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

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

Epigenetic malleability at core promoter initiates tobacco PR-1a expression post salicylic acid treatment.

BACKGROUND: Tobacco's PR-1a gene is induced by pathogen attack or exogenous application of salicylic acid (SA). Nucleosome mapping and chromatin immunoprecipitation assay were used to delineate the histone modifications on the PR-1a promoter. However, the epigenetic modifications of the inducible promoter of the PR-1a gene are not fully understood yet. METHODS AND RESULTS: Southern approach was used to scan the promoter of PR-1a to identify presence of nucleosomes, ChIP assays were performed using anti-histones antibodies of repressive chromatin by di- methylated at H3K9 and H4K20 or active chromatin by acetylated H3K9/14 and H4K16 to find epigenetic malleability of nucleosome over core promoter in uninduced or induced state post SA treatment. Class I and II mammalian histone deacetylase (HDAC) inhibitor TSA treatment was used to enhance the expression of PR-1a by facilitating the histone acetylation post SA treatment. Here, we report correlated consequences of the epigenetic modifications correspond to disassembly of the nucleosome (spans from - 102 to + 55 bp, masks TATA and transcription initiation) and repressor complex from core promoter, eventually initiates the transcription of PR-1a gene post SA treatment. While active chromatin marks di and trimethylation of H3K4, acetylation of H3K9 and H4K16 are increased which are associated to the transcription initiation of PR-1a following SA treatment. However, in uninduced state constitutive expression of a negative regulator (SNI1) of AtPR1, suppresses AtPR1 expression by six-fold in Arabidopsis thaliana. Further, we report 50-to-1000-fold increased expression of AtPR1 in uninduced lsd1 mutant plants, up to threefold increased expression of AtPR1 in uninduced histone acetyl transferases (HATs) mutant plants, SNI1 dependent negative regulation of AtPR1, all together our results suggest that inactive state of PR-1a is indeed maintained by a repressive complex. CONCLUSION: The study aimed to reveal the mechanism of transcription initiation of tobacco PR-1a gene in presence or absence of SA. This is the first study that reports nucleosome and repressor complex over core promoter region maintains the inactivation of gene in uninduced state, and upon induction disassembling of both initiates the downstream gene activation process.

Atmospheric temperature and humidity demonstrated strong correlation with productivity in tropical moist deciduous forests.

Tropical forests sequester six times higher carbon than that released by humans annually into the atmosphere. These biodiversity-rich tropical forests have high net primary productivity (NPP), which differs among constituent plant communities. Tropical moist deciduous forests occupy 179,335 km2 of India's geographical area and constitute 44% of the country's total protected area (PA) forests. The productivity of these forests has neither been estimated specifically nor precisely. We measured the annual NPP of three predominant distinct community types, viz., mixed (DM), sal (SM), and teak (TP), in a tropical moist deciduous forest in northern India. The NPP was estimated from tree biomass data collected from nine long-term ecological research (LTER) plots of 1 ha each representing the above three community types. The estimated annual NPP were 10.28, 6.25, and 9.79 Mg ha-1 year-1 in DM; 8.93, 7.09, and 10.59 Mg ha-1 year-1 in SM; and 14.57, 7.14, and 13.56 Mg ha-1 year-1 in TP for the years 2010, 2011, and 2012, respectively. The NPP was correlated with tree density, height and DBH, species richness, diversity, microclimatic and edaphic variables, and leaf area index (LAI) using principal component analysis (PCA) and generalized linear modeling (GLM). Air temperature and humidity were strongly related to NPP in all the community types, while "complementarity" and "selection effects" contributed to the NPP in both the sal and mixed forest communities with equal importance, and the NPP in teak plantation ould point to "dominance effect."

Transcriptome analysis at mid-stage seed development in litchi with contrasting seed size.

Litchi is a sub-tropical fruit crop with genotypes that bear fruits with variable seed size. Small seed size is a desirable trait in litchi, as it improves consumers' preference and facilitates fruit processing. Seed specific transcriptome analysis was performed in two litchi genotypes with contrasting seed size to identify the genes associated with seed development. The transcriptomic sequence data from seeds at mid-development stages (16-28 days after anthesis) were de-novo assembled into 1,39,608 Trinity transcripts. Out of these, 6325 transcripts expressed differentially between the two contrasting genotypes. Several putative genes for salicylic acid, jasmonic acid and brassinosteriod pathways were down-regulated in seeds of the small-seeded litchi. The putative regulators of seed maturation and seed storage were down-regulated in the small-seeded genotype. Embryogenesis, cell expansion, seed size and stress related Trinity transcripts exhibited differential expression. Further studies on gene characterization will reveal the early regulators of seed size in litchi. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03098-8.

Predicting the growth and trend of COVID-19 pandemic using machine learning and cloud computing.

The outbreak of COVID-19 Coronavirus, namely SARS-CoV-2, has created a calamitous situation throughout the world. The cumulative incidence of COVID-19 is rapidly increasing day by day. Machine Learning (ML) and Cloud Computing can be deployed very effectively to track the disease, predict growth of the epidemic and design strategies and policies to manage its spread. This study applies an improved mathematical model to analyse and predict the growth of the epidemic. An ML-based improved model has been applied to predict the potential threat of COVID-19 in countries worldwide. We show that using iterative weighting for fitting Generalized Inverse Weibull distribution, a better fit can be obtained to develop a prediction framework. This has been deployed on a cloud computing platform for more accurate and real-time prediction of the growth behavior of the epidemic. A data driven approach with higher accuracy as here can be very useful for a proactive response from the government and citizens. Finally, we propose a set of research opportunities and setup grounds for further practical applications.

Characterization of functionalized multiwalled carbon nanotubes and comparison of their cellular toxicity between HEK 293 cells and zebra fish in vivo.

Carbon nanotubes (CNTs) hold tremendous potential due to their unique and modifiable properties. Their robust biological applications necessitate minimizing their cytotoxicity and increasing the solubilization. In the present manuscript, we have functionalized multiwalled carbon nanotubes (MWCNTs) using defect functionalization methodology to covalently bind carboxy and amino groups on their walls. This functionalization was reassured through fourier-transform infrared spectroscopy (FTIR), energy dispersive x-ray analysis (EDX), elemental and field emission scanning electron microscopy (FE-SEM) analysis. The observations demonstrated that addition of carboxy as well as amino groups on MWCNTs, besides enabling MWCNTs solubilization also significantly ameliorated the cytotoxicity and the oxidative stress in comparison to pristine MWCNTs. It is envisaged that changes in agglomeration of the functionalized MWCNTs and the acquired surface charge is the reason for the reduction of cytotoxicity. Zebra fish embryo model test system employed for in vivo analysis of the MWCNTs showed no significant toxicity on account of any nanoparticle tested pointing towards intrinsic mechanisms in place for deterring the damage in complex organisms. Overall, the observations besides pointing towards functionalized MWCNTs effectiveness towards weakening the toxicity of pristine MWCNTs also caution for extrapolating in vitro data to in vivo observations. The observations further lend credibility for exploiting the zebra fish as a model system for analyzing the effects of MWCNTs functionalization.

Pathway Editing Targets for Thiamine Biofortification in Rice Grains.

Thiamine deficiency is common in populations consuming polished rice as a major source of carbohydrates. Thiamine is required to synthesize thiamine pyrophosphate (TPP), an essential cofactor of enzymes of central metabolism. Its biosynthesis pathway has been partially elucidated and the effect of overexpression of a few genes such as thi1 and thiC, on thiamine accumulation in rice has been reported. Based on current knowledge, this review focuses on the potential of gene editing in metabolic engineering of thiamine biosynthesis pathway to improve thiamine in rice grains. Candidate genes, suitable for modification of the structural part to evolve more efficient versions of enzymes in the pathway, are discussed. For example, adjacent cysteine residues may be introduced in the catalytic domain of thi4 to improve the turn over activity of thiamine thiazole synthase 2. Motif specific editing to modify promoter regulatory regions of genes is discussed to modulate gene expression. Editing cis acting regulatory elements in promoter region can shift the expression of transporters and thiamine binding proteins to endosperm. This can enhance dietary availability of thiamine from rice grains. Differential transcriptomics on rice varieties with contrasting grain thiamine and functional genomic studies will identify more strategic targets for editing in future. Developing functionally enhanced foods by biofortification is a sustainable approach to make diets wholesome.

Corrigendum: Pathway Editing Targets for Thiamine Biofortification in Rice Grains.

[This corrects the article DOI: 10.3389/fpls.2018.00975.].

Transcriptional changes during ovule development in two genotypes of litchi (Litchi chinensis Sonn.) with contrast in seed size.

Litchi chinensis is a subtropical fruit crop, popular for its nutritional value and taste. Fruits with small seed size and thick aril are desirable in litchi. To gain molecular insight into gene expression that leads to the reduction in the size of seed in Litchi chinensis, transcriptomes of two genetically closely related genotypes, with contrasting seed size were compared in developing ovules. The cDNA library constructed from early developmental stages of ovules (0, 6, and 14 days after anthesis) of bold- and small-seeded litchi genotypes yielded 303,778,968 high quality paired-end reads. These were de-novo assembled into 1,19,939 transcripts with an average length of 865 bp. A total of 10,186 transcripts with contrast in expression were identified in developing ovules between the small- and large- seeded genotypes. A majority of these differences were present in ovules before anthesis, thus suggesting the role of maternal factors in seed development. A number of transcripts indicative of metabolic stress, expressed at higher level in the small seeded genotype. Several differentially expressed transcripts identified in such ovules showed homology with Arabidopsis genes associated with different stages of ovule development and embryogenesis.

Expression of an insecticidal fern protein in cotton protects against whitefly.

Whitefly (Bemisia tabaci) damages field crops by sucking sap and transmitting viral diseases. None of the insecticidal proteins used in genetically modified (GM) crop plants to date are effective against whitefly. We report the identification of a protein (Tma12) from an edible fern, Tectaria macrodonta (Fee) C. Chr., that is insecticidal to whitefly (median lethal concentration = 1.49 µg/ml in in vitro feeding assays) and interferes with its life cycle at sublethal doses. Transgenic cotton lines that express Tma12 at ∼0.01% of total soluble leaf protein were resistant to whitefly infestation in contained field trials, with no detectable yield penalty. The transgenic cotton lines were also protected from whitefly-borne cotton leaf curl viral disease. Rats fed Tma12 showed no detectable histological or biochemical changes, and this, together with the predicted absence of allergenic domains in Tma12, indicates that Tma12 might be well suited for deployment in GM crops to control whitefly and the viruses it carries.

Senna leaf curl virus: a novel begomovirus identified in Senna occidentalis.

Begomoviruses are whitefly-transmitted, single-stranded DNA viruses that infect a variety of cultivated (crop) and non-cultivated (weed) plants. The present study identified a novel begomovirus and satellites (alpha- and betasatellite) in Senna occidentalis (syn. Cassia occidentalis) showing leaf curl symptoms. The begomovirus shared a maximum sequence identity of 88.6 % with french bean leaf curl virus (JQ866297), whereas the alphasatellite and the betasatellite shared identities of 98 % and 90 % with ageratum yellow vein India alphasatellite (LK054802) and papaya leaf curl betasatellite (HM143906), respectively. No other begomovirus or satellites were detected in the suspected plants. We propose to name the virus "senna leaf curl virus" (SenLCuV).

De novo assembly and characterization of transcriptomes of early-stage fruit from two genotypes of Annona squamosa L. with contrast in seed number.

BACKGROUND: Annona squamosa L., a popular fruit tree, is the most widely cultivated species of the genus Annona. The lack of transcriptomic and genomic information limits the scope of genome investigations in this important shrub. It bears aggregate fruits with numerous seeds. A few rare accessions with very few seeds have been reported for Annona. A massive pyrosequencing (Roche, 454 GS FLX+) of transcriptome from early stages of fruit development (0, 4, 8 and 12 days after pollination) was performed to produce expression datasets in two genotypes, Sitaphal and NMK-1, that show a contrast in the number of seeds set in fruits. The data reported here is the first source of genome-wide differential transcriptome sequence in two genotypes of A. squamosa, and identifies several candidate genes related to seed development. RESULTS: Approximately 1.9 million high-quality clean reads were obtained in the cDNA library from the developing fruits of both the genotypes, with an average length of about 568 bp. Quality-reads were assembled de novo into 2074 to 11004 contigs in the developing fruit samples at different stages of development. The contig sequence data of all the four stages of each genotype were combined into larger units resulting into 14921 (Sitaphal) and 14178 (NMK-1) unigenes, with a mean size of more than 1 Kb. Assembled unigenes were functionally annotated by querying against the protein sequences of five different public databases (NCBI non redundant, Prunus persica, Vitis vinifera, Fragaria vesca, and Amborella trichopoda), with an E-value cut-off of 10(-5). A total of 4588 (Sitaphal) and 2502 (NMK-1) unigenes did not match any known protein in the NR database. These sequences could be genes specific to Annona sp. or belong to untranslated regions. Several of the unigenes representing pathways related to primary and secondary metabolism, and seed and fruit development expressed at a higher level in Sitaphal, the densely seeded cultivar in comparison to the poorly seeded NMK-1. A total of 2629 (Sitaphal) and 3445 (NMK-1) Simple Sequence Repeat (SSR) motifs were identified respectively in the two genotypes. These could be potential candidates for transcript based microsatellite analysis in A. squamosa. CONCLUSION: The present work provides early-stage fruit specific transcriptome sequence resource for A. squamosa. This repository will serve as a useful resource for investigating the molecular mechanisms of fruit development, and improvement of fruit related traits in A. squamosa and related species.

Expression of rabies glycoprotein and ricin toxin B chain (RGP-RTB) fusion protein in tomato hairy roots: a step towards oral vaccination for rabies.

Transgenic hairy roots of Solanum lycopersicum were engineered to express a recombinant protein containing a fusion of rabies glycoprotein and ricin toxin B chain (rgp-rtxB) antigen under the control of constitutive CaMV35S promoter. Asialofetuin-mediated direct ELISA of transgenic hairy root extracts was performed using polyclonal anti-rabies antibodies (Ab1) and epitope-specific peptidal anti-RGP (Ab2) antibodies which confirmed the expression of functionally viable RGP-RTB fusion protein. Direct ELISA based on asialofetuin-binding activity was used to screen crude protein extracts from five transgenic hairy root lines. Expressions of RGP-RTB fusion protein in different tomato hairy root lines varied between 1.4 and 8 µg in per gram of tissue. Immunoblotting assay of RGP-RTB fusion protein from these lines showed a protein band on monomeric size of ~84 kDa after denaturation. Tomato hairy root line H03 showed highest level of RGP-RTB protein expression (1.14 %) and was used further in bench-top bioreactor for the optimization of scale-up process to produce large quantity of recombinant protein. Partially purified RGP-RTB fusion protein was able to induce the immune response in BALB/c mice after intra-mucosal immunization. In the present investigation, we have not only successfully scaled up the hairy root culture but also established the utility of this system to produce vaccine antigen which subsequently will reduce the total production cost for implementing rabies vaccination programs in developing nations. This study in a way aims to provide consolidated base for low-cost preparation of improved oral vaccine against rabies.

Comparative transcriptional profiling of two wheat genotypes, with contrasting levels of minerals in grains, shows expression differences during grain filling.

Wheat is one of the most important cereal crops in the world. To identify the candidate genes for mineral accumulation, it is important to examine differential transcriptome between wheat genotypes, with contrasting levels of minerals in grains. A transcriptional comparison of developing grains was carried out between two wheat genotypes- Triticum aestivum Cv. WL711 (low grain mineral), and T. aestivum L. IITR26 (high grain mineral), using Affymetrix GeneChip Wheat Genome Array. The study identified a total of 580 probe sets as differentially expressed (with log2 fold change of ≥2 at p≤0.01) between the two genotypes, during grain filling. Transcripts with significant differences in induction or repression between the two genotypes included genes related to metal homeostasis, metal tolerance, lignin and flavonoid biosynthesis, amino acid and protein transport, vacuolar-sorting receptor, aquaporins, and stress responses. Meta-analysis revealed spatial and temporal signatures of a majority of the differentially regulated transcripts.

ßC1 is a pathogenicity determinant: not only for begomoviruses but also for a mastrevirus.

ßC1 proteins, encoded by betasatellites, are known to be pathogenicity determinants, and they are responsible for symptom expression in many devastating diseases caused by begomoviruses. We report the involvement of ßC1 in pathogenicity determination of a mastrevirus. Analysis of field samples of wheat plants containing wheat dwarf India virus (WDIV) revealed the presence of a full-length and several defective betasatellite molecules. The detected betasatellite was identified as ageratum yellow leaf curl betasatellite (AYLCB). No begomovirus was detected in any of the samples. The full-length AYLCB contained an intact ßC1 gene, whereas the defective molecules contained complete or partial deletions of ßC1. Agroinoculation of wheat with the full-length AYLCB and WDIV or of tobacco with ageratum enation virus enhanced the pathogenicity and accumulation of the respective viruses, whereas the defective molecules could not. This study indicates that ßC1 is a pathogenicity determinant for WDIV and can interact functionally not only with begomoviruses but also with a mastrevirus.

Differential expression of structural genes for the late phase of phytic acid biosynthesis in developing seeds of wheat (Triticum aestivum L.).

In cereals, phytic acid (PA) or inositol hexakisphosphate (IP6) is a well-known phosphate storage compound as well as major chelator of important micronutrients (iron, zinc, calcium, etc.). Genes involved in the late phases of PA biosynthesis pathway are known in crops like maize, soybeans and barley but none have been reported from wheat. Our in silico analysis identified six wheat genes that might be involved in the biosynthesis of inositol phosphates. Four of the genes were inositol tetraphosphate kinases (TaITPK1, TaITPK2, TaITPK3, and TaITPK4), and the other two genes encode for inositol triphosphate kinase (TaIPK2) and inositol pentakisphosphate kinase (TaIPK1). Additionally, we identified a homolog of Zmlpa-1, an ABCC subclass multidrug resistance-associated transporter protein (TaMRP3) that is putatively involved in PA transport. Analyses of the mRNA expression levels of these seven genes showed that they are differentially expressed during seed development, and that some are preferentially expressed in aleurone tissue. These results suggest selective roles during PA biosynthesis, and that both lipid-independent and -dependent pathways are active in developing wheat grains. TaIPK1 and TaMRP3 were able to complement the yeast ScΔipk1 and ScΔycf1 mutants, respectively, providing evidence that the wheat genes have the expected biochemical functions. This is the first comprehensive study of the wheat genes involved in the late phase of PA biosynthesis. Knowledge generated from these studies could be utilized to develop strategies for generating low phyate wheat.

Spatial X-ray fluorescence micro-imaging of minerals in grain tissues of wheat and related genotypes.

MAIN CONCLUSION: Wheat and its related genotypes show distinct distribution patterns for mineral nutrients in maternal and filial tissues in grains. X-ray-based imaging techniques are very informative to identify genotypes with contrasting tissue-specific localization of different elements. This can help in the selection of suitable genotypes for nutritional improvement of food grain crops. Understanding mineral localization in cereal grains is important for their nutritional improvement. Spatial distribution of mineral nutrients (Mg, P, S, K, Ca, Fe, Zn, Mn and Cu) was investigated between and within the maternal and filial tissues in grains of two wheat cultivars (Triticum aestivum Cv. WH291 and WL711), a landrace (T. aestivum L. IITR26) and a related wild species Aegilops kotschyi, using micro-proton-induced X-ray emission (µ-PIXE) and micro-X-ray fluorescence (µ-XRF). Aleurone and scutellum were major storage tissues for macro (P, K, Ca and Mg) as well as micro (Fe, Zn, Cu and Mn) nutrients. Distinct elemental distribution patterns were observed in each of the four genotypes. A. kotschyi, the wild relative of wheat and the landrace, T. aestivum L. IITR26, accumulated more Zn and Fe in scutellum and aleurone than the cultivated wheat varieties, WH291 and WL711. The landrace IITR26, accumulated far more S in grains, Mn in scutellum, aleurone and embryo region, Ca and Cu in aleurone and scutellum, and Mg, K and P in scutellum than the other genotypes. Unlike wheat, lower Mn and higher Fe, Cu and Zn concentrations were noticed in the pigment strand of A. kotschyi. Multivariate statistical analysis, performed on mineral distribution in major grain tissues (aleurone, scutellum, endosperm and embryo region) resolved the four genotypes into distinct clusters.

Association of satellites with a mastrevirus in natural infection: complexity of Wheat dwarf India virus disease.

UNLABELLED: In contrast to begomoviruses, mastreviruses have not previously been shown to interact with satellites. This study reports the first identification of the association of satellites with a mastrevirus in field-grown plants. Two alphasatellite species were detected in different field samples of wheat infected with Wheat Dwarf India Virus (WDIV), a Cotton leaf curl Multan alphasatellite (CLCuMA) and a Guar leaf curl alphasatellite (GLCuA). In addition to the alphasatellites, a betasatellite, Ageratum yellow leaf curl betasatellite (AYLCB), was also identified in the wheat samples. No begomovirus was detected in the wheat samples, thus establishing association of the above-named satellites with WDIV. Agrobacterium-mediated inoculation of WDIV in wheat, in the presence of either of the alphasatellites or the betasatellite, resulted in infections inducing more severe symptoms. WDIV efficiently maintained each of the alphasatellites and the betasatellite in wheat. The satellites enhanced the level of WDIV DNA in wheat. Inoculation of the satellites isolated from wheat with various begomoviruses into Nicotiana tabacum demonstrated that these remain capable of interacting with the viruses with which they were first identified. Virus-specific small RNAs accumulated in wheat upon infection with WDIV but were lower in abundance in plants coinfected with the satellites, suggesting that both the alphasatellites and the betasatellite suppress RNA silencing. These results suggest that the selective advantage for the maintenance of the alphasatellites and the betasatellite by WDIV in the field is in overcoming RNA silencing-mediated host defense. IMPORTANCE: Wheat is the most widely cultivated cereal crop in the world. A number of viruses are important pathogens of wheat, including the viruses of the genus Mastrevirus, family Geminiviridae. This study reports the association of subgenomic components, called satellites (alpha- and betasatellites), with a mastrevirus, Wheat Dwarf India Virus (WDIV), isolated from two distant locations in India. This study reports the first identification of the satellites in a monocot plant. The satellites enhanced accumulation of WDIV and severity of disease symptoms. The satellites lowered the concentration of virus-specific small RNAs in wheat plants, indicating their silencing suppressor activity. The involvement of the satellites in symptom severity of the mastrevirus can have implications in the form of economic impact of the virus on crop yield. Understanding the role of the satellites in disease severity is important for developing disease management strategies.

Virus-induced gene silencing using a modified betasatellite: a potential candidate for functional genomics of crops.

Betasatellites are geminivirus-associated single-stranded DNA molecules that play an important role in symptom modulation. A VIGS vector was developed by modifying cotton leaf curl Multan betasatellite (CLCuMB). CLCuMB DNA was modified by replacing the ßC1 gene with a multiple cloning site. The silencing ability of the modified CLCuMB was investigated by cloning a fragment of a host gene (Su) or a reporter transgene (uidA) into the modified CLCuMB and co-agroinoculation with cotton leaf curl Multan virus, cotton leaf curl Kokhran virus, and ageratum enation virus, separately. The inoculated Nicotiana tabacum, N. benthamiana, Solanum lycopersicum, Arabidopsis thaliana and Gossypium hirsutum plants showed efficient silencing of the cognate genes.

Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

BACKGROUND: Expression of double strand RNA (dsRNA) designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi), thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci) upon oral feeding. The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. METHODOLOGY/PRINCIPAL FINDINGS: Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. CONCLUSIONS/SIGNIFICANCE: Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops.