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Key Points Quantitative evaluation of the tumor load in patients with IgM monoclonal gammopathies in tDNA and cfDNA samples., Visual Abstract, We describe a novel method for the detection of MYD88L265P mutation using a competitive allele-specific polymerase chain reaction (Cast-PCR) assay. This assay has a sensitivity of 1 × 10−3, is applicable in reactions containing very low amounts of DNA (as low as 20 pg), and allowed the detection of MYD88L265P somatic mutation in both tumor-derived DNA (tDNA) and cell-free DNA (cfDNA). In addition, using the Cast-PCR assay, we were able to determine the mutation allele fraction (MAF) in each tested sample. We then analyzed baseline tDNA and cfDNA samples from 163 patients (53 with immunoglobulin M monoclonal gammopathy of undetermined significance and 110 with Waldenström’s macroglobulinemia [WM], of whom 54 were asymptomatic and 56 were symptomatic) and also in sequential samples of 37 patients. MAF in both cfDNA and tDNA was higher among patients with symptomatic compared with asymptomatic WM and in those with asymptomatic WM compared with those with immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance. In addition, the evaluation of sequential samples showed that MAF decreased after treatment, whereas it increased in patients who relapsed or progressed to symptomatic WM. Thus, Cast-PCR is a highly sensitive, cost-effective diagnostic tool for MYD88L265P detection, applicable in both tDNA and cfDNA samples, that also provides a quantitative evaluation of the tumor load in patients with IgM monoclonal gammopathies.

A novel electrochemical DNA sensor was designed to detect Helicobacter pylori based on accordion-like Ti3C2Tx. Here the multilayer Ti3C2Tx obtained by DMSO delamination was used to modify the glass carbon electrode, with a large specific surface area and excellent conductivity. Au nanoparticles were supported on the modified electrode and worked as an effective carrier to fix the capture probe (cpDNA) with sulfhydryl group through the firm binding of Au-S bond. Such an accordion-like Ti3C2Tx structure provides an ultrahigh electroactive surface area and ample binding sites for accommodating Au nanoparticles, which is advantageous for the signal amplification during the detection. And further, the sandwich structure formed by hybridizing cpDNA with target DNA sequence (tDNA) and rpDNA (rpDNA is a strand of DNA that can be base-paired with the tested tDNA) increases greatly the current signal and enhances the sensitivity of the electrochemical DNA sensor. Under optimal conditions, the developed electrochemical DNA sensor showed a wide linear range from 10−11 to 10−14 M and a low detection limit of 1.6 × 10−16 M and exhibited good sensitivity, reproducibility, and stability.

Agrobacterium T-DNA (transfer DNA) integration into the plant genome relies mostly on host proteins involved in the DNA damage repair pathways. However, conflicting results have been obtained using plants with mutated or down-regulated genes involved in these pathways. Here, we chose a different approach by following the expression of a series of genes, encoding proteins involved in the DNA damage response, during early stages of Agrobacterium infection in tobacco. First, we identified tobacco homologs of Arabidopsis genes induced upon DNA damage and demonstrated that their expression was activated by bleomycin, a DNA-break causing agent. Then, we showed that Agrobacterium infection induces the expression of several of these genes markers of the host DNA damage response, with different patterns of transcriptional response. This induction largely depends on Agrobacterium virulence factors, but not on the T-DNA, suggesting that the DNA damage response activation may rely on Agrobacterium-encoded virulence proteins. Our results suggest that Agrobacterium modulates the plant DNA damage response machinery, which might facilitate the integration of the bacterial T-DNA into the DNA breaks in the host genome.

Summary Targeted mutagenesis via CRISPR/Cas9 is now widely used, not only in model plants but also in agriculturally important crops. However, in vegetative crop propagation, CRISPR/Cas9 expression cassettes cannot be segregated out in the resulting progenies, but must nevertheless be eliminated without leaving unnecessary sequences in the genome. To this end, we designed a piggyBac‐mediated transgenesis system for the temporary expression of CRISPR/Cas9 in plants. This system allows integration into the host genome of piggyBac carrying both CRISPR/Cas9 and positive selection marker expression cassettes from an extrachromosomal double‐stranded transfer DNA (dsT‐DNA), with subsequent excision of the transgenes by the re‐transposition of piggyBac from the host genome after successful induction of targeted mutagenesis via CRISPR/Cas9. Here, we demonstrate that the transgenesis system via piggyBac transposition from T‐DNA works to deliver transgenes in rice. Following positive–negative selection to exclude transgenic cells randomly transformed with T‐DNA, piggyBac‐mediated transgenesis from the extrachromosomal dsT‐DNA was successful in ca. 1% of transgenic callus lines. After temporary expression of CRISPR/Cas9 within piggyBac, we confirmed, in a proof‐of‐concept experiment, that piggyBac could be excised precisely from the genome via the stably transformed transposase PBase. Even after excision of piggyBac, CRISPR/Cas9‐induced targeted mutations could be detected in the endogenous gene in regenerated rice plants. These results suggest that our piggyBac‐mediated transgenesis system will be a valuable tool in establishing efficient CRISPR/Cas9‐mediated targeted mutagenesis in vegetatively propagated crops.

The rapid development of the CRISPR–Cas9, –Cas12a and –Cas12b genome editing systems has greatly fuelled basic and translational plant research1–6. DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G)7, limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant8. Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. 9) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for secondary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox. An engineered SpRY Cas9 variant enables efficient gene editing without PAM requirement in rice transgenic lines and Dahurian larch protoplasts, and its derived base editors can edit the rice genome efficiently in a PAM-less fashion too.

VirE2 from Agrobacterium tumefaciens is a single-stranded (ss) DNA-binding protein involved in delivery of ssT-DNA (single-stranded transfer DNA) from the agrobacterial Ti plasmid into the eukaryotic cell nucleus. The crystallized part of VirE2 was studied by X-ray diffraction, and the noncrystallized parts of the C- (40 amino acid residues [aars]) and N- (111 aars) termini of the protein, which are presumably disordered, were evaluated by computational methods. We did a molecular dynamics simulation of VirE2 without VirE1 and observed no large changes in domain orientation. The interaction of VirE2 with ssDNA and formation of ssDNA-VirE2 complexes in silico were studied. We also used computer-aided methods to design model complexes consisting from two- and four-subunit VirE2 proteins. We examined the implication of disordered sites in formation of two- and four-subunit VirE2 complexes. Formation of VirE2 dimers and tetramers within ssDNA-VirE2 complexes was demonstrated by computational methods. Using the Platinum program, we found that hydrophilic amino acids were predominant on the surface of the four-subunit VirE2 complex.

Metal-organic frameworks(MOFs)-based nanozyme plays an important role in biosensing, therapy and catalysis. In this study, the effects of single-stranded DNA(ssDNA) with programmable sequences and its complementary DNA(TDNA) on the intrinsic peroxidase-like activity of hemin loaded MOFs(UiO-66-2), denoted as hemin@UiO-66-NH2, were investigated. The hemin@UiO-66-2 exhibited improved catalytic activity compared with free hemin. However, the catalytic activity is inhibited in the presence of ssDNA, as ssDNA can be adsorbed by MOFs and therefore protected the active sites from contact with substrates. Upon the addition of the TDNA, double-stranded DNA(dsDNA) was formed and detached from the MOFs, resulting in the recovery of catalytic activity. Sequentially adding ssDNA or its complementary DNA strands can achieve the reversible regulation of the catalytic activity of MOFs nanozymes. Moreover, the DNA hybridization-based regulation was further applied to a cascaded catalytic system composed of the nanozyme, hemin@UiO-66-NH2, and glucose oxidase. These nanozyme based programmable and reversibly regulated catalytic systems may have potential applications in future smart biosensing and catalysis systems.

An ultrasensitive fluorescence biosensor for detecting cytokeratin fragment antigen 21-1 (CYFRA 21-1) DNA of non-small cell lung carcinoma (NSCLC) is designed using polysaccharide and activator regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) signal amplification strategy. Thiolated peptide nucleic acid (PNA) is fixed on magnetic nanoparticles (MNPs) by a cross-linking agent and hybridized with CYFRA 21-1 DNA. Hyaluronic acid (HA) is linked to PNA/tDNA heteroduplexes in the form of carboxy-Zr4+-phosphate. Subsequently, multiple 2-bromo-2-methylpropionic acid (BMP) molecules are linked with HA to initiate ARGET ATRP reaction. Finally, a large number of fluorescein o-acrylate (FA) monomers are polymerized on the macro-initiators, and the fluorescence signal is significantly amplified. Under optimal conditions, this biosensor shows a significant linear correlation between the fluorescence intensity and logarithm of CYFRA 21-1 DNA concentration (0.1 fM to 0.1 nM), and the limit of detection is as low as 78 aM. Furthermore, the sensor has a good ability to detect CYFRA 21-1 DNA in serum samples and to recognize mismatched bases. It suggests that the strategy has broad application in early diagnosis by virtue of its high sensitivity and selectivity.

Herein, a highly sensitive electrochemical genosensor is proposed by the construction of an innovative DNA walking machine. Generally, a number of tetrahedral DNA (TDNA)-supported tracks and walkers are comodified on the electrode surface. DNA walking is inhibited in the absence of target DNA. After the interaction between a DNA walker strand and target DNA, a single-stranded primer sequence could be released, which initiates subsequent rolling circle amplification (RCA). The generated long single-stranded product contains multiple DNAzyme cores, which facilitate highly efficient cleavage of track strands and subsequent DNA walking. The electrode then loses the ability to localize silver nanoparticles (AgNPs) as the electrochemical species. Thus, when the reduced silver stripping current is recorded, a highly sensitive method for the detection of DNA is fabricated. Under optimal conditions, it achieves an admirable sensitivity with the limit of detection as low as 0.1 fM. Satisfactory specificity is also guaranteed. In addition, the practicality is further confirmed by applying human serum samples, which show great potential utility for clinical diagnosis.

A fast and simple strategy for the detection of DNA based on a new type of electrochemical Zr(IV) signaling probe is reported. Briefly, this DNA biosensor was fabricated by covalently coupling the amino groups of peptide nucleic acid (PNA) probes with aldehyde groups on the surface of a glassy carbon electrode (GCE), for specifically capturing target DNA (tDNA). After this, Zr(IV) probes were attached to the phosphate groups of DNA directly with strong coordination interaction, and stoichiometrically labeled Zr(IV) probes can be detected via differential pulse voltammetry (DPV). Under optimized conditions, this strategy showed a good linear response from 0.01 nM to 100 nM tDNA, and the detection limit was 0.81 pM (R2 = 0.998). Moreover, this DNA biosensor is also successfully applied to the DNA detection in serum samples with satisfactory results and shows great potential in clinical analysis.

Agrobacterium species transfer DNA (T−DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T−DNA often contains, at its junctions with plant DNA, deletions of T−DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T−DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T−DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T−DNA integration into the plant genome.

Genetic transformation of host plants by Agrobacterium tumefaciens and related species represents a unique model for natural horizontal gene transfer. Almost five decades of studying the molecular interactions between Agrobacterium and its host cells have yielded countless fundamental insights into bacterial and plant biology, even though several steps of the DNA transfer process remain poorly understood. Agrobacterium spp. may utilize different pathways for transferring DNA, which likely reflects the very wide host range of Agrobacterium. Furthermore, closely related bacterial species, such as rhizobia, are able to transfer DNA to host plant cells when they are provided with Agrobacterium DNA transfer machinery and T-DNA. Homologs of Agrobacterium virulence genes are found in many bacterial genomes, but only one non- Agrobacterium bacterial strain, Rhizobium etli CFN42, harbors a complete set of virulence genes and can mediate plant genetic transformation when carrying a T-DNA-containing plasmid.

The present study evaluated the potential of 2100 MHz radiofrequency radiations to act as cytotoxic and genotoxic agent. Fresh onion (Allium cepa L.) roots were exposed to electromagnetic field radiations (EMF-r) for different durations (1 h and 4 h) and evaluated for mitotic index (MI), phase index, chromosomal aberrations, and DNA damage. DNA damage was investigated with the help of the comet assay by assessing various parameters like % head DNA (HDNA), % tail DNA (TDNA), tail moment (TM), and olive tail moment (OTM). Effects of EMF-r exposure were also compared with that of methyl methanesulfonate (MMS; 90 μM), which acted as a positive control. The post-exposure effects of EMF-r after providing the test plants with an acclimatization period of 24 h were also evaluated. Compared to the control, a significant increase in the MI and aberration percentage was recorded upon 4 h of exposure. However, no specific trend of phase index in response to exposure was detected. EMF-r exposure incited DNA damage with a significant decrease in HDNA accompanied by an increase in TDNA upon exposure of 4 h. However, TM and OTM did not change significantly upon exposure as compared to that of control. Analysis of the post-exposure effects of EMF-r did not show any significant change/recovery. Our data, thus, suggest the potential cytotoxic and genotoxic nature of 2100 MHz EMF-r. Our study bears great significance in view of the swiftly emergent EMF-r in the surrounding environment and their potential for inciting aberrations at the chromosomal level, thus posing a genetic hazard.

—Changes in the level of DNA damage induced by atmospheric factors were evaluated from the percentage of tail DNA (%TDNA) in the comet assay of mouse cells for blood leukocytes, Ehrlich ascites carcinoma, and P388 lymphocytic leukemia. It was shown that (1) the values of DNA damage of blood leukocytes (%TDNA) were high within narrow ranges of atmospheric temperature (from –4°С to +3°С) and atmospheric partial oxygen contents within 298–304 mg/L; (2) the range of variations in %TDNA of leukocytes increased with increasing geomagnetic activity; (3) the changes in %TDNA of intact and X-irradiated (4 Gy) leukocytes and of tumor cells were associated with changes in air temperature; and (4) %TDNA correlated with atmospheric temperature in both types of intact tumor cells and irradiated P388 cells. Thus, variations in %TDNA appear to be caused by reactive oxygen species generated in the environment at temperatures of phase transitions of water. It is conceivable that changes in the basal %TDNA of cells modulate their radiation response.

Based on target DNA (tDNA)-mediated proximity binding to develop unique DNA assembly as output translator and enzyme-cleaved recycle for signal amplification, an electrochemical impedimetric biosensor is developed. The DNA assembly is constructed by the proximal ligation of tDNA with two affinity probes (A1 and A2) on Au-Fe3O4 magnetic microspheres as substrate previously conjugated with A1, DNA linker (L1) and DNA transduction probe (TP). This binding brings the domain C2* of A2 close to the formed dsDNA TP:L1, facilitating the displacement of TP by C2* (C2*:L1). Under the T7-powered catalysis, the cleavage reaction, C2* liberation and next displacement of TP are initiated and successively recycled. In response to a single-target binding event, hundreds of output TP are released and are recognizable to hairpin DNA (NH2-HP) modified in the electrode surface. After exonuclease III (ExoIII) catalyzing the cleavage recycle, the retained C-rich repeats are converted into stable i-motif configuration when decreasing pH, generating decreased electron transfer impedance (Ret) owing to increased electrostatic attraction to [Fe(CN)6]3−/4−. Thus, the impedimetric biosensor is highly specific and sensitive with a limit of detection of 31 fM. This strategy would be promising to open up a new analytical route for disease diagnosis and bioanalysis.

Agrobacterium tumefaciens causes crown gall disease in plants by transferring a portion of the tumor-inducing plasmid, transfer DNA, into the plant genome. To examine the physiological changes induced by A. tumefaciens in cherry rootstock ‘Gisela 6’, we determined the activity of defense-related enzymes, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO), phenylalanine ammonialyase (PAL), lipoxygenase (LOX), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), and the content of phytohormones, salicylic acid (SA) and jasmonic acid (JA), in cherry plants before and after infection. Furthermore, we assayed the expression of genes encoding these enzymes and SA and JA biosynthesis genes using quantitative real-time PCR, and examined the morphology of the infected tissue surface. Infection with A. tumefaciens increased the activity of POD, SOD, PPO, APX, MDHAR, and GR, and upregulated the expression of the corresponding genes. It also elevated the JA content of cherry plants. No significant difference was noted in CAT and PAL activity between the infected and control groups. In the treatment group, a slight increase in LOX activity was observed at 15 days post-infection (dpi), whereas DHAR activity declined by almost 50% at 10 dpi. The total SA content showed a general upward trend in infected plants but did not show a clear difference compared with the control. Overall, our data suggest that Agrobacterium infection did not elicit a hypersensitive response in ‘Gisela 6’ but altered the expression level of genes involved in defense responses and phytohormone biosynthesis.

Efficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the expression of its type 4 secretion system (T4SS) genes, controlled by the PQ promoter. Transcription from the PQ promoter is tightly regulated, partially to limit cell toxicity caused by overproduction of PrgB, a T4SS adhesin. PrgU plays an important role in regulating this toxicity by decreasing PrgB levels. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. We used a combination of in vivo methods to quantify PrgU effects on prgQ transcripts at both single-cell and population levels. PrgU function requires a specific RNA sequence within an intergenic region (IGR) about 400 bp downstream of PQ. PrgU interaction with the IGR reduces levels of downstream transcripts. Single-cell expression analysis showed that cells expressing prgU decreased transcript levels more rapidly than isogenic prgU-minus cells. PrgU bound RNA in vitro without sequence specificity, suggesting that PrgU requires a specific RNA structure or one or more host factors for selective binding in vivo. PrgU binding to its IGR target might recruit RNase(s) for targeted degradation of downstream transcripts or reduce elongation of nascent transcripts beyond the IGR. IMPORTANCE Bacteria utilize type 4 secretion systems (T4SS) to efficiently transfer DNA between donor and recipient cells, thereby spreading genes encoding antibiotic resistance as well as various virulence factors. Regulation of expression of the T4SS proteins and surface adhesins in Gram-positive bacteria is crucial, as some of these are highly toxic to the cell. The significance of our research lies in identifying the novel mechanism by which PrgU performs its delicate fine-tuning of the expression levels. As prgU orthologs are present in various conjugative plasmids and transposons, our results are likely relevant to understanding of diverse clinically important transfer systems.

Plant biotechnology provides a means for the rapid genetic improvement of crops including the enhancement of complex traits like yield and nutritional quality through the introduction and coordinated expression of multiple genes. GAANTRY (gene assembly in Agrobacterium by nucleic acid transfer using recombinase technology) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid transfer DNA (T-DNA) region. The system provides a simple and efficient method for assembling and stably maintaining large stacked constructs within the GAANTRY ArPORT1 Agrobacterium rhizogenes strain. The assembly process utilizes unidirectional site-specific recombinases in vivo and an alternating bacterial selection scheme to sequentially assemble multiple genes into a single transformation construct. A detailed description of the procedures used for bacterial transformation, selection, counter selection, and genomic PCR validation with the GAANTRY system are presented. The methods described facilitate the efficient assembly and validation of large GAANTRY T-DNA constructs. This powerful, yet simple to use, technology will be a convenient tool for transgene stacking and plant genetic engineering of rice and other crop plants.

Rhizobium rhizogenes exploits horizontal gene transfer as part of its mechanism of pathogenesis. In this respect, genetic material, transfer DNA (T-DNA) from the bacterium, is transferred transiently to the host plant genome. However, plant species within Nicotiana, Linaria and Ipomea genera contain genomic traces of ancient bacterial T-DNA. To determine if ancient bacterial T-DNA is present in uncultivated plants of European genera within Scrophulariaceae, seeds of Linaria, Antirrhinum, Digitalis, and Veronica were analysed for the presence of one of the root oncogenic loci genes; rolC of R. rhizogenes. This study discloses remnants of ancient rolC haplotypes in several species of Linaria, Antirrhinum, Digitalis and Veronica. The distribution of plant species harbouring rolC sequences within each genus was not uniform. In total, 7 of the 16 investigated species were found to be positive for at least one of the rolC haplotypes, where 6 of these are new additions to the group of naturally transformed plants. Sequence alignment showed high interspecies homology of rolC. Five unique rolC haplotypes (rolCa-rolCe) were found in several plant species. The most abundant, rolCa, was identified in all the plant species holding ancient bacterial DNA. Transcripts of rolC were not detected in leaves, which indicates that rolC may not have a function in leaves under non-stress conditions. This study adds Antirrhinum, Digitalis and Veronica to the list of plant genera within Scrophulariaceae which have been subjected to ancient transformation events via horizontal gene transfer from bacteria to plants.

The remarkable ability of Agrobacterium tumefaciens to transfer DNA to plant cells has allowed the generation of important transgenic crops. One challenge of A. tumefaciens-mediated transformation is eliminating the bacteria after plant transformation to prevent detrimental effects to plants and the release of engineered bacteria to the environment. Here we use a reverse genetics approach to identify genes involved in ampicillin resistance with the goal of utilizing these antibiotic-sensitive strains for plant transformations. We show that treating A. tumefaciens C58 with ampicillin led to increased β-lactamase production, a response dependent on the broad-spectrum β-lactamase AmpC and its transcription factor AmpR. Loss of the putative ampD orthologue, atu2113, led to constitutive production of AmpC-dependent β-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpC-dependent β-lactamase activity and its loss elicited ampicillin and carbenicillin sensitivity in the A. tumefaciens C58 and GV3101 strains. Furthermore, GV3101 ΔmltB3 transforms plants with comparable efficiency to wildtype but can be cleared with sub-lethal concentrations of ampicillin. The functional characterization of the genes involved in the inducible ampicillin resistance pathway of A. tumefaciens constitutes a major step forward in efforts to reduce the intrinsic antibiotic resistance of this bacterium.IMPORTANCEAgrobacterium tumefaciens, a significant biotechnological tool for production of transgenic plant lines, is highly resistant to a wide variety of antibiotics, posing challenges for various applications. One challenge is the efficient elimination of A. tumefaciens from transformed plant tissue without using levels of antibiotics that are toxic to the plants. Here, we present the functional characterization of genes involved in β-lactam resistance in A. tumefaciens. Knowledge about proteins that promote or inhibit β-lactam resistance will enable the development of strains to improve the efficiency of Agrobacterium-mediated plant genetic transformations. Effective removal of Agrobacterium from transformed plant tissue has the potential to maximize crop yield and food production, improving the outlook for global food security.

We showed that sepiolite can transfer DNA into mammalian cells, opening alluring avenues for biothechnological and biomedical applications. Importantly, mammalian cells spontaneously internalize sepiolite, facilitating the spontaneous transport and delivery of bound molecules, but increasing the risks of potential toxicity. In addition, the fibrous nature of sepiolite raised health concerns about possible asbestos-like effects, especially because sepiolite can transfer DNA into bacteria through Yoshida effect. In spite that extrapolations from bacteria to mammalian cells could correspond to over-interpretations and the classification by IARC as non-hazardous and non-carcinogenic, we addressed here the response of human cells to interactions with sepiolite. We tested three classical cell responses to stress. We show that mammalian cells respond to sepiolite exposure, inducing the production of reactive oxygen species and the expression of inflammatory cytokines. This shows that cells detect sepiolite contamination and respond. Remarkably, sepiolite exposure did not alter the cell cycle distribution and triggers neither the DNA damage response program nor apoptosis, suggesting that it does not significantly assault the genetic material in mammalian cells. The potential toxicity of chronic versus transient exposure to sepiolite is discussed.

A novel photoelectrochemical (PEC) aptasensor was fabricated for DNA detection based on the coupling of cosensitization and peroxidase-like catalytic activity. Specifically, the surfaces of branched-TiO2 nanorods (B-TiO2 NRs) were modified with Cd2+ and S2+ to obtain B-TiO2 NRs/CdS hybrid structures, which were subsequently used as matrices to immobilize hairpin DNA (hDNA) probes. CdTe/TCPP (TCPP = meso-tetra(4-carboxyphenyl)-porphine) used for signal amplification was labeled on the terminal of the hDNA probe. Without the target DNA (tDNA) presence, the immobilized hDNA probe with CdTe/TCPP possessed a hairpin form and was located near the B-TiO2 NRs/CdS electrode surface, forming a cosensitized structure formation and then generating strong photocurrent with H2O2 as the electron donor. During detection, the specific recognition of tDNA by the sensing hDNA probe triggered the formation of the G-quadruplex/hemin DNAzyme, which effectively catalyzed the decomposition of H2O2. Meanwhile, cosensitization disappeared when the hDNA probe hybridized with tDNA, further reducing the photocurrent. With a double-signal amplification strategy, the sensing platform designed in this work demonstrated a linear detection ability in the 0.5 fM-5 nM range with a detection limit equal to 0.14 fM. Notably, through encoding in the base sequences of the hDNA and marking it, a versatile PEC platform could be structured for the detection of various DNA targets, which could promise applications in point-of-care diagnostic fields.

Efficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the sex pheromone cCF10, which induces the expression of the Type 4 Secretion System (T4SS) genes controlled by the PQ promoter. The pheromone responsive PQ promoter is strictly regulated to prevent overproduction of the prgQ operon, which contains the T4SS, and to limit the cell toxicity caused by overproduction of PrgB, a T4SS adhesin involved in cellular aggregation. PrgU plays an important role in regulating this toxicity by decreasing PrgB production. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. With a combination of lacZ reporter fusion, northern blot, and RNAseq analyses, we provide evidence that PrgU binds a specific RNA sequence within the intergenic region (IGR), ca 400 bp downstream of the PQ promoter. PrgU-IGR binding reduces levels of downstream transcripts, with the strongest decrease seen for prgB messages. Consistent with these findings, we determined that pCF10-carrying cells expressing prgU decreased transcript levels more rapidly than isogenic cells deleted of prgU. Finally, purified PrgU bound RNA in vitro, but without sequence specificity, suggesting that PrgU requires a specific RNA structure or one or more host factors to bind its RNA target in vivo. Together, our results support a working model where PrgU binding to the IGR serves to recruit RNase(s) for targeted degradation of downstream transcripts.ImportanceBacteria utilize Type 4 Secretion Systems (T4SS) to efficiently transfer DNA from donor to recipient cells, thereby spreading genes encoding for antibiotic resistance as well as various virulence factors. The conjugative plasmid pCF10 from Enterococcus faecalis, originally isolated from clinical isolates, serves as a model system for these processes in Gram-positive bacteria. It is very important to strictly regulate the expression of the T4SS proteins for the bacteria, as some of these proteins are highly toxic to the cell. Here, we identify the mechanism by which PrgU performs its delicate fine tuning of the expression levels. As prgU genes are present in various conjugative plasmids and transposons, this provides an important new insight into the bacterial repertoire of regulation mechanisms of these clinically important systems.

SUMMARY Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium‐mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium‐mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas‐mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies., Significance Statement The tissue culture and transformation technology developed herein represents a significant advance in Agrobacterium‐mediated transformation and CRISPR/Cas‐mediated genome editing of an important orphan crop, cowpea (Vigna unguiculata (L.) Walp.). The principles established in this study have the potential to improve the transformation and editing efficiencies not only for cowpea, but also for other legume species, such as soybean (Glycine max) and common bean (Phaseolus vulgaris).

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

In this paper we present a rapid electrochemical enzyme-linked DNA hybridization assay using disposable pencil graphite electrodes (PeGE) to detect target DNA (tDNA) sequences in DNA fragments amplified by polymerase chain reaction. the procedure consists of several short (1-2 min) incubation steps, including adsorption of the tDNA at unpretreated PeGE from denaturing medium, surface blocking with milk proteins, hybridization with a biotinylated oligonucleotide probe and binding of streptavidin-alkaline phosphatase conjugate to the biotin tags. Then the PeGE is transferred into background electrolyte solution containing 1-naphthyl phosphate, which is enzymatically dephosphorylated to give electrochemically oxidizable indicator 1-naphthol. the assay, which can be performed within 7-8 min, offers a perfect discrimination between specific and nonspecific DNA amplicons and easy detection of about similar to 40 femtomoles of tDNA in large excesses of non-complementary DNA. An application on the detection of p53 gene deletion in a human cell culture, featuring a real biologicalmodel, is presented. the designed setup has a potential to be applied as one of simple, fast, robust ultralow cost "do-it-yourself "instruments recently introduced as diagnostic tools for third world countries. (c) 2020 Elsevier B.V. All rights reserved., Czech Science FoundationGrant Agency of the Czech Republic [P206/11/1638]; Czech Academy of SciencesCzech Academy of Sciences [RVO 68081707]; Turkish Scientific and Technological Research Council (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [111 T050], This work has been supported by a grant by the Czech Science Foundation (P206/11/1638 to M.F.) and by the Czech Academy of Sciences (RVO 68081707). A.E acknowledges the grant of international joint project by Turkish Scientific and Technological Research Council (TUBITAK, Project No. 111 T050). the authors thank Dr. Jan Coufal for providing cultured cells for experiments shown in Fig. 6.

Agrobacterium tumefaciens is an important tool in plant biotechnology due to its natural ability to transfer DNA into the genomes of host plants. Genetic manipulations of A. tumefaciens have yielded considerable advances in increasing transformational efficiency in a number of plant species and cultivars. Moreover, there is overwhelming evidence that modulating the expression of various mediators of A. tumefaciens virulence can lead to more successful plant transformation; thus, the application of synthetic biology to enable targeted engineering of the bacterium may enable new opportunities for advancing plant biotechnology. In this review, we highlight engineering targets in both A. tumefaciens and plant hosts that could be exploited more effectively through precision genetic control to generate high-quality transformation events in a wider range of host plants. We then further discuss the current state of A. tumefaciens and plant engineering with regard to plant transformation and describe how future work may incorporate a rigorous synthetic biology approach to tailor strains of A. tumefaciens used in plant transformation.

This work described a sensitive electrochemical sensing method for detection of invA gene sequence of Salmonella by integrating direct signal inducer of silver nanoclusters (AgNCs) and cystine-rich (C-rich) signal probe DNA assembled gold nanoparticls (sDNA-AuNPs). Thiolated capture probe was covalently immobilized onto the glassy carbon electrode, and further hybridized with target DNA sequence (tDNA) and sDNA-AuNPs onto form a sandwich-type structure. The C-rich sDNA in the structure was then used as template for the formation of AgNCs, which can produce the direct voltametric signal during the detection of tDNA. Under optimal conditions, the fabricated biosensor showed good linearity from 1 fM to 0.1 nM with a detection limit of 0.162 fM (S/N = 3). It exhibited good selectivity, stability and reproducibility and can be applied to detect Salmonella effectively by detecting the polymerase chain reaction products of invA gene.

A major limitation of molecular studies in powdery mildew fungi (Erysiphales) is their genetic intractability. This is because they are obligate biotrophs. In these parasites, biotrophy is determined by the presence of haustoria, which are specialized structures of parasitism that play an essential role in the acquisition of nutrients and the deliverance of effectors. Podosphaera xanthii is the main causal agent of cucurbit powdery mildew and a major limitation for crop productivity. In a previous study using P. xanthii conidia, we showed, for the first time, the transformation of powdery mildew fungi by Agrobacterium tumefaciens. In this work, we hypothesized that the haustorium could also act as a natural route for the acquisition of DNA. To test our hypothesis, melon cotyledons were agro-infiltrated with A. tumefaciens that contained diverse transfer DNA (T-DNA) constructs harbouring different marker genes under the control of fungal promoters and, after elimination of the bacterium, the cotyledons were subsequently inoculated with P. xanthii conidia. Our results conclusively demonstrated the transfer of different T-DNAs from A. tumefaciens to P. xanthii, including two fungicide resistance markers (hph and tub2), a reporter gene (gfp) and a translational fusion (cfp-PxEC2). These results were further supported by the co-localization of translational fluorescent fusions of A. tumefaciens VirD2 and P. xanthii Rab5 proteins into small vesicles of haustorial and hyphal cells, suggesting endocytosis as the mechanism for T-DNA uptake, presumably by the haustorium. From our perspective, transformation by growth onto agro-infiltrated tissues (TGAT) is the easiest and most reliable method for the transient transformation of powdery mildew fungi.

Engineering microorganisms to promote human or plant health will require manipulation of robust bacteria that are capable of surviving in harsh, competitive environments. Genetic engineering of undomesticated bacteria can be limited by an inability to transfer DNA into the cell. Here we developed an approach based on the integrative and conjugative element from Bacillus subtilis (ICEBs1) to overcome this problem. A donor strain (XPORT) was built to transfer miniaturized integrative and conjugative elements (mini-ICEBs1) to undomesticated bacteria. The strain was engineered to enable inducible control over conjugation, to integrate delivered DNA into the chromosome of the recipient, to restrict spread of heterologous DNA through separation of the type IV secretion system from the transferred DNA, and to enable simple isolation of engineered bacteria through a D-alanine auxotrophy. Efficient DNA transfer (10-1 to 10-7 conjugation events per donor) is demonstrated using 35 Gram-positive strains isolated from humans (skin and gut) and soil. Mini-ICEBs1 was used to rapidly characterize the performance of an isopropyl-β-D-thiogalactoside (IPTG)-inducible reporter across dozens of strains and to transfer nitrogen fixation to four Bacillus species. Finally, XPORT was introduced to soil to demonstrate DNA transfer under non-ideal conditions.

Collecting sufficient template DNA from a crime scene sample is often challenging, especially with low quantity samples such as touch DNA (tDNA). Traditional DNA collection methods such as double swabbing have limitations, in particular when used on certain substrates which can be found at crime scenes, thus a better collection method is advantageous. Here, the effectiveness of the M-Vac® Wet-Vacuum System is evaluated as a method for DNA recovery on tiles and bricks. It was found that the M-Vac® recovered 75% more DNA than double swabbing on bricks. However, double swabbing collected significantly more DNA than the M-Vac® on tiles. Additionally, it was found that cell-free DNA is lost in the filtration step of M-Vac® collection. In terms of peak height and number of true alleles detected, no significant difference was found between the DNA profiles obtained through M-Vac® collection versus double swabbing of tDNA depositions from 12 volunteers on bricks. The results demonstrate that the M-Vac® has potential for DNA collection from porous surfaces such as bricks, but that alterations to the filter apparatus would be beneficial to increase the amount of genetic material collected for subsequent DNA profiling. These results are anticipated to be a starting point to validate the M-Vac® as a DNA collection device, providing an alternative method when DNA is present on a difficult substrate, or if traditional DNA collection methods have failed.

Sensitive and specific detection of DNA is of great significance for clinical diagnosis. In this paper, an effective cascade signal amplification strategy was introduced into photoelectrochemical (PEC) biosensor for ultrasensitive detection of human T-cell lymphotropic virus type I (HTLV-I) DNA. This proposed signal amplification strategy integrates λ-exonuclease (λ-Exo) aided target recycling with hybridization chain reaction (HCR) and enzyme catalysis. In the presence of target DNA (tDNA) of HTLV-I, the designed hairpin DNA (h1DNA) hybridized with tDNA, subsequently recognized and cleaved by λ-Exo to set free tDNA. With the λ-Exo aided tDNA recycling, an increasing number of DNA fragments (output DNA, oDNA) were released from the digestion of h1DNA. Then, triggered by the hybridization of oDNA with capture DNA (cDNA), numerous biotin-labeled hairpin DNAs (h2DNA and h3DNA) could be loaded onto the photoelectrode via the HCR. Finally, avidin-labeled alkaline phosphatase (avidin-ALP) could be introduced onto the electrode by specific interaction between biotin and avidin. The ALP could catalyze dephosphorylation of phospho-L-ascorbic acid trisodium salt (AAP) to generate an efficient electron donor of ascorbic acid (AA), and thereby greatly increasing the photocurrent signal. By utilizing the proposed cascade signal amplification strategy, the fabricated PEC biosensor exhibited an ultrasensitive and specific detection of HTLV-I DNA down to 11.3 aM, and it also offered an effective strategy to detect other DNAs at ultralow levels.

Acquisition of foreign DNA by natural transformation is an important mechanism of adaptation and evolution in diverse microbial species. Here, we characterize the mechanism of ComM, a broadly conserved AAA+ protein previously implicated in homologous recombination of transforming DNA (tDNA) in naturally competent Gram-negative bacterial species.In vivo, we found that ComM was required for efficient comigration of linked genetic markers inVibrio choleraeandAcinetobacter baylyi, which is consistent with a role in branch migration. Also, ComM was particularly important for integration of tDNA with increased sequence heterology, suggesting that its activity promotes the acquisition of novel DNA sequences.In vitro, we showed that purified ComM binds ssDNA, oligomerizes into a hexameric ring, and has bidirectional helicase and branch migration activity. Based on these data, we propose a model for tDNA integration during natural transformation. This study provides mechanistic insight into the enigmatic steps involved in tDNA integration and uncovers the function of a protein required for this conserved mechanism of horizontal gene transfer.

Noninvasive diagnosis of Helicobacter pylori (H. pylori) infection is very attractive. This study investigated the single strand DNA (ssDNA) acquisition method from H. pylori in dental plaque, and the integration of our previously developed 43-mer H. pylori DNA biosensor with the obtained target ssDNA (tDNA). Dental plaque samples were collected from 34 patients/volunteers, whose gastric H. pylori infection statuses were tested with the 13C urea breath test (UBT). The samples were treated with colony polymerase chain reaction (PCR) to obtain double strand DNA (dsDNA) of 104 basepairs (bp) long. A blocker ssDNA was designed and used in thermal treatment of the dsDNA to release the 104-mer tDNA, which contains the 43-mer DNA sequence in the middle. PCR primers were designed, and the tDNA releasing and detection conditions with the biosensor were optimized. The limit of detection with the biosensor was 12 fM dsDNA. The dental plaque detection results correlated quite well with the UBT results, with a sensitivity of 100%, and specificity of 97%. These results indicate that the residence of H. pylori in dental plaque is highly associated with gastric H. pylori infection, and detection of dental plaque samples with our DNA biosensor is promisingly applicable in noninvasive diagnosis of H. pylori infection.

Here, we demonstrate a powerful method to discriminate DNA mismatches at single-nucleotide resolution from 0 to 5 mismatches (χ0 to χ5) using Kelvin probe force microscopy (KPFM). Using our previously developed method, we quantified the surface potentials (SPs) of individual DNA-capped nanoparticles (DCNPs, ∼100 nm). On each DCNP, DNA hybridization occurs between ∼2200 immobilized probe DNA (pDNA) and target DNA with mismatches (tDNA, ∼80 nM). Thus, each DCNP used in the bioassay (each pDNA-tDNA interaction) corresponds to a single ensemble in which a large number of pDNA-tDNA interactions take place. Moreover, one KPFM image can scan at least dozens of ensembles, which allows statistical analysis (i.e., an ensemble average) of many bioassay cases (ensembles) under the same conditions. We found that as the χn increased from χ0 to χ5 in the tDNA, the average SP of dozens of ensembles (DCNPs) was attenuated owing to fewer hybridization events between the pDNA and the tDNA. Remarkably, the SP attenuation vs. the χn showed an inverse-linear correlation, albeit the equilibrium constant for DNA hybridization exponentially decreased asymptotically as the χn increased. In addition, we observed a cascade reaction at a 100-fold lower concentration of tDNA (∼0.8 nM); the average SP of DCNPs exhibited no significant decrease but rather split into two separate states (no-hybridization vs. full-hybridization). Compared to complementary tDNA (i.e., χ0), the ratio of no-hybridization/full-hybridization within a given set of DCNPs became ∼1.6 times higher in the presence of tDNA with single mismatches (i.e., χ1). The results imply that our method opens new avenues not only in the research on the DNA hybridization mechanism in the presence of DNA mismatches but also in the development of a robust technology for DNA mismatch detection.

Hybrid varieties can provide the boost needed to increase stagnant wheat yields through heterosis. The lack of an efficient hybridization system, which can lower the cost of goods of hybrid seed production, has been a major impediment to commercialization of hybrid wheat varieties. In this review, we discuss the progress made in characterization of nuclear genetic male sterility (NGMS) in wheat and its advantages over two widely referenced hybridization systems, i.e., chemical hybridizing agents (CHAs) and cytoplasmic male sterility (CMS). We have characterized four wheat genes, i.e., Ms1, Ms5, TaMs26 and TaMs45, that sporophytically contribute to male fertility and yield recessive male sterility when mutated. While Ms1 and Ms5 are Triticeae specific genes, analysis of TaMs26 and TaMs45 demonstrated conservation of function across plant species. The main features of each of these genes is discussed with respect to the functional contribution of three sub-genomes and requirements for complementation of their respective mutants. Three seed production systems based on three genes, MS1, TaMS26 and TaMS45, were developed and a proof of concept was demonstrated for each system. The Tams26 and ms1 mutants were maintained through a TDNA cassette in a Seed Production Technology-like system, whereas Tams45 male sterility was maintained through creation of a telosome addition line. These genes represent different options for hybridization systems utilizing NGMS in wheat, which can potentially be utilized for commercial-scale hybrid seed production.

A novel and sensitive Helicobacter pylori assay strategy based on linear isothermal amplification reaction (LIAR) and DNAzymes catalyzed DNA sensor was developed. A 17 nt target DNA (tDNA) specific in the H. pylori genome was selected and used as primer in the LIAR. A template DNA was designed to produce an amplicon with Mg2+-dependent and RNA-cleaving DNAzyme (Md-Dz) activity. The amplicon (i.e. Md-Dz) was then detected with the DNA sensor. The DNA sensor was fabricated simply through immobilization of a hairpin DNA that contains both Md-Dz substrate and G-quadruplex DNAzyme (Gq-Dz), on gold electrodes. The Gq-Dz possesses peroxidase activity, catalyzing the electro-redox of the electrochemical signal probe hydroquinon. The LIAR, Md-Dz catalytic reaction, and DNA sensor fabrication and detection processes were characterized and optimized. The LIAR amplification efficiency is as high as ~5 × 105 fold, and the assay strategy is very sensitive, selective and repetitive. The differential pulse voltammetry (DPV) detection signals linearly correlate to the logarithmic tDNA concentration in the range of 0.1–10,000 fM, with a limit of detection (LOD) of 34 aM. The detection linear range for H. pylori genomic DNA is 2.1–67.2 pg, with an LOD of 1.3 pg. Muted DNA sequences and other pathogenic bacteria did not result in interferences. In addition, the DNA sensor is very stable in storage. More importantly, the DNA sensor and the assay strategy could be universally applicable to detect any biological sample and short DNA sequence of interests.

Rapeseed (Brassica napus L.) is an important edible oil-producing crop worldwide. However, abiotic stresses such as cold in winter can adversely affect plant growth at the seedling stage and subsequently, cause poor seed yield and quality at the mature stage. To understand the underlying molecular mechanisms for cold-stress response in rapeseed, we comprehensively analyzed the profiles of transcripts by using RNA-Seq and proteins by iTRAQ (Isobaric Tag for Relative and Absolute Quantification) in a cold-tolerant (C18) and a cold-sensitive (C6) rapeseed line under cold stress at the four-leaf stage. Based on integrated analysis of transcriptomics and proteomics, 48 differentially expressed genes (DEGs) were found to be corresponded to 17 differentially expressed proteins (DEPs) in C18 during cold stress treatment, whereas 82 DEGs corresponded to 38 DEPs in C6. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that 8 key genes were involved in photosynthesis, followed by 3 in antioxidant enzymes, and 11 in energy metabolism. To verify that these genes function in cold response, Arabidopsis transfer DNA (T-DNA) insertion lines for homologs of the three candidates (nir, cml, and cat) were obtained; the three mutant lines differed significantly in levels of freezing response. The integrated analysis here provides insights into molecular mechanisms of the cold response in rapeseed, and valuable information for functional characterization of cold-related genes in rapeseed.

The development of convenient and efficient strategies without involving any complex nanomaterials or enzymes for signal amplification is of great importance in bioanalytical applications. In this work, we report the use of electrochemically mediated surface-initiated atom transfer radical polymerization (SI-eATRP) as a novel amplification strategy based on the de novo growth of polymers (dnGOPs) for the electrochemical detection of DNA. Specifically, the capture of target DNA (tDNA) by the immobilized peptide nucleic acid (PNA) probes provides a high density of phosphate groups for the subsequent attachment of ATRP initiators onto the electrode surface by means of the phosphate-Zr4+-carboxylate chemistry, followed by the de novo growth of electroactive polymer via the SI-eATRP. De novo growth of long polymeric chains enables the labeling of numerous electroactive probes, which in turn greatly improves the electrochemical response. Moreover, it circumvents the slow kinetics and poor coupling efficiency en...

In this article we report the identification of T-DNA (transfer DNA) insertion sites within two different gene regions in the genome of an Arabidopsis mutant line, SALK_084889. The T-DNA positions are in the 3′ UTR (untranslated region) of DREB2A (Dehydration-responsive element-binding protein 2A) (AT5G05410) and promoter of LOX1 (Lipoxygenase 1) (AT1G55020) as determined by DNA-PCR and sanger sequencing. The expression levels of DREB2A and LOX1 were also analyzed using quantitative realtime PCR (qPCR) in SALK_084889 and wild type Arabidopsis (Col, Columbia). Further, the comparison of drought and heat tolerance between Col and SALK_084889 were conducted by stress treatments. The present data indicate that in SALK_084889, the expression of DREB2A is not downregulated under normal growth conditions but can be affected only in roots under drought treatment, while LOX1 is significantly downregulated in both roots and shoots under all tested conditions. These data are original and have not been published elsewhere. Keywords: Arabidopsis, DREB2A, LOX1, SALK_084889, T-DNA

T-DNA tagging lines are useful for analyzing the functions of genes and regulatory elements. We have previously generated approximately 100,000 insertional mutants in japonica rice (Oryza sativa), using T-DNA vectors carrying the promoter-less GUS reporter gene. In this study, we conducted GUS assays of seedlings from 430 lines in which TDNA was inserted into transcription factor genes. Among the 75 lines that showed GUS signals, nine displayed an endospermpreferential expression pattern; two lines demonstrated GUS signals in both endosperm and roots; 21 lines had GUS expression mainly in leaves; 19 lines showed GUS signal in both leaves and roots; and 24 lines expressed GUS predominantly in the roots. Co-segregation analyses of 49 homozygous lines indicated that the GUS expression patterns observed from 38 lines were due to the T-DNA insertion. We also identified fusion transcripts between tagged genes and the GUS reporter in six lines. Quantitative RT-PCR confirmed that the GUS expression patterns of those tagged lines indeed represent organ- and tissue-preferential expression of the tagged genes. The GUS-tagged transcription factor lines identified here will be useful for functional analysis of these candidates.

Display Omitted The conformational properties of the pores inside the two and four-subunit VirE2 complexes have been explored by the molecular dynamics and normal modes methods.The possible functioning mechanism of the two and four-subunit VirE2 pore complexes has been predicted.Predicted complexes remain stable and do not decompose. The single-stranded transfer DNA from the Ti plasmid of the soil bacteria Agrobacterium nonspecifically integrates into the plant chromosome and is inherited at subsequent cell divisions. How it is transferred across host membranes is unknown, but it is believed that VirE2 proteins form a membrane-spanning pore or channel in a lipid bilayer and possibly mediate the delivery of the single-stranded transfer DNAVirD2VirE2 complex to the plant cell chromosomes. The aim of this work was to perform a computer simulation of VirE2s pore-forming capacity and an evaluation of constructed VirE2 complexes. The oscillating motions of complexes consisting of two and four VirE2 subunits were estimated by the molecular dynamics and normal modes methods. We did not predict any large changes in domain orientation for two and four-subunit VirE2 complexes within simulation times of 1ns. A possible gating mechanism similar to that seen in the ion channels of the complex formed from two VirE2 proteins was proposed, whereas no conformational changes were predicted inside the pore in the complex formed from four VirE2 proteins.

Background: Gene electrotrotransfer describes the use of electric pulses to transfer DNA to cells. Particularly skeletal muscle has potential for systemic secretion of therapeutic proteins. Gene electrotransfer to muscle using the integrin inhibitor plasmid AMEP (Antiangiogenic MEtargidin Peptide) was investigated in a phase I dose escalation study. Primary objective was safety. Material and methods: Patients with metastatic or locally advanced solid tumors, without further standard treatments available, were treated with once-only gene electrotransfer of plasmid AMEP to the femoral muscle. Safety was monitored by adverse events registration, visual analog scale (VAS) after procedure and magnetic resonance imaging (MRI) of treated muscles. Pharmacokinetics of plasmid AMEP in plasma and urine was determined by quantitative polymerase chain reaction. Response was evaluated by positron emission tomography–computed tomography (PET–CT) scans. Results: Seven patients were enrolled and treated at dose levels from 50 to 250 μg of plasmid AMEP, the study was terminated early due to cessation of plasmid production. Minimal systemic toxicity was observed and only transient mild pain was associated with the delivery of the electric pulses. MRI of the treated muscles revealed discrete intramuscular edema 24 h after treatment. The changes in the muscle tissue resolved within 2 weeks after treatment. Peak concentrations of plasmid AMEP was detected only in plasma within the first 24 hours after injection. Protein AMEP could not be detected, which could be due to the limit of detection. No objective responses were seen. Conclusions: Gene electrotransfer of plasmid AMEP was found to be safe and tolerable. No objective responses were observed but other DNA drugs may be tested in the future using this procedure.

The development of next generation sequencing (NGS) technologies has enabled the determination of whole genome sequences in many non-model plant species. However, genome sequencing in sweetpotato (Ipomoea batatas (L.) Lam) is still difficult because of the hexaploid genome structure. Previous studies suggested that a diploid wild relative, I. trifida (H.B.K.) Don., is the most possible ancestor of sweetpotato. Therefore, the genetic and genomic features of I. trifida have been studied as a potential reference for sweetpotato. Meanwhile, several research groups have begun the challenging task of directly sequencing the sweetpotato genome. In this manuscript, we review the recent results and activities of large-scale genome and transcriptome analysis related to genome sequence dissection in sweetpotato under the sections as follows: I. trifida genome and transcript sequencing, genome sequences of I. nil (Japanese morning glory), transcript sequences in sweetpotato, chloroplast sequences, transposable elements and transfer DNA. The recent international activities of de novo whole genome sequencing in sweetpotato are also described. The large-scale publically available genome and transcript sequence resources and the international genome sequencing streams are expected to promote the genome sequence dissection in sweetpotato.

Bacteria undergo horizontal gene transfer via various mechanisms. We recently reported that cell-to-cell transfer of nonconjugative plasmids occurs between strains of Escherichia coli in co-cultures, and that a specific strain (CAG18439) causes frequent plasmid transfer involving a DNase-sensitive mechanism, which we termed "cell-to-cell transformation". Here we found that CAG18439 is a type of P1 bacteriophage lysogen that continuously releases phages. We tested the ability of P1vir bacteriophage to induce horizontal plasmid transfer and demonstrated that such a horizontal plasmid transfer was caused by adding culture supernatants of P1vir-infected cells harboring plasmids to other plasmid-free cells. This plasmid transfer system also reproduced the major features of plasmid transfer involving CAG18439, suggesting that P1vir-induced plasmid transfer is equivalent or very similar to plasmid transfer involving CAG18439. We further revealed that approximately two-thirds of the P1vir-induced plasmid transfer was DNase-sensitive, but that complete abolition of plasmid transfer was observed when proteins were denatured or removed, despite the presence or absence of DNase. Therefore, we concluded that P1vir-induced plasmid transfer is largely due to the occurrence of cell-to-cell transformation, which involves the assistance of some proteinaceous factor, and partly due to the occurrence of plasmid transduction, which is mediated by phage virions. This is the first demonstration of the P1-phage-induced cell-to-cell transformation.

Agrobacterium tumefaciens is a rhizosphere bacterium that can infect wound sites on plants. The bacterium transfers a segment of DNA (T-DNA) from the Ti plasmid to the plant host cell via a type IV secretion system where the DNA becomes integrated into the host cell chromosomes. The expression of T-DNA in the plant results in tumor formation. Although the binding of the bacteria to plant surfaces has been studied previously, there is little work on possible interactions of the bacteria with the plant cell wall. Seven of the 48 genes encoding putative glycoside hydrolases (Atu2295, Atu2371, Atu3104, Atu3129, Atu4560, Atu4561, and Atu4665) in the genome of A. tumefaciens C58 were found to play a role in virulence on tomato and Bryophyllum daigremontiana. Two of these genes (pglA and pglB; Atu3129 and Atu4560) encode enzymes capable of digesting polygalacturonic acid and, thus, may play a role in the digestion of pectin. One gene (arfA; Atu3104) encodes an arabinosylfuranosidase, which could remove arabinose from the ends of polysaccharide chains. Two genes (bglA and bglB; Atu2295 and Atu4561) encode proteins with β-glycosidase activity and could digest a variety of plant cell wall oligosaccharides and polysaccharides. One gene (xynA; Atu2371) encodes a putative xylanase, which may play a role in the digestion of xylan. Another gene (melA; Atu4665) encodes a protein with α-galactosidase activity and may be involved in the breakdown of arabinogalactans. Limited digestion of the plant cell wall by A. tumefaciens may be involved in tumor formation on tomato and B. daigremontiana. IMPORTANCEA. tumefaciens is used in the construction of genetically engineered plants, as it is able to transfer DNA to plant hosts. Knowledge of the mechanisms of DNA transfer and the genes required will aid in the understanding of this process. Manipulation of glycoside hydrolases may increase transformation and widen the host range of the bacterium. A. tumefaciens also causes disease (crown gall tumors) on a variety of plants, including stone fruit trees, grapes, and grafted ornamentals such as roses. It is possible that compounds that inhibit glycoside hydrolases could be used to control crown gall disease caused by A. tumefaciens.

Insertional mutagenesis is an indispensable tool for engendering a mutant population using exogenous DNA as the mutagen. The advancement in the next-generation sequencing platform has allowed for faster screening and analysis of generated mutated populations. Rice is a major staple crop for more than half of the world’s population; however, the functions of most of the genes in its genome are yet to be analyzed. Various mutant populations represent extremely valuable resources in order to achieve this goal. Here, we have reviewed different insertional mutagenesis approaches that have been used in rice, and have discussed their principles, strengths, and limitations. Comparisons between transfer DNA (T-DNA), transposons, and entrapment tagging approaches have highlighted their utilization in functional genomics studies in rice. We have also summarised different forward and reverse genetics approaches used for screening of insertional mutant populations. Furthermore, we have compiled information from several efforts made using insertional mutagenesis approaches in rice. The information presented here would serve as a database for rice insertional mutagenesis populations. We have also included various examples which illustrate how these populations have been useful for rice functional genomics studies. The information provided here will be very helpful for future functional genomics studies in rice aimed at its genetic improvement.