Summary
As a key virulence strategy to cause bacterial leaf blight, Xanthomonas oryzae pv. oryzae (Xoo) injects into the plant cell DNA‐binding proteins called transcription activator‐like effectors ...(TALEs) that bind to effector‐binding elements (EBEs) in a sequence‐specific manner, resulting in host gene induction. TALEs AvrXa7, PthXo3, TalC and Tal5, found in geographically distant Xoo strains, all target OsSWEET14, thus considered as a pivotal TALE target acting as major susceptibility factor during rice–Xoo interactions. Here, we report the generation of an allele library of the OsSWEET14 promoter through stable expression of TALE‐nuclease (TALEN) constructs in rice. The susceptibility level of lines carrying mutations in AvrXa7, Tal5 or TalC EBEs was assessed. Plants edited in AvrXa7 or Tal5 EBEs were resistant to bacterial strains relying on the corresponding TALE. Surprisingly, although indels within TalC EBE prevented OsSWEET14 induction in response to BAI3 wild‐type bacteria relying on TalC, loss of TalC responsiveness failed to confer resistance to this strain. The TalC EBE mutant line was, however, resistant to a strain expressing an artificial SWEET14‐inducing TALE whose EBE was also edited in this line. This work offers the first set of alleles edited in TalC EBE and uncovers a distinct, broader range of activities for TalC compared to AvrXa7 or Tal5. We propose the existence of additional targets for TalC beyond SWEET14, suggesting that TALE‐mediated plant susceptibility may result from induction of several, genetically redundant, host susceptibility genes by a single effector.
As natural dominant pigments, carotenoids and their derivatives not only contribute to fruit color and flavor quality but are regarded as phytochemicals beneficial to human health because of various ...bioactivities. Tomato is one of the most important vegetables as well as a main dietary source of carotenoids. So, it's of great importance to generate carotenoid-biofortified tomatoes. The carotenoid biosynthesis pathway is a network co-regulated by multiple enzymes and regulatory genes. Here, we assembled four binary constructs containing different combinations of four endogenous carotenoids metabolic-related genes, including SlORHis, SlDXS, SlPSY, and SlBHY by using a high efficiency multi-transgene stacking system and a series of fruit-specific promotors. Transgenic lines overexpression SlORHis alone, three genes (SlORHis/SlDXS/SlPSY), two genes (SlORHis/SlBHY), and all these four genes (SlORHis/SlDXS/SlPSY/SlBHY) were enriched with carotenoids to varying degrees. Notably, overexpressing SlORHis alone showed comparable effects with simultaneous overexpression of the key regulatory enzyme coding genes SlDXS, SlPSY, and SlORHis in promoting carotenoid accumulation. Downstream carotenoid derivatives zeaxanthin and violaxanthin were detected only in lines containing SlBHY. In addition, the sugar content and total antioxidant capacity of these carotenoids-enhanced tomatoes was also increased. These data provided useful information for the future developing of biofortified tomatoes with different carotenoid profiles, and confirmed a promising system for generation of nutrients biofortified tomatoes by multiple engineering genes stacking strategy.
•A multi-transgene stacking strategy was used to produce carotenoid biofortified tomatoes.•All the transgenic lines were enriched with carotenoids to varying degrees.•Zeaxanthin was enriched in the fruit of transgenic plants overexpressing SlBHY.•Carotenoid-enhanced tomatoes had higher sugar content and total antioxidant capacity.
AaPIF3, a bHLH transcription factor phylogenetically associated with Arabidopsis PIF3, positively regulates artemisinin biosynthesis in Artemisia annua. Overexpression of AaPIF3 significantly ...promotes the production of artemisinin and its related metabolites.
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•AaPIF3 is a bHLH transcription factor that is phylogenetically related with Arabidopsis PIF3.•AaPIF3 is able to transactivate artemisinin biosynthesis genes, according to dual-luciferase assays.•Overexpression of AaPIF3 markedly increased the production of artemisinin in Artemisia annua.
Artemisia annua is a Chinese traditional herbal plant that produces artemisinin, the potent anti-malarial drug. It is a common goal for the artemisinin industry to develop plants of A. annua with high yields of artemisinin. In this study, a bHLH transcription factor of A. annua (AaPIF3) was characterized and used to promote the production of artemisinin. AaPIF3 was highly expressed in flower buds and glandular secretory trichomes and its coding protein was localized to the nucleus. Dual-luciferase assays indicated that AaPIF3 was able to significantly enhance the promoter activities of artemisinin biosynthesis genes, including ADS, CYP71AV1, DBR2, and ALDH1, suggesting that AaPIF3 positively regulated artemisinin biosynthesis. Overexpression of AaPIF3 markedly upregulated artemisinin biosynthesis genes at the transcriptional level, and consequently the production of artemisinin was significantly promoted in transgenic A. annua plants. On the contrary, suppression of AaPIF3 led to significantly decreased transcript levels of artemisinin biosynthesis genes, with overall production of artemisinin significantly reduced. In summary, AaPIF3 plays a positive role in regulating artemisinin biosynthesis and transgenic plants of A. annua with high yields of artemisinin have been successfully developed through overexpression of AaPIF3.
The relatively recent discovery of CRISPR/Cas has led to a revolution in our ability to efficiently manipulate the genome of eukaryotic cells. We describe here a protocol that employs CRISPR ...technology to precisely knock-in a PET imaging reporter transgene into a specific genetic locus of interest. Resulting transcription of the targeted reporter will more accurately mimic physiologic expression of the endogenous allele than conventional approaches, and so this method has the potential to become an efficient way to generate a new generation of "gold-standard" reporter transgenes. We break down the protocol into three experimental stages: how to identify the genomic location that the reporter transgene will be inserted, how to practically insert the reporter transgene into the genome, and how to screen resultant clones for the correct targeted event.
Transgene integration typically takes place in an easy-to-transform laboratory variety before the transformation event is introgressed through backcrosses to elite cultivars. As new traits are added ...to existing transgenic lines, site-specific integration can stack new transgenes into a previously created transgenic locus.
site-specific integration minimizes the number of segregating loci to assemble into a breeding line, but cannot break genetic linkage between the transgenic locus and nearby undesirable traits. In this study, we describe an additional feature of an
gene-stacking scheme, in which the Cre (control of recombination) recombinase not only deletes transgenic DNA no longer needed after transformation but also mediates recombination between homologous or non-homologous chromosomes. Although the target site must first be introgressed through conventional breeding, subsequent transgenes inserted into the same locus would be able to use Cre-mediated translocation to expedite a linkage drag-free introgression to field cultivars.
To feed 10 billion people in 30 years from now will heavily depend on new higher yield, yet safe, biotech crop varieties, which are developed using biotechnologies, particularly genetic ...transformation and genome editing. A clean transgenic plant (CTP) contains only the defined transgene, whilst a clean edited plant (CEP) is transgene-free. Currently, it is difficult and time-consuming to identify and locate transgenes within plant genomes due to the complex nature of transfer DNA (T-DNA) or plasmid backbone integration, which has led to a major obstacle in mass scale production of clean transgenic/edited plants (CTREPs). Here we have built a web-based portal, CTREP-finder, for fast detection, characterization and visualization of foreign DNA fragments in plants, based on next-generation whole genome sequencing data. We invented a novel bioinformatics strategy to handle three scenarios of transgene integration and successfully identified CTREPs by testing 132 samples. Furthermore, we compared the CTREP-finder with three public programs, FED, transgeneR and TDNAscan, and it showed that the CTREP-finder outperforms those programs particularly on the localization of transgene integration. The use of CTREP-finder needs little requirement for bioinformatics expertise. Therefore, it is a user-friendly web-based portal that could be used by plant breeders and regulatory agencies for the rapid selection/detection of CTREPs for crop breeding and production.
•CTREP-finder is designed for fast detection, characterization and visualization of clean transgenic or genome-edited plants.•It’s more efficient and sensitive than existing tools, especially in localization and visualization of transgene insertion.•It’s valuable for plant breeders and regulators with limited bioinformatics training in research, development and regulation.
Summary
Biotechnology provides a means for the rapid genetic improvement of plants. Although single genes have been important in engineering herbicide and pest tolerance traits in crops, future ...improvements of complex traits like yield and nutritional quality will likely require the introduction of multiple genes. This research reports a system (GAANTRY; Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) for the flexible, in vivo stacking of multiple genes within an Agrobacterium virulence plasmid Transfer‐DNA (T‐DNA). The GAANTRY system utilizes in vivo transient expression of unidirectional site‐specific recombinases and an alternating selection scheme to sequentially assemble multiple genes into a single transformation construct. To demonstrate GAANTRY's capabilities, 10 cargo sequences were sequentially stacked together to produce a 28.5‐kbp T‐DNA, which was used to generate hundreds of transgenic events. Approximately 90% of the events identified using a dual antibiotic selection screen exhibited all of the introduced traits. A total of 68% of the tested lines carried a single copy of the selection marker transgene located near the T‐DNA left border, and only 8% contained sequence from outside the T‐DNA. The GAANTRY system can be modified to easily accommodate any method of DNA assembly and generate high‐quality transgenic plants, making it a powerful, yet simple to use tool for plant genetic engineering.
Significance Statement
A highly efficient, inexpensive and easy to use system for assembling large T‐DNAs (transfer‐DNAs) within the Agrobacterium virulence plasmid was developed and shown to frequently generate high quality transgenic plants.
Gene Therapy in Hemophilia: Recent Advances Rodríguez-Merchán, E. Carlos; De Pablo-Moreno, Juan Andres; Liras, Antonio
International journal of molecular sciences,
07/2021, Letnik:
22, Številka:
14
Journal Article
Recenzirano
Odprti dostop
Hemophilia is a monogenic mutational disease affecting coagulation factor VIII or factor IX genes. The palliative treatment of choice is based on the use of safe and effective recombinant clotting ...factors. Advanced therapies will be curative, ensuring stable and durable concentrations of the defective circulating factor. Results have so far been encouraging in terms of levels and times of expression using mainly adeno-associated vectors. However, these therapies are associated with immunogenicity and hepatotoxicity. Optimizing the vector serotypes and the transgene (variants) will boost clotting efficacy, thus increasing the viability of these protocols. It is essential that both physicians and patients be informed about the potential benefits and risks of the new therapies, and a register of gene therapy patients be kept with information of the efficacy and long-term adverse events associated with the treatments administered. In the context of hemophilia, gene therapy may result in (particularly indirect) cost savings and in a more equitable allocation of treatments. In the case of hemophilia A, further research is needed into how to effectively package the large factor VIII gene into the vector; and in the case of hemophilia B, the priority should be to optimize both the vector serotype, reducing its immunogenicity and hepatotoxicity, and the transgene, boosting its clotting efficacy so as to minimize the amount of vector administered and decrease the incidence of adverse events without compromising the efficacy of the protein expressed.
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