Agrobacterium-mediated Transient Assay Research Articles

CsCuAO1 Associated with CsAMADH1 Confers Drought Tolerance by Modulating GABA Levels in Tea Plants.

Our previous study showed that COPPER-CONTAINING AMINE OXIDASE (CuAO) and AMINOALDEHYDE DEHYDROGENASE (AMADH) could regulate the accumulation of γ-aminobutyric acid (GABA) in tea through the polyamine degradation pathway. However, their biological function in drought tolerance has not been determined. In this study, Camellia sinensis (Cs) CsCuAO1 associated with CsAMADH1 conferred drought tolerance, which modulated GABA levels in tea plants. The results showed that exogenous GABA spraying effectively alleviated the drought-induced physical damage. Arabidopsis lines overexpressing CsCuAO1 and CsAMADH1 exhibited enhanced resistance to drought, which promoted the synthesis of GABA and putrescine by stimulating reactive oxygen species' scavenging capacity and stomatal movement. However, the suppression of CsCuAO1 or CsAMADH1 in tea plants resulted in increased sensitivity to drought treatment. Moreover, co-overexpressing plants increased GABA accumulation both in an Agrobacterium-mediated Nicotiana benthamiana transient assay and transgenic Arabidopsis plants. In addition, a GABA transporter gene, CsGAT1, was identified, whose expression was strongly correlated with GABA accumulation levels in different tissues under drought stress. Taken together, CsCuAO1 and CsAMADH1 were involved in the response to drought stress through a dynamic GABA-putrescine balance. Our data will contribute to the characterization of GABA's biological functions in response to environmental stresses in plants.

Cloning and characterization of vacuolar Na+/H+ antiporter gene promoter from wheat (Triticum aestivum) cv. Kharchia Local

Soil salinity is one of the significant abiotic stresses in plants. Few efficient stress-inducible promoters from plants, especially from monocotyledonous crops like wheat (Triticum aestivum L.) are available to drive stress-inducible expression of transgene. Therefore, an experiment was conducted at ICAR-National Institute for Plant Biotechnology, New Delhi, during 2017–18 to clone and characterize stress-inducible promoter from wheat. Results found a 418 bp long NHX1 promoter (TaUNHX) from a bread wheat landrace, Kharchia Local. In silico analysis of TaUNHX predicted several cis-acting regulatory elements, including CAAT, DOF, GATA motifs and some essential stress-responsive elements. To analyze the activity of TaUNHX, Agrobacterium-mediated transient GUS assay in tobacco (Nicotiana tabacum L.) leaves and stems was studied under various abiotic stresses like salt (300 mM NaCl), drought (20% PEG) and ABA (100 μM). Stress-responsive nature of this promoter can be used to drive the expression of transgene following exposure to various stresses. The study's finding has significant implications as the characterized promoter can be used to develop transgenics where the transgene expression can be induced following exposure to stress.

The Citrus yellow mosaic badnavirus ORFI functions as a RNA-silencing suppressor.

Citrus yellow mosaic badnavirus (CMBV) causes mosaic disease in all economically important citrus cultivars of India, with losses reaching up to 70%. CMBV belongs to the genus Badnavirus, family Caulimoviridae, possessing a circular double-stranded (ds) DNA genome with six open reading frames (ORFs I to VI), whose functions are yet to be deciphered. The RNA-silencing suppressor (RSS) activity has not been assigned to any CMBV ORF as yet. In the present study, it was found that ORFI exhibited RSS activity among all the six CMBV ORFs tested. Studies were done by employing the well-established Agrobacterium-mediated transient assay based on the transgenic Nicotiana benthamiana 16c plant line expressing the green fluorescent protein (GFP). The RSS activity of ORFI was confirmed by the analysis of the GFP visual expression in the agroinfiltrated leaves, further supported by quantification of GFP expression by RT-PCR. Based on the GFP visual expression, the CMBV ORFI was a weak RSS when compared to the p19 protein of tomato bushy stunt virus. In contrast, the ORFII, ORFIV, ORFV, ORFVI, and CP gene did not exhibit any RSS activity. Hence, ORFI is the first ORF of CMBV to be identified with RNA-silencing suppression activity.

Effects of early cold stress on gene expression in Chlamydomonas reinhardtii

Cold stress imposes a great impact on the growth of nearly all photosynthetic organisms, including Chlamydomonas reinhardtii (C. reinhardtii). Despite prior studies on the mechanism of stress acclimation in plants, little has been done on the early events of cold sensing in C. reinhardtii. Here, we used C. reinhardtii as a model to study early events of cold signal transduction. By analyzing transcriptomic changes of C. reinhardtii exposed to cold, we found that 3471 genes were differentially expressed after 1 h of cold exposure. These genes were associated with a wide range of biological events and processes such as protein synthesis, cell cycle and protein kinase-based phosphorylation. Besides, the promoter of one gene (named as crAP2) which belongs to AP2/EREBP family and was significantly induced by cold was cloned, and functional analysis was conducted using GUS activity analysis through Agrobacterium-mediated transient assay in tobacco leaves.

Subtractive hybridization-assisted screening and characterization of genes involved in the rice-Magnaporthe oryzae interaction

Transcription profiling assays have revealed substantial changes in gene expression during plant-microbe interactions, but it is often time- and labor-consuming to define the causative roles of the differentially expressed genes that finetune the plant responses to diverse pathogens. We improved the duplex-specific nuclease-mediated transcriptome subtraction method and integrated it with SMART cDNA library construction technology to generate normalized libraries consisting of full-length cDNAs derived from transcripts upregulated in the rice-Magnaporthe oryzae interaction. By adding BP recombination sites to the sequence of primers used for the synthesis of full-length cDNAs, we were able to transfer the full-length cDNAs of the library to an expression binary vector with CaMV 35S promoter, which allowed a subsequent screening for candidate genes potentially involved in the disease process by Agrobacterium-mediated transient assay. Results showed that the newly established approach preferentially suppressed the cDNA abundance of most constitutive genes and enriched or normalized that of the upregulated ones. Subsequent screening with transient expression in planta isolated 61 clones, which carry full-length cDNAs of 32 distinct genes, were capable of causing cell death on Nicotiana benthamiana. When transiently expressed in barley leaf, five of the identified genes were able to induce chlorosis, and additional 11 genes were found to promote disease symptoms caused by infection with the blast pathogen while the others did not enhance the symptoms of pathogen infection. These observations demonstrate that the subtractive hybridization-assisted functional screening is an efficient approach that provides initial leads to the role of candidate genes in the complex process of plant-microbe interactions.

Agrobacterium-Mediated Transient Assay of the Gus Gene Expression in Sugar Beet

Agrobacterium-Mediated Transient Assay of the Gus Gene Expression in Sugar Beet

MicroR408 regulates defense response upon wounding in sweet potato.

MiRNAs play diverse roles in plant development and defense responses by binding to their mRNA targets based on sequence complementarity. Here, we investigated a wound-related miR408 and its target genes in sweet potato (Ipomoea batatas) by small RNA deep sequencing and transcriptome analysis. The expression patterns of miR408 and the miR408 precursor were significantly repressed by wounding and jasmonate (JA). In contrast, expression of the putative target genes IbKCS (3-ketoacyl-CoA synthase 4), IbPCL (plantacyanin), and IbGAUT (galacturonosyltransferase 7-like) of miR408 was increased following wounding, whereas only IbKCS was increased after JA treatment. Target cleavage site mapping and Agrobacterium-mediated transient assay demonstrated that IbKCS, IbPCL, and IbGAUT were the targets of miR408. The expression of miR408 target genes was repressed in transgenic sweet potatoes overexpressing miR408. These data indicated a relationship between miR408 and its target genes. Notably, miR408-overexpressing plants showed a semi-dwarf phenotype and attenuated resistance to insect feeding, while transgenic plants overexpressing IbKCS exhibited more insect resistance than plants overexpressing only the empty vector. Collectively, sweet potato reduces the abundance of miR408 upon wounding to elevate the expression of IbKCS, IbPCL, and IbGAUT. The expression of IbKCS enhances the defense system against herbivore wounding.

Terpene profiling, transcriptome analysis and characterization of cis-β-terpineol synthase from Ocimum

Ocimum species produces a varied mix of different metabolites that imparts immense medicinal properties. To explore this chemo-diversity, we initially carried out metabolite profiling of different tissues of five Ocimum species and identified the major terpenes. This analysis broadly classified these five Ocimum species into two distinct chemotypes namely, phenylpropanoid-rich and terpene-rich. In particular, β-caryophyllene, myrcene, limonene, camphor, borneol and selinene were major terpenes present in these Ocimum species. Subsequently, transcriptomic analysis of pooled RNA samples from different tissues of Ocimum gratissimum, O. tenuiflorum and O. kilimandscharicum identified 38 unique transcripts of terpene synthase (TPS) gene family. Full-length gene cloning, followed by sequencing and phylogenetic analysis of three TPS transcripts were carried out along with their expression in various tissues. Terpenoid metabolite and expression profiling of candidate TPS genes in various tissues of Ocimum species revealed spatial variances. Further, putative TPS contig 19414 (TPS1) was selected to corroborate its role in terpene biosynthesis. Agrobacterium-mediated transient over-expression assay of TPS1 in the leaves of O. kilimandscharicum and subsequent metabolic and gene expression analyses indicated it as a cis-β-terpineol synthase. Overall, present study provided deeper understanding of terpene diversity in Ocimum species and might help in the enhancement of their terpene content through advanced biotechnological approaches.

Engineering resistance to a resistance-breaking strain of Cucumber mosaic virus in plants utilizing viral dsRNA

Cucumber mosaic virus Ca-P1 strain, a P0 resistance-breaking virus, was isolated from the leaves of virus-infected Capsicum annuum ‘Manidda’ (a P0 resistant cultivar) in South Korea. Since CMV-Ca-P1 was first reported in 2006, this virus causing damage on pepper production has been constantly detected in South Korea. We constructed three CMV RNAi vectors based on the post-transcriptional gene silencing of defense system against virus infection. These independent vectors (hpCMV1, hpCMV2, and hpCMV2 + 1 RNAi vectors) were designed to produce dsRNAs containing each hpCMV1 (1270–1629 nt), hpCMV2 (1–300 nt), and hpCMV2 + 1 (fused hpCMV2 and hpCMV1) fragments, in RNA-1 (replicase gene) of CMV-Ca-P1, which were then confirmed by Agrobacterium-mediated transformation transient assay. Among these, dsRNAs expressed from the hpCMV2 + 1 vector showed resistance to both CMV-Ca-P1 and CMV-Fny (ordinary strain). To obtain high level of resistance to both CMV-Ca-P1 and CMV-Fny, transgenic Nicotiana benthamiana plants containing hpCMV2 + 1 vector were developed and conferred resistance to both CMV-Ca-P1 and CMV-Fny. This study contributes to the effective selection of target sequences that may inhibit CMV infection.

Optimization of Agrobacterium-mediated transient expression of heterologous genes in spinach

The Agrobacterium-mediated transient assay is a relatively rapid technique and a promising approach for assessing the expression of a gene of interest. Despite the successful application of this transient expression system in several plant species, it is not well understood in spinach. In this study, we analyzed various factors, including infiltration method, Agrobacterium strain and density, and co-infiltration of an RNA silencing suppressor (p19), that affect transient expression following agroinfiltration in spinach. To evaluate the effects of these factors on the transient expression system, we used the β-glucuronidase (GUS) reporter gene construct pB7WG2D as a positive control. The vacuum-based infiltration method was much more effective at GUS gene expression than was the syringe-based infiltration method. Among the three Agrobacterium strains examined (EHA105, LBA4404, and GV2260), infiltration with the GV2260 strain suspension at a final optical cell density (OD600) of 1.0 resulted in the highest gene expression. Furthermore, co-expression of suppressor p19 also increased the efficiency and duration of gene expression and protein accumulation. The results indicate that the use of optimized conditions for transient gene expression could be a simple, rapid, and effective tool for functional genomics in spinach.

Comparative analysis of the genome of the field isolate V86010 of the rice blast fungus Magnaporthe oryzae from Philippines

AbstractGenome dynamics of pathogenic organisms are driven by plant host and pathogenic organism co-evolution, in which pathogen genomes are used to overcome stresses imposed by hosts with various genetic backgrounds through generation of a range of field isolates. This model also applies to the rice host and its fungal pathogen Magnaporthe oryzae. To better understand genetic variation of M. oryzae in nature, the field isolate V86010 from the Philippines was sequenced and analyzed. Genome annotation found that the assembled V86010 genome was composed of 1 931 scaffolds with a combined length of 38.9 Mb. The average GC ratio is 51.3% and repetitive elements constitute 5.1% of the genome. A total of 11 857 genes including 616 effector protein genes were predicted using a combined analysis pipeline. All predicted genes and effector protein genes of isolate V86010 distribute on the eight chromosomes when aligned with the assembled genome of isolate 70-15. Effector protein genes are located disproportionately at several chromosomal ends. The Pot2 elements are abundant in V86010. Seven V86010-specific effector proteins were found to suppress programmed cell death induced by BAX in tobacco leaves using an Agrobacterium-mediated transient assay. Our results may provide useful information for further study of the molecular and genomic dynamics in the evolution of M. oryzae and rice host interactions, and for characterizing novel effectors and AVR genes in the rice blast pathogen.

Testing the utility of fluorescent proteins in Mimulus lewisii by an Agrobacterium-mediated transient assay.

The Agrobacterium -mediated transient expression assay by leaf infiltration in Mimulus lewisii is robust. Fluorescent proteins EGFP, EYFP and DsRed give bright fluorescence signals in the infiltrated tissue. Mimulus lewisii is an emerging developmental genetic model system. Recently developed genomic and genetic resources and a stable transformation protocol have greatly facilitated the identification and functional characterization of genes controlling the development of ecologically important floral traits using this species. To further expedite gene and protein function analyses in M. lewisii, we adopted and simplified the Agrobacterium-mediated transient gene expression method routinely used in tobacco plants. With the validated transient assay, we examined the performance of fluorescent proteins EGFP, EYFP and DsRed in M. lewisii. All three proteins gave bright fluorescence signals when transiently expressed in agroinfiltrated leaves. Furthermore, we demonstrated the utility of fluorescent proteins in M. lewisii by showing the nuclear localization of Reduced Carotenoid Pigmentation 1 (RCP1), a recently discovered R2R3-MYB transcription factor that regulates carotenoid pigmentation during flower development. Both the transient assay and the fluorescent proteins are valuable additions to the M. lewisii toolbox, making this emerging genetic and developmental model system even more powerful.

Tobacco rattle virus 16K silencing suppressor binds ARGONAUTE 4 and inhibits formation of RNA silencing complexes.

The cysteine-rich 16K protein of tobacco rattle virus (TRV), the type member of the genus Tobravirus, is known to suppress RNA silencing. However, the mechanism of action of the 16K suppressor is not well understood. In this study, we used a GFP-based sensor strategy and an Agrobacterium-mediated transient assay in Nicotiana benthamiana to show that 16K was unable to inhibit the activity of existing small interfering RNA (siRNA)- and microRNA (miRNA)-programmed RNA-induced silencing effector complexes (RISCs). In contrast, 16K efficiently interfered with de novo formation of miRNA- and siRNA-guided RISCs, thus preventing cleavage of target RNA. Interestingly, we found that transiently expressed endogenous miR399 and miR172 directed sequence-specific silencing of complementary sequences of viral origin. 16K failed to bind small RNAs, although it interacted with ARGONAUTE 4, as revealed by bimolecular fluorescence complementation and immunoprecipitation assays. Site-directed mutagenesis demonstrated that highly conserved cysteine residues within the N-terminal and central regions of the 16K protein are required for protein stability and/or RNA silencing suppression.

Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses.

Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression.

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.

The nuclear immune receptor RPS4 is required for RRS1SLH1-dependent constitutive defense activation in Arabidopsis thaliana.

Plant nucleotide-binding leucine-rich repeat (NB-LRR) disease resistance (R) proteins recognize specific “avirulent” pathogen effectors and activate immune responses. NB-LRR proteins structurally and functionally resemble mammalian Nod-like receptors (NLRs). How NB-LRR and NLR proteins activate defense is poorly understood. The divergently transcribed Arabidopsis R genes, RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1), function together to confer recognition of Pseudomonas AvrRps4 and Ralstonia PopP2. RRS1 is the only known recessive NB-LRR R gene and encodes a WRKY DNA binding domain, prompting suggestions that it acts downstream of RPS4 for transcriptional activation of defense genes. We define here the early RRS1-dependent transcriptional changes upon delivery of PopP2 via Pseudomonas type III secretion. The Arabidopsis slh1 (sensitive to low humidity 1) mutant encodes an RRS1 allele (RRS1SLH1) with a single amino acid (leucine) insertion in the WRKY DNA-binding domain. Its poor growth due to constitutive defense activation is rescued at higher temperature. Transcription profiling data indicate that RRS1SLH1-mediated defense activation overlaps substantially with AvrRps4- and PopP2-regulated responses. To better understand the genetic basis of RPS4/RRS1-dependent immunity, we performed a genetic screen to identify suppressor of slh1 immunity (sushi) mutants. We show that many sushi mutants carry mutations in RPS4, suggesting that RPS4 acts downstream or in a complex with RRS1. Interestingly, several mutations were identified in a domain C-terminal to the RPS4 LRR domain. Using an Agrobacterium-mediated transient assay system, we demonstrate that the P-loop motif of RPS4 but not of RRS1SLH1 is required for RRS1SLH1 function. We also recapitulate the dominant suppression of RRS1SLH1 defense activation by wild type RRS1 and show this suppression requires an intact RRS1 P-loop. These analyses of RRS1SLH1 shed new light on mechanisms by which NB-LRR protein pairs activate defense signaling, or are held inactive in the absence of a pathogen effector.

Transient hairpin RNAi-induced silencing in floral tissues of Dendrobium Sonia ‘Earsakul’ by agroinfiltration for rapid assay of flower colour modification

Agroinfiltration is an Agrobacterium-mediated transient assay for the analysis of gene function and genetic modification in leaves, flowers and fruit tissues of various plants, and transient RNAi-induced silencing by agroinfiltration has been developed in leaves and fruits of several plant species. Here we report the establishment of a transient hairpin RNAi-induced silencing system for colour modification assay in floral tissues of Dendrobium Sonia ‘Earsakul’, a tropical hybrid orchid with purple and white bi-coloured flowers. Portions of the D. Sonia ‘Earsakul’ anthocyanin-related genes chalcone synthase (DseCHS-B) and dihydroflavonol 4-reductase (DseDFR) which are required for anthocyanin synthesis, were cloned into the hairpin-based RNAi vectors, pSTARGATE and pWATERGATE, under the control of the maize ubiquitin and Arabidopsis Rubisco small subunit (ARbcS) promoters, respectively. Characterisation of DseCHS-B and DseDFR expression profiles revealed that their expression was developmentally regulated in the flower and associated with flower pigmentation. Transient RNA silencing of DseCHS-B and DseDFR in the floral tissues of D. Sonia ‘Earsakul’ was induced by delivering 436-bp DseCHS-B and 470-bp DseDFR hairpin RNAs (hpRNAs) into the sepals and petals of flower buds at early developmental stages using agroinfiltration. Impaired anthocyanin accumulation and reduction of endogenous mRNAs of the corresponding targets were observed in the infiltrated areas of the sepals and petals. Silencing of the endogenous DseCHS-B and DseDFR mRNAs was highly effective at the early stages of mRNA synthesis and anthocyanin accumulation. This transient silencing system is a prototype for modification of the anthocyanin biosynthetic pathway in D. Sonia Earsakul and other orchids through gene suppression.

Cloning and functional characterization of the promoter of PsSEOF1 gene from Pisum sativum under different stress conditions using Agrobacterium-mediated transient assay

PsSEOF1, a SEO (sieve element occlusion) gene family protein (forisome) is calcium powered motor protein and is located close to plasma membrane of sieve element. In sieve element (SE) it senses the calcium ion levels and undergoes ATP-independent conformational shifts. Forisome, meaning gate-bodies (Latin foris: wing of a gate; Greek soma: body). Recent reports show that SEO gene family protein can prevent the loss of nutrient rich photoassimilate upon wound injury. The regulation of SEO protein forisome under abiotic/ biotic stress is still unknown. The analysis of cis-regulatory element present in the upstream region is not well understood. Tissue specific promoters guarantee correct expression when it perceives particular stimuli. Here we report isolation of tissue specific promoter of PsSEOF1 was isolated by gene walking PCR from P. sativum (pea) genomic DNA library constructed by BD genome walker kit. In silico analysis revealed several putative cis element within this promoter sequence like wound response, cold, dehydration. Putative elements which might be required for its vascular tissue specificity has also been identified. The GUS activities of PsSEOF1 promoter-GUS chimeric construct in the agroinfiltrated leaves under different environmental stress abiotic and biotic like wound, cold, salt and phytohormones has shown high level of GUS activity. To identify the activity of PsSEOF1 promoter under different stress condition an Agrobacterium-mediated transient expression of tobacco plants were subjected to histochemical GUS staining. Stress-inducible nature of PsSEOF1 promoter opens possibility for the study of the PsSEOF1 gene regulation under stress condition. The isolated promoter sequence could serve as an important candidate for tissue specific promoter in genetic engineering of plant under stress conditions.

Isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 using Agrobacterium-mediated transient assay

Helicases are molecular motor proteins that perform a variety of cellular functions including transcription, translation, DNA replication and repair, RNA maturation, ribosome synthesis, nuclear export and splicing processes. The p68 is an evolutionarily conserved protein which plays pivotal roles in all aspect RNA metabolism processes. It is well established that helicases provides abiotic stress adaptation in plants but analysis of cis-regulatory elements present in the upstream regions is still infancy. Here we report isolation and functional characterization of the promoter of a DEAD-box helicase Psp68 in response to abiotic stress and hormonal regulation. The promoter of Psp68 was isolated by gene walking PCR from pea genomic DNA library constructed in BD genome walker kit. In silico analysis revealed that promoter of Psp68 contained a TATA, a CAAT motif and also harbors some important stress and hormone associated cis regulatory elements, including E-box, AGAAA, GATA-box, ACGT, GAAAA and GTCTC. Functional analyses were performed by Agrobacterium-mediated transient assay in tobacco leaves. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the Psp68 promoter::GUS, subjected to abiotic stress and exogenous hormonal treatments. Stress-inducible nature of Psp68 promoter opens possibility for the study of the gene regulation under stress condition. Therefore, may be useful in the field of agriculture and biotechnology.

Functional Characterization of a Bidirectional Plant Promoter from Cotton Leaf Curl Burewala Virus Using an Agrobacterium-Mediated Transient Assay

The C1 promoter expressing the AC1 gene, and V1 promoter expressing the AV1 gene are located in opposite orientations in the large intergenic region of the Cotton leaf curl Burewala virus (CLCuBuV) genome. Agro-infiltration was used to transiently express putative promoter constructs in Nicotiana tabacum and Gossypium hirsutum leaves, which was monitored by a GUS reporter gene, and revealed that the bidirectional promoter of CLCuBuV transcriptionally regulates both the AC1 and AV1 genes. The CLCuBuV C1 gene promoter showed a strong, consistent transient expression of the reporter gene (GUS) in N. tabacum and G. hirsutum leaves and exhibited GUS activity two- to three-fold higher than the CaMV 35S promoter. The CLCuBuV bidirectional genepromoter is a nearly constitutive promoter that contains basic conserved elements. Many cis-regulatory elements (CREs) were also analyzed within the bidirectional plant promoters of CLCuBuV and closely related geminiviruses, which may be helpful in understanding the transcriptional regulation of both the virus and host plant.