CRISPR/Cas9-mediated editing of phytoene desaturase (PDS) gene in an important staple crop, potato.

The gene editing using the CRISPR/Cas9 system has become an important biotechnological tool for studying gene function and improving crops. In this study, we have used CRISPR/Cas9 system for editing the phytoene desaturase gene (PDS) in popular Indian potato cultivar Kufri Chipsona-I. A construct (pHSE401) carrying two target gRNAs with glycine tRNA processing system under the control of Arabidopsis U6 promoter and the Cas9 protein was constructed and transformed in potato plants using Agrobacterium-mediated genetic transformations. The regeneration efficiency of 45% was observed in regenerated plants, out of which 81% of the putative transformants shoot lines exhibited mutant or bleached phenotype (albinism). The deletion mutations were detected within the StPDS gene in the genotyped plants and a mutation efficiency of 72% for gRNA1 and gRNA2 has been detected using Sanger sequencing. Hence, we set up a CRISPR/Cas9-mediated genome editing protocol which is efficient and generates mutations (deletions) within StPDS gene in potato. The bleached phenotype is easily detectable after only few weeks after Agrobacterium-mediated transformation. This is the first report as a proof of concept for CRISPR/Cas9-based editing of PDS gene in Indian potato cv. Kufri Chipsona-I. This study demonstrates that CRISPR/Cas9 can be used to edit genes at high frequency within the genome of the potato for various traits. Therefore, this study will aid in creating important mutants for modifying molecular mechanisms controlling traits of agronomic importance.

Spraying of dsRNA molecules derived from Phytophthora infestans, along with nanoclay carriers as a proof of concept for developing novel protection strategy for potato late blight.

BACKGROUND: Phytophthora infestans is a late blight-causing oomycetes pathogen. It rapidly evolves and adapts to the host background and new fungicide molecules within a few years of their release, most likely because of the predominance of transposable elements in its genome. Frequent applications of fungicides cause environmental concerns. Here, we developed target-specific RNA interference (RNAi)-based molecules, along with nanoclay carriers, that when sprayed on plants are capable of effectively reducing late blight infection. RESULTS: Targeted the genes unique to sporulation, early satge infection and the metabolism pathway stages based on in an our own microarray data. We used nanoclay as a carrier for sorbitol dehydrogenase, heat shock protein 90, translation elongation factor 1-α, phospholipase-D like 3 and glycosylphosphatidylinositol-anchored acidic serine-threonine-rich HAM34-like protein double-stranded (ds)RNAs, which were assessed by culture bioassay, detached leaf assay and spray methods, and revealed a reduction in growth, sporulation and symptom expression. Plants sprayed with multigene targeted dsRNA-nanoclay showed enhanced disease resistance (4% disease severity) and less sporulation (<1 × 103 ) compared with plants sprayed with dsRNA alone. CONCLUSION: The use of nanoclay with multigene targeted dsRNA was assumed to be involved in effective delivery, protection and boosting the action of RNAi as a spray-induced gene silencing approach (SIGS). A significant reduction in growth, sporulation, disease severity and decreased gene expression authenticates the effects of SIGS on late blight progression. This study demonstrated as a proof of concept the dsRNA-nanoclay SIGS approach, which could be used as an alternative to chemical fungicides and transgenic approaches to develop an environmentally friendly novel plant protection strategy for late blight. © 2022 Society of Chemical Industry.

Validation of molecular response of tuberization in response to elevated temperature by using a transient Virus Induced Gene Silencing (VIGS) in potato.

Temperature plays an important role in potato tuberization. The ideal night temperature for tuber formation is ~17 °C while temperature beyond 22 °C drastically reduces the tuber yield. Moreover, high temperature has several undesirable effects on the plant and tubers. Investigation of the genes involved in tuberization under heat stress can be helpful in the generation of heat-tolerant potato varieties. Five genes, including StSSH2 (succinic semialdehyde reductase isoform 2), StWTF (WRKY transcription factor), StUGT (UDP-glucosyltransferase), StBHP (Bel1 homeotic protein), and StFLTP (FLOWERING LOCUS T protein), involved in tuberization and heat stress in potato were investigated. The results of our microarray analysis suggested that these genes regulate and function as transcriptional factors, hormonal signaling, cellular homeostasis, and mobile tuberization signals under elevated temperature in contrasting KS (Kufri Surya) and KCM (Kufri Chandramukhi) potato cultivars. However, no detailed report is available which establishes functions of these genes in tuberization under heat stress. Thus, the present study was designed to validate the functions of these genes in tuber signaling and heat tolerance using virus-induced gene silencing (VIGS). Results indicated that VIGS transformed plants had a consequential reduction in StSSH2, StWTF, StUGT, StBHP, and StFLTP transcripts compared to the control plants. Phenotypic observations suggest an increase in plant senescence, reductions to both number and size of tubers, and a decrease in plant dry matter compared to the control plants. We also establish the potency of VIGS as a high-throughput technique for functional validation of genes.

Functional genomic approaches to improve crop plant heat stress tolerance.

Heat stress as a yield limiting issue has become a major threat for food security as global warming progresses. Being sessile, plants cannot avoid heat stress. They respond to heat stress by activating complex molecular networks, such as signal transduction, metabolite production and expressions of heat stress-associated genes. Some plants have developed an intricate signalling network to respond and adapt it. Heat stress tolerance is a polygenic trait, which is regulated by various genes, transcriptional factors, proteins and hormones. Therefore, to improve heat stress tolerance, a sound knowledge of various mechanisms involved in the response to heat stress is required. The classical breeding methods employed to enhance heat stress tolerance has had limited success. In this era of genomics, next generation sequencing techniques, availability of genome sequences and advanced biotechnological tools open several windows of opportunities to improve heat stress tolerance in crop plants. This review discusses the potential of various functional genomic approaches, such as genome wide association studies, microarray, and suppression subtractive hybridization, in the process of discovering novel genes related to heat stress, and their functional validation using both reverse and forward genetic approaches. This review also discusses how these functionally validated genes can be used to improve heat stress tolerance through plant breeding, transgenics and genome editing approaches.

Recent trends and perspectives of molecular markers against fungal diseases in wheat.

Wheat accounts for 19% of the total production of major cereal crops in the world. In view of ever increasing population and demand for global food production, there is an imperative need of 40-60% increase in wheat production to meet the requirement of developing world in coming 40 years. However, both biotic and abiotic stresses are major hurdles for attaining the goal. Among the most important diseases in wheat, fungal diseases pose serious threat for widening the gap between actual and attainable yield. Fungal disease management, mainly, depends on the pathogen detection, genetic and pathological variability in population, development of resistant cultivars and deployment of effective resistant genes in different epidemiological regions. Wheat protection and breeding of resistant cultivars using conventional methods are time-consuming, intricate and slow processes. Molecular markers offer an excellent alternative in development of improved disease resistant cultivars that would lead to increase in crop yield. They are employed for tagging the important disease resistance genes and provide valuable assistance in increasing selection efficiency for valuable traits via marker assisted selection (MAS). Plant breeding strategies with known molecular markers for resistance and functional genomics enable a breeder for developing resistant cultivars of wheat against different fungal diseases.