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

Abstract

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.

Mechanisms of plant defense against insect herbivores

“…; jack fruit), OSA (Oryza sativa L.; rice), ASAL (Allium sativum L.), and UDA (Urtica dioica L.; stinging nettle). 58,60,63 The Arum maculatum L. lectin has been found effective against the aphids Lipaphis erysimi (Kalt.) and A. craccivora when incorpoated in an artificial diet.…”

“…Lectins are carbohydrate-binding (glyco) proteins, ubiquitous in nature, and have protective function against a range of pests. 58,59 The insecticidal activities of different plant lectins have been utilized as naturally occurring insecticides against insect pests ( Table 2). 60 One of the most important properties of lectins is their survival in the digestive system of herbivores that gives them a strong insecticidal potential.…”

Computational and Real-Time RT-PCR Expression Analyses of Garlic Lectin Gene

“…Garlic lectins remained the focus of various transformation studies. Studies such as pyramided rice lines (Foissac et al, 2000;Nagadhara et al, 2003); marker genefree transgenic tobacco (Bharathi et al, 2011); chickpea (Chakraborti et al, 2009); rice (Yarasi et al, 2008) show effectiveness of lectins against sucking insect population. The efficacy of snowdrop (G. nivalis) and garlic lectins have been studied against phloem feeding Aphis craccivora, cotton aphids A. gossypii (Sauvion et al, 1996), red spider mite of tea (Roy et al, 2008), cotton leafworm and sapsucking planthoppers (Powell, 2001).…”

“…Previous studies have only focused on the glycosylation patterns and protein structural analysis of lectins (Pilobello et al, 2005;Chakraborti et al, 2009), lectin protein expression and insecticidal efficacy based studies via expression in Pichia pastoris (Aslam and Gilani, 2000), and statistical study of multivalent nature of lectins (Damme et al, 1992). Transformation studies have also been conducted to study efficacy of lectins against sucking insects in pyramided rice lines (Bharathi et al, 2011), marker gene-free transgenic rice (Yarasi et al, 2008;Sengupta et al, 2010), tobacco (Sadeghi et al, 2008) and chickpea (Chakraborti et al, 2009).…”

“…Transformation studies have also been conducted to study efficacy of lectins against sucking insects in pyramided rice lines (Bharathi et al, 2011), marker gene-free transgenic rice (Yarasi et al, 2008;Sengupta et al, 2010), tobacco (Sadeghi et al, 2008) and chickpea (Chakraborti et al, 2009). However, studies have not been conducted on the gene expression levels of lectin and roles of its protein structure in insecticidal activity.…”