor of TOPP. TOPP directly dephosphorylated ATG13a in vitro and in vivo. We identified 18 phosphorylation sites in ATG13a by LC-MS. Phospho-dead ATG13a at these 18 sites significantly promoted autophagy and increased the tolerance of the atg13ab mutant to fixed-C starvation. The dephosphorylation of ATG13a facilitated ATG1a-ATG13a complex formation. Consistently, the recruitment of ATG13a for ATG1a was markedly inhibited in topp-7m-1. Finally, TOPP-controlled dephosphorylation of ATG13a boosted ATG1a phosphorylation. Taken together, our study reveals the crucial role of TOPP in regulating autophagy by stimulating the formation of the ATG1a-ATG13a complex by dephosphorylating ATG13a in Arabidopsis.

Vacuolar nitrate efflux requires multiple functional redundant nitrate transporter in Arabidopsis thaliana.

Nitrate in plants is preferentially stored in vacuoles; however, how vacuolar nitrate is reallocated and to which biological process(es) it might contribute remain largely elusive. In this study, we functionally characterized three nitrate transporters NPF5.10, NPF5.14, and NPF8.5 that are tonoplast-localized. Ectopic expression in Xenopus laevis oocytes revealed that they mediate low-affinity nitrate transport. Histochemical analysis showed that these transporters were expressed preferentially in pericycle and xylem parenchyma cells. NPF5.10, NPF5.14, and NPF8.5 overexpression significantly decreased vacuolar nitrate contents and nitrate accumulation in Arabidopsis shoots. Further analysis showed that the sextuple mutant (npf5.10 npf5.14 npf8.5 npf5.11 npf5.12 npf5.16) had a higher 15NO3-uptake rate than the wild-type Col-0, but no significant difference was observed for nitrate accumulation between them. The septuple mutant (npf5.11 npf5.12 npf5.16 npf5.10 npf5.14 npf8.5 clca) generated by using CRISPR/Cas9 showed essentially decreased nitrate reallocation compared to wild type when exposed to nitrate starvation, though no further decrease was observed when compared to clca. Notably, NPF5.10, NPF5.14, and NPF8.5 as well as NPF5.11, NPF5.12, and NPF5.16 were consistently induced by mannitol, and more nitrate was detected in the sextuple mutant than in the wild type after mannitol treatment. These observations suggest that vacuolar nitrate efflux is regulated by several functional redundant nitrate transporters, and the reallocation might contribute to osmotic stress response other than mineral nutrition.

Local conjugation of auxin by the GH3 amido synthetases is required for normal development of roots and flowers in Arabidopsis

Gretchen Hagen 3 (GH3) amido synthetases conjugate amino acids to a carboxyl group of small molecules including hormones auxin, jasmonate, and salicylic acid. The Arabidopsis genome harbors 19 GH3 genes, whose exact roles in plant development have been difficult to define because of genetic redundancy among the GH3 genes. Here we use CRISPR/Cas9 gene editing technology to delete the Arabidopsis group II GH3 genes, which are able to conjugate indole-3-acetic acid (IAA) to amino acids. We show that plants lacking the eight group II GH3 genes (gh3 octuple mutants) accumulate free IAA and fail to produce IAA-Asp and IAA-Glu conjugates. Consequently, gh3 octuple mutants have extremely short roots, long and dense root hairs, and long hypocotyls. Our characterization of gh3 septuple mutants, which provide sensitized backgrounds, reveals that GH3.17 and GH3.9 play prominent roles in root elongation and seed production, respectively. We show that GH3 functions correlate with their expression patterns, suggesting that local deactivation of auxin also contributes to maintaining auxin homeostasis. Moreover, this work provides a method for elucidating functions of individual members of a gene family, whose members have overlapping functions.

Nitrate-responsive NIN-like protein transcription factors perform unique and redundant roles in Arabidopsis.

Upon sensing nitrate, NODULE INCEPTION (NIN)-like protein (NLP) transcription factors alter gene expression to promote nitrate uptake and utilization. Of the nine NLPs in Arabidopsis, the physiological roles of only three NLPs (NLP6-NLP8) have been characterized to date. To evaluate the unique and redundant roles of Arabidopsis NLPs, we assessed the phenotypes of single and higher order nlp mutants. Unlike other nlp single mutants, nlp2 and nlp7 single mutants showed a reduction in shoot fresh weight when grown in the presence of nitrate as the sole nitrogen source, indicating that NLP2, like NLP7, plays a major role in vegetative growth. Interestingly, the growth defect of nlp7 recovered upon the supply of ammonium or glutamine, whereas that of nlp2 did not. Furthermore, complementation assays using chimeric constructs revealed that the coding sequence, but not the promoter region, of NLP genes was responsible for the differences between nlp2 and nlp7 single mutant phenotypes, suggesting differences in protein function. Importantly, nitrate utilization was almost completely abolished in the nlp septuple mutant (nlp2 nlp4 nlp5 nlp6 nlp7 nlp8 nlp9), suggesting that NLPs other than NLP2 and NLP7 also assist in the regulation of nitrate-inducible gene expression and nitrate-dependent promotion of vegetative growth in Arabidopsis.

TCP transcription factors suppress cotyledon trichomes by impeding a cell differentiation-regulating complex.

Trichomes are specialized epidermal cells that act as barriers against biotic and abiotic stresses. Although the formation of trichomes on hairy organs is well studied, the molecular mechanisms of trichome inhibition on smooth organs are still largely unknown. Here, we demonstrate that the CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors inhibit the formation of trichomes on cotyledons in Arabidopsis (Arabidopsis thaliana). The tcp2/3/4/5/10/13/17 septuple mutant produces cotyledons with ectopic trichomes on the adaxial sides. The expression patterns of TCP genes are developmentally regulated during cotyledon development. TCP proteins directly interact with GLABRA3 (GL3), a key component of the MYB transcription factor/basic helix-loop-helix domain protein/WD40-repeat proteins (MYB-bHLH-WD40, MBW) complex essential for trichome formation, to interfere with the transactivation activity of the MBW complex in cotyledons. TCPs also disrupt the MBW complex-R3 MYB negative feedback loop by directly promoting the expression of R3 MYB genes, which enhance the repression of the MBW complex. Our findings reveal a molecular framework in which TCPs suppress trichome formation on adaxial sides of cotyledons by repressing the activity of the MBW complex at the protein level and the transcripts of R3 MYB genes at the transcriptional level.

Roasted coffee wastes as a substrate for Escherichia coli to grow and produce hydrogen.

After brewing roasted coffee, spent coffee grounds (SCGs) are generated being one of the daily wastes emerging in dominant countries with high rate and big quantity. Escherichia coli BW25113 wild-type strain, mutants with defects in hydrogen (H2)-producing/oxidizing four hydrogenases (Hyd) (ΔhyaB ΔhybC, ΔhycE, ΔhyfG) and septuple mutant (ΔhyaB ΔhybC ΔhycA ΔfdoG ΔldhA ΔfrdC ΔaceE) were investigated by measuring change of external pH, bacterial growth and H2 production during the utilization of SCG hydrolysate. In wild type, H2 was produced with rate of 1.28mL H2 (g sugar)-1 h-1 yielding 30.7mL H2 (g sugar)-1 or 2.75L (kg SCG)-1 during 24h. In septuple mutant, H2 production yield was 72mL H2 (g sugar)-1 with rate of 3mL H2 (g sugar)-1 h-1. H2 generation was absent in hycE single mutant showing the main role of Hyd-3 in H2 production. During utilization of SCG wild type, specific growth rate was 0.72±0.01 h-1 with biomass yield of 0.3g L-1. Genetic modifications and control of external parameters during growth could lead to prolonged and enhanced microbiological H2 production by organic wastes, which will aid more efficiently global sustainable energy needs resulting in diversification of mobile and fixed energy sources.

What Are the Functions of Chitin Deacetylases in Aspergillus fumigatus?

Deacetylation of chitin by chitin deacetylases (Cda) results in the formation of chitosan. Chitosan, a polymer of β1,4 linked glucosamine, plays multiple roles in the function of the fungal cell wall, including virulence and evasion of host immune responses. In this study, the roles of chitosan and putative CDAs in cell wall structure and virulence of Aspergillus fumigatus were investigated. Low levels of chitosan were found in the conidial and cell wall of A. fumigatus. Seven putative CDA genes were identified, disrupted and the phenotype of the single mutants and the septuple mutants were investigated. No alterations in fungal cell wall chitosan levels, changes in fungal growth or alterations in virulence were detected in the single or septuple Δcda1-7 mutant strains. Collectively, these results suggest that chitosan is a minority component of the A. fumigatus cell wall, and that the seven candidate Cda proteins do not play major roles in fungal cell wall synthesis or virulence. However, Cda2 is involved in conidiation, suggesting that this enzyme may play a role in N-acetyl-glucosamine metabolism.

Type one protein phosphatases (TOPPs) contribute to the plant defense response in Arabidopsis.

Plant immunity must be tightly controlled to avoid activation of defense mechanisms in the absence of pathogen attack. Protein phosphorylation is a common mechanism regulating immune signaling. In Arabidopsis thaliana, nine members of the type one protein phosphatase (TOPP) family (also known as protein phosphatase 1, PP1) have been identified. Here, we characterized the autoimmune phenotype of topp4-1, a previously identified dominant-negative mutant of TOPP4. Epistasis analysis showed that defense activation in topp4-1 depended on NON-RACE-SPECIFIC DISEASE RESISTANCE1, PHYTOALEXIN DEFICIENT4, and the salicylic acid pathway. We generated topp1/4/5/6/7/8/9 septuple mutants to investigate the function of TOPPs in plant immunity. Elevated defense gene expression and enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 in the septuple mutant indicate that TOPPs function in plant defense responses. Furthermore, TOPPs physically interacted with mitogen-activated protein kinases (MAPKs) and affected the MAPK-mediated downstream defense pathway. Thus, our study reveals that TOPPs are important regulators of plant immunity.

TCP7 functions redundantly with several Class I TCPs and regulates endoreplication in Arabidopsis

TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR) proteins, a family of plant-specific transcription factors, play important roles in many developmental processes. However, genetic and functional redundancy among class I TCP limits the analysis of their biological roles. Here, we identified a dominant-negative mutant of Arabidopsis thaliana TCP7 named leaf curling-upward (lcu), which exhibits smaller leaf cells and shorter hypocotyls than the wild type, due to defective endoreplication. A septuple loss-of-function mutant of TCP7, TCP8, TCP14, TCP15, TCP21, TCP22, and TCP23 displayed similar developmental defects to those of lcu. Genome-wide RNA-sequencing showed that lcu and the septuple mutant share many misexpressed genes. Intriguingly, TCP7 directly targets the CYCLIN D1;1 (CYCD1;1) locus and activates its transcription. We determined that the C-terminus of TCP7 accounts for its transcriptional activation activity. Furthermore, the mutant protein LCU exhibited reduced transcriptional activation activity due to the introduction of an EAR-like repressive domain at its C-terminus. Together, these observations indicate that TCP7 plays important roles during leaf and hypocotyl development, redundantly, with at least six class I TCPs, and regulates the expression of CYCD1;1 to affect endoreplication in Arabidopsis.

Arabidopsis ICK/KRP cyclin-dependent kinase inhibitors function to ensure the formation of one megaspore mother cell and one functional megaspore per ovule.

In most plants, the female germline starts with the differentiation of one megaspore mother cell (MMC) in each ovule that produces four megaspores through meiosis, one of which survives to become the functional megaspore (FM). The FM further develops into an embryo sac. Little is known regarding the control of MMC formation to one per ovule and the selective survival of the FM. The ICK/KRPs (interactor/inhibitor of cyclin-dependent kinase (CDK)/Kip-related proteins) are plant CDK inhibitors and cell cycle regulators. Here we report that in the ovules of Arabidopsis mutant with all seven ICK/KRP genes inactivated, supernumerary MMCs, FMs and embryo sacs were formed and the two embryo sacs could be fertilized to form two embryos with separate endosperm compartments. Twin seedlings were observed in about 2% seeds. Further, in the mutant ovules the number and position of surviving megaspores from one MMC were variable, indicating that the positional signal for determining the survival of megaspore was affected. Strikingly, ICK4 fusion protein with yellow fluorescence protein was strongly present in the degenerative megaspores but absent in the FM, suggesting an important role of ICKs in the degeneration of non-functional megaspores. The absence of or much weaker phenotypes in lower orders of mutants and complementation of the septuple mutant by ICK4 or ICK7 indicate that multiple ICK/KRPs function redundantly in restricting the formation of more than one MMC and in the selective survival of FM, which are critical to ensure the development of one embryo sac and one embryo per ovule.

Rational Re-Engineering of the O-Dealkylation of 7-Alkoxycoumarin Derivatives by Cytochromes P450 2B from the Desert Woodrat Neotoma lepida.

On the basis of recent functional and structural characterization of cytochromes P450 2B from the desert woodrat (Neotoma lepida), the 7-alkoxycoumarin and 7-alkoxy-4-(trifluoromethyl)coumarin O-dealkylation profiles of CYP2B35 and CYP2B37 were re-engineered. Point mutants interchanging residues at seven positions in the enzyme active sites were created and purified from an Escherichia coli expression system. In screens for O-dealkylation activity, wild-type CYP2B35 metabolized long-chain 7-alkoxycoumarins but not 7-alkoxy-4-(trifluoromethyl)coumarins or short-chain 7-alkoxycoumarins. Wild-type CYP2B37 metabolized short-chain substrates from both series of compounds. CYP2B35 A367V showed maximal activity with 7-butoxycoumarin as opposed to 7-heptoxycoumarin in the parental enzyme, and CYP2B35 A363I/A367V produced an activity profile like that generated by CYP2B37. CYP2B35 A363I/A367V/I477F showed 7-ethoxycoumarin and 7-ethoxy-4-(trifluoromethyl)coumarin O-dealkylation rates similar to those of CYP2B37 and higher than those of the double mutant. A CYP2B35 septuple mutant retained a CYP2B37-like activity profile. In contrast, the CYP2B37 septuple mutant produced very low rates of O-dealkylation of all substrates. As mutating residue 108 in either enzyme was detrimental, this change was removed from both septuple mutants. Remarkably, the CYP2B35 sextuple mutant produced an activity profile that was a hybrid of that of CYP2B35 and CYP2B37, whereas the CYP2B37 sextuple mutant had almost no O-dealkylation activity. Docking of 7-substituted coumarin derivatives into a model of the CYP2B35 sextuple mutant based on a previous crystal structure of the 4-(4-chlorophenyl)imidazole wild-type complex revealed how the mutant can exhibit activities of both CYP2B35 and CYP2B37.

Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.

SNAC-As, stress-responsive NAC transcription factors, mediate ABA-inducible leaf senescence.

Leaf senescence is the terminal phenotype of plant leaf development, and ethylene is a major plant hormone inducing leaf senescence. Recent studies have shown that abscisic acid (ABA) also induces leaf senescence. However, the detailed mechanisms of ABA-induced leaf senescence remain unclear. We focused on the A subfamily of stress-responsive NAC (SNAC-A) transcription factors, the expression of which is induced by abiotic stresses, particularly ABA. Gene expression analysis revealed that seven SNAC-A genes including ANAC055, ANAC019, ANAC072/RD26, ANAC002/ATAF1, ANAC081/ATAF2, ANAC102 and ANAC032 were induced by long-term treatment with ABA and/or during age-dependent senescence. The SNAC-A septuple mutant clearly showed retardation of ABA-inducible leaf senescence. Microarray analysis indicated that SNAC-As induce ABA- and senescence-inducible genes. In addition, comparison of the expression profiles of the downstream genes of SNAC-As and ABA-responsive element (ABRE)-binding protein (AREB)/ABRE-binding factor (ABF) (AREB/ABFs) indicates that SNAC-As induce a different set of ABA-inducible genes from those mediated by AREB/ABFs. These results suggest that SNAC-As play crucial roles in ABA-induced leaf senescence signaling. We also discuss the function of SNAC-As in the transcriptional change of leaf senescence as well as in ABA response under abiotic stress conditions.

High-level Plasmodium falciparum sulfadoxine-pyrimethamine resistance with the concomitant occurrence of septuple haplotype in Tanzania.

BackgroundTanzania abandoned sulfadoxine-pyrimethamine (SP) as the first-line treatment for uncomplicated malaria in 2006 due to high levels Plasmodium falciparum resistance. However, SP is still being used for intermittent preventive treatment during pregnancy (IPTp-SP). This study aimed to assess the pattern of P. falciparumdihydrofolate reductase(Pfdhfr) and dihydropteroate synthetase (Pfdhps) mutations and associated haplotypes in areas with different malaria transmission intensities in mainland Tanzania, 6 years after withdrawal of SP as a first-line treatment regimen for uncomplicated malaria.MethodsA total of 264 samples were collected during cross-sectional surveys in three districts of Muheza, Muleba and Nachingwea in Tanga, Kagera and Lindi regions, respectively. Parasite genomic DNA was extracted from P. falciparum positive samples. The Pfdhfr, Pfdhps single nucleotide polymorphisms (SNPs) were amplified using nested polymerase chain reaction and detected by sequence specific oligonucleotide probe-enzyme linked immunosorbent assay (SSOP-ELISA).ResultsThe prevalence of the mutant Pfdhfr-Pfdhps haplotypes was heterogenous and transmission dependent. The triple Pfdhfr mutant haplotypes (CIRNI) were predominant in all sites with significantly higher frequencies at Muheza (93.3 %) compared to Muleba (75.0 %) and Nachingwea districts (70.6 %), (p < 0.001). Overall, the prevalence of the wild-type Pfdhps (SAKAA) haplotype was lowest at Muheza (1.3 %), (p = 0.002). Double Pfdhps haplotype SGEAA was significantly high at Muheza (27.2 %) and Muleba (20.8 %) while none (0 %) was detected at Nachingwea (p < 0.001). The prevalence of triple Pfdhps SGEGA haplotype was significantly higher at Muheza compared to Muleba and Nachingwea (p < 0.001). In contrast, Nachingwea and Muleba had significantly higher prevalence of another triple PfdhpsAGEAA haplotype (χ2 = 39.9, p < 0.001). Conversely, Pfdhfr-Pfdhps as quintuple and sextuple haplotypes were predominant including the emergence of a septuple mutant haplotype CIRNI-AGEGA (n = 11) observed at Muheza and Muleba.ConclusionThese results ascertain the high prevalence and saturation of Pfdhfr and Pfdhps haplotypes conferring SP resistance in areas with changing malaria epidemiology; and this could undermine the use of IPTp-SP in improving pregnancy outcomes. In these settings where high level SP resistance is documented, additional control efforts are needed and evaluation of an alternative drug for IPTp is an urgent priority.