scholarly journals The use of detached leaf inoculation for selecting Cercospora kikuchii resistance in soybean genotypes

2021 ◽  
Author(s):  
Takeshi Kashiwa ◽  
Miguel Angel Lavilla ◽  
Antonio Diaz Paleo ◽  
Antonio Juan Gerardo Ivancovich ◽  
Naoki Yamanaka
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Post Inoculation ◽  
Soybean Genotype ◽  
Resistant Varieties ◽  
Soybean Genotypes ◽  
Soybean Resistance ◽  
Soybean Leaves ◽  
Resistant Genotypes

Cercospora leaf blight (CLB) causes extensive losses in soybean production in worldwide, including major soybean producing countries such as Argentina. C. kikuchii, C. cf. sigesbeckiae, C. cf. flagellaris, and C. cf. nicotianae are identified as pathogen of CLB. Soybean resistance for CLB is still unknown. Also, chemical control for CLB is losing effectiveness by fungicide resistance of the pathogen, such as C. kikuchii. We urgently need to breed a CLB resistant cultivar. Unfortunately, efficient methods for the screening of a resistant soybean genotype have not yet been established. In this study, we designed new, high-throughput inoculation method for identifying resistance against one of the CLB pathogen, C. kikuchii. We used liquid-cultured mycelia of the pathogen C. kikuchii on detached soybean leaves. Lesions on soybean leaflets appeared nine days post inoculation by this method. We used this method to select four C. kikuchii resistant genotypes from 80 genotypes in the World Soybean Core Collection. High-throughput screening method developed in this study can contribute to the research about C. kikuchii resistance by facilitating identification of resistant varieties.

scholarly journals Genotypes A and B of Cadophora gregata Differ in Ability to Colonize Susceptible Soybean

Plant Disease ◽  
2007 ◽  
Vol 91 (5) ◽  
pp. 574-580 ◽  
Author(s):  
G. M. Tabor ◽  
G. L. Tylka ◽  
C. R. Bronson
Keyword(s):  
Screening Method ◽  
Stem Length ◽  
Brown Stem Rot ◽  
Phialophora Gregata ◽  
Foliar Symptoms ◽  
Soybean Genotypes ◽  
Soybean Resistance ◽  
Soybean Plants ◽  
Resistant Genotypes ◽  
Genotype A

Growth chamber experiments were conducted to determine if extent of colonization of soybean stems by genotypes A and B of Cadophora gregata (Phialophora gregata), the causal agent of brown stem rot (BSR) of soybean, is similar in soybean plants resistant or susceptible to genotype A. Upon introduction of the two genotypes separately into the base of stems of 2-week-old seedlings, genotype A advanced with the growing tips of susceptible but not resistant genotypes. In contrast, genotype B did not advance with the growing tips of either resistant or susceptible soybean. In similar experiments, 5 weeks after introduction of genotype A, both mean percent stem length colonized by C. gregata and mean percentage of symptomatic trifoliate leaflets were significantly less for resistant than for susceptible genotypes. For genotype B, there was no or a slight difference between resistant and susceptible soybean genotypes in mean percent stem length colonized and no difference in mean percentage of symptomatic trifoliate leaflets 5 weeks after introduction of the pathogen. These results indicate that genotype A and genotype B differ not only in the severity of foliar symptoms they cause on genotype A-susceptible soybean plants, but also in how severely they colonize the stems of these soybean plants. In our experiments, genotype A and genotype B did not differ consistently in their ability to cause internal stem discoloration. The two genotypes of C. gregata can be distinguished based on how severely they colonize stems of genotype A-susceptible soybean. Thus, a BSR resistance screening method, which relies on assessment of stem colonization by C. gregata, works only for screening soybean lines resistance to genotype A. In light of these results, it is important to distinguish soybean resistance to genotype A versus genotype B of C. gregata. Whether genotype B causes yield loss and whether soybean plants can be distinguished as resistant or susceptible to genotype B needs to be investigated.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

scholarly journals A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

2020 ◽  
Author(s):  
Yuru Wang ◽  
Christopher D Katanski ◽  
Christopher Watkins ◽  
Jessica N Pan ◽  
Qing Dai ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Targeted Mutagenesis ◽  
Rna Repair ◽  
Dna And Rna ◽  
Modified Dna ◽  
Dna Substrates ◽  
Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

scholarly journals A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

2021 ◽  
Vol 22 (6) ◽  
pp. 3041
Author(s):  
Gheorghita Menghiu ◽  
Vasile Ostafe ◽  
Radivoje Prodanović ◽  
Rainer Fischer ◽  
Raluca Ostafe
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Screening Method ◽  
Enzymatic Reaction ◽  
Hydrolytic Enzymes ◽  
Screening System ◽  
Fluorescence Activated Cell Sorting ◽  
Fungal Cell ◽  
Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

A new high-throughput screening method for determining active and enantioselective hydrolases

2009 ◽  
Vol 46 (3) ◽  
pp. 345-349 ◽  
Author(s):  
Bo Wang ◽  
Xiaoling Tang ◽  
Gangfeng Ren ◽  
Ji Liu ◽  
Hongwei Yu
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method

scholarly journals Pathogenic Variation of Phakopsora pachyrhizi Isolates on Soybean in the United States from 2006 to 2009

Plant Disease ◽  
2012 ◽  
Vol 96 (1) ◽  
pp. 75-81 ◽  
Author(s):  
M. Twizeyimana ◽  
G. L. Hartman
Keyword(s):  
United States ◽  
Resistance Genes ◽  
The United States ◽  
Soybean Rust ◽  
Phakopsora Pachyrhizi ◽  
Soybean Genotype ◽  
Pathogenic Variation ◽  
Differential Set ◽  
Soybean Genotypes ◽  
Soybean Leaves

The introduction of Phakopsora pachyrhizi, the cause of soybean rust, into the United States is a classic case of a pathogen introduction that became established in a new geographical region overwintering on a perennial host (kudzu, Pueraria lobata). The objective of our study was to classify the pathogenic variation of P. pachyrhizi isolates collected in the United States, and to determine the spatial and temporal associations. In total, 72 isolates of P. pachyrhizi collected from infected kudzu and soybean leaves in the United States were purified, then established and increased on detached soybean leaves. These isolates were tested for virulence and aggressiveness on a differential set of soybean genotypes that included six genotypes with known resistance genes (Rpp), one resistant genotype without any known characterized resistance gene, and a susceptible genotype. Three pathotypes were identified among the 72 U.S. P. pachyrhizi isolates based on the virulence of these isolates on the genotypes in the differential set. Six aggressiveness groups were established based on sporulating-uredinia production recorded for each isolate on each soybean genotype. All three pathotypes and all six aggressiveness groups were found in isolates collected from the southern region and from both hosts (kudzu or soybean) in 2008. Shannon's index based on the number of pathotypes indicated that isolates from the South region were more diverse (H = 0.83) compared with the isolates collected in other regions. This study establishes a baseline of pathogenic variation of P. pachyrhizi in the United States that can be further compared with variation reported in other regions of the world and in future studies that monitor P. pachyrhizi virulence in association to deployment of rust resistance genes.

scholarly journals A high throughput optical method for studying compositional effects in electrocatalysts for CO2 reduction

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jeremy L. Hitt ◽  
Yuguang C. Li ◽  
Songsheng Tao ◽  
Zhifei Yan ◽  
Yue Gao ◽  
...  
Keyword(s):  
Hydrogen Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Co2 Reduction ◽  
Fold Increase ◽  
Faradaic Efficiency ◽  
Atomic Pair Distribution Function ◽  
Earth Abundant ◽  
Ternary Catalysts

AbstractIn the problem of electrochemical CO2 reduction, the discovery of earth-abundant, efficient, and selective catalysts is essential to enabling technology that can contribute to a carbon-neutral energy cycle. In this study, we adapt an optical high throughput screening method to study multi-metallic catalysts for CO2 electroreduction. We demonstrate the utility of the method by constructing catalytic activity maps of different alloyed elements and use X-ray scattering analysis by the atomic pair distribution function (PDF) method to gain insight into the structures of the most active compositions. Among combinations of four elements (Au, Ag, Cu, Zn), Au6Ag2Cu2 and Au4Zn3Cu3 were identified as the most active compositions in their respective ternaries. These ternary electrocatalysts were more active than any binary combination, and a ca. 5-fold increase in current density at potentials of −0.4 to −0.8 V vs. RHE was obtained for the best ternary catalysts relative to Au prepared by the same method. Tafel plots of electrochemical data for CO2 reduction and hydrogen evolution indicate that the ternary catalysts, despite their higher surface area, are poorer catalysts for the hydrogen evolution reaction than pure Au. This results in high Faradaic efficiency for CO2 reduction to CO.

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