scholarly journals Parasitic Fitness of Fungicide-Resistant and -Sensitive Isolates of Alternaria solani

Plant Disease ◽  
2018 ◽  
Vol 102 (3) ◽  
pp. 666-673 ◽  
Author(s):  
Mitchell J. Bauske ◽  
Neil C. Gudmestad
Keyword(s):  
Spore Germination ◽  
Point Mutations ◽  
Alternaria Solani ◽  
The United States ◽  
Wild Type ◽  
Parasitic Fitness ◽  
Fitness Parameters ◽  
Multiple Chemical

Resistance to chemistries of the succinate dehydrogenase inhibiting (SDHI) and quinone outside inhibiting (QoI) fungicides has developed rapidly in populations of Alternaria solani, the cause of early blight of potato. Reduced sensitivity to the anilinopyrimidine (AP) fungicide pyrimethanil has also been identified recently, determining that resistance to three chemical classes of fungicides is present within the A. solani population. Although no mutations have been characterized to confer resistance to APs, in A. solani five point mutations on three AsSdh genes have been determined to convey resistance to SDHIs, and the substitution of phenylalanine with leucine at position 129 (F129L) in the cytb gene confers resistance to QoIs. The objective of this study was to investigate the parasitic fitness of A. solani isolates with resistance to one or more of these chemical classes. A total of 120 A. solani isolates collected from various geographical locations around the United States were chosen for in vitro assessment, and 60 of these isolates were further evaluated in vivo. Fitness parameters measured were (i) spore germination in vitro, (ii) mycelial expansion in vitro, and (iii) aggressiveness in vivo. No significant differences in spore germination or mycelial expansion (P = 0.44 and 0.51, respectively) were observed among wild-type and fungicide-resistant isolates in vitro. Only A. solani isolates possessing the D123E mutation were shown to be significantly more aggressive in vivo (P < 0.0001) compared with wild-type isolates. These results indicate that fungicide-resistant A. solani isolates have no significant fitness penalties compared with sensitive isolates under the parameters evaluated regardless of the presence or absence of reduced sensitivity to multiple chemical classes. Results of these studies suggest that A. solani isolates with multiple fungicide resistances may compete successfully with wild-type isolates under field conditions.

scholarly journals Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

1998 ◽  
Vol 42 (7) ◽  
pp. 1811-1814 ◽  
Author(s):  
Leonardo K. Basco ◽  
Rachida Tahar ◽  
Pascal Ringwald
Keyword(s):  
Dihydrofolate Reductase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Adult Patients ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Reductase Gene ◽  
Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

scholarly journals Cubitus interruptus Requires DrosophilaCREB-Binding Protein To Activate wingless Expression in theDrosophila Embryo

2000 ◽  
Vol 20 (5) ◽  
pp. 1616-1625 ◽  
Author(s):  
Yang Chen ◽  
R. H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Point Mutations ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Interaction Domain ◽  
Cubitus Interruptus

ABSTRACT CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophilahomologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophiladevelopment, including the hedgehog (hh),decapentaplegic (dpp), and Tollpathways. Although dCBP is required for the expression of thehh target genes, wingless (wg) andpatched (ptc) in vivo, and potentiatesci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by cibinding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary forci-mediated transactivation of wg duringDrosophila embryogenesis.

scholarly journals Spatial and Temporal Distribution of Mutations Conferring QoI and SDHI Resistance in Alternaria solani Across the United States

Plant Disease ◽  
2018 ◽  
Vol 102 (2) ◽  
pp. 349-358 ◽  
Author(s):  
Mitchell J. Bauske ◽  
Ipsita Mallik ◽  
S. K. R. Yellareddygari ◽  
Neil C. Gudmestad
Keyword(s):  
Temporal Frequency ◽  
Point Mutations ◽  
Temporal Distribution ◽  
Alternaria Solani ◽  
The United States ◽  
Future Research ◽  
Wide Range ◽  
Parasitic Fitness ◽  
Polymerase Chain ◽  
Prevalent Mutation

The application of succinate dehydrogenase inhibiting (SDHI) and quinone outside inhibiting (QoI) fungicide chemistries is a primary tactic in the management of early blight of potato, caused by Alternaria solani. Resistance to QoIs in A. solani has been attributed to the F129L mutation, while resistance to SDHIs is conferred by five different known point mutations on three AsSdh genes. In total, 1,323 isolates were collected from 2013 through 2015 across 11 states to determine spatial and temporal frequency distribution of these mutations. A real-time polymerase chain reaction (PCR) was used to detect the presence of the F129L mutation. Molecular detection of SDHI-resistant isolates was performed using SDH multiplex PCR specific for point mutations in AsSdhB, AsSdhC, or AsSdhD genes and mismatch amplification analysis PCR detecting the point mutations in AsSdhB. Previous work in our research group determined that substitutions of histidine for tyrosine (H278Y) or arginine (H278R) at codon 278 on the AsSdhB gene were the most prevalent mutations, detected in 46 and 21% of A. solani isolates, respectively, collected in 2011 to 2012, and uniformly distributed among six sampled states. In contrast, the substitution of histidine for arginine (H134R) at codon 134 in the AsSdhC gene was the most prevalent mutation in 2013 through 2015, identified in 36% of isolates, compared with 7.5% of isolates recovered in 2011 to 2012. Substitutions of histidine for arginine (H133R) at codon 133 and aspartic acid for glutamic acid (D123E) at codon 123 in the AsSdhD gene were detected in 16 and 12%, respectively, in the A. solani population by 2015 and were recovered across a wide range of states, compared with 15 and 1.5% of isolates collected in 2011 to 2012, respectively. Overall, SDHI- and QoI-resistant isolates were detected at high frequencies across all years, with evidence of significant spatial variability. Future research will investigate whether these results are due to differences in parasitic fitness.

scholarly journals Mutant alcohol dehydrogenase (ADH III) presequences that affect both in vitro mitochondrial import and in vitro processing by the matrix protease.

1990 ◽  
Vol 10 (6) ◽  
pp. 2801-2808 ◽  
Author(s):  
D T Mooney ◽  
D B Pilgrim ◽  
E T Young
Keyword(s):  
Point Mutations ◽  
Wild Type ◽  
Urea Treatment ◽  
Amino Terminal ◽  
In Vitro Processing ◽  
The Matrix ◽  
Terminal Amino ◽  
Processing Protease

Point mutations in the presequence of the mitochondrial alcohol dehydrogerase isoenzyme (ADH III) have been shown to affect either the import of the precursor protein into yeast mitochondria in vivo or its processing within the organelle. In the present work, the behavior of these mutants during in vitro import into isolated mitochondria was investigated. All point mutants tested were imported with a slower initial rate than that of the wild-type precursor. This defect was corrected when the precursors were treated with urea prior to import. Once imported, the extent of processing to the mature form of mutant precursors varied greatly and correlated well with the defects observed in vivo. This result was not affected by prior urea treatment. When matrix extracts enriched for the processing protease were used, this defect was shown to be due to failure of the protease to efficiently recognize or cleave the presequence, rather than to a lack of access to the precursor. The rate of import of two ADH III precursors bearing internal deletions in the leader sequence was similar to those of the point mutants, whereas a deletion leading to the removal of the 15 amino-terminal amino acids was poorly imported. The mature amino terminus of wild-type ADH III was determined to be Gln-25. Mutant m01 (Ser-26 to Phe), which reduced the efficiency of cleavage in vitro by 80%, was cleaved at the correct site.

scholarly journals Specific protein binding to the simian virus 40 enhancer in vitro.

1986 ◽  
Vol 6 (6) ◽  
pp. 2098-2105 ◽  
Author(s):  
A G Wildeman ◽  
M Zenke ◽  
C Schatz ◽  
M Wintzerith ◽  
T Grundström ◽  
...  
Keyword(s):  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Specific Protein ◽  
Dnase I ◽  
Wild Type ◽  
Dnase I Footprinting ◽  
Nuclear Extracts

HeLa cell nuclear extracts and wild-type or mutated simian virus 40 enhancer DNA were used in DNase I footprinting experiments to study the interaction of putative trans-acting factors with the multiple enhancer motifs. We show that these nuclear extracts contain proteins that bind to these motifs. Because point mutations which are detrimental to the activity of a particular enhancer motif in vivo specifically prevent protection of that motif against DNase I digestion in vivo, we suggest that the bound proteins correspond to trans-acting factors involved in enhancement of transcription. Using mutants in which the two domains A and B of the simian virus 40 enhancer are either separated by insertion of DNA fragments or inverted with respect to their natural orientation, we also demonstrate that the trans-acting factors bind independently to the two domains.

scholarly journals Why Does Escherichia coli Grow More Slowly on Glucosamine than on N-Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

2005 ◽  
Vol 187 (9) ◽  
pp. 2974-2982 ◽  
Author(s):  
Laura I. Álvarez-Añorve ◽  
Mario L. Calcagno ◽  
Jacqueline Plumbridge
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Sugar Transport ◽  
Point Mutations ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Allosteric Activation

ABSTRACT Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

scholarly journals Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung

2016 ◽  
Vol 39 (2) ◽  
pp. 544-553 ◽  
Author(s):  
Sabrina V. Martini ◽  
Adriana L. Silva ◽  
Debora Ferreira ◽  
Rafael Rabelo ◽  
Felipe M. Ornellas ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Point Mutations ◽  
Lung Mechanics ◽  
Mouse Lung ◽  
Control Group ◽  
Polymorphonuclear Cells ◽  
Wild Type ◽  
Tyrosine Residues

Background/Aims: Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. Results: No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Conclusion: Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.

scholarly journals Potential Impact of Fluopyram on the Frequency of the D123E Mutation in Alternaria solani

Plant Disease ◽  
2018 ◽  
Vol 102 (3) ◽  
pp. 656-665 ◽  
Author(s):  
Mitchell J. Bauske ◽  
S. K. R. Yellareddygari ◽  
Neil C. Gudmestad
Keyword(s):  
Disease Control ◽  
Solanum Tuberosum L ◽  
Management Strategies ◽  
Point Mutations ◽  
Fungal Growth ◽  
Alternaria Solani ◽  
Selective Advantage ◽  
Field Trials

Succinate dehydrogenase-inhibiting (SDHI) fungicides have been widely applied in commercial potato (Solanum tuberosum L.) fields for the control of early blight, caused by Alternaria solani Sorauer. Five-point mutations on three AsSdh genes in A. solani have been identified as conferring resistance to SDHI fungicides. Recent work in our laboratory determined that A. solani isolates possessing the D123E mutation, or the substitution of aspartic acid for glutamic acid at position 123 in the AsSdhD gene, were collected at successively higher frequencies throughout a 3-year survey. In total, 118 A. solani isolates previously characterized as possessing the D123E mutation were evaluated in vitro for boscalid and fluopyram sensitivity. Over 80% of A. solani isolates with the D123E mutation evaluated were determined to be highly resistant to boscalid in vitro. However, effective concentration at which the fungal growth is inhibited by 50% values of isolates with the D123E mutation to fluopyram, ranging from 0.2 to 3 µg/ml, were sensitive and only slightly higher than those of baseline isolates to fluopyram, which ranged from 0.1 to 0.6 µg/ml. Five A. solani isolates with the D123E mutation were further evaluated in vivo for percent disease control obtained from boscalid and fluopyram compared with two wild-type isolates, three isolates possessing the F129L mutation, two isolates possessing the H134R mutation, two isolates possessing the H133R mutation, and one isolate with the H278R mutation. Relative area under the dose response curve values for boscalid and fluopyram were significantly lower for all five D123E-mutant isolates, demonstrating reduced disease control in vivo. In field trials, the frequency of A. solani isolates with the D123E mutation recovered from treatments receiving an in-furrow application of fluopyram ranged from 5 to 37%, which was significantly higher compared with treatments receiving foliar applications of standard protectants, in which the frequency of the D123E mutation in isolates ranged from 0 to 2.5%. Results suggest that A. solani isolates possessing the D123E mutation have a selective advantage under the application of fluopyram compared with SDHI-sensitive isolates, as well as isolates possessing other mutations conferring SDHI resistance. These data illustrate the importance of implementing fungicide resistance management strategies and cautions the use of fluopyram for in-furrow applications that target other pathogens of potato.

scholarly journals Occurrence of sdh Mutations in German Alternaria solani Isolates and Potential Impact on Boscalid Sensitivity In Vitro, in the Greenhouse, and in the Field

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3065-3071 ◽  
Author(s):  
Nicole Metz ◽  
Birgit Adolf ◽  
Nicole Chaluppa ◽  
Ralph Hückelhoven ◽  
Hans Hausladen
Keyword(s):  
Succinate Dehydrogenase ◽  
Early Blight ◽  
Solanum Tuberosum L ◽  
Point Mutations ◽  
Alternaria Solani ◽  
Field Trials ◽  
Sensitivity Testing ◽  
Development Of Resistance

The fungus Alternaria solani is the main pathogen causing early blight on potatoes (Solanum tuberosum L.). An increase in the development of resistance to the succinate dehydrogenase inhibitor (SDHI) boscalid, one of the main active ingredients for the control of early blight, has been reported. For this study, monitoring data from Germany were collected between 2013 and 2016 and an increase in the occurrence of A. solani succinate dehydrogenase (SDH) mutant isolates was observed. In addition to the known point mutations in sdh complex II, a new mutation in subunit C was found in German isolates (SdhC-H134Q). SDHI fungicide sensitivity testing was performed in the laboratory, greenhouse, and field. Reduced boscalid sensitivity was shown for mutant isolates (SdhB-H278Y and SdhC-H134R) both in vitro and in vivo. In addition, field trials with artificial inoculation were performed in 2016 and 2017. In both years, fungicide efficacy was significantly reduced after mutant inoculation compared with wild-type inoculation.

scholarly journals Glycosylation Tunes Neuroserpin Physiological and Pathological Properties

2020 ◽  
Vol 21 (9) ◽  
pp. 3235 ◽  
Author(s):  
Cristina Visentin ◽  
Luca Broggini ◽  
Benedetta Maria Sala ◽  
Rosaria Russo ◽  
Alberto Barbiroli ◽  
...  
Keyword(s):  
Pathological Condition ◽  
Expression System ◽  
Point Mutations ◽  
Tryptophan Fluorescence ◽  
Biochemical Properties ◽  
Eukaryotic Expression ◽  
Cell Models ◽  
Wild Type

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

Sign in / Sign up

Export Citation Format

Share Document