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Biology subjects

Vu, T. V.

Publications and source records attributed to Vu, T. V..

8 recordsLinked to original sources

A novel GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE 13 is involved in the Phosphate starvation-induced lipid remodeling in rice

Phosphorus (P) is one of the most vital macronutrient determinants in plant development and productivity. However, the bioavailability of inorganic phosphate (Pi), the only form that plants can assimilate, is limited in the soil, thus significantly affecting plant development. Plants have adopted various specialized strategies to modify their morphological, physiological, and biochemical properties for better adaptation to Pi deficiency conditions. GLYCEROPHOSPHODIESTER PHOSPHODIESTERASES (GDPDs), generally known as phospholipid remodeling proteins, have been suggested to play essential roles in maintaining phosphate homeostasis. The previous genome-wide association studies (GWAS) in a Vietnamese rice collection led to the discovery of a robust QTL named qRST9.14 associating with the phosphate adaption in rice, in which OsGDPD13 is located within this locus. Interestingly, we discovered an absence of OsGDPD13 from the Indica reference genome. A subcellular localization study showed that the GDPD13 protein localizes to the plasma membrane, cytoplasmic speckles, and plasmodesmata. The osgdpd13 knockout mutant was generated in the Japonica cv Kitaake background to characterize its function. Phenotypic analysis indicated that mutation of OsGDPD13 significantly lowered crown root number and reduced the plant height under Pi deficient condition. Moreover, osgdpd13 lines reduce the capability to degrade total phospholipids compared to the wild type under Pi starvation conditions. This finding suggests the involvement of the OsGDPD13 gene in rice growth and the Phosphate starvation-induced lipid remodeling under P deficiency. This work will provide valuable information for developing crop plants with higher Phosphate use efficiency.

plant biology↗

Redirecting DNA repair for efficient CRISPR-Cas-based gene targeting in tomato

The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical nonhomologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented various approaches to shift the repair pathway preference toward HR by using a dominant-negative KU80 mutant protein (KUDN) to disrupt the initiation of cNHEJ and enhance DSB end resection through nucleases. Our results show from 1.71- to 3.55-fold improvement of the GT efficiency at the callus stage and a more remarkable, up to 9.84-fold, increase in GT efficiency at two specific tomato loci, SlHKT1;2 and SlEPSPS1, when we screened transformants obtained from the KUDN-mediated cNHEJ suppression approach. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated GT at an additional locus, SlCAB13. These findings provide a promising method for more efficient and precise plant breeding in the future.

plant biology↗

Breakthrough in Dicot Prime Editing: Enabling Heritable Desired Edits in Tomato and Arabidopsis

Prime editing (PE) enables almost all types of precise genome editing in animals and plants. It has been successfully adapted to edit several plants at variable efficiency and versatility. However, this technique is inefficient for dicots for unknown reasons. Here, by employing novel combinations of PE components, including an RNA chaperone and modified epegRNAs driven by a PolII-PolIII composite promoter and a viral replicon system, we obtained up to 9.7% of the desired PE efficiency at the callus stage assessed by targeted deep sequencing. Subsequently, we identified that up to 38.2% of transformants contained desired PE alleles in tomatoes and Arabidopsis, marking the first successful heritable PE transmission in dicots. Our PE tools also showed high accuracy, specificity, and multiplexing capability, which unlocked the potential for practical PE applications in dicots, paving the way for transformative advancements in plant sciences.

synthetic biology↗

Generation of Parthenocarpy tomato on multiple commercial cultivars utilizing CRIPR/Cas9 system

Tomato (Solanum lycopersicum L.) is one of the most important crops in the world for its fruit production. Advances in cutting-edge techniques have enabled the development of numerous critical traits related to the quality and quantity of tomatoes. Genetic engineering techniques, such as gene transformation and gene editing, have emerged as powerful tools for generating new plant varieties with superior traits. In this study, we induced parthenocarpic traits in a population of elite tomato (ET) lines. At first, the adaptability of ET lines to genetic transformation was evaluated to identify the best-performing lines by transforming the SlANT1 gene overexpression cassette and then later used to produce the SlIAA9 knockout lines using the CRISPR/Cas9 system. ET5 and ET8 emerged as excellent materials for these techniques and showed higher efficiency. Typical phenotypes of knockout sliaa9 were clearly visible in G0 and G1 plants, in which simple leaves and parthenocarpic fruits were observed. The high efficiency of the CRISPR/Cas9 system in developing new tomato varieties with desired traits in a short period was demonstrated by generating T-DNA-free homozygous sliaa9 knockout plants in the G1 generation. Additionally, a simple artificial fertilization method was successfully applied to recover seed production from parthenocarpic plants, securing the use of these varieties as breeding materials.

plant biology↗

Characterization of yield and fruit quality parameters of Vietnamese elite tomato lines generated through phenotypic selection and conventional breeding methods

Tomato (Solanum lycopersicum L.) is the second most important vegetable crop after potatoes, and global demands have been steadily increasing in recent years. Conventional breeding has been applied to breed and domesticate tomato varieties to meet the need for higher yield or superior agronomical traits that allow to sustain under different climatic conditions. In the current study, we applied bulk population breeding by crossing eight tomato accessions procured from the Asian Vegetable Research and Development Center (AVRDC) with three heat-resistant tomato inbred lines from Vietnam and generated ten elite tomato (ET) lines in the F8 generation. The individual F8 lines exhibited robust vigor and adaptability to Vietnamese climate conditions. Among the ten lines, ET1 and ET3 displayed indeterminate growth. ET2 showed semi-determinate, while all the other lines had determinate growth. The different ET lines showed distinctive superior agronomical traits, including early maturing (ET4, ET7, and ET10), highly efficient fruit set (ET1), higher yield (ET1, ET8, ET10), jointless pedicels (ET2), and partial parthenocarpy (ET9). Molecular analysis revealed that the ET3 line consisted of Ty-1 and Ty-3 loci that positively contribute to Tomato yellow leaf curl virus (TYCLV) resistance in tomato plants. The elite tomato lines developed in this study would contribute significantly to the Vietnamese and Asian gene pool for improved tomato production and may be a valuable resource for various breeding goals.

plant biology↗

CRISPR/Cas-based precision gene replacement in plants via homologous recombination-independent approaches

Precise gene or allele replacement is a desirable technology, but implementing it in plants remains challenging. CRISPR-Cas-based approaches, such as gene targeting (GT) and prime editing (PE), have opened up new possibilities for precise gene replacement in plants. However, their editing size and efficiency still need improvement. Recently, strategies using canonical nonhomologous end-joining (cNHEJ) and microhomology-mediated end joining (MMEJ) have been considered promising alternatives for precise gene replacement in yeast and mammals. However, these approaches have not been extensively explored and applied to plants. Here, we proposed and tested a CRISPR-Cas-based tool, termed PREMJ (precision gene replacement via microhomology-mediated end joining), for precision gene replacement in plants. The PREMJ strategy employing 20-bp microhomology MMEJ donors ([~]100 bp lengths) and a canonical nonhomologous end-joining (cNHEJ) inhibitor, NU7441, produced high targeted gene replacement efficiencies, up to 1.60 {+/-} 0.14, 4.47 {+/-} 1.98, and 8.98 {+/-} 4.73 % in protoplasts of tomato, lettuce, and cabbage, respectively. Our data also revealed the critical impacts of the microhomology length and NU7441 concentration on PREMJ-based precision gene editing in plants. Although obtaining edited plants remains challenging due to inefficient protoplast regenerations and Agrobacterium-mediated delivery, PREMJ may significantly contribute to precision gene editing in plants competent to PREMJ complex delivery and plant regeneration. Key MessageWe designed and tested a method, termed PREMJ, for precise gene replacement using the CRISPR-Cas-mediated double-stranded break (DSB) formation and repairing the DSBs via microhomology-mediated end joining (MMEJ) with MMEJ donor template carrying desired base changes and microhomologies flanking the DSB ends. PREMJ showed feasibility in precise gene replacement in protoplasts of tomatoes, lettuce, and cabbage, albeit its efficacy in Agrobacterium-mediated plant transformation requires further optimization.

synthetic biology↗

A potential clue for the application of prime editing in tomato, a dicot plant

Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement.

molecular biology↗

Homology-directed repair using transient CRISPR/Cpf1-geminiviral replicon in tomato

Genome editing via the homology-directed repair (HDR) pathway in somatic plant cells is very inefficient compared to error-prone repair by nonhomologous end joining (NHEJ). Here, we increased HDR-based genome editing efficiency approximately 3-fold compared to a Cas9-based single-replicon system via the use of de novo multi-replicon systems equipped with CRISPR/LbCpf1 in tomato and obtained replicon-free but stable HDR alleles. The efficiency of CRISPR/LbCpf1-based HDR was significantly modulated by physical culture conditions such as temperature and light. Ten days of incubation at 31{degrees}C under a light/dark cycle after Agrobacterium-mediated transformation resulted in the best performance among the tested conditions. Furthermore, we developed our single-replicon system into a multi-replicon system that effectively increased HDR efficiency. Although this approach is still challenging, we showed the feasibility of HDR-based genome editing of a salt-tolerant SlHKT1;2 allele without genomic integration of antibiotic markers or any phenotypic selection. Self-pollinated offspring plants carrying the HKT1;2 HDR allele showed stable inheritance and germination tolerance in the presence of 100 mM NaCl. Our work may pave the way for transgene-free editing of alleles of interest in asexually as well as sexually reproducing plants.

molecular biology↗