Development of CS-TPP-dsRNA Nanoparticles to Enhance RNAi Efficiency in the Yellow Fever Mosquito, \u3cem\u3eAedes aegypti\u3c/em\u3e

Mosquito-borne diseases are a major threat to human health and are responsible for millions of deaths globally each year. Vector control is one of the most important approaches used in reducing the incidence of these diseases. However, increasing mosquito resistance to chemical insecticides presents challenges to this approach. Therefore, new strategies are necessary to develop the next generation vector control methods. Because of the target specificity of dsRNA, RNAi-based control measures are an attractive alternative to current insecticides used to control disease vectors. In this study, Chitosan (CS) was cross-linked to sodium tripolyphosphate (TPP) to produce nano-sized polyelectrolyte complexes with dsRNA. CS-TPP-dsRNA nanoparticles were prepared by ionic gelation method. The encapsulation efficiency, protection of dsRNA from nucleases, cellular uptake, in vivo biodistribution, larval mortality and gene knockdown efficiency of CS-TPP-dsRNA nanoparticles were determined. The results showed that at a 5:1 weight ratio of CS-TPP to dsRNA, nanoparticles of less than 200 nm mean diameter and a positive surface charge were formed. Confocal microscopy revealed the distribution of the fed CS-TPP-dsRNA nanoparticles in midgut, fat body and epidermis of yellow fever mosquito, Aedes aegypti larvae. Bioassays showed significant mortality of larvae fed on CS-TPP-dsRNA nanoparticles. These assays also showed knockdown of a target gene in CS-TPP-dsRNA nanoparticle fed larvae. These data suggest that CS-TPP nanoparticles may be used for delivery of dsRNA to mosquito larvae

Double-Stranded RNA Binding Protein, Staufen, Is Required for the Initiation of RNAi in Coleopteran Insects

RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, Leptinotarsa decemlineata and Tribolium castaneum, showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing L. decemlineata, Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects

Soil and Herbage Heavy Metal/ Trace Element Variability and Relationships at Farm and Regional Level.

End of Project ReportVariability of heavy metal trace/element amounts in soil and herbage from 3 farms encompassing 26.5, 33.6 and 21.6 ha and in a 230 km2 surrounding area has been estimated. Variability was least in the farm with the greatest soil physical uniformity. It increased in line with increasing sampling area. In soil, variability was least (CV 10%) for sesquioxide metals, iron and aluminium, and elements chromium and nickel. It was highest where there was evidence of geochemical contamination with cadmium and selenium. In general, variability was higher (>20% CV) for extractable components including copper, zinc and manganese. Regression analysis of elements in soil showed up a number of useful associations particularly for the volatiles selenium, sulphate sulphur, mercury, cobalt, vanadium and fluorine with soil organic matter. Associations were also found between fluorine, vanadium and chromium. • Elements in herbage exhibited two types of associations (i) those reckoned to be within the plant and confined mostly to major elements and (ii) those involving metals associated primarily with soil and indicative of the extent to which herbage was contaminated by soil. • Elements that showed lower variability in soil exhibited large variability in herbage. These included iron, aluminium, chromium, vanadium and fluorine, where the cause was attributed to contamination of herbage by soil. It was suggested that iron could be used almost equally as well as titanium as an indicator of the degree to which herbage has been contaminated by soil. • Long term monitoring of herbage for selenium content highlighted the unreliability of relying on a single determination to characterise the selenium status of pasture

Effects of Deficiency and Excess of Zinc on Morphophysiological Traits and Spatiotemporal Regulation of Zinc-Responsive Genes Reveal Incidence of Cross Talk between Micro- and Macronutrients

Zinc (Zn) is an essential micronutrient which affects plant growth and development in deficiency and can be toxic when present in excess. In Arabidopsis thaliana, different families of cation transporters play pivotal roles in Zn homeostasis. In the present study, we evaluated the effects of Zn in its deficiency (0 μM; Zn−) and excess (75 μM; Zn++) on various morphophysiological and molecular traits. Primary root length was reduced in Zn– seedlings, whereas there were significant increases in the number and length of lateral roots under Zn– and Zn++ conditions, respectively. Concentration of various macro- and microelements showed variations under different Zn regimes and notable among them was the reduced level of iron (Fe) in Zn++ seedlings compared to Zn+. Certain members of the ZIP family (<i>ZIP4</i>,<i> ZIP9</i>, and <i>ZIP12</i>) showed significant induction in roots and shoots of the Zn– seedlings. Their suppression under Zn++ condition indicated their transcriptional regulation by Zn and their roles in the maintenance of its homeostasis. Zn-deficiency-mediated induction of <i>HMA2</i> in roots and shoots suggested its role in effluxing Zn into xylem for long-distance transport. Attenuation in the expression of Fe-responsive <i>FRO2</i> and <i>IRT1</i> in Zn– roots and their induction in Zn++ roots provided empirical evidence toward the prevalence of a cross talk between Zn and Fe homeostasis. Variable effects of Zn– and Zn++ on the expression of subset of genes involved in the homeostasis of phosphate (Pi), potassium (K), and sulfur (S) further highlighted the prevalence of cross talk between the sensing and signaling cascades of Zn and macronutrients. Further, the inducibility of <i>ZIP4</i> and <i>ZIP12</i> in response to cadmium (cd) treatment could be harnessed by tailoring them in homologous or heterologous plant system for removing pollutant toxic heavy metals from the environment

Not Available

Not AvailableWeed infestation is one of the major biotic stress factors that is responsible for yield loss in direct-seeded rice (DSR). Herbicide-resistant rice has potential to improve the efficiency of weed management under DSR. Hence, the popular indica rice cultivar IR64, was genetically modified using Agrobacterium-mediated transformation with a codon-optimized CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, with N-terminal chloroplast targeting peptide from Petunia hybrida. Integration of the transgenes in the selected rice plants was confirmed by Southern hybridization and expression by Northern and herbicide tolerance assays. Transgenic plants showed EPSPS enzyme activity even at high concentrations of glyphosate, compared to untransformed control plants. T 0, T 1 and T 2 lines were tested by herbicide bioassay and it was confirmed that the transgenic rice could tolerate up to 1 % of commercial Roundup, which is five times more in dose used to kill weeds under field condition. All together, the transgenic rice plants developed in the present study could be used efficiently to overcome weed menace.Not Availabl

Delineating the glycoproteome of elongating cotton fiber cells

The data presented here delineates the glycoproteome component in the elongating cotton fiber cells attained using complementary proteomic approaches followed by protein and N-linked glycosylation site identification (Kumar et al., 2013) [1]. Utilizing species specific protein sequence databases in proteomic approaches often leads to additional information that may not be obtained using cross-species databases. In this context we have reanalyzed our glycoproteome dataset with the Gossypium arboreum, Gossypium raimondii (version 2.0) and Gossypium hirsutum protein databases that has led to the identification of 21 N-linked glycosylation sites and 18 unique glycoproteins that were not reported in our previous study. The 1D PAGE and solution based glycoprotein identification data is publicly available at the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) [2] using the dataset identifier PXD000178 and the 2D PAGE based protein identification and glycopeptide approach based N-linked glycosylation site identification data is available at the ProteomeXchange Consortium via the PRIDE partner repository (Vizcaíno et al., 2013) [2] using the dataset identifier PXD002849

Silencing of <i>HaAce1</i> gene by host-delivered artificial microRNA disrupts growth and development of <i>Helicoverpa armigera</i>

<div><p>The polyphagous insect-pest, <i>Helicoverpa armigera</i>, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of <i>Bt</i> transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement <i>Bt</i> to tackle insect-pests menace. In this study, we report host-delivered silencing of <i>HaAce1</i> gene, encoding the predominant isoform of <i>H</i>. <i>armigera</i> acetylcholinesterase, by an artificial microRNA, <i>HaAce1</i>-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with <i>HaAce1</i>-amiR1/<i>HaAce1</i>-amiR1* sequences. The recombinant <i>HaAce1</i>-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing <i>HaAce1</i>-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of <i>HaAce1</i> mRNA was 70–80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of <i>HaAce1</i> gene for <i>H</i>. <i>armigera</i> management.</p></div

Additional file 3: Table S3. of Transcript profiling of genes expressed during fibre development in diploid cotton (Gossypium arboreum L.)

Differentially expressed transcripts encoding transcription factors (TFs) in Gossypium arboreum fuzzy-lintless line (Fl) as compared to fuzzy-linted (FL) at fibre itiation stage (0 dpa) and fibre elongation stage (10 dpa). (DOC 75 kb

Designing and construction of pBI::HAR1.

<p>Primer extension PCR was done with a pair of overlapping primers, <i>HAR-</i>F & <i>HAR</i>-R (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194150#pone.0194150.s006" target="_blank">S1 Table</a>) containing <i>Xba</i>I and <i>Sac</i>I recognition sequences at their 5′ ends. <i>Xba</i>I and <i>Sac</i>I digested PCR product was ligated onto pUC19. pBI121 backbone was generated by digestion with <i>Xba</i>I and <i>Sac</i>I that released <i>GUS</i> coding sequence. After sequence confirmation, <i>HaAce1</i>-preamiRNA1 DNA fragment was released from recombinant pUC19 and ligated onto pBI121 backbone to get pBI::HAR1.</p