Identification of target genes for RNAi-mediated control of the Twospotted Spider Mite.

RNA interference (RNAi) is being developed for the management of pests that destroy crops. The twospotted Spider Mite (TSSM), Tetranychus urticae is a worldwide pest due to its unique physiological and behavioral characteristics including extraordinary ability to detoxify a wide range of pesticides and feed on many host plants. In this study, we conducted experiments to identify target genes that could be used for the development of RNAi-based methods to control TSSM. Leaf disc feeding assays revealed that knockdown in the expression genes coding for proteins involved in the biosynthesis and action of juvenile hormone (JH) and action of ecdysteroids [Methoprene-tolerant (Met), retinoid X receptor ß, farnesoic acid O-methyltransferase, and CREB-binding protein] caused 35-56% mortality. Transgenic tobacco plants expressing hairpin dsRNA targeting Met gene were generated and tested. About 48% mortality was observed in TSSM raised on transgenic tobacco plants expressing dsMet. These studies not only broaden our knowledge on understanding hormone action in TSSM but also identified target genes that could be used in RNAi-mediated control of TSSM.

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.

RETRACTED: WRKY71 and TGA1a physically interact and synergistically regulate the activity of a novel promoter isolated from Petunia vein-clearing virus.

Caulimoviral promoters have become excellent tools for efficient transgene expression in plants. However, the transcriptional framework controlling their systematic regulation is poorly understood. To understand this regulatory mechanism, we extensively studied a novel caulimoviral promoter, PV8 (-163 to +138, 301 bp), isolated from Petunia vein-clearing virus (PVCV). PVCV was found to be Salicylic acid (SA)-inducible and 2.5-3.0 times stronger than the widely used CaMV35S promoter. In silico analysis of the PV8 sequence revealed a unique clustering of two stress-responsive cis-elements, namely, as-11 and W-box1-2, located within a span of 31 bp (-74 to -47) that bound to the TGA1a and WRKY71 plant transcription factors (TFs), respectively. We found that as-1 (TTACG) and W-box (TGAC) elements occupied both TGA1a and WRKY71 on the PV8 backbone. Mutational studies demonstrated that the combinatorial influence of as-1 (-57) and W-box1-2 (-74 and -47) on the PV8 promoter sequence largely modulated its activity. TGA1a and WRKY71 physically interacted and cooperatively enhanced the transcriptional activity of the PV8 promoter. Biotic stress stimuli induced PV8 promoter activity by ~1.5 times. We also established the possible pathogen-elicitor function of AtWRKY71 and NtabWRKY71 TFs. Altogether, this study elucidates the interplay between TFs, biotic stress and caulimoviral promoter function.

Structural characterization of a novel full-length transcript promoter from Horseradish Latent Virus (HRLV) and its transcriptional regulation by multiple stress responsive transcription factors.

KEY MESSAGE: The promoter fragment described in this study can be employed for strong transgene expression under both biotic and abiotic stress conditions. Plant-infecting Caulimoviruses have evolved multiple regulatory mechanisms to address various environmental stimuli during the course of evolution. One such mechanism involves the retention of discrete stress responsive cis-elements which are required for their survival and host-specificity. Here we describe the characterization of a novel Caulimoviral promoter isolated from Horseradish Latent Virus (HRLV) and its regulation by multiple stress responsive Transcription factors (TFs) namely DREB1, AREB1 and TGA1a. The activity of full length transcript (Flt-) promoter from HRLV (- 677 to + 283) was investigated in both transient and transgenic assays where we identified H12 (- 427 to + 73) as the highest expressing fragment having ~ 2.5-fold stronger activity than the CaMV35S promoter. The H12 promoter was highly active and near-constitutive in the vegetative and reproductive parts of both Tobacco and Arabidopsis transgenic plants. Interestingly, H12 contains a distinct cluster of cis-elements like dehydration-responsive element (DRE-core; GCCGAC), an ABA-responsive element (ABRE; ACGTGTC) and as-1 element (TGACG) which are known to be induced by cold, drought and pathogen/SA respectively. The specific binding of DREB1, AREB1 and TGA1a to DRE, ABRE and as-1 elements respectively were confirmed by the gel-binding assays using H12 promoter-specific probes. Detailed mutational analysis of the H12 promoter suggested that the presence of DRE-core and as-1 element was indispensable for its activity which was further confirmed by the transactivation assays. Our studies imply that H12 could be a valuable genetic tool for regulated transgene expression under diverse environmental conditions.

Interaction of Arabidopsis TGA3 and WRKY53 transcription factors on Cestrum yellow leaf curling virus (CmYLCV) promoter mediates salicylic acid-dependent gene expression in planta.

MAIN CONCLUSION: This paper highlighted a salicylic acid-inducible Caulimoviral promoter fragment from Cestrum yellow leaf curling virus (CmYLCV). Interaction of Arabidopsis transcription factors TGA3 and WRKY53 on CmYLCV promoter resulted in the enhancement of the promoter activity via NPR1-dependent salicylic acid signaling. Several transcriptional promoters isolated from plant-infecting Caulimoviruses are being presently used worldwide as efficient tools for plant gene expression. The CmYLCV promoter has been isolated from the Cestrum yellow leaf curling virus (Caulimoviruses) and characterized more than 12 years ago; also we have earlier reported a near-constitutive, pathogen-inducible CmYLCV promoter fragment (-329 to +137 from transcription start site; TSS) that enhances stronger (3×) expression than the previously reported fragments; all these fragments are highly efficient in monocot and dicot plants (Sahoo et al. Planta 240: 855-875, 2014). Here, we have shown that the full-length CmYLCV promoter fragment (-729 to +137 from TSS) is salicylic acid (SA) inducible. In this context, we have performed an in-depth study to elucidate the factors responsible for SA-inducibility of the CmYLCV promoter. We found that the as-1 1 and W-box1 elements (located at -649 and -640 from the TSS) of the CmYLCV promoter are required for SA-induced activation by recruiting Arabidopsis TGA3 and WRKY53 transcription factors. Consequently, as a nascent observation, we established the physical interaction between TGA3 and WYKY53; also demonstrated that the N-terminal domain of TGA3 is sufficient for the interaction with the full-length WRKY53. Such interaction synergistically activates the CmYLCV promoter activity in planta. Further, we found that activation of the CmYLCV promoter by SA through TGA3 and WRKY53 interaction depends on NPR1. Finally, the findings presented here provide strong support for the direct regulatory roles of TGA3 and WRKY53 in the SA and NPR1-dependent activation of a Caulimoviral promoter (CmYLCV).

Enrichment of apoplastic fluid with therapeutic recombinant protein for efficient biofarming.

OBJECTIVE: For efficient biofarming we attempted to enrich plant interstitial fluid (IF)/apoplastic fluid with targeted recombinant therapeutic protein. We employed a synthetic human Glucocerebrosidase (GCB), a model biopharmaceutical protein gene in this study. RESULTS: Twenty one Nicotiana varieties, species and hybrids were initially screened for individual IF recovery and based on the findings, we selected Nicotiana tabacum NN (S-9-6), Nicotiana tabacum nn (S-9-7) and Nicotiana benthamiana (S-6-6) as model plants for raising transgenic expressing GCB via Agrobacterium mediated transformation under the control of M24 promoter; GCB specific activity in each transgenic lines were analyzed and we observed higher concentration of recombinant GCB in IF of these transgenic lines (S-9-6, S-9-7, and S-6-6) in comparison to their concentration in crude leaf extracts. CONCLUSION: Recovery of valuable therapeutics in plant IF as shown in the present study holds great promise for promoting plant based biofarming. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:726-736, 2017.

Novel Synthetic Promoters from the Cestrum Yellow Leaf Curling Virus.

Constitutive promoters direct gene expression uniformly in most tissues and cells at all stages of plant growth and development; they confer steady levels of transgene expression in plant cells and hence their demand is high in plant biology. The gene silencing due to promoter homology can be avoided by either using diverse promoters isolated from different plant and viral genomes or by designing synthetic promoters. The aim of this chapter was to describe the basic protocols needed to develop and analyze novel, synthetic, nearly constitutive promoters from Cestrum yellow leaf curling virus (CmYLCV) through promoter/leader deletion and activating cis-sequence analysis. We also describe the methods to evaluate the strength of the promoters efficiently in various transient expression systems like agroinfiltration assay, gene-gun method, and assay in tobacco protoplasts. Besides, the detailed methods for developing transgenic plants (tobacco and Arabidopsis) for evaluation of the promoter using the GUS reporter gene are also described. The detailed procedure for electrophoretic mobility shift assay (EMSA) coupled with super-shift EMSA analysis are also described for showing the binding of tobacco transcription factor, TGA1a to cis-elements in the CmYLCV distal promoter region.

Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells.

The gene Par-4 (Prostate Apoptosis Response 4) was originally identified in prostate cancer cells undergoing apoptosis and its product Par-4 showed cancer specific pro-apoptotic activity. Particularly, the SAC domain of Par-4 (SAC-Par-4) selectively kills cancer cells leaving normal cells unaffected. The therapeutic significance of bioactive SAC-Par-4 is enormous in cancer biology; however, its large scale production is still a matter of concern. Here we report the production of SAC-Par-4-GFP fusion protein coupled to translational enhancer sequence (5' AMV) and apoplast signal peptide (aTP) in transgenic Nicotiana tabacum cv. Samsun NN plants under the control of a unique recombinant promoter M24. Transgene integration was confirmed by genomic DNA PCR, Southern and Northern blotting, Real-time PCR, and Nuclear run-on assays. Results of Western blot analysis and ELISA confirmed expression of recombinant SAC-Par-4-GFP protein and it was as high as 0.15% of total soluble protein. In addition, we found that targeting of plant recombinant SAC-Par-4-GFP to the apoplast and endoplasmic reticulum (ER) was essential for the stability of plant recombinant protein in comparison to the bacterial derived SAC-Par-4. Deglycosylation analysis demonstrated that ER-targeted SAC-Par-4-GFP-SEKDEL undergoes O-linked glycosylation unlike apoplast-targeted SAC-Par-4-GFP. Furthermore, various in vitro studies like mammalian cells proliferation assay (MTT), apoptosis induction assays, and NF-κB suppression suggested the cytotoxic and apoptotic properties of plant-derived SAC-Par-4-GFP against multiple prostate cancer cell lines. Additionally, pre-treatment of MAT-LyLu prostate cancer cells with purified SAC-Par-4-GFP significantly delayed the onset of tumor in a syngeneic rat prostate cancer model. Taken altogether, we proclaim that plant made SAC-Par-4 may become a useful alternate therapy for effectively alleviating cancer in the new era.

Synthetic promoters in planta.

MAIN CONCLUSION: This paper reviews the importance, prospective and development of synthetic promoters reported in planta. A review of the synthetic promoters developed in planta would help researchers utilize the available resources and design new promoters to benefit fundamental research and agricultural applications. The demand for promoters for the improvement and application of transgenic techniques in research and agricultural production is increasing. Native/naturally occurring promoters have some limitations in terms of their induction conditions, transcription efficiency and size. The strength and specificity of native promoter can be tailored by manipulating its 'cis-architecture' by the use of several recombinant DNA technologies. Newly derived chimeric promoters with specific attributes are emerging as an efficient tool for plant molecular biology. In the last three decades, synthetic promoters have been used to regulate plant gene expression. To better understand synthetic promoters, in this article, we reviewed promoter structure, the scope of cis-engineering, strategies for their development, their importance in plant biology and the total number of such promoters (188) developed in planta to date; we then categorized them under different functional regimes as biotic stress-inducible, abiotic stress-inducible, light-responsive, chemical-inducible, hormone-inducible, constitutive and tissue-specific. Furthermore, we identified a set of 36 synthetic promoters that control multiple types of expression in planta. Additionally, we illustrated the differences between native and synthetic promoters and among different synthetic promoter in each group, especially in terms of efficiency and induction conditions. As a prospective of this review, the use of ideal synthetic promoters is one of the prime requirements for generating transgenic plants suitable for promoting sustainable agriculture and plant molecular farming.

Comparative analysis of synthetic DNA promoters for high-level gene expression in plants.

MAIN CONCLUSION: We have designed two near- constitutive and stress-inducible promoters (CmYLCV9.11 and CmYLCV4); those are highly efficient in both dicot and monocot plants and have prospective to substitute the CaMV 35S promoter. We performed structural and functional studies of the full-length transcript promoter from Cestrum yellow leaf curling virus (CmYLCV) employing promoter/leader deletion and activating cis-sequence analysis. We designed a 465-bp long CmYLCV9.11 promoter fragment (-329 to +137 from transcription start site) that showed enhanced promoter activity and was highly responsive to both biotic and abiotic stresses. The CmYLCV9.11 promoter was about 28-fold stronger than the CaMV35S promoter in transient and stable transgenic assays using ß-glucuronidase (GUS) reporter gene. The CmYLCV9.11 promoter also demonstrated stronger activity than the previously reported CmYLCV promoter fragments, CmpC (-341 to +5) and CmpS (-349 to +59) in transient systems like maize protoplasts and onion epidermal cells as well as transgenic systems. A good correlation between CmYLCV9.11 promoter-driven GUS-accumulation/enzymatic activities with corresponding uidA-mRNA level in transgenic tobacco plants was shown. Histochemical (X-Gluc) staining of transgenic seedlings, root and floral parts expressing the GUS under the control of CmYLCV9.11, CaMV35S, CmpC and CmpS promoters also support the above findings. The CmYLCV9.11 promoter is a constitutive promoter and the expression level in tissues of transgenic tobacco plants was in the following order: root > leaf > stem. The tobacco transcription factor TGA1a was found to bind strongly to the CmYLCV9.11 promoter region, as shown by Gel-shift assay and South-Western blot analysis. In addition, the CmYLCV9.11 promoter was regulated by a number of abiotic and biotic stresses as studied in transgenic Arabidopsis and tobacco plants. The newly derived CmYLCV9.11 promoter is an efficient tool for biotechnological applications.

Biomass derived from transgenic tobacco expressing the Arabidopsis CESA3ixr1-2 gene exhibits improved saccharification.

Studies in Arabidopsis thaliana and Nicotiana tabacum L. variety Samsun NN demonstrated that expression of the CESA3 cellulose synthase gene that contains a point mutation, named ixr1-2, results in greater conversion of plant-derived cellulose to fermentable sugars. The present study was designed to examine the improved enzymatic saccharification efficiency of lignocellulosic biomass of tobacco plants expressing AtCESA3ixr1-2. Three-month-old AtCESA3ixr1-2 transgenic and wild-type tobacco plants (Nicotiana tabacum L. variety Samsun NN) were grown in the presence and absence of isoxaben. Biomass obtained from leaf, stem, and root tissues were analyzed for enzymatic saccharification rates. During enzymatic saccharification, 45% and 25% more sugar was released from transgenic leaf and stem samples, respectively, when compared to the wild-type samples. This gain in saccharification efficiency was achieved without chemical or heat pretreatment. Additionally, leaf and stem biomass from transgenic AtCESA3ixr1-2 requires a reduced amount of enzyme for saccharification compared to biomass from wild-type plants. From a practical standpoint, a similar strategy could be employed to introduce the mutated CESA into energy crops like poplar and switchgrass to improve the efficiency of biomass conversion.

Overexpression of the synthetic chimeric native-T-phylloplanin-GFP genes optimized for monocot and dicot plants renders enhanced resistance to blue mold disease in tobacco (N. tabacum L.).

To enhance the natural plant resistance and to evaluate the antimicrobial properties of phylloplanin against blue mold, we have expressed a synthetic chimeric native-phylloplanin-GFP protein fusion in transgenic Nicotiana tabacum cv. KY14, a cultivar that is highly susceptible to infection by Peronospora tabacina. The coding sequence of the tobacco phylloplanin gene along with its native signal peptide was fused with GFP at the carboxy terminus. The synthetic chimeric gene (native-phylloplanin-GFP) was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. The chimeric gene, expressed in transgenic tobacco, was stably inherited in successive plant generations as shown by molecular characterization, GFP quantification, and confocal fluorescent microscopy. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effects due to transgene expression. Blue mold-sensitivity assays of tobacco lines were performed by applying P. tabacina sporangia to the upper leaf surface. Transgenic lines expressing the fused synthetic native-phyllopanin-GFP gene in the leaf apoplast showed resistance to infection. Our results demonstrate that in vivo expression of a synthetic fused native-phylloplanin-GFP gene in plants can potentially achieve natural protection against microbial plant pathogens, including P. tabacina in tobacco.

Development of an intra-molecularly shuffled efficient chimeric plant promoter from plant infecting Mirabilis mosaic virus promoter sequence.

We developed an efficient chimeric promoter, MUASMSCP, with enhanced activity and salicylic acid (SA)/abscisic acid (ABA) inducibility, incorporating the upstream activation sequence (UAS) of Mirabilis mosaic virus full-length transcript (MUAS, -297 to -38) to the 5' end of Mirabilis mosaic virus sub-genomic transcript (MSCP, -306 to -125) promoter-fragment containing the TATA element. We compared the transient activity of the MUASMSCP promoter in tobacco/Arabidopsis protoplasts and in whole plant (Petunia hybrida) with the same that obtained from CaMV35S and MUAS35SCP promoters individually. The MUASMSCP promoter showed 1.1 and 1.5 times stronger GUS-activities over that obtained from MUAS35SCP and CaMV35S promoters respectively, in tobacco (Xanthi Brad) protoplasts. In transgenic tobacco (Nicotiana tabacum, var. Samsun NN), the MUASMSCP promoter showed 1.1 and 2.2 times stronger activities than MUAS35SCP and CaMV35S(2) promoters respectively. We observed a fair correlation between MUASMSCP-, MUAS35SCP- and CaMV35S(2)-driven GUS activities with the corresponding uidA-mRNA level in transgenic plants. X-gluc staining of transgenic germinating seed-sections and whole seedlings also support above findings. Protein-extracts made from tobacco protoplasts expressing GFP and human-IL-24 genes driven individually by the MUASMSCP promoter showed enhanced expression of the reporters compared to that obtained from the CaMV35S promoter. Furthermore, MUASMSCP-driven protoplast-derived human IL-24 showed enhanced cell inhibitory activity in DU-145 prostate cancer cells compared to that obtained from the CaMV35S promoter. We propose chimeric MUASMSCP promoter developed in the study could be useful for strong constitutive expression of transgenes in both plant/animal cells and it may become an efficient substitute for CaMV35S/CaMV35S(2) promoter.

Efficient chimeric plant promoters derived from plant infecting viral promoter sequences.

In the present study, we developed a set of three chimeric/hybrid promoters namely FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt incorporating different important domains of Figwort Mosaic Virus sub-genomic transcript promoter (FSgt, -270 to -60), Mirabilis Mosaic Virus sub-genomic transcript promoter (MSgt, -306 to -125) and Peanut Chlorotic Streak Caulimovirus full-length transcript promoter (PFlt-, -353 to +24 and PFlt-UAS, -353 to -49). We demonstrated that these chimeric/hybrid promoters can drive the expression of reporter genes in different plant species including tobacco, Arabidopsis, petunia, tomato and spinach. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 4.2, 1.5 and 1.2 times stronger GUS activities compared to the activity of the CaMV35S promoter, respectively, in tobacco protoplasts. Protoplast-derived recombinant promoter driven GFP showed enhanced accumulation compared to that obtained under the CaMV35S promoter. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 3.0, 1.3 and 1.0 times stronger activities than the activity of the CaMV35S² (a modified version of the CaMV35S promoter with double enhancer domain) promoter, respectively, in tobacco (Nicotiana tabacum, var. Samsun NN). Alongside, we observed a fair correlation between recombinant promoter-driven GUS accumulation with the corresponding uidA-mRNA level in transgenic tobacco. Histochemical (X-gluc) staining of whole transgenic seedlings and fluorescence images of ImaGene Green™ treated floral parts expressing the GUS under the control of recombinant promoters also support above findings. Furthermore, we confirmed that these chimeric promoters are inducible in the presence of 150 µM salicylic acid (SA) and abscisic acid (ABA). Taken altogether, we propose that SA/ABA inducible chimeric/recombinant promoters could be used for strong expression of gene(s) of interest in crop plants.

Calmodulin-mediated signal transduction pathways in Arabidopsis are fine-tuned by methylation.

Calmodulin N-methyltransferase (CaM KMT) is an evolutionarily conserved enzyme in eukaryotes that transfers three methyl groups to a highly conserved lysyl residue at position 115 in calmodulin (CaM). We sought to elucidate whether the methylation status of CaM plays a role in CaM-mediated signaling pathways by gene expression analyses of CaM KMT and phenotypic characterization of Arabidopsis thaliana lines wherein CaM KMT was overexpressed (OX), partially silenced, or knocked out. CaM KMT was expressed in discreet spatial and tissue-specific patterns, most notably in root tips, floral buds, stamens, apical meristems, and germinating seeds. Analysis of transgenic plants with genetic dysfunction in CaM KMT revealed a link between the methylation status of CaM and root length. Plants with suppressed CaM methylation had longer roots and CaM KMT OX lines had shorter roots than wild type (Columbia-0). CaM KMT was also found to influence the root radial developmental program. Protein microarray analyses revealed a number of proteins with specificity for methylated forms of CaM, providing candidate functional intermediates between the observed phenotypes and the target pathways. This work demonstrates that the functionality of the large CaM family in plants is fine-tuned by an overarching methylation mechanism.

Expression of an apoplast-directed, T-phylloplanin-GFP fusion gene confers resistance against Peronospora tabacina disease in a susceptible tobacco.

KEY MESSAGE: Phylloplanins are plant-derived, antifungal glycoproteins produced by leaf trichomes. Expression of phylloplanin-GFP fusion gene to the apoplast of a blue mold susceptible tobacco resulted in increased resistance to this pathogen. ABSTRACT: Tobaccos and certain other plants secrete phylloplanin glycoproteins to aerial surfaces where they appear to provide first-point-of-contact resistance against fungi/fungi-like pathogens. These proteins can be collected by water washing of aerial plant surfaces, and as shown for tobacco and a sunflower phylloplanins, spraying concentrated washes onto, e.g., turf grass aerial surfaces can provide resistance against various fungi/fungi-like pathogens, in the laboratory. These results suggest that natural-product, phylloplanins may be useful as broad-selectivity fungicides. An obvious question now is can a tobacco phylloplanin gene be introduced into a disease-susceptible plant to confer endogenous resistance. Here we demonstrate that introduction of a tobacco phylloplanin gene--as a fusion with the GFP gene--targeted to the apoplasm can increase resistance to blue mold disease in a susceptible host tobacco.

Manipulating cellulose biosynthesis by expression of mutant Arabidopsis proM24::CESA3(ixr1-2) gene in transgenic tobacco.

Manipulation of the cellulose biosynthetic machinery in plants has the potential to provide insight into plant growth, morphogenesis and to create modified cellulose for anthropogenic use. Evidence exists that cellulose microfibril structure and its recalcitrance to enzymatic digestion can ameliorated via mis-sense mutation in the primary cell wall-specific gene AtCELLULOSE SYNTHASE (CESA)3. This mis-sense mutation has been identified based on conferring drug resistance to the cellulose inhibitory herbicide isoxaben. To examine whether it would be possible to introduce mutant CESA alleles via a transgenic approach, we overexpressed a modified version of CESA3, AtCESA3(ixr1-2) derived from Arabidopsis thaliana L. Heynh into a different plant family, the Solanceae dicotyledon tobacco (Nicotiana tabacum L. variety Samsun NN). Specifically, a chimeric gene construct of CESA3(ixr1-2) , codon optimized for tobacco, was placed between the heterologous M24 promoter and the rbcSE9 gene terminator. The results demonstrated that the tobacco plants expressing M24-CESA3(ixr1-2) displayed isoxaben resistance, consistent with functionality of the mutated AtCESA3(ixr1-2) in tobacco. Secondly, during enzymatic saccharification, transgenic leaf- and stem-derived cellulose is 54%-66% and 40%-51% more efficient, respectively, compared to the wild type, illustrating translational potential of modified CESA loci. Moreover, the introduction of M24-AtCESA3(ixr1-2) caused aberrant spatial distribution of lignified secondary cell wall tissue and a reduction in the zone occupied by parenchyma cells.

Development of an efficient bi-directional promoter with tripartite enhancer employing three viral promoters.

We have developed a novel bi-directional promoter (FsFfCBD) by placing two heterogeneous core-promoters from the Figwort mosaic virus sub-genomic transcript promoter (FsCP, -69 to +31) and Cauliflower mosaic virus 35S promoter (CCP, -89 to +1) respectively on upstream (5') and downstream (3') ends of a tri-hybrid enhancer (FsEFfECE), in reverse orientation. The FsEFfECE domain encompasses three heterologous enhancer fragments from Figwort mosaic virus sub-genomic transcript promoter (FsE, 101 bp, -70 to -170), Figwort mosaic virus full-length transcript promoter (FfE, 196 bp, -249 to -54) and Cauliflower mosaic virus 35S promoter (CE, 254 bp, -343 to -90). The bi-directional nature of the FsFfCBD promoter (coupled to GFP and GUS) was established both in transient systems (onion epidermal cells and tobacco protoplasts) and transgenic plant (Nicotiana tabacum samsun NN) by monitoring the simultaneous expression of GFP and GUS employing fluorescence (for GFP) and biochemical (for GUS) based assays. In transgenic plants, the FsFfCBD promoter was found to be 6.8 and 2.5 times stronger than two parent promoters; Fs and FfC respectively. The bi-directional compound promoter FsFfCBD, composed of three heterologous enhancers with enhanced activity could become a valuable additional tool for efficient plant metabolic engineering and molecular pharming.

Development of a salicylic acid inducible minimal sub-genomic transcript promoter from Figwort mosaic virus with enhanced root- and leaf-activity using TGACG motif rearrangement.

In Figwort mosaic virus sub-genomic transcript promoter (F-Sgt), function of the TGACG-regulatory motif, was investigated in the background of artificially designed promoter sequences. The 131bp (FS, -100 to +31) long F-Sgt promoter sequence containing one TGACG motif [FS-(TGACG)] was engineered to generate a set of three modified promoter constructs: [FS-(TGACG)(2), containing one additional TGACG motif at 7 nucleotides upstream of the original one], [FS-(TGACG)(3), containing two additional TGACG motifs at 7 nucleotides upstream and two nucleotides downstream of the original one] and [FS-(TGCTG)(mu), having a mutated TGACG motif]. EMSA and foot-printing analysis confirmed binding of tobacco nuclear factors with modified TGACG motif/s. The transcription-activation of the GUS gene by the TGACG motif/s in above promoter constructs was examined in transgenic tobacco and Arabidopsis plants and observed that the transcription activation was affected by the spacing/s and number/s of the TGACG motif/s. The FS-(TGACG)(2) promoter showed strongest root-activity compared to other modified and CaMV35S promoters. Also under salicylic acid (SA) stress, the leaf-activity of the said promoter was further enhanced. All above findings were confirmed by real-time and semi-qRT PCR analysis. Taken together, these results clearly demonstrated that the TGACG motif plays an important role in inducing the root-specific expression of the F-Sgt promoter. This study advocates the importance of genetic manipulation of functional cis-motif for amending the tissue specificity of a plant promoter. SA inducible FS-(TGACG)(2) promoter with enhanced activity could be a useful candidate promoter for developing plants with enhanced crop productivity.