T1 Generation Research Articles

A rapid and highly efficient sorghum transformation strategy using GRF4-GIF1/ternary vector system.

Sorghum is an important crop for food, forage, wine and biofuel production. To enhance its transformation efficiency without negative developmental by-effects, we investigated the impact of GRF4-GIF1 chimaera and GRF5 on sorghum transformation. Both GRF4-GIF1 and GRF5 effectively improved the transformation efficiency of sorghum and accelerated the transformation process of sorghum to less than 2 months which was not observed when using BBM-WUS. As agrobacterium effectors increase the ability of T-DNA transfer into plant cells, we checked whether ternary vector system can additively enhance sorghum transformation. The combination of GRF4-GIF1 with helper plasmid pVS1-VIR2 achieved the highest transformation efficiency, reaching 38.28%, which is 7.71-fold of the original method. Compared with BBM-WUS, overexpressing GRF4-GIF1 caused no noticeable growth defects in sorghum. We further developed a sorghum CRISPR/Cas9 gene-editing tool based on this GRF4-GIF1/ternary vector system, which achieved an average gene mutation efficiency of 41.36%, and null mutants were created in the T0 generation.

Diabetes Modulates Iodothyronine Deiodinase 2 Expression in the Mouse Retina: A Role for Thyroid Hormone in the Pathogenesis of Diabetic Retinopathy.

Clinical investigations associate hypothyroidism with an increased risk for microvascular complications, yet the mechanism by which thyroid hormone regulates the development of diabetic retinopathy is not clearly understood. We investigated the role of iodothyronine deiodinase 2 (DIO2) in the pathogenesis of diabetic retinopathy. Retinas from streptozotocin-induced diabetic and nondiabetic mice were evaluated by RNA sequencing, RT-PCR, and immunostaining. Media and cell lysates from mouse retinal microvascular endothelial cells and retinal astrocytes exposed to physiologic (5mM) and high glucose (25 mM) containing media were assessed by liquid chromatography-tandem mass spectrometry to measure tetraiodothyronine (T4) and tri-iodothyronine (T3) concentrations and by Western blot analysis to determine the relationship of T4/T3 to oxidative stress and inflammatory mediators. Cell death was determined by Trypan Blue exclusion assay. At 12 weeks of diabetes duration, retinas from diabetic mice compared with nondiabetic mice demonstrated a significant decrease in Dio2 transcripts and Dio2 gene and protein (P < 0.05) expression. When cultured in the presence of high glucose, both mouse retinal astrocytes and microvascular endothelial cells demonstrated a significant reduction of DIO2 protein compared with cells cultured in physiologic glucose. High glucose inhibited generation of T3, leading to a significantly increased T4/T3 (P < 0.0079). Supplementation of cells with T3, but not T4, prevented the high glucose-induced rise in endothelial nitric oxide synthase, intercellular cell adhesion molecule 1, and endothelial cell death (P < 0.0079). Decreased intraretinal T3 owing to diabetes-induced loss of DIO2 may lead to dysfunction and death of cells in the retina, thereby contributing to the pathogenesis of early diabetic retinopathy.

Pyramided transgenic jute (Corchorus capsularis) with biotic stress resistance and herbicide tolerance

Jute (Corchorus sp.) is a commercially important bast (stem) fibre–producing crop. The natural, long, lustrous, and strong jute fibres are globally popular for their domestic and industrial utility. Unfortunately, jute fibre production is under continuous threat from three major agricultural challenges: insect infestation (predominantly lepidopteran insects), fungal infection, and weed management issues. Creating jute plants that could themselves overcome these three challenges would be spectacular. Hence, multi–trait transgenic lines were developed by incorporating three genes—cry1Ab/Ac (encodes Bt δ–endotoxin for Lepidopteran insect resistance), chi11 (encodes chitinase for fungus resistance), and bar (encodes phosphinothricin acetyltransferase (PAT) for glufosinate herbicide tolerance)—into the white jute (C. capsularis) cultivar JRC–321 through Agrobacterium–mediated co–transformation. A bioassay–based event selection strategy was applied for the selection of the transformed jute lines. Each plant was tested through consecutive bioassays involving herbicide, insects, and fungus, followed by molecular and biochemical validation. The functions of the three genes, incorporated in the greenhouse–grown transgenic progeny plants, were confirmed for every generation (till the T2 generation). The plants were able to detoxify the glufosinate herbicide Basta®, resist the two devastating lepidopteran pests tested: hairy caterpillars (Spilarctia obliqua) and semiloopers (Anomis sabulifera), and recover from the stem rot–causing fungal pathogen Macrophomina phaseolina, without compromising agronomic characteristics or fibre quality. The white jute lines thus developed may be effectively used to increase the production of quality jute fibres, meet local and global demands, and minimise input cultivation costs.

Functional analysis of transgenic cry1Ah-1 maize

Maize is an important food crop in the world, but the yield and quality of maize have been significantly reduced due to the impact of insect pests. In order to address this issue, the cry1Ah gene was subjected to error-prone PCR for mutagenesis, and subsequently, the mutant cry1Ah-1 gene was introduced into maize inbred line GSH9901 callus using the Agrobacterium-mediated method. The T2 generation transformed plants were obtained by subculture, and 9 transgenic positive plants were obtained by molecular detection which was carried out by PCR, qRT-PCR, Bt gold-labeled immunoassay test strips, Western blot and ELISA. It was found that the Cry1Ah-1 gene could be transcribed normally in maize leaves, of which OE1 and OE3 had higher relative expression levels and could successfully express proteins of 71.94 KD size. They were expressed in different tissues at the 6-leaf stage, heading stage and grain-filling stage, and could ensure the protection of maize from corn borer throughout the growth period. The biological activities of OE1 and OE3 were tested indoors and in the field, and the results showed that in indoors, the corn borer that fed on OE1 and OE3 corn leaves had a mortality rate of 100 % after 3 days; in the field, OE1 and OE3 had strong insecticidal activity against corn borer, reaching a high resistance level. In conclusion, the transgenic cry1Ah-1 maize has a strong insecticidal effect on corn borer, and has a good prospect of commercialization.

Cloning of the Soybean GmNHL1 Gene and Functional Analysis under Salt Stress.

When encountered in the soybean seedling stage, salt stress has serious impacts on plant growth and development. This study explores the role of the soybean NDR1/HIN1-like family gene GmNHL1 under salt stress. First, the GmNHL1 gene was successfully cloned, and bioinformatic analysis revealed multiple cis-acting elements which are related to adversity stress and involved in the oxidative response in the promoter region. Sub-cellular localization analysis indicated that the protein expressed by GmNHL1 was localized on the cell membrane. An over-expression vector of the target gene and a CRISPR/Cas9 gene-editing vector were constructed, and the recipient soybean variety Jinong 74 was genetically transformed using the Agrobacterium tumefaciens-mediated method. By analyzing the performance of the different plants under salt stress, the results showed that GmNHL1 was over-expressed in the T2 generation. The germination potential, germination rate, germination index, and vitality index of the strain were significantly higher than those of the recipient control JN74. Under salt stress conditions, the root microanatomical structure of the GmNHL1 over-expressing material remained relatively intact, and its growth was better than that of the recipient control JN74. Measurement of physiological and biochemical indicators demonstrated that, compared with the receptor control JN74, the malondialdehyde and O2- contents of the GmNHL1 over-expressing material were significantly reduced, while the antioxidant enzyme activity, proline content, and chlorophyll content significantly increased; however, the results for GmNHL1 gene-edited materials were the opposite. In summary, over-expression of GmNHL1 can improve the salt tolerance of plants and maintain the integrity of the root anatomical structure, thereby more effectively and rapidly reducing the accumulation of malondialdehyde and O2- content and increasing antioxidant enzyme activity. This reduces cell membrane damage, thereby improving the salt tolerance of soybean plants. These results help to better understand the mechanism of salt tolerance in soybean plants, laying a theoretical foundation for breeding new stress-resistant varieties of soybean.

Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage

Abstract Flavonols are the major class of flavonoids of green Chinese cabbage (Brassica rapa subsp. pekinensis). The B. rapa genome harbors seven flavonol synthase genes (BrFLSs), but they have not been functionally characterized. Here, transcriptome analysis showed four BrFLSs mainly expressed in Chinese cabbage. Among them, only BrFLS1 showed major FLS activity and additional flavanone 3β-hydroxylase (F3H) activity, while BrFLS2 and BrFLS3.1 exhibited only marginal F3H activities. We generated BrFLS1-knockout (BrFLS1-KO) Chinese cabbages using CRISPR/Cas9-mediated genome editing and obtained transgene-free homozygous plants without off-target mutation in the T1 generation, which were further advanced to the T2 generation showing normal phenotype. UPLC-ESI-QTOF-MS analysis revealed that flavonol glycosides were dramatically decreased in the T2 plants, while dihydroflavonol glycosides accumulated concomitantly to levels corresponding to the reduced levels of flavonols. Quantitative PCR analysis revealed that the early steps of phenylpropanoid and flavonoid biosynthetic pathway were upregulated in the BrFLS1-KO plants. In accordance, total phenolic contents were slightly enhanced in the BrFLS1-KO plants, which suggests a negative role of flavonols in phenylpropanoid and flavonoid biosynthesis in Chinese cabbage. Phenotypic surveys revealed that the BrFLS1-KO Chinese cabbages showed normal head formation and reproductive phenotypes, but subtle morphological changes in their heads were observed. In addition, their seedlings were susceptible to osmotic stress compared to the controls, suggesting that flavonols play a positive role for osmotic stress tolerance in B.rapa seedling. In this study, we showed that CRISPR/Cas9-mediated BrFLS1-KO successfully generated a valuable breeding resource of Chinese cabbage with distinctive metabolic traits and that CRISPR/Cas9 can be efficiently applied in functional Chinese cabbage breeding.

Boosting Dye-Sensitized Luminescence by Enhanced Short-Range Triplet Energy Transfer.

Dye-sensitization can enhance lanthanide-based upconversion luminescence, but is hindered by interfacial energy transfer from organic dye to lanthanide ion Yb3+ . To overcome these limitations, modifying coordination sites on dye conjugated structures and minimizing the distance between fluorescence cores and Yb3+ in upconversion nanoparticles (UCNPs) are proposed. The specially designed near-infrared (NIR) dye, disulfo-indocyanine green (disulfo-ICG), acts as the antenna molecule and exhibits a 2413-fold increase in luminescence under 808nm excitation compared to UCNPs alone using 980nm irradiation. The significant improvement is attributed to the high energy transfer efficiency of 72.1% from disulfo-ICG to Yb3+ in UCNPs, with majority of energy originating from triplet state (T1 ) of disulfo-ICG. Shortening the distance between the dye and lanthanide ions increases the probability of energy transfer and strengthens the heavy atom effect, leading to enhanced T1 generation and improved dye-triplet sensitization upconversion. Importantly, this approach also applies to 730nm excitation Cy7-SO3 sensitization system, overcoming the spectral mismatch between Cy7 and Yb3+ and achieving a 52-fold enhancement in luminescence. Furthermore, the enhancement of upconversion at single particle level through dye-sensitization is demonstrated. This strategy expands the range of NIR dyes for sensitization and opens new avenues for highly efficient dye-sensitized upconversion systems.

Enhancing powdery mildew resistance in soybean by targeted mutation of MLO genes using the CRISPR/Cas9 system

BackgroundPowdery mildew is a major disease that causes great losses in soybean yield and seed quality. Disease-resistant varieties, which are generated by reducing the impact of susceptibility genes through mutation in host plants, would be an effective approach to protect crops from this disease. The Mildew Locus O (MLO) genes are well-known susceptibility genes for powdery mildew in plant. In this study, we utilized the CRISPR/Cas9 system to induce targeted mutations in the soybean GmMLO genes to improve powdery mildew resistance.ResultsA dual-sgRNA CRISPR/Cas9 construct was designed and successfully transferred into the Vietnamese soybean cultivar DT26 through Agrobacterium tumefaciens-mediated transformation. Various mutant forms of the GmMLO genes including biallelic, chimeric and homozygous were found at the T0 generation. The inheritance and segregation of CRISPR/Cas9-induced mutations were confirmed and validated at the T1 and T2 generations. Out of six GmMLO genes in the soybean genome, we obtained the Gmmlo02/Gmmlo19/Gmmlo23 triple and Gmmlo02/Gmmlo19/Gmmlo20/Gmmlo23 quadruple knockout mutants at the T2 generation. When challenged with Erysiphe diffusa, a fungus that causes soybean powdery mildew, all mutant plants showed enhanced resistance to the pathogen, especially the quadruple mutant. The powdery mildew severity in the mutant soybeans was reduced by up to 36.4% compared to wild-type plants. In addition, no pleiotropic effect on soybean growth and development under net-house conditions was observed in the CRISPR/Cas9 mutants.ConclusionsOur results indicate the involvement of GmMLO02, GmMLO19, GmMLO20 and GmMLO23 genes in powdery mildew susceptibility in soybean. Further research should be conducted to investigate the roles of individual tested genes and the involvement of other GmMLO genes in this disease infection mechanism. Importantly, utilizing the CRISPR/Cas9 system successfully created the Gmmlo transgene-free homozygous mutant lines with enhanced resistance to powdery mildew, which could be potential materials for soybean breeding programs.

Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice

Prime editing (PE) technology enables precise alterations in the genetic code of a genome of interest. PE offers great potential for identifying major agronomically important genes in plants and editing them into superior variants, ideally targeting multiple loci simultaneously to realize the collective effects of the edits. Here, we report the development of a modular assembly-based multiplex PE system in rice and demonstrate its efficacy in editing up to four genes in a single transformation experiment. The duplex PE (DPE) system achieved a co-editing efficiency of 46.1% in the T0 generation, converting TFIIAγ5 to xa5 and xa23 to Xa23SW11. The resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae (Xoo) strains in the T1 generation. In addition, we successfully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele, achieving a co-editing rate of 57.14%. Furthermore, with the quadruple PE (QPE) system, we edited four genes—two for herbicide tolerance (OsEPSPS1 and OsALS1) and two for Xoo resistance (TFIIAγ5 and OsSWEET11a)—using one construct, with a co-editing efficiency of 43.5% for all four genes in the T0 generation. We performed multiplex PE using five more constructs, including two for triplex PE (TPE) and three for QPE, each targeting a different set of genes. The editing rates were dependent on the activity of pegRNA and/or ngRNA. For instance, optimization of ngRNA increased the PE rates for one of the targets (OsSPL13) from 0% to 30% but did not improve editing at another target (OsGS2). Overall, our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents, making it feasible for more labs to utilize PE for their editing experiments. These findings have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.

A 4.43-Kb deletion of chromosomal segment containing an ovate family protein confers long capsule in sesame (Sesamum indicum L.).

A 4.43-Kb structural variation in the sesame genome results in the deletion of the Siofp1 gene and induces the long capsule length trait. Capsule length (CL) has a positive effect on seed weight and yield in various agronomically important species; however, the molecular mechanism underlying long capsule trait regulation in sesame remains unknown. The inheritance analysis showed that long capsule traits (CL > 4.0cm) were dominant over normal length (average CL = 3.0cm) and were controlled by a single gene pair. Association mapping with a RIL population and 259 natural sesame germplasm accessions indicated that the target interval was 52,830-730,961bp of SiChr.10 in sesame. Meanwhile, the structural variation (SV) of the association mapping revealed that only SV_414325 on chromosome 10 was significantly associated with the CL trait, with a P value of 1.1135E-19. SV_414325 represents a 4430-bp deletion from 414,325 to 418,756bp on SiChr.10, covering Sindi_2155000 (named SiOFP1). In the normal length type, Siofp1 encodes 411 amino acids of the ovate family proteins and is highly expressed in the leaf, stem, bud, and capsule tissues of sesame. In accordance with the transcriptional repressor character, Siofp1 overexpression in transgenic Arabidopsis (T0 and T1 generations) induced a 25-39% greater shortening of silique length than the wild type (P < 0.05), as well as round cauline leaves and short carpels. These results confirm that SiOFP1 plays a key role in regulating CL trait in sesame and other flowering plants. These findings provide a theoretical and material basis for sesame capsule development and high-yield breeding research.

An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different T-DNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research.

The effects and mechanisms of the new brominated flame retardant BTBPE on thyroid toxicity.

As an alternative to octabromodiphenyl ether (octa-BDE), 1, 2-bis (2,4, 6-tribromophenoxy) ethane (BTBPE) has been widely used in a variety of combustible materials, such as plastics, textiles and furniture. Previous studies have demonstrated the thyroid toxicity of traditional brominated flame retardants for example octa-BDE clearly. Nevertheless, little is known about the thyroid toxicity of alternative novel brominated flame retardants BTBPE. In this study, it was demonstrated that BTBPE in vivo exposure induced FT4 reduction in 2.5, 25 and 250mg/kg bw treated group and TT4 reduction in 25mg/kg bw treated group. TG, TPO and NIS are key proteins of thyroid hormone synthesis. The results of Western blot and RT-PCR from thyroid tissue showed decreased protein levels and gene expression levels of TG, TPO and NIS as well as regulatory proteins PAX8 and TTF2. To investigate whether the effect also occurred in humans, anthropogenic Nthy-ori 3-1cells were selected. Similar results were seen in vitro condition. 2.5mg/L BTBPE reduced the protein levels of PAX8, TTF1 and TTF2, which in turn inhibited the protein levels of TG and NIS. The results in vitro experiment were consistent with that in vivo, suggesting possible thyrotoxic effects of BTBPE on humans. It was indicated that BTBPE had the potential interference of T4 generation and the study provided more evidence of the effects on endocrine disorders.

Transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation via a co-editing strategy.

Transgene-free plant genome editing in the T0 generation is highly desirable but challenging1,2. Here we achieved such a goal using a co-editing strategy via Agrobacterium-mediated transient expression of cytosine base editor to edit ALS encoding acetolactate synthase to confer herbicide chlorsulfuron resistance as a selection marker, Cas12a/CRISPR RNA for editing gene(s) of interest, and green fluorescent protein for selecting transgene-free transformants. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 1.9% to 42.1% in tomato, tobacco, potato and citrus. This co-editing strategy is particularly useful for transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation.

Synthesis of Crocin I and Crocin II by Multigene Stacking in Nicotiana benthamiana.

Crocins are a group of highly valuable water-soluble carotenoids that are reported to have many pharmacological activities, such as anticancer properties, and the potential for treating neurodegenerative diseases including Alzheimer's disease. Crocins are mainly biosynthesized in the stigmas of food-medicine herbs Crocus sativus L. and Gardenia jasminoides fruits. The distribution is narrow in nature and deficient in resources, which are scarce and expensive. Recently, the synthesis of metabolites in the heterologous host has opened up the potential for large-scale and sustainable production of crocins, especially for the main active compounds crocin I and crocin II. In this study, GjCCD4a, GjALDH2C3, GjUGT74F8, and GjUGT94E13 from G. jasminoides fruits were expressed in Nicotiana benthamiana. The highest total content of crocins in T1 generation tobacco can reach 78,362 ng/g FW (fresh weight) and the dry weight is expected to reach 1,058,945 ng/g DW (dry weight). Surprisingly, the primary effective constituents crocin I and crocin II can account for 99% of the total crocins in transgenic plants. The strategy mentioned here provides an alternative platform for the scale-up production of crocin I and crocin II in tobacco.

Transcriptional and Metabolic Profiling of Arabidopsis thaliana Transgenic Plants Expressing Histone Acetyltransferase HAC1 upon the Application of Abiotic Stress-Salt and Low Temperature.

Augmented knowledge of plant responses upon application of stress could help improve our understanding of plant tolerance under abiotic stress conditions. Histone acetylation plays an important role in gene expression regulation during plant growth and development and in the response of plants to abiotic stress. The current study examines the level of transcripts and free metabolite content in transgenic Arabidopsis thaliana plants expressing a gene encoding histone acetyltransferase from Medicago truncatula (MtHAC1) after its heterologous expression. Stable transgenic plants with HAC1 gain and loss of function were constructed, and their T5 generation was used. Transgenic lines with HAC1-modified expression showed a deviation in root growth dynamics and leaf area compared to the wild-type control. Transcriptional profiles were evaluated after the application of salinity stress caused by 150 mM NaCl at four different time points (0, 24, 48, and 72 h) in treated and non-treated transgenic and control plants. The content and quantity of free metabolites-amino acids, mono- and dicarbohydrates, organic acids, and fatty acids-were assessed at time points 0 h and 72 h in treated and non-treated transgenic and control plants. The obtained transcript profiles of HAC1 in transgenic plants with modified expression and control were assessed after application of cold stress (low temperature, 4 °C).

Improved forage quality and biomass yield of alfalfa (Medicago sativa L.) by Arabidopsis QQS orphan gene

Improving the forage quality of alfalfa in terms of digestibility and crude content is essential for any alfalfa quality breeding programs. Arabidopsis thaliana orphan gene QQS (Qua-Quine Starch) has been shown to improve protein content and alter carbohydrate composition in different food crops. However, there are significant differences in agronomic traits and nutritional conditions between alfalfa and other food crops. To explore the biological function and molecular mechanisms of QQS in alfalfa, we generated QQS transgenic plants and their segregated population (T1 generation), and evaluated their performance under normal- and nitrogen-deficient conditions. Our findings indicate that QQS can significantly enhance the total nitrogen and crude protein content of alfalfa and increase nodule weight under low-nitrogen conditions. Furthermore, QQS transgenic lines also showed reduced levels of neutral detergent fiber (NDF) and lignin, improving forage digestibility. By RNA sequencing and RT-qPCR analysis, we found that QQS affected the expression of genes involved in carbon and nitrogen metabolism, lignin biosynthesis and amino acid biosynthesis and degradation pathways in alfalfa. In addition, QQS also improved alfalfa biomass yield by increasing branch number and plant height in both greenhouse and field conditions. Our results demonstrate that QQS as a useful molecular tool can improve alfalfa biomass yield and overall forage quality and could have significant implications for the alfalfa breeding industry in satisfying the constant demands for high-quality and high-yielding forage.

Co-expression of nitrogenase proteins in cotton (Gossypium hirsutum L.).

Chemical nitrogen fertilizer can maintain crop productivity, but overuse of chemical nitrogen fertilizers leads to economic costs and environmental pollution. One approach to reduce use of nitrogen fertilizers is to transfer nitrogenase biosynthetic pathway to non-legume plants. Fe protein encoded by nifH and MoFe protein encoded by nifD and nifK are two structural components of nitrogenase. NifB encoded by nifB is a critical maturase that catalyzes the first committed step in the biosynthesis of nitrogenase FeMo-cofactor that binds and reduces N2. Expression of the nifB, nifH, nifD and nifK is essential to generate plants that are able to fix atmospheric N2. In this study, the four genes (nifB, nifH, nifD and nifK) from Paenibacillu polymyxaWLY78 were assembled in plant expression vector pCAMBIA1301 via Cre/LoxP recombination system, yielding the recombinant expression vector pCAMBIA1301-nifBHDK. Then, the four nif genes carried in the expression vector were co-introduced into upland cotton R15 using Agrobacterium tumefaciens-mediated transformation. Homozygous transgenic cotton lines B2, B5 and B17 of T3 generation were selected by PCR and RT-PCR. qRT-PCR showed that nifB, nifH, nifD and nifK were co-expressed in the transgenic cottons at similar levels. Western blotting analysis demonstrated that NifB, NifH, NifD and NifK were co-produced in the transgenic cottons. Co-expression of the four critical Nif proteins (NifB, NifH, NifD and NifK) in cottons represents an important step in engineering nitrogenase biosynthetic pathway to non-legume plants.

CRISPR/Cas9-mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV-U1 strain in regenerated Nicotiana tabacum.

CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.

Functional analysis of a susceptibility gene (HIPP27) in the Arabidopsis thaliana-Meloidogyne incognita pathosystem by using a genome editing strategy

BackgroundPlant-parasitic root-knot nematodes cause immense yield declines in crop plants that ultimately obviate global food security. They maintain an intimate relationship with their host plants and hijack the host metabolic machinery to their own advantage. The existing resistance breeding strategies utilizing RNAi and resistance (R) genes might not be particularly effective. Alternatively, knocking out the susceptibility (S) genes in crop plants appears to be a feasible approach, as the induced mutations in S genes are likely to be long-lasting and may confer broad-spectrum resistance. This could be facilitated by the use of CRISPR/Cas9-based genome editing technology that precisely edits the gene of interest using customizable guide RNAs (gRNAs) and Cas9 endonuclease.ResultsInitially, we characterized the nematode-responsive S gene HIPP27 from Arabidopsis thaliana by generating HIPP27 overexpression lines, which were inoculated with Meloidogyne incognita. Next, two gRNAs (corresponding to the HIPP27 gene) were artificially synthesized using laboratory protocols, sequentially cloned into a Cas9 editor plasmid, mobilized into Agrobacterium tumefaciens strain GV3101, and transformed into Arabidopsis plants using the floral dip method. Apart from 1–3 bp deletions and 1 bp insertions adjacent to the PAM site, a long deletion of approximately 161 bp was documented in the T0 generation. Phenotypic analysis of homozygous, ‘transgene-free’ T2 plants revealed reduced nematode infection compared to wild-type plants. Additionally, no growth impairment was observed in gene-edited plants.ConclusionOur results suggest that the loss of function of HIPP27 in A. thaliana by CRISPR/Cas9-induced mutagenesis can improve host resistance to M. incognita.

The Amplification and Activity Analysis of the Small Rubber Particle Protein (SRPP) Promoter of Eucommia ulmoides (EuSRPP) Revealed That Its Activity Was Regulated by MeJA, GA3, and Drought Pathways

As an important temperate gum source plant, Eucommia ulmoides is widely distributed in China, but the low yield of Eucommia ulmoides gum considerably affects its application as a natural rubber in practical production. The small rubber particle protein (SRPP) gene is an influential participant in the Eucommia ulmoides gum biosynthesis process, and its expression affects the gum content. In this study, the promoter activity of the Eucommia ulmoides SRPP (EuSRPP) gene was analyzed by molecular biology and bioinformatics. In order to understand the molecular regulation mechanism of the EuSRPP genes at the transcriptional level, we first obtained the promoter sequences of the EuSRPP1, 3, 4, 5, 6, and 7 genes via genome walking and PCR amplification experiments. Then, the T3 generation of the transgenic homozygous line was obtained via a genetic transformation of Arabidopsis thaliana mediated by Agrobacterium. The six EuSRPP promoters were expressed in transgenic plants and were stably expressed in the leaves, pollinated flowers, and mature pods. As the transgenic plant grows and develops, promoter activity in the root is barely expressed. In addition, after the transgenic Arabidopsis was treated with methyl jasmonate (1 mmol/L MeJA), gibberellin (1 mmol/L GA3), and drought (20% PEG6000), the activity expression of the six EuSRPP promoters increased first and then decreased. The difference, however, is that EuSRPP1, 3, and 4 reach their strongest GUS activity at 3 h of plant treatment, while EuSRPP5, 6, and 7 reach their strongest activity at 6 h of treatment. Based on all experimental results, for the first time, it has been shown that the expression loci of the six EuSRPP gene promoters were relatively consistent. Second, the expression activity of the promoters of the six EuSRPP genes was different under MeJA, GA3, and drought treatment, suggesting that the promoter activity of the EuSRPP genes was regulated by endogenous hormones and drought pathways.