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Wolbachia is a maternally inherited, intercellular bacterial symbiont of insects and some other invertebrates. Here, we investigated the effect of two different Wolbachia strains, differing in a large chromosomal inversion, on the differential expression of genes in D. melanogaster females. We revealed significant changes in the transcriptome of the infected flies compared to the uninfected ones, as well as in the transcriptome of flies infected with the Wolbachia strain, wMelPlus, compared to flies infected with the wMelCS 112 strain. We linked differentially expressed genes (DEGs) from two pairwise comparisons, "uninfected-wMelPlus-infected" and "uninfected-wMelCS 112 -infected", into two gene networks, in which the following functional groups were designated: "Proteolysis", "Carbohydrate transport and metabolism", "Oxidation-reduction process", "Embryogenesis", "Transmembrane transport", "Response to stress" and "Alkaline phosphatases". Our data emphasized similarities and differences between infections by different strains under study: a wMelPlus infection results in more than double the number of upregulated DEGs and half the number of downregulated DEGs compared to a wMelCS 112 infection. Thus, we demonstrated that Wolbachia made a significant contribution to differential expression of host genes and that the bacterial genotype plays a vital role in establishing the character of this contribution.

The maternally transmitted endocellular bacteria Wolbachia is a well-known symbiont of insects, demonstrating both negative and positive effects on host fitness. The previously found Wolbachia strain wMelPlus is characterized by a positive effect on the stress-resistance of its host Drosophila melanogaster, under heat stress conditions. This investigation is dedicated to studying the genomic underpinnings of such an effect. We sequenced two closely related Wolbachia strains, wMelPlus and wMelCS112, assembled their complete genomes, and performed comparative genomic analysis engaging available Wolbachia genomes from the wMel and wMelCS groups. Despite the two strains under study sharing very close gene-composition, we discovered a large (>1/6 of total genome) chromosomal inversion in wMelPlus, spanning through the region that includes the area of the inversion earlier found in the wMel group of Wolbachia genotypes. A number of genes in unique inversion blocks of wMelPlus were identified that might be involved in the induction of a stress-resistant phenotype in the host. We hypothesize that such an inversion could rearrange established genetic regulatory-networks, causing the observed effects of such a complex fly phenotype as a modulation of heat stress resistance. Based on our findings, we propose that wMelPlus be distinguished as a separate genotype of the wMelCS group, named wMelCS3.

A number of methods for extracting the DNA of maternally inherited obligate intracellular bacteria Wolbachia from an insect host and its subsequent purification have been described in previous scholarship. As Wolbachia is present in the hosts' organisms in rather low quantities, these techniques used to be quite labor-intensive. For this paper, we analyzed them in detail, searched for a possibility to simplify and accelerate the protocol, and proposed an easy and effective method for isolating Wolbachia DNA from Drosophila melanogaster with a purity sufficient for genomic sequencing. Our method involves the centrifugation of homogenized flies or just their ovaries, as the most Wolbachia -enriched tissue, followed by the filtration of homogenate and extraction of DNA using a modified version of the Livak buffer protocol. The proportion of Wolbachia DNA in the total DNA was quantified based on the results of sequencing with the use of the Illumina MiSeq platform and a pipeline of bioinformatic analysis. For the two analyzed D. melanogaster lines infected with two different Wolbachia strains, the proportion was at least 68 and 94%, respectively.

Barley ( Hordeum vulgare L.) grain pigmentation is caused by two types of phenolic compounds: anthocyanins (which are flavonoids) give a blue or purple color, and melanins (which are products of enzymatic oxidation and polymerization of phenolic compounds) give a black or brown color. Genes Ant1 and Ant2 determine the synthesis of purple anthocyanins in the grain pericarp, whereas melanins are formed under the control of the Blp1 gene in hulls and pericarp tissues. Unlike anthocyanin synthesis, melanin synthesis is poorly understood. The objective of the current work was to reveal features of the phenylpropanoid biosynthesis pathway functioning in melanin-accumulating barley grains. For this purpose, comparative transcriptomic and metabolomic analyses of three barley near-isogenic lines accumulating anthocyanins, melanins, or both in the grain, were performed. A comparative analysis of mRNA libraries constructed for three stages of spike development (booting, late milk, and early dough) showed transcriptional activation of genes encoding enzymes of the general phenylpropanoid pathway in all the lines regardless of pigmentation; however, as the spike matured, unique transcriptomic patterns associated with melanin and anthocyanin synthesis stood out. Secondary activation of transcription of the genes encoding enzymes of the general phenylpropanoid pathway together with genes of monolignol synthesis was revealed in the line accumulating only melanin. This pattern differs from the one observed in the anthocyanin-accumulating lines, where - together with the genes of general phenylpropanoid and monolignol synthesis pathways - flavonoid biosynthesis genes were found to be upregulated, with earlier activation of these genes in the line accumulating both types of pigments. These transcriptomic shifts may underlie the observed differences in concentrations of phenylpropanoid metabolites analyzed in the grain at a late developmental stage by high-performance liquid chromatography. Both melanin-accumulating lines showed an increased total level of benzoic acids. By contrast, anthocyanin-accumulating lines showed higher concentrations of flavonoids and p -coumaric and ferulic acids. A possible negative effect of melanogenesis on the total flavonoid content and a positive influence on the anthocyanin content were noted in the line accumulating both types of pigments. As a conclusion, redirection of metabolic fluxes in the phenylpropanoid biosynthesis pathway occurs when melanin is synthesized., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Glagoleva, Vikhorev, Shmakov, Morozov, Chernyak, Vasiliev, Shatskaya, Khlestkina and Shoeva.)

Plastids and mitochondria have their own small genomes, which do not undergo meiotic recombination and may have evolutionary fates different from each other and that of the nuclear genome. For the first time, we sequenced mitochondrial genomes of pea (Pisum L.) from 42 accessions mostly representing diverse wild germplasm from throughout the wild pea geographical range. Six structural types of the pea mitochondrial genome were revealed. From the same accessions, plastid genomes were sequenced. Phylogenetic trees based on the plastid and mitochondrial genomes were compared. The topologies of these trees were highly discordant, implying not less than six events of hybridisation between diverged wild peas in the past, with plastids and mitochondria differently inherited by the descendants. Such discordant inheritance of organelles could have been driven by plastid-nuclear incompatibility, which is known to be widespread in crosses involving wild peas and affects organellar inheritance. The topology of the phylogenetic tree based on nucleotide sequences of a nuclear gene, His5, encoding a histone H1 subtype, corresponded to the current taxonomy and resembled that based on the plastid genome. Wild peas (Pisum sativum subsp. elatius s.l.) inhabiting Southern Europe were shown to be of hybrid origin, resulting from crosses of peas related to those presently inhabiting the eastern Mediterranean in a broad sense. These results highlight the roles of hybridisation and cytonuclear conflict in shaping plant microevolution., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Viroids belong to a very interesting class of molecules attracting researchers in phytopathology and molecular evolution. Here we review recent literature data concerning the genetics of Potato spindle tuber viroid (PSTVd) and the mechanisms related to its pathological effect on the host plants. PSTVd can be transmitted vertically through microspores and macrospores, but not with pollen from another infected plant. The 359 nucleotidelong genomic RNA of PSTVd is highly structured and its 3D-conformation is responsible for interaction with host cellular factors to mediate replication, transport between tissues during systemic infection and the severity of pathological symptoms. RNA replication is prone to errors and infected plants contain a population of mutated forms of the PSTVd genome. Interestingly, at 7 DAI, only 25 % of the newly synthesized RNAs were identical to the master copy, but this proportion increased to up to 70 % at 14 DAI and remained the same afterwards. PSTVd infection induces the immune response in host plants. There are PSTVd strains with a severe, a moderate or a mild pathological effect. Interestingly, viroid replication itself does not necessarily induce strong morphological or physiological symptoms. In the case of PSTVd, disease symptoms may occur due to RNA-interference, which decreases the expression levels of some important cellular regulatory factors, such as, for example, potato StTCP23 from the gibberellic acid pathway with a role in tuber morphogenesis or tomato FRIGIDA-like protein 3 with an early flowering phenotype. This association between the small segments of viroid genomic RNAs complementary to the untranslated regions of cellular mRNAs and disease symptoms provides a way for new resistant cultivars to be developed by genetic editing. To conclude, viroids provide a unique model to reveal the fundamental features of living systems, which appeared early in evolution and still remain undiscovered., (Copyright © AUTHORS.)

Background: Globodera rostochiensis belongs to major potato pathogens with a sophisticated mechanism of interaction with roots of the host plants. Resistance of commercial varieties is commonly based on specific R genes introgressed from natural populations of related wild species and from native potato varieties grown in the Andean highlands. Investigation of molecular resistance mechanisms and screening the natural populations for novel R genes are important for both fundamental knowledge on plant pathogen interactions and breeding for durable resistance. Here we exploited the Solanum phureja accessions collected in South America with contrasting resistance to G. rostochiensis., Results: The infestation of S. phureja with G. rostochiensis juveniles resulted in wounding stress followed by activation of cell division and tissue regeneration processes. Unlike the susceptible S. phureja genotype, the resistant accession reacted by rapid induction of variety of stress response related genes. This chain of molecular events accompanies the hypersensitive response at the juveniles' invasion sites and provides high-level resistance. Transcriptomic analysis also revealed considerable differences between the analyzed S. phureja genotypes and the reference genome., Conclusion: The molecular processes in plant roots associated with changes in gene expression patterns in response to G. rostochiensis infestation and establishment of either resistant or susceptible phenotypes are discussed. De novo transcriptome assembling is considered as an important tool for discovery of novel resistance traits in S. phureja accessions.

Organellar genomes may shed light on complicated patterns of plant evolution at inter- and intraspecies level. Primary structure of plastid genomes sequenced in this study and taken from public databases was characterised and compared in 22 diverse, mostly wild representatives of the genus Pisum (peas). Phylogenetic trees reconstructed via Bayesian approach on the basis of entire plastid genomes resembled those reconstructed on the basis of a nuclear gene His5 coding for a minor histone H1 subtype. They reveal Pisum fulvum as an early divergence of the genus but do not support other taxonomical subdivisions. The positions of three accessions, classified as P. sativum subsp. elatius (the wild subspecies of the common pea), appeared quite unexpected. On the entire plastid genome tree, two accessions, from the Black Sea area of Turkey and Georgia, clustered with representatives of another species, P. fulvum, while the other, from Greece, was the first divergence of the P. sativum branch. We suppose these unusual plastid genomes to be ancient lineages ascending to a 'missing link' between P. fulvum and P. sativum, represented by accession Pe 013 from Turkey. Accessions with common pea appearance but deeply diverged plastids could occur through occasional crossing of diverged pea lines in the past and biparental plastid inheritance, both events being possible in peas., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The characterization of major resistance genes (R genes) in the potato remains an important task for molecular breeding. However, R genes are rapidly evolving and frequently occur in genomes as clusters with complex structures, and their precise mapping and identification are complicated and time consuming., Results: Comparative analysis of root transcriptomes of Solanum phureja genotypes with contrasting resistance to Globodera rostochiensis revealed a number of differentially expressed genes. However, compiling a list of candidate R genes for further segregation analysis was hampered by their scarce annotation. Nevertheless, combination of transcriptomic analysis with data on predicted potato NBS-LRR-encoding genes considerably improved the quality of the results and provided a reasonable number of candidate genes that provide S. phureja with strong resistance to the potato golden cyst nematode., Conclusion: Combination of comparative analyses of tissue-specific transcriptomes in resistant and susceptible genotypes may be used as an approach for the rapid identification of candidate potato R genes for co-segregation analysis and may be used in parallel with more sophisticated studies based on genome resequencing.

Background: Some plant species have 'melanin-like' black seed pigmentation. However, the chemical and genetic nature of this 'melanin-like' black pigment have not yet been fully explored due to its complex structure and ability to withstand almost all solvents. Nevertheless, identification of genetic networks participating in trait formation is key to understanding metabolic processes involved in the expression of 'melanin-like' black seed pigmentation. The aim of the current study was to identify differentially expressed genes (DEGs) in barley near-isogenic lines (NILs) differing by allelic state of the Blp (black lemma and pericarp) locus., Results: RNA-seq analysis of six libraries (three replicates for each line) was performed. A total of 957 genome fragments had statistically significant changes in expression levels between lines BLP and BW, with 632 fragments having increased expression levels in line BLP and 325 genome fragments having decreased expression. Among identified DEGs, 191 genes were recognized as participating in known pathways. Among these were metabolic pathways including 'suberin monomer biosynthesis', 'diterpene phytoalexins precursors biosynthesis', 'cutin biosynthesis', 'cuticular wax biosynthesis', and 'phenylpropanoid biosynthesis, initial reactions'. Differential expression was confirmed by real-time PCR analysis of selected genes., Conclusions: Metabolic pathways and genes presumably associated with black lemma and pericarp colour as well as Blp-associated resistance to oxidative stress and pathogens, were revealed. We suggest that the black pigmentation of lemmas and pericarps is related to increased level of phenolic compounds and their oxidation. The effect of functional Blp on the synthesis of ferulic acid and other phenolic compounds can explain the increased antioxidant capacity and biotic and abiotic stress tolerance of black-grained cereals. Their drought tolerance and resistance to diseases affecting the spike may also be related to cuticular wax biosynthesis. In addition, upregulated synthesis of phytoalexins, suberin and universal stress protein (USP) in lemmas and pericarps of the Blp carriers may contribute to their increased disease resistance. Further description of the DEGs haplotypes and study of their association with physiological characteristics may be useful for future application in barley pre-breeding.

Background: Albinism in plants is characterized by lack of chlorophyll and results in photosynthesis impairment, abnormal plant development and premature death. These abnormalities are frequently encountered in interspecific crosses and tissue culture experiments. Analysis of albino mutant phenotypes with full or partial chlorophyll deficiency can shed light on genetic determinants and molecular mechanisms of albinism. Here we report analysis of RNA-seq transcription profiling of barley (Hordeum vulgare L.) near-isogenic lines, one of which is a carrier of mutant allele of the Alm gene for albino lemma and pericarp phenotype (line i:BwAlm)., Results: 1221 genome fragments have statistically significant changes in expression levels between lines i:BwAlm and Bowman, with 148 fragments having increased expression levels in line i:BwAlm, and 1073 genome fragments, including 42 plastid operons, having decreased levels of expression in line i:BwAlm. We detected functional dissimilarity between genes with higher and lower levels of expression in i:BwAlm line. Genes with lower level of expression in the i:BwAlm line are mostly associated with photosynthesis and chlorophyll synthesis, while genes with higher expression level are functionally associated with vesicle transport. Differentially expressed genes are shown to be involved in several metabolic pathways; the largest fraction of such genes was observed for the Calvin-Benson-Bassham cycle. Finally, de novo assembly of transcriptome contains several transcripts, not annotated in current H. vulgare genome version., Conclusions: Our results provide the new information about genes which could be involved in formation of albino lemma and pericarp phenotype. They demonstrate the interplay between nuclear and chloroplast genomes in this physiological process.

A system for delivery of analogues of AZT-triphosphates (AZT*TP) based on SiO₂ nanoparticles was proposed. For this purpose, a simple and versatile method was developed for the preparation of SiO₂∼dNTP conjugates using the 'click'-reaction between AZTTP and premodified nanoparticles containing the alkyne groups. The substrate properties of SiO₂∼AZT*TP were tested using Klenow fragment and HIV reverse transcriptase. The 3'-triazole derivatives of thymidine triphosphate being a part of the SiO₂∼AZT*TP nanocomposites were shown to be incorporated into the growing DNA chain. It was shown by confocal microscopy that the proposed SiO₂∼AZT*TP nanocomposites penetrate into cells. These nanocomposites were shown to inhibit the reproduction of POX and Herpes viruses at nontoxic concentrations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized.

A system for delivery of analogues of 2'-deoxyribonucleoside triphosphate (dNTP) based on SiO(2) nanoparticles was proposed. A simple and versatile method was developed for the preparation of SiO(2)-dNTP conjugates using the 'click'-reaction between premodified nanoparticles containing the azido groups and dNTP containing the alkyne-modified γ-phosphate group. The substrate properties of SiO(2)-dNTP were tested using Klenow fragment and HIV reverse transcriptase. Nucleoside triphosphates being a part of the SiO(2)-dNTP nanocomposites were shown to be incorporated into the growing DNA chain. The rate of polymerization with the use of SiO(2)-dNTP or common dNTP in case of HIV reverse transcriptase differed insignificantly. It was shown by confocal microscopy that the proposed SiO(2)-dNTP nanocomposites bearing the fluorescent label penetrate into cells and even into cellular nuclei., (Copyright © 2012 Elsevier Ltd. All rights reserved.)