Allium Sativum Leaf Agglutinin Research Articles

Characterization of vascular-specific RSs1 and rolC promoters for their utilization in engineering plants to develop resistance against hemipteran insect pests

Rice sucrose synthase1, RSs1 (isolated from rice) and rolC (isolated from Agrobacterium rhizogenes) promoters were evaluated by binding analyses of their respective cis-elements with host nuclear transcription factors. The expression profile of an insecticidal protein driven by these promoters in transgenic plants was monitored. Motif-search analysis with available phloem-specific promoter sequences revealed the presence of two BoxII elements in RSs1. An octopine synthase element, a stem-specific, a root-specific and a light-responsive element were found in the rolC promoter, whereas the ASL box, GATA and 13 bp motifs were detected in both promoters. Binding analysis of these cis-elements (both in native and mutant forms) with the trans-factors present in the nuclear extracts from rice, tobacco and chickpea, followed by electrophoretic mobility shift assay, documented a highly specific cis-trans interaction. Both promoters were utilized to express Allium sativum leaf agglutinin (ASAL) gene in the three aforementioned plant systems. By immunohistochemistry and immunohistofluorescence, specific patterns of ASAL accumulation were detected in vascular tissues of single copy transgenic plants. Transgenic plants expressing ASAL in a phloem-specific manner demonstrated about 60-65% more insecticidal activity than control plants. The two promoters, which evolved independently from two distinctly unrelated origins, were found to maintain their functionality in a conserved manner. They were able to express the insecticidal protein coding ASAL as transgene both in monocot and dicot hosts. Thus, the two promoters are valuable as prospective phloem-specific promoters for use in plant biotechnological programmes.

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests.

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be approximately 12.1%+/-0.351 (mean +/- SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T0 and T1 plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P < 0.01), 32% (P < 0.05) and 40.5, 29.5% (P < 0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T1 progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.

Constitutive and phloem specific expression of Allium sativum leaf agglutinin (ASAL) to engineer aphid ( Lipaphis erysimi) resistance in transgenic Indian mustard ( Brassica juncea)

Lipaphis erysimi, commonly known as mustard aphid is a seriously damaging pest of important oilseed crop, Brassica juncea. In previous study we reported using artificial diet bioassay that Allium sativum leaf lectin, ASAL shows insecticidal activity against sap sucking L. erysimi. A gut receptor protein having binding affinity to ASAL was purified from L. erysimi. The receptor protein was further identified as a SymL, playing role in aphid mediated virus transmission. To develop resistance against the above pest, ASAL had been expressed in mustard both in constitutive and phloem tissue specific manner. Stable integration and inheritance of ASAL gene has been shown through Southern hybridization. Expression of ASAL has been confirmed in T 0 and T 1 plants through northern and western blot analyses. The segregation pattern of ASAL transgene and hygromycin selection marker gene were observed in T 1 progenies which followed the 3:1 Mendelian ratio. The phloem tissue specific expression of ASAL gene driven by rice sucrose synthase promoter (RSs-1) has also been monitored by immunohistochemical analysis of mature stem and petiole sections. In-planta bioassays on T 0 plants and their progenies exhibited the efficacy of expressed ASAL to reduce the survival and fecundity of L. erysimi. This is the first report showing sustainable resistance in transgenic B. juncea with ASAL gene against the target pest, mustard aphid.

The Interactions of Allium sativum Leaf Agglutinin with a Chaperonin Group of Unique Receptor Protein Isolated from a Bacterial Endosymbiont of the Mustard Aphid

The homopteran sucking insect, Lipaphis erysimi (mustard aphid) causes severe damage to various crops. This pest not only affects plants by sucking on the phloem, but it also transmits single-stranded RNA luteoviruses while feeding, which cause disease and damage in the crop. The mannose-binding Allium sativum (garlic) leaf lectin has been found to be a potent control agent of L. erysimi. The lectin receptor protein isolated from brush border membrane vesicle of insect gut was purified to determine the mechanism of lectin binding to the gut. Purified receptor was identified as an endosymbiotic chaperonin, symbionin, using liquid chromatography-tandem mass spectrometry. Symbionin from endosymbionts of other aphid species have been reported to play a significant role in virus transmission by binding to the read-through domain of the viral coat protein. To understand the molecular interactions of the said lectin and this unique symbionin molecule, the model structures of both molecules were generated using the Modeller program. The interaction was confirmed through docking of the two molecules forming a complex. A surface accessibility test of these molecules demonstrated a significant reduction in the accessibility of the complex molecule compared with that of the free symbionin molecule. This reduction in surface accessibility may have an effect on other molecular interactive processes, including "symbionin virion recognition", which is essential for such symbionin-mediated virus transmission. Thus, garlic leaf lectin provides an important component of a crop management program by controlling, on one hand, aphid attack and on the other hand, symbionin-mediated luteovirus transmission.