K. Chandrashekar

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.

siRNA machinery in whitefly (Bemisia tabaci).

Background RNA interference has been emerged as an utmost tool for the control of sap sucking insect pests. Systemic response is necessary to control them in field condition. Whitefly is observed to be more prone to siRNA in recent studies, however the siRNA machinery and mechanism is not well established. Methodology/Principal Findings To identify the core siRNA machinery, we curated transcriptome data of whitefly from NCBI database. Partial mRNA sequences encoding Dicer2, R2D2, Argonaute2 and Sid1 were identified by tblastn search of homologous sequences from Aphis glycines and Tribolium castaneum. Complete encoding sequences were obtained by RACE, protein sequences derived by Expasy translate tool and confirmed by blastp analysis. Conserved domain search and Prosite-Scan showed similar domain architecture as reported in homologs from related insects. We found helicase, PAZ, RNaseIIIa, RNaseIIIb and double-stranded RNA-binding fold (DSRBF) in Dicer2; DsRBD in R2D2; and PAZ and PIWI domains in Argonaute2. Eleven transmembrane domains were detected in Sid1. Sequence homology and phylogenetic analysis revealed that RNAi machinery of whitefly is close to Aphids. Real-time PCR analysis showed similar expression of these genes in different developmental stages as reported in A. glycines and T. castaneum. Further, the expression level of above genes was quite similar to the housekeeping gene actin. Conclusions/Significance Availability of core siRNA machinery including the Sid1 and their universal expression in reasonable quantity indicated significant response of whitefly towards siRNA. Present report opens the way for controlling whitefly, one of the most destructive crop insect pest.

SUMO fusion facilitates expression and purification of garlic leaf lectin but modifies some of its properties.

Over expression of lectin genes in E. coli often gives inclusion bodies that are solubilised to characterize lectins. We made N-terminal fusion of the Allium sativum leaf agglutinin (ASAL) with SUMO (small ubiquitin related modifier) peptide. The SUMO peptide allowed expression of the recombinant lectin in E. coli, predominantly in soluble form. The soluble fusion protein could be purified by immobilized metal affinity column (IMAC), followed by size exclusion chromatography. The SUMO protease failed to cleave the SUMO peptide from ASAL. This may be due to steric hindrance caused by the homodimer structure of the chimeric ASAL. Some properties like dimerization, haemagglutination and insecticidal properties of the recombinant SUMO-ASAL fusion protein were comparable to the plant derived native lectin. However, glycan array analysis revealed that the carbohydrate binding specificity of the recombinant SUMO-ASAL was altered. Further, the fusion protein was not toxic to E. coli (native ASAL exhibited toxicity). The recombinant lectin was more thermo-labile as compared to the native lectin. Three important findings of this study are: (1) sugar specificity of ASAL can be altered by amino-terminal fusion; (2) anti-E. coli activity of ASAL can be eliminated by N-terminal SUMO fusion and (3) SUMO-ASAL may be a preferred candidate insecticidal protein for the development of transgenic plants.

Interaction of Allium sativum leaf agglutinin with midgut brush border membrane vesicles proteins and its stability in Helicoverpa armigera.

Allium sativum leaf agglutinin (ASAL) binds to several proteins in the midgut of Helicoverpa armigera and causes toxicity. Most of these were glycosylated. Six ASAL‐binding proteins were selected for identification. PMF and MS/MS data showed their similarity with midgut aminopeptidase APN2, polycalins and alkaline phosphatase of H. armigera, cadherin‐N protein (partial AGAP009726‐PA) of Acyrthosiphon pisum, cytochrome P450 (CYP315A1) of Manduca sexta and alkaline phosphatase of Heliothis virescens. Some of the ASAL‐binding midgut proteins were similar to the larval receptors responsible for the binding of δ‐endotoxin proteins of Bacillus thuringiensis. Galanthus nivalis agglutinin also interacted with most of the ASAL‐binding proteins. The ASAL showed resistance to midgut proteases and was detected in the larval hemolymph and excreta. Immunohistochemical staining revealed the presence of ASAL in the body tissue also.

Enhanced Methanol Production in Plants Provides Broad Spectrum Insect Resistance

Plants naturally emit methanol as volatile organic compound. Methanol is toxic to insect pests; but the quantity produced by most of the plants is not enough to protect them against invading insect pests. In the present study, we demonstrated that the over-expression of pectin methylesterase, derived from Arabidopsis thaliana and Aspergillus niger, in transgenic tobacco plants enhances methanol production and resistance to polyphagous insect pests. Methanol content in the leaves of transgenic plants was measured using proton nuclear spectroscopy (1H NMR) and spectra showed up to 16 fold higher methanol as compared to control wild type (WT) plants. A maximum of 100 and 85% mortality in chewing insects Helicoverpa armigera and Spodoptera litura larvae was observed, respectively when fed on transgenic plants leaves. The surviving larvae showed less feeding, severe growth retardation and could not develop into pupae. In-planta bioassay on transgenic lines showed up to 99 and 75% reduction in the population multiplication of plant sap sucking pests Myzus persicae (aphid) and Bemisia tabaci (whitefly), respectively. Most of the phenotypic characters of transgenic plants were similar to WT plants. Confocal microscopy showed no deformities in cellular integrity, structure and density of stomata and trichomes of transgenic plants compared to WT. Pollen germination and tube formation was also not affected in transgenic plants. Cell wall enzyme transcript levels were comparable with WT. This study demonstrated for the first time that methanol emission can be utilized for imparting broad range insect resistance in plants.

Whitefly genome expression reveals host-symbiont interaction in amino acid biosynthesis.

Background Whitefly (Bemisia tabaci) complex is a serious insect pest of several crop plants worldwide. It comprises several morphologically indistinguishable species, however very little is known about their genetic divergence and biosynthetic pathways. In the present study, we performed transcriptome sequencing of Asia 1 species of B. tabaci complex and analyzed the interaction of host-symbiont genes in amino acid biosynthetic pathways. Methodology/Principal Findings We obtained about 83 million reads using Illumina sequencing that assembled into 72716 unitigs. A total of 21129 unitigs were annotated at stringent parameters. Annotated unitigs were mapped to 52847 gene ontology (GO) terms and 131 Kyoto encyclopedia of genes and genomes (KEGG) pathways. Expression analysis of the genes involved in amino acid biosynthesis pathways revealed the complementation between whitefly and its symbiont partner Candidatus Portiera aleyrodidarum. Most of the non-essential amino acids and intermediates of essential amino acid pathways were supplied by the host insect to its symbiont. The symbiont expressed the pathways for the essential amino acids arginine, threonine and tryptophan and the immediate precursors of valine, leucine, isoleucine and phenyl-alanine. High level expression of the amino acid transporters in the whitefly suggested the molecular mechanisms for the exchange of amino acids between the host and the symbiont. Conclusions/Significance Our study provides a comprehensive transcriptome data for Asia 1 species of B. tabaci complex that focusses light on integration of host and symbiont genes in amino acid biosynthesis pathways.

Pectin Methylesterase of Datura species, purification, and characterization from Datura stramoniumand its application

Pectin methylesterases (PME; EC 3.1.1.11) involved in de-esterification of pectin and have applicability in food, textiles, wines, pulp, and paper industries. In the present study, we compared PME activity of different parts of 3 Datura species and found that fruit coat showed maximum PME activity followed by leaf and seed. PME from leaves of D. stramonium (DsPME) was purified and characterized. DsPME showed optimum activity at 60 °C and pH 9 in the presence of 0.3 M NaCl. DsPME was stable at 70 °C and retained more than 40% activity after 60 min of incubation. However, enzyme activity completely abolished at 80 after 5 min of incubation. It follows Michaelis-Menten enzyme kinetics. Km and Vmax with citrus pectin were 0.008 mg/ml and 16.96 µmol/min, respectively. DsPME in combination with polygalactourenase (PGA) increased the clarity of orange, apple, pomegranate and pineapple juices by 2.9, 2.6, 2.3, and 3.6 fold, respectively in comparison to PGA alone. Due to very high de-esterification activity, easy denaturation and significant efficacy in incrementing clarification of fruit juice makes DsPME useful for industrial application.

Compatibility of garlic (Allium sativum L.) leaf agglutinin and Cry1Ac δ-endotoxin for gene pyramiding

Abstractδ-Endotoxins produced by Bacillus thuringiensis (Bt) have been used as bio-pesticides for the control of lepidopteran insect pests. Garlic (Allium sativum L.) leaf agglutinin (ASAL), being toxic to several sap-sucking pests and some lepidopteran pests, may be a good candidate for pyramiding with δ-endotoxins in transgenic plants for enhancing the range of resistance to insect pests. Since ASAL shares the midgut receptors with Cry1Ac in Helicoverpa armigera, there is possibility of antagonism in their toxicity. Our study demonstrated that ASAL increased the toxicity of Cry1Ac against H. armigera while Cry1Ac did not alter the toxicity of ASAL against cotton aphids. The two toxins interacted and increased binding of each other to brush border membrane vesicle (BBMV) proteins and to the two important receptors, alkaline phosphatase (ALP) and aminopeptidase N (APN). The results indicated that the toxins had different binding sites on the ALP and APN but influenced mutual binding. We conclude that ASAL can be safely employed with Cry1Ac for developing transgenic crops for wider insect resistance.