scholarly journals A functional genomics approach to dissect spotted alfalfa aphid resistance in Medicago truncatula

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Silke Jacques ◽  
Jana Sperschneider ◽  
Gagan Garg ◽  
Louise F. Thatcher ◽  
Ling-Ling Gao ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Medicago Truncatula ◽  
Stress Responses ◽  
Target Genes ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Aphid Resistance ◽  
Exome Capture ◽  
Protein Kinase Activity ◽  
Ap2 Domain

AbstractAphids are virus-spreading insect pests affecting crops worldwide and their fast population build-up and insecticide resistance make them problematic to control. Here, we aim to understand the molecular basis of spotted alfalfa aphid (SAA) or Therioaphis trifolii f. maculata resistance in Medicago truncatula, a model organism for legume species. We compared susceptible and resistant near isogenic Medicago lines upon SAA feeding via transcriptome sequencing. Expression of genes involved in defense and stress responses, protein kinase activity and DNA binding were enriched in the resistant line. Potentially underlying some of these changes in gene expression was the finding that members of the MYB, NAC, AP2 domain and ERF transcription factor gene families were differentially expressed in the resistant versus susceptible lines. A TILLING population created in the resistant cultivar was screened using exome capture sequencing and served as a reverse genetics tool to functionally characterise genes involved in the aphid resistance response. This screening revealed three transcription factors (a NAC, AP2 domain and ERF) as important regulators in the defence response, as a premature stop-codon in the resistant background led to a delay in aphid mortality and enhanced plant susceptibility. This combined functional genomics approach will facilitate the future development of pest resistant crops by uncovering candidate target genes that can convey enhanced aphid resistance.

scholarly journals Biofilm Growth By Listeria Monocytogenes On Stainless Steel and Expression of Biofilm-Related Genes Under Stressing Conditions

2021 ◽  
Author(s):  
Danilo Augusto Lopes da Silva ◽  
Rafaela de Melo Tavares ◽  
Anderson Carlos Camargo ◽  
Ricardo Seiti Yamatogi ◽  
Elaine Cristina Pereira De Martinis ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Stress Responses ◽  
Biofilm Formation ◽  
Gene Networks ◽  
Stop Codon ◽  
Regulatory Gene ◽  
Premature Stop Codon ◽  
Biofilm Growth ◽  
Adverse Conditions

Abstract This research was carried out to assess the ability of L. monocytogenes for adhesion and growth in biofilm on stainless steel coupons under different stressing conditions (NaCl, curing salts and quaternary ammonium compounds - QAC), besides determining the expression of different genes involved in biofilm formation and stress response. Results from crystal violet assay revealed that one isolate carrying a premature stop codon (PMSC) in agrC gene formed high-density biofilms in the presence of QAC or cure salts (7.5% and 10%). Reverse Transcriptase-qPCR results revealed that isolates of L. monocytogenes lineages I and II presented differences in transcriptional profile of genes related to biofilm formation and adaptation to environmental conditions. In conclusion, our results demonstrated how L. monocytogenes can survive, multiply and form biofilm under adverse conditions related to food processing environments. Differences in transcriptional expression were observed, highlighting the role of regulatory gene networks for particular serotypes under different stress responses.

scholarly journals Biological Characteristics and Molecular Mechanisms of Fludioxonil Resistance in Fusarium graminearum in China

Plant Disease ◽  
2020 ◽  
Vol 104 (9) ◽  
pp. 2426-2433
Author(s):  
F. Zhou ◽  
D. X. Li ◽  
H. Y. Hu ◽  
Y. L. Song ◽  
Y. C. Fan ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Stop Codon ◽  
Point Mutations ◽  
Premature Stop Codon ◽  
Cross Resistance ◽  
Alternative Mechanism ◽  
Head Blight ◽  
Resistant Mutants

Fusarium graminearum is the primary causal agent of Fusarium head blight (FHB) of wheat. The phenylpyrrole fungicide fludioxonil is not currently registered for the management of FHB in China. The current study assessed the fludioxonil sensitivity of a total of 53 F. graminearum isolates collected from the six most important wheat-growing provinces of China during 2018 and 2019. The baseline fludioxonil sensitivity distribution indicated that all of the isolates were sensitive, exhibiting a unimodal cure with a mean effective concentration for 50% inhibition value of 0.13 ± 0.12 μg/ml (standard deviation). Five fludioxonil-resistant mutants were subsequently induced by exposure to fludioxonil under laboratory conditions. Ten successive rounds of subculture in the absence of the selection pressure indicated that the mutation was stably inherited. However, the fludioxonil-resistant mutants were found to have reduced pathogenicity, higher glycerol accumulation, and higher osmotic sensitivity than the parental wild-type isolates, indicating that there was a fitness cost associated with fludioxonil resistance. In addition, the study also found a positive cross resistance between fludioxonil, procymidone, and iprodione, but not with other fungicides such as boscalid, carbendazim, tebuconazole, and fluazinam. Sequence analysis of four candidate target genes (FgOs1, FgOs2, FgOs4, and FgOs5) revealed that the HBXT2R mutant contained two point mutations that resulted in amino acid changes at K223T and K415R in its FgOs1 protein, and one point mutation at residue 520 of its FgOs5 protein that resulted in a premature stop codon. Similarly, the three other mutants contained point mutations that resulted in changes at the K192R, K293R, and K411R residues of the FgOs5 protein but none in the FgOs2 and FgOs4 genes. However, it is important to point out that the FgOs2 and FgOs4 expression of all the fludioxonil-resistant mutants was significantly (P < 0.05) downregulated compared with the sensitive isolates (except for the SQ1-2 isolate). It was also found that one of the resistant mutants did not have changes in any of the sequenced target genes, indicating that an alternative mechanism could also lead to fludioxonil resistance.

scholarly journals Highly multiplexed AmpliSeq technology identifies novel variation of flowering time-related genes in soybean (Glycine max)

DNA Research ◽  
2019 ◽  
Vol 26 (3) ◽  
pp. 243-260 ◽  
Author(s):  
Eri Ogiso-Tanaka ◽  
Takehiko Shimizu ◽  
Makita Hajika ◽  
Akito Kaga ◽  
Masao Ishimoto
Keyword(s):  
Flowering Time ◽  
Core Collection ◽  
Target Genes ◽  
Sequence Data ◽  
Stop Codon ◽  
Response Regulator ◽  
Premature Stop Codon ◽  
Mini Core Collection ◽  
Novel Variants ◽  
Two Component

Abstract Whole-genome re-sequencing is a powerful approach to detect gene variants, but it is expensive to analyse only the target genes. To circumvent this problem, we attempted to detect novel variants of flowering time-related genes and their homologues in soybean mini-core collection by target re-sequencing using AmpliSeq technology. The average depth of 382 amplicons targeting 29 genes was 1,237 with 99.85% of the sequence data mapped to the reference genome. Totally, 461 variants were detected, of which 150 sites were novel and not registered in dbSNP. Known and novel variants were detected in the classical maturity loci—E1, E2, E3, and E4. Additionally, large indel alleles, E1-nl and E3-tr, were successfully identified. Novel loss-of-function and missense variants were found in FT2a, MADS-box, WDR61, phytochromes, and two-component response regulators. The multiple regression analysis showed that four genes—E2, E3, Dt1, and two-component response regulator—can explain 51.1–52.3% of the variation in flowering time of the mini-core collection. Among them, the two-component response regulator with a premature stop codon is a novel gene that has not been reported as a soybean flowering time-related gene. These data suggest that the AmpliSeq technology is a powerful tool to identify novel alleles.

scholarly journals Interplay of Efflux System, ampC, and oprD Expression in Carbapenem Resistance of Pseudomonas aeruginosa Clinical Isolates

2006 ◽  
Vol 50 (5) ◽  
pp. 1633-1641 ◽  
Author(s):  
John Quale ◽  
Simona Bratu ◽  
Jyoti Gupta ◽  
David Landman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Target Genes ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Carbapenem Resistance ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Carbapenem Resistant ◽  
High Level ◽  
Increased Activity

ABSTRACT Carbapenems are important agents for the therapy of infections due to multidrug-resistant Pseudomonas aeruginosa; the development of carbapenem resistance hampers effective therapeutic options. To assess the mechanisms leading to resistance, 33 clinical isolates with differing degrees of carbapenem susceptibility were analyzed for the expression of the chromosomal β-lactamase (ampC), the porin that is important for the entry of carbapenems (oprD), and the proteins involved in four efflux systems (mexA, mexC, mexE, and mexX). Real-time reverse transcriptase PCR was performed using primers and fluorescent probes for each of the target genes. The sequencing of regulatory genes (ampR, mexR, nalC, nalD, mexT, and mexZ) was also performed. Diminished expression of oprD was present in all imipenem- and meropenem-resistant isolates but was not required for ertapenem resistance. Increased expression of ampC was not observed in several isolates that were overtly resistant to carbapenems. Increased expression of several efflux systems was observed in many of the carbapenem-resistant isolates. Increased efflux activity correlated with high-level ertapenem resistance and reduced susceptibility to meropenem and aztreonam. Most isolates with increased expression of mexA had mutations affecting nalC and/or nalD. Two isolates with mutations leading to a premature stop codon in mexZ had markedly elevated mexX expressions, although mutations in mexZ were not a prerequisite for overexpression. β-Lactam resistance in clinical isolates of P. aeruginosa is a result of the interplay between diminished production of oprD, increased activity of ampC, and several efflux systems.

Ectopic Transcript Analysis Indicates that Allelic Exclusion is an Important Cause of Type I Protein C Deficiency in Patients with Nonsense and Frameshift Mutations in the PROC Gene

1996 ◽  
Vol 75 (06) ◽  
pp. 870-876 ◽  
Author(s):  
José Manuel Soria ◽  
Lutz-Peter Berg ◽  
Jordi Fontcuberta ◽  
Vijay V Kakkar ◽  
Xavier Estivill ◽  
...  
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Allelic Exclusion ◽  
Polymorphism Analysis ◽  
Type I ◽  
Protein C Deficiency ◽  
Nonsense Mutations ◽  
Stop Codons

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

Induction of Translational Readthrough across the Thalassemia-Causing Premature Stop Codon in β-Globin-Encoding mRNA

Biochemistry ◽  
2019 ◽  
Vol 59 (1) ◽  
pp. 80-84 ◽  
Author(s):  
Debaleena Kar ◽  
Karthi Sellamuthu ◽  
Sangeetha Devi Kumar ◽  
Sandeep M. Eswarappa
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Translational Readthrough

scholarly journals A Novel Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive DFNA68 Hearing Loss in a Spanish Family

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 411
Author(s):  
María Lachgar ◽  
Matías Morín ◽  
Manuela Villamar ◽  
Ignacio del Castillo ◽  
Miguel Ángel Moreno-Pelayo
Keyword(s):  
Hearing Loss ◽  
Stop Codon ◽  
Coiled Coil ◽  
Premature Stop Codon ◽  
Scaffolding Protein ◽  
Common Mechanism ◽  
Chinese Family ◽  
Truncating Mutation ◽  
Spanish Family ◽  
Sensory Defect

Nonsyndromic hereditary hearing loss is a common sensory defect in humans that is clinically and genetically highly heterogeneous. So far, 122 genes have been associated with this disorder and 50 of them have been linked to autosomal dominant (DFNA) forms like DFNA68, a rare subtype of hearing impairment caused by disruption of a stereociliary scaffolding protein (HOMER2) that is essential for normal hearing in humans and mice. In this study, we report a novel HOMER2 variant (c.832_836delCCTCA) identified in a Spanish family by using a custom NGS targeted gene panel (OTO-NGS-v2). This frameshift mutation produces a premature stop codon that may lead in the absence of NMD to a shorter variant (p.Pro278Alafs*10) that truncates HOMER2 at the CDC42 binding domain (CBD) of the coiled-coil structure, a region that is essential for protein multimerization and HOMER2-CDC42 interaction. c.832_836delCCTCA mutation is placed close to the previously identified c.840_840dup mutation found in a Chinese family that truncates the protein (p.Met281Hisfs*9) at the CBD. Functional assessment of the Chinese mutant revealed decreased protein stability, reduced ability to multimerize, and altered distribution pattern in transfected cells when compared with wild-type HOMER2. Interestingly, the Spanish and Chinese frameshift mutations might exert a similar effect at the protein level, leading to truncated mutants with the same Ct aberrant protein tail, thus suggesting that they can share a common mechanism of pathogenesis. Indeed, age-matched patients in both families display quite similar hearing loss phenotypes consisting of early-onset, moderate-to-profound progressive hearing loss. In summary, we have identified the third variant in HOMER2, which is the first one identified in the Spanish population, thus contributing to expanding the mutational spectrum of this gene in other populations, and also to clarifying the genotype–phenotype correlations of DFNA68 hearing loss.

scholarly journals Use of Oxidative Stress Responses to Determine the Efficacy of Inactivation Treatments on Cryptosporidium Oocysts

2021 ◽  
Vol 9 (7) ◽  
pp. 1463
Author(s):  
Tamirat Tefera Temesgen ◽  
Kristoffer Relling Tysnes ◽  
Lucy Jane Robertson
Keyword(s):  
Oxidative Stress ◽  
Stress Responses ◽  
Target Genes ◽  
Cryptosporidium Oocyst ◽  
Drinking Water Treatment ◽  
Proof Of Concept ◽  
Environmental Matrices ◽  
Oocyst Wall ◽  
Novel Approach ◽  
Cryptosporidium Oocysts

Cryptosporidium oocysts are known for being very robust, and their prolonged survival in the environment has resulted in outbreaks of cryptosporidiosis associated with the consumption of contaminated water or food. Although inactivation methods used for drinking water treatment, such as UV irradiation, can inactivate Cryptosporidium oocysts, they are not necessarily suitable for use with other environmental matrices, such as food. In order to identify alternative ways to inactivate Cryptosporidium oocysts, improved methods for viability assessment are needed. Here we describe a proof of concept for a novel approach for determining how effective inactivation treatments are at killing pathogens, such as the parasite Cryptosporidium. RNA sequencing was used to identify potential up-regulated target genes induced by oxidative stress, and a reverse transcription quantitative PCR (RT-qPCR) protocol was developed to assess their up-regulation following exposure to different induction treatments. Accordingly, RT-qPCR protocols targeting thioredoxin and Cryptosporidium oocyst wall protein 7 (COWP7) genes were evaluated on mixtures of viable and inactivated oocysts, and on oocysts subjected to various potential inactivation treatments such as freezing and chlorination. The results from the present proof-of-concept experiments indicate that this could be a useful tool in efforts towards assessing potential technologies for inactivating Cryptosporidium in different environmental matrices. Furthermore, this approach could also be used for similar investigations with other pathogens.

scholarly journals Alternative splicing landscapes in Arabidopsis thaliana across tissues and stress conditions highlight major functional differences with animals

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Guiomar Martín ◽  
Yamile Márquez ◽  
Federica Mantica ◽  
Paula Duque ◽  
Manuel Irimia
Keyword(s):  
Gene Expression ◽  
Arabidopsis Thaliana ◽  
Alternative Splicing ◽  
Stress Responses ◽  
Target Genes ◽  
Intron Retention ◽  
Single Gene ◽  
Exon Skipping ◽  
Environmental Response ◽  
Biotic Stresses

Abstract Background Alternative splicing (AS) is a widespread regulatory mechanism in multicellular organisms. Numerous transcriptomic and single-gene studies in plants have investigated AS in response to specific conditions, especially environmental stress, unveiling substantial amounts of intron retention that modulate gene expression. However, a comprehensive study contrasting stress-response and tissue-specific AS patterns and directly comparing them with those of animal models is still missing. Results We generate a massive resource for Arabidopsis thaliana, PastDB, comprising AS and gene expression quantifications across tissues, development and environmental conditions, including abiotic and biotic stresses. Harmonized analysis of these datasets reveals that A. thaliana shows high levels of AS, similar to fruitflies, and that, compared to animals, disproportionately uses AS for stress responses. We identify core sets of genes regulated specifically by either AS or transcription upon stresses or among tissues, a regulatory specialization that is tightly mirrored by the genomic features of these genes. Unexpectedly, non-intron retention events, including exon skipping, are overrepresented across regulated AS sets in A. thaliana, being also largely involved in modulating gene expression through NMD and uORF inclusion. Conclusions Non-intron retention events have likely been functionally underrated in plants. AS constitutes a distinct regulatory layer controlling gene expression upon internal and external stimuli whose target genes and master regulators are hardwired at the genomic level to specifically undergo post-transcriptional regulation. Given the higher relevance of AS in the response to different stresses when compared to animals, this molecular hardwiring is likely required for a proper environmental response in A. thaliana.

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