Biological Activity of Artificial Plant Peptides Corresponding to the Translational Products of Small ORFs in Primary miRNAs and Other Long "Non-Coding" RNAs.

Generally, lncPEPs (peptides encoded by long non-coding RNAs) have been identified in many plant species of several families and in some animal species. Importantly, molecular mechanisms of the miPEPs (peptides encoded by primary microRNAs, pri-miRNAs) are often poorly understood in different flowering plants. Requirement for the additional studies in these directions is highlighted by alternative findings concerning positive regulation of pri-miRNA/miRNA expression by synthetic miPEPs in plants. Further extensive studies are also needed to understand the full set of their roles in eukaryotic organisms. This review mainly aims to consider the available data on the regulatory functions of the synthetic miPEPs. Studies of chemically synthesized miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed. Brief description of the studies to identify lncORFs (open reading frames of long non-coding RNAs) and the encoded protein products is also provided.

Rolling circle amplification with fluorescently labeled dUTP-balancing the yield and degree of labeling.

Detection methods based on rolling circle amplification (RCA) have been applied to a large number of targets in molecular biology. The key feature of RCA-based methods as well as other nucleic acid amplification methods is their exceptional sensitivity, which allows the detection of molecules at low concentrations, achieved by signal amplification due to nucleic acid magnification and subsequent detection. Variations on the method, such as immuno-RCA, extend the range of potential targets that can be detected. Employing fluorescently labeled nucleotides for direct incorporation into an amplification product is an attractive method for RCA product detection. However, the effectiveness of this approach remains doubtful. In our study, we utilized different modified dUTPs, including sulfo-cyanine3-dUTP, sulfo-cyanine5-dUTP, sulfo-cyanine5.5-dUTP, BDP-FL-dUTP, and amino-11-dUTP, to investigate whether the properties of the fluorophore used for modification affected the reaction yield and effectiveness of incorporation of nucleotide analogs by phi29 DNA polymerase. Among the modified dUTPs, sulfo-cyanine3-dUTP demonstrated the highest incorporation effectiveness, equal to 4-9 labels per 1000 nucleotides. The mean length of the RCA product was estimated to be approximately 175,000 nucleotides. The total increase in fluorescence from a single target/product complex was 850 times. The results obtained in the study illustrate the possibility of successful application of nucleotide analogs for RCA detection and present quantitative characteristics of fluorescently labeled dUTPs to be incorporated into RCA products.

Diversity of Harpin-Like Defense Peptides from Barnyard Grass (Echinochloa crusgalli L.) Seeds.

The results of the study of the structure and function of harpin-like peptides (alpha-harpinins) of the EcAMP group from the barnyard grass (E. crusgalli) seeds and the possibility of their involvement in the innate immunity to biotic stresses are presented.

A Novel Fluorescent GFP Chromophore Analog-Based Dye for Quantitative PCR.

This is the first report describing the possibility of using a green fluorescent protein chromophore synthetic analog, P-HOBDI-BF2, as a fluorescent dye for a linear hydrolysis probe used in qPCR. The study was carried out on a system for detection of the plant pathogenic fungus Fusarium avenaceum using a plasmid containing translation elongation factor 1α fragment as a template. To estimate fluorogenic properties of P-HOBDI-BF2, 6-FAM- and BDP-FL-labeled probes were used. It was demonstrated that a synthetic dye based on the P-HOBDI-BF2 chromophore can be used for labeling hydrolysis probes for qPCR, but fluorescence increase levels for P-HOBDI-BF2-labeled probes were slightly lower than those for 6-FAM-labeled ones. At the same time, the sensitivity of P-HOBDI-BF2-based assays remained high, and this fact together with acceptable fluorescence levels suggests that this dye can be considered as an efficient alternative for reporters traditionally used for fluorescence detection in the FAM channel.

Phylogenetic Analysis and Molecular Typing of Trichothecene-Producing Fusarium Fungi from Russian Collections.

We performed a three-locus phylogenetic analysis of Fusarium strains presumably capable of trichothecene production, which were deposited in the Russian national collections. The intra- and interspecific polymorphism of partial sequences of the translation elongation factor 1 alpha (TEF1α) gene and two genes from the trichothecene cluster TRI5 and TRI14 was studied. A study of 60 strains of different origins using DNA markers confirmed, and in the case for several strains, clarified their taxonomic characteristics. As a result, a strain of F. commune (F-900) was identified in Russia for the first time. Furthermore, the strain F-846 proved to be phylogenetically distinct from any of the known Fusarium species. F. equiseti strains from Northwest Russia were found to belong to the North European group (I), whereas a strain from the North Caucasus - to the South European one (II). Partial TRI14 sequences from 9 out of 12 species were determined for the first time. Their comparative analysis demonstrated a relatively high level of intraspecific variability in F. graminearum and F. sporotrichioides, but no correlation between the sequence polymorphism and the geographic origin of the strains or their chemotype was found. Specific chemotypes of trichothecene B producers were characterized using two primer sets. The chemotyping results were verified by HPLC.

[Immuno-PCR Assay for Quantitation of Antibodies to Epstein-Barr Virus].

Successful disease prevention and therapy critically depend on timely diagnosis of infections. Quantitative immuno-PCR (qiPCR) technology improves the sensitivity in the detection of antibodies to pathogens. A qiPCR-based assay was developed to determine IgG antibodies to Epstein-Barr virus (EBV) in the human blood serum. EBV nuclear protein 1 fragment (pEBV) was expressed in Escherichia coli. A synthetic single-stranded deoxyribonucleotide was conjugated to streptavidin, and the conjugate was used to detect рEBV-IgG1-biotin complexes by qiPCR. The IgG1 titers determined by qiPCR were compared to the results of enzyme-linked immunosorbent assay (ELISA). The sensitivity of qiPCR was one order of magnitude higher than that of ELISA. Thus, a highly sensitive qiPCR-based assay was developed to quantitate antibodies specific to the recombinant EBV antigen.

Immuno-PCR: Achievements and Perspectives.

The immuno-PCR (iPCR) method combines advantages of enzyme-linked immunosorbent assay and polymerase chain reaction, which is used in iPCR as a method of "visualization" of antigen-antibody interaction. The use of iPCR provides classical PCR sensitivity to objects traditionally detected by ELISA. This method could be very sensitive and allow for detection of quantities of femtograms/ml order. However, iPCR is still not widely used. The aim of this review is to highlight the special features of the iPCR method and to show the main aspects of its development and application in recent years.

High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis.

The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation.

[Detection of Staphylococcus aureus toxins using immuno-PCR].

A highly sensitive test system, based on the method of immuno-PCR, was developed for the detection of two staphylococcal toxins: enterotoxin A (SEA) and toxic shock syndrome toxin (TSST). A key element of the developed systems was obtaining supramolecular complexes of bisbiotinylated oligodeoxynucleotides and streptavidin, which were used as DNA tags. Specificity studies showed no cross-reactivity when determining SEA and TSST. The sensitivity of detection of these toxins in the culture supernatants S. aureus was not less than 10 pg/mL.

[[Expression of house dust mite allergens Der f1 and Der f2 in Nicotiana benthamiana leaves].

The class E immunoglobulins (IgE) is known to recognize conformational epitopes and therefore the native conformation of recombinant allergens is essential for their using in test-systems. Recombinant Dermatophagoides farinae house dust mite (HDM) allergens Der f1 and Der f2 were expressed in bacteria Escherichia coli and Nicotiana benthamiana plants. It has been shown that IgE in sera from children allergic to HDM recognizes Der f2 expressed both in E. coli and N. benthamiana. Mature form of Der f1 expressed in E. coli does not interact with IgE while the protein purified from N. benthamiana is able to recognize IgE as a native allergen.

[Phosphate permease gene as a marker for the specific identification of toxigenic fungus Fusarium cerealis].

We have developed phosphate permease gene sequence-based PCR detection system of Fusarium cerealis phytopathogenic fungus. Sequencing and analysis revealed that the gene displayed unique polymorphism and could serve to establish phylogenetic relations as well as a marker to design specific primers. The specificity assay has confirmed the absence of cross reactions with DNAs of closely related Fusarium species. The qPCR assay demonstrated the 10 pg detection limit of specific DNA per reaction.

Defense peptides from barnyard grass (Echinochloa crusgalli L.) seeds.

A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.

[Novel DNA markers reveal for taxonomic characterization and identification of the Fusarium fungi species].

RAPD analysis for ten F. sporotrichioides strains of different geographical origin was done for DNA loci, potentially suitable as a new markers for taxonomic characterization and identification of toxigenic Fusarium fungi. Three selected monomorphic fragments--products of amplification with one of standard RAPD primers were sequenced that allowed creating SCAR markers for identification of Fusarium fungi on the species group level with similar profiles of produced mycotoxins.

[Efficient approach for potato viral pathogen sensitive diagnostic and identification].

Potato, one of the most widespread agricultural plants in Russia, is strongly affected by various pathogens of viral, bacterial, and fungal origin as well as by pests. Their simple and accurate diagnostics and identification sound rather important both for production of virus free planting material and to perform monitoring of the phytosanitary state of planting areas. Based on qualitative Fluorescent Amplification--based Specific Hybridization Polymerase Chain Reaction (FLASH-PCR) we have developed the diagnostic systems, which provided fast, careful, and with the minimum risk of contamination in the working zone by amplification products, detection of the major potato pathogens, i. e. A, Y, X, M, S potato viruses, potato leafroll virus, potato mop top virus, as well as potato spindle tuber viroids.

[FLASH-PCR diagnostics of toxigenic fungi of the genus Fusarium].

A test system for the diagnostics and identification of seven toxigenic fungi causing fusarioses of cereals (Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides, F. langsethiae, F. avenaceum, and F. tricinctum) was developed using PCR. The identification of pathogen is based on the specific amplification of a DNA fragment of the gene of translation elongation factor 1 alpha (tef-1alpha) and subsequent detection of the results by the fluorescent amplification-based specific hybridization method. The system was tested on 38 isolates of different fungi of the genus Fusarium.

[Diagnostics of phytopathogen fungi Septoria tritici and Stagonospora nodorum by fluorescent amplification-based specific hybridization (FLASH) PCR].

A PCR system in the fluorescent amplification-based specific hybridization (FLASH) format was developed for the detection and identification of two important wheat pathogenic fungi Septoria tritici (teleomorph of Mycosphaerella graminicola and Stagonospora nodorum (teleomorph of Phaeosphaeria nodorum), which cause spots on leaves and glumes, respectively. The pathogen detection system is based on the amplification of a genome fragment in the internal transcribed spacer 1 (ITS 1) region and a site encoding the 5.8S ribosomal RNA. The forward primers to ITS1 and a universal reverse primer and a Beacon type probe to the 5.8S ribosomal RNA region were chosen to provide the detection of the products in the FLASH format. This system was tested on different isolates of the pathogens, and on infected soil, leaf, and seed samples.

[Interaction of cysteine-rich protein of Carlavirus with plant defense system].

Viruses of genus Carlavirus encode a small cysteine-rich protein (CRP) of unknown function. To investigate the role of CRP of carlavirus chrysanthemum virus B (CVB), a recombinant potato virus X (PVX) genome was constructed, which carried the CVB CRP gene. Expression of CVB CRP in the PVX genetic background drastically changed the PVX symptom phenotype in N. benthamiana. Instead of symptomless infection and mild mosaic, which are characteristic of PVX in this plant host, the recombinant virus expressing CVB CRP induced formation of necrotic local lesions on inoculated leaves and necrosis of the apical leaves. In N. tabacum, the infection pattern depended on the host genotype: the recombinant PVX was able to spread systemically only in N gene-carrying plants. In agroinfiltration-mediated transient expression assay, CVB CRP did not exhibit the properties of avirulence factor in N. benthamiana and was unable to suppress post-transcriptional gene silencing. Thus, CVB CRP is the viral pathogenicity determinant controlling the virus interaction with plant hosts in a manner which depends on plant defense mediated by resistance genes such as the N gene.

The new plant virus family Flexiviridae and assessment of molecular criteria for species demarcation.

The new plant virus family Flexiviridae is described. The family is named because its members have flexuous virions and it includes the existing genera Allexivirus, Capillovirus, Carlavirus, Foveavirus, Potexvirus, Trichovirus and Vitivirus, plus the new genus Mandarivirus together with some related viruses not assigned to any genus. The family is justified from phylogenetic analyses of the polymerase and coat protein (CP) sequences. To help to define suitable molecular criteria for demarcation of species, a complete set of pairwise comparisons was made using the nucleotide (nt) and amino acid (aa) sequences of each fully-sequenced gene from every available accession in the family. Based on the distributions and on inspection of the data, it was concluded that, as a general rule, distinct species have less than ca. 72% identical nt or 80% identical aa between their entire CP or replication protein genes.

[A point mutation in the coat protein gene affects long distance transport of the tobacco mosaic virus]. / Tochechnaia mutatsiia v gene belka obolochki vliiaet na dal'nii transport virusa tabachnoi mozaiki.

A mutation resulting in substitution of positively charged Lys53 with negatively charged Glu in the coat protein was introduced in the infectious cDNA copy of the genome of wild-type tobacco mosaic virus strain U1. Kinetic analysis of long-distance virus transport in plants showed that systemic distribution of the mutant virus was delayed by 5-6 days as compared with the wild-type one. On evidence of RNA sequencing in the mutant progeny, Glu50 of the coat protein was substituted with Lys after passage I to compensate for the loss of the positive charge at position 53. Electron microscopy revealed atypical inclusions (rodlike structures, multiple electron-dense globular particles) in the nuclear interchromatin space of leaf mesophyll cells infected with the mutant but not with the wild-type virus.