Phylogenetic analysis and functional characterisation of strictosidine synthase-like genes in Arabidopsis thaliana

2009 ◽  
Vol 36 (12) ◽  
pp. 1098 ◽  
Author(s):  
Natalie A. J. Kibble ◽  
M. Mehdi Sohani ◽  
Neil Shirley ◽  
Caitlin Byrt ◽  
Ute Roessner ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Phylogenetic Analysis ◽  
Plant Protection ◽  
Pcr Analysis ◽  
Strictosidine Synthase ◽  
Metabolomics Data ◽  
Unknown Compound ◽  
Class A ◽  
Functional Characterisation ◽  
Arabidopsis Protein

Monoterpenoid indole alkaloids (MIA) are a diverse class of secondary metabolites important for plant protection and are drugs for treating human diseases. Arabidopsis thaliana (L.) is not known to produce MIAs, yet its genome has 15 genes with similarity to the periwinkle (Catharanthus roseus (L.) G. Don) strictosidine synthase (STR) gene. Phylogenetic analysis of strictosidine synthase-like (SSL) proteins reveals four well supported classes of SSLs in Arabidopsis. To determine if Arabidopsis produces active strictosidine synthase, Arabidopsis protein extracts were assayed for enzymatic activity and cDNAs were expressed in Escherichia coli. Arabidopsis protein extracts from leaves and hairy roots do not make strictosidine at levels comparable to C. roseus, but they metabolise one substrate, secologanin, a precursor of strictosidine in other plant species, and produce an ‘unknown’ compound proposed to be a dimer of secologanic acid. Recombinant Arabidopsis proteins expressed in E. coli were not active STRs. Quantitative PCR analysis was performed on class A Ssls and showed they are upregulated by salt, ultraviolet light and salicylic acid treatment. RNAi mutants of Arabidopsis with reduced expression of all four class A Ssls, suggest that class A SSL proteins can modify secologanin. Gene expression and metabolomics data suggests that class A Ssl genes may have a role in plant protection.

Tissue specificity of expression of heat shock gene ATHSP70-10 in Arabidopsis thaliana seedlings under normal and stress conditions

2020 ◽  
Vol 2020 (3) ◽  
pp. 37-47
Author(s):  
L.Ye. Kozeko ◽  
◽  
E.L. Kordyum ◽  
Keyword(s):  
Arabidopsis Thaliana ◽  
Heat Shock ◽  
Water Deficit ◽  
Tissue Specificity ◽  
Abiotic Factors ◽  
Root Apex ◽  
Stress Conditions ◽  
Heat Shock Gene ◽  
Organ Specificity ◽  
Pcr Analysis

Mitochondrial heat shock proteins of HSP70 family support protein homeostasis in mitochondria under normal and stress conditions. They provide folding and complex assembly of proteins encoded by mitochondrial genome, as well as import of cytosolic proteins to mitochondria, their folding and protection against aggregation. There are reports about organ-specificity of mitochondrial HSP70 synthesis in plants. However, tissue specificity of their functioning remains incompletely characterized. This problem was studied for mitochondrial AtHSP70-10 in Arabidopsis thaliana seedlings using a transgenic line with uidA signal gene under normal conditions, as well as high temperature and water deficit. Under normal conditions, histochemical GUS-staining revealed the expression of AtHSP70-10 in cotyledon and leaf hydathodes, stipules, central cylinder in root differentiation and mature zones, as well as weak staining in root apex and root-shoot junction zone. RT-PCR analysis of wild-type seedlings exposed to 37°C showed rapid upregulation of AtHSP70-10, which reached the highest level within 2 h. In addition, the gradual development of water deficit for 5 days caused an increase in transcription of this gene, which became more pronounced after 3 days and reached a maximum after 5 days of dehydration. Histochemical analysis showed complete preservation of tissue localization of AtHSP70-10 expression under both abiotic factors. The data obtained indicate the specific functioning of mitochondrial chaperone AtHSP70-10 in certain plant cellular structures.

scholarly journals The carbon source-dependent pattern of antimicrobial activity and gene expression in Pseudomonas donghuensis P482

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marta Matuszewska ◽  
Tomasz Maciąg ◽  
Magdalena Rajewska ◽  
Aldona Wierzbicka ◽  
Sylwia Jafra
Keyword(s):  
Gene Expression ◽  
Antimicrobial Activity ◽  
Carbon Source ◽  
Reference Genes ◽  
Plant Pathogens ◽  
Plant Protection ◽  
Carbon Sources ◽  
Unknown Compound ◽  
Fungal Plant Pathogens ◽  
The Impact

AbstractPseudomonas donghuensis P482 is a tomato rhizosphere isolate with the ability to inhibit growth of bacterial and fungal plant pathogens. Herein, we analysed the impact of the carbon source on the antibacterial activity of P482 and expression of the selected genes of three genomic regions in the P482 genome. These regions are involved in the synthesis of pyoverdine, 7-hydroxytropolone (7-HT) and an unknown compound (“cluster 17”) and are responsible for the antimicrobial activity of P482. We showed that the P482 mutants, defective in these regions, show variations and contrasting patterns of growth inhibition of the target pathogen under given nutritional conditions (with glucose or glycerol as a carbon source). We also selected and validated the reference genes for gene expression studies in P. donghuensis P482. Amongst ten candidate genes, we found gyrB, rpoD and mrdA the most stably expressed. Using selected reference genes in RT-qPCR, we assessed the expression of the genes of interest under minimal medium conditions with glucose or glycerol as carbon sources. Glycerol was shown to negatively affect the expression of genes necessary for 7-HT synthesis. The significance of this finding in the light of the role of nutrient (carbon) availability in biological plant protection is discussed.

scholarly journals Conserved Water Molecules Stabilize the Ω-Loop in Class A β-Lactamases

2008 ◽  
Vol 52 (3) ◽  
pp. 1072-1079 ◽  
Author(s):  
Fabian Bös ◽  
Jürgen Pleiss
Keyword(s):  
Phylogenetic Analysis ◽  
High Resolution ◽  
Evolutionary Relationship ◽  
Water Molecules ◽  
Protein Residues ◽  
Class A ◽  
Conserved Water Molecules ◽  
Dense Cluster ◽  
The Relationship

ABSTRACT A set of 49 high-resolution (≤2.2 Å) structures of the TEM, SHV, and CTX-M class A β-lactamase families was systematically analyzed to investigate the role of conserved water molecules in the stabilization of the Ω-loop. Overall, 13 water molecules were found to be conserved in at least 45 structures, including two water positions which were found to be conserved in all structures. Of the 13 conserved water molecules, 6 are located at the Ω-loop, forming a dense cluster with hydrogen bonds to residues at the Ω-loop as well as to the rest of the protein. This layer of conserved water molecules is packed between the Ω-loop and the rest of the protein and acts as structural glue, which could reduce the flexibility of the Ω-loop. A correlation between conserved water molecules and conserved protein residues could in general not be detected, with the exception of the conserved water molecules at the Ω-loop. Furthermore, the evolutionary relationship between the three families, derived from the number of conserved water molecules, is similar to the relationship derived from phylogenetic analysis.

scholarly journals Phylogenetic Analysis Indicates That Evasin-Like Proteins of Ixodid Ticks Fall Into Three Distinct Classes

2021 ◽  
Vol 11 ◽  
Author(s):  
Shoumo Bhattacharya ◽  
Patricia Anne Nuttall
Keyword(s):  
Phylogenetic Analysis ◽  
Blood Feeding ◽  
Ixodid Ticks ◽  
Cysteine Residues ◽  
Tick Saliva ◽  
Class A ◽  
Leucocyte Migration ◽  
Biochemical Characterisation ◽  
Class B ◽  
Functional Classes

Chemokines are structurally related proteins that activate leucocyte migration in response to injury or infection. Tick saliva contains chemokine-binding proteins or evasins which likely neutralize host chemokine function and inflammation. Biochemical characterisation of 50 evasins from Ixodes, Amblyomma and Rhipicephalus shows that they fall into two functional classes, A and B, with exclusive binding to either CC- or CXC- chemokines, respectively. Class A evasins, EVA1 and EVA4 have a four-disulfide-bonded core, whereas the class B evasin EVA3 has a three-disulfide-bonded “knottin” structure. All 29 class B evasins have six cysteine residues conserved with EVA3, arrangement of which defines a Cys6-motif. Nineteen of 21 class A evasins have eight cysteine residues conserved with EVA1/EVA4, the arrangement of which defines a Cys8-motif. Two class A evasins from Ixodes (IRI01, IHO01) have less than eight cysteines. Many evasin-like proteins have been identified in tick salivary transcriptomes, but their phylogenetic relationship with respect to biochemically characterized evasins is not clear. Here, using BLAST searches of tick transcriptomes with biochemically characterized evasins, we identify 292 class A and 157 class B evasins and evasin-like proteins from Prostriate (Ixodes), and Metastriate (Amblyomma, Dermacentor, Hyalomma, Rhipicephalus) ticks. Phylogenetic analysis shows that class A evasins/evasin-like proteins segregate into two classes, A1 and A2. Class A1 members are exclusive to Metastriate ticks and typically have a Cys8-motif and include EVA1 and EVA4. Class A2 members are exclusive to Prostriate ticks, lack the Cys8-motif, and include IHO01 and IRI01. Class B evasins/evasin-like proteins are present in both Prostriate and Metastriate lineages, typically have a Cys6-motif, and include EVA3. Most evasins/evasin-like proteins in Metastriate ticks belong to class A1, whereas in Prostriate species they are predominantly class B. In keeping with this, the majority of biochemically characterized Metastriate evasins bind CC-chemokines, whereas the majority of Prostriate evasins bind CXC-chemokines. While the origin of the structurally dissimilar classes A1 and A2 is yet unresolved, these results suggest that class B evasin-like proteins arose before the divergence of Prostriate and Metastriate lineages and likely functioned to neutralize CXC-chemokines and support blood feeding.

scholarly journals Molecular Detection and Phylogenetic Analysis of Anaplasma Spp. in Cattle in Al-Qadisiyah Province of Iraq

2019 ◽  
Vol 42 (2) ◽  
pp. 181-188
Author(s):  
Hayder N. Ayyez ◽  
Yahia I. Khudhair ◽  
Qassim Haleem Kshash
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Control Strategies ◽  
Age Groups ◽  
Cattle Breed ◽  
16S Rrna Gene Sequencing ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

AbstractAnaplasma spp. are widely spread rickettsial bacteria transmitted by ticks and placing high impacts on veterinary and public health. A limited number of studies have been carried out on Anaplasmosis in the central part of Iraq. This study was conducted to determine the presence of Anaplasma spp. in cattle in Al-Qadisiyah province, Iraq. A total of 400 blood specimens were collected from cattle suffering from heavy tick infestation. Cattle were blood-sampled from four hyper-endemic areas with ticks. Blood samples were screened using microscopic and polymerase chain reaction (PCR) methods. Diff-quick stained blood smears revealed Anaplasma-like inclusion bodies in 254 (63.5%) samples. According to the 16S rRNA-gene-based PCR analysis, Anaplasma spp. was detected in 124 of the 400 (31%) samples, divided as 96/254 (37.8%) among the microscopical positive samples and 28/146 (19.17%) among the microscopical negative samples. Phylogenetic analysis based on the partial 16S rRNA gene sequencing of ten-PCR positive samples were 99–97% identical to sequences deposited in the GenBank, revealing presence of A. phagocytophilum, A. marginale and unnamed Anaplasma spp. in 40%, 20%, and 40% samples, respectively. Relationships among Anaplasma spp. infections and cattle breed, age, and sex were analyzed. Calves less than one year old showed significantly higher rates (p<0.005) than those from other age groups, whereas sex and breed demonstrated no significant differences (p˃0.001). This study shows that a variety of Anaplasma spp., were endemic in central part of Iraq and is still a hidden problem in cattle in the hyperendemic areas of tick, which requires serious control strategies.

scholarly journals Mutations within the Piga gene in patients with paroxysmal nocturnal hemoglobinuria

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2418-2422 ◽  
Author(s):  
RE Ware ◽  
WF Rosse ◽  
TA Howard
Keyword(s):  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Clinical Manifestations ◽  
Surface Protein ◽  
Convincing Evidence ◽  
Pcr Analysis ◽  
Single Nucleotide ◽  
Class A ◽  
Hematologic Disorder ◽  
Polymerase Chain ◽  
Protein Attachment

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder with multiple and varied clinical manifestations. The biochemical defect in PNH resides in the incomplete enzymatic assembly of glycosylphosphatidylinositol (GPI) anchors used for surface protein attachment. In all patients tested thus far, the defect is at the level of N-acetylglucosamine attachment to phosphatidylinositol (complementation class A defect). A human cDNA, Piga, that repairs cell lines with the class A defect has been recently cloned, making Piga a candidate gene for PNH. In the current study, using highly purified GPI- deficient granulocytes, we have performed Northern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analysis of Piga in four patients with PNH. In each case, we have identified a mutation in the Piga coding sequence: three frameshift mutations were found, and a single nucleotide substitution (missense) mutation was identified. Our results provide convincing evidence that alterations in the Piga gene are responsible for PNH.

scholarly journals Differentiation of monocytes to macrophages induced by influenza virus-infected apoptotic cells

2002 ◽  
Vol 83 (4) ◽  
pp. 747-751 ◽  
Author(s):  
Noboru Uchide ◽  
Kunio Ohyama ◽  
Bo Yuan ◽  
Toshio Bessho ◽  
Toshio Yamakawa
Keyword(s):  
Influenza Virus ◽  
Culture Supernatant ◽  
Scavenger Receptor ◽  
Pcr Analysis ◽  
Adhesion Assay ◽  
Apoptotic Cells ◽  
Rt Pcr ◽  
Class A ◽  
Monocytic Leukaemia ◽  
Class A Scavenger Receptor

The effect of the culture supernatant of influenza virus (IV)-infected apoptotic and non-apoptotic cells on the differentiation of monocytes to macrophages was investigated. IV infection induced apoptotic DNA fragmentation in cultured chorion cells but not in amnion cells prepared from human foetal membrane tissue. To examine the differentiation of monocytes to macrophages, an adhesion assay was employed using the human monocytic leukaemia THP-1 cell line. THP-1 cells became adherent to a substrate by incubation with the culture supernatant of IV-infected chorion cells, but not with that of amnion cells. The spreading THP-1 cells were morphologically characteristic of macrophages and they phagocytosed latex particles. RT–PCR analysis revealed that the expression of class A scavenger receptor mRNA was induced in THP-1 cells by incubation with the culture supernatant of IV-infected chorion cells. These results suggested that monocytic THP-1 cells were morphologically and functionally differentiated to macrophages by IV-infected apoptotic cells due to a soluble factor released from the apoptotic cells.

scholarly journals Genomic Organization and Comparative Phylogenic Analysis of NBS-LRR Resistance Gene Family in Solanum pimpinellifolium and Arabidopsis thaliana

2020 ◽  
Vol 16 ◽  
pp. 117693432091105
Author(s):  
Huawei Wei ◽  
Jia Liu ◽  
Qinwei Guo ◽  
Luzhao Pan ◽  
Songlin Chai ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Phylogenetic Analysis ◽  
Gene Family ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Chromosome Mapping ◽  
Gene Families ◽  
Gene Clusters ◽  
Motif Analysis ◽  
Solanum Pimpinellifolium

NBS-LRR (nucleotide-binding site and leucine-rich repeat) is one of the largest resistance gene families in plants. The completion of the genome sequencing of wild tomato Solanum pimpinellifolium provided an opportunity to conduct a comprehensive analysis of the NBS-LRR gene superfamily at the genome-wide level. In this study, gene identification, chromosome mapping, and phylogenetic analysis of the NBS-LRR gene family were analyzed using the bioinformatics methods. The results revealed 245 NBS-LRRs in total, similar to that in the cultivated tomato. These genes are unevenly distributed on 12 chromosomes, and ~59.6% of them form gene clusters, most of which are tandem duplications. Phylogenetic analysis divided the NBS-LRRs into 2 subfamilies (CNL-coiled-coil NBS-LRR and TNL-TIR NBS-LRR), and the expansion of the CNL subfamily was more extensive than the TNL subfamily. Novel conserved structures were identified through conserved motif analysis between the CNL and TNL subfamilies. Compared with the NBS-LRR sequences from the model plant Arabidopsis thaliana, wide genetic variation occurred after the divergence of S. pimpinellifolium and A thaliana. Species-specific expansion was also found in the CNL subfamily in S. pimpinellifolium. The results of this study provide the basis for the deeper analysis of NBS-LRR resistance genes and contribute to mapping and isolation of candidate resistance genes in S. pimpinellifolium.

scholarly journals A single Proteus mirabilis lineage from human and animal sources: a hidden reservoir of OXA-23 or OXA-58 carbapenemases in Enterobacterales

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Rémy A. Bonnin ◽  
Delphine Girlich ◽  
Agnès B. Jousset ◽  
Lauraine Gauthier ◽  
Gaëlle Cuzon ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Proteus Mirabilis ◽  
Long Period ◽  
Class A ◽  
Class D ◽  
Class B ◽  
Long Read ◽  
Major Clone ◽  
Reference Strains

Abstract In Enterobacterales, the most common carbapenemases are Ambler’s class A (KPC-like), class B (NDM-, VIM- or IMP-like) or class D (OXA-48-like) enzymes. This study describes the characterization of twenty-four OXA-23 or OXA-58 producing-Proteus mirabilis isolates recovered from human and veterinary samples from France and Belgium. Twenty-two P. mirabilis isolates producing either OXA-23 (n = 21) or OXA-58 (n = 1), collected between 2013 and 2018, as well as 2 reference strains isolated in 1996 and 2015 were fully sequenced. Phylogenetic analysis revealed that 22 of the 24 isolates, including the isolate from 1996, belonged to a single lineage that has disseminated in humans and animals over a long period of time. The blaOXA-23 gene was located on the chromosome and was part of a composite transposon, Tn6703, bracketed by two copies of IS15∆II. Sequencing using Pacbio long read technology of OXA-23-producing P. mirabilis VAC allowed the assembly of a 55.5-kb structure encompassing the blaOXA-23 gene in that isolate. By contrast to the blaOXA-23 genes, the blaOXA-58 gene of P. mirabilis CNR20130297 was identified on a 6-kb plasmid. The acquisition of the blaOXA-58 gene on this plasmid involved XerC-XerD recombinases. Our results suggest that a major clone of OXA-23-producing P. mirabilis is circulating in France and Belgium since 1996.

scholarly journals CISGENIC BIOLISTIC TRANSFORMATION FOR OBTAINING NEW FORMS OF POTATOES WITH IMPROVED RESISTANCE TO LATE BLIGHT

REPORTS ◽  
2020 ◽  
Vol 6 (334) ◽  
pp. 14-21
Author(s):  
N.P. Malakhova ◽  
◽  
Y.A. Skiba ◽  
E.R. Maltseva ◽  
G.A. Iskakova ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Solanum Tuberosum ◽  
Late Blight ◽  
Expression Level ◽  
Biolistic Transformation ◽  
Pcr Analysis ◽  
Potato Varieties ◽  
Polyubiquitin Gene ◽  
Common Potato ◽  
Gene Promotor

This article presents the results of application of cisgenic biolistic transformation for the accelerated production of new forms of potato with increased resistance to late blight. The reason for late blight development is the parasitic organism Phytophthora infestans, belonging to oomycetes (pseudo-fungi), which infects valuable agricultural plants. In this study, with the aim of combating P. infestans, a number of experiments on the biolistic transformation of the most common potato varieties Aksor and Nevskiy were carried out in Kazakhstan. Two potato genes – Rpi-vnt1.1 and StREM1.3 – were selected as targets for introduction. Expression of the first gene should be activated, and the expression of the REMORIN1.3 gene should be suppressed. Rpi-vnt1.1 was under the control of Solanum tuberosum polyubiquitin gene promotor (Pat) and Arabidopsis thaliana polyubiquitin 5 gene terminator (ubq5). Knock-down double stranded RNA-hairpin gene construction for StREM1.3 silencing was under the control of Solanum tuberosum phytochrome B gene promotor (phyB) and Arabidopsis thaliana hot-shock protein 18.2 terminator (HSP18.2). Three series of biolistic transformation were carried out, as a result of which 636 regenerated plants of potato varieties Aksor and Nevskiy were obtained. DNA was extracted from the plant material of potato transformant plants in the quality and quantity suitable for PCR analysis for the presence of an insert. PCR analysis was carried out, revealing 52 plants carrying the VNT insert. StREM1.3 silencing gene construction was detected in plant lines by qPCR, based on comparative analysis of of gene expression level and revealed 6 lines with reliably lower StREM1.3 expression level in comparison with wild-type plants.

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