Purification, Characterization and Bioefficacy of Legume Lectins against Mustard Aphid

Lectins are carbohydrate-binding proteins which bind to glycoproteins, glycolipids, and also polysaccharides. They recognize specific carbohydrate structures and agglutinate a variety of animal cells by binding to their cell-surface glycoproteins and glycolipids (Van Damme et al., 1998; Bharathi, 2017). Lectins are highly diverse in structure, molecular weight, composition, and number of sugar binding sites present per molecule (Laija et al., 2010). They are widely distributed in nature and can be found in plants, animals and microorganisms (Jawade et al., 2016) with most abundance in plants, especially in legume seeds, where they constitute 15% of the total proteins (Loris et al., 1998; Sagar and Dhall, 2018).
       
Lectins play an important role in nitrogen fixation, plant defense and stress physiology, symbiotic interactions between the plants and microorganisms, carbohydrate metabolism and packaging of storage proteins (Ayesha and Rao, 2020; Thakur et al., 2013). Lectins are highly resistant to proteolysis, can bind to insect proteins mainly in the gut, thus retain inside the insect body (Lagarda-Diaz et al., 2017). The anti-insect activity of plant lectins against a broad range of insect species has been identified (Fitches et al., 2010). Thus, lectins could act as one of the promising agents against insect pests under integrated pest management strategies. Several insect-resistant transgenics have been developed in economically-important crops like cotton, maize, rice and potato, which primarily carry genes of bacterial origin encoding Bacillus thuringiensis toxins (Hussain et al., 2008; Wang et al., 2005). Now other sources of potential insecticidal gene products are also being studied, mainly from plant defense proteins such as lectins or protease inhibitors or both as fusion gene (Singh et al., 2006).
       
Mustard aphid (Lipaphis erysimi) is a major pest of oilseed crops. It is a phloem-sap sucking pest that can cause up to 75% loss in crop yield (Kumar et al., 2011). Parasitic feeding in Brassica oilseeds leads to yellowing and curling of leaves, followed by wilting and stunting of plant parts, that eventually results in retarded growth, poor seed formation and low oil content. The aphids are currently being controlled with the indiscriminate use of insecticidal sprays. The focus has recently moved towards developing the alternatives for example, entomotoxic proteins so as to curtail down the usage of chemical pesticides (Jaber et al., 2010). Lectins as entomotoxic proteins could offer a promising strategy that needs to be explored against insect pests (Paul and Das, 2020; Sathyapriya et al., 2012).
       
There are a few scientific reports on purification of lectins isolated from soybean and lentil, but the information about the insecticidal effect of purified lectins against mustard aphid is still lacking. Hence, the main objective of the present study was to isolate and purify lectins from the seeds of Soybean variety SL 525 and Lentil variety LL 931, respectively and assess their efficacy against mustard aphid (Lipaphis erysimi).

Plant material
 
Seeds of pulses (Glycine max- Soybean variety SL 525 and Lens culinaris- Lentil variety LL 931) were procured from Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana. Experiments were conducted during 2012-13 at Department of Biochemistry, Punjab Agricultural University, Ludhiana.
 
Protein purification
 
For isolation, the seedflour (20g) was defatted with petroleum ether (60°-80ºC). The defatted seedmeal (10g) treated with 0.9% sodium chloride solution (100ml), was shaken for 2 hours and kept overnight at 4ºC. After centrifugation at 10,000 rpm (4ºC), the supernatant was subjected to (NH₄)₂SO₄ fractionation and dialyzed against normal saline solution (Bajaj et al., 2001). The dialysed ammonium sulfate fraction (2.5 ml) was loaded on treated ion exchange resin Toyopearl DEAE-650 M column (13 cm x 1.0 cm) equilibrated with 0.01 M PBS (pH 7.4) and eluted the fractions (2 ml each) with stepwise NaCl gradient, 0-500 mM (Bala, 1998; Oliveira et al., 2008). The haemagglutination activity of the preparations was determined following serial dilution technique (Liener and Hill, 1953) and the protein content was also estimated (Lowry et al., 1951). The same purification protocol was followed for both the legume samples.
 
Assay for haemagglutination activity
 
A serial two-fold dilutions of the lectin solution in microtiter U plates were mixed with 50 µl of 2% suspension of red blood cells in 0.9% normal saline, and were allowed to stand for two hours at 37º C. Agglutination and the clumping of cells was then observed. A tube containing 50 µl of saline and 50 µl of 2% RBC suspension served as a negative control. One haemagglutination unit (HU) was assigned to the tube with maximum dilution showing haemagglutination. Lectin activity was expressed as HU/ml. Specific activity was expressed as HU/mg protein.
 
Sodium dodecyl sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of lectins
 
The molecular mass of purified lectin preparation was studied by SDS-PAGE using the standardized protocol of Walker (1996) with 10% resolving gel and 5% stacking gel composition. Protein bands were coomassie brilliant blue stained to detect proteins after electrophoretic separation (Merril et al., 1981).
Temperature, pH, metal ion stability and sugar specificity of lectins
 
The samples were incubated at different temperatures viz. 40°C, 50°C, 60°C, 70°C, 80°C and 90°C for 30 min to find out the thermostability of lectins (Bala, 1998). Lectin stability at different pH was studied using PBS with a pH range of 5.0-9.0 (Bala, 1998; Oliveira et al., 2008). The metal ion requirement for lectin activity was examined by demetalizing the sample and then treating with different metal ions (Kawagishi et al., 1990). Sugar specificity of lectins was estimated by determining the inhibition of agglutination with specific sugars. Negative agglutination indicated the specificity for that sugar (Bala et al., 2010).
 
Artificial diet bioassay
 
Liquid artificial diet mixed with different concentrations (0- 50 µg/ml) of purified lectin were prepared and introduced between the two layers of parafilm forming a diet sachet (Fig  1). Liquid diet consisted of leaf tissue extract of mustard plant and 15% sucrose (Wille and Hartman, 2008). The diet was changed every 24 hours. The insects were reared in the incubator at 22°C at a photoperiod of 16 hours light/8 hours dark. The percent mortality of the aphid was recorded after every 24 hours for a total period of 96 hours.
 

 
Aphid Mortality
 
Considering the natural death of the insect, the percentage of insect mortality was determined using Abbott’s formula:

  Where,
X = Percentage of survivability in the control where no toxin is present.
Y = Percentage of survivability in treated sample.

The data was subjected to Standard Probit Analysis to find out the values of LC₅₀ (median lethal concentration). Percentage of mortality values were further converted to probability unit (probit) with the help of computerized program POLO (Russell et al., 1977). A linearregression was obtained by plotting probit values vs. log₁₀ of doses to get the LC₅₀ values.

Isolation, purification and characterization
 
Lectins were extracted and purified from seeds of soybean (Glycine max) and lentil (Lens culinaris). The purification chart of purified lectins represented in Table 1 and Fig 2. The haemagglutination activity in the elution graph was plotted by considering maximum agglutination activity as 100% (Suseelan et al., 1997). In case of Glycine max, the dialyzed protein sample fractionated into two peaks, F1 (fraction 11-20) and F2 (fractions 40-50), on ion exchange chromatography using Toyopearl DEAE 650 M column (Fig 2). The haemagglutination active proteins separated into two isoforms corresponding to respective protein peaks. The first isoform designated as GMA-I (Glycine max agglutinin-I) eluted at 100 mM NaCl concentration.  The second isoform eluted at 200 mM NaCl concentration and was designated as GMA-II (Glycine max agglutinin-II). The recovery of GMA-I was 32.1% with a purification fold of 9.3 and that of GMA-II was 9.4% with a purification fold of 4.6. In Lens culinaris, the dialyzed ammonium sulfate fraction when subjected to ion exchange chromatography on Toyopearl 650 M column separated into two protein peaks viz. F1 (fractions 14-20) and F2 (fractions 33-42). The haemagglutination activity corresponded with F1 protein peak (fraction 11-21). The peak corresponding to lectin activity eluted at 100 mM NaCl concentration and was designated as LCA-I (Lens culinaris agglutinin-I). LCA-I was purified to 8.7-fold with a yield of 27.1%.
 

 

           
The homogeneity of purified lectins was checked on SDS-PAGE. Purified Glycine max agglutinin (GMA-I) gave a single band of approx 11 kDa on SDS-PAGE while Lens culinaris agglutinin (LCA-I) showed two bands of approx. 14 and 22 kDa respectively (Fig 3). A study conducted by Lin et al., (2008) reported that yellow soybean lectin was a tetramer of 30 kDa, while black soybean lectin a dimer of 25 kDa.
 

 
Stability studies and sugar specificity of purified lectins
 
Lectins purified from both the sources were stable upto 40ºC with complete inactivation at 70ºC in case of GMA- I while Lens culinaris lectin lost haemagglutination activity completely at 80ºC (Fig 4). The pH sensitivity profile of the lectins is shown in (Fig 5). Glycine max agglutinin (GMA-I) was stable in the pH range of 7.0 to 8.5. pH optimum for GMA-I and LCA-I was found out to be 7.5-8.0 and 7.0-7.5, respectively, corresponding to maximum haemagglutination activity in their respective pH range.
 

 

       
The incubation of Glycine max and Lens culinaris lectin with 10 mM EDTA abolished the haemagglutination activity which was later restored completely with the addition of divalent cations viz. 1 mM MnCl₂ in Glycine max lectins and 1 mM MgCl₂, 1mM MnCl₂ in Lens culinaris lectins (Table 2). These results are in agreement with the findings of Rao et al., (1998) who reported that soybean agglutinin (SBA) contained a single carbohydrate binding site and required Ca²⁺ and Mn²⁺ ions for haemagglutination activity. Similarly, Bhattacharyya et al., (1985) also reported the metalloprotein nature of lentil lectin (LcH) that requires the metal ions (Ca²⁺ and Mn²⁺) for its saccharide binding activity.
 

       
Sugar specificity of lectins was evaluated by determining the inhibition of agglutination by different sugars. Minimum inhibitory concentration (MIC) is the lowest concentration of sugar capable of complete inhibition of agglutination. Glycine max agglutinin showed specificity towards D-Galactose and N-Acetyl D-galactosamine. Both these sugars were effective for inhibiting the agglutination of rabbit erythrocytes at concentration of 0.01 M (Table 3). Bashir et al., (2010) reported the carbohydrate specificity of purified soybean lectin towards N-acetyl galactoasmine, galactose and other carbohydrates containing galactopyranosyl residue. In case of Lens culinaris agglutinin, the agglutination was readily inhibited by D-mannose at 0.01 M; D-glucose and sucrose at 0.02 M concentration.
 
 

 
Bioefficacy against mustard aphid (Lipaphis erysimi kalt.)
 
Effect of liquid artificial diet mixed with different concentrations (0-50 µg/ml) of purified lectin was studied against mustard aphid. Mortality of aphids was monitored after 24 hours interval upto 96 hours. The data of corrected % mortality at 48 hours is presented in Table 4, Fig 6. The results revealed that mortality increased with increase in lectin concentration. The percent mortality at different concentrations ranged from 0 to 81.3 for both the purified lectins, GMA-I & LCA-I respectively. The LC50 value of the purified lectin against L.erysimi was calculated by Probit analysis with a 95% confidence interval and are presented in Table 5. The LC50 values obtained were 32.1 and 19.1 µg/ml for GMA-I & LCA-I respectively. It is evident that lower the value of LC50, the more toxic the lectin is. Thus, mannose-specific LCA-I is more effective against mustard aphid as compared to galactose-specific GMA-I. Our findings are consistent with the results of other workers who reported that lectins with mannose binding specificity were most effective against hemipteran insects (Hussain et al., 2008; Majumder et al., 2004; Van Damme 2008; Zapata et al., 2016). A lucrative solution to protect crop plants from sap-sucking insects would be the production of transgenic plants expressing lectin genes compared to routine chemical insecticides used to date.
 

 



           
Further, there are some reports which shows that genetic engineering of crop plants based on lectin gene confers wide resistance against whitefly, aphids, lepidopterans and hemipteran insects (Dias et al., 2015; Dutta et al., 2005; Boddupally et al., 2018).

The lectins from seeds of two important legumes viz. Glycine max and Lens culinaris have been purified to homogeneity. Sugar specificity for Glycine max agglutinin and Lens culinaris agglutinin was found to be D-Galactose and D-Mannose respectively. Lectin from lentil seeds showed higher mortality rate in aphids in artificial diet bioassay. Based on our results it could be proposed that the lectin gene from lentil could be a potential candidate for the development of transgenic plants which may help to reduce the losses caused by sap-sucking insects in oilseed brassicas.