Multiplex Cas9-based excision of CLCuV betasatellite and DNA-A revealed reduction of viral load with asymptomatic cotton plants.

MAIN CONCLUSION: Multiplexed Cas9-based genome editing of cotton resulted in reduction of viral load with asymptomatic cotton plants. In depth imaging of proteomic dynamics of resulting CLCuV betasatellite and DNA-A protein was also performed. The notorious  cotton leaf curl virus (CLCuV), which is transmitted by the sap-sucking insect whitefly, continuously damages cotton crops. Although the application of various toxins and RNAi has shown some promise, sustained control has not been achieved. Consequently, CRISPR_Cas9 was applied by designing multiplex targets against DNA-A (AC2 and AC3) and betasatellite (ßC1) of CLCuV using CRISPR direct and ligating into the destination vector of the plant using gateway ligation method. The successful ligation of targets into the destination vector was confirmed by the amplification of 1049 bp using a primer created from the promoter and target, while restriction digestion using the AflII and Asc1 enzymes determined how compact the plasmid developed and the nucleotide specificity of the plasmid was achieved through Sanger sequencing. PCR confirmed the successful introduction of plasmid into CKC-1 cotton variety. Through Sanger sequencing and correlation with the mRNA expression of DNA-A and betasatellite in genome-edited cotton plants subjected to agroinfiltration of CLCuV infectious clone, the effectiveness of knockout was established. The genome-edited cotton plants demonstrated edited efficacy of 72% for AC2 and AC3 and 90% for the (ßC1) through amplicon sequencing, Molecular dynamics (MD) simulations were used to further validate the results. Higher RMSD values for the edited ßC1 and AC3 proteins indicated functional loss caused by denaturation. Thus, CRISPR_Cas9 constructs can be rationally designed using high-throughput MD simulation technique. The confidence in using this technology to control plant virus and its vector was determined by the knockout efficiency and the virus inoculation assay.

Tissue specific expression of bacterial cellulose synthase (Bcs) genes improves cotton fiber length and strength.

Fiber growing inside the cotton bolls is a highly demandable product and its quality is key to the success of the textile industry. Despite the various efforts to improve cotton fiber staple length Pakistan has to import millions of bales to sustain its industrial needs. To improve cotton fiber quality Bacterial cellulose synthase (Bcs) genes (acsA, acsB) were expressed in a local cotton variety CEMB-00. In silico studies revealed a number of conserved domains both in the cotton-derived and bacterial cellulose synthases which are essential for the cellulose synthesis. Transformation efficiency of 1.27% was achieved by using Agrobacterium shoot apex cut method of transformation. The quantitative mRNA expression analysis of the Bcs genes in transgenic cotton fiber was found to be many folds higher during secondary cell wall synthesis stage (35 DPA) than the expression during elongation phase (10 DPA). Average fiber length of the transgenic cotton plant lines S-00-07, S-00-11, S-00-16 and S-00-23 was calculated to be 13.02% higher than that of the non-transgenic control plants. Likewise, the average fiber strength was found to be 20.92% higher with an enhanced cellulose content of 22.45%. The mutated indigenous cellulose synthase genes of cotton generated through application of CRISPR/Cas9 resulted in 6.03% and 12.10% decrease in fiber length and strength respectively. Furthermore, mature cotton fibers of transgenic cotton plants were found to have increased number of twists with smooth surface as compared to non-transgenic control when analyzed under scanning electron microscope. XRD analysis of cotton fibers revealed less cellulose crystallinity index in transgenic cotton fibers as compared to control fibers due to deposition of more amorphous cellulose in transgenic fibers as a result of Bcs gene expression. This study paved the way towards unraveling the fact that Bcs genes influence cellulose synthase activity and this enzyme helps in determining the fate of cotton fiber length and strength.

CRISPR/Cas-mediated knockdown of vacuolar invertase gene expression lowers the cold-induced sweetening in potatoes.

MAIN CONCLUSION: VInv gene editing in potato using CRISPR/Cas9 resulted in knockdown of expression and a lower VInv enzymatic activity resulting in a decrease in post-harvest cold-storage sugars formation and sweetening in potatoes. CRISPR-Cas9-mediated knockdown of vacuolar invertase (VInv) gene was carried out using two sgRNAs in local cultivar of potato plants. The transformation efficiency of potatoes was found to be 11.7%. The primary transformants were screened through PCR, Sanger sequencing, digital PCR, and ELISA. The overall editing efficacy was determined to be 25.6% as per TIDE analysis. The amplicon sequencing data showed maximum indel frequency for potato plant T12 (14.3%) resulting in 6.2% gene knockout and 6% frame shift. While for plant B4, the maximum indel frequency of 2.0% was found which resulted in 4.4% knockout and 4% frameshift as analyzed by Geneious. The qRT-PCR data revealed that mRNA expression of VInv gene was reduced 90-99-fold in edited potato plants when compared to the non-edited control potato plant. Following cold storage, chips analysis of potatoes proved B4 and T12 as best lines. Reducing sugars' analysis by titration method determined fivefold reduction in percentage of reducing sugars in tubers of B4 transgenic lines as compared to the control. Physiologically genome-edited potatoes behaved like their conventional counterpart. This is first successful report of knockdown of potato VInv gene in Pakistan that addressed cold-induced sweetening resulting in minimum accumulation of reducing sugars in genome edited tubers.

Early Stage Development of a Newcastle Disease Vaccine Candidate in Corn.

Newcastle disease (ND) is a viral disease that causes labored breathing, periorbital oedema, and ataxia in the majority of avian species. The available vaccines against Newcastle disease virus (NDV) are limited, owing to their low reactivity and multiple dosage requirements. Plant-based machinery provides an attractive and safe system for vaccine production. In the current study, we attempted to express fusion (F) and hemagglutinin-neuraminidase (HN) proteins (the protective antigens against NDV) under constitutive 35S and seed-specific Zein promoters, respectively. Almost 2-7.1-fold higher expression of F gene mRNA in transgenic corn leaves and 8-28-fold higher expression of HN gene mRNA in transgenic corn seeds were observed, when the expression was analyzed by real-time PCR on a relative basis as compared to non-transgenic control plant material (Leaves and seeds). Similarly, 1.66 µg/ml of F protein in corn leaves, i.e., 0.5% of total soluble protein, and 2.4 µg/ml of HN protein in corn seed, i.e., 0.8% of total seed protein, were found when calculated through ELISA. Similar levels of immunological response were generated in chicks immunized through injection of E. coli-produced pET F and pET HN protein as in chickens orally fed leaves and seeds of maize with expressed immunogenic protein. Moreover, the detection of anti-NDV antibodies in the sera of chickens that were fed maize with immunogenic protein, and the absence of these antibodies in chickens fed a normal diet, confirmed the specificity of the antibodies generated through feeding, and demonstrated the potential of utilizing plants for producing more vaccine doses, vaccine generation at higher levels and against other infectious diseases.

Comparative analysis of Constitutive and fiber-specific promoters under the expression pattern of Expansin gene in transgenic Cotton.

Promoters are specified segments of DNA that lead to the initiation of transcription of a specific gene. The designing of a gene cassette for plant transformation is significantly dependent upon the specificity of a promoter. Constitutive Cauliflower mosaic virus promoter, CaMV35S, due to its developmental role, is the most commonly used promoter in plant transformation. While Gossypium hirsutum (Gh) being fiber-specific promoter (GhSCFP) specifically activates transcription in seed coat and fiber associated genes. The Expansin genes are renowned for their versatile roles in plant growth. The overexpression of Expansin genes has been reported to enhance fiber length and fineness. Thus, in this study, a local Cotton variety was transformed with Expansin (CpEXPA1) gene, in the form of two separate cassettes, each with a different promoter, named as 35SEXPA1 and FSEXPA1 expressed under CaMV35S and GhSCFP promoters respectively. Integration and Spatiotemporal relative expression of the transgene were studied in an advanced generation. GhSCFP bearing transgene expression was significantly higher in Cotton fiber than other plant parts. While transgene with CaMV35S promoter was found to be continually expressing in all tissues but the expression was lower in fiber than that expressed under GhSCFP. The temporal expression profile was quite interesting with a gradual increasing pattern of both constructs from 1DPA (days post anthesis) to 18DPA and decreased expression from 24 to 30 DPA. Besides the relative expression of promoters, fiber cellulose quantification and fluorescence intensity were also observed. The study significantly compared the two most commonly used promoters and it is deduced from the results that the GhSCFP promoter could be used more efficiently in fiber when compared with CaMV35S which being constitutive in nature preferred for expression in all parts of the plant.