Explore the vast range of titles available.

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.

Neomycin phosphotransferase II ( nptII ) is a selectable marker gene that is commonly used in plant molecular genetics and crop improvement, helping researchers to identify and select transgenically modified plants. The NPTII enzyme binds to and phosphorylates the aminoglycoside family of antibiotics, which are known translation inhibitors. Once the aminoglycoside is phosphorylated it is unable to bind to the ribosome and can no longer disrupt translation. Currently, the most widely used selection agent for screening NPTII expressing seedlings is kanamycin. Because the nptII transgene is frequently silenced epigenetically, kanamycin can be too toxic to seedlings that weakly express NPTII, leading to false negatives and making it harder to accurately identify transgenic plants. In this study we investigate the related aminoglycoside, paromomycin, as an alternative, non-lethal, selection agent to kanamycin across a series of transgenic Arabidopsis lines with varying NPTII expression. We investigated phenotypes of transgenic seedlings during and after antibiotic exposure. Seedling pigmentation and size are useful phenotypes for selecting seedlings with low nptII expression. Additionally, monitoring the photosynthetic efficiency and flowering time can help reduce the risk of false-positive results when treating seedlings with paromomycin.

Recent research has shown that plants can uptake long dsRNAs and dsRNA-derived siRNAs that target important genes of infecting fungi or viruses when applied on the surface of plant leaves. The external RNAs were capable of local and systemic movement inducing plant resistance against the pathogens. Few studies have been made for plant gene regulation by foliar application of RNAs. In this study, several types of ssRNA and siRNA duplexes targeting the neomycin phosphotransferase II (NPTII) transgene were in vitro-synthesized and externally applied to the leaf surface of 4-week-old transgenic Arabidopsis thaliana plants. External application of the synthetic NPTII-encoding siRNAs down-regulated NPTII transcript levels in transgenic A. thaliana 1 and 7 days post-treatment with a higher and more consistent effect being observed for siRNAs methylated at 3′ ends. We also analyzed the effects of external NPTII-encoding dsRNA precursors and a dsRNA-derived heterogenous siRNA mix. Digestion of the NPTII-dsRNA to the heterogeneous siRNAs did not improve efficiency of the transgene suppression effect.Key Points• Foliar application of siRNAs down-regulated a commonly used transgene in Arabidopsis.• A more consistent effect was observed for methylated siRNAs.• The findings are important for development of plant gene regulation approaches.

Efficient selection of transgenic plants is essential in plant biotechnology, and development of effective selectable marker systems plays a crucial role, especially as multiple rounds of transformation and gene stacking may require multiple selectable markers. The present study aims to develop and evaluate an enhanced spectinomycin resistance construct to provide robust selection for transformation events. The new construct was compared in multiple plant species to the standard spectinomycin selectable marker gene currently used for soybean transformation and to the neomycin phosphotransferase ( nptII ) gene that confers kanamycin resistance. In Arabidopsis thaliana , potato, and citrus, the transformation efficiency provided by the enhanced spectinomycin marker was approximately equal to that of kanamycin and considerably better than that observed for the standard spectinomycin marker. In soybean, the enhanced spectinomycin resistance marker preformed as well as the standard spectinomycin gene. These results demonstrate that the enhanced spectinomycin construct provides strong resistance, comparable to kanamycin in multiple species, offering an alternative tool for transgenic plant selection.

Aminoglycoside-modifying enzymes (AMEs) play an essential role in bacterial resistance to aminoglycoside antimicrobials. With the development of sequencing techniques, more bacterial genomes have been sequenced, which has aided in the discovery of an increasing number of novel resistance mechanisms. The bacterial species was identified by 16S rRNA gene homology and average nucleotide identity (ANI) analyses. The minimum inhibitory concentration (MIC) of each antimicrobial was determined by the agar dilution method. The protein was expressed with the pCold I vector in BL21, and enzyme kinetic parameters were examined. The whole-genome sequence of the bacterium was obtained via the Illumina and PacBio sequencing platforms. Reconstruction of the phylogenetic tree, identification of conserved functional residues, and gene context analysis were performed using the corresponding bioinformatic techniques. A novel aminoglycoside resistance gene, designated , which confers resistance to ribostamycin, kanamycin, sisomicin and paromomycin, was identified in the chromosome of the animal bacterium DW61, which exhibited a multidrug resistance phenotype. APH(3')-Ie showed the highest amino acid identity of 74.90% with the functionally characterized enzyme APH(3')-Ia. Enzyme kinetics analysis demonstrated that it had phosphorylation activity toward four aminoglycoside substrates, exhibiting the highest affinity ( , 4.22 ± 0.88 µM) and the highest catalytic efficiency [ / , (32.27 ± 8.14) × 10 ] for ribomycin. Similar to the other APH(3') proteins, APH(3')-Ie contained all the conserved functional sites of the APH family. The homologous genes were present in isolates from different sources, including some of clinical significance. In this work, a novel chromosomal aminoglycoside resistance gene, designated , conferring resistance to aminoglycoside antimicrobials, was identified in a rabbit isolate DW61. The elucidation of the novel resistance mechanism will aid in the effective treatment of infections caused by pathogens carrying such resistance genes.

Key messageTwo intercistronic regions were identified as functional intercistronic expression elements (IEE) for the simultaneous expression of aphA-6 and gfp in a synthetic operon in the chloroplast of C. reinhardtii.Chlamydomonas reinhardtii, a biflagellate photosynthetic microalga, has been widely used in basic and applied science. Already three decades ago, Chlamydomonas had its chloroplast genome transformed and to this day constitutes the only alga routinely used in transplastomic technology. Despite the fact that over a 100 foreign genes have been expressed from the chloroplast genome, little has been done to address the challenge of expressing multiple genes in the form of operons, a development that is needed and crucial to push forward metabolic engineering and synthetic biology in this organism. Here, we studied five intercistronic regions and investigated if they can be used as intercistronic expression elements (IEE) in synthetic operons to drive the expression of foreign genes in the chloroplast of C. reinhardtii. The intercistronic regions were those from the psbB-psbT, psbN-psbH, psaC-petL, petL-trnN and tscA-chlN chloroplast operons, and the foreign genes were the aminoglycoside 3′-phosphotransferase (aphA-6), which confers resistance to kanamycin, and the green fluorescent protein gene (gfp). While all the intercistronic regions yielded lines that were resistant to kanamycin, only two (obtained with intercistronic regions from psbN-psbH and tscA-chlN) were identified as functional IEEs, yielding lines in which the second cistron (gfp) was translated and generated GFP. The IEEs we have identified could be useful for the stacking of genes for metabolic engineering or synthetic biology circuits in the chloroplast of C. reinhardtii.

Ulva prolifera is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of U. prolifera cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in U. prolifera based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene ( aph7” ). Integration of aph7” in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of aph7” . The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of aph7” cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase ( UpAPT ). APT gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance U. prolifera as a genetic platform, enabling functional research to elucidate Ulva biology, and to bring forth biotechnological utilization of algal resources.

DNA methylation and repressive histone Histone3 Lysine9 (H3K9) dimethylation correlate with chromatin silencing in plants and mammals. To identify factors required for DNA methylation and H3K9 dimethylation, we screened for suppressors of the repressor of silencing1 (ros1) mutation, which causes silencing of the expression of the RD29A (RESPONSE TO DESSICATION 29A) promoter-driven luciferase transgene (RD29A-LUC) and the 35S promoter-driven NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) transgene (35S-NPTII). We identified the folylpolyglutamate synthetase FPGS1 and the known factor DECREASED DNA METHYLATION1 (DDM1). The fpgs1 and ddm1 mutations release the silencing of both RD29A-LUC and 35S-NPTII. Genome-wide analysis indicated that the fpgs1 mutation reduces DNA methylation and releases chromatin silencing at a genome-wide scale. The effect of fpgs1 on chromatin silencing is correlated with reduced levels of DNA methylation and H3K9 dimethylation. Supplementation of fpgs1 mutants with 5-formyltetrahydrofolate, a stable form of folate, rescues the defects in DNA methylation, histone H3K9 dimethylation, and chromatin silencing. The competitive inhibitor of methyltransferases, S-adenosylhomocysteine, is markedly upregulated in fpgs1, by which fpgs1 reduces S-adenosylmethionine accessibility to methyltransferases and accordingly affects DNA and histone methylation. These results suggest that FPGS1-mediated folate polyglutamylation is required for DNA methylation and H3K9 dimethylation through its function in one-carbon metabolism. Our study makes an important contribution to understanding the complex interplay among metabolism, development, and epigenetic regulation.

ObjectivesIn the plant transformation process, marker genes play a vital role in identifying transformed cells from non-transformed cells. However, once transgenic plants have been obtained, the presence of marker genes may provoke public concern about environmental or biosafety issues. In our previous study, a double T-DNA vector system has been developed to obtain marker-free transgenic plants, but the T-DNA left border (LB) and right border (RB) of the vector showed an RB–LB–RB–LB pattern and led to high linkage integration between the selectable marker gene (SMG) and the gene of interest (GOI). To improve this double T-DNA vector system, we inverted the first T-DNA direction such that a LB–RB–RB–LB pattern resulted to avoid transcriptional read-through at the LB and the subsequent linkage transfer of the SMG and GOI.ResultsWe separately inserted the green fluorescent protein (GFP) gene as the GOI and the neomycin phosphotransferase II (NPTII) gene as the SMG in both optimized and original vectors and carried out Agrobacterium-mediated tobacco transformation. Statistical analysis revealed that the linkage frequency was 25.6% in T0 plants transformed with the optimized vector, which is a 42.1% decrease compared with that of the original vector (44.2%). The frequency of obtaining marker-free transgenic plants was 66.7% in T1 plants transformed with the optimized vector, showing a 33.4% increase compared with that of the original vector (50.0%).ConclusionOur results demonstrate that the optimized double T-DNA binary vector system is a more effective, economical and time-saving approach for obtaining marker-free transgenic plants.

ABSTRACT Multidrug-resistant (MDR) Klebsiella pneumoniae, particularly the lineages resistant to carbapenems and aminoglycosides, is an escalating global public health threat across human, animal, and environmental reservoirs. We examined phenotypic and genetic features of MDR K. pneumoniae isolates. A total of 70 K. pneumoniae strains were collected from clinical (n=55), environmental (n=7), and animal (n=8) sources. To better understand the evolutionary relationship between these isolates, a phylogenetic analysis was performed alongside 35 publicly available K. pneumoniae genomes from NCBI and Pathogenwatch. Whole-genome sequencing (WGS) revealed that 43 isolates carried the blaKPC gene, including blaKPC-2 and blaKPC-3 variants, with different susceptibility profiles to aminoglycosides. Among all isolates, 84% (n = 59/70) were resistant to amikacin and 53% (n = 37/70) were resistant to gentamicin. Aminoglycoside resistance was primarily associated with aminoglycoside-modifying enzymes, including aph(3’)-Ia (52%), aac(3)-IIa/aadA2 (49%), and aac(6’)-Ib-cr (37%). Additionally, 16S rRNA methyltransferases rmtB and rmtG were detected in 14% of isolates and were associated with high-level amikacin MICs. Overall, 81% of strains were non-susceptible to at least one aminoglycoside, underscoring the clinical importance of these determinants. Phylogenetic analysis based on WGS data showed two main clusters (A and B), and the multilocus sequence type ST11 predominated among Brazilian isolates. Our findings showed a heterogeneous distribution of sequence type profiles across the two clusters and a close relationship between K. pneumoniae strains from human, animal, and environmental sources, highlighting the need for integrated One Health surveillance.