MethSemble-6mA: an ensemble-based 6mA prediction server and its application on promoter region of LBD gene family in Poaceae.

The Lateral Organ Boundaries Domain (LBD) containing genes are a set of plant-specific transcription factors and are crucial for controlling both organ development and defense mechanisms as well as anthocyanin synthesis and nitrogen metabolism. It is imperative to understand how methylation regulates gene expression, through predicting methylation sites of their promoters particularly in major crop species. In this study, we developed a user-friendly prediction server for accurate prediction of 6mA sites by incorporating a robust feature set, viz., Binary Encoding of Mono-nucleotide DNA. Our model,MethSemble-6mA, outperformed other state-of-the-art tools in terms of accuracy (93.12%). Furthermore, we investigated the pattern of probable 6mA sites at the upstream promoter regions of the LBD-containing genes in Triticum aestivum and its allied species using the developed tool. On average, each selected species had four 6mA sites, and it was found that with speciation and due course of evolution in wheat, the frequency of methylation have reduced, and a few sites remain conserved. This obviously cues gene birth and gene expression alteration through methylation over time in a species and reflects functional conservation throughout evolution. Since DNA methylation is a vital event in almost all plant developmental processes (e.g., genomic imprinting and gametogenesis) along with other life processes, our findings on epigenetic regulation of LBD-containing genes have dynamic implications in basic and applied research. Additionally, MethSemble-6mA (http://cabgrid.res.in:5799/) will serve as a useful resource for a plant breeders who are interested to pursue epigenetic-based crop improvement research.

Identification, characterization, and comprehensive expression profiling of floral master regulators in pigeon pea (Cajanus cajan [L.] Millspaugh).

Pigeon pea is an important protein-rich pulse crop. Identification of flowering master regulators in pigeon pea is highly imperative as indeterminacy and late flowering are impediments towards yield improvement. A genome-wide analysis was performed to explore flowering orthologous groups in pigeon pea. Among the 412 floral orthologs identified in pigeon pea, 148 genes belong to the meristem identity, photoperiod-responsive, and circadian clock-associated ortholog groups. Our comparative genomics study revealed purifying selection pressures (ka/ks) on floral orthologs, and duplication patterns and evolution through synteny with other model species. Phylogenetic analysis of floral genes substantiated a connection between pigeon pea plant architecture and flowering time as all the PEBP domain-containing genes belong to meristem identity floral networks of pigeon pea. Expression profiling of eleven major orthologs in contrasting determinate and indeterminate genotypes indicated that these orthologs might be involved in flowering regulation. Expression of floral inducer, FT, and floral repressor, TFL1, was non-comparable in indeterminate genotypes across all the developmental stages of pigeon pea. However, dynamic FT/TFL1 expression ratio detected in all tissues of both the genotypes suggested their role in floral transition. One TFL1 ortholog having high sequence conserveness across pigeon pea genotypes showed differential expression indicating genotype-dependent regulation of this ortholog. Presence of conserved 6mA-methylation patterns in light-responsive elements and in other cis-regulatory elements of FT and TFL1 across different plant genotypes indicated possible involvement of epigenetic regulation in flowering.

Host-Delivered RNA Interference for Durable Pest Resistance in Plants: Advanced Methods, Challenges, and Applications.

Insect-pests infestation greatly affects global agricultural production and is projected to become more severe in upcoming years. There is concern about pesticide application being ineffective due to insect resistance and environmental toxicity. Reduced effectiveness of Bt toxins also made the scientific community shift toward alternative strategies to control devastating agricultural pests. With the advent of host-delivered RNA interference, also known as host-induced gene silencing, targeted insect genes have been suppressed through genetic engineering tools to deliver a novel insect-pest resistance strategy for combating a number of agricultural pests. This review recapitulates the possible mechanism of host-delivered RNA interference (HD-RNAi), in particular, the silencing of target genes of insect-pests. We emphasize the development of the latest strategies against evolving insect targets including designing of artificial microRNAs, vector constructs, and the benefit of using plastid transformation to transform target RNA-interfering genes. Advantages of using HD-RNAi over other small RNA delivery modes and also the supremacy of HD-RNAi over the CRISPR-Cas system particularly for insect resistance have been described. However, the broader application of this technology is restricted due to its several limitations. Using artificial miRNA designs, the host-delivered RNAi + Bt combinatorial approach and chloroplast transformation can overcome limitations of RNAi. With careful design and delivery approaches, RNAi promises to be extremely valuable and effective plant protection strategy to attain durable insect-pest resistance in crops. Development of transgenic plant using novel strategies to achieve durable resistance against the target insect.

Amelioration of cold-induced sweetening in potato by RNAi mediated silencing of StUGPase encoding UDP-glucose pyrophosphorylase.

Cold-induced sweetening (CIS) is an unwanted physiological phenomenon in which reducing sugars (RS) get accumulated in potato (Solanum tuberosum) upon cold storage. High RS content makes potato commercially unsuitable for processing due to the unacceptable brown color in processed products like chips, fries, etc., and the production of a potential carcinogen, acrylamide. UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose towards the synthesis of sucrose and is also involved in the regulation of CIS in potato. The objective of the present work was RNAi-mediated downregulation of the StUGPase expression level in potato for the development of CIS tolerant potato. Hairpin RNA (hpRNA) gene construct was developed by placing UGPase cDNA fragment in sense and antisense orientation intervened by GBSS intron. Internodal stem explants (cv. Kufri Chipsona-4) were transformed with hpRNA gene construct, and 22 transgenic lines were obtained by PCR screening of putative transformants. Four transgenic lines showed the highest level of RS content reduction following 30 days of cold storage, with reductions in sucrose and RS (glucose & fructose) levels of up to 46% and 57.5%, respectively. Cold stored transgenic potato of these four lines produced acceptable chip colour upon processing. The selected transgenic lines carried two to five copies of the transgene. Northern hybridization revealed an accumulation of siRNA with a concomitant decrease in the StUGPase transcript level in these selected transgenic lines. The present work demonstrates the efficacy of StUGPase silencing in controlling CIS in potato, and the strategy can be employed for the development of CIS tolerant potato varieties.

Strategic transgene-free approaches of CRISPR-based genome editing in plants.

Genome editing through the alteration of nucleotide sequence has already revolutionized the field of site-directed mutagenesis for a decade. However, research in terms of precision and efficacy in targeting the loci and reduction in off-target mutation has always been a priority when DNA is involved. Therefore, recent research interest lies in utilizing the same precision technology but results in non-transgenic. In this review article, different technological advancements have been explained, which may provide a holistic concept of and need for transgene-free genome editing. The advantage and lacunas of each technology have been critically discussed to deliver a transparent view to the readers. A systematic analysis and evaluation of published research articles implied that researchers across the globe are putting continuous efforts in this direction to eliminate the hindrance of transgenic regulation. Nevertheless, this approach has severe implications legitimate for mitigating the conflict of acceptance, reliability, and generosity of gene-editing technology and sustainably retorting to the expanding global population feeding challenges.