scholarly journals Genome Editing in Cowpea Vigna unguiculata Using CRISPR-Cas9

2019 ◽  
Vol 20 (10) ◽  
pp. 2471 ◽  
Author(s):  
Jie Ji ◽  
Chunyang Zhang ◽  
Zhongfeng Sun ◽  
Longlong Wang ◽  
Deqiang Duanmu ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Genome Editing ◽  
Vigna Unguiculata ◽  
Symbiotic Nitrogen Fixation ◽  
Nodule Formation ◽  
Guide Rnas ◽  
Legume Crop ◽  
Receptor Like Kinase ◽  
Agronomical Traits

Cowpea (Vigna unguiculata) is widely cultivated across the world. Due to its symbiotic nitrogen fixation capability and many agronomically important traits, such as tolerance to low rainfall and low fertilization requirements, as well as its high nutrition and health benefits, cowpea is an important legume crop, especially in many semi-arid countries. However, research in Vigna unguiculata is dramatically hampered by the lack of mutant resources and efficient tools for gene inactivation in vivo. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We applied the CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the representative symbiotic nitrogen fixation (SNF) gene in Vigna unguiculata. Our customized guide RNAs (gRNAs) targeting symbiosis receptor-like kinase (SYMRK) achieved ~67% mutagenic efficiency in hairy-root-transformed plants, and nodule formation was completely blocked in the mutants with both alleles disrupted. Various types of mutations were observed near the PAM region of the respective gRNA. These results demonstrate the applicability of the CRISPR/Cas9 system in Vigna unguiculata, and therefore should significantly stimulate functional genomics analyses of many important agronomical traits in this unique crop legume.

Nitrogen Nutrition of Cowpea (Vigna unguiculata)

1977 ◽  
Vol 13 (3) ◽  
pp. 241-252 ◽  
Author(s):  
P. J. Dart ◽  
P. A. Huxley ◽  
A. R. J. Eaglesham ◽  
F. R. Minchin ◽  
R. J. Summerfield ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Vigna Unguiculata ◽  
Symbiotic Nitrogen Fixation ◽  
Nitrogen Nutrition ◽  
Combined Nitrogen ◽  
Nitrogen Treatments ◽  
Seed Yields ◽  
Applied Nitrogen

SUMMARYAverage seed yields of effectively nodulated cowpea plants were 38% greater than those of non-nodulated plants when both received applied nitrogen at concentrations ranging from 60 to 240 ppm during one of three periods: emergence to first flower, first flower to mid pod-fill, or mid pod-fill to maturity. Nodulation increased seed yields by 45% when plants received a ‘basal’ level of 30 ppm N throughout growth. None of the combined nitrogen treatments could compensate non-nodulated plants for the loss of symbiotic nitrogen fixation. Non-nodulated plants relying on applied N branched less, produced fewer peduncles and set fewer pods on each peduncle than nodulated plants.

scholarly journals Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

2021 ◽  
Vol 22 (19) ◽  
pp. 10570
Author(s):  
Yuya Kato ◽  
Hirotaka Tabata ◽  
Kumiko Sato ◽  
Mariko Nakamura ◽  
Izumu Saito ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Genome Editing ◽  
Adenovirus Vector ◽  
Target Sequence ◽  
Guide Rna ◽  
Guide Rnas ◽  
B Virus ◽  
X Gene

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.

Influence of chlorsulfuron on rhizobial growth, nodule formation, and nitrogen fixation with chickpea

2004 ◽  
Vol 55 (10) ◽  
pp. 1059 ◽  
Author(s):  
A. Anderson ◽  
J. A. Baldock ◽  
S. L. Rogers ◽  
W. Bellotti ◽  
G. Gill
Keyword(s):  
Nitrogen Fixation ◽  
Symbiotic Nitrogen Fixation ◽  
Defined Medium ◽  
Application Rate ◽  
Field Application ◽  
Nodule Formation ◽  
Negative Effects ◽  
Legume Crops ◽  
Cell Inoculum

Sulfonylurea residues have been found to inhibit the growth of some legume crops and pastures in seasons following application. Negative effects of these herbicides on symbiotic nitrogen fixation by legume crops and pastures have been demonstrated. Reductions in nitrogen fixation may result from a direct effect of the herbicide on rhizobial growth and/or an indirect effect on plant growth. In this study the influence of chlorsulfuron on the growth of chickpea rhizobia [Mesorhizobium ciceri (CC1192)], the growth of chickpea plants, and the extent of nodulation and nitrogen fixation by the chickpea/rhizobia symbiosis were examined. In vitro studies (in yeast mannitol broth and a defined medium) showed that chlorsulfuron applied at double the recommended field application rate did not influence the growth of chickpea rhizobia. An experiment using 14C-labelled chlorsulfuron was conducted to determine if rhizobial cells exposed to chlorsulfuron could deliver the herbicide to the point of root infection and nodule formation. Approximately 1% of the herbicide present in the rhizobial growth medium remained with the cell/inoculum material after rinsing with 1/4 strength Ringer’s solution. This was considered unlikely to affect chickpea growth, nodulation, or nitrogen fixation. A pot experiment was used to define the influence of chlorsulfuron on the growth, nodulation, and nitrogen fixation of chickpeas. The presence of chlorsulfuron in the soil reduced the nodulation and nitrogen fixation of the chickpea plants. Pre-exposing rhizobia to chlorsulfuron before inoculating them into pots with germinating chickpea seeds, reduced the number of nodules formed by 51%. Exposure of chickpeas and chickpea rhizobia to chlorsulfuron can adversely affect the formation and activity of symbiotic nitrogen-fixing nodules, even when only the rhizobial inoculant is exposed briefly to the herbicide.

Metabolite Regulation of Phosphenolpyruvate Carboxylase in Legume Root Nodules

1996 ◽  
Vol 23 (4) ◽  
pp. 413 ◽  
Author(s):  
KC Woo ◽  
S Xu
Keyword(s):  
Nitrogen Fixation ◽  
Glycine Max ◽  
Vigna Unguiculata ◽  
Symbiotic Nitrogen Fixation ◽  
Feedback Inhibition ◽  
Root Nodules ◽  
Psophocarpus Tetragonolobus ◽  
Glycine Max L ◽  
Fructose 1,6 Bisphosphate ◽  
Metabolite Regulation

The effects of metabolic activators and inhibitors on phosphoenolpyruvate carboxylase (PEPC) activity were examined at pH 7 in partially purified enzyme from nodules of soybean (Glycine max (L.) Merr.), Psophocarpus tetragonolobus DC. and Vigna unguiculata ssp. sesquipedalis (L.) Verdc. Glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate, fructose 1-phosphate, fructose 1,6- bisphosphate and phosphoglycerate stimulated the activity about 2-fold at low (0.5 mM) but not saturating (2.5 mM) PEP concentration. Glc 6-P and fru 6-P were the most effective activators and they increased the affinity of the enzyme for PEP by 2-4-fold. The dicarboxylates, malate, succinate, malonate, 2-oxoglutarate and aspartate inhibited PEPC activity. Malate was the most inhibitory, and strongly inhibited PEPC activity even at saturating PEP concentration. The Ki values for malate were 0.3-0.4 mM for soybean and P. tetragonolobus. However, glc 6-P and fru 6-P alleviated maiate inhibition and increased the Ki values by 11- to 28-fold in these two species. We propose that glc 6-P (fru 6-P) activates PEPC in a feedforward regulation and protects it against feedback inhibition by malate and thus coordinates the supply of photosynthate availability with malate synthesis required by the bacteroids to support symbiotic nitrogen fixation in nodules.

INFLUENCE OF ROOT TEMPERATURE ON THE EARLY GROWTH AND SYMBIOTIC NITROGEN FIXATION OF NODULATED LOTUS CORNICULATUS PLANTS

1970 ◽  
Vol 50 (5) ◽  
pp. 569-575 ◽  
Author(s):  
H. T. KUNELIUS ◽  
K. W. CLARK
Keyword(s):  
Nitrogen Fixation ◽  
Ammonium Nitrate ◽  
Environmental Conditions ◽  
Symbiotic Nitrogen Fixation ◽  
Lotus Corniculatus ◽  
Early Growth ◽  
Birdsfoot Trefoil ◽  
Nodule Formation ◽  
Root Temperature ◽  
Nitrogen Yields

Three birdsfoot trefoil (Lotus corniculatus L.) cultivars, inoculated with one of six Lotus rhizobia strains or dependent on ammonium nitrate, were grown in diSPo growth pouches under controlled environmental conditions at five root temperatures (9–30 C) for 35 days after nodule formation. When the plants were dependent on symbiotic nitrogen fixation, the highest dry weights and nitrogen yields per plant were obtained at 18 or 24 C depending on symbiotic combination. At 9 and 12 C, nitrogen fixation was depressed and the growth was poor. The dry weights of plants at 9 C were 19 to 45% of those at 24 C. At 30 C the growth and nitrogen fixation were generally depressed. At all root temperatures the growth of plants dependent on symbiotic nitrogen fixation was inferior to that of plants receiving combined nitrogen (NH4NO3). Significant interactions indicate that the nitrogen fixing ability of cultivars was dependent on both root temperature and the strain of Lotus rhizobia.

scholarly journals Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system

2020 ◽  
Vol 6 (8) ◽  
pp. eaay6812 ◽  
Author(s):  
Yu Zhang ◽  
Hui Li ◽  
Yi-Li Min ◽  
Efrain Sanchez-Ortiz ◽  
Jian Huang ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genome Editing ◽  
Dystrophin Gene ◽  
Adeno Associated Virus ◽  
Guide Rnas ◽  
Dystrophin Expression ◽  
Cas9 Nuclease ◽  
High Doses

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the dystrophin gene (DMD). Previously, we applied CRISPR-Cas9–mediated “single-cut” genome editing to correct diverse genetic mutations in animal models of DMD. However, high doses of adeno-associated virus (AAV) are required for efficient in vivo genome editing, posing challenges for clinical application. In this study, we packaged Cas9 nuclease in single-stranded AAV (ssAAV) and CRISPR single guide RNAs in self-complementary AAV (scAAV) and delivered this dual AAV system into a mouse model of DMD. The dose of scAAV required for efficient genome editing were at least 20-fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression and improved muscle contractility. These findings show that the efficiency of CRISPR-Cas9–mediated genome editing can be substantially improved by using the scAAV system. This represents an important advancement toward therapeutic translation of genome editing for DMD.

Symbiotic Nitrogen Fixation at the Late Stage of Nodule Formation in Lotus japonicus and Other Legume Plants

2000 ◽  
Vol 113 (4) ◽  
pp. 467-473 ◽  
Author(s):  
Shigeyuki Tajima ◽  
Kenichi Takane ◽  
Mika Nomura ◽  
Hiroshi Kouchi
Keyword(s):  
Nitrogen Fixation ◽  
Late Stage ◽  
Symbiotic Nitrogen Fixation ◽  
Lotus Japonicus ◽  
Nodule Formation ◽  
Legume Plants

scholarly journals A Convenient, Soil-Free Method for the Production of Root Nodules in Soybean to Study the Effects of Exogenous Additives

2018 ◽  
Author(s):  
Swarup Roy Choudhury ◽  
Sarah M. Johns ◽  
Sona Pandey
Keyword(s):  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Root Nodules ◽  
Growth Conditions ◽  
Nodule Development ◽  
Nodule Formation ◽  
Legume Crop ◽  
Simple Protocol ◽  
Biological Nitrogen

Legumes develop root nodules that harbour endosymbiotic bacteria, rhizobia. These rhizobia convert nitrogen to ammonia by biological nitrogen fixation. A thorough understanding of the biological nitrogen fixation in legumes and its regulation is key to develop sustainable agriculture. It is well known that plant hormones affect nodule formation; however, most studies are limited to model legumes due to their suitability for in vitro, plate-based assays. Specifically, it is almost impossible to measure the effects of exogenous hormones or other additives during nodule development in crop legumes such as soybean as they have huge root system in soil. To circumvent this issue, the present research develops suitable media and growth conditions for efficient nodule development under in vitro, soil free conditions in an important legume crop, soybean. Moreover, we also evaluate the effects of all major phytohormones during soybean nodulation under identical conditions. This versatile, inexpensive, scalable and simple protocol provides several advantages over previously established methods. It is extremely time-and resource-efficient, does not require special training or equipment, and produces highly reproducible results. The approach is expandable to other large legumes as well as for other exogenous additives.

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