"Genome-wide identification of bZIP gene family in Pearl millet and transcriptional profiling under abiotic stress, phytohormonal treatments; and functional characterization of PgbZIP9".

Abiotic stresses are major constraints in crop production, and are accountable for more than half of the total crop loss. Plants overcome these environmental stresses using coordinated activities of transcription factors and phytohormones. Pearl millet an important C4 cereal plant having high nutritional value and climate resilient features is grown in marginal lands of Africa and South-East Asia including India. Among several transcription factors, the basic leucine zipper (bZIP) is an important TF family associated with diverse biological functions in plants. In this study, we have identified 98 bZIP family members (PgbZIP) in pearl millet. Phylogenetic analysis divided these PgbZIP genes into twelve groups (A-I, S, U and X). Motif analysis has shown that all the PgbZIP proteins possess conserved bZIP domains and the exon-intron organization revealed conserved structural features among the identified genes. Cis-element analysis, RNA-seq data analysis, and real-time expression analysis of PgbZIP genes suggested the potential role of selected PgbZIP genes in growth/development and abiotic stress responses in pearl millet. Expression profiling of selected PgbZIPs under various phytohormones (ABA, SA and MeJA) treatment showed differential expression patterns of PgbZIP genes. Further, PgbZIP9, a homolog of AtABI5 was found to localize in the nucleus and modulate gene expression in pearl millet under stresses. Our present findings provide a better understanding of bZIP genes in pearl millet and lay a good foundation for the further functional characterization of multi-stress tolerant PgbZIP genes, which could become efficient tools for crop improvement.

Protocol for imbibed seed piercing for Agrobacterium-mediated transformation of jute.

Here, we present a streamlined Agrobacterium-mediated transformation protocol for jute (Corchorus sp.). We describe steps to pierce and vacuum infiltrate imbibed jute seeds with Agrobacterium suspension. 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. For complete details on the use and execution of this protocol, please refer to Majumder et al. (2020).1.

Unraveling the involvement of WRKY TFs in regulating plant disease defense signaling.

MAIN CONCLUSION: This review article explores the intricate role, regulation, and signaling mechanisms of WRKY TFs in response to biotic stress, particularly emphasizing their pivotal role in the trophism of plant-pathogen interactions. Transcription factors (TFs) play a vital role in governing both plant defense and development by controlling the expression of various downstream target genes. Early studies have shown the differential expression of certain WRKY transcription factors by microbial infections. Several transcriptome-wide studies later demonstrated that diverse sets of WRKYs are significantly activated in the early stages of viral, bacterial, and fungal infections. Furthermore, functional investigations indicated that overexpression or silencing of certain WRKY genes in plants can drastically alter disease symptoms as well as pathogen multiplication rates. Hence the new aspects of pathogen-triggered WRKY TFs mediated regulation of plant defense can be explored. The already recognized roles of WRKYs include transcriptional regulation of defense-related genes, modulation of hormonal signaling, and participation in signal transduction pathways. Some WRKYs have been shown to directly bind to pathogen effectors, acting as decoys or resistance proteins. Notably, the signaling molecules like salicylic acid, jasmonic acid, and ethylene which are associated with plant defense significantly increase the expression of several WRKYs. Moreover, induction of WRKY genes or heightened WRKY activities is also observed during ISR triggered by the beneficial microbes which protect the plants from subsequent pathogen infection. To understand the contribution of WRKY TFs towards disease resistance and their exact metabolic functions in infected plants, further studies are required. This review article explores the intrinsic transcriptional regulation, signaling mechanisms, and hormonal crosstalk governed by WRKY TFs in plant disease defense response, particularly emphasizing their specific role against different biotrophic, hemibiotrophic, and necrotrophic pathogen infections.

CRISPR-mediated iron and folate biofortification in crops: advances and perspectives.

Micronutrient 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.

Synthetic sub-genomic transcript promoter from Horseradish Latent Virus (HRLV).

MAIN CONCLUSION: We characterized an efficient chimeric sub-genomic transcript promoter from Horseradish Latent Virus, FHS4, active in both dicot and monocot plants, and it could be a potential tool for plant biotechnology. Plant pararetroviruses are a rich source of novel plant promoters widely used for biotechnological applications. Here, we comprehensively characterized a unique sub-genomic transcript (Sgt) promoter of Horseradish Latent Virus (HRLV) and identified a fragment (HS4; - 340 to + 10; 351 bp) that showed the highest expression of reporter genes in both transient and transgenic assays as evidenced by biochemical, histochemical GUS reporter assay and transcript analysis of uidA gene by qRT-PCR. Phylogenetic analysis showed that the HSgt promoter was closely related to the sub-genomic promoter of the Cauliflower Mosaic Virus (CaMV19S). We found that the as-1 element and W-box played an important role in the transcriptional activity of the HS4 promoter. Furthermore, the HS4 promoter was also induced by salicylic acid. Alongside, we enhanced the activity of the HS4 promoter by coupling the enhancer region from Figwort Mosaic Virus (FMV) promoter to the upstream region of it. This hybrid promoter FHS4 was around 1.1 times stronger than the most commonly used promoter, 35S (Cauliflower Mosaic Virus full-length transcript promoter), and was efficient in driving reporter genes in both dicot and monocot plants. Subsequently, transgenic tobacco plants expressing an anti-microbial peptide BrLTP2.1 (Brassica rapa lipid transport protein 2.1), under the control of the FHS4 promoter, were developed. The in vitro anti-fungal assay revealed that the plant-derived BrLTP2.1 protein driven by an FHS4 promoter manifested increased resistance against an important plant fungal pathogen, Alternaria alternata. Finally, we concluded that the FHS4 promoter can be used as an alternative to the 35S promoter and has a high potential to become an efficient tool in plant biotechnology.

MYB Transcription Factor Family in Pearl Millet: Genome-Wide Identification, Evolutionary Progression and Expression Analysis under Abiotic Stress and Phytohormone Treatments.

Transcription factors (TFs) are the regulatory proteins that act as molecular switches in controlling stress-responsive gene expression. Among them, the MYB transcription factor family is one of the largest TF family in plants, playing a significant role in plant growth, development, phytohormone signaling and stress-responsive processes. Pearl millet (Pennisetum glaucum L.) is one of the most important C4 crop plants of the arid and semi-arid regions of Africa and Southeast Asia for sustaining food and fodder production. To explore the evolutionary mechanism and functional diversity of the MYB family in pearl millet, we conducted a comprehensive genome-wide survey and identified 279 MYB TFs (PgMYB) in pearl millet, distributed unevenly across seven chromosomes of pearl millet. A phylogenetic analysis of the identified PgMYBs classified them into 18 subgroups, and members of the same group showed a similar gene structure and conserved motif/s pattern. Further, duplication events were identified in pearl millet that indicated towards evolutionary progression and expansion of the MYB family. Transcriptome data and relative expression analysis by qRT-PCR identified differentially expressed candidate PgMYBs (PgMYB2, PgMYB9, PgMYB88 and PgMYB151) under dehydration, salinity, heat stress and phytohormone (ABA, SA and MeJA) treatment. Taken together, this study provides valuable information for a prospective functional characterization of the MYB family members of pearl millet and their application in the genetic improvement of crop plants.

Vignette of Vigna domestication: From archives to genomics.

The genus Vigna comprises fast-growing, diploid legumes, cultivated in tropical and subtropical parts of the world. It comprises more than 200 species among which Vigna angularis, Vigna radiata, Vigna mungo, Vigna aconitifolia, Vigna umbellata, Vigna unguiculata, and Vigna vexillata are of enormous agronomic importance. Human selection along with natural variability within these species encompasses a vital source for developing new varieties. The present review convokes the early domestication history of Vigna species based on archeological pieces of evidence and domestication-related traits (DRTs) together with genetics of domestication. Traces of early domestication of Vigna have been evidenced to spread across several temperate and tropical regions of Africa, Eastern Asia, and few parts of Europe. Several DRTs of Vigna species, such as pod shattering, pod and seed size, dormancy, seed coat, seed color, maturity, and pod dehiscence, can clearly differentiate wild species from their domesticates. With the advancement in next-generation high-throughput sequencing techniques, exploration of genetic variability using recently released reference genomes along with de novo sequencing of Vigna species have provided a framework to perform genome-wide association and functional studies to figure out different genes related to DRTs. In this review, genes and quantitative trait loci (QTLs) related to DRTs of different Vigna species have also been summarized. Information provided in this review will enhance the in-depth understanding of the selective pressures that causes crop domestication along with nature of evolutionary selection made in unexplored Vigna species. Furthermore, correlated archeological and domestication-related genetic evidence will facilitate Vigna species to be considered as suitable model plants.

Further Characterization of MUAS35SCP and FUAS35SCP Recombinant Promoters and Their Implication in Translational Research.

Recombinant promoters are of high value in translational research. Earlier, we developed two recombinant promoters, namely MUAS35SCP and FUAS35SCP, and their transcriptional activities were found to be stronger than that of the most widely used CaMV35S promoter in dicot plants. Presently, we are reporting constitutive expression of both GUS and GFP reporters under the control of these promoters in several monocots, including rice, wheat, and pearl millet. We observed that these promoters could express the reporter genes constitutively, and their expression abilities were almost equal to that of the CaMV35S2 promoter. Plant-derived enriched PaDef (Persea americana var. drymifolia defensin) and NsDef2 (Nigella sativa L. defensin 2) antimicrobial peptides expressed under the control of these promoters arrest the growth of devastating phytopathogens like Pseudomonas syringae, Rhodococcus fascians, and Alternaria alternata. We observed that plant-derived NsDef2 and PaDef under control of these promoters showed approximately 80-90% inhibitory activity against Pseudomonas syringae. Hence, these promoters were constitutive and universal, as they can drive the expression of transgenes in both dicot and monocot plants. Alongside, these promoters could become a valuable tool for raising genetically modified plants with in-built resistance toward phytopathogens.

Engineering of Plants for Efficient Production of Therapeutics.

Plants are becoming useful platforms for recombinant protein production at present time. With the advancement of efficient molecular tools of genomics, proteomics, plants are now being used as a biofactory for production of different life saving therapeutics. Plant-based biofactory is an established production system with the benefits of cost-effectiveness, high scalability, rapid production, enabling post-translational modification, and being devoid of harmful pathogens contamination. This review introduces the main challenges faced by plant expression system: post-translational modifications, protein stability, biosafety concern and regulation. It also summarizes essential factors to be considered in engineering plants, including plant expression system, promoter, post-translational modification, codon optimization, and fusion tags, protein stabilization and purification, subcellular targeting, and making vaccines in an edible way. This review will be beneficial and informative to scholars and readers in the field of plant biotechnology.

Comprehensive identification and expression analysis of GRAS gene family under abiotic stress and phytohormone treatments in Pearl millet.

Pearl millet is an important C4 cereal plant that possesses enormous capacity to survive under extreme climatic conditions. It serves as a major food source for people in arid and semiarid regions of south-east Asia and Africa. GRAS is an important transcription factor gene family of plant that play a critical role in regulating developmental processes, stress responses and phytohormonal signalling. In the present study, we have identified a total number of 57 GRAS members in pearl millet. Phylogenetic analysis clustered all the PgGRAS genes into eight groups (GroupI-GroupVIII). Motif analysis has shown that all the PgGRAS proteins had conserved GRAS domains and gene structure analysis revealed a high structural diversity among PgGRAS genes. Expression patterns of PgGRAS genes in different tissues (leaf, stem and root) and under various abiotic stress (drought, heat and salinity) were determined. Further, expression analysis was also carried out in response to various hormones (SA, MeJA, GA and ABA). The results provide a clear understanding of GRAS transcription factor family in pearl millet, and lay a good foundation for the functional characterisation of GRAS genes in pearl millet.

Synthetic promoters from blueberry red ringspot virus (BRRV).

MAIN CONCLUSION: We analyzed the synthetic full-length transcript promoter of Blueberry red ringspot virus (BRRV) and developed two chimeric promoters (MBR3 and FBR3). Transcriptional activities of these chimeric promoters were found equivalent to that of the CaMV35S2 promoter. Chimeric promoters driven plant-derived PaDef protein showed high antimicrobial activities against several pathogens. Blueberry red ringspot virus (BRRV) is a pararetrovirus under the genus, Soymovirus belongs to the Caulimoviridae family. We have made a synthetic version of the BRRV-Flt promoter and analyzed its activity in detail. A 372 bp promoter fragment BR3 (- 212 to + 160) showed the strongest transcriptional activity compared with other fragments in both transient and transgenic assays; its activity was found near equivalent to that of the CaMV35S promoter. We constructed two chimeric promoters; MBR3 and FBR3 by fusing the UASs (Upstream activation sequences) of Mirabilis mosaic virus (MUAS; - 297 to - 38; 335 bp) and Figwort mosaic virus (FUAS; - 249 to - 54; 303 bp) respectively to the core promoter domain of BR3 (BR3; - 212 to + 160; 372 bp). The activities of MBR3 and FBR3 promoters were found equivalent to that of the activity of the CaMV35S2 promoter and approximately 4.0 (four) times stronger than that of the CaMV35S promoter. Histochemical and fluorometric GUS assays confirmed the above observation. The transcriptional efficacies of these recombinant promoters were tested by evaluating the antibacterial and antifungal activities of recombinant plant-derived antimicrobial peptide Persea americana var. drymifolia defensin (PaDef) driven under these promoters. Bioassays showed promising antifungal activities of the plant made PaDef against Alternaria alternata and antibacterial property against Gram-positive (S. aureus and R. fascians) and Gram-negative bacteria (E. coli and P. aeruginosa). Based upon the above results, MBR3 and FBR3 could be useful promoters for plant genetic engineering and can become useful substitutes for the widely used CaMV35S2 promoter in plant biology.

Synthetic Promoters from Strawberry Vein Banding Virus (SVBV) and Dahlia Mosaic Virus (DaMV).

We have constructed two intra-molecularly shuffled promoters, namely S100 and D100. The S100 recombinant promoter (621 bp) was generated by ligation of 250 bp long upstream activation sequence (UAS) of Strawberry vein banding virus (SV10UAS; - 352 to - 102 relative to TSS) with its 371 bp long TATA containing core promoter domain (SV10CP; - 352 to + 19). Likewise, 726 bp long D100 promoter was constructed by fusion of 170 bp long UAS of Dahlia mosaic virus (DaMV14UAS; - 203 to - 33) with its 556 bp long core promoter domain (DaMV4CP; - 474 to + 82). S100 and D100 promoters showed 1.8 and 2.2 times stronger activities than that of the CaMV35S promoter. The activity of the promoters is comparable to that of the CaMV35S2 promoter. Transcript analysis employing qRT-PCR and histochemical assays supported the above findings. Abscisic acid and salicylic acid induce the activity of the D100 promoter. Leaf protein obtained from Nicotiana tabacum plant expressing NSD2 gene (Nigella sativa L. defensin 2) driven by the D100 promoter showed antifungal activity against Alternaria alternata and Phoma exigua var. exigua and antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus. Strong S100 and D100 promoters have potential to become efficient candidates for plant metabolic engineering and molecular pharming.

Development of efficient synthetic promoters derived from pararetrovirus suitable for translational research.

MAIN CONCLUSION: In this study, useful hybrid promoters were developed for efficient ectopic gene expression in monocot and dicot plants, and they hold strong prominence in both transgenic research and biotech industries. This study deals with developing novel synthetic promoters derived from Rice Tungro Bacilliform Virus (RTBV) and Mirabilis Mosaic Virus (MMV). Despite numerous availability, there is a severe scarcity of promoters universally suitable for monocot and dicot plants. Here, eight chimeric promoter constructs were synthesized as gBlocks gene fragments through domain swapping and hybridization by incorporating important domains of previously characterized RTBV and MMV promoters. The developed promoter constructs were assessed for transient GUS expression in tobacco protoplast (Xanthi Brad) and agro-infiltrated tobacco, petunia, rice and pearl millet. Protoplast expression analysis showed that two promoter constructs, namely pUPMA-RP1-MP1GUS and pUPMA-RP4-MP1GUS exhibited 3.56 and 2.5 times higher activities than that of the CaMV35S promoter. We had observed the similar type of expression patterns of these promoters in agroinfiltration-based transient studies. RP1-MP1 and RP4-MP1 promoters exhibited 1.87- and 1.68-fold increase expression in transgenic tobacco plants; while, a 1.95-fold increase was found in RP1-MP1 transgenic rice plants when compared their activities with CaMV35S promoter. Furthermore, on evaluating these promoter constructs for their expression in the bacterial system, pUPMA-RP1-MP1GFP was found to have the highest GFP expression. Moreover, the promoter construct was also evaluated for its capacity to express the HMP3 gene. Biobeads of encapsulated bacterial cells expressing HMP3 gene under control of the pUPMA-RP4-MP1 promoter were found to reduce 72.9% copper and 29.2% zinc concentration from wastewater. Our results had demonstrated that the developed promoter constructs could be used for translational research in dicot, monocot plants and bacterial systems for efficient gene expression.

Genome-wide identification and expression analysis of WRKY transcription factors in pearl millet (Pennisetum glaucum) under dehydration and salinity stress.

BACKGROUND: Plants have developed various sophisticated mechanisms to cope up with climate extremes and different stress conditions, especially by involving specific transcription factors (TFs). The members of the WRKY TF family are well known for their role in plant development, phytohormone signaling and developing resistance against biotic or abiotic stresses. In this study, we performed a genome-wide screening to identify and analyze the WRKY TFs in pearl millet (Pennisetum glaucum; PgWRKY), which is one of the most widely grown cereal crops in the semi-arid regions. RESULTS: A total number of 97 putative PgWRKY proteins were identified and classified into three major Groups (I-III) based on the presence of WRKY DNA binding domain and zinc-finger motif structures. Members of Group II have been further subdivided into five subgroups (IIa-IIe) based on the phylogenetic analysis. In-silico analysis of PgWRKYs revealed the presence of various cis-regulatory elements in their promoter region like ABRE, DRE, ERE, EIRE, Dof, AUXRR, G-box, etc., suggesting their probable involvement in growth, development and stress responses of pearl millet. Chromosomal mapping evidenced uneven distribution of identified 97 PgWRKY genes across all the seven chromosomes of pearl millet. Synteny analysis of PgWRKYs established their orthologous and paralogous relationship among the WRKY gene family of Arabidopsis thaliana, Oryza sativa and Setaria italica. Gene ontology (GO) annotation functionally categorized these PgWRKYs under cellular components, molecular functions and biological processes. Further, the differential expression pattern of PgWRKYs was noticed in different tissues (leaf, stem, root) and under both drought and salt stress conditions. The expression pattern of PgWRKY33, PgWRKY62 and PgWRKY65 indicates their probable involvement in both dehydration and salinity stress responses in pearl millet. CONCLUSION: Functional characterization of identified PgWRKYs can be useful in delineating their role behind the natural stress tolerance of pearl millet against harsh environmental conditions. Further, these PgWRKYs can be employed in genome editing for millet crop improvement.

Enhancement of ABA Sensitivity Through Conditional Expression of the ARF10 Gene in Brassica juncea Reveals Fertile Plants with Tolerance Against Alternaria brassicicola.

Concomitant increase of auxin-responsive factors ARF16 and ARF17, along with enhanced expression of ARF10 in resistant Sinapis alba compared with that in susceptible Brassica juncea upon challenge with Alternaria brassicicola, revealed that abscisic acid (ABA)-auxin crosstalk is a critical factor for resistance response. Here, we induced the ABA response through conditional expression of ARF10 in B. juncea using the A. brassicicola-inducible GH3.3 promoter. Induced ABA sensitivity caused by conditional expression of ARF10 in transgenic B. juncea resulted in tolerance against A. brassicicola and led to enhanced expression of several ABA-responsive genes without affecting the auxin biosynthetic gene expression. Compared with ABI3 and ABI4, ABI5 showed maximum upregulation in the most tolerant transgenic lines upon pathogen challenge. Moreover, elevated expression of ARF10 by different means revealed a direct correlation between ARF10 expression and the induction of ABI5 protein in B. juncea. Through in vitro DNA-protein experiments and chromosome immunoprecipitation using the ARF10 antibody, we demonstrated that ARF10 interacts with the auxin-responsive elements of the ABI5 promoter. This suggests that ARF10 may function as a modulator of ABI5 to induce ABA sensitivity and mediate the resistance response against A. brassicicola.

Identification of Novel Pararetroviral Promoters for Designing Efficient Plant Gene Expression Systems.

Plant-infecting viruses, particularly the Pararetroviruses, have been used for many years as versatile genetic resources to design efficient plant expression vectors. The Pararetroviruses (members of the Caulimoviridae) typically contain two transcriptional promoters (the sub-genomic transcript promoter and the full-length transcript promoter) and 6-7 overlapping open reading frames (ORFs) with a genome size of 7-9 kB. Their promoter elements have been extensively exploited during the last two decades to construct effective gene expression systems. At the same time, the caulimoviral promoters have also been genetically manipulated with different molecular approaches to develop synthetic "chimeras" exhibiting precise functionality. Native and "tailor-made" synthetic promoters of Pararetroviruses are particularly attractive for formulating unique gene expression cassettes that perform extremely well in gene-stacking and gene-pyramiding in plant cells. In this chapter, we will mainly discuss important protocols associated with identifying novel/unique pararetroviral promoters that have optimal lengths with appropriate activities for developing efficient plant gene expression systems.

Biochemical and Molecular Characterization of Novel Pararetroviral Promoters in Plants.

Special attention needs to be given to defining and studying the regulatory apparatus of different pararetroviral promoters under various physiological conditions because they have significant sequence heterogeneity and unique distributions of stress-responsive cis-elements. Transcriptional regulation studies of a pararetroviral promoter involve both gene expression analyses and investigation of its structural/regulatory framework. The expression of reporter genes such as ß-Glucuronidase (GUS) or Luciferase (LUC) transcriptionally fused to a promoter usually determines the strength or function of a target promoter. In parallel, DNA-protein interaction studies are employed to assess the functional relevance of predicted transcription factor binding sites in target pararetroviral promoter sequences. In this chapter, we will describe protocols used to determine the transgene integration and expression in transgenic plant systems. Alongside, we will also discuss the fusion reporter assays that can determine the promoter activity and DNA-protein interaction studies that aid in the evaluation of its transcriptional regulation.

Synthetic Salicylic acid inducible recombinant promoter for translational research.

In the present study, we have developed an inter-molecularly shuffled caulimoviral promoter for protein over-expression by placing the Upstream Activation Sequence (UAS) of Figwort Mosaic Virus (FMV; -249 to -54) at the 5'-end of the Cassava Vein Mosaic Virus (CsVMV) promoter fragment 8 (CsVMV8; -215 to +166) to design a hybrid promoter; FUASCsV8CP. The FUASCsV8CP promoter exhibited approximately 2.1 and 2.0 times higher GUS-activities than that obtained from the CaMV35S promoter, in tobacco (Xanthi Brad) protoplasts and in Agroinfiltration assays respectively. Hereto, when FUASCsV8CP was assayed using transgenic tobacco plants (T2- generation), it showed 2.0 times stronger activity than CaMV35S promoter and almost equivalent activity to that of CaMV35S2 promoter. The promoter displayed Salicylic acid (SA) inducibility and hence can also be used for ensuring effective gene expression in plants under constitutive as well as specific inducible conditions. Furthermore, FUASCsV8CP was used to drive the expression of victoviral Vin gene (encoding Victoriocin) transiently in tobacco. The recombinant Victoriocin could be successfully detected by western blotting three days post infiltration. Also, the in vitro Agar-based killing zone assays employing plant-derived Victoriocin-His (obtained from transient expression of Vin) revealed enhanced antifungal activity of Victoriocin against hemi-biotrophic pathogen Phoma exigua Desm. var. exigua.

cis-trans Engineering: Advances and Perspectives on Customized Transcriptional Regulation in Plants.

Coordinated transcriptional control employing synthetic promoters and transcription factors (TFs) can be used to achieve customized regulation of gene expression in planta. Synthetic promoter technology has yielded a series of promoters with modified cis-regulatory elements that provide useful tools for efficient modulation of gene expression. In addition, the use of zinc fingers (ZFs), transcription activator-like effectors (TALEs), and catalytically inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) has made it feasible to engineer TFs that can produce targeted gene expression regulation; these approaches are particularly effective when artificial TFs are coupled with transcriptional activators or repressors. This review focuses on strategies used to engineer both promoters and TFs in the context of targeted transcriptional regulation. We also discuss the creation of synthetic inducible platforms, which can be used to impart stress tolerance to plants. We propose that combinatorial "cis-trans engineering" using a CRISPR-dCas9-based bipartite module could be used to regulate the expression of multiple target genes. This approach provides an attractive tool for introduction of specific qualitative traits into plants, thus enhancing their overall environmental adaptability.

Recombinant Promoter (MUASCsV8CP) Driven Totiviral Killer Protein 4 (KP4) Imparts Resistance Against Fungal Pathogens in Transgenic Tobacco.

Development of disease-resistant plant varieties achieved by engineering anti-microbial transgenes under the control of strong promoters can suffice the inhibition of pathogen growth and simultaneously ensure enhanced crop production. For evaluating the prospect of such strong promoters, we comprehensively characterized the full-length transcript promoter of Cassava Vein Mosaic Virus (CsVMV; -565 to +166) and identified CsVMV8 (-215 to +166) as the highest expressing fragment in both transient and transgenic assays. Further, we designed a new chimeric promoter 'MUASCsV8CP' through inter-molecular hybridization among the upstream activation sequence (UAS) of Mirabilis Mosaic Virus (MMV; -297 to -38) and CsVMV8, as the core promoter (CP). The MUASCsV8CP was found to be ∼2.2 and ∼2.4 times stronger than the CsVMV8 and CaMV35S promoters, respectively, while its activity was found to be equivalent to that of the CaMV35S2 promoter. Furthermore, we generated transgenic tobacco plants expressing the totiviral 'Killer protein KP4' (KP4) under the control of the MUASCsV8CP promoter. Recombinant KP4 was found to accumulate both in the cytoplasm and apoplast of plant cells. The agar-based killing zone assays revealed enhanced resistance of plant-derived KP4 against two deuteromycetous foliar pathogenic fungi viz. Alternaria alternata and Phoma exigua var. exigua. Also, transgenic plants expressing KP4 inhibited the growth progression of these fungi and conferred significant fungal resistance in detached-leaf and whole plant assays. Taken together, we establish the potential of engineering "in-built" fungal stress-tolerance in plants by expressing KP4 under a novel chimeric caulimoviral promoter in a transgenic approach.