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ABSTRACTMicronutrient deficiency conditions, such as anemia, are the most prevalent global health problem due to inadequate iron and folate in dietary sources. Biofortification advancements can propel the rapid amelioration of nutritionally beneficial components in crops that are required to combat the adverse effects of micronutrient deficiencies on human health. To date, several strategies have been proposed to increase micronutrients in plants to improve food quality, but very few approaches have intrigued `clustered regularly interspaced short palindromic repeats’ (CRISPR) modules for the enhancement of iron and folate concentration in the edible parts of plants. In this review, we discuss two important approaches to simultaneously enhance the bioavailability of iron and folate concentrations in rice endosperms by utilizing advanced CRISPR-Cas9-based technology. This includes the ‘tuning of cis-elements’ and ‘enhancer re-shuffling’ in the regulatory components of genes that play a vital role in iron and folate biosynthesis/transportation pathways. In particular, base-editing and enhancer re-installation in native promoters of selected genes can lead to enhanced accumulation of iron and folate levels in the rice endosperm. The re-distribution of micronutrients in specific plant organs can be made possible using the above-mentioned contemporary approaches. Overall, the present review discusses the possible approaches for synchronized iron and folate biofortification through modification in regulatory gene circuits employing CRISPR-Cas9 technology.

Here, we present a streamlined Agrobacterium-mediated transformation protocol for jute (Corchorussp.). We describe steps to pierce and vacuum infiltrate imbibed jute seeds with Agrobacteriumsuspension. We then detail procedures for selecting transformed seeds by using a hygromycin-B-supplemented medium. This approach can achieve transformation efficiencies of 20.44% ± 1.17% and 15.55% ± 0.58% for tossa (C. olitorius) and white (C. capsularis) jute, respectively. Demanding minimal resources and time, this protocol can elevate genetic engineering research in jute fiber crops.

ABSTRACTWe successfully produced two human β-defensins (hBD-1 and hBD-2) in bacteria as functional peptides and tested their antibacterial activities against Salmonella entericaserovar Typhi, Escherichia coli, and Staphylococcus aureusemploying both spectroscopic and viable CFU count methods. Purified peptides showed approximately 50% inhibition of the bacterial population when used individually and up to 90% when used in combination. The 50% lethal doses (LD50) of hBD-1 against S.Typhi, E. coli, and S. aureuswere 0.36, 0.40, and 0.69 μg/μl, respectively, while those for hBD-2 against the same bacteria were 0.38, 0.36, and 0.66 μg/μl, respectively. Moreover, we observed that bacterium-derived antimicrobial peptides were also effective in increasing survival time and decreasing bacterial loads in the peritoneal fluid, liver, and spleen of a mouse intraperitoneally infected with S.Typhi. The 1:1 hBD-1/hBD-2 combination showed maximum effectiveness in challenging the Salmonellainfection in vitroand in vivo. We also observed less tissue damage and sepsis formation in the livers of infected mice after treatment with hBD-1 and hBD-2 peptides individually or in combination. Based on these findings, we conclude that bacterium-derived recombinant β-defensins (hBD-1 and hBD-2) are promising antimicrobial peptide (AMP)-based substances for the development of new therapeutics against typhoid fever.

Mutants in the maize defective kernel1 (dek1) gene are blocked in embryogenesis and the endosperm is chalky and lacks an aleurone layer. Here we show that intermediate alleles result in embryos that lack a shoot axis while weak alleles result in endosperms with mosaic aleurone and deformed plants with epidermal cells that resemble bulliform cells, a specialized epidermal cell type. This indicates that dek1 functions in embryonic pattern formation, cell fate specification and pattern formation in the leaf epidermis, and cell fate specification in the endosperm. Thus, thedek1 gene product appears to control different cellular-developmental processes depending on cellular context. The phenotype of the weakdek1-Dooner allele resembles the crinkly4 (cr4)mutant phenotype. Double mutants between dek1 and cr4 showed elements of epistasis, additivity and synergy, suggesting that the gene products may function in overlapping developmental processes. cr4transcript was detectable in dek1 mutant kernels indicating that DEK1 was not required for Cr4 transcript accumulation. To test whether DEK1 regulated the ligand for the CR4 receptor kinase, a genetic mosaic analysis was performed. The dek1 phenotype appeared to be generally cell-autonomous, leading to the conclusion that it was not likely to produce a diffusible signal molecule, and therefore was not likely to regulate the CR4 ligand.

A full-length transcript (FLt) promoter fragment was isolated from a genomic clone of mirabilis mosaic virus (MMV), a double-stranded DNA plant pararetrovirus belonging to the caulimovirus family. The boundaries required for maximal promoter expression were defined by 5′ and 3′ deletion analysis of the MMV promoter fragments coupled to a GUS reporter gene. The expression patterns of these chimeric gene constructs were evaluated both in transgenic Nicotiana tabacum cv. Samsun NN plants and in protoplast transient expression experiments. A 360 bp FLt promoter fragment (sequence −297 to +63 from the transcription start site) was found sufficient for strong promoter activity. The transcription start site (TSS) of the MMV FLt promoter was determined by primer extension analysis using total RNA isolated from transgenic plants containing a MMV promoter:uidA fusion gene. Analysis of the 5′ and 3′ deletion constructs showed that an upstream region (sequence −248 to −193 from the transcription start site) is required for the MMV FLt promoter activity along with the as-1, TATA box regions. In addition, a 31 bp sequence (+33 to +63 from the transcription start site) located downstream of a TATA box is also essential for the maximum expression of the MMV FLt promoter. Analysis of transcripts (mRNA) from these chimeric constructs also indicated that the MMV FLt promoter fragment (−297 to +63 from the transcription start site) has the highest promoter activity. In a comparative analysis the MMV FLt promoter showed much greater activity than the CaMV 35S promoter.