Effect of gibberellin on crown root development in the mutant of the rice plasmodesmal Germin-like protein OsGER4.

Rice is an essential but highly stress-susceptible crop, whose root system plays an important role in plant development and stress adaptation. The rice root system architecture is controlled by gene regulatory networks involving different phytohormones including auxin, jasmonate, and gibberellin. Gibberellin is generally known as a molecular clock that interacts with different pathways to regulate root meristem development. The exogenous treatment of rice plantlets with Gibberellin reduced the number of crown roots, whilst the exogenous jasmonic acid treatment enhanced them by involving a Germin-like protein OsGER4. Due to those opposite effects, this study aims to investigate the effect of Gibberellin on crown root development in the rice mutant of the plasmodesmal Germin-like protein OsGER4. Under exogenous gibberellin treatment, the number of crown roots significantly increased in osger4 mutant lines and decreased in the OsGER4 overexpressed lines. GUS staining showed that OsGER4 was strongly expressed in rice root systems, particularly crown and lateral roots under GA3 application. Specifically, OsGER4 was strongly expressed from the exodermis, epidermis, sclerenchyma to the endodermis layers of the crown root, along the vascular bundle and throughout LR primordia. The plasmodesmal protein OsGER4 is suggested to be involved in crown root development by maintaining hormone homeostasis, including Gibberillin.

CRISPR/Cas9 targeted mutations of OsDSG1 gene enhanced salt tolerance in rice.

Salinization is one of the leading causes of arable land shrinkage and rice yield decline, recently. Therefore, developing and utilizing salt-tolerant rice varieties have been seen as a crucial and urgent strategy to reduce the effects of saline intrusion and protect food security worldwide. In the current study, the CRISPR/Cas9 system was utilized to induce targeted mutations in the coding sequence of the OsDSG1, a gene involved in the ubiquitination pathway and the regulation of biochemical reactions in rice. The CRISPR/Cas9-induced mutations of the OsDSG1 were generated in a local rice cultivar and the mutant inheritance was validated at different generations. The OsDSG1 mutant lines showed an enhancement in salt tolerance compared to wild type plants at both germination and seedling stages indicated by increases in plant height, root length, and total fresh weight as well as the total chlorophyll and relative water contents under the salt stress condition. In addition, lower proline and MDA contents were observed in mutant rice as compared to wild type plants in the presence of salt stress. Importantly, no effect on seed germination and plant growth parameters was recorded in the CRISRP/Cas9-induced mutant rice under the normal condition. This study again indicates the involvement of the OsDSG1 gene in the salt resistant mechanism in rice and provides a potential strategy to enhance the tolerance of local rice varieties to the salt stress.

The Germin-like protein OsGER4 is involved in promoting crown root development under exogenous jasmonic acid treatment in rice.

In rice (Oryza sativa L.), crown roots (CRs) have many important roles in processes such as root system expansion, water and mineral uptake, and adaptation to environmental stresses. Phytohormones such as auxin, cytokinin, and ethylene are known to control CR initiation and development in rice. However, the role of jasmonic acid (JA) in CR development remained elusive. Here, we report that JA promotes CR development by regulating OsGER4, a rice Germin-like protein. Root phenotyping analysis revealed that exogenous JA treatment induced an increase in CR number in a concentration-dependent manner. A subsequent genome-wide association study and gene expression analyses pinpointed a strong association between the Germin-like protein OsGER4 and the increase in CR number under exogenous JA treatment. The ProGER4::GUS reporter line showed that OsGER4 is a hormone-responsive gene involved in various stress responses, mainly confined to epidermal and vascular tissues during CR primordia development and to vascular bundles of mature crown and lateral roots. Notable changes in OsGER4 expression patterns caused by the polar auxin transport inhibitor NPA support its connection to auxin signaling. Phenotyping experiments with OsGER4 knockout mutants confirmed that this gene is required for CR development under exogenous JA treatment. Overall, our results provide important insights into JA-mediated regulation of CR development in rice.

The Germin-like protein gene OsGER4 is involved in heat stress response in rice root development.

Rice (Oryza sativa L.) is one of the most important dietary carbohydrate sources for half of the world's population. However, it is not well adapted to environmental stress conditions, necessitating to create new and improved varieties to help ensure sufficient rice production in the face of rising populations and shrinking arable land. Recently, the development of the CRISPR/Cas9 gene editing system has allowed researchers to study functional genomics and engineer new rice varieties with great efficiency compared to conventional methods. In this study, we investigate the involvement of OsGER4, a germin-like protein identified by a genome-wide association study that is associated with rice root development under a stress hormone jasmonic acids treatment. Analysis of the OsGER4 promoter region revealed a series of regulatory elements that connect this gene to ABA signaling and water stress response. Under heat stress, osger4 mutant lines produce a significantly lower crown root than wild-type Kitaake rice. The loss of OsGER4 also led to the reduction of lateral root development. Using the GUS promoter line, OsGER4 expression was detected in the epidermis of the crown root primordial, in the stele of the crown root, and subsequently in the primordial of the lateral root. Taken together, these results illustrated the involvement of OsGER4 in root development under heat stress by regulating auxin transport through plasmodesmata, under control by both ABA and auxin signaling.

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

BACKGROUND: Powdery 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. RESULTS: A 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. CONCLUSIONS: Our 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.

Disrupting Sc-uORFs of a transcription factor bZIP1 using CRISPR/Cas9 enhances sugar and amino acid contents in tomato (Solanum lycopersicum).

MAIN CONCLUSION: Induced mutations in the SC-uORF of the tomato transcription factor gene SlbZIP1 by the CRISPR/Cas9 system led to the high accumulation of sugar and amino acid contents in tomato fruits. Tomato (Solanum lycopersicum) is one of the most popular and consumed vegetable crops in the world. Among important traits for tomato improvement such as yield, biotic and abiotic resistances, appearance, post-harvest shelf life and fruit quality, the last one seems to face more challenges because of its genetic and biochemical complexities. In this study, a dual-gRNAs CRISPR/Cas9 system was developed to induce targeted mutations in uORF regions of the SlbZIP1, a gene involved in the sucrose-induced repression of translation (SIRT) mechanism. Different induced mutations in the SlbZIP1-uORF region were identified at the T0 generation, stably transferred to the offspring, and no mutation was found at potential off-target sites. The induced mutations in the SlbZIP1-uORF region affected the transcription of SlbZIP1 and related genes in sugar and amino acid biosynthesis. Fruit component analysis showed significant increases in soluble solid, sugar and total amino acid contents in all SlbZIP1-uORF mutant lines. The accumulation of sour-tasting amino acids, including aspartic and glutamic acids, raised from 77 to 144%, while the accumulation of sweet-tasting amino acids such as alanine, glycine, proline, serine, and threonine increased from 14 to 107% in the mutant plants. Importantly, the potential SlbZIP1-uORF mutant lines with desirable fruit traits and no impaired effect on plant phenotype, growth and development were identified under the growth chamber condition. Our result indicates the potential utility of the CRISPR/Cas9 system for fruit quality improvement in tomato and other important crops.

Enhancing plant growth and biomass production by overexpression of GA20ox gene under control of a root preferential promoter.

Overexpression of GA20 oxidase gene has been a recent trend for improving plant growth and biomass. Constitutive expression of GA20ox has successfully improved plant growth and biomass in several plant species. However, the constitutive expression of this gene causes side-effects, such as reduced leaf size and stem diameter, etc. To avoid these effects, we identified and employed different tissue-specific promoters for GA20ox overexpression. In this study, we examined the utility of At1g promoter to drive the expression of GUS (ß-glucuronidase) reporter and AtGA20ox genes in tobacco and Melia azedarach. Histochemical GUS assays and quantitative real-time-PCR results in tobacco showed that At1g was a root-preferential promoter whose expression was particularly strong in root tips. The ectopic expression of AtGA20ox gene under the control of At1g promoter showed improved plant growth and biomass of both tobacco and M. azedarach transgenic plants. Stem length as well as stem and root fresh weight increased by up to 1.5-3 folds in transgenic tobacco and 2 folds in transgenic M. azedarach. Both tobacco and M. azedarach transgenic plants showed increases in root xylem width with xylem to phloem ratio over 150-200% as compared to WT plants. Importantly, no significant difference in leaf shape and size was observed between At1g::AtGA20ox transgenic and WT plants. These results demonstrate the great utility of At1g promoter, when driving AtGA20ox gene, for growth and biomass improvements in woody plants and potentially some other plant species.

The columbamine O-methyltransferase gene (CoOMT) is capable of increasing alkaloid content in transgenic tobacco plants.

BACKGROUND: In the alkaloid biosynthetic pathways of Stephania and Rannunculaceae, columbamine O-methyltransferase (CoOMT) is an important enzyme that catalyses the formation of the tetrahydropalmatin (rotundin) biosynthesis pathway. In this study, the transgenic construct pBI121-35S-CoOMT-cmyc-Kdel was designed successfully. METHODS AND RESULTS: The real-time RT-PCR results proved that the CoOMT transgene was successfully introduced into Nicotiana tabacum L. plants and produced mRNA. Its transcription levels in three transgenic tobacco lines, T0-7, T0-9, and T0-20, in the T0 generation were higher than those in wild-type tobacco plants. By analysing Western blots and ELISAs, three T0 generation transgenic tobacco lines also expressed recombinant CoOMT (rCoOMT) protein with a molecular weight of approximately 40 kDa, and its contents ranged from 0.048 µg mg-1 to 0.177 µg mg-1. These data illustrated that the CoOMT transgene was expressed; thus, the rCoOMT protein synthesis efficiency increased significantly in comparison with that of the wild-type tobacco plants. The total alkaloid contents ranged from 2.12 g 100 g-1 (of dry weight) to 3.88 g 100 g-1 (of dry weight). The T0-20 plant had the highest total alkaloid content (3.88 g 100 g-1 of dry weight), followed by the T0-7 line (2.75 g 100 g-1 of dry weight). The total alkaloid contents of the CoOMT transgenic tobacco lines increased by approximately 1.09-1.83-fold compared to the wild-type tobacco plants. CONCLUSIONS: This is the first study on the transformation and expression of the CoOMT gene in N. tabacum plants. Initial results of the analysis of transgenic plants proved that the transgenic structure pBI121- CoOMT-Cmyc-Kdel can be used for transformation into Stephania plants.

Effects of Supplemental Light Spectra on the Composition, Production and Antimicrobial Activity of Ocimum basilicum L. Essential Oil.

This study was performed to investigate the effects of different supplemental light spectra and doses (duration and illuminance) on the essential oil of basil (Ocimum basilicum L.) cultivated in the net-house in Vietnam during four months. Ten samples of basil aerial parts were hydrodistilled to obtain essential oils which had the average yields from 0.88 to 1.30% (v/w, dry). The oils analyzed using GC-FID and GC-MS showed that the main component was methyl chavicol (87.4−90.6%) with the highest values found in the oils of basil under lighting conditions of 6 h/day and 150−200 µmol·m−2·s−1. Additional lighting conditions caused the significant differences (p < 0.001) in basil biomass and oil production with the highest values found in the oils of basil under two conditions of (1) 71% Red: 20% Blue: 9.0% UVA in at 120 µmol·m−2·s−1 in 6 h/day and (2) 43.5% Red: 43.5% Blue: 8.0% Green: 5.0% Far-Red at 100 µmol·m−2·s−1 in 6 h/day. The oils of basil in some formulas showed weak inhibitory effects on only the Bacillus subtilis strain. Different light spectra affect the biomass and essential oil production of basil, as well as the concentrations of the major components in the oil.

Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds.

Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.

A sequential transformation method for validating soybean genome editing by CRISPR/Cas9 system.

This study was performed to evaluate the sequential transformation for soybean genome editing using the CRISPR/Cas9 system as well as to show a strategy for examining the activity of CRISPR/Cas9 constructs, especially the designed guide RNAs (gRNAs). The gRNAs for targeted mutations of an exogenous gene and multiple endogenous genes were constructed and transferred into a stably-overexpressed-Cas9 soybean line using Agrobacterium rhizogenes-mediated hairy root induction system. The targeted mutations were identified and characterized by the poly-acrylamide gel electrophoresis (PAGE) heteroduplex method and by sequencing. Induced mutations of the exogenous gene (gus) were observed in 57% of tested transgenic hairy roots, while 100% of the transgenic root lines showed targeted mutations of the endogenous (SACPD-C) gene. Multiple gRNAs targeting two endogenous genes (SACPD-C and SMT) induced mutation rates of 75% and 67%, respectively. Various indels including small and large deletions as well as insertions were found in target sites of the tested genes. This sequential transformation method could present the targeting efficacy of different gRNAs of each tested gene. Additionally, in this study differences in gRNA ratings were found between bioinformatics predictions and actual experimental results. This is the first successful application of the sequential transformation method for genome editing in soybean using the hairy root system. This method could be potentially useful for validating CRISPR/Cas9 constructs, evaluating gRNA targeting efficiencies, and could be applied for other research directions.

Simultaneously induced mutations in eIF4E genes by CRISPR/Cas9 enhance PVY resistance in tobacco.

Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVYO accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVYO confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVYO in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes.

An Efficient Hairy Root System for Validation of Plant Transformation Vector and CRISPR/Cas Construct Activities in Cucumber (Cucumis sativus L.).

Hairy root induction system has been applied in various plant species as an effective method to study gene expression and function due to its fast-growing and high genetic stability. Recently, these systems have shown to be an effective tool to evaluate activities of CRISPR/Cas9 systems for genome editing. In this study, Rhizobium rhizogenes mediated hairy root induction was optimized to provide an effective tool for validation of plant transformation vector, CRISPR/Cas9 construct activities as well as selection of targeted gRNAs for gene editing in cucumber (Cucumis sativus L.). Under the optimized conditions including OD650 at 0.4 for infection and 5 days of co-cultivation, the highest hairy root induction frequency reached 100% for the cucumber variety Choka F1. This procedure was successfully utilized to overexpress a reporter gene (gus) and induce mutations in two Lotus japonicus ROOTHAIRLESS1 homolog genes CsbHLH66 and CsbHLH82 using CRISPR/Cas9 system. For induced mutation, about 78% of transgenic hairy roots exhibited mutant phenotypes including sparse root hair and root hair-less. The targeted mutations were obtained in individual CsbHLH66, CsbHLH82, or both CsbHLH66 and CsbHLH82 genes by heteroduplex analysis and sequencing. The hairy root transformation system established in this study is sufficient and potential for further research in genome editing of cucumber as well as other cucumis plants.

GmDREB6, a soybean transcription factor, notably affects the transcription of the NtP5CS and NtCLC genes in transgenic tobacco under salt stress conditions.

Soil is contaminated with salinity, which inhibits plant growth and development and reduces crop yields. The DREB (dehydration responsive element binding protein) gene responds to salt stresses through enhanced transcriptional expression and activation of genes involved in plant salinity resistance. In this study, we present the results of the analysis of the expression of the GmDREB6 transgene, a gene that encodes the soybean DREB6 transcription factor, regulating the transcription of the NtP5CS and NtCLC genes in transgenic tobacco under salt stress conditions. The transcription of GmDREB6, NtP5CS, and NtCLC in transgenic tobacco lines was confirmed by qRT-PCR. Under salt stress conditions, the GmDREB6 gene transcription levels in the transgenic tobacco lines L1 and L9 had increased from 2.40- to 3.22- fold compared with the condition without salinity treatment. Two transgenic lines, L1 and L9, had transcription levels of the P5CS gene that had increased from 1.24- to 3.60- fold compared with WT plants. For the NtCLC gene, under salt stress conditions, the transgenic lines had transcription levels that had increased by 3.65-4.54 (fold) compared with WT plants (P < 0.05). The L1-transgenic tobacco line showed simultaneous expression of both the GmDREB6 transgene and two intrinsic genes, the NtP5CS and NtCLC genes. This study demonstrated that expression of the GmDREB6 gene from soybean increases the transcription levels of the NtP5CS and NtCLC genes in transgenic tobacco plants under salt stress conditions. The analysis results have suggested that the GmDREB6 gene is a potential candidate for improving the salt tolerance of plants, opening up research and development opportunities for salt stress-tolerant crops to respond to climate change and the rise in sea levels.

CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds.

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.

Rapid and Efficient Genetic Transformation of Sorghum via Agrobacterium-Mediated Method.

Genetic transformation via Agrobacterium-mediated methodology has been used in many sorghum studies. However, the transformation efficiency still varies significantly due to high dependence on sorghum genotypes and technical expertise. In this article, we describe a sorghum transformation procedure in sufficient detail using a public genotype, P898012. This system utilizes a standard binary transgenic vector carrying the bar gene as a selectable marker and immature embryos as starting explants. Glufosinate is employed as the selective agent during callus and shoot induction. This procedure is relatively rapid, efficient, highly reproducible, and should be applicable for many other sorghum genotypes. © 2018 by John Wiley & Sons, Inc.