The Role of Carbonic Anhydrase αCA4 in Photosynthetic Reactions in Arabidopsis thaliana Studied, Using the Cas9 and T-DNA Induced Mutations in Its Gene.

An homozygous mutant line of Arabidopsis thaliana with a knocked out At4g20990 gene encoding thylakoid carbonic anhydrase αCA4 was created using a CRISPR/Cas9 genome editing system. The effects of the mutation were compared with those in two mutant lines obtained by the T-DNA insertion method. In αCA4 knockouts of all three lines, non-photochemical quenching of chlorophyll a fluorescence was lower than in the wild type (WT) plants due to a decrease in its energy-dependent component. The αCA4 knockout also affected the level of expression of the genes encoding all proteins of the PSII light harvesting antennae, the genes encoding cytoplasmic and thylakoid CAs and the genes induced by plant immune signals. The production level of starch synthesis during the light period, as well as the level of its utilization during the darkness, were significantly higher in these mutants than in WT plants. These data confirm that the previously observed differences between insertional mutants and WT plants were not the result of the negative effects of T-DNA insertion transgenesis but the results of αCA4 gene knockout. Overall, the data indicate the involvement of αCA4 in the photosynthetic reactions in the thylakoid membrane, in particular in processes associated with the protection of higher plants' photosynthetic apparatus from photoinhibition.

Increasing the Efficiency of the Accumulation of Recombinant Proteins in Plant Cells: The Role of Transport Signal Peptides.

The problem with increasing the yield of recombinant proteins is resolvable using different approaches, including the transport of a target protein to cell compartments with a low protease activity. In the cell, protein targeting involves short-signal peptide sequences recognized by intracellular protein transport systems. The main systems of the protein transport across membranes of the endoplasmic reticulum and endosymbiotic organelles are reviewed here, as are the major types and structure of the signal sequences targeting proteins to the endoplasmic reticulum and its derivatives, to plastids, and to mitochondria. The role of protein targeting to certain cell organelles depending on specific features of recombinant proteins and the effect of this targeting on the protein yield are discussed, in addition to the main directions of the search for signal sequences based on their primary structure. This knowledge makes it possible not only to predict a protein localization in the cell but also to reveal the most efficient sequences with potential biotechnological utility.

CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of Arabidopsis thaliana suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.

Optimization of Genome Knock-In Method: Search for the Most Efficient Genome Regions for Transgene Expression in Plants.

Plant expression systems are currently regarded as promising alternative platforms for the production of recombinant proteins, including the proteins for biopharmaceutical purposes. However, the accumulation level of a target protein in plant expression systems is still rather low compared with the other existing systems, namely, mammalian, yeast, and E. coli cells. To solve this problem, numerous methods and approaches have been designed and developed. At the same time, the random nature of the distribution of transgenes over the genome can lead to gene silencing, variability in the accumulation of recombinant protein, and also to various insertional mutations. The current research study considered inserting target genes into pre-selected regions of the plant genome (genomic "safe harbors") using the CRISPR/Cas system. Regions of genes expressed constitutively and at a high transcriptional level in plant cells (housekeeping genes) that are of interest as attractive targets for the delivery of target genes were characterized. The results of the first attempts to deliver target genes to the regions of housekeeping genes are discussed. The approach of "euchromatization" of the transgene integration region using the modified dCas9 associated with transcription factors is considered. A number of the specific features in the spatial chromatin organization allowing individual genes to efficiently transcribe are discussed.

Three Parts of the Plant Genome: On the Way to Success in the Production of Recombinant Proteins.

Recombinant proteins are the most important product of current industrial biotechnology. They are indispensable in medicine (for diagnostics and treatment), food and chemical industries, and research. Plant cells combine advantages of the eukaryotic protein production system with simplicity and efficacy of the bacterial one. The use of plants for the production of recombinant proteins is an economically important and promising area that has emerged as an alternative to traditional approaches. This review discusses advantages of plant systems for the expression of recombinant proteins using nuclear, plastid, and mitochondrial genomes. Possibilities, problems, and prospects of modifications of the three parts of the genome in light of obtaining producer plants are examined. Examples of successful use of the nuclear expression platform for production of various biopharmaceuticals, veterinary drugs, and technologically important proteins are described, as are examples of a high yield of recombinant proteins upon modification of the chloroplast genome. Potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated. Although these opportunities have not yet been exploited, potential utility of plant mitochondria as an expression system for the production of recombinant proteins and its advantages over the nucleus and chloroplasts are substantiated.

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.

Cytological Techniques to Study Cytomixis in Plant Male Meiosis.

In this chapter we describe cytological techniques to study cytomixis, a process of nuclear migration between plant cells, in squashed plant male meiocytes of Nicotiana tabacum and Secale cereale. To perform immunostaining or fluorescence in situ hybridization (FISH) on meiotic cells involved in cytomixis common protocols are modified. During preparation of specimens for subsequent cytological analysis, it is necessary not only to make DNA and proteins accessible to DNA probes and antibodies, but also to preserve cell cytoplasm. There are also some important modifications in the protocols applied for meiocytes of different plant species. Here we describe protocols for immunostaining and FISH in rigid tobacco male meiocytes with dense cytoplasm and thick callose wall, that tolerate hard squashing, and in soft rye male meiocytes, that are easily damaged upon squashing, both to study cytomixis.

CRISPR/Cas9-mediated gfp gene inactivation in Arabidopsis suspension cells.

Targeted genome editing using CRISPR/Cas9 is a promising technology successfully verified in various plant species; however, it has hardly been used in plant cell suspension cultures. Here, we describe a successful knockout of a green fluorescent protein (gfp) reporter gene in Arabidopsis cell culture. We transformed seven transgenic suspension cell lines carrying one to three gfp gene copies with a binary vector containing genes coding for Cas9 and guide RNAs targeting the gfp gene. We detected the site-specific mutations by restriction analysis of a gfp amplicon. DNA sequencing of the PCR products confirmed high diversity of insertion-deletion mutations in the cell lines after the editing. We also analyzed gfp mRNA expression by real-time PCR and observed a decrease in gfp transcription after the target site modification. We can conclude that the CRISPR/Cas9 system can be successfully used for introducing site-specific mutations into the genome of cultured suspension cells of Arabidopsis.

The Problem of the Low Rates of CRISPR/Cas9-Mediated Knock-ins in Plants: Approaches and Solutions.

The main number of genome editing events in plant objects obtained during the last decade with the help of specific nucleases zinc finger (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas are the microindels causing frameshift and subsequent gene knock-out. The knock-ins of genes or their parts, i.e., the insertion of them into a target genome region, are between one and two orders of magnitude less frequent. First and foremost, this is associated with the specific features of the repair systems of higher eukaryotes and the availability of the donor template in accessible proximity during double-strand break (DSB) repair. This review briefs the main repair pathways in plants according to the aspect of their involvement in genome editing. The main methods for increasing the frequency of knock-ins are summarized both along the homologous recombination pathway and non-homologous end joining, which can be used for plant objects.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein ESAT6-CFP10-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute tuberculosis. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of animals with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized animals do not develop the symptoms of acute tuberculosis and their body weight gain was five times more as compared with the non-immunized-infected animals. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

FlowerMorphology: fully automatic flower morphometry software.

MAIN CONCLUSION: The software FlowerMorphology is designed for automatic morphometry of actinomorphic flowers. The novel complex parameters of flowers calculated by FlowerMorphology allowed us to quantitatively characterize a polyploid series of tobacco. Morphological differences of plants representing closely related lineages or mutants are mostly quantitative. Very often, there are only very fine variations in plant morphology. Therefore, accurate and high-throughput methods are needed for their quantification. In addition, new characteristics are necessary for reliable detection of subtle changes in morphology. FlowerMorphology is an all-in-one software package to automatically image and analyze five-petal actinomorphic flowers of the dicotyledonous plants. Sixteen directly measured parameters and ten calculated complex parameters of a flower allow us to characterize variations with high accuracy. The program was developed for the needs of automatic characterization of Nicotiana tabacum flowers, but is applicable to many other plants with five-petal actinomorphic flowers and can be adopted for flowers of other merosity. A genetically similar polyploid series of N. tabacum plants was used to investigate differences in flower morphology. For the first time, we could quantify the dependence between ploidy and size and form of the tobacco flowers. We found that the radius of inner petal incisions shows a persistent positive correlation with the chromosome number. In contrast, a commonly used parameter-radius of outer corolla-does not discriminate 2n and 4n plants. Other parameters show that polyploidy leads to significant aberrations in flower symmetry and are also positively correlated with chromosome number. Executables of FlowerMorphology, source code, documentation, and examples are available at the program website: https://github.com/Deyneko/FlowerMorphology .

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10-ESAT6-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of guinea pigs with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized guinea pigs do not develop the symptoms of acute TB and their body weight gain was five times more as compared with the non-immunized infected guinea pigs. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

Cytomixis in the cereal (Gramineae) microsporogenesis.

The specific features in behavior of the nuclei and chromatin migrating through cytomictic channels as well as in formation of micronuclei in the cereal microsporogenesis have been studied. Immunofluorescence microscopy has allowed for demonstration that the tubulin cytoskeleton does not play a significant role in the intercellular migration of nuclei. Potential involvement of the actin cytoskeleton and SUN-KASH linker complexes in cytomixis is discussed. Comparative analysis of the published and own data suggests that the cytological patterns of cytomixis in monocots and dicots are conserved. As has been shown, each higher ploidy level in the polyploid series of the family Gramineae is accompanied by an increase in the rate of cytomixis independently of individual species. The results confirm the assumption on a correlation between the rate of cytomixis, ploidy level, and genome balance.

Transgenic carrot expressing fusion protein comprising M. tuberculosis antigens induces immune response in mice.

Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L.) genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

MatrixCatch--a novel tool for the recognition of composite regulatory elements in promoters.

BACKGROUND: Accurate recognition of regulatory elements in promoters is an essential prerequisite for understanding the mechanisms of gene regulation at the level of transcription. Composite regulatory elements represent a particular type of such transcriptional regulatory elements consisting of pairs of individual DNA motifs. In contrast to the present approach, most available recognition techniques are based purely on statistical evaluation of the occurrence of single motifs. Such methods are limited in application, since the accuracy of recognition is greatly dependent on the size and quality of the sequence dataset. Methods that exploit available knowledge and have broad applicability are evidently needed. RESULTS: We developed a novel method to identify composite regulatory elements in promoters using a library of known examples. In depth investigation of regularities encoded in known composite elements allowed us to introduce a new characteristic measure and to improve the specificity compared with other methods. Tests on an established benchmark and real genomic data show that our method outperforms other available methods based either on known examples or statistical evaluations. In addition to better recognition, a practical advantage of this method is first the ability to detect a high number of different types of composite elements, and second direct biological interpretation of the identified results. The program is available at http://gnaweb.helmholtz-hzi.de/cgi-bin/MCatch/MatrixCatch.pl and includes an option to extend the provided library by user supplied data. CONCLUSIONS: The novel algorithm for the identification of composite regulatory elements presented in this paper was proved to be superior to existing methods. Its application to tissue specific promoters identified several highly specific composite elements with relevance to their biological function. This approach together with other methods will further advance the understanding of transcriptional regulation of genes.

New insights into cytomixis: specific cellular features and prevalence in higher plants.

The phenomenon of intercellular migration of nuclei in plant tissues (cytomixis) was discovered over a century ago, which has been followed by numerous attempts to clarify the essence of this process as well as to determine its causes and consequences. Most attention of researchers has been paid to cytomixis in microsporogenesis, since the transfer of part of genetic material between microsporocytes may influence the ploidy level of the produced pollen and, presumably, have an evolutionary significance. This review compiles the data on cytological pattern of cytomixis and proposes a scheme as to how cytomictic channels are formed and function in angiosperms. The prevalence of cytomixis in different plant taxa is analyzed using the published data. The causes, mechanisms, and consequences of the nuclear migration between cells in plant tissues are discussed.

Oral Immunogenicity of plant-made Mycobacterium tuberculosis ESAT6 and CFP10.

Two lines of transgenic carrot plants producing Mycobacterium tuberculosis proteins (ESAT6 and CFP10) have been constructed. The target proteins are present in carrot storage roots at a level not less than 0.056% of the total storage protein (TSP) for ESAT6 and 0.002% of TSP for CFP10. As has been shown, oral immunization of mice induces both the cell-mediated and humoral immunities. These data suggest that the proteins in question are appropriate as a candidate edible vaccine against tuberculosis.

An ultrastructural study of cytomixis in tobacco pollen mother cells.

Intercellular chromatin migration (cytomixis) in the pollen mother cells of two tobacco (Nicotiana tabacum L.) lines was analyzed by electron microscopy during the first meiotic prophase. The maximal manifestation of cytomixis was observed in the pachytene. As a rule, several cells connected with one another by cytomictic channels wherein the nuclei migrated were observable at this stage. In the majority of cases, nuclei passed from cell to cell concurrently through several closely located cytomictic channels. Chromatin migrated between cells within the nuclear envelope, and its disintegration was unobservable. The nucleus, after passing through cytomictic channels into another cell, can be divided into individual micronuclei or, in the case of a direct contact with another nucleus, can form a nuclear bridge. It has been demonstrated that the chromatin structure after intracellular migration visually matches the chromatin structure before it passed through the cytomictic channel. No signs of pyknosis were observable in the chromatin of the micronuclei formed after cytomixis, and the synaptonemal complex was distinctly seen. The dynamics of changes in the nucleoli during cytomixis was for the first time monitored on an ultrastructural level. Possible mechanisms determining cytomixis are discussed and the significance of this process in plant development is considered.

Spontaneous spectinomycin resistance mutations detected after biolistic transformation of Daucus carota L.

Spectinomycin resistant mutant carrot (Daucus carota L.) callus lines detected in the experiments on biolistic transformation of plastome were analyzed. It has been found that this antibiotic resistance is determined by point nucleotide substitutions at two distinct sites of the chloroplast gene rrn16, coding for 16S rRNA, namely, G1012T, G1012C, and A1138G. The detected mutations are localized to the 16S rRNA region forming helix h34, which contains spectinomycin binding site, and lead to its destabilization by several kilocalories per mole. Comparative analysis of rrn16 gene sequences has demonstrated conservation of the positions of the nucleotide substitutions determining this antibiotic resistance in carrot (D. carota L.), tobacco (Nicotiana tabacum L.), and bladder pod (Lesquerella fendleri L.), as well as in Escherichia coli.