Allium Sativum Leaf Agglutinin Research Articles

Transgenic cotton expressing Allium sativum leaf agglutinin exhibits resistance to whiteflies and aphids without negative effects on ladybugs

Cotton (Gossypium hirsutum L.) is of great economic importance as a cultivated crop in many parts of the world. In addition to being a pillar of the textile industry, cotton and its byproducts are used for livestock feed, seed oil, and other products. Bacillus thuringiensis crystal toxin (Bt) expression in cotton provides effective protection against chewing insects but does not defend plants from piercing/sucking insect pests. With the aim to create transgenic plants with resistance against piercing/sucking pests, we used Agrobacterium-mediated genetic transformation of cotton cultivar Coker 312 to express the Allium sativum leaf agglutinin (ASLA) gene from the phloem-specific rolC promoter. The ASLA transgene was stably inherited and showed Mendelian segregation in the T1 generation. Transgenic lines, expressing the ASLA gene, showed explicit resistance against major sap-sucking pests. Green peach aphid (Myzus persicae Sulzer) choice assays showed that 75% of aphids preferred untransformed cotton plants relative to those expressing the ASLA gene. In detached leaf bioassays, plants expressing ASLA caused 82% aphid mortality and 44–53% reduction in fecundity. Clip cage bioassays with whiteflies (Bemisia tabaci Gennadius) showed 74–82% mortality and 44–60% decrease in fecundity due to ASLA gene expression. In whole plant bioassays, whiteflies showed 77% mortality and a 54% decrease in fecundity on ASLA transgenics. Importantly, we did not observe a negative effect of the ASLA gene on ladybugs (Coccinella septempunctata) that consumed these whiteflies. Together, our findings demonstrate the potential of ASLA-transgenic cotton for providing protection against two devastating insect pests, whiteflies and aphids. The ASLA-transgenic cotton appears promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.

Transformation of Allium sativum Leaf Agglutinin (ASAL) Gene in Cotton through Pollen Tube Method to Enhance Resistance against Whitefly

Transformation of Allium sativum Leaf Agglutinin (ASAL) Gene in Cotton through Pollen Tube Method to Enhance Resistance against Whitefly

Inhibition of Bemisia tabaci vectored, GroEL mediated transmission of tomato leaf curl New Delhi virus by garlic leaf lectin (Allium sativum leaf agglutinin)

GroEL or symbionin synthesized by the endosymbionts of whitefly (Bemisia tabaci)/ aphids play a cardinal role in the persistent, circulative transmission of plant viruses by binding to viral coat protein/ read-through protein. Allium sativum leaf agglutinin (ASAL), a Galanthus nivalis agglutinin (GNA)- related mannose-binding lectin from garlic leaf has been reported as a potent controlling agent against hemipteran insects including whitefly and aphids. GroEL related chaperonin- symbionin was previously identified as a receptor of ASAL by the present group in the brush border membrane vesicle (BBMV) of mustard aphid. In the present study similar GroEL receptor of ASAL has been identified through LC-MS/MS in the BBMV of B. tabaci which serves as a vector for several plant viruses including tomato leaf curl New Delhi virus (ToLCNDV). Ligand blot analysis of ASAL-fed B. tabaci showed that when GroEL is pre-occupied by ASAL, it completely blocks its further binding to ToLCNDV coat protein (ToLCNDV-CP). Prior feeding of ASAL hindered the co-localization of ToLCNDV-CP and GroEL in the midgut of B. tabaci. Immunoprecipitation followed by western blot with ASAL-fed B. tabaci yielded similar result. Moreover, ASAL feeding inhibited viral transmission by B. tabaci. Together, these results confirmed that the interaction of ASAL with GroEL interferes with the binding of ToLCNDV-CP and inhibits further B. tabaci mediated viral transmission.

Development of broad-spectrum and sustainable resistance in cotton against major insects through the combination of Bt and plant lectin genes.

Second generation Btinsecticidaltoxinin comibination with Allium sativum leaf agglutinin gene has been successfully expressed in cotton to develop sustainable resistance against major chewing and sucking insects. The first evidence of using the Second-generationBt gene in combination with Allium sativumplant lectin to develop sustainable resistance against chewing and sucking insects has been successfully addressed in the current study. Excessive use of Bt δ-endotoxins in the field is delimiting its insecticidal potential. Second-generation Bt Vip3Aa could be the possible alternative because it does not share midgut receptor sites with any known cry proteins. Insecticidal potential of plant lectins against whitefly remains to be evaluated. In this study, codon-optimized synthetic Bt Vip3Aa gene under CaMV35S promoter and Allium sativum leaf agglutinin gene under phloem-specific promoter were transformed in a local cotton variety. Initial screening of putative transgenic cotton plants was done through amplification, histochemical staining and immunostrip assay. The mRNA expression of Vip3Aa gene was increased to be ninefold in transgenic cotton line L6P3 than non-transgenic control while ASAL expression was found to be fivefold higher in transgenic line L34P2 as compared to non-transgenic control. The maximum Vip3Aa concentration was observed in transgenic line L6P3. Two copy numbers in homozygous form at chromosome number 9 and one copy number in hemizygous form at chromosome number 10 was observed in transgenic line L6P3 through fluorescent in situ hybridization. Significant variation was observed in transgenic cotton lines for morphological characteristics, whereas physiological parameters of plants and fiber characteristics (as assessed by scanning electron microscopic) remained comparable in transgenic and non-transgenic cotton lines. Leaf-detach bioassay showed that all the transgenic lines were significantly resistant to Helicoverpa armigera showing mortality rates between 78% and 100%. Similarly, up to 95% mortality of whiteflies was observed in transgenic cotton lines when compared with non-transgenic control lines.

The potential and efficacy of Allium sativum leaf lectin (ASAL) against sap-sucking insect pests of transgenic maize

The gene sequence encoding the mannose-binding homodimeric protein Allium sativum leaf agglutinin (ASAL) was introduced into maize inbred lines to achieve resistance against sap-sucking corn leaf aphids in transgenic lines. Maize transformants were generated after the co-cultivation of immature maize embryos with the Agrobacterium strain LB4404 harbouring a recombinant Ti-binary ASAL construct driven by the polyubiquitin promoter. The preliminary screening of maize transformants was performed via GUS histochemical analysis, and PCR and Southern blotting confirmed the insertion of the transgene and its stable integration in five transgenic maize lines. Transcript abundance was quantified by a quantitative real-time PCR assay, which revealed variable expression of the ASAL transgene among five transgenic maize lines. The highest mRNA expression of the ASAL gene was found in the A23 transgenic maize line, while the lowest expression was found in the AU1 transgenic maize line. In planta bioassays in the T1 progeny of the transgenic maize lines revealed high resistance against corn leaf aphids compared to the control non-transgenic line. The mortality of the infesting aphids (Rhopalosiphum maidi) was found to vary from 40 to 71% compared to that of the non-transgenic control maize line.

Monitoring the efficacy of mutated Allium sativum leaf lectin in transgenic rice against Rhizoctonia solani.

BackgroundRice sheath blight, caused by Rhizoctonia solani is one of the most devastating diseases of rice. It is associated with significant reduction in rice productivity worldwide. A mutant variant of mannose binding Allium sativum leaf agglutinin (mASAL) was previously reported to exhibit strong antifungal activity against R. solani. In this study, the mASAL gene has been evaluated for its in planta antifungal activity in rice plants.ResultsmASAL was cloned into pCAMBIA1301 binary vector under the control of CaMV35S promoter. It was expressed in an elite indica rice cv. IR64 by employing Agrobacterium tumefaciens-mediated transformation. Molecular analyses of transgenic plants confirmed the presence and stable integration of mASAL gene. Immunohistofluorescence analysis of various tissue sections of plant parts clearly indicated the constitutive expression of mASAL. The segregation pattern of mASAL transgene was observed in T1 progenies in a 3:1 Mendelian ratio. The expression of mASAL was confirmed in T0 and T1 plants through western blot analysis followed by ELISA. In planta bioassay of transgenic lines against R. solani exhibited an average of 55 % reduction in sheath blight percentage disease index (PDI).ConclusionsThe present study opens up the possibility of engineering rice plants with the antifungal gene mASAL, conferring resistance to sheath blight.

Deciphering the mode of action of a mutant Allium sativum Leaf Agglutinin (mASAL), a potent antifungal protein on Rhizoctonia solani.

BackgroundMutant Allium sativum leaf agglutinin (mASAL) is a potent, biosafe, antifungal protein that exhibits fungicidal activity against different phytopathogenic fungi, including Rhizoctonia solani.MethodsThe effect of mASAL on the morphology of R.solani was monitored primarily by scanning electron and light microscopic techniques. Besides different fluorescent probes were used for monitoring various intracellular changes associated with mASAL treatment like change in mitochondrial membrane potential (MMP), intracellular accumulation of reactive oxygen species (ROS) and induction of programmed cell death (PCD). In addition ligand blot followed by LC-MS/MS analyses were performed to detect the putative interactors of mASAL.ResultsKnowledge on the mode of function for any new protein is a prerequisite for its biotechnological application. Detailed morphological analysis of mASAL treated R. solani hyphae using different microscopic techniques revealed a detrimental effect of mASAL on both the cell wall and the plasma membrane. Moreover, exposure to mASAL caused the loss of mitochondrial membrane potential (MMP) and the subsequent intracellular accumulation of reactive oxygen species (ROS) in the target organism. In conjunction with this observation, evidence of the induction of programmed cell death (PCD) was also noted in the mASAL treated R. solani hyphae.Furthermore, we investigated its interacting partners from R. solani. Using ligand blots followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses, we identified different binding partners including Actin, HSP70, ATPase and 14-3-3 protein.ConclusionsTaken together, the present study provides insight into the probable mode of action of the antifungal protein, mASAL on R. solani which could be exploited in future biotechnological applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0549-7) contains supplementary material, which is available to authorized users.

Development of Transgenic Rice Harbouring Mutated Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase (Os-mEPSPS) and Allium sativum Leaf Agglutinin (ASAL) Genes Conferring Tolerance to Herbicides and Sap-Sucking Insects

Globally, weeds and sap-sucking insects are major constrains in rice production. We have combined two desired traits for resistance to glyphosate and sapsuckers by transgenic overexpression of mutated rice 5-enolpyruvylshikimate3-phosphate synthase (Os-mEPSPS) and garlic leaf lectin (Allium sativum leaf agglutinin, ASAL) in a high yielding IR-64 rice cultivar. Polymerase chain reaction (PCR), southern and genome walking analyses showed stable integration of transgenes in genomes of transgenic lines. The molecular analysis of transgenic lines revealed effective expression of both Os-mEPSPS and ASAL transcripts and their respective proteins and imparted resistance to glyphosate and brown planthopper. In planta brown hopper assays in the progenies of independent rice transgenic lines exhibit enhanced ASAL expression that imparts substantial level of resistance, reduced survival and fecundity of brown hoppers by ~74 and ~68 %, respectively. The glyphosate and sap-sucking insect pest-resistant transgenic rice lines showed normal phenotype and no growth penalty or fitness costs. The double transgenic traits co-segregated as a single unit in the subsequent generations. In spite of the presence of several herbicide- and insect-resistant GM rice crops in the market, there is a need to develop new transgenic rice lines by selecting the transgenes from homologous and/or edible crop sources that circumvent the biosafety issues raised by regulatory bodies regarding the acceptance of these products for human consumption. In this scenario, the transgenic rice varieties generated in this study serve as a novel genetic resource in rice breeding aimed at durable resistance to glyphosate and sap-sucking insect pests.

Biological Safety Assessment of Mutant Variant of Allium sativum Leaf Agglutinin (mASAL), a Novel Antifungal Protein for Future Transgenic Application

Genetic engineering has established itself to be an important tool for crop improvement. Despite the success, there is always a risk of food allergy induced by alien gene products. The present study assessed the biosafety of mutant Allium sativum leaf agglutinin (mASAL), a potent antifungal protein generated by site directed mutagenesis of Allium sativum leaf agglutinin (ASAL). mASAL was cloned in pET28a+ and expressed in E. coli, and the safety assessment was carried out according to the FAO/WHO guideline (2001). Bioinformatics analysis, pepsin digestion, and thermal stability assay showed the protein to be nonallergenic. Targeted sera screening revealed no significant IgE affinity of mASAL. Furthermore, mASAL sensitized Balb/c mice showed normal histopathology of lung and gut tissue. All results indicated the least possibility of mASAL being an allergen. Thus, mASAL appears to be a promising antifungal candidate protein suitable for agronomical biotechnology.

Development of selectable marker free, insect resistant, transgenic mustard (Brassica juncea) plants using Cre/loxmediated recombination

BackgroundAntibiotic/ herbicide resistant marker genes have been proven to be very useful in plant transformation for the initial selection of desired transgenic events. However, presence of these genes in the genetically modified crops may render the crop less acceptable to the consumers. Among several different approaches, the effectiveness of Cre/lox mediated recombination strategy for selectable marker gene (SMG) elimination has previously been demonstrated by different groups in several plants including Brassica. In the present study exploiting Cre/lox mediated recombination strategy, attempt has been made for selectable marker gene elimination from Allium sativum leaf agglutinin (ASAL) expressing Brassica plants with hemipteran insect resistant phenotype.ResultsAllium sativum leaf agglutinin (ASAL) linked with lox flanked hygromycin resistant (hpt) gene was introduced in mustard. Cre recombinase gene cassette was also integrated in separate event. A Cre/lox mediated recombination using crossing strategy was adopted to remove the hpt gene from the subsequent generation of selected hybrid events. Reciprocal crosses were made between T1ASAL-lox-hpt-lox and cre-bar plants. Marker gene elimination was confirmed in the resulting F1 hybrid progenies by PCR analysis, using hpt, cre and ASAL specific primers followed by Southern hybridization. In marker free plants, expression of ASAL was also confirmed by western blotting and ELISA analysis. Retention of functionality of expressed ASAL was investigated by agglutination assay using rabbit erythrocytes. Expressed ASAL was also found to be thermo-sensitive. In planta insect bioassay on F1 hybrid progenies exhibited detrimental effect on the performance of devastating target pest, Lipaphis erysimi. The F1 hybrid hpt negative, ASAL positive plants were allowed to self- fertilize to obtain F2 progeny plants. In some of these plants cre gene was found to be segregated out of the ASAL gene by genetic segregation yielding completely marker free plants.ConclusionsThe present study establishes the efficient expression of the newly introduced insect resistant ASAL gene even after Cre/lox mediated recombination resulting in elimination of selectable marker gene.

Development of Transgenic Cotton Lines Expressing Allium sativum Agglutinin (ASAL) for Enhanced Resistance against Major Sap-Sucking Pests

Mannose-specific Allium sativum leaf agglutinin encoding gene (ASAL) and herbicide tolerance gene (BAR) were introduced into an elite cotton inbred line (NC-601) employing Agrobacterium-mediated genetic transformation. Cotton transformants were produced from the phosphinothricin (PPT)-resistant shoots obtained after co-cultivation of mature embryos with the Agrobacterium strain EHA105 harbouring recombinant binary vector pCAMBIA3300-ASAL-BAR. PCR and Southern blot analysis confirmed the presence and stable integration of ASAL and BAR genes in various transformants of cotton. Basta leaf-dip assay, northern blot, western blot and ELISA analyses disclosed variable expression of BAR and ASAL transgenes in different transformants. Transgenes, ASAL and BAR, were stably inherited and showed co-segregation in T1 generation in a Mendelian fashion for both PPT tolerance and insect resistance. In planta insect bioassays on T2 and T3 homozygous ASAL-transgenic lines revealed potent entomotoxic effects of ASAL on jassid and whitefly insects, as evidenced by significant decreases in the survival, development and fecundity of the insects when compared to the untransformed controls. Furthermore, the transgenic cotton lines conferred higher levels of resistance (1–2 score) with minimal plant damage against these major sucking pests when bioassays were carried out employing standard screening techniques. The developed transgenics could serve as a potential genetic resource in recombination breeding aimed at improving the pest resistance of cotton. This study represents the first report of its kind dealing with the development of transgenic cotton resistant to two major sap-sucking insects.

Allergenicity Assessment of Allium sativum Leaf Agglutinin, a Potential Candidate Protein for Developing Sap Sucking Insect Resistant Food Crops

BackgroundMannose-binding Allium sativum leaf agglutinin (ASAL) is highly antinutritional and toxic to various phloem-feeding hemipteran insects. ASAL has been expressed in a number of agriculturally important crops to develop resistance against those insects. Awareness of the safety aspect of ASAL is absolutely essential for developing ASAL transgenic plants.Methodology/Principal FindingsFollowing the guidelines framed by the Food and Agriculture Organization/World Health Organization, the source of the gene, its sequence homology with potent allergens, clinical tests on mammalian systems, and the pepsin resistance and thermostability of the protein were considered to address the issue. No significant homology to the ASAL sequence was detected when compared to known allergenic proteins. The ELISA of blood sera collected from known allergy patients also failed to show significant evidence of cross-reactivity. In vitro and in vivo assays both indicated the digestibility of ASAL in the presence of pepsin in a minimum time period.Conclusions/SignificanceWith these experiments, we concluded that ASAL does not possess any apparent features of an allergen. This is the first report regarding the monitoring of the allergenicity of any mannose-binding monocot lectin having insecticidal efficacy against hemipteran insects.

Purification and Characterization of a Lectin with High Hemagglutination Property Isolated from Allium altaicum

A lectin was purified from the leaves of Allium altaicum and corresponding gene was cloned. The lectin namely Allium altaicum agglutinin (AAA) was ~24kDa homodimeric protein and similar to a typical garlic leaf lectin. It was synthesized as 177 amino acid residues pre-proprotein, which consisted of 28 and 43 amino acid long N and C-terminal signal peptides, respectively. The plant expressed this protein more in scapes and flowers in comparison to the bulbs and leaves. Hemagglutination activity (with rabbit erythrocytes) was 1,428 fold higher as compared to Allium sativum leaf agglutinin (ASAL) although, the insecticidal activity against cotton aphid (Aphis gossypii) was relatively low. Glycan array revealed that AAA had higher affinity towards GlcAb1-3Galb as compared to ASAL. Homology analysis showed 57-94% similarity with other Allium lectins. The mature protein was expressed in E. coli as a fusion with SUMO peptide in soluble and biologically active form. Recombinant protein retained high hemagglutination activity.

Functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status.

Background Allium sativum leaf agglutinin (ASAL) is a 25-kDa homodimeric, insecticidal, mannose binding lectin whose subunits are assembled by the C-terminal exchange process. An attempt was made to convert dimeric ASAL into a monomeric form to correlate the relevance of quaternary association of subunits and their functional specificity. Using SWISS-MODEL program a stable monomer was designed by altering five amino acid residues near the C-terminus of ASAL.Methodology/Principal FindingsBy introduction of 5 site-specific mutations (-DNSNN-), a β turn was incorporated between the 11th and 12th β strands of subunits of ASAL, resulting in a stable monomeric mutant ASAL (mASAL). mASAL was cloned and subsequently purified from a pMAL-c2X system. CD spectroscopic analysis confirmed the conservation of secondary structure in mASAL. Mannose binding assay confirmed that molecular mannose binds efficiently to both mASAL and ASAL. In contrast to ASAL, the hemagglutination activity of purified mASAL against rabbit erythrocytes was lost. An artificial diet bioassay of Lipaphis erysimi with mASAL displayed an insignificant level of insecticidal activity compared to ASAL. Fascinatingly, mASAL exhibited strong antifungal activity against the pathogenic fungi Fusarium oxysporum, Rhizoctonia solani and Alternaria brassicicola in a disc diffusion assay. A propidium iodide uptake assay suggested that the inhibitory activity of mASAL might be associated with the alteration of the membrane permeability of the fungus. Furthermore, a ligand blot assay of the membrane subproteome of R. solani with mASAL detected a glycoprotein receptor having interaction with mASAL.Conclusions/SignificanceConversion of ASAL into a stable monomer resulted in antifungal activity. From an evolutionary aspect, these data implied that variable quaternary organization of lectins might be the outcome of defense-related adaptations to diverse situations in plants. Incorporation of mASAL into agronomically-important crops could be an alternative method to protect them from dramatic yield losses from pathogenic fungi in an effective manner.

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.

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.

Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers

Rice, the major food crop of world is severely affected by homopteran sucking pests. We introduced coding sequence of Allium sativum leaf agglutinin, ASAL, in rice cultivar IR64 to develop sustainable resistance against sap-sucking planthoppers as well as eliminated the selectable antibiotic-resistant marker gene hygromycin phosphotransferase (hpt) exploiting cre/lox site-specific recombination system. An expression vector was constructed containing the coding sequence of ASAL, a potent controlling agent against green leafhoppers (GLH, Nephotettix virescens) and brown planthopper (BPH, Nilaparvata lugens). The selectable marker (hpt) gene cassette was cloned within two lox sites of the same vector. Alongside, another vector was developed with chimeric cre recombinase gene cassette. Reciprocal crosses were performed between three single-copy T(0) plants with ASAL- lox-hpt-lox T-DNA and three single-copy T(0) plants with cre-bar T-DNA. Marker gene excisions were detected in T(1) hybrids through hygromycin sensitivity assay. Molecular analysis of T(1) plants exhibited 27.4% recombination efficiency. T(2) progenies of L03C04(1) hybrid parent showed 25% cre negative ASAL-expressing plants. Northern blot, western blot and ELISA showed significant level of ASAL expression in five marker-free T(2) progeny plants. In planta bioassay of GLH and BPH performed on these T(2) progenies exhibited radical reduction in survivability and fecundity compared with the untransformed control plants.

Tissue specific expression of potent insecticidal, Allium sativum leaf agglutinin (ASAL) in important pulse crop, chickpea (Cicer arietinum L.) to resist the phloem feeding Aphis craccivora

The phloem sap-sucking hemipteran insect, Aphis craccivora, commonly known as cowpea aphid, cause major yield loss of important food legume crop chickpea. Among different plant lectins Allium sativum leaf agglutinin (ASAL), a mannose binding lectin was found to be potent antifeedant for sap sucking insect A. craccivora. Present study describes expression of ASAL in chickpea through Agrobacterium-mediated transformation of "single cotyledon with half embryo" explant. ASAL was expressed under the control of CaMV35S promoter for constitutive expression and phloem specific rolC promoter for specifically targeting the toxin at feeding site, using pCAMBIA2301 vector containing plant selection marker nptII. Southern blot analysis demonstrated the integration and copy number of chimeric ASAL gene in chickpea and its inheritance in T(1) and T(2) progeny plants. Expression of ASAL in T(0) and T(1) plants was confirmed through northern and western blot analysis. The segregation pattern of ASAL transgene was observed in T(1) progenies, which followed the 3:1 Mendelian ratio. Enzyme linked immunosorbant assay (ELISA) determined the level of ASAL expression in different transgenic lines in the range of 0.08-0.38% of total soluble protein. The phloem tissue specific expression of ASAL gene driven by rolC promoter has been monitored by immunolocalization analysis of mature stem sections. Survival and fecundity of A. craccivora decreased to 11-26% and 22-42%, respectively when in planta bioassay conducted on T(1) plants compared to untransformed control plant which showed 85% survival. Thus, through unique approach of phloem specific expression of novel insecticidal lectin (ASAL), aphid resistance has been successfully achieved in chickpea.

Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests.

BackgroundRice (Oryza sativa) productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which ~21% is attributed to the attack of insect pests. In this paper we report the isolation, cloning and characterization of Allium sativum leaf agglutinin (asal) gene, and its expression in elite indica rice cultivars using Agrobacterium-mediated genetic transformation method. The stable transgenic lines, expressing ASAL, showed explicit resistance against major sap-sucking pests.ResultsAllium sativum leaf lectin gene (asal), coding for mannose binding homodimeric protein (ASAL) from garlic plants, has been isolated and introduced into elite indica rice cultivars susceptible to sap-sucking insects, viz., brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH). Embryogenic calli of rice were co-cultivated with Agrobacterium harbouring pSB111 super-binary vector comprising garlic lectin gene asal along with the herbicide resistance gene bar, both under the control of CaMV35S promoter. PCR and Southern blot analyses confirmed stable integration of transgenes into the genomes of rice plants. Northern and western blot analyses revealed expression of ASAL in different transgenic rice lines. In primary transformants, the level of ASAL protein, as estimated by enzyme-linked immunosorbent assay, varied between 0.74% and 1.45% of the total soluble proteins. In planta insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on BPH, GLH and WBPH insects, as evidenced by significant decreases in the survival, development and fecundity of the insects.ConclusionIn planta insect bioassays were carried out on asal transgenic rice lines employing standard screening techniques followed in conventional breeding for selection of insect resistant plants. The ASAL expressing rice plants, bestowed with high entomotoxic effects, imparted appreciable resistance against three major sap-sucking insects. Our results amply demonstrate that transgenic indica rice harbouring asal exhibit surpassing resistance against BPH, GLH and WBPH insects. The prototypic asal transgenic rice lines appear promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.

Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect

A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T(0) lox plants was crossed with T(0) Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T(1) plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T(1) hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T(2) plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view.