Rubina Mushtaq (M.Phil)

Production of Bacillus thuringiensis Recombinant Cry Proteins and Analysis of Mode of Action of their Toxicity

Thesis Submitted to University of the Punjab, Lahore for the Award of Degree of Doctor of Philosophy in Biological Sciences

Research Supervisor Prof. Dr. A.R. Shakoori Aizaz-i-Kamal, Tamgha-i-Imtiaz, ECO Laureate Distinguished National Professor, Professor Emeritus School of Biological Sciences, University of the Punjab

School of Biological Sciences, University of the Punjab, Lahore, Pakistan 2018

DEDICATION

I would like to dedicate this work to my parents, my husband and to my loving children who helped me unconditionally

SUMMARY

The Cry insecticidal proteins of Bacillus thuringiensis (Bt) are produced by transgenic crops for effective and environmentally-safe insect pest control. These transgenic Bt crops are considered to be the most successful agricultural biotechnology for insect control, yet their sustainability is threatened by the evolution of resistance in targeted pests. The evidence suggests that this resistance is probably due to the alterations in recognition of Cry toxin receptors in the insect midgut membrane. Consequently, information is needed on determinants of receptor recognition for designing improved toxins and to adopt effective insect resistance management practices. In this study four Cry proteins, Cry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 were expressed either in E. coli expression system or in native Bt strain. Insoluble Cry1Ac and Cry2Ac7 proteins were refolded and purified prior to use. Bioassays of Cry1Ac (C-terminally truncated version) and Cry2Ac7 protoxins were performed with an armyworm, Spodoptera litura Fabricius (Lepidoptera: Noctuidae), a polyphagous cosmopolitan insect which is a serious crop pest of many Asian countries including Pakistan. Cry2Ac7 was found to be toxic to this pest whereas tnCry1ac (truncated Cry1Ac) was nontoxic. To activate the protoxins, the proteins were trypsin digested and further purified by anion-exchange chromatography. Trypsin activated proteins (toxins) were assayed against velvetbean caterpillar, Anticarsia gemmatalis and soybean looper, Chrysodeixes includens (Pseudoplusia includens) which are lepidopteran pests of crops of great economic importance especially soybean. All these proteins were found to be toxic to both of these pests. Amongst all these toxins Cry1Ac was the most highly toxic protein. In general, C. includens larvae were always less susceptible compared to larvae of A. gemmatalis to these toxins which is in agreement with the previous findings of the relative susceptibilities of these soybean pests for Bt pesticides.

The Bt insecticidal proteins Cry1Ac and Cry2Ac7 belong to the three domain family of Bt toxins. Commercial transgenic soybean hybrids produce Cry1Ac to control larvae of the soybean looper and the velvetbean caterpillar. Specificity of Cry proteins is known to be majorly determined by domain II and domain III of the toxin. In this study, we constructed a hybrid toxin (H1.2Ac) containing domains I and II of Cry1Ac and domain III of Cry2Ac7, in an attempt to obtain a protein with enhanced toxicity compared to parent toxins. H1.2Ac protein was expressed in E. coli expression system and was refolded and purified using His- tagged chromatography. Recombinant H1.2Ac protein was also trypsin activated prior to the bioassays. Bioassays with H1.2Ac revealed toxicity to larvae of A. gemmatalis but not to C. iii includens. Saturation binding assays with radiolabeled toxins and midgut brush border membrane vesicles demonstrated no specific H1.2Ac binding to C. includens, while binding to A. gemmatalis was specific and saturable. The competition binding assays showed that Cry1Ac specificity against A. gemmatalis was mainly dictated by domain II. The binding assay in the presence of N-acetylgalactosamine (GalNAc) further clarifies the significance of domain III of Cry1Ac in binding to the receptors of C. includens. Taken together, these distinct interactions with binding sites explain the differential susceptibility of C. includens and A. gemmatalis to Cry1Ac and may provide guidelines for designing the improved toxins against soybean pests.

The significance of this work is in identifying Cry2Ac7 as toxic to S. litura and Cry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 as toxic to C. includens and A. gemmatalis. Cry1Ie2 can be pyramided with any or all of Cry1Ac, Cry2Ac7 and Cry1Fa proteins in transgenic plants to enhance their efficiency to combat insect pests. While the present study presents evidence for the importance of Cry1Ac domain III for toxicity against C. includens, further research would be needed to identify lethal Cry1Ac receptors and determine their interactions with domains II and/or III of Cry1Ac. This information contributes to the design of more active insecticidal proteins against this pest and our general understanding of the Cry mode of action in Lepidoptera.

ACKNOWLEDGEMENTS

All praise is for Allah Almighty, the most Merciful and the most Beneficent, for giving me inspiration, opportunity and stoutheartedness along this journey.

I will be indebted to my thesis advisor, Dr. A. R. Shakoori, for being a kind mentor. His guidance and expertise were invaluable in all aspects of my research and my grooming as a researcher. I am thankful to him for all his precious time, constructive discussions and encouragement. I am highly grateful to Dr. Juan Luis Jurat-Fuentes, University of Tennessee, USA, for providing me the opportunity to work in his laboratory. I acknowledge his patience to understand my questions and obliged for his support and guidance. I am thankful to him for review of my manuscripts and also grateful to his lab members for their help and support.

I would like to acknowledge Dr. M. Akhtar, Director General School of Biological Sciences for providing excellent facilities and encouraging atmosphere towards scientific work. I am grateful to Dr. Javed Iqbal for his fatherly kindness for me. I am thankful to Dr. Naeem Rashid who always supported the students and acknowledge his suggestions. I am grateful to all the faculty of SBS for the knowledge they shared with us.

My deepest thanks are to all my friends, lab members and SBS fellows. I want to express my gratitude to all those who helped me in any form to complete my work. I do not have words to say thanks to my mother and husband who suffered a lot during my work. Without their support I could not be able to get admission in PhD and complete my research. Special thanks are to my children who overlooked my absence from home and understood my passion for research and granted me permission to live away from them for seven months.

Rubina Mushtaq

ABBREVIATIONS NOT DESCRIBED IN THE TEXT

Abbreviation Name

bp base pair

Da Dalton

DNA Deoxyribonucleic acid

EDTA Ethylene diamine tetra acetic acid

FPLC Fast protein liquid chromatography

M Molar

MgCl2 Magnesium Chloride

(NH4)2SO4 Ammonium Sulfate

OD Optical density

PCR Polymerase chain reaction

TEMED N,N,N′,N′-tetramethylethylene-diamine

TPCK Tosyl-sulfonyl-phenylalanyl chloromethyl ketone

X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

CONTENTS

Dedication ...... i

Summary ...... ii

Acknowledgements ...... iv

Abbreviations not described in the text ...... v

List of Figures ...... xi

List of tables...... xiv

Chapter 1 ...... 1

Introduction and Literature Review ...... 1 Bacillus thuringiensis...... 1 Introduction and discovery ...... 1 Bt transgenic crops ...... 2 Cry protein nomenclature ...... 4 Crystal protein structure ...... 5 Domains functions ...... 6 Cry-proteins receptors ...... 7 Aminopeptidase-N (APN) ...... 8 Alkaline phosphatase (ALP) ...... 9 Cadherin-like proteins ...... 9 Glycolipids ...... 12 Mode of action of Cry toxins ...... 12 Pore formation model 1: Formation of pre-pore involves toxin interaction with different receptors in sequential manner ...... 12 Pore formation model 2: Formation of pre-pore involves toxin interaction with single receptor and insertion into membrane in monomeric form ...... 14 Signal transduction model: Monomeric toxin interaction with cadherin receptor initiates a signal transduction pathway ...... 15 Important findings for mode of action ...... 16 Dual model of action ...... 16 vii

Role of aquaporin ...... 16 Role of ABC proteins ...... 16 Resistance emergence in target insects ...... 17 Resistance evolution ...... 17 Resistance management ...... 18 Aims and objective of this work ...... 19

Chapter 2 ...... 20

General Materials and Methods ...... 20 Chemicals and kits ...... 20 Cultivation culture media ...... 20 Bacterial strains ...... 20 Plasmid DNA isolation ...... 21 Recovery of DNA from agarose gel ...... 22 Preparation of competent cells ...... 22 Transformation of competent cells with recombinant vector ...... 23 Analysis of protein samples through SDS-PAGE ...... 23

Chapter 3 ...... 24

Production of Purified Cry Proteins and Analysis of Their Activity

Against Crop Pests ...... 24

Abstract ...... 24

Introduction ...... 24

Materials and methods ...... 26 Cloning of cry1Ac and cry2Ac7 in pET28a(+) ...... 26 Sub-cloning of tncry1Ac and cry2Ac7 into pET28a(+) expression vector ..... 26 Double restriction digestion of recombinant vectors with gene inserts and the sub-cloning vector ...... 28 Ligation of cry1Ac and cry2Ac7 gene inserts ...... 28 Confirmation of clones through PCR amplification ...... 29 Over-expression and purification of recombinant proteins ...... 30 Transformation of host cells for gene expression ...... 30 viii

Refolding and purification of tnCry1Ac and Cry2Ac7 protoxins ...... 31 Refolding and purification of His-tagged tnCry1Ac and Cry2Ac7 protoxins 32 Production and purification of Cry1Fa protoxin ...... 33 Production and purification of Cry1Ie2 protoxin...... 33 Trypsin activation and purification of proteins ...... 34 Bioassays of tnCry1Ac and Cry2Ac7 against S. litura ...... 35 Bioassays of Cry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 toxins against A. gemmatalis and C. includens ...... 36

Results ...... 37 tncry1Ac and cry2Ac7 sub-clones ...... 37 tnCry1Ac and Cry2Ac7 untagged proteins ...... 38 tnCry1Ac, Cry2Ac7 and Cry1Ie2 His-tagged proteins ...... 39 Purification and trypsin activation of tnCry1Ac, Cry2AC7, Cry1Ie2 and Cry1Fa proteins ...... 41 Purification of trypsin activated proteins ...... 43 Bioassays of tnCry1Ac and Cry2Ac7 protoxin proteins against susceptible S. litura ...... 45 Bioassays of Cry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 activated toxins against susceptible C. includens and A. gemmatalis ...... 46

Discussion ...... 47

Chapter 4 ...... 49

Analysis of Mode of Action of Cry1Ac, Cry2Ac and Hybrid H1.2Ac by Binding Affinity to Receptors in Chrysodeixes includens and

Anticarsia gemmatalis ...... 49

Abstract ...... 49

Introduction ...... 49

Materials and Methods ...... 52 Cloning of hybrid H1.2Ac ...... 52 Selection of gene fragments ...... 52 Oligonucleotide primer designing ...... 53 ix

PCR amplification of selected gene segments ...... 53 TA cloning of the amplified gene segments ...... 54 Cloning of gene segments in pET28a(+) to construct H1.2Ac ...... 56 Over-expression and purification of recombinant protoxins ...... 57 Over-expression of hybrid protein ...... 57 Refolding of H1.2Ac protein ...... 58 Trypsin Activation and purification of the recombinant toxins ...... 58 Insect bioassays ...... 59 Preparation of BBMVs ...... 60 Binding assays with radiolabeled proteins ...... 60 Radiolabeling of Cry1Ac and Hybrid protein ...... 60 125I-H1.2Ac and 125I-Cry1Ac binding assays with BBMVs of A. gemmatalis and C. includens ...... 61 125I-H1.2Ac or 125I-Cry1Ac competition binding assays with BBMVs of A. gemmatalis ...... 61 Binding assays with biotin labeled proteins...... 62 Biotin labeling of Cry proteins ...... 62 Ligand blots of biotinylated toxins ...... 62 Western blotting of biotinylated toxins using GalNAc and GlcNAc competitors ...... 63

Results ...... 63 Construction of hybrid toxin H1.2Ac ...... 63 Identification of domain segments to construct H1.2Ac ...... 63 PCR amplification of selected DNA with designed primers ...... 65 TA cloning: screening and confirmation ...... 66 Construction of H1.2Ac: Fusion of DNA segments in pET expression vector ...... 66 Over-expression and purification of H1.2AC ...... 69 His-tag purification and trypsin activation of H1.2Ac...... 69 BBMV homogenate analysis ...... 70 Anion exchange purification and labelling of protein ...... 71 Labeling of Cry1Ac and Cry2Ac7 activated proteins...... 72 Insecticidal activity of hybrid protein ...... 74 x

Binding assays with radiolabeled Cry toxins ...... 74 Binding affinity of radiolabeled toxins ...... 74 Competition assays with radiolabeled toxins ...... 76 Ligand blots of biotinylated toxins ...... 77 Ligand blots with A. gemmatalis BBMVs ...... 78 Ligand blots with C. includens BBMVs ...... 78 Effect of GalNAc and GlcNAc on toxin binding to receptors ...... 79

Discussion ...... 80

Chapter 5 ...... 84

Identification of Proteins for Pyramiding with Cry1Ie2 in Transgenic

Crops for Resistance to Soybean Looper and Velvetbean Caterpillar . 84

Abstract ...... 84

Introduction ...... 84

Materials and Methods ...... 85 Biotin Labeling of the Cry1Ie2 protein ...... 85 Binding Competition assays ...... 86 Binding of toxins to receptor proteins ...... 86 Electroblotting of membranes ...... 89 Washing and probing the membranes ...... 90 Detection and analysis of fluorescence ...... 90

Results ...... 90 Biotinylation of protein ...... 90 Binding competition analysis of proteins ...... 91

Discussion ...... 94

Chapter 6 ...... 96

Discussion and Conclusions ...... 96

References ...... 100 xi

LIST OF FIGURES

Figure Page No.

Fig. 1. Global area planted to Bt crops ...... 3

Fig. 2. Three-dimensional structures of insecticidal toxins ...... 5

Fig. 3. Activated Cry1Ab toxin structure (Lucena et al., 2014)...... 7

Fig. 4. The role of lepidopteran BtRs (Bacillus thuringiensis Cry toxin cadherin receptors) in

the mode of action of Cry1 toxins...... 11

Fig. 5. Different models of the possible mechanisms of action of 3d-Cry proteins (A–D). .... 14

Fig. 6. Schematic representation of the mechanism of action of 3d-Cry toxins in Lepidoptera

at the molecular level ...... 15

Fig. 7. Dichotomous flow chart detailing seven steps in the mode of action of Cry insecticidal

proteins that determine toxin specificity...... 17

Fig. 8. A, Army worm, Spodoptera litura larva and adult; B, velvetbean caterpillar,

Anticarsia gemmatalis larva and adult; C, Soybean looper, Chrysodeixes includens,

larva (making its signature looping motion) and adult...... 25

Fig. 9. Circle map of the pET-28a(+) expression vector showing various sequence landmarks

of the expression system ...... 27

Fig. 10. Flowchart showing the sequential steps adopted in the refolding and purification of

tnCry1Ac and Cry2Ac7 insoluble proteins...... 32

Fig. 11. 1% Agarose gel showing restriction analysis of recombinant plasmids tncry1Acp22

(A) and cry2Ac7p22 (B)...... 37

Fig. 12. PCR amplification of tncry1Ac and cry2Ac7 genes from tncry1Ac28 and cry2Ac28

plasmids...... 38 xii

Fig. 13. 10% SDS-PAGE gel showing fractions from refolding and purification steps of

tnCry1Ac and Cry2Ac7 protoxin ...... 39

Fig. 14. Expression profile of his-tagged tnCry1Ac and Cry2Ac7 ...... 40

Fig. 15. SDS- PAGE analysis of Cry1Ie2 protein expression ...... 40

Fig. 16. Chromatogram of affinity chromatography purification of Cry1Ie2 protoxin by using

His-Trap HP 1ml column mounted on FPLC system...... 41

Fig. 17. Coomassie stained SDS-PAGE gel showing the his-tag purified and trypsin activated

toxins ...... 42

Fig. 18. SDS- PAGE analysis of Cry1Fa production and activation ...... 42

Fig. 19. Chromatogram of anion-exchange chromatography purification of activated toxins

Cry1Ac (A), Cry2Ac7 (B), Cry1Ie2 (C) and Cry1Fa (D) using an FPLC system...... 44

Fig. 20. SDS-PAGE analysis of activated toxins ...... 45

Fig. 21 . Structure of three domains of cry1Aa (taken from Bravo et al., 2005)...... 50

Fig. 22. Schematic representation for the construction of hybrid protein H1.2Ac ...... 52

Fig. 23. TA cloning vector and pictorial procedure ...... 55

Fig. 24. Domain architecture of cry proteins showing three domains and signature matches 64

Fig. 25. PCR amplified clearly defined DNA bands stained with EtBr on 1% agarose gel .... 65

Fig. 26. Restriction analysis of DIDIIAcpTZ and DIII2AcpTZ ...... 66

Fig. 27. 1% agarose gels showing different step of DIDIIAc cloninig in pET28a(+) ...... 67

Fig. 28. PCR amplified and restricted fragments of H1.2Ac construct ...... 68

Fig. 29. Confirmation of fused inserts in pET28a(+) ...... 68

Fig. 30. 10% SDS-PAGE gel showing over-expression of H1.2Ac protein ...... 69

Fig. 31. Coomassie stained SDS-PAGE gel showing the his-tag purified and trypsin activated

toxin...... 70 xiii

Fig. 32. Early fifth instar larvae BBMVs homogenate of A. gemmatalis and C. includens

resolved on 10% SDS-PAGE gel ...... 70

Fig. 33. Chromatogram of anion exchange chromatography purification of H1.2Ac toxin on

FPLC...... 71

Fig. 34. Biotin-labeling and iodination of H1.2Ac active toxin on SDS-PAGE and western

blots...... 72

Fig. 35. Purification, biotinylation and iodination images of Cry1Ac toxin ...... 73

Fig. 36. Purification, biotinylation images of Cry2Ac7 toxin...... 73

Fig. 37. Specific binding of 125I-H1.2Ac and 125I-Cry1Ac to different concentrations of

BBMVs of C. includens (A) and A. gemmatalis (B) ...... 75

Fig. 38. % Binding of radiolabeled toxin in the presence of homologous or heterologous

competitors (A) Cry1Ac and (B) H1.2Ac...... 77

Fig. 39. Ligand blot of A. gemmatalis early fifth instar BBMVs with biotinylated proteins .. 78

Fig. 40. Ligand blot of C. includens early fifth instar BBMVs homogenate with biotinylated

proteins ...... 79

Fig. 41. Western blots of biotinylated Cry toxins in the presence of GalNAc or GlcNAc ..... 80

Fig. 42. SDS-PAGE gel and western blot of biotin labeled-Cry1Ie2...... 91

Fig. 43. Biotinylated Cry1Ie2 binding competition assays...... 92

Fig. 44. Biotinylated Cry1Ie2 binding competition assays...... 93

xiv

LIST OF TABLES

Table Page No.

Table I. Toxicity of selected Bt protoxins against neonates of S. litura ...... 45

Table II. Toxicity of purified Bt toxins against neonates...... 46

Table III. Primers used for PCR amplification of selected domains of Cry toxins ...... 53

Table IV. DNA insert and pTZ57R/T vector ligation reaction components ...... 55

Table V. Reaction components of double restriction digestion of DNA plasmids ...... 56

Table VI. Toxicity of hybrid activated protein against neonates ...... 74

Table VII. Biotinylated-Cry1Ie2 to Cry1Ie2 binding competition reaction mixture...... 87

Table VIII. Biotinylated-Cry1Ie2 to Cry1Ac binding competition reaction mixture...... 87

Table IX. Biotinylated-Cry1Ie2 to Cry1Fa binding competition reaction mixture...... 88

Table X. Biotinylated-Cry1Ie2 to Cry2Ac7 binding competition reaction mixture...... 88

CHAPTER 1

Introduction and Literature Review

Since the evolution of crop plants from their wild progenitors, man is continuously trying to increase the crop yield by employing different physical and molecular techniques and protecting them by killing the animals and insects that feed on them. But with the evolution of crop plants its natural enemies like insect pests are also acquiring such characteristics that resulted in development of insect resistance to the existing chemical insecticides. For this, it is becoming more important to utilize newer pesticides which are not only effective but also can be used safely without upsetting the ecosystem. One of the safest and important methods of controlling the insect pests is the use of bio-pesticides based on the bacterium Bacillus thuringiensis.

Bacillus thuringiensis

Introduction and discovery Bacillus thuringiensis (Bt) is a wide spread endospore forming Gram-positive bacterium. During sporulation phase of growth this bacterium produces a large number of virulence factors like β-exotoxins (Levinson, 1990), cytolytic toxins (Cyt toxins; Crickmore et al., 1998), vegetative insecticidal proteins (VIPs; Estruch et al., 1996), phospholipases (Zhang et al., 1993) and crystal proteins (Cry proteins; Höfte and Whiteley, 1989). Due to these protein and other factors it has the ability to kill some devastating insect pests of plants but without toxicity to other organisms mostly. For this most of these proteins and toxins are being used as controlling agent for many insects (reviewed in Pardo-López et al., 2013).

B. thuringiensis was isolated by Ishiwata from diseased larvae of Bombyx mori (silkworm) in 1901 and named it Bacillus sotto (Ishiwata, 1901). The name ‘Bacillus thuringiensis’ was given by Berliner in 1915 on the name of the German region Thuringia from where the bacterium was isolated from Mediterranean flour moth, Ephestia kuehniella.

These crystal inclusions can be composed of one or more Cry proteins and have insecticidal properties (Schnepf et al., 1998). However, the insecticidal activity to the observed parasporal bodies (crystal inclusions) was associated later after five decades of its isolation (Angus, 1956). The ability of B. thuringiensis to produce parasporal crystal inclusions 2 differentiates it from Bacillus cereus, a closely related species (Schnepf et al., 1998). The first crystal protein encoding gene was cloned and sequenced in early 1980s and many are being characterized and classified according to the revised nomenclature of B. thuringiensis crystal proteins (Crickmore et al., 1998). The cry genes which encode Cry proteins are mostly located on the large plasmids in B. thuringiensis (González et al., 1981), while some have been found on chromosomal DNA (Carlson and Kolstø, 1993).

Many Bt proteins possess insecticidal activities against diverse number of insect orders like Lepidoptera, Hymenoptera, Coleoptera, Orthoptera, Mallophaga and Homoptera (Schnepf et al., 1998). A number of Bt strains having toxicity to protozoa, mites and nematodes have also been identified ( Wei et al., 2016; Crickmore et al., 1998). The evolutionary advantage to B. thuringiensis over other entomopathogens is the capability to produce parasporal crystal proteins and colonization in insect midgut which results in midgut disruption favoring the spore germination of B. thuringiensis (Schnepf et al., 1998). B. thuringiensis is commercially the most important insect pathogen and δ-endotoxins or Cry toxins are most widely and well- studied entomocidal proteins. Because of their easy production and different formulations Cry toxins are widely used bio-pesticides that are being used either as sprays or in transgenic crops for environment friendly pest control (Betz et al., 2000). The introduction of cry genes in transgenic plants have enabled them to produce their own pesticidal proteins (Adang et al., 1993; De Cosa et al., 2001).

Bt transgenic crops In the native bacterium, the Cry proteins are formed in the form of crystalline inclusion bodies and are stored as insoluble crystal proteins in the spores. Some important Cry proteins have been expressed in plants by cloning cry genes in them. In the transgenic plants B. thuringiensis DNA, which is rich in A-T bases, could not produce significant levels of Cry proteins due to the RNA processing in the eukaryotic plant cells. So, the Cry proteins encoding DNA was modified by eliminating A-T rich sequences and using plant preferred codons (Adang et al.,1993). The first cry transgenic plant was tobacco which produced Cry toxin successfully after which many major crops like maize, potato, rice, cotton and soybean were transformed to express Cry proteins (Betz et al., 2000; James, 2013).

During 1995-1996 Bt transgenic corn, cotton and potato were commercialized (reviewed in de Maagd et al., 1999b). With the popularity of cry transgenic crops producing 3

Cry toxins the area of its plantation has been increased since 1996 from 1 million ha to 76 million ha till 2013 (Fig. 1).

Fig. 1. Global area planted to Bt crops (black) and cumulative number of insect species with practical resistance to Bt crops (grey). The asterisks indicate that the number of species with resistant populations may be underestimated for 2011 to 2013 because reports of field-evolved resistance typically are published 2 or more years after resistance is first detected (Adapted from Tabashnik et al., 2013).

Initially, the formation of Cry proteins in Bt transgenic plants was through nucleus directed expression but now high levels of Cy proteins have been analyzed to obtain through chloroplast directed expression (De Cosa et al., 2001; Kota et al., 1999) and without imposing crop yield (Reddy et al., 2002). Now the plants are being transformed with more than one cry genes in order to increase the target insect number. Very recently, Ghosh et al. (2017) reported that pigeon pea events co-transformed with two cry genes successfully expressed Cry1Ac and Cry2Aa proteins that conferred resistance to transgenic plant against Helicoverpa armigera. However, pyramiding of more than one Bt toxins in the transgenic crops may result in cross resistance of the toxins in at least two of them. For this reason, it is necessary to check for the cross resistance of the two toxins against particular target insect(s) before pyramiding them in crops (Carrière et al., 2016a; Yang et al., 2016) 4

These transgenic crops possess some important qualities and advantages over the conventional ones which include: reduction in the use of Cry proteins as sprays, reduction in the use of chemical pesticides which not only saves money and time but also are non-toxic to other organisms, no disturbance in ecosystem due to safety to flora and fauna other than the specific target organisms, continuous production of the toxin by the plant enabling it to combat occasional pest attacks and the very important advantage is that they reduce health risks to consumers and farmers (Betz et al., 2000). Commercial benefit of Bt transgenic crops have been established by many studies, for example, the use of insecticidal chemicals has been reduced significantly by growing Bt cotton in USA and saved almost 1,870,000 pounds in 2001 (Carpenter et al., 2002; Edge et al., 2001). Indirect good effect of Bt crops (transgenic) is the reduction in custom use of broad range chemical insecticides effecting beneficial insects (Head et al., 2001).

However, the emergence of resistance in the insects against Bt toxins calls for other strategies like co-transformation of cry genes, tissue specific expression and the use of newly isolated and identified effective Bt protein genes to transgene crops. Use of refuge fields around the Bt transgenic crops can be another strategy to prolong the effectiveness of insect resistant crops (reviewed in Kumar et al., 2008).

Cry protein nomenclature The first systematic nomenclature based on biological specificity of Cry proteins was given by Höfte and Whiteley in 1989 who assigned Roman numerals to the cry genes consistent with the toxicity of that crystal protein. According to this system cry genes encoding crystal proteins having insecticidal activity against lepidopterans were classified as cryI genes; the proteins toxic to lepidopterans and dipterans were encoded from cryII genes; cry genes encoding insecticidal protein to coleopterans were named as cryIII genes; the genes only encoding dipterans specific proteins were named as cryIV genes (Höfte and Whiteley, 1989). But there were inconsistencies in classifying those cry genes encoding Cry toxins having insecticidal activities against more than one pest orders different from the classified ones.

This issue was resolved by assigning the names according to their evolutionary divergence based upon cry genes phylogenetic analysis and amino acid sequences analysis of Cry proteins (Crickmore et al., 1998). Ranks were made to classify the Cry proteins and Arabic numerals replaced Roman numerals in the primary rank. On the basis of sequence identity boundaries of ranks were made. The sequences similar up to 45 % were included in first rank, 5 the toxins with 46-78 % sequence identity were given the second rank denoted by the first capital letter. The third rank was denoted by the second lower case letter which groups toxins with 78 to 96 % identities. The cry gene sequences having sequence identity more than 96 % were determined as the alleles of the same genes (Crickmore et al., 1998). Present system of Cry protein nomenclature is solely based on the amino acid sequence identity (Crickmore et al., 2016).

Crystal protein structure Cry proteins phylogenetic analysis separated them in four families which are non- phylogenetically related. These families are Cry proteins (three domain), mosquitocidal Cry proteins (Mtx), binary like (Bin) and Cyt toxin (Bravo et al., 2005). Among these families the 3D Cry family is the largest and 3-D structure of some Cry toxins have been resolved through X-ray crystallography: Cry1Aa (Grochulski et al., 1995), Cry3Aa (Li et al., 1991), Cry4Ba (Boonserm et al., 2005), Cry2Aa (Morse et al. 2001) and Cry6Aa (Huang et al., 2016) as shown in Fig. 2.

Fig. 2. Three-dimensional structures of insecticidal toxins produced by Bacillus thuringiensis Cry1Aa, Cry2Aa, Cry3Aa, Cry3Bb, Cry4Aa and Cry4Bb (taken from Bravo et al., 2007).

The amino acid sequences of these Cry proteins are about 27 to 36 % identical but are similar in three dimensional structures (Crickmore et al., 1998; Morse et al., 2001). But the most recently resolved crystal structure of Cry6Aa reveals the presence of only two-domain architecture (Huang et al., 2016). Most Cry toxins have three structurally delimited domains; domain I, domain II and domain III. Domain I is composed of seven α-helix bundles comprising six helices (amphipatic) surrounding the helix α-5 (hydrophobic). Domain I is known to oligomerize the toxin, inserts in membrane and forms a pore in it. Eleven β-sheets with exposed loop regions comprise domain II which binds to specific midgut proteins of insect larva. Domain III is composed of beta sandwich with a jelly like structure that performs receptor recognition function like domain III (Bravo et al., 2011).

The proteins produced during sporulation are called protoxins in which some produce large protoxins of almost 130 kDa (e.g. Cry1Aa), others synthesize short protoxins of almost 65–70 kDa (e.g. Cry11Aa). These large protoxins when acted upon by proteases of the insect midgut, are trimmed majorly from C-terminal end and minorly from N-terminal end to become biological active toxins of approximately 60 kDa (Bravo et al., 2013; de Maagd et al., 2001).

Domains functions Cry toxin domains are not restricted to Cry toxins but have structural similarities with other proteins also, for example the α-toxin of Staphylococcus aureus, colicin from Escherichia coli and jacalin, a plant lectin (Burton et al., 1999; English and Slatin, 1992; Li et al., 1991). Thus, the Cry toxin domains were studied and their role in the mode of action has been investigated on the basis of functional analogy with other structurally similar proteins. Cry toxins have insect species specificity for its action, domain I is not generally considered as a major determinant of this specificity whereas domain II is thought to be involved in determining insect specificity. However, these two domains have evolved together due to structural constraints (de Maagd et al., 2001; Grochulski et al., 1995). The amphipathic helices present making domain I have been found to be similar to colicin-A having pore forming domains (Li et al., 1991). Binding specificity is highly influenced by the three protruding loops within domain II and loop II is suggested to be critical in recognition of insect receptors (Fig. 3).

Fig. 3. Activated Cry1Ab toxin structure (Lucena et al., 2014). The three Domains are colored as follows: Domain I (red), Domain II (green), and Domain III (blue). Loop 1 is shown in cyan, loop 2 is shown in magenta, loop 3 is shown in black and loop α8 is shown in orange.

Domain III has a different topology from domain I and II in the phylogenetic trees. It has been determined from many studies that in Cry toxins domain II and domain III are the main determinants of binding specificity to insect receptors ( Xu et al., 2014; Dean et al., 1996). If the sequences of two Cry toxins have similarities it results in cross resistance between these toxins (Carrière et al., 2015). The role of domain III in toxin binding specificity is also clearly evident by domain swapping experiments (de Maagd et al., 1996). It was hypothesized that domain III may be involved in Cry toxin stability and protect it from proteolytic degradation (Li, 1991). N-acetylgalactosamine (GalNAc) residues are considered as Cry1Ac binding recognition epitope (Knowles, 1991). The domain III has a structural pocket that was identified as GalNAc binding site (Burton et al., 1999).

Cry-proteins receptors

Receptors are the binding molecules that recognizes an invading molecule and transduce binding to a response. The correlation between the binding of Cry proteins to the receptors present on the midgut epithelial membrane of specific insects and the toxicity of Cry proteins is very important. Many Cry toxin putative receptors have been reported by probing blots of isolated BBMV proteins with labeled Cry toxins (Jurat-Fuentes and Adang, 2001; Lee et al., 1999) among them the important and best characterized are aminopeptidases, alkaline phosphatases and cadherins. The other important class of Cry toxin putative receptors are 8 glycolipids although its toxin binding interaction is less elaborated. The summarized introduction of these important putative receptors is discussed here.

Aminopeptidase-N (APN) This belongs to zinc binding metalloprotease superfamily of enzyme which cleaves amino acid residues (neutral) present at the N-terminus of polypeptide. In larval midguts of lepidopteran insects APN works along with carboxypeptidases and endopeptidases to digest proteins (Wang et al., 2005b; Taylor, 1996). Some Cry proteins have affinity to bind multiple APNs and some APNs have binding ability to multiple Cry toxins and many classes have been identified for Lepidopteran insects. Class-1 to class-4 APNs were found bound to the Cry1Aa and Cry1Ab in ligand blot analysis which suggests the presence of a conserved and common binding site in receptors isolated from Bombyx mori and Plutella xylostella BBMVs (Nakanishi et al., 2002). The study of many different APNs revealed that they have many features in common among them, for example, they are proteins of almost 90 to 170 kDa size and have a signal peptide on N-terminus of polypeptide to export to the cytoplasmic membrane outer surface where they get attached by glycosylphosphatidylinositol (GPI) anchor (Agarwal et al., 2002; Denolf et al., 1997; Lu and Adang, 1996; Knight et al., 1995; Takesue et al., 1992). Several Cry1 toxins like Cry1Aa, Cry1Ac, Cry1Ab, Cry1Ca, Cry1Ba and Cry1Fa were found to bind APNs but with different patterns of binding (reviewed in Pigott and Ellar, 2007). Binding of Cry1Ac protein to APN (class 3) of H. virescens was analyzed where this binding could be blocked by using GalNAc as competitor (Gill et al., 1995). However the binding of the Cry1Ac toxin with class 3 APN protein (120 kDa) from H. armigera was not affected by GalNAc (Rajagopal et al., 2003).

In brief, the binding of Cry toxins to APN is very complex and is affected by other factors also. The purified APN protein can be contaminated with another class APN as for example, the purified APN (class 1) on mass spectrometry analysis detected the contamination of APNs from class 3 and 4 (Stephens et al., 2004). The other major problem is the degradation products of cadherins, a receptor for Cry toxins, at about 120 kDa size could be misinterpreted as a band of 120 kDa APN in a ligand-blot assay (Candas et al., 2002; Yaoi et al., 1999; Martinezramirez et al., 1994). All the binding APNs to Cry toxins may not be responsible for the susceptibility of the insect to the toxin. To determine the functionality of the receptors in vitro and in vivo studies have been performed (Garner et al., 1999; Gill and Ellar, 2002) and till now a few APNs have been identified as functional receptors by these studies. 9

Alkaline phosphatase (ALP) Alkaline phosphatases (ALPs) have been identified as Cry protein binding receptors. These are anchored to lipid raft membrane by glycosylphosphatidyl-inositol (GPI) proteins (Arenas et al., 2010; Pacheco et al., 2009). The role of ALP receptor as putative binding protein was analyzed in a resistant strain of H. virescens, where it was demonstrated that the resistance of this strain to Cry1Ac was linked to the lower expression levels of ALP binding protein (Jurat-Fuentes et al., 2002). A 68 kDa ALP membrane protein anchored by GPI in H. virescens was detected to bind Cry1Ac protein. This binding was demonstrated to depend on an oligosaccharide (N-linked) that contain a GalNAc residue on its terminus (Jurat-Fuentes and Adang, 2004). While detecting the reasons of resistance of insects to the Cry toxins, reduced levels of membrane bound alkaline phosphatase (mALP) have been found as a common feature in the midguts of different resistant strains of H. armigera, H. virescens, and Spodoptera frugiperda as compared to the higher levels in susceptible strains (Jurat-Fuentes et al., 2011). In a very recent study, Yuan et al (2017) analyzed a high affinity binding of Cry2Aa toxin with ALP2 partial peptide of the larvae of Spodoptera exigua. On the bases of ALP2 knocking down in vivo ALP2 was proposed as a functional receptor in S. exigua for Cry2Aa (Yuan et al., 2017).

Cadherin-like proteins Cadherin proteins are proteins that have an extracellular region of cadherin repeat (CR) domains, an intracellular cytoplasmic (IC) domain and a transmembrane domain. Classic cadherins are single span transmembrane proteins that perform cell-cell adhesion in the presence of Ca2+ ions by forming cis and trans dimers (Brasch et al., 2012; Isacke and Horton, 2000). These proteins play role in tissue morphogenesis and take part to maintain the epithelium, and provide extracellular information to the cell. Cadherin like proteins are mainly localized in adhesion junctions of mature epithelial-cells. Although, cadherin like proteins are exposed on the surface of the cell they also possess a C-terminus domain on the cytoplasmic side. These domains interact with actin cytoskeleton in intracellular signal transduction (Isacke and Horton, 2000). The schematic picture of the role of lepidopteran BtRs in Cry1 toxin intoxication is given in Fig. 4.

The cadherins found in Lepidoptera, Diptera and Coleoptera are phylogenetically unique and are known as ‘cadherin like proteins’, ‘CADR’, ‘Cad’ or ’12-cadherin domain’. Bacterium B. thuringiensis co-opted these proteins as receptors for Bt endotoxins (Midboe et al., 2003). Insect midgut cadherins are also known as ‘BtRs’ and their role as functional 10 receptors for Bt have been extensively studied. The BtRs are the transmembrane proteins ranging in size from 175 to 250 kDa with CRs, membrane proximal region (MPR), a single transmembrane domain and an IC domain. The correlation of Cry toxin binding sites with BtRs came from the expression of these proteins within the midgut epithelial-microvilli in B. mori,

Aedes aegypti, and M. sexta. BT-R1 from M. sexta was the first BtR that was cloned and characterized (Hua et al., 2004; Dorsch et al., 2002; Vadlamudi et al., 1995).

Subsequently, the BtRs functioning as Cry receptors were identified from three different insect orders which include: HevCaLP from H. virescens (Gahan et al., 2010; Xie et al., 2005); HaCad from H. armigera (Wang et al., 2005a; Zhang et al., 2012); OnBt-R1 from Ostrinia nubilalis (Flannagan et al., 2005); BtR175 from B. mori (Atsumi et al., 2008; Nagamatsu et al., 1998); SeCad1b from S. exigua (Chen et al., 2014; Park and Kim, 2013; Ren et al., 2013); PgCad1 from Pectinophora gossypiella (Fabrick and Tabashnik, 2007; Morin et al., 2003); AdCad1 from Alphitobius diaperinus (Hua et al., 2014); TmCad1 from Tenebrio molitor (Fabrick et al., 2009); AgCad2 and AgCad1 that is also known as BT-R3 from Anopheles gambiae (Hua et al., 2013; Ibrahim et al., 2013); and AaeCad from Ae. aegypti (Chen et al., 2009). Cry toxin binding sites in cadherin have been found to be located mainly on the CR domains which is adjacent to membrane proximal regions of the protein (Ibrahim et al., 2013; Fabrick et al., 2009; Gómez et al., 2001, 2002, 2003; Dorsch et al., 2002; Nagamatsu et al., 1999).

It has been discovered that a cadherin fragment increases the oligomerization of Cry toxins that may result in increased formation of the pre-pore oligomers and consequently enhance the cytotoxicity through pore-formation (Peng et al., 2010; Liu et al., 2009; Fabrick et al., 2009; Gómez et al., 2002). In the pore formation model, it was proposed that toxin oligomerization is promoted by cadherin like receptors which helps the binding of oligomer to the GPI-anchored proteins (APN and ALP) whereas, the APN and ALP proteins act as scaffolding molecules during the process of pore-formation (Jurat-Fuentes and Adang, 2004).

Fig. 4. The role of lepidopteran BtRs (Bacillus thuringiensis Cry toxin cadherin receptors) in the mode of action of Cry1 toxins. The five steps for Bt intoxication (1–5) include the following: (1) Ingestion of Bt Cry protoxin either solubilized from the Bt bacterium or from a transgenic Bt plant; (2) proteolytic activation of the protoxin by midgut endopeptidases; (3) binding of monomeric Cry1 toxin to GPI (glycosylphosphatidylinositol) anchored aminopeptidase N (APN) and/or alkaline …………………………………………………………………………………..(continued on next page) 12

phosphatase (ALP); (4) binding of monomeric Cry toxin to BtR and proteolytic removal of the domain I α-helix; and (5), either (5a) oligomerization of Cry monomers and binding of the oligomer to GPI-anchored APN or ALP, which leads to insertion of the pre-pore toxin oligomer via ABC transporter into the cell membrane to form pores and ultimately leads to cell death (sequential/pore formation model), or (5b) binding of monomeric Cry toxin to cadherin, which activates an intracellular signal transduction pathway that leads to cell death (cell signaling model) (5b) (taken from Fabrick and Wu, 2015).

Glycolipids Another significant class of Bt (Cry) protein receptors is glycolipids. In 1986, interaction between Cry toxins and glycosphingolipids was first time reported (Dennis et al., 1986). Because of the presence of many study tools (genomic, cell biological and genetic) for Caenorhabditis elegans in comparison for the Cry toxin susceptible insects, importance of this putative receptor was described in this nematode (Griffitts et al., 2003). C. elegance models can be used for Cry toxin susceptible insects because nematocidal Cry toxins and insecticidal Cry toxins share similarities in sequence except Cry6Aa toxin. The glycolipids were identified as Cry toxin receptors by the characterization of mutant (chemically) strains of C. elegance selected for Cry5Ba resistance in them (Griffitts et al., 2003; Marroquin et al., 2000). The extracted glycolipids from the M. sexta midguts showed binding to Cry1Ab , Cry1Aa and Cry1Ac, for this the role of glycolipids as receptors for Cry toxins was postulated (Griffitts et al., 2005). The Cry1Ac binding to glycolipids in other experiments classify them as potential receptors for Cry toxins (Garczynski and Adang, 2000).

Mode of action of Cry toxins

Three different models to explain the mechanism of action of 3d Cry toxins have been proposed with the same initial steps. During sporulation of B. thuringiensis bacteria accumulation of 3d-Cry protoxins takes place in the form of inclusion bodies. On ingestion by the susceptible insect, protoxin proteins are solubilized by the action of mid gut juices in the gut lumen of insect to produce active toxins. The next steps towards the death of the insect can be explained by three models as under.

Pore formation model 1: Formation of pre-pore involves toxin interaction with different receptors in sequential manner The pore formation model suggested that the 3d-Cry toxins destroys the gut tissue by forming pores in the midgut cells. The disruption of gut tissue kills the larvae. The toxin inserts 13 the membrane after some sequential interactions with different receptors located in the apical membrane of midgut cells of insects (Fig. 6; Pardo-López et al., 2013). It was proposed that in the case of Cry1A toxins, loop-3 of domain-II and β-16 of domain-III interact with low affinity to APN and ALP, respectively. The ALP and APN proteins are abundant on surface of the cell and are glycosylphosphatidylinositol (GPI) anchored to the membrane. This binding step is very important to localize the toxin in close proximity of the membrane. The importance of this binding step with ALP and APN has been predicted when mutant Cry1A toxins showed altered toxicity due to the affected binding to these proteins (Arenas et al., 2010; Pacheco et al., 2009). The other important membrane protein is cadherin like transmembrane protein which binds with high affinity to loops 2, 3 and α-8 of domain II of Cry1A toxin. Cry1A toxins with mutations in these regions resulted in proteins non-toxic to insects (Rodríguez-Almazán et al., 2009; Gómez et al., 2006). This binding of toxin to cadherin eliminates N-terminal end with helix α1 of domain I by inducing an extra cleavage (Gómez et al., 2002). This results in exposure of toxin’s buried hydrophobic regions which triggers toxin oligomerization. The oligomerized protein forms a pre-pore oligomer structure outside of the membrane. The affinity of pre-pore oligomer to ALP and APN receptors increase up to 100-200-fold (Arenas et al., 2010; Pacheco et al., 2009). The final step is the insertion of the pre-pore into the membrane forming a pore which affects the cells permeability and cause consequent death of insect larva (Gómez et al., 2014; Bravo et al., 2015a). The failure to oligomerize and membrane insertion by mutant toxins are non-toxic although they have ability to bind receptor proteins (Girard et al., 2008; Vachon et al., 2002). Recently, the high affinity of both the protoxin and trypsin activated toxin to bind cadherin was detected with the formation of two different pre-pores. These two pre-pores assemble together before the insertion in the gut membrane (Gómez et al., 2014). The interaction of 3d-Cry proteins with different receptors indicated the presence of different receptors in different insects that might have different proteases in insect gut lumen that triggers formation of two oligomeric structure. This model proposes the functional role of protoxin region also (Pathway B, Fig. 5).

Fig. 5. Different models of the possible mechanisms of action of 3d-Cry proteins (A–D). In the sequential binding and pore formation model of activated Cry toxin (a), activated 3-d Cry1A toxin binds to alkaline phosphatase/aminopeptidase N (ALP/APN), then to cadherin, facilitating oligomerization and binding to ALP/APN before membrane insertion. In the sequential binding and pore formation model of Cry protoxin (b), Cry1A protoxin binding to cadherin facilitates oligomer pre-pore formation, binding to ALP/APN receptors and membrane insertion. In the pore formation model of monomeric activated toxin (c), activated 3-d Cry1A toxin binds to ALP, APN or cadherin, inserts into the membrane and oligomerizes in the membrane plane. In the signal transduction model (d), the interaction of Cry activated toxin with cadherin induces an intracellular signal transduction death mechanism. (taken from Bravo et al., 2015b).

Pore formation model 2: Formation of pre-pore involves toxin interaction with single receptor and insertion into membrane in monomeric form This model proposes the insertion of trypsin activated Cry protein into gut membrane of insect takes place after its binding to one of the different receptors for Cry toxins. This binding of the Cry toxin to any of ALP, APN or cadherin is an irreversible fashion. In the membrane plane several membrane molecules interact to form oligomerized structure which forms pore. The ability of Cry1A activated protein to induce pore formation activity when no receptor is present in planar lipid-bilayer supports this model (Vachon et al., 2012). However, the pore formation ability of Cry toxin in the presence of receptors has been demonstrated to be increased significantly (Schwartz et al., 1997). Other experiments to measure conductance in the presence of BBMVs containing toxin receptors have shown improvement in monomeric 15 toxin activity of pore formation resulting in 2-40 times higher conductance (Peyronnet et al., 2001; Martin and Wolfersberger, 1995). Schematic representation of the pore formation model at the molecular level showing the role of different Cry toxin receptors APN, ALP and cadherin is shown in Fig. 6 taken from a review article by Pardo-López, 2013.

Fig. 6. Schematic representation of the mechanism of action of 3d-Cry toxins in Lepidoptera at the molecular level. 1, the larvae ingest the 3d-Cry protoxin, which is solubilized in the midgut lumen of the larvae due to high pH and reducing conditions and activated by gut proteases, generating the toxin fragment. 2, the monomeric 3d-Cry toxin binds ALP and APN receptors; in a low-affinity interaction, the toxin is then located in close proximity to the membrane. 3, the monomeric 3d-Cry toxin binds the CAD receptor in a high-affinity interaction and this interaction induces proteolytic cleavage of the N-terminal end of the toxin, including helix a-1 of domain I. 4, the cleaved 3d-Cry toxin is then able to oligomerize in a toxin prepore oligomer. 5, the oligomeric 3d-Cry structure binds to ALP and APN receptors with high affinity. 6, the prepore inserts into the membrane causing pore formation (adopted from Pardo-López, 2013). Signal transduction model: Monomeric toxin interaction with cadherin receptor initiates a signal transduction pathway The Trichoplusi ni originated cell line (TnH5) modified to express cadherin protein of Manduca sexta showed susceptibility towards Cry1Ab toxin (Zhang et al., 2006) which formed the basis of this model. In this model, it was proposed that the cadherin receptor interaction with the 3d-Cry toxin activates an intracellular death-signal transduction pathway. Binding of the toxin with cadherin activates a protein ‘G’ that causes activation of adenylate 16 cyclase, which in turn increases the cyclic-AMP levels. The cyclic-AMP results in the activation of protein kinase A (PKA) which is responsible for the induction of cell necrosis (Zhang et al., 2006). Pathway D in Fig. 5 shows the steps in this mode of action. However, more recently Portugal et al. (2017) analyzed the mode of action of native and mutant forms of Cry1Ac and Cry1Ab toxins in the CF1 insect cell line from Choristoneura fumiferana , a sensitive insect to these toxins. Their results showed the induction of permeability of K+ ions into the CF1 cells. It was proposed that induction of apoptotic death response is not related with PKA/AC activation and pore formation by toxins triggers cell death response in CF1 cells (Portugal et al., 2017).

Important findings for mode of action

Dual model of action Recently, a dual model was proposed for the mechanism of action of Cry toxins. It was proposed that the Cry1Ac and Cry1Ab protoxins were more potent to seven resistant strains of major crop pests than their activated forms. Moreover, higher resistance of the insects was recorded to the activated forms in many cases of study. The dual model states that the protoxin and activated proteins both kill insects through different pathways (Tabashnik et al., 2015).

Role of aquaporin Endo et al. (2016) demonstrated that the Cry toxin induces water influx which mediates aquaporins (AQPs) and not by the Cry toxin pore. They suggested that this Cry toxin induced water influx directly determines cell death by necrosis.

Role of ABC proteins Although most widely mechanism for the cytotoxicity of Cry toxins has been the pore formation model but the explanations of pore insertion into the cell membrane have remained unclear. Through the genetic studies of Cry toxins resistant strains of many Lepidoptera species, the role of ABC proteins in the pore insertion step has been detected (reviewed in Heckel, 2015). A frameshift mutation in ABCC2 protein from the C subfamily of ABC proteins caused its truncation which conferred the insect resistance against Cry1Ac and Cry1Ab proteins due to their loss of binding to epithelial membrane (Gahan et al., 2010).

The specificity determinants for insecticidal Cry proteins playing important role in their mode of action shown in the form of flow chart in Fig. 7 summarizes the discussion about different models for the mode of action of Cry proteins. 17

Fig. 7. Dichotomous flow chart detailing seven steps in the mode of action of Cry insecticidal proteins that determine toxin specificity. Each specificity determining step is shown as a dichotomous key in roman numeral. Cry proteins produced by transgenic Bt crops are not subjected to the two first specificity determinants (taken from Jurat-Fuentes and Crickmore, 2016).

Resistance emergence in target insects

Resistance evolution It has been a century since the emergence of resistance in insects against pesticide sprays (Melander, 1914) and the first case of insect resistance development to Bt toxins was reported in 1985 (McGaughey, 1985). Till now many cases of emergence of resistance in a number of insect species from the fields of Bt crop have also been reported (Gassmann et al., 2014; Wan et al., 2012; Van Rensburg, 2007; Janmaat and Myers, 2003; Shelton et al., 1993; 18

Tabashnik et al., 1990) and it has become a serious threat to the effectiveness and usefulness of Bt crops (Tabashnik et al., 2013).

Many insect populations selected in the laboratories for resistance to Bt toxins helped to understand mode of action of toxins and mechanism of resistance in them ( Pardo-López et al., 2013; Oppert et al., 1997). This selection showed that the insect resistance to Bt toxins occur at different levels for different insects and different toxins have cross resistance for other toxins in a particular insect (Tabashnik et al., 2003; Ferré and Van Rie, 2002). In lepidopteran pest such as P. xylostella, Busseola fusca, T. ni, S. frugiperda, H. armigera, H. zea, P. gossypiella and H. punctigera resistant alleles were frequently detected (Wan et al., 2012; Dhurua and Gujar, 2011; Janmaat and Myers, 2003; Tabashnik et al., 1990). The evolution of resistance in Bt target insects may be due to the alterations in any one or more than one steps in mode of action of Cry toxins. Schnepf et al. (1998) reported reduction in toxicity of Bt toxins by poor solubilization of crystals of the Cry protein. Excessive degradation or insufficient activation of Bt toxins can alter susceptibility of insects to toxins (Karumbaiah et al., 2007; Li et al., 2004; Oppert et al., 1997). Lower level of permeability of the peritrophic membrane in the midgut to the toxin is another factor for resistance emergence (Hayakawa et al., 2004). Enhanced sequestering of Cry toxins in the insect midgut (Gunning et al., 2005) and elevated immune responses in insect (Rahman et al., 2004) may be other causes of resistance emergence. In addition to these factors the highly reported primary reason is the reduced binding of the Cry toxin to the BBMV membranes (Pardo-López et al., 2013; Heckel et al., 2007). Moreover, mutations in ABCC2 (ATP binding cassette transporter subfamily C member 2) protein which have been detected as Cry toxin binding protein earlier was found to be linked to resistance emergence (Tanaka et al., 2016).

Resistance management Physical methods to deal with resistance is the use of Bt crop fields with surrounding areas of non-Bt crops which can act as refuge crops for insects and the use of different varieties of Cry transgene plants. Pyramiding of different Cry toxins with different target insects in the crops is another important tool to increase the efficiency of transgenic crops against resistant insects (Gould, 1998). Some molecular methods are very important to make Cry toxins more toxic and with increased range of target insects. Site directed mutagenesis is one of the molecular method for the modification of Cry toxins. Different mutations in helices, loops and domains (mostly domain II and III) have been reported worthy to produce more toxic Cry proteins (Rajamohan et al., 1996a, b; Schnepf et al., 1998; Wu et al., 2000). Mutations in the 19 toxin binding sited were found to have potential for the development of more potent Cry toxins even with specificity changes (reviewed in Bravo et al., 2013; Pardo-López et al., 2009).

Construction of hybrid toxin mainly by the substitution of domain III of Cry toxins with that of other toxins have become a useful tool to produce novel insecticides either with wider target spectrum or/and possess higher toxicity related to the parent toxin. A number of hybrid toxins have been generated by domain III shuffling with toxicity to those insects which were not the target insects of the parent toxins (reviewed in Lucena et al., 2014). This method also helps scientist to understand the role of receptors and mechanism of action of Bt toxins.

Aims and objective of this work

There are three main objectives of the research work presented in this thesis.

Objective I is to explore additional target insects in addition to known susceptible insects for the Cry proteins used in this study.

Objective II is the construction of a hybrid toxin through domain substitution in order to see its effect on potency of the parent toxin for target insects. This hybrid toxin may also help to explore the importance of substituted domain and its binding ability to specific insect receptors which on the whole may point at the difference in mode of action of the toxins for the particular target insects.

Objective III is to check whether the detected active Cry toxins in this study effective against the two target insects could be candidates to pyramid in a transgenic crop to control these insects in addition to the control of already known susceptible insects for these proteins.

CHAPTER 2

General Materials and Methods

Chemicals and kits

General materials used in routine molecular biology labs were purchased from scientific vendors like Sigma-Aldrich (USA) and Thermo Fisher Scientific (UK, USA) depending upon the quality and availability of the required chemical. Taq DNA polymerase and buffers for PCR were from ThermoFisher Scientific (USA). T4 DNA ligase kits and restriction endonucleases were from Fermentas Life Sciences. DNA bands after agarose gel electrophoresis were extracted from agarose gel with Vivantis GF-1 gel DNA Recovery kit. Other kits and chemicals specifically used for a particular reaction are mentioned where ever used.

Cultivation culture media

Luria Bertani (LB) medium (low salt formulation): 1% Trypton (10 g/L), 0.5% NaCl (5 g/L), and 0.5% Yeast extract (5 g/L) dissolved in distilled water. Agar plates (LB) were prepared by adding 1.5% agar (15g/L) in LB broth.

Tryptic Soy Broth (TSB) medium: 1/3X Tryptic Soy Broth (10g/L) in distilled water. TSB agar plates were prepared with the addition of 1.5% agar in 1/3X TSB broth.

Culture media were autoclaved prior to use and were inoculated/streaked when their temperature dropped to 37ºC approximately.

Bacterial strains

E. coli DH5α and BL21 CodonPLus (DE3)-RIPL (Stratagene, USA) were used in this work. DH5α bacterial cells were used for cloning because of their high efficiency of transformation and reliable preservation of the transformed cells in glycerol. For overexpression of the cloned gene, BL21 CodonPLus (DE3)-RIPL cells were used since they contain lac promoter controlled gene for T7 RNA polymerase transcription. This lacUV5 promoter is inducible by IPTG addition in the culture medium. 21

Plasmid DNA isolation

Glycerol stocks of clones from stocks of School of Biological Sciences, PU were streaked on LB agar plates and incubated at 37ºC without shaking for 16-18 h. One colony from each plate was picked and inoculated in sterile LB broth. The culture flask was incubated in a shaking incubator at 37ºC overnight. Both the LB agar plates and broth were supplemented with specific antibiotic for the cloning vector. This culture was processed further to isolate plasmid DNA by a modified alkaline lysis method of Birnboim and Dolly (1979) as detailed below:

Five milliliter overnight culture was centrifuged at 5,000 ×g in a microfuge tube (1 ml/spin) at 4ºC for 1 min. To the pellet, 250 µl of solution I (25 mM Tris-Cl, 50 mM glucose, 10 mM EDTA pH 8.0) was added and vortexed until fully suspended. Solution II was prepared freshly by mixing the two components. Five hundred microliter of freshly prepared solution II (1% SDS, 0.2 N NaOH) was added to the sample. The microfuge tube was inverted five times to mix the contents of the tube after which it was left on ice for not more than 5 minutes. Then 375 µl solution III {glacial acetic acid (11.5 ml), 5 M potassium acetate (60 ml) and distilled water (28.5 ml)} was added. The contents of the tube were mixed by inverting the tubes carefully and incubated on ice for 3-5 min. After incubation the tube was centrifuged at 6,000 ×g for 5 min at 4ºC. The clear supernatant was carefully transferred to a new vial and pellet (white debris) was discarded. An equal volume of phenol: chloroform (1:1) mixture (freshly mixed) was added to this supernatant and vortexed to mix properly. The tube was centrifuged at 8,000 ×g for 5 min. The supernatant (uppermost layer) was pipetted to a fresh centrifuge tube and an equal volume of chloroform was added. After vigorous mixing of the contents, the tube was centrifuged for 5 min at 8,000 ×g. The upper layer of the supernatant was carefully picked by not taking any debris stuck to the walls of the vial and transferred to new microfuge vial. To the one volume of the supernatant taken, double volume of absolute ethanol/ isopropanol was added, mixed by vortexing and left at room temperature for about 20-30 min for DNA precipitation. The vials were centrifuged at high speed for 5 min. The white pellet was washed after discarding the supernatant with 70% ethanol. After centrifugation and discarding the supernatant (ethanol) the DNA pellet was air dried completely. The dried DNA pellets were either dissolved in small volume of TE buffer (1X) or in nuclease free water. After the dissolution of DNA in the solvent, was added to it was incubated at 37 ºC for half an hour after the addition of 1 µl DNase free RNase (20 mg/ml). The purity of isolated plasmid DNA 22 was detected by electrophoresing the sample on 1% agarose gel. The plasmids were stored at - 20 ºC for further us

Agarose gel electrophoresis was used to analyze DNA following the method described in Sambrook and Russell (2001). The concentration of required DNA band in agarose gels were estimated by comparing its density with the equivalent or nearly equivalent sized DNA ladder band when both the sample and DNA ladder were loaded in equal volumes.

Recovery of DNA from agarose gel

The PCR products and the products of restriction digestion of DNA were to be recovered from the agarose gel in order to ligate them into the cloning vectors. The required DNA band marked under UV light was cut and transferred to a pre-weighed microfuge vial. Further protocol was used as described in the instructions mentioned in the DNA recovery kit (GF-1 gel DNA Recovery Kit, Vivantis). Briefly, one volume of solubilizing buffer (buffer GB) to one volume of gel (0.1g= 0.1 ml) was added and incubated in 50 ºC water bath until complete melting of the gel. Extracted purified DNA was analyzed on agarose gel (1%). The sample was transferred to column provided in the kit and centrifuged at 10,000 ×g for 1 min. Flow through was discarded and the column was washed with wash buffer (650 µl) and after centrifugation the flow through was discarded and centrifuged again to completely dry the column. The DNA was eluted in a sterile microcentrifuge vial with 30-50 µl of sterile nuclease free water and 2 min incubation followed by centrifugation. All the centrifugation was performed at 10,000 ×g for 1 min.

The DNA concentration was calculated by measuring OD values at 260 nm and 280 nm in spectrophotometer (Eppendorf biophotometer, Germany) and applying these values in formula for the calculation of DNA amount (Amount of DNA= OD260 x Dilution factor x 50).

The OD280 values indicated the contamination of proteins in the DNA samples. Quantified DNA samples were stored at -20.

Preparation of competent cells

Competent cells of E. coli (DH5α and BL21 strains) were prepared and transformation of these competent cells with the recombinant genes or gene segments was performed as given in Sambrook and Russell (2001). 23

The competent cells were preserved in DMSO for later use. Filtered DMSO (75 µl) was added to 2 ml competent cells and kept on ice for 15 min. DMSO (70 µl) was added and again placed on ice for 5 min. the preserved competent cells were stored in aliquots (200 µl) at -80 ºC after snap freezing in liquid nitrogen.

Transformation of competent cells with recombinant vector

All the steps involving the direct handling of competent cells (with open vials) were performed under sterile conditions in laminar air flow cabinet. To 200 µl of E. coli competent cells, 1 µl of plasmid DNA or 10-20 µl of ligation mixture (vector + insert) was gently mixed and kept on ice for 40 min. The cells were given heat shock for 90 sec in water bath set at 42 ºC and then transferred immediately to ice for 5 min. Autoclaved LB broth (0.8 ml) was added in it and incubated in a shaking incubator at 37 ºC for 1-1.5 h. The cells were pellet down at 1500 ×g for 1 min and almost 800-850 µl of supernatant was removed. The settled cells were resuspended and spread on LB agar plates supplemented with or without appropriate antibiotic according to the cloning vector. The plates were incubated at 37 ºC without shaking for 16-18 h and analyzed for the presence of bacterial colonies.

Analysis of protein samples through SDS-PAGE

SDS-PAGE was used for the electrophoresis and analysis of the protein samples according to the protocols by Sambrook and Russell (2006). In this study, 10% SDS-PAGE gels were used according to the recommendation for the masses of the proteins being analyzed.

CHAPTER 3

Production of Purified Cry Proteins and Analysis of Their Activity Against Crop Pests

Abstract

tnCry1Ac, Cry2Ac7, Cry1Ie2 and Cry1Fa proteins were expressed, purified and used either as protoxins or activated toxins to check their toxicities to important crop pests. Bioassays of Spodoptera litura with tnCry1Ac and Cry2ac7 protoxins determined its susceptibility to Cry2Ac7 only. Activated toxins of all these proteins were found to be toxic to Anticarsia gemmatalis and Chrysodeixes includens in accordance with the previous reports of 5 times higher susceptibility of A. gemmatalis to that of C. includens to Cry proteins with some exceptions.

Introduction

Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is a polyphagous cosmopolitan insect which is also known as common cutworm, tobacco cutworm (Rehan and Freed, 2014; Shivayogeshwara et al., 1991). It is a serious crop pest of many Asian countries and is also notorious in Pakistan and in Indo-Pak region it is also called armyworm for its army like movement ( Shad et al., 2012; Ahmad et al., 2007; Shivayogeshwara et al., 1991). Its host range is very wide for example it feeds on beet, chickpea, soybean, cabbage, cotton, okra, tomato, rice, citrus plants, sunflowers and tobacco. (Ullah et al., 2016; Ahmad et al., 2013; Qin et al., 2003; Brown and Dewhurst, 1975). With its high rate of reproduction and gregarious feeding first on leaves and then the other plant parts resulting in crop loss, it categorizes itself as major destructive pest of economically important crops (Ahmad et al., 2007). It highly inflicted cotton and soybean growing regions and its outbreak resulted in almost 90% sunflower defoliation during 2005 in India (Dhaliwal et al., 2010; Sujatha and Lakshminarayana, 2007). In Punjab during 2003 to 2004 it became a serious pest of cotton and was responsible for great economic loss by lowering cotton crop mass (Arshada et al., 2009). Chemical pesticide control of this pest in the past was effective but now it has evolved resistance to many chemical pesticides (Shad et al., 2012; Saleem et al., 2008; Ahmad et al., 2007; Kranthi et al., 2002). 25

Anticarsia gemmatalis, velvetbean caterpillar and Chrysodeixes includens (Psuedoplusia includens), soybean looper, are significant lepidopteran pests of many economically important crops especially soybean. Soybean crop is one of these important crops which is widely grown in Asian coast, Europe, southeastern United States and South America as vegetable and oil seed crop (Formentini et al., 2015; McPherson and Macrae, 2009). Soybean looper (larva) attacks plants of different families like tomato, beans and cotton. It is also found on mild weather crops like cabbage, watercress, broccoli and some other plants of family Brassicaceae (Moscardi et al., 2012). It is suspected that Soybean looper can emerge as significant cotton pest in the South America countries (Blanco et al., 2016) and southern area (Ashfaq et al., 2001). Velvetbean caterpillar (larva) attacks crops of peanut, bean, pea, alfalfa, rice and wheat (Stürmer et al., 2013). These pests greatly damage the crop by reducing the leaf area and effecting productivity with reduced photosynthesis and hence less mass (Morales et al., 1995). On heavy infestation larvae of A. gemmatalis can defoliate soybean foliage by consuming 110 cm2 per larva (Aragón et al., 1997) leaving only the leaf veins intact only after consuming the epidermis and mesophyll of the whole leaf (Bundy and McPherson, 2007). The last instar larvae of S. litura, A. gemmatalis and C. includens are shown in Fig. 8.

Fig. 8. A, Army worm, Spodoptera litura larva and adult (https://wiki.bugwood.org/Spodoptera_litura); B, velvetbean caterpillar, Anticarsia gemmatalis larva and adult (http://entnemdept.ufl.edu/creatures/field/velvetbean.htm); C, Soybean looper, Chrysodeixes includens, larva (making its signature looping motion) and adult (http://entnemdept.ufl.edu/creatures/field/soybean_looper.htm). 26

These important pests have been controlled with chemical insecticides mainly but now Bt transgenic plants are being used for their control. Since 2013, cry1Ac transgenic soybean crop has been available commercially in Brazil (Schünemann et al., 2014). It has been reported earlier that Cry1A group of Cry toxins are non-toxic to Spodoptera species but later on Cry1C toxin found to be effective against it (Sanchis and Ellar, 1993). However, S. frugiperda (Smith) is susceptible to more Cry toxins like Cry1B, Cry1C and Cry1D (Monnerat et al., 2006). In another experiment by Monnerat et al. (2007) Bt isolates S0550 and S0845 possessed greater toxicity to S. frugiperda which was supposed to be enrichment or presence of either Cry1 or Cry2 alone or together in the crystal mixtures. The emergence of resistance of S. exigua to Cry1C toxin calls for to look for newer and potent Cry toxin for better pest management (Moar et al., 1995). The reports of toxicity of Cry toxins especially of Cry2 activated toxins against S. litura are not available according to our knowledge. In order to explore more Cry toxins effective for the control of these economically important Lepidopteran pests in the present study tnCry1Ac, Cry2Ac7 protoxins have been tested for toxicity to control S. litura and tnCry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 activated toxins for having toxicities against A. gemmatalis and C. includens.

Materials and methods

Cloning of cry1Ac and cry2Ac7 in pET28a(+)

Sub-cloning of tncry1Ac and cry2Ac7 into pET28a(+) expression vector The cry1Ac gene (GenBank accession no. EU250285.1) N-terminal domain encoding the activated toxin core and the cry2Ac7 gene (GenBank accession no. CAL18690.1) clones in pET22b(+) expression vectors were obtained from our lab stocks at School of Biological Sciences, University of the Punjab (Lahore, Pakistan). Another expression vector, pET28a(+) (Novagen, Madison, USA) was chosen to sub-clone these genes for the presence of His-tag coding sequence in it (Fig. 9).

Fig. 9. Circle map of the pET-28a(+) expression vector showing various sequence landmarks of the expression system; T7 promoter, T7 transcription start site, His-tag coding sequence, Multiple cloning sites (MCS) and Kanamycin (Kan) coding sequence (http://www.merckmillipore.com/INTL/en/product/pET-28a+-DNA-Novagen,EMD_BIO- 69864#anchor_USP).

On ligation using NdeI restriction site, the expressed protein contains His6-tag at the N- terminal end which is used for the purification of protein through affinity chromatography. 28

This expression vector contains kanamycin antibiotic resistance marker which facilitates selection and identification of the particular clone.

Double restriction digestion of recombinant vectors with gene inserts and the sub-cloning vector The RestrictionMapper version 3 online tool (http://www.restrictionmapper.org/) to map sites for the restriction enzymes were used to find appropriate enzymes from the available restriction sites of the cloned vectors. Two endonuclease restriction enzymes for tncry1Ac (NdeI and XhoI) and cry2Ac7 (NdeI and HindIII) which do not cut within the genes were selected for this purpose. The pET28a(+) vector DNA plasmid was restriction digested with selected endonucleases to linearize the plasmid and to generate sticky overhangs. Thirty milliliter restriction mixtures were prepared by adding nuclease free water (12 µl), 10X Tango buffer (6 µl to make 2X final concentration), NdeI and XhoI/HindIII restriction enzymes (1, 1 µl each) and vector plasmid DNA (10 µl). The reactions were completed in 16 h incubation at 37 ºC without shaking.

The pET22b(+) recombinant vector plasmids containing the tncry1Ac and cry2Ac7 gene inserts were restricted with the same selected enzymes to isolate the DNA of inserts. The restriction mixture contained nuclease free water (12 µl), 10X Tango buffer (6 µl to make 2X final concentration), NdeI and XhoI/HindIII restriction enzymes (1, 1 µl each), tncry1Ac recombinant vector plasmid DNA (7 µl) or cry2Ac7 recombinant vector plasmid DNA (10 µl) and the volume was made up to 30 µl with nuclease free water. The reaction vial was incubated at 37 ºC without shaking for 16 h.

Whole volume of all the restricted samples were electrophoresed and analyzed on agarose gel (1%) under UV light. The required restricted bands of the linearized vector (pET28a(+) and the inserts (cry1Ac and cry2Ac7) were cut and placed in a pre-weighed sterile microfuge vial. DNA recovery kit (Vivantis) was used to recover DNA from the agarose bands as detailed in general methods section.

Ligation of cry1Ac and cry2Ac7 gene inserts The purified (gel cleaned) double digested DNA fragments with sticky overhangs were ligated into gel cleaned linearized pET28a(+) with the same enzymes generated cohesive overhangs. The volumes of vector and insert DNA (having required pmol quantities of DNA) were calculated using the measured concentration and DNA fragment size. The reaction was set up with the use of 1:10 ratio of vector to insert which was calculated as 0.0225: 0.2 pmols 29

for both the reactions. The reaction mixture contained 1X ligation buffer (2 µl), T4 DNA ligase (10 units in 1 µl), vector plasmid DNA (5 µl), cry1Ac DNA (8 µl) or cry2Ac7 DNA (7 µl) and nuclease free sterile water was used to make the final volume up to 20 µl. The vials containing the ligation mixtures were incubated at 22 ºC for 2-3 h.

Transformation of DH5α (E. coli) competent cells with the ligation mixtures (10 µl each) was separately performed following the transformation method described in general method section. The transformed cell cultures were plated on LB agar plates supplemented with 50 µg/ml kanamycin antibiotic for selection and screening purpose. The colonies appeared on LB agar plates were used for recombinant plasmid DNA isolation following the protocol detailed previously in general method section. The plasmids were named as tncry1Ac28 and cry2Ac28 and were stored at -20 ºC for further use.

Confirmation of clones through PCR amplification The isolated plasmid DNA was used to confirm cloning of genes in pET28a(+) vector through PCR amplification which is a logarithmic procedure for the amplification of DNA template. DNA was amplified through logarithmic procedure of PCR. Forward primer cry1AcF: 5′-TGAGGA GGTAACATATGGATAACAATCCGAACATC-3′ and the reverse primer cry1AcR: 5′-GCAGCGCTCGAGTCATGCAGTAACT-3′ were used for amplification of tncry1Ac template DNA. NdeI and XhoI restriction site are underlined in the forward and reverse primers, respectively. Forward primer cry2Ac7F: 5′-CAC ACATATGAATACTGTATTGAATAAC-3′ and the reverse primer cry2Ac7R: 5′-GGC AAGCTTTTAATAAAGTGGTGGAAG-3′ were used for amplification of cry2Ac7 template DNA. NdeI and HindIII restriction sites are underlined in the forward and reverse primers for cry2Ac7, respectively.

Reaction mixture for PCR contained; taq buffer (1X), dNTP mixture (0.25 mM), magnesium chloride (2 mM), forward and reverse primers (100 pmol each), taq DNA polymerase (2.5 units), plasmid DNA as template (1ng) and the sterile nuclease free water to make final reaction volume 25 µl. The PCR tubes used were made sterile by autoclaving them and the reaction mixtures were prepared by keeping the PCR tubes on ice all the time. Prior to start of the amplification the vials were short spun to bring the contents of the tube at bottom.

In Applied Biosystems thermocycler (2720) PCR reactions were performed separately. Gene segment (tncry1Ac) was initially denatured at 95 °C for 5 min and amplified over 30 cycles; each of denaturation at 95 °C for 1 min, annealing at 58 °C for 45 s and extension at 30

72 °C for 1.30 min, with a final extension at 72 °C for 4 min. Polymerase chain reaction for the amplification of cry2Ac7 was carried out using these conditions: initial denaturation at 95 °C for 5 min; 30 cycles of each of denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min and extension at 72 °C for 1.30 min; a final extension at 72 °C for 5 min. The PCR products were resolved by electrophoresing on 1% agarose gel and analyzed under UV light.

Over-expression and purification of recombinant proteins

Transformation of host cells for gene expression DNA plasmids (tncry1Ac28 and cry2Ac28) and original recombinant plasmids of tncry1Ac and cry2Ac7 clones in pET22b(+) from School of Biological Sciences, PU, Lahore were used to transform E.coli (BL21-CodonPlus (DE3)-RIL host cells for expression vectors. The transformed competent cell cultures were spread on LB agar plates supplemented with kanamycin (50 µg/ml) for clones in pET28a(+) and ampicillin (100 µg/ml). The colonies appeared after overnight incubation at 37 ºC were picked and inoculated separately in LB broth (25 ml) supplemented with appropriate antibiotic and incubated overnight in a shaking incubator set at 37 ºC. LB broth (500 ml) in 2 L flasks were inoculated with 3-5 % of the specific primary cultures individually. Each flask was incubated at 37 ºC with shaking at 180 rpm until OD600 reached 0.8-1 at which 1 ml un-induced culture was drawn out for comparison with the induced one later. The gene expression was induced by adding IPTG for a final concentration of 1mM for the production of Cry1Ac and 0.1-0.5 mM for Cry2Ac7 proteins and the incubation was continued under the same conditions for 6-8 hours further. OD600 of the induced cell cultures was measured and the cell cultures were transferred to 1L centrifuge buckets. Cells were pellet down by centrifuging at 5,000 ×g for 20 min in a Beckman centrifuge. The cell pellets were washed with distilled water after discarding the supernatant. the pellets were washed and resuspended in 50 mM Tris-Cl, 0.5 M NaCl buffer pH 8.0 (suspension buffer). Cells were lysed ultrasonically with 30 s On 90 s Off per cycle for a total of 10 cycles at 65% amplitude (Cell Disruptor SONIF M250, Fisher Scientific). The lysed cells were separated by centrifuging again at 4 ºC for 10 min at 8,000 ×g and resultant pellet was sonicated twice for 5 more cycles with 0.2% Triton X-100 (v/v) in suspension buffer with change of buffer in between. Semi purified inclusion bodies were washed and sonicated on ice resuspended in the suspension buffer to get rid of Triton-x and the DNA contamination until its 260 nm:280nm ratio becomes around 0.64: 1. Total cell proteins, soluble and insoluble fractions of the cell lysate were analyzed on 10% SDS-PAGE gels. 31

Refolding and purification of tnCry1Ac and Cry2Ac7 protoxins tnCry1Ac and Cry2Ac7 protein localized in inclusion bodies were solubilized in small volume of solubilization buffer (50 mM Na2CO3, 0.5 M NaCl, 8 M urea and 0.2 % β- mercaptoethanol, pH 10.0) and incubated at 37 °C for 45 min to achieve complete reduction of disulfide bridges. The solubilized proteins were separated from the insoluble materials by centrifuging at 12,000 × g for 15 min at 4 °C. The clear solution of solubilized proteins was added dropwise (0.1ml/10 min) by using peristaltic pump in its 10 times (v/v) refolding buffer

(50 mM Na2CO3, 0.5 M NaCl, 5 mM cysteine and 0.5 mM cystine, pH 10.0). The refolding buffer was kept on ice and set on constant stirring during the addition of solubilized proteins. The diluted protein in the refolding buffer was added in a 10 kDa MWCO dialysis tube (SnakeSkin, Thermo Scientific) and was dialyzed against the same refolding buffer for 2 h and once overnight against 50 mM Na2CO3, 0.5 M NaCl, pH 9.0 buffer at 4 °C. Each step of dialysis was performed on magnetic stirrer at 4 ºC. Solubilized protein was separated from the precipitated proteins and contaminants by centrifuging at 20,000 ×g for 20 min at 4 ºC in between each buffer change. The refolded proteins through dialysis were very dilute and still contained other contaminant proteins. Diluted protein solutions were concentrated and purified by centrifuging at 3,000 ×g for 10 min at 25 ºC in a centrifugal concentrator of 50,000 MWCO (Millipore). Briefly, 15 ml of dilute protein solution was centrifuged to reduce the volume to 5 ml and then again brought to 15 ml by the addition of native buffer with one more centrifuge step until the reduction of volume to 2 ml. A brief overview of the refolding and purification is given in Fig. 10. This concentrating step was repeated until all dilute protein solution was concentrated. The final concentrate was washed one more time and was quantified by recording the absorbance at 260 nm and 280 nm in a UV spectrophotometer and corrected concentration of protein was calculated (Layne, 1957). The purified proteins (protoxins) were analyzed by running the samples on SGS-PAGE (10%) and stored at -80 °C for use in bioassays against insect pests.

Fig. 10. Flowchart showing the sequential steps adopted in the refolding and purification of tnCry1Ac and Cry2Ac7 insoluble proteins. Refolding and purification of His-tagged tnCry1Ac and Cry2Ac7 protoxins Inclusion bodies were denatured in the denaturing buffer (20mM Tris-cl, 0.5 M NaCl, 20 mM Imidazole, 8 M Urea, 0.2 % β-mercaptoethanol, pH 8.8). The denaturing reaction was incubated in shaking incubator at 37 °C for 30-60 min to denature the misfolded proteins and 33 to expose the his-tag (6 histidine residues) at the N-terminal end of proteins due to cloning in pET28a(+). Ni-NTA agarose resin (Thermo scientific) was resuspended in its bottle and 1.5 ml resin slurry (called 1 column volume) was pipetted into 10 ml purification column. The resin was allowed to settle down by gravity and was washed with sterile distilled water. Supernatant obtained after high speed centrifugation was loaded on Ni-NTA resin pre-equilibrated with 5 column volume (CV) binding buffer (50 mM Tris-Cl, 0.5 M NaCl, pH 8.8). The his-tag bound protein was on column refolded by using step gradient of urea (8 M, 6 M, 4 M, 2 M and 0 M) in binding buffer to get the protein refolded. Briefly, to the protein bound to resin, 5 CV of refolding buffer (decreasing concentration of urea in binding buffer) was passed through the column under gravitational force. Then a final washing was performed with 5 CV of binding buffer alone. The bound refolded protein (protoxin) was eluted with elution buffer (20 mM Tris-Cl, 0.5 M NaCl, 250 mM Imidazole, pH 8.8) and the fractions were analyzed on SDS- PAGE. Proteins were quantified by Bradford assay, bovine serum albumin (BSA) was used as a standard (Bradford 1976). Proteins were stored at 4 °C for immediate use or for use within a week.

Production and purification of Cry1Fa protoxin

A recombinant Bt strain harbouring the cry1Fa gene (Zhao et al., 2015) was used for the production of Cry1Fa protoxin following the protocol described elsewhere (Luo et al., 1999). Briefly, the Bt strain was grown at 28 ºC in 500 ml TSB medium for 3 days until sporulation. The cell culture was centrifuged at 7,000 for 30 min to pellet down the spores, cell debris and protein crystals. The supernatant was discarded and the pellet was washed with 0.1% Triton X-100 in 1M NaCl solution. After centrifugation the supernatant was discarded and the pellet was washed with distilled water. The pellet was resuspended in 25 ml of 50 mM Na2CO3 (pH 9.6) buffer to which 0.1% 2-mercaptoethanol was added and incubated at room temperature for 2 h. The dissolved crystals were separated from the debris by centrifuging the preparation at 20,000 ×g for 30 min. The supernatant contained dissolved Cry1Fa protoxin protein which was saved and analyzed on 10 % SDS-PAGE.

Production and purification of Cry1Ie2 protoxin

The full-length cry1Ie2 gene (GenBank accession no. HM439636) from Bt strain T03B001 cloned in a recombinant E. coli (Rosetta DE3) strain for production of Cry1Ie2 toxin was obtained from Dr. Juan Luis Jurat-Fuentes, University of Tennessee, Knoxville, TN, USA. 34

The method used for the production and purification is as described elsewhere (Zhao et al., 2015) with some modifications. Fresh colony was inoculated in 20 ml LB medium with 100 µg/ml ampicillin and 24 µg/ml chloramphenicol and incubated over night at 37 ºC with shaking at 200 rpm. In a 2L flask 400 ml LB medium was inoculated with 2% overnight primary culture along with the addition of 100 µg/ml ampicillin and 24 µg/ml chloramphenicol. The flask was incubated in a shaking incubator with 200 rpm at 37 ºC until the OD600 reaches 0.8-1. Over- expression of protein was induced with 0.4 mM IPTG incubating at 20 ºC for 16 h. After 16 h the cells were harvested by centrifuging the culture media in 500 ml Beckman bottles in a Beckman ultracentrifuge at 6,000 ×g. The supernatant was discarded and the cell pellet was washed with Na2CO3 buffer pH 10. The cells were re-suspended in 25 ml of 50 mM Na2CO3, 0.1 M NaCl pH 10.0 buffer and 0.1% 2-Mercaptoethanol was added freshly to it. The cells were lysed by sonication at 75% amplitude, 5 sec on and 40 sec off for 20 cycles. The lysed cells were incubated overnight at 28 ºC on shaking at 150 rpm. After centrifugation in 50 ml bottles at 10,000 ×g the supernatant was collected and the cell debris was discarded. The overexpression of protein was checked by running the samples on 10% SDS-PAGE gel.

The Cry1Ie2 protein was expressed in soluble form and has a N-terminus 6X His-tag so, His-Trap HP 1ml column was used on ÄKTA FPLC (GE Healthcare) to purify the protein.

His-Trap HP column was not compatible with Na2CO3 buffer, the protein was dialyzed against 20 mM Tris-Cl buffer with 50 mM Imidazole pH 8.8. The same buffer was used as binding buffer and the protein was eluted at 100% elution buffer (20 mM Tris-Cl, 0.5 M Imidazole pH 8.8) in a linear gradient of 100%. The peak fractions were checked on 10% SDS-PAGE. Affinity purified protein fractions were pooled and protein was quantified using Qubit Protein Assay Kits (Life Technologies) according to the assay protocol given by manufacturer. Briefly, 200 µl working solution was made by adding 198 µl of the buffer (provided in the kit), 1 µl of the fluorescent dye (provided in the kit) and 1 µl of the protoxin in a 0.5 ml thin walled PCR tube. After 15 min of incubation at room temperature the protein concentration was quantified in a Qubit fluorometer (Thermo Fisher Scientific). The quantified fractions were aliquoted and stored at -80 ºC for use when needed.

Trypsin activation and purification of proteins

To activate the tnCry1Ac and Cry2Ac7 proteins, they were incubated with 5:1 (w/w) or 10:1 (w/w) toxin: trypsin (TCPK treated bovine trypsin, Sigma) ratios, respectively, for 90- 120 min at 37 °C. Cry1Fa and Cry1Ie2 protoxin were also activated using TPCK treated trypsin 35 in a toxin to trypsin ratio of 5:1 (w/w) and incubated at 37 ºC for 1h. The reactions were stopped by centrifuging the contents at 10,000 ×g for 5 min and the tryptic digestion of protoxin was checked by running the sample on 10% SDS-PAGE. Activation was assessed by SDS-10%PAGE and activated toxins were dialyzed against

20 mM Na2CO3 pH 9.8 buffer (binding buffer) overnight at 4 ºC. Toxins were then bound to an anion exchange HiTrap Q HP column (GE Healthcare) pre-equilibrated with binding buffer and connected to an ÄKTA Pure FPLC. After washing the unbound proteins with binding buffer, toxins were eluted with a linear gradient of 100 % elution buffer (20 mM Na2CO3, 1 M NaCl, pH 9.8). The peak fractions were analyzed on 10%SDS-PAGE gel and fractions containing protein were pooled and saved at -80 for further use.

Bioassays of tnCry1Ac and Cry2Ac7 against S. litura

Rearing of S. litura

S. litura (Lepidoptera: Noctuidae) moths were captured from the vegetable crop (adjacent tomato, and pea plots) fields of Quaid-e-Azam Campus, University of the Punjab and colonies were raised from seven adults (two male and five female). The bucket was lined and covered by muslin cloth after placing a plastic cup containing a cotton wool ball soaked in 10% honey solution to feed moths (Ahmad et al., 2007). The bucket was kept at 28 ºC almost for a week for egg laying. The pieces of muslin cloth with laid eggs were collected and placed in an incubator with 26 ± 2 ºC, 68 ± 5 % relative humidity and 14 h light # 10 h dark photoperiod. Three cultures were separately reared in the laboratory on the basis of diet. Two cultures were reared on natural diet of previously washed and dried mulberry and spinach leaves in glass jars covered with muslin cloth (Ahmad et al., 2013). The third population was reared on chickpea based semi-synthetic diet as described elsewhere (Ahmad et al., 2003). It was made sure before putting the larvae that the diet did not contain any moisture. The larvae were shifted to new diet after every 24 h until start of pupation. Healthy pupae were selected to obtain next generation. This S. litura population was reared in our laboratory (insectary) for two generations in order to acclimatize them to laboratory conditions and to maintain a sufficient number of larvae for bioassay replicas.

tnCry1Ac and Cry2Ac7 protoxin bioassays against S. litura

Third generation larvae of the field collected S. litura were used to check the insecticidal activity of tnCry1Ac and Cry2Ac7 protein. Bioassays were conducted by diet 36 incorporation method, test protein was diluted and mixed in the prepared diet in different concentrations just before pouring in the petri plates. The seven concentrations of Cry2Ac7 protein in the diet were 0.025 µg/ml, 0.050 µg/ml, 0.075 µg/ml, 0.10 µg/ml, 0.15 µg/ml, 0.30 µg/ml and 0.6 µg/ml. The Cry1Ac protein concentrations were 0.10 µg/ml, 0.15 µg/ml, 0.30 µg/ml, 0.6 µg/ml, 1.2 µg/ml, 1.80 µg/ml and 2.4 µg/ml. Autoclaved distilled water was used for protein dilutions and as negative control. The diet was solidified at room temperature and dried in laminar air flow cabinet. One day old active starving larvae were placed in the plates (1 larvae/plate) with fine camel hair brush using 10 larvae per tested concentration. Petri plates were covered with polystyrene lids and the lids were perforated with needle. The plates were incubated in incubator as described for rearing and mortality was scored from third to seventh days of incubation. Experiment was repeated thrice with duplicates each time. Abbott’s formula (Abbott, 1925) was used to correct the calculated control larval mortality percentage where necessary. Fifty percent Lethal Concentration (LC50) and their 95% fiducial limits (FL) were estimated employing Polo-Pc (LeOra, 2005). 95% fiducial limits were considered significant at 5 % significance level after pairwise comparisons of LC50 (Litchfield and Wilcoxon, 1949).

Bioassays of Cry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 toxins against A. gemmatalis and C. includens

To determine the toxicity of activated Cry1Ac and Cry2Ac7 proteins bioassays were performed against A. gemmatalis and C. includens. For bioassays susceptible insect larvae eggs of A. gemmatalis and P. includens were purchased from Benzon Research (Carlisle, PA). Five concentrations were tested for each purified toxin: 20, 60, 180, 400 and 600 ng/cm2 for A. gemmatalis and 30, 90, 270, 810, 2430 ng/cm2 for P. includens. For Cry1Ie2 one more toxin concentration (4860 ng/cm2) was tested against P. includens. On the solidified meridic diet (General Purpose Lepidoptera diet; BioServ) in 128 well trays 75 µl/well test protein solution was evenly poured and dried in laminar flow cabinet. Controls included diet contaminated with the buffer used for toxin dilutions (20 mM Na2CO3, pH 9.8). Newly emerged larvae were transferred to wells (one neonate/well) with fine camel hair brush and placed at 27 °C, 70-80 % humidity and 14: 10 h light: dark photoperiod. Mortality was recorded after 7 days. The bioassays were repeated thrice with 16 larvae tested in each bioassay per toxin concentration 37

(n=48). Lethal concentrations causing 50 % mortality (LC50s) and its 95 % fiducial limits were estimated by Probit analysis (Finney, 1971) using POLO-PC program (LeOra, 2005).

Results tncry1Ac and cry2Ac7 sub-clones

The restriction of recombinant plasmids (pET22b+ recombinant vectors) containing tncry1Ac and cry2Ac7 genes produced two bands after double restriction with NdeI and HindIII, and NdeI and XhoI, respectively. Fig. 11 shows the linearized vector (6 kb) and the gene (1.8 kb) DNA bands.

Fig. 11. 1% Agarose gel showing restriction analysis of recombinant plasmids tncry1Acp22 (A) and cry2Ac7p22 (B). Lane 1, recombinant plasmids of cry1Ac (A) and cry2Ac7 (B) in pET22b(+); Lane 2, double digested recombinant plasmid; M, DNA size marker (GeneRuler DNA ladder mix, ThermoScientific # SM0331), reference bands base pairs are shown on left of each panel.

The transformed cell culture (DH5α) gave many colonies on LB agar plates supplemented with kanamycin. The appearance of colonies for the selection of recombinant clones on kanamycin plates proved the presence of pET28a(+) vector with kanamycin 38 resistance gene in it. However, the presence of tncry1Ac and cry2Ac7 gene inserts in pET28a(+) vectors (tncry1Ac28 and cry2Ac28 plasmids) was confirmed by PCR amplification of the inserts in the two recombinant plasmids. The amplified bands were seen around 1.8 kb band size position when compared with the DNA molecular size markers ran along with the samples (Fig. 12).

Fig. 12. PCR amplification of tncry1Ac and cry2Ac7 genes from tncry1Ac28 and cry2Ac28 plasmids. Lane 1, 1.8 kb PCR amplified band of tncry1Ac; Lane 2, 1.8 kb PCR amplified band of cry2Ac7; M, DNA size marker (GeneRuler 1 kb DNA ladder, ThermoScientific, SM0311); reference bands bp are shown on left of panel.

tnCry1Ac and Cry2Ac7 untagged proteins

The tnCry1Ac and Cry2Ac7 proteins expressed as inclusion bodies were partially purified by washing with Triton-X 100 inclusion bodies were solubilized in urea. Addition of β-mercaptoethanol helped solubilization by cleaving di-sulfide bridges formed by the cysteine residue of the protein polypeptide. Cysteine and cystine helped correct refolding of the misfolded proteins. Fast dilution and dialysis method for tnCry1Ac and Cry2Ac7 protein removed contaminants and resulted in 45-55% pure proteins (Fig. 13, lane 3 (Cry2Ac7) and lane 6 (tnCry1Ac). The repeated dialysis and centrifuging the proteins at high speed removed more contaminant proteins. However, the fast dilution method of protein refolding resulted in very dilute protein solutions that could not be detected through SDS-PAGE. The repeated centrifuging of the protein solution in the centrifugal concentrators concentrated the proteins by reducing the volume of refolding buffer. The washing of the protein solutions by adding 39 fresh native buffer each time washed the proteins and the native buffer replaced the refolding buffer completely. Heavy washing of the proteins in centrifugal concentrators also removed remaining contaminants from the proteins bringing the purity level to 75-80 % (Fig. 13, lanes marked PP).

Fig. 13. 10% SDS-PAGE gel showing fractions from refolding and purification steps of tnCry1Ac and Cry2Ac7 protoxin. Lanes 1 and 4, extensively washed inclusion bodies with Triton-X 100; fast diluted and dialyzed refolded proteins of Cry2Ac7 (lanes 2 and 3) and tncry1Ac (lanes 5 and 6); UI, uninduced cell lysate fraction; IB, inclusion bodies; PP, purified proteins; M, protein molecular weight marker (PageRuler 26616, Thermo Scientific) shown by values (kDa) to the extreme left of panel.

tnCry1Ac, Cry2Ac7 and Cry1Ie2 His-tagged proteins

Over-expression of tncry1Ac and cry2Ac7 cloned in pET28a(+) expression vectors produced the proteins with same molecular weight excepts with the added 6- His amino acid residues to the N-terminal end of the proteins. The proteins were expressed in the insoluble fraction (inclusion bodies) of the cell proteins. Protein bands at 68 kDa and 69 kDa in Fig. 14 represent tnCry1Ac and Cry2Ac7 proteins, respectively. 40

Fig. 14. Expression profile of his-tagged tnCry1Ac and Cry2Ac7. Lane 1, uninduced fraction of cell protein; Lanes 3 and 5, soluble protein fraction of cell lysate; Lanes 2 and 4, tnCry1Ac and Cry2Ac7 insoluble protein fractions, respectively, closed ended arrow show the expressed protein bands (~68-69 kDa); M, protein molecular weight marker (Fermentas, SM0661) shown by values (kDa) to the extreme left of panel.

The Cry1Ie2 protoxin protein was expressed as soluble protein and was detected as 81 kDa band when analyzed through 10% SDS-PAGE. Cry1Ie2 protein was not detected in the insoluble fraction of the cell lysate (Fig. 15).

Fig. 15. SDS- PAGE analysis of Cry1Ie2 protein expression. Lane 1, uninduced fraction of total cell lysate; Lane 2, insoluble fraction of cell lysate; Lane 3, insoluble fraction of induced cell lysate; lane 4, soluble fraction of induces cell lysate; M, protein molecular size marker. The numbers on the left are molecular masses (in kilodaltons). 41

Purification and trypsin activation of tnCry1Ac, Cry2AC7, Cry1Ie2 and Cry1Fa proteins

The tagging of the proteins with polyhistidine (6xHis) sequence at N-terminus made it very convenient, cost effective and time saving to refold and purify these proteins simultaneously. The employment of nickel-charged affinity resin (Ni-NTA) to bind the tagged proteins allowed rapid refolding with lesser volumes of refolding buffers and the bound refolded proteins were easily eluted by competition with imidazole. The elution of his-tagged Cry1Ie2 protein from the column mounted on FPLC was eluted at 100% elution buffer in good protein yield as shown in chromatogram (Fig. 16).

Fig. 16. Chromatogram of affinity chromatography purification of Cry1Ie2 protoxin by using His-Trap HP 1ml column mounted on FPLC system.

About 80-90 % pure proteins were obtained as shown in Fig. 17. Affinity chromatographically purified proteins were successfully activated by trypsin digestion. Tryptic digestion removed His-tag from the proteins which might hinder the activity of the proteins and cleaved almost 10-25 amino acid residues from the N-terminus of the proteins which generated required trypsin resistant active cores of the proteins (Fig 17, lanes 2, 3 and 6). After 42 trypsin activation tnCry1Ac was named as Cry1Ac as it generates trypsin resistant core of similar molecular weight as that of full-length Cry1Ac.

Fig. 17. Coomassie stained SDS-PAGE gel showing the his-tag purified and trypsin activated toxins. Lanes 1, 4 and 5, his-tag purified tnCry1Ac, Cry2Ac7 and Cry1Ie2; lanes 2, 3 and 6, trypsin resistant Cry1Ac, Cry2Ac7 and Cry1Ie2 core proteins; M, protein molecular weight marker (PageRuler 26616, Thermo Scientific) shown by values (kDa) to the extreme left of panel. Close ended arrows show the activated toxins of 60 kDa (Cry1Ac), 58 kDa (Cry2Ac7) and 55 kDa (Cry1Ie2).

Cry1Fa protoxin crystal inclusions were solubilized and the soluble protein (120 kDa) obtained was in partially purified form which was directly treated with trypsin. On tryptic digestion, a protease resistant core of 62 kDa was analyzed on SDS-Page gel (Fig. 18).

Fig. 18. SDS- PAGE analysis of Cry1Fa production and activation. Lane 1, solubilized Cry1Fa proteins; lane 2, trypsin digested Cry1Fa; M, protein molecular size marker. The numbers on the left are molecular masses (in kilodaltons). 43

Purification of trypsin activated proteins After protoxin activation with trypsin digestion, trypsin was removed from and proteins were further purified by anion-exchange chromatography on an FPLC system (Fig. 19 A-D). All the proteins were eluted at 100% elution buffer.

Fig. 19. Chromatogram of anion-exchange chromatography purification of activated toxins Cry1Ac (A), Cry2Ac7 (B), Cry1Ie2 (C) and Cry1Fa (D) using an FPLC system. 45

The pooled fractions of proteins purified through anion-exchange chromatography are shown in Fig. 20.

Fig. 20. SDS-PAGE analysis of activated toxins purified through anion-exchange chromatography. Lane 1, Cry1Ac (60 kDa); lane 2, Cry2Ac7 (55 kDa); lane 3, Cry1Fa (62 kDa); lane 4, Cry1Ie2 (55 kDa); M, protein molecular size marker. The numbers on the left are molecular masses (in kilodaltons).

Bioassays of tnCry1Ac and Cry2Ac7 protoxin proteins against susceptible S. litura

The extent of susceptibility of S. litura for tnCry1Ac and Cry2Ac7 protoxins was detected by feeding the larvae on diet containing these protoxins. Biological bioassay results predicted Cry2Ac7 protoxin protein toxic to S. litura. However, even the higher amounts of tnCry1Ac protoxin protein could not distinctly kill laboratory reared third generation S. litura larvae used for bioassay. The bioassay results categorized tnCry1Ac as non-toxic protein for

S. litura. as shown by the LC50s of the two proteins in Table I.

Table I. Toxicity of selected Bt protoxins against neonates of S. litura from diet surface-contamination bioassays.

Insect Toxin LC50 (ng/ml) Lower 95% CI Upper 95% CI

tnCry1Ac NA NA NA Spodoptera litura Cry2Ac7 227.24 167.23 312.6 46

Bioassays of Cry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 activated toxins against susceptible C. includens and A. gemmatalis

After 7 days of incubation only the dead larvae were scored and the larvae with stunted growth were not counted for the mortality calculations. The quantitative bioassay results (LC50s and its 95% confidence intervals) with Cry1Ac, Cry2Ac7, Cry1Fa and Cry1Ie2 are shown (Table II).

Table II. Toxicity of purified Bt toxins against neonates of Chrysodeixis includens and Anticarsia gemmatalis from diet surface-contamination bioassays.

2 Insect Toxin LC50 (ng/cm ) Lower 95% CI Upper 95% CI

Cry1Ac 109.49 52.86 179.56

Cry2Ac7 214.18 109.72 315.91 Chrysodeixis includens Cry1Ie2 3,467.79 1,829.36 4,480.92

Cry1Fa 242.82 183.23 322.39

Cry1Ac 20.31 11.09 27.96

Cry2Ac7 46.56 5.71 106.60 Anticarsia gemmatalis Cry1Ie2 454.17 123.76 693.37

Cry1Fa 42.47 17.98 69.52

In general, C. includens larvae were always less susceptible than A. gemmatalis to all the tested toxins. Toxicity values (Table II) were substantially higher, Cry1Ac being the most active toxin against both insects. In contrast, Cry1Ie2 was 10-fold less active against both insect species. Cry2Ac7 and Cry1Fa toxins displayed almost equal toxicity to A. gemmatalis whereas for C. includens Cry2Ac7 exhibited enhanced toxicity than Cry1Fa toxin. However, almost the similar values of upper 95% confidence interval of these toxins for C. includens made their 47

toxicities not statistically different. The difference in the LC50 values of Cry2Ac7 and Cry1Fa for both the insects is almost twice to that of Cry1Ac. Among all the proteins tested, Cry1Ie2 was the least toxic protein with considerable LC50 value for A. gemmatalis but a higher LC50 value for C. incudens (Table II).

Discussion

Bt Cry toxins are effective in the control of insect pests of economically important crops like cotton, maize and soybean. The rearing of S. litura culture in lab under controlled conditions was successful with good development on mulberry leaves and semi synthetic diets. The larvae population reared on spinach leaves was lost between 4th-5th instar and after repeated efforts we could not get second generation of S. litura larvae fed on spinach leave. This suggests that leaf worm has high survival rate on mulberry leaves as compared to almost zero survival on spinach leaves emphasizing the status of spinach as rare host plant by Ahmad et al. (2013). Semi synthetic diet was used for bioassays to make sure of the final concentration of the test protein solution. Bioassay results characterized tnCry1Ac as nontoxic and Cry2Ac7 protein as toxic to S. litura insect pest. Initially, larvae fed on Cry2Ac7 contaminated protein became slow and stunted growth was noticed after 48 h. Mortality of larvae started after 72 h of their exposure to Cry2Ac7 protein. The proteins we used were protoxins as they were not activated by trypsin digestion or gut juices prior to use. These protoxins are activated by larval midgut juices as like other Cry1 and Cry2 protoxins (de Maagd et al., 2001; Bravo et al., 2005) to become active toxins in the insect gut. C-terminally truncated Cry1Ac could not be effective to suppress larval growth and hence LC50 value was not established. The LC50 of Cry2Ac7 protoxin is 310 µg/ml as shown in table I. The highest concentration tested was 0.6 µg/ml which resulted in 100 % mortality. Cry2Ac7 can be a good choice to be used as a bio pesticide for the control of S. litura. The transformation of crop plants with cry2Ac7 gene along with other Bt insecticidal gene could help to combat against this and other susceptible insects

The molecular masses of the trypsin activated Cry1Ac (60 kDa) and Cry2Ac7 (58 kDa) are in agreement with the molecular masses of the protease resistant toxin cores for the long and short Cry protoxins (de Maagd et al., 2001; Hernandez-Rodriguez et al., 2008; Pardo-

López et al., 2013). Considering the LC50 values in the Table II imply the findings of Bernardi et al. (2012), that the toxicity of Cry1Ac protein to A gemmatalis is more than to P. includens. While looking for the variation of susceptibilities of lepidopteran larvae to the Bt toxins, 48

Luttrell et al. (1999) reported that P. includens with LC50 of 0.31 µg/ ml of diet is 4-fold less susceptible to Cry1Ac than H. virescens (F) with LC50 of 0.07 µg/ ml of diet. Bobrowski et al.

(2002) found that Cry1A has four times lesser LC50 for A. gemmatalis in comparison with LC50 for H. virescens reported earliear by Rie et al. (1989). Taking H. virescens as a reference it means that velvetbean caterpillar is four times more susceptible to Cry1A(C) than soybean looper. A previous study reported 1.5-5.6 times higher susceptibility of A. gemmatalis than P. includens to Bt biological insecticides (Morales et al., 1995). In the present work, the LC50 of Cry1Ac for P. includens is 109.48 ng/cm2 is almost 5-fold higher than A. gemmatalis (20.31 ng/cm2) and Cry1Fa displays 5.7-fold more toxicity to A. gemmatalis which is in agreement with the previous findings. However, the difference in susceptibility of these insects is more than 7-fold for Cry1Ie2. This is the first report of the efficacy of Cry2Ac7 toxin against A. 2 gemmatalis and P. includens. Cry2Ac7 toxin, with LC50 of 46.53 ng/cm against A. gemmatalis which is almost 4-5 times lesser than that of 214 ng/cm2 against P. includens, shows the similar behavior as that of Cry1 insecticidal proteins for these insects. Toxicity values (Table II) were substantially higher than in recent reports for Cry1Ac and Cry1Fa (Bel et al., 2017), which may be due to different toxin production systems and strains being used. Nevertheless, the same pattern of relative toxicity was observed, with Cry1Ac being the most active toxin against both insects, as previously described (Bel et al., 2017; Bernardi et al., 2012).These observations support Cry2Ac for pyramiding with Cry1Ac or Cry1Fa in soybeans to manage A. gemmatalis and P. includens more effectively and delay evolution of resistance, as these toxins do not share binding sites with Cry1 toxins in lepidopteran pests (Jakka et al., 2015).

CHAPTER 4

Analysis of Mode of Action of Cry1Ac, Cry2Ac and Hybrid H1.2Ac by Binding Affinity to Receptors in Chrysodeixes includens and Anticarsia gemmatalis

Abstract

Cry1Ac is one of the most widely used 3d-Cry proteins. Domain-II and Domain-III recognize different insect gut proteins and determine the target specificity. We constructed a hybrid protein with domain-I and II of Cry1Ac and domain-III of Cry2Ac7 to check the effect of domain substitution on specificity and toxicity of Cry1Ac. Hybrid (H1.2Ac) activated toxin was bioassayed against A. gemmatalis and C. includens and was found to be highly toxic to A. gemmatalis but nontoxic to C. includens contrary to the parent protein. Binding assays further emphasizes the importance of Cry1Ac domain-III for its activity against C. includens.

Introduction

The biggest group of Cry families is 3d-Cry toxin family (Bravo et al., 2015b) and is structurally best characterized. Domain I of 3d-toxins is composed of seven amphipathic alpha helices found to be involved in toxin oligomerization, membrane insertion and pore formation. Domain II has highly diverse organization; it is like a beta prism, composed of three antiparallel beta sheets with exposed loop regions. It contributes in specific toxicity and high-affinity binding (Schnepf et al., 1998). Domain III consists of two beta sheets and its organization among other Cry proteins is not very diverse (Grochulski et al., 1995; Morse et al., 2001). The structure of these domains is shown in Fig. 21.

Fig. 21 . Structure of three domains of cry1Aa (taken from Bravo et al., 2005).

Domain II and domain III recognize different insect gut proteins and determine the target specificity. Bt crystal solubilization, formation of activated toxin and binding to receptors on insect BBMVs are the key steps which result in pore formation in the membranes and collapse of the gut epithelium (Jurat-Fuentes and Crickmore, 2016). High level of pest resistance to Cry toxins is most commonly due to alteration in the binding mechanism to the specific insect receptor (Jurat-Fuentes and Adang, 2001; Pardo-López et al., 2013; Tabashnik et al., 2013).

The efficacy of Bt toxins has been reduced gradually for the evolution of resistance by the target pest insects against the Bt Cry toxins either in the fields or in the laboratories. In several cases, the emergence of resistance to Bt Cry toxins is due to the changes in insect receptors (Ferré et al., 1995). In order to understand mechanism of resistance and designing different strategies to modify Cry toxins, it is important to identify Cry toxin receptor proteins and understand mode of action of Bt toxins. Construction of hybrid toxins with domain substitution not only results in novel toxins with higher toxicities and wider target range than the parent toxins but also is a good tool to determine the domain specificity and the importance of conserved amino acid sequences (Bosch et al., 1994; Lee et al., 1999; Schnepf et al., 1990). Cry1Aa domain III residues substitution by Cry1Ac residues produced a toxin having 300 times higher toxicity for Heliothis virescens (Caramori et al., 1991). The toxicity of Cry1Ab toxin enhanced six fold when its domain III was replaced by domain III of Cry1C (de Maagd et al., 51

2000). The coleopteran active Cry3A toxin was modified by exchanging its domain III with that of Cry1A lepidopteran active toxin to produce eCry3.1Ab active toxin (Walters et al., 2010) and was successful shuffling within two distantly related Cry toxins.

The involvement of specific domains in specific receptor binding is confirmed by alteration in the binding pattern on ligand blots of the new recombinant toxin formed by domain substitution (de Maagd et al., 1996). The presence of a unique N-acetylgalactosamine (GalNAc) binding cavity in domain III of Cry1Ac is evident by many experiments (Derbyshire et al., 2001; Li et al., 2001) which recognises GalNAc residues in receptor proteins (Burton et al., 1999). Aminopeptidase-N (APN) isoforms were identified as Cry1Ac receptors (Garczynski et al., 1991; Gill et al., 1995; Hua et al., 1998) and the binding of Cry1Ac toxin to APN inhibition by the presence of GalNAc is evident from previous research (Knowles et al., 1991; Masson et al., 1995). Many alkaline phosphatases (ALPs) have also been identified as Cry toxin receptors which in case of H. virescence is a GPI-anchored membrane 68 kDa glycoprotein (Jurat-Fuentes and Adang, 2004) and for the presence of a carbohydrate moity it is similar to APN interaction with Cry1Ac (reviewed in Pigott and Ellar, 2007). It was demonstrated by Wei et al. (2016) that Helicoverpa zea APN1 is a functional receptor for Cry1Ac but not for Cry2Ab which indicates that the receptors for different Cry types in the same insect may differ accordingly.

Domain III swapping has been used extensively in many substitution experiments and has been proved important in implicating toxin binding and host specificity (reviewed in Deist et al., 2014). We constructed a hybrid protein with domain I and II of Cry1Ac and domain III of Cry2Ac7 in order to check the effect of domain swapping on the target range and extent of toxicity of Cry1Ac. Cry1Ac and Cry2Ac7 active toxins have significant activity against the two pests tested. We substituted domain-III of the more active toxin Cry1Ac with lesser toxic Cry2Ac7 domain III to test that whether the lesser toxic protein segment could enhance the toxicity of the more active parent toxin or impart a negative effect on its toxicity and affinity of the target insects. We tested the activity of Hybrid toxin on two insects C. includens and A. gemmatalis which are susceptible for both Cry1Ac and Cry2Ac protein toxins. The bioassay results of H1.2Ac along with ligand blot binding assays and binding inhibition experiment in the presence of GalNAc and GlcNAc demonstrated that the presence of Cry1Ac domain-III is critical for toxicity against C. includens but not for A. gemmatalis. 52

Materials and Methods

Cloning of hybrid H1.2Ac

Selection of gene fragments The SBS-Bt isolates tncry1Ac (GenBank accession no. EU250285) and cry2Ac7 (accession no. CAL18690.1) gene sequences were translated by ExPASy translate tool (Gasteiger et al., 2003) to get the amino acid sequence of these Cry proteins. The deduced amino acid residues sequences were scanned using EMBL-EBI InterProScan tool (Goujon et al., 2010; Quevillon et al., 2005) to locate the domains of these Cry toxins. The InterProScan tool was used to detect any signature sequences like specific folds through alignment to other structurally known Cry toxins. The selected regions of the tnCry1Ac and Cry2Ac7 proteins were reverse-translated to nucleotide sequences by using online Expasy translate tool. After joining of the selected nucleotides from the two Cry genes the sequence of this proposed hybrid DNA was analyzed on InterProScan tool to make sure the presence of all the three domains of 3d-Cry toxins important for their activity. The schematic construction of the fusion protein is as shown in Fig. 22.

Fig. 22. Schematic representation for the construction of hybrid protein H1.2Ac. The bars represent the polypeptides; different patterns indicate different domain segments of each protein while the numbers indicate the amino acid residues forming distinct domains of the proteins. 53

Oligonucleotide primer designing Oligonucleotide primers were designed using online tools like NEB cutter V2.0 (Vincze et al., 2003), OligoCalc (Kibbe, 2007) and Primer 3.0 (Untergasser et al., 2012). Selected restriction sites were introduced in the primers for the correct orientation and ligation of the gene segments. NdeI restriction enzyme recognition site was introduced in the forward primer 1AcFD1. In the reverse primer 1AcRD2 and forward primer 2AcFD3 SalI restriction enzyme recognition site was introduced. The reverse primer 2AcRD3 was constructed with a XhoI restriction enzyme recognition site and a stop codon were introduces in it.

The primers were named according to the type of the primer and on the name of the gene segments to be amplified with them. The primer sequences with underlined restriction sites and their melting temperature are given in Table III.

Table III. Primers used for PCR amplification of selected domains of Cry toxins with restriction sites underlined.

PCR amplification of selected gene segments Domain I and II encoding DNA nucleotides segment of tncry1Ac and domain III of cry2Ac7 were amplified by the logarithmic procedure (PCR) in thermo cycler (Applied Biosystems 2720). The gene fragment containing the first and second domains of template tncry1Ac DNA was amplified by primers 1AcFD1 and 1AcRD2. The third domain containing DNA segment of template cry2Ac7 DNA was amplified by using the primers 2AcFD3 and 2AcRD3. PCR reaction mixtures contained 1 ng of template plasmid DNA, 1X Taq buffer with

(NH4)2SO4, 2.0 mM MgCl2, 0.25 mM dNTP mix, 100 pmol of each primer, 1 U of Taq DNA Polymerase (Fermentas, ThermoFisher). The reaction mixture was made up to 50 µl with sterile 54 nuclease free water. The PCR reaction conditions were set as: initial denaturation at 95ºC for 5 min followed by 35 cycles denaturation at 94ºC for 90 s, annealing at 67ºC (for tncry1Ac template DNA) and 68ºC (for cry2Ac7 template DNA) for 45 s, extension at 72ºC for 60 s and then a 10 min final extension at 72 ºC.

Amplified PCR products were named according to the name of amplified gene segments as DIDIIAC for the first two domains of tnCry1Ac and DII2Ac for the third domain of Cry2Ac7 proteins. PCR products were visualized by running them on 1 % agarose gel and the correct sized DNA bands were cut under UV light. The cut bands of DNA were weighed in pre-weighed microfuge tubes and cleaned from agarose gel according to the protocols described in chapter 2.

TA cloning of the amplified gene segments The PCR amplified DNA segments had single 3´- dA overhangs at both ends. These 3´- dA overhangs were added by the terminal transferase activity of the Taq DNA Polymerase enzyme. The 3´- dA overhangs are utilized to ligate with the linearized cloning vector pTZ57R/T (InsTAcloneTM PCR Cloning Kit, Thermo Scientific) with ddT-tail (Fig. 23A).

Fig. 23. TA cloning vector and pictorial procedure. A, Map of pTZ57R/T TA cloning vector showing the 3´-ddT overhangs and multiple restriction sites; B, Pictorial representation of TA cloning procedure (Thermo Scientific).

The agarose purified DNA segments were ligated to pTZ57R/T linearized vector with optimal 3:1 ratio of insert to vector. Contents and concentrations of the ligation reactions were according to the table IV.

Table IV. DNA insert and pTZ57R/T vector ligation reaction components

Components DIDIIAc (µl) DIII2Ac (µl)

Ligation buffer (5X) 5 5

Linearized vector 3 3

PCR DNA product 4 2

T4 DNA ligase (10U/µl) 1 1

Nuclease free water 12 14

Total volume 25 25

The ligation reactions were incubated for 1 h in water bath with set temperature of 22 ºC or at 16 ºC for overnight. The sequential steps for the cloninig procedure were performed as shown in Fig. 23B. Briefly, 200 µl of second day old competent cells were transformed with the ligation mixtures according to the protocol described in chapter 2. Transformed bacterial 56 cultures were spreaded on LB agar plates suplemented with Ampicilli (100 µg/ml), X-gal (2%) and IPTG (0.1 mM). The plates were screened for the presence of white bacterial colonies after 16 h incubation at 37 ºC.

White colonies were picked and inoculated in ampicillin suplemented LB media and grown overnight. The overnight bacterial cultures were used to isolate DNA plasmids as described previously. The plasmid DNA of the two clones were named on the name of the inserts as DIDIIAcpTZ and DIII2AcpTZ. The DIDIIACpTZ plasmid DNA was restriction digested with NdeI and SalI restriction enzymes. DIII2AcpTZ plasmid DNA was digested with SalI and XhoI restriction enzymes. All the enzymes used for digestion were conventional enzymes (Fermentas, Thermo Fisher) and the reactions were incubated at optimal 37ºC for 16 hours. The contents of the double reaction mixtures and their concentration are shown in Table V.

Table V. Reaction components of double restriction digestion of DNA plasmids

Components DIDIIAc (µl) DIII2Ac (µl)

10X Tango buffer 10 10

Each restriction enzyme 4 4

Plasmid DNA 20 15

Nuclease free water 16 21

Total volume 50 50

The restriction reactions were run on 1% agarose gel to confirm the presence of inserts and to isolate and purify the restricted DNA bands for further cloning.

Cloning of gene segments in pET28a(+) to construct H1.2Ac Expression vector pET28a(+) was chosen to clone the hybrid toxin to get good expression level of overexpressed insoluble protein. The plasmid DNA of pET vector was double digested with NdeI and SalI according to the digestion reaction for DIDIIAc in Table III. The linearized vector was purified from the agarose gel and both the insert and vector were quantified by running the cleaned DNA products on agarose gel along with equal volume of the DNA ladder marker. The ligation reaction was set as given in Table II and incubated at 22 57

ºC for 16 h. The DH5α competent cells were transformed with the ligation product and plated on kanamycin (50 µg/ml) supplemented LB agar plates and incubated at 37 ºC for 16 h. From the inoculated culture media with the selected colony plasmid DNA of the clone DIDIIACp28 was prepared.

For confirmation of DIDIIAc insert in DIDIIAcp28 plasmid double restriction of the plasmid was performed. For double restriction of the recombinant plasmid and DIII2AcpTZ, restriction enzymes SalI and XhoI were selected. The reaction mixture contained 2 µl of SalI and 4 µl of XhoI enzymes. The other reaction ingredients were added in the same proportions as given in Table V. This digestion reaction generated linearized vector and the insert with sticky ends. These restricted fragments were purified from agarose gel as described previously. The ligation of DIII2Ac (SalI-XhoI digested) into linearised DIDIIAcp28 (SalI-XhoI digested) vector resulted in DIDIIAc.DIII2Acp28 plasmid. Twenty microliter of the ligation mixture was used for the transformation of DH5α (E.coli) competent cells. The transformed bacterial culture was plated on LB agar plates supplemented with kanamycin. The colonies were picked and inoculated in LB media with kanamycin for plasmid DNA isolation.

For the confirmation of proper ligation of the second insert into DIDIIAcp28, the colonies were checked by the PCR amplification of both the inerts in new clone. The new construct was named as H1.2Ac and was single restriction digested with SalI restriction enzyme for confirmation of clone. For further confirmation, H1.2Ac was also double restriction digested as described above with the same restriction enzymes used for their ligation together. The hybrid construct H1.2Ac was also restricted with NdeI and XhoI to generate a band of almost 1.8 kb size which comprises of both the DNA segments joint together. The orientation of cloned segments of DNA and their presence was also confirmed by DNA sequencing on Beckman Coulter CEQ8000 Genetic Analyzer at School of Biological Sciences, Pakistan by chain termination method (Sanger et al., 1977).

Over-expression and purification of recombinant protoxins

Over-expression of hybrid protein Recombinant DNA plasmid of H1.2Ac was used to transform E. coli Bl21 CodonPlus (DE3)-RIPL chemically competent cells. 500ml LB media supplemented with Kanamycin (50µg/ml final concentration) was used for the expression of the recombinant Hybrid proteins.

Hybrid protein over-expression was induced with 0.25 mM IPTG at cell OD600 0.8 and 58 incubated at 37 °C for 6 h. The cells were harvested by centrifugation at 5,000 x g for 15 min in Avanti centrifuge. The cell pellet was washed with Tris-Cl buffer pH 8.8 to get rid of any culture media and resuspended in the same buffer. The cells were sonicated for 20 cycles 5 sec on and 55 sec off at 70% amplitude. A fraction from the whole cell lysate was saved for analysis and then the sonicated suspension was centrifuged at 8,000 x g for 20 min to separate the soluble and insoluble fractions of cell lysate. All these fractions were run on 10% SDS-PAGE gel and after coomassie brilliant blue staining gel was analyzed for the protein of interest.

Refolding of H1.2Ac protein Inclusion bodies were harvested and washed with Tris-Cl buffer containig 0.2% Triton- X and 0.5 M NaCl, pH 8.8. The wash step was repeated twice and then centrifuged at 8,000 x g for 15 min. Washed inclusion bodies were resuspended in the solubilization buffer (20 mM Tris-Cl, 0.5 mM NaCl, 8 M urea and 10 mM imidazole, pH 9) and incubated at room temperature for 1 h for complete denaturation of the protein. The denatured protein solution was separated from the insoluble contaminants by centrifuging at 15,000 x g for 20 min. The solubilized proteins were filtered through 0.45 µm microfilter prior to use further. The washed and settled resin was equilibrated with binding buffer (20mM Tris-cl, 0.5M NaCl pH 9). The settled pre-equilibrated resin was incubated with 10 ml of solubilized protein at room temperature on gentle rotation on a tube rotator. The column was clamped in vertical position and unbound proteins were washed by 5 CV of denaturing buffer. The bound proteins were on- column refolded by washing with 5 CV of step gradient of 6 M, 4 M, and 2 M urea in binding buffer. After the final wash with 10 CV of binding buffer, refolded protein was eluted first with 100 mM and then with 250 mM imidazole in 20 mM Tris-cl, 20mM NaCl pH 9.0 buffer. The fractions were analyzed on 10% SDS-PAGE. The positive fractions containing the refolded soluble protein were pooled, quantified and stored at -80 ºC.

Trypsin Activation and purification of the recombinant toxins

tnCry1Ac, Cry2Ac7 proteins were purified as described in chapter 2. All the three proteins purified by affinity chromatography were pooled and quantified using Qubit Protein Assay kit and measuring in Qubit fluorometer (Life Technologies). These proteins were trypsin activated as described in chapter 2. Protoxin H1.2Ac was activated by incubating the proteins with optimal concentration of freshly prepared trypsin (TPCK-treated) for particular protein at 37 ºC. Protoxins H1.2Ac was digested with 5:1 (w/w) protein to trypsin ratio for 75 min. The trypsin digestion reaction was stopped by spinning the tubes at 8,000 ×g for 10 min at 4 ºC and 59 keeping the decants in new tubes on ice. Tryptic digestion of protoxin was confirmed by running fractions on 10% SDS-PAGE gel. In order to remove trypsin and buffer exchange the activated protein was dialyzed against 20 mM Na2CO3 pH 9.8 buffer overnight. The activated proteins were further purified through anion exchange chromatography on FPLC (ÄKTA pure, GE Healthcare Life Sciences). The activated toxins were separately loaded on Hi Trap Q HP-

1ml column (GE Healthcare), pre-equilibrated by binding buffer (20 mM Na2CO3 pH 9.8). After washing unbound proteins with 5 column volume binding buffer, protein was eluted at 100% elution buffer (binding buffer + 1M NaCl pH 9.8) in a linear gradient of 100% elution buffer. The peek fractions from the anion exchange purification process for all the proteins were collected and analyzed on 1% SDS-PAGE gel. The best purified fractions were pooled, quantified and then stored at -80ºC for further use.

Insect bioassays

Eggs of A. gemmatalis and C. includens were purchased from Benzon Research (Carlisle, PA) and neonate larvae were used for bioassays against Cry1Ac, Cry2Ac7 and H1.2Ac active toxins. The procedure for bioassays is same for all the toxins as described below. Freshly prepared General-Purpose Lepidoptera diet (BioServ) was solidified in 128 wells trays in a laminar air flow cabinet. The solidified diet was surface contaminated with 75 µl test solution per well. 50 mM Na2CO3 pH 9.8 buffer was used as control solution and to dilute the test proteins. After the adsorption of the solution at bench top the bioassay trays were placed in laminar air flow cabinet to make the diet surface moisture free. Insect eggs were kept in the incubator at 27(±2) °C, 75 % humidity with 12:12 h light:dark photoperiod until hatching. One day old actively moving larvae were placed on the surface of diet (1 larva/well) with fine camel hair brush. The wells were carefully covered with adhesive porous plastic sheets so that each well contained larva without sticking to the sticky edges of the cover. Bioassay trays were placed in incubator with 70-80 % humidity and 14: 10 h light: dark photoperiod at 27(±1) °C. Sixteen larvae per concentration were assayed per replicate and bioassays were performed in duplicates and replicated three times. Plates were checked daily for the appearance of any fungal growth and to count the dead larvae. Mortality was scored after 7 days from the start of bioassays and Abbott’s formula (Abbott, 1925) was applied to correct for natural deaths. For the calculation of LC50s (50% lethal concentrations) of the toxins POLO software (LeOra- Software, 2005) was used for probit analysis. 60

Preparation of BBMVs

Untreated larvae of A. gemmatalis and C. includens were reared under the same incubating conditions as described above for bioassays until fifth instar. Healthy fifth instar larvae of both insects were anesthetized on ice and dissected to remove midguts which were immediately frozen on dry ice and stored at -80°C for less than a week. Wolfersberg et al. (1987) differential centrifugation method with some modifications (Jurat-Fuentes et al., 2002) was used to prepare BBMVs. BBMV homogenate proteins were analyzed on 10% SDS-PAGE gel and quantified with qubit fluorometer as described above and were stored in aliquots at - 80°C for further use.

Binding assays with radiolabeled proteins

Radiolabeling of Cry1Ac and Hybrid protein Purified activated H1.2ac and the parent activated Cry1Ac toxins were radiolabeled1 using chloramine T method (Hunter) as described elsewhere (Gouffon et al., 2011). Briefly, two PD10 desalting columns (GE healthcare) were equilibrated by passing 25 mL column buffer (20 mM Tris-HCl, pH 8.6 with 150 mM NaCl and 0.1% BSA) from the columns. Activated purified H1.2Ac and Cry1Ac proteins (1 µg each) were mixed in 59 µl labelling buffer (20 mM Tris-HCl, pH 8.6 and 150 mM NaCl) to which 5 µl of 0.5 mCi Na125I (Perkin Elmer, Boston, MA) was added and mixed. To initiate the iodination of proteins 32 µl of chloramine T (18 mM) in labelling buffer was added to it and the final reaction (96 µl) was kept for 1 minute at room temperature. Thirty-two microliter of 1M NaI and 24 µl of 23 mM

Na2S2O5 in labelling buffer was added in the reaction mixture to stop the reaction. Free iodine from the labelled protein solutions was removed by loading the proteins onto preequilibrated PD10 desalting columns. The bound contents were eluted with 10 ml column buffer and eluted proteins were collected in total 20 fractions of 0.5 ml each. The radioiodination of proteins was confirmed by counting the radioactivity of eluted fractions in a Wizard2 gamma counter (Perkin Elmer) and by autoradiography of the dried gels (10% SDS-PAGE) on which positive radiolabeled fractions were run. The impressions of the radiations were taken on X-Ray film by exposing it to the dried gels for 7 days at -80 °C. To analyze the radiographic image, the radiograph was developed it in the dark room according to the protocol for developing X-Ray

1 Handling of radioisotope, radiolabeling and gamma counting was performed by Dr. Juan Luis Jurat- Fuentes. 61 films. The concentrations of the radiolabeled toxins were estimated and the positive fractions (1.5 ml) were stored at 4 °C for use within one month of radiolabeling.

125I-H1.2Ac and 125I-Cry1Ac binding assays with BBMVs of A. gemmatalis and C. includens The optimal concentration of BBMV of A. gemmatalis and C. includnes for use in competition experiments was determined by binding experiments. For this series of increasing concentration of BBMVs (0, 10, 20, 30, 40, 50 and 60 µg) were used keeping the ligand concentration (radiolabeled proteins) constant (1 nM). In prelabeled microfuge vials 1.0 nM 125I-H1.2Ac or 125I-Cry1Ac were incubated for 1 h at room temperature with six different concentrations of BBMVs of A. gemmatalis and C. includes in binding buffer (PBS, pH 7.5, 0.1% BSA) in final reaction volume of 0.1 ml. Pellets obtained after centrifuging (14,000 × g) these reactions for 15 min were washed with ice-cold binding buffer (1 ml) two times and radioactivity was measured in a Wizrad2 gamma counter. Nonspecific binding was determined by adding 500-fold of unlabeled toxin (500 nM) to the reaction mixture in the presence of radiolabeled toxin and proceeded as for radiolabeled toxin alone above. Specific bindings of 125I-H1.2Ac and 125I-Cry1Ac were determined by subtracting nonspecific bindings from total bindings and each experiment was repeated thrice with two replicas.

125I-H1.2Ac or 125I-Cry1Ac competition binding assays with BBMVs of A. gemmatalis Competition binding assays were performed using H1.2Ac or Cry1Ac as homologous (competitive protein is the same as the radiolabeled protein) and heterologous (competitive protein is different from the labeled protein) competitors. Cry2Ac7 was used as heterologous competitor for both 125I-H1.2Ac and 125I-Cry1Ac in the competition assays. In competition binding assays 20 µg of BBMVs of A. gemmatalis was incubated 1 nM 125I-H1.2Ac or 125I- Cry1Ac activated toxins in the presence of increasing concentrations of competitor (0, 1, 3, 5, 10, 30, 50, 100, 300, 500, 1000-fold of radiolabeled toxin) at room temperature in a final reaction volume of 0.1 ml in binding buffer and processed to count radioactivity as described above for binding assays. Percent bound radiolabeled toxin in presence of competitor was determined by taking the radiolabeled bound toxin count without competitor as 100%. Binding experiments were performed in duplicates and repeated twice. 62

Binding assays with biotin labeled proteins

Biotin labeling of Cry proteins Activated H1.2Ac, Cry1Ac and Cry2Ac7 were biotinylated using EZ-Link Sulfo-NHS- LC-Biotin (Thermo Scientific). Purified activated proteins were dialyzed at 4 °C against phosphate buffered saline (PBS) buffer (137 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4,

Na2HPO4, pH 7.4) for 6 h with exchange of old buffer with fresh buffer after every 2 h. The amount of biotin to be used for each protein was calculated according to the manufacturer’s instructions. A 30- fold molar excess of biotin reagent for all the proteins was selected. Ten millimolar solution of biotin reagent was prepared by adding 180 µl of ultrapure water to 1.0 mg of biotin reagent in a vial just prior to use. Twenty-five microliter of freshly prepared 10 mM biotin was added to each of 1 ml Cry1Ac (0.5 µg/µl) and H1.2Ac (0.5 µg/µl), whereas 27.27 µl of it was added to 1 ml Cry2Aac7 (0.5 µg/µl). The reactions were incubated for 2 h on ice after which the contents were injected into 0.5-3 ml Slide-A-Lyzers (ThermoFisher) with a molecular weight cut off 2,000 daltons (2K MWCO). The Slide-A-Lyzer cassette float buoys (ThermoFisher) were used to keep the cassettes floated in dialyzing buffer. Excess and hydrolyzed non-reacted biotin was removed from the biotin labelled proteins by dialyzing it against 50 mM Na2Co3, 50 mM NaCl pH 10.0 buffer in a total of 2 L buffer. The dialyzing buffer was replaced three times with fresher one after every 2 hours and after last exchange the reaction was allowed to complete overnight. Incorporation of biotin molecules to proteins was confirmed by running the labeled toxin on 10% SDS-PAGE gel and performing western blots according to the protocol described below. The biotinylated proteins were quantified, aliquoted and stored at -80 °C for ligand and western blots.

Ligand blots of biotinylated toxins Thirty microgram BBMV proteins of A. gemmatalis and C. includes were resuspended in 2X SDS-PAGE loading buffer to make the total volume 15 µl and boiled at 100 °C for 5 min. The samples and prestained marker were run on 10 % SDS-PAGE gels and were transferred onto PVDF membranes in the trans-blot cell (Biorad) according to manufacturer’s instructions at 60 V for 4 h. The membranes were blocked for 1 h with blocking buffer (PBS pH 7.4, 0.1% Tween-20, 3% BSA) on orbital shaker. Biotin labeled H1.2Ac (0.16 nM), Cry1Ac (0.5 nM), and Cry2Ac7 (0.50 nM) activated toxins were added in 10 ml blocking buffer containing the blocked membranes and were incubated for 1 h with shaking. The membranes were washed with washing buffer (PBS pH 7.4, 0.1% Tween-20, 0.1% BSA) 6 times with 10 63 min incubation each time on orbital shaker. In the new wash buffer (20 ml) the membranes were incubated with streptavidin-HRP conjugate (Thermo Scientific) at 1: 15,000 dilution for 1 h. The membranes were again washed 6 times with wash buffer and were treated on saran wrap with enhanced chemiluminescence (ECL; Amersham-pharmacia) reagents following the manufacturer’s instructions. The filters were exposed to X-ray films to get impressions in dark room. The films were developed with developer and rinsed with water followed by fixing in fixer and rinsing in water in red light. The films were dried and visualized for the blots of bound proteins.

Western blotting of biotinylated toxins using GalNAc and GlcNAc competitors BBMVs (30 µg) of A. gemmatalis and C. includens were incubated with biotin labeled Cry1Ac (0.3 µg) and H1.2Ac (0.1 µg) in the presence of 100 mM GalNAc or GlcNAc separately for 1 h at room temperature in binding buffer (PBS pH 7.4, 0.1% BSA) to final total volume of 100 µl in microfuge tubes. The microfuge tubes were centrifuged at 10,000 ×g and supernatants were discarded. The pellets were washed with 1 ml ice-cold binding buffer and centrifuged. The washing step was repeated one more time and the final pallets were resuspended in 2x SDS-PAGE loading buffer and boiled at 100 °C for 5 min and transferred to PVDF filters from the electrophoresed gels. The filters were blocked with blocking buffer for 1 h and then incubated for 1 h with streptavidin-HRP conjugate (Thermo Scientific) at 1: 15,000 dilution for 1 h. The electroblotted filters were treated further as described for the ligand blots with radiolabeled toxins. The X-ray films were exposed and developed under red light in dark room and visualized it.

Results

Construction of hybrid toxin H1.2Ac

Identification of domain segments to construct H1.2Ac The DNA translation of tncry1Ac, cry2Ac7 by ExPASy translate tool generated a sequence of 610 and 623 amino acid residues polypeptides, respectively. The deduced protein sequences on scan with InterProScan tool detected three domains in tnCry1Ac and Cry2Ac7 proteins. The tool also marked the position of these domains in the protein. Domain I and II of tnCry1Ac spanned from 3-251 and 256-462 amino acid residues respectively and its third 64 domain extended from 454 amino acid residues to 609 amino acid residues. In Cry2Ac7 the first two domains spanned from 8 to 471 amino acid residues, whereas the third domain was located from 485 to 623 amino acid residues. The DNA encoding the first two domains of tnCry1Ac and third domain of Cry2Ac7 were joined together. Scanning of the resultant recombinant DNA with InterProScan software showed the presence of all the three domains. In H1.2Ac, the N-terminal and the central domains spanned from 3 to 461 amino acid residues which was similar to that of tnCry1Ac. The third C-terminal domain of H1.2Ac was predicted to start from 453 and end at 602 amino acid residues. The software also searched for signature matches in the proteins as shown in Fig. 24. The use of this software to predict domain location confirmed the presence of all the three domains important for the activity of Cry toxins in hybrid protein. However, Cry2Ac7 protein sequence has not been reported to contain galactose binding site signature in its third domain contrary to the signature match prediction by this software. The software prediction of presence of galactose binding site like signature in H1.2Ac is also suspected to be absent because of its absence in the parent Cry2Ac7 third domain. This might be because the software calculates the signature matches against the protein family members database’s signatures.

Fig. 24. Domain architecture of cry proteins showing three domains and signature matches. Domains are represented by different color bars and constituting amino acid numbers above bars. The number below the bars show the length of the polypeptides. 65

PCR amplification of selected DNA with designed primers The selected gene segment DIDIIAc was PCR amplified from tnCry1Acp28 plasmid DNA with forward and reverse primers designed with care not to form dimers and self- annealing sites. No stop codon was introduced in the reverse primer of DIDIIAc so the amplified DNA did not contain any stop codon. The forward and reverse primers used to amplify DIII2Ac from Cry2Ac7p22 plasmid DNA added specific restriction enzymes recognition sites in its N-terminus and C-terminus. The reverse primer to amplify this DNA segment contained a stop codon so the amplified DNA encodes a stop codon.

Fig. 25. PCR amplified clearly defined DNA bands stained with EtBr on 1% agarose gel. Lane 1, DIII2Ac 400 bp DNA band; lane 2, DIDIIAc 1400 bp DNA band; M, DNA size marker (GeneRuler 1 kb DNA Ladder, ThermoScientific, SM0311), reference bands bp are shown on left of panel.

The amplified DNA segment DIDIIAc had added NdeI restriction site at its N-terminus and SalI restriction site at its C-terminus and on 1% agarose gel it positioned below 1500 bp showing its size of almost 1406 bp (Fig. 25). DIII2Ac amplified product band was visualized at 400 bp position based on size compared to molecular size marker and have additional restriction sites of SalI at N-terminus and XhoI at C-terminus. 66

TA cloning: screening and confirmation Out of many blue and some white colonies appeared on LB agar plates spreaded with the transformed bacterial cultures only the white colonies were picked and checked for the presence of insert. Double restriction digestion of the isolated plasmid DNAs confirmed the presence of cloned inserts in vector as shown in the Fig. 26.

Fig. 26. Restriction analysis of DIDIIAcpTZ and DIII2AcpTZ. Lane 1, double restricted recombinant plasmid DIII2AcpTz; Lane 2, double restricted recombinant plasmid DIDIIAcpTZ; M, DNA size marker (GeneRuler DNA ladder mix, ThermoScientific # SM0331), reference bands base pairs are shown on left of panel.

The NdeI and SalI restricted plasmid DIDIIAcpTZ generated two DNA bands one of 2.8 kb of linearized pTZ vector and the other of 1.4 kb insert band (Fig. 26, lane 1). Double restriction of DIII2AcpTZ recombinant plasmid with SalI and XhoI resulted in a band of vector (2.8 kb) and a 0.4 kb (400 bp) band of insert as shown in 1% agarose gel image (Fig. 26, lane 2).

Construction of H1.2Ac: Fusion of DNA segments in pET expression vector The restriction of DIDIIAcpTZ plasmid DNA with NdeI and SalI restriction enzymes added 5´ and 3´ overhangs to the DNA molecule (Fig. 26, lane 1). The double restriction of pET28a(+) with NdeI and SalI restriction enzymes also added 5´ and 3´ overhangs to it (Fig. 27A, lane 1) needed for the ligation of vector and the insert molecules. 67

Fig. 27. 1% agarose gels showing different step of DIDIIAc cloninig in pET28a(+). (A) double restricted pET28a(+) in lane 1; (B) amplified DIDIIAc DNA band (lane 1) from DIDIIAcp28 plasmid (lane 2); (C) double restricted DIDIIAcp28 plasmid in lane 1; M, DNA size marker (GeneRuler 1 kb DNA Ladder, ThermoScientific, SM0311); reference bands bp are shown on left of panel. The restricted and amplified products are shown in kilo bases (kb).

The transformed E. cloi (DH5α) colonies on kanamycin supplemented LB agar plates were positive for the presence of DIDIIAc fragment in pET checked through PCR amplification (Fig. 27B, lane 1). The DIDIIAcp28 plasmid (Fig. 27B, lane 2) double restricted with SalI and XhoI not only confirmed the presence of insert in it but also generated sticky ends on the DNA molecule required for ligation to DIII2Ac segment DNA restricted with the same enzymes.

The bacterial colonies obtained after the transformation of DH5α cells with the ligation product of DIDIIAcp28 and DIII2Ac confirmed the presence of both the inserts in the expression vector (Fig. 28, lanes 3, 4). Single restriction digestion of the recombinant plasmid with SalI resulted in 7.1 kb DNA molecule (Fig. 28, lane 1). Double digestion of H1.2Ac with NdeI and SalI resulted in 5.7 kb DIII2Acp28 and 1.4 kb DIDIIAc DNA molecules (Fig. 28, lane 5). The double restriction of H1.2Ac with SalI and XhoI cleaved it into 7.1 kb DIDIIAcp28 and 0.4 kb DIII2Ac DNA molecules (Fig. 28, lane 2).

Fig. 28. PCR amplified and restricted fragments of H1.2Ac construct ran on 1% agarose gel. Lane 1, single restricted H1.2Ac recombinant plasmid; lanes 2 and 5, double restricted H1.2Ac recombinant plasmid; lanes 3 and 4, PCR amplified DNA segments from H1.2Ac template DNA; lane 6, recombinant DNA plasmid H1.2Ac; M, DNA size marker (GeneRuler 1 kb DNA Ladder, Thermo Scientific, SM0311), reference bands bp are shown on left of panel. The restricted and amplified products are shown in kilo bases (kb).

Successful cloning of fused gene segments in pET28(+) were further confirmed by double digestion of the recombinant H1.2Ac plasmid NdeI and XhoI which restricted the plasmid into two fragments. The DNA fragment of linearized vector band electrophoresed at 5.3 kb size position and the other (of fused insert) at 1.8 kb size position (Fig. 29, lane 1).

Fig. 29. Confirmation of fused inserts in pET28a(+). Lane 1, double restricted H1.2Ac recombinant DNA plasmid ran on 1% agarose gel; M, DNA size marker (GeneRuler DNA ladder mix, ThermoScientific # SM0331), reference bands base pairs are shown on left of panel. Kb, size of the restricted vector and fused insert in H1.2Ac. 69

DNA sequencing confirmed the correct orientation and sequence of both the inserts (1809 bp) in H1.2Ac recombinant DNA plasmid clone.

Over-expression and purification of H1.2AC

The H1.2Ac protoxin was overexpressed from E. cloi BL21-CodonPlus-(DE3)-RIPL harbouring DIDIIAc.DIII2Ac-pET28 upon induction with IPTG. The SDS-PAGE (10 %) showed the band of 68 kDa corresponding to the size of H1.2Ac predicted protein. Coomassie blue stained gel showed the protein in total cell protein and in insoluble fraction of E. cloi expressed proteins. H1.2Ac was expressed at the level of 28 % of the total cell proteins of E. cloi proteins. No H1.2Ac protein was detected in the soluble fraction of the cell lysate after careful separation of the soluble and insoluble fractions of E. cloi proteins (Fig. 30).

Fig. 30. 10% SDS-PAGE gel showing over-expression of H1.2Ac protein. Lane 1, uninduced cell lysate; lane 2, induced cell lysate; lane 3, soluble fraction of cell lysate; lane 4, insoluble fraction of cell lysate. MWM, protein molecular weight marker (BenchMark protein ladder 10747, Thermo Sceintific), reference band sizes in kDa at extreme left.

His-tag purification and trypsin activation of H1.2Ac

The added 6X-his tag to the N-terminus of the protein due to the chosen vector pET28a(+) made it easier to refold the misfolded protein through affinity chromatography. The 70 protein was eluted at 250 mM imidazole concentration. The eluted protein was much pure and still contained His tag on it but was very dilute. The use of His-tag for purification of protein allowed to refold the protein attached to Ni-resin without employing many steps of dialysis for many hours. On column refolded protein (68-kDa) on trypsin activation yielded a trypsin resistant core of 60 kDa (Fig. 31).

Fig. 31. Coomassie stained SDS-PAGE gel showing the his-tag purified and trypsin activated toxin. Lane 1, his tag purified H1.2Ac; lane 2, trypsin resistant H1.2Ac core protein; M, protein molecular weight marker (PageRuler 26616, Thermo Scientific) shown by values (kDa) to the extreme left of panel.

BBMV homogenate analysis

BBMV homogenate ran on the SDS-PAGE gel showed clear protein bands (Fig. 32).

Fig. 32. Early fifth instar larvae BBMVs homogenate of A. gemmatalis and C. includens resolved on 10% SDS-PAGE gel. Ag, A. gemmatalis; Ci, C. includens. 71

Anion exchange purification and labelling of protein

The trypsin activated protein was further purified from the trypsin and other contaminants with a Hi-trap Q Hp 1ml column on FPLC. The sample protein purified by affinity chromatography was dilute and it became more dilute after dialysis on trypsin activation of the protoxin. Maximum binding of the sample protein to the column was achieved by passing the sample through the column twice. The bound protein fractions were eluted at 100% elution buffer (Fig. 33).

Fig. 33. Chromatogram of anion exchange chromatography purification of H1.2Ac toxin on FPLC.

The four fractions were kept separate according to the concentration and purity level. More dilute eluted samples found good for bioassays and concentrated purified samples were good to label with biotin and radioiodine. Only small quantities of purified protein were used to analyze on SDS-PAGE in accordance with the amounts loaded for the analysis of labelled protein samples. The purified activated toxin and iodine labeled samples stored at -80ºC in aliquots saved protein from being precipitated when stored for long time at 4 ºC. Biotin labeled protein band was detected slightly above 60-kDa due to incorporation of biotin molecules in the protein (Fig. 34, lane 2). The western blot of unlabeled and labeled toxin showed no blot 72 for unlabeled and a distinguishable blot of labeled toxin (Fig. 34, lanes 3 and 4). The radiolabeled H1.2Ac toxin was not precipitated on radiolabeling and a big blot was analyzed on radiogram (Fig. 34, lane 5).

Fig. 34. Biotin-labeling and iodination of H1.2Ac active toxin on SDS-PAGE and western blots. Lane 1, SDS-PAGE analysis showing anion exchange purified protein through FPLC; lane 2, biotin labeled protein on SDS-PAGE; lanes 3 and 4, western blot of H1.2Ac and biotin-H1.2Ac respectively; lane 5, radiolabeled H1.2Ac protein bloted on western blot; M, protein molecular weight markers shown in values (kDa) to the extreme left of panel (PageRuler 26616, Thermo Scientific).

Labeling of Cry1Ac and Cry2Ac7 activated proteins

Biotin labeling of Cry1Ac and Cry2Ac7 were confirmed by analyzing the SDS-PAGE gel. The biotin labeled protein of both the toxins was slightly higher due to the incorporation of biotin in these toxins (Fig. 35, lane 2; Fig. 36, lane 2). A blot at 60-kDa of biotinylated Cry1Ac was visualized after developing the exposed X-ray film but no blot of unlabeled protein was visible (Fig. 35, lane 3 and 4). On the radiolabeled toxin radiogram, a 60-kDa was also detected showing that the protein was not precipitated and was actively radiolabeled (Fig. 35, lane 5).

Fig. 35. Purification, biotinylation and iodination images of Cry1Ac toxin. Lane 1, SDS-PAGE analysis showing anion exchange purified Cry1Ac protein through FPLC; lane 2, biotin labeled protein on SDS-PAGE gel; lanes 3 and 4, western blot of Cry1Ac and biotin-Cry1Ac respectively; lane 5, radiolabeled Cry1Ac protein bloted on western blot; M, protein molecular weight markers shown in values (kDa) to the extreme left of panel (PageRuler 26616, Thermo Scientific).

Cry2Ac7 trypsin activated protein showed a clear blot on the developed film (Fig. 36, lane 4). The concentration of radiolabeled and biotin labeled proteins was good and the quantity was enough to use in further experiments. The biotin labeled toxins were used upto 3 months while stored in smaller aliquots at -80 ºC. The radiolabeled toxins were used within a month stored at 4 ºC.

Fig. 36. Purification, biotinylation images of Cry2Ac7 toxin. Lane 1, SDS-PAGE gel showing purified protein through FPLC; lane 2, biotin labeled protein on SDS-PAGE; lanes 3 and 4, western blot of Cry2Ac7 and biotin-Cry2Ac7 respectively; M, protein molecular weight markers shown in values (kDa) to the extreme left of panel (PageRuler 26616, Thermo Scientific). 74

Insecticidal activity of hybrid protein

Toxicity of hybrid H1.2Ac against A. gemmatalis and C. includens larvae were counted for mortality after 7 days of bioassay and only dead larvae were scored. No fungal growth was detected due to the sterilized buffers and drying of diet in air flow cabinet. H1.2Ac activated toxin was found highly active against A. gemmatalis but showed no toxicity to C. includens. It did not inhibit growth of C. includens larvae, even at the highest concentration (5 µg) tested. No significant number of larvae with stunted growth was observed. The insecticidal activity of hybrid toxin was compared with insecticidal activities of its parent Cry toxins Cry1Ac and Cry2Ac7 in Table VI.

Table VI. Toxicity of hybrid activated protein against neonates of A. gemmatalis and C. includens.

a Protein concentration in ng/cm2.

b LC50 values of Cry1Ac and Cry2Ac7 are referenced here for comparison from chapter 3, bioassays with the three proteins were performed simultaneously. c Not available, as no toxicity was detected.

Cry1Ac was having LC50 of 20 ng against A. gemmatalis but when its third domain was substituted with the same domain of Cry2Ac7, it became more toxic to it. The insecticidal activity of Cry1Ac against C. includens was lost when it became H1.2Ac. So, the bioassay results showed that H1.2Ac was the highly active toxin against velvet bean caterpillar in contrast to soybean looper for which it was detected nontoxic.

Binding assays with radiolabeled Cry toxins

Binding affinity of radiolabeled toxins H1.2Ac and Cry1Ac radiolabeled toxins were found active for binding experiments however, some non-specific binding was also detected. Nonspecific binding was calculated for 75 both 125I-H1.2Ac or 125I-Cry1Ac by using 500-fold excess unlabeled toxin with radiolabeled toxin in binding experiment. Specific binding was determined by subtracting nonspecific binding from total binding (radiolabeled toxin only) to C. includens and A. gemmatalis BBMVs. Radiation count per minute (cpm) of Cry1Ac showed that it exhibited strong binding affinity to proteins on BBMVs of both C. includens (Fig. 37A) and A. gemmatalis (Fig. 37B). Hybrid toxin, 125I-H1.2Ac, also displayed high binding affinity for BBMV proteins of A. gemmatalis (Fig. 37B) whereas cpm of 125I-H1.2Ac was very low and for most of the replicates it was in negative range and a high non-specific binding was detected rather than the specific binding to C. includens BBMV proteins (Fig. 37A).

Fig. 37. Specific binding of 125I-H1.2Ac and 125I-Cry1Ac to different concentrations of BBMVs of C. includens (A) and A. gemmatalis (B). Specific binding of the labeled toxins to the increasing concentrations of BBMVs (-0 to 60- µg) of the two insects was calculated by subtracting the nonspecific binding in the presence of excess of unlabeled H1.2Ac or Cry1Ac from the total binding in the presence of 125I-H1.2Ac or 125I-Cry1Ac. The data points are the means of two independent experiments performed in duplicates for each. The error bars are the standard error of the mean calculate from two independent experiments. Symbol keys are given at the right bottom of the figures. 76

Competition assays with radiolabeled toxins Figures 37A and 37B show plots of binding competition assays of radiolabeled Cry1Ac and H1.2Ac in the presence of homologous or heterologous unlabeled competitors. Analysis of the plots displayed distinctly different behavior in binding to BBMV between the two radiolabeled proteins was observed. Binding curves in the presence of -0 to 100- fold nM of Cry1Ac competitor displaced the binding of 125I-Cry1Ac which showed the specific binding of radiolabeled Cry1Ac. Hybrid H1.2Ac as a competitor at its lower concentrations displaced binding of 125I-Cry1Ac more than Cry1Ac and at its higher concentration, it displaced all 125I- Cry1Ac more than unlabeled Cry1Ac. Cry2Ac7 as a competitor did not displace 125I-Cry1Ac binding to BBMV of A. gemmatalis to a noticeable extent. The binding of 125I-H1.2Ac was specifically displaced by H1.2Ac to much extent and was not displaced by Cry1Ac at lower concentrations but at its higher concentrations it partially displaced the binding of hybrid labeled toxin. The Cry2Ac7as a competitor exhibited a uniform lesser competition with labeled 125I-H1.2Ac but at its -100-fold concentration it competes partially for the binding sites.

Competition assays tested sharing of binding sites between radiolabeled Cry1Ac or H1.2Ac and unlabeled competitors in BBMV from A. gemmatalis and the binding kinetics were calculated by defining curves by KELL program (Ferguson, MO, USA). Binding of 125I- Cry1Ac was displaced by unlabeled Cry1Ac (Fig. 38A), allowing estimation of an apparent dissociation constant (Kd) of 0.33 ± 0.09 nM and a concentration of binding sites (Bmax) of 108 ± 30 nmol/mg of BBMV protein. In comparison, H1.2Ac displaced all 125I-Cry1Ac binding, with similar affinity (Kd = 0.20 ± 0.04 nM) but lower concentration of binding sites (Bmax = 64 ± 11 nmol/mg BBMV protein). No displacement of 125I-Cry1Ac binding was observed when using Cry2Ac7 as competitor (Fig. 38A).

In competition assays with 125I-H1.2Ac, we observed displacement of binding by unlabeled H1.2Ac, with a Kd of 0.20 ± 0.04 nM and a Bmax of 82 ± 16 nmol/mg of BBMV protein. When Cry1Ac or Cry2Ac7 were used as unlabeled competitors, partial (20%) displacement of 125I-H1.2Ac binding at the highest concentration of Cry1Ac or Cry2Ac7 competitors was detected (Fig. 38B).

Fig. 38. % Binding of radiolabeled toxin in the presence of homologous or heterologous competitors (A) Cry1Ac and (B) H1.2Ac. Symbol keys are given in the figure.

Ligand blots of biotinylated toxins

The electrotransferred BBMVs on PVDF filters were incubated with the Cry toxins for 1 h followed by washing for another 1 h. The filters were probed with streptavidin-avidin and the bands were visualized by exposing the films just after the ECL chemiluminescence treatment. Biotin labeled Cry1Ac, H1.2Ac and Cry2Ac7 might bind to denatured BBMVs protein specifically and non-specifically due to the destruction of important binding site or exposure of some non-binding sites under denaturing conditions (Daniel et al., 2002). Smear 78 blots were seen with the lower molecular mass proteins of BBMVs however, the blotted bands above 40 kDa were distinct.

Ligand blots with A. gemmatalis BBMVs The biotin-labeled Cry1Ac bond mostly to a ~90-kDa protein band in A. gemmatalis BBMV (Fig. 39, lane 1), but binding was also observed to a protein band of approximately 57 kDa. In the case of Cry2Ac7, the biotinylated toxin recognized the 90-kDa protein as well like Cy1Ac, but it also bound to protein bands of approximately 75-, 55-, 50-and <30-kDa protein bands in A. gemmatalis BBMV (Fig. 39, lane 2). In contrast, the H1.2Ac toxin recognized all protein bands recognized by Cry1Ac and Cry2Ac7, in addition to interactions with protein bands between 35- to 50-kDa (Fig. 39, lane 3). Week bands around 60- to 68-kDa for Cry2Ac7 and H1.2Ac were also detected (Fig. 39, lanes 2 and 3)

Fig. 39. Ligand blot of A. gemmatalis early fifth instar BBMVs with biotinylated proteins. Lane 1, Cry1Ac blot; lane 2, Cry2Ac7 blot; lane 3, Hybrid (H1.2Ac) blot. Molecular mass markers are shown at the left of the figure. Ligand blots with C. includens BBMVs The binding pattern of biotin labeled Cry1Ac and Cry2Ac7 found more distinct and toxin specific with C. includens BBMV proteins. For Cry1Ac blots at around 45-, 75-, 115- and 200-kDa were detected (Fig. 40, lane 1). While for Cry2Ac7 the blotted bands were 79 localized approximately around 50-, 75-, 95- and 165-kDa (Fig. 40, lane 2). Hybrid H1.2Ac labeled protein showed binding pattern almost similar to Cry1Ac except two prominent differences. H1.2Ac bound to the membrane protein at almost 165-kDa like Cry2Ac7 and no binding band to BBMV protein around 115 kDa was detected as visualized for Cry1Ac blot with C. includens (Fig. 40, lane 3).

Fig. 40. Ligand blot of C. includens early fifth instar BBMVs homogenate with biotinylated proteins. Lane 1, Cry1Ac blot; lane 2, Cry2Ac7 blot; lane 3, Hybrid (H1.2Ac) blot. Molecular mass markers are shown at the left of the figure.

Effect of GalNAc and GlcNAc on toxin binding to receptors

Western blots analysis showed the inhibition of binding of Cry1Ac to binding site(s) on the BBMV membranes of C. includens in the presence of GalNAc Whereas, the binding of Cry1Ac to receptor site was not inhibited by the presence of GlcNAc (Fig. 41A). The presence of GalNAc inhibited the binding only partially and GlcNAc did not inhibit the binding of Cry1Ac labeled toxin to the BBMV membranes of A. gemmatalis (Fig. 41B). We could not detect specific binding of H1.2Ac in the presence or absence of GalNAc and GlcNAc to the 80 binding sites on BBMV membranes of C. includens. Hybrid protein binding to BBMVs of A. gemmatalis was not inhibited by GalNAc but was partially inhibited by GlcNAc (Fig. 41C).

Fig. 41. Western blots of biotinylated Cry toxins in the presence of GalNAc or GlcNAc. Binding of Cry1Ac to BBMVs from C. includens (A) and A. gemmatalis (B); and of H1.2 to BBMVs from A. gemmatalis in the presence of 50 mM GalNAc or GlcNAc were visualized by exposing the film to the ECL treated PVDF membranes after electrotransfer of proteins from SDS-10% PAGE.

Discussion

High toxicities of Cry1Ac and Cry2Ac7 against A.gemmatalis and C. includens make them good candidate for use against these insect pests.. The protein expression encoded protein of correct size (68 kDa) which shows correct orientation of the cloned fragments. Trypsin activation of hybrid protein complies with Cry1Ac tryptic digestion and produces a protease resistant core of almost 60 kDa. Bioassay experiments with A. gemmatalis and C. includens larvae against which both the parent Cry toxins are active shows that activated H1.2Ac has insecticidal activity against A. gemmatalis only. The activity against A. gemmatalis indicate that the hybrid construct has been processed appropriately and is a stable active toxin in its molecular confirmation. Substitution of domain III Cry1Ac with that of Cry2Ac7 enhanced the 2 2 activity of parent Cry1Ac from LC50 of 20 ng/cm to LC50 of 10 ng/cm against A gemmatalis. This shows that the toxicity of a toxic toxin can be enhanced by a lesser toxic toxin by using molecular approaches. However, bioassays with C. includens show that domain substitution deprives Cry1Ac from its toxicity and it could not kill or halt the growth of C. includens larvae even at higher concentrations. The non-toxicity of H1.2Ac towards C. includens is also verified 81 by the binding experiments with the radiolabeled toxin H1.2Ac that could not bind to the receptor proteins of insect BBMVs whereas its parent toxin Cry1Ac bound to the C. includens receptors specifically as shown in Fig. 37A.The loss of activity of an active protein upon domain substitution with the same domain of another toxin lethal to C. includens emphasizes the importance of each domain participating in the sequential steps to cross the peritrophic matrix (Lertcanawanichakul et al., 2004) and binding to appropriate receptors (Wolfersberger, 1990).

The results of our competition binding experiments using 125I-Cry1Ac shows that Cry1Ac does not share binding sites with Cry2Ac7 for A. gemmatalis is the same behavior as predicted for Helicoverpa species in a previous study demonstrating that Cry2A proteins have binding sites different from Cry1Ac (Hernández-Rodríguez et al., 2008). For the insects under study the it has been reported in a very recent study (Bel et al., 2017) that for binding sites in BBMVs of A. gemmatalis and C. includens Cry2Aa and Cry1Ca do not compete with Cry1Ac or Cry1Fa. However, competition binding experiment with 125I-H1.2Ac a modified form of Cry1Ac may share binding sites on BBMVs of A. gemmatalis (Fig. 38B) with Cry1Ac or with Cry2Ac. However, the extent of competing for binding sites with Cry2Ac7 is less than it does for Cry1Ac. These shared binding sites may involve some relevant sites and this partial competition of H1.2Ac may be revealing the presence of more than one binding sites in A. gemmatalis. This adds to the findings suggesting that both domain II and domain III are determinants for binding specificity of Cry toxins to receptors on gut cells of A. gemmatalis as reviewed elsewhere (Xu et al., 2014). The enhanced toxicity of H1.2Ac against A. gemmatalis may be due to the dual binding affinity of the protein distinctly added by the parent proteins as recently found that Cry1Ac have a single population of binding sites in A. gemmatalis which is not shared by Cry2Ac7 (Bel et al., 2017). As it is proposed that Cry1Ac may bind with different modes of binding to many binding sites in many lepidopteran insects (Herrero et al., 2016; Jakka et al., 2015), our ligand blots also support this hypothesis and suggest more than one binding sites for all the proteins under talk. Analysis of the ligand blots with biotinylated Cry1Ac, Cry2Ac and H1.2Ac with BBMV proteins of A. gemmatalis (Fig.4.18) shows difference in binding pattern between Cry1Ac and Cry2Ac7. The ligand blotted protein band for Cry1Ac is at almost 57 kDa and for Cry2Ac7 and H1.2Ac it is almost at 55 kDa. H1.2Ac binds to the BBMV proteins of A. gemmatalis more like Cry2Ac7 for proteins at almost 55- and 50 kDa with stronger interaction like Cry2Ac7. These proteins of almost 60- to 68-kDa could be isoforms of ALP as identified for Cry1Ac in Menduca sexta (McNall and Adang, 82

2003) or for Ostrinia furnacalis (Jin et al., 2016) and for Cry2Ac7 toxin the blotted bands could be ALP proteins as putative receptors. This can be explained also by the results of binding experiments in the presence of GalNAc or GlcNAc with BBMV proteins of A. gemmatalis which shows the binding of Cry1Ac to putative receptors is slightly inhibited in the presence of GalNAc and displays very low binding supression in the presence of GlcNAc (Fig. 41B). Whereas H1.2 binding was not inhibited by the presence of GalNAc but is slightly inhibited by GlcNAc (Fig. 41C). These putative ALP receptors of Cry2Ac7 toxin along with the other ALP/APN receptors for domain II of Cry1Ac may have synergistically enhanced the activity of H1.2Ac. The binding patterns in ligand blots of Cry1Ac, Cry2Ac and H1.2Ac with BBMV proteins of C. includens (Fig. 40) are more distinct between Cry1Ac and Cry2Ac7. Out of these, Cry2Ac7 bound BBMV protein bands at approximately 50-kDa and 165-kDa could be very critical for its activity against C. includens. For Cry1Ac the putative protein receptor bands at about 115-kDa and 200-kDa are distinct and we could not locate it in H1.2Ac blot (Fig 40, lanes 1 and 3). However, limitations of the ligand blot should be kept in mind (Daniel et al., 2002). The absence of 115-kDa band in the ligand blot of H1.2Ac suggests that domain III of Cry1Ac specifically binds to this putative ANP receptor and this might be the crucial step for the activity of this toxin. This suggestion of the presence of two binding sites for Cry1Ac in C. includens can be supported by the very recent finding of Bel et al. (2017) who through Cry1Ac homologous competition experiment with 125I-Cry1Ac hypothesized the presence of two receptors in C. includens for it. Their statistical analysis suggests that one of the receptor (receptor 1) has high affinity for Cry1Ac but with lower number of binding sites in comparison to the other receptor (receptor 2) with more binding sites but lower protein affinity. Our results support this finding and we propose that the binding of Cry1Ac to the one of the suggested receptors is determined by domain III of the protein. The strong inhibition of Cry1Ac binding to BBMV proteins of C. includens in the presence of GalNAc (Fig. 41A) suggest that this binding site is recognized by domain III of Cry1Ac having a lectin like pocket which is previously found to bind with 120-kDa APN protein and a 210-kDa receptor protein on BBMV proteins of M. sexta and the binding is inhibited by GalNAc but not with GlcNAc (De Maagd et al., 1999a). Our finding of Cry1Ac inhibition in the presence of sugars is in agreement with the previous findings that some binding sites for Cry1 toxin in target insects may be glycosylated which may play important role in binding interactions (Garczynski et al., 1991; Jurat-Fuentes et al., 2002; Sangadala et al., 2001). The slight inhibition of Cry1Ac binding to receptors in A. gemmatalis and significant inhibition of Cry1Ac binding to C. includens receptors in the presence of GalNAc has also been reported recently (Bel et al., 2017) 83

Further research is needed to identify the putative receptors important for activity of Cry1Ac and Cry2Ac7 proteins against A. gemmatalis and C. includens and to understand the mode of action of these proteins for these pests. This study proposes that the putative binding receptors of A. gemmatalis and C. includens for Cry1Ac and Cry2Ac7 are not shared. The activity of H1.2Ac against A. gemmatalis suggests that domain III recognized receptors for Cry1AC are not crucial for its toxicity against this insect or the receptors may be not glycosylated or inhibited by GalNAc. Domain III replacement of Cry1Ac with the same domain of Cry2Ac7 do recognises receptors with slight inhibition of binding in the presence of GlcNAc, this proposes that domain II of Cry1Ac and domain III of Cry2Ac in H1.2Ac have affinity for the putative receptors and showed enhanced activity. The current research work puts forward the hypothesis that domain III of Cry1Ac is necessary for toxicity against C. includens suggesting that binding to GalNAc is critical for C. includens but not for A. gemmatalis. These findings also suggest different mode of actions of the same toxin depending on the insect. However, the above hypothesis should be verified further by performing experiments with different approaches and by taking other important models for the action of Cry proteins into consideration.

CHAPTER 5

Identification of Proteins for Pyramiding with Cry1Ie2 in Transgenic Crops for Resistance to Soybean Looper and Velvetbean Caterpillar

Abstract

Transgenic crops pyramided with different Bt toxins having no shared binding sites on the gut membranes of target insects has been a good approach to increase the range of target insect pests. The presence or absence of shared binding sites on the BBMVs of A. gemmatalis and C. includens between Cry1Ie2 and Cry1Ac, Cry2Ac7 and Cry1Fa was detected by binding competition assays. Lack of shared binding sites for Cry1Ie2 and the competitor proteins makes it a good candidate to pyramid it with any or all of these proteins to increase the efficacy of the transgenic crop.

Introduction

B. thuringiensis Cry proteins are being used in agriculture and forestry as sprays or as transgenic plants to control pests of economic importance (James, 2013; Ferré et al., 2008; Schnepf et al., 1998). The first successfully transformed soybean with B. thuringiensis Cry1Ab gene expressed Cry protein in 1994 (Parrott et al., 1994). In 1996 soybean was transformed with biologically effective insect resistant Cry1Ac gene and was characterized as fertile transgene (Stewart Jr et al., 1996). B. thuringiensis insecticidal gene transgene crops like Bt soybean, Bt cotton, and Bt maize in ten Latin American countries have been grown successfully. In USA Bt transgene crops of rice, cotton, potato, alfalfa, wheat and tomato have been planted which are resistant to specific lepidopteran and coleopteran pests (ISAAA, 2016).

However, emergence of resistance by pest insects to the Bt toxins threatens the long term environmental efficacy and economic benefits of the use of these toxins in insect pest management (Tabashnik et al., 2013; Van den Berg et al., 2013; Gould, 1998). Isolation of novel Cry toxins is a preliminary step to combat different insect pests (Bravo et al., 2007). 85

Evolution of resistance by pests against single cry gene transgenic crops (Tabashnik et al., 2013) is delayed by pyramiding two or more Bt toxins in a specific crop which also broadens the spectrum of pest (Carrière et al., 2015). The use of Bt pyramids has been prevalent since 2003, Carrière et al. (2016b) has tabulated the current multi-toxin corn expressing from one to five Bt toxins with efficacy for lepidopteran and/or coleopteran pests. Transgenic tobacco expressing Cry1Ac and Cry1Ie proteins showed great efficacy against Cry1Ac-resistant or susceptible H. armigera in comparison to the plants overexpressing a single Cry toxin (Lian et al., 2008) However, cross resistance of one toxin to other can occur and a strong cross- resistance can reduce the efficacy of both the toxins (Tabashnik et al., 2014). In field trials Cry1Ie1 expressing transgenic maize was found effective against H. armigera (Cry1Ac resistant) and Ostrinia furnacalis (Zhang et al., 2013). Cry1Ie2 has been identified effective against O. nubilalis with no shared binding sites on its midgut brush border membrane vesicles with Cry1Ab and Cry1Fa (Zhao et al., 2015). Identification of new potent toxins and probability of cross-resistance should be checked and for this Cry1Ie2 toxic protein to A. gemmatalis and C. includens pests was analyzed for the presence of shared binding sites with the other more potent Cry1Ac, Cry2Ac7 and Cry1Fa proteins. The absence of such shared binding sites could make these proteins as candidate to pyramid in transgenic crops to target O. nubilalis along with other known pests of Cry1Ac, Cry2Ac and Cry1Fa. Probability of presence or absence of shared binding sites of these proteins against Cry1Ie2 on BBMVs of A. gemmatalis and C. includens was examined through qualitative binding competition assays.

Materials and Methods

Biotin Labeling of the Cry1Ie2 protein

Activated Cry1Ie2 protein was biotinylated as described by Zhao et al. (2015) with some modifications. Briefly, 0.5 mg/mL of trypsin activated purified Cry1Ie2 toxin was dialyzed against 2 L of 50 mM Na2CO3 and 50 mM NaCl, pH 8 buffer overnight. For complete buffer exchange, the dialysant was replaced two more times with 2 h interval between each change. The amount of EZ-Link™ Sulfo-NHS-LC-Biotin (thermos Scientific) to be used was calculated according to the manufacturer’s instructions. Briefly, for a 30-fold molar excess of biotin in the labeling reaction, 26 µl of 10 mM biotin solution was required to label 0.5 mg of the protein. Ten millimolar biotin solution was freshly prepared by adding 0.5 mg biotin in 60 µl ultrapure water. Fifty two microliter biotin solution was added in 1 mL of protein(1mg) and 86 incubated on ice for 2 h. Excess and hydrolyzed non-reacted biotin was removed from the labeled protein by dialyzing it against 50 mM Na2CO3, 50 mM NaCl pH 10 buffer (2 L) and replacing twice with fresh buffer for total 4 h (2 h each). Finally, the labeled protein was dialyzed overnight against the same buffer. All the dialysis reactions were performed at 4 ºC. Western blotting was performed to analyze the biotinylation of Cry1Ie2 protein and to estimate the minimum amount of biotin labeled protein to be used in the competition binding assays. One microgram of labeled-Cry1Ie2 (for coomassie staining), 0.3 µg of labeled-Cry1Ie2 (for western blot) along with unlabeled toxin (4 µg) on both sides were electrophoresed. On completion of electrophoresis the gel was cut into two halves and one side of SDS-PAGE gel was stained with coomassie blue stain and the other side was used for electro-transference of proteins on Hybond low fluorescence PVDF membranes (GE Healthcare, Life Sciences). Further process and detection was performed as described in the following section (Detection and analysis of fluorescence). Quantified biotinylated toxin was aliquoted and stored at -80 until further use.

Binding Competition assays

Binding of toxins to receptor proteins Biotinylated Cry1Ie2 was used to perform binding competition experiments with BBMVs of C. includens and A. gemmatalis. Activated Cry1Ac, Cry2Ac7 and Cry1Fa were used as heterologous competitor proteins to analyze the extent of biotin labeled Cry1Ie2 binding to the putative receptors of C. includens and A. gemmatalis in the presence of 0-to-500 fold of the competitor proteins. Unlabeled Cry1Ie2 was used as homologous competitor protein in binding competition experiments. The brush border membrane vesicles (BBMVs) of C. includens and A. gemmatalis stored in aliquots at -80 ºC were thawed on ice and the buffer was replaced with fresh PBS (pH 7.4) and quantified. The BBMVs of C. includens and A. gemmatalis were diluted to the concentration of 6 mg/ml for ease of calculations for all the binding experiments. All the proteins used as competitor were thawed on ice and quantified just prior to use. The volumes of the reaction ingredients (in µl) are given in Table VII for Cry1Ie2, Table VIII for Cry1Ac, Table IX for Cry1Fa and Table X for Cry2Ac7 binding competition experiments.

Table VII. Biotinylated-Cry1Ie2 to Cry1Ie2 binding competition reaction mixture.

Competitor fold 0 3 10 30 50 100 300 500 Reagent

BBMVs (4µg/µl) 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00

Biotin-Cry1Ie2 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 (0.1µg/µl)

Cry1Ie2(2.0µg/µl) ___ 0.45 1.50 4.50 7.50 15.00 45.00 75.00

Buffer 92 91.55 90.50 87.50 84.50 77.00 47.00 17.00

Total volume 100 100 100 100 100 100 100 100

Table VIII. Biotinylated-Cry1Ie2 to Cry1Ac binding competition reaction mixture.

Competitor fold 0 3 10 30 50 100 300 500 Reagent

BBMVs (4µg/µl) 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00

Biotin-Cry1Ie2 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 (0.1µg/µl)

Cry1Ac (1.7µg/µl) ___ 0.53 1.76 5.29 8.82 17.65 52.94 88.24

Buffer 92 91.47 90.24 86.71 83.18 74.35 39.06 3.76

Total volume 100 100 100 100 100 100 100 100

Table IX. Biotinylated-Cry1Ie2 to Cry1Fa binding competition reaction mixture.

Competitor fold 0 3 10 30 50 100 300 500 Reagent

BBMVs (4µg/µl) 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00

Biotin-Cry1Ie2 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 (0.1µg/µl)

Cry1Fa (1.8µg/µl) ___ 0.50 1.67 5.00 8.33 16.67 50.00 83.33

Buffer 92 91.50 90.33 87.00 83.67 75.33 42.00 8.67

Total volume 100 100 100 100 100 100 100 100

Table X. Biotinylated-Cry1Ie2 to Cry2Ac7 binding competition reaction mixture.

Competitor fold 0 3 10 30 50 100 300 500 Reagent

BBMVs (4µg/µl) 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00

Biotin-Cry1Ie2 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.00 (0.1µg/µl)

Cry2Ac7(1.65µg/µl) ___ 0.55 1.82 5.45 9.09 18.18 54.55 90.91

Buffer 92 91.45 90.18 86.55 82.91 73.82 37.45 1.09

Total volume 100 100 100 100 100 100 100 100

Tables VII-X shows reaction mixture ingredients for binding competition assays analyzed through western blots. The amount of biotin-labeled Cry1Ie2 and BBMVs of C. includens or A. gemmatalis was constant for all reactions with different homologous and heterologous competitors. Column 1 shows reactants and the concentration of proteins used to calculate required volume (in µl) of the ingredient. Row 1 indicates the fold-competitor against biotinylated Cry1Ie2. Binding buffer was PBS with 0.1% BSA, pH 7.4.

In pre-labeled microfuge tubes calculated volume of binding buffer (PBS buffer, pH 7.4 with 0.1 % BSA) was added. To the binding buffer unlabeled homologous and heterologous competitor proteins were added according to the calculated volume for the equivalent fold of the labeled-toxin. Three microliters of the biotin labeled Cry1Ie2 toxin was added to each tube and mixed gently. Finally, 30 µg of BBMV proteins prepared from midguts of C. includens or A. gemmatalis were added to appropriate tubes. The reactions were incubated for 1 h at room temperature. Reactions were stopped by centrifuging at 15,000 ×g for 10 min and the pellets were washed with 1 ml of ice-cold binding buffer. Washing with binding buffer was repeated one more time and the pellet formed after centrifugation was denatured for 5 min at 100 ºC in 2x SDS-PAGE loading buffer (15 µl). The evaporated contents of the denatured sample proteins were brought to bottom by short spin and loaded into wells along with pre-stained protein molecular weight marker in one well. Electrophoresis was carried out at 120 V until completion. All the toxin binding competition experiments with BBMVs preparations of both the insect guts were performed in duplicates.

Electroblotting of membranes After the completion of electrophoresis, the gels and filter papers were washed with water and soaked in transference buffer (25 mM Tris, 192 mM glycine, 0.1 % SDS and 20 % methanol) for 30 seconds. Hybond low fluorescence PVDF membranes (GE Healthcare, Life Sciences) were soaked in methanol for 15-to-30 sec because of their hydrophobic nature. Blotting sandwiches were prepared by putting the gels in contact with the blotting filter membranes covered by wet filter papers on both sides. Precaution was taken not to introduce or leave any air bubble in the sandwiches by gently pressing it with glass rod rolling lever. The sandwiches were placed on cassettes which were closed and placed into the western blotting tank apparatus (Bio-Rad) so that the gel faced towards the negative (-) pole of the blotting tank. Cold transference buffer was added in the tank up to the maximum level mark indicated on the tank and electro-transference was done at 20 V overnight at 4 ºC. 90

Washing and probing the membranes The membranes were blocked in blocking buffer (PBS, 0.1% Tween-20 plus 5% non- fat dry milk powder) for 1 h on orbital shaker. In the same blocking buffer streptavidine-Alexa Fluor 594 conjugate (Invitrogen) was added to the membranes at a dilution of 1:5,000 and probed for 2 h at room temperature with constant shaking. The membranes were first washed twice with wash buffer (PBS, 0.1% Tween-20) and then 6 times after every 10-min shaking on orbital shaker in between each wash. After the last washing step, the filters were put on filter paper and dried in air for 5 min.

Detection and analysis of fluorescence The air-dried filter membranes were wrapped in saran wraps for scanning. Typhoon Trio scanner (GE Healthcare) was turned on 15 min prior to use as directed by the manufacturer. The saran wrapped membrane filters were scanned for 633 nm red fluorescence and the images of the fluorescence were captured. The images were analyzed by ImageJ software (Abramoff et al., 2004) to quantify the density of the fluorescent bands. The densiometric data of the two replicates were used to get average fluorescence of the bound biotinylated Cry1Ie2 toxin and the curves were plotted using SigmaPlot v.11.0 (Systat Software).

Results

Biotinylation of protein

Activated Cry1Ie2 toxin was successfully biotinylated as shown in Fig. 42. The trypsin activated toxin is almost at 55 kDa and on the SDS-PAGE gel the biotin labeled toxin (1µg) shows a clear band. The biotin-labeled toxin (0.3 µg) shows detectable fluorescence as shown in the lane 4 of Fig. 42.

Fig. 42. SDS-PAGE gel and western blot of biotin labeled-Cry1Ie2. 1, anion exchange chromatography purified trypsin activated toxin; 2, biotin labeled toxin; 3, unlabeled toxin showing no fluorescence; 4, biotin labeled toxin fluorescence band at 55kDa detected with 633 nm red light using Typhoon Trio scanner (GE Healthcare); M, protein molecular weight marker, the values on the left of the panel are the molecular mass of proteins in kDa.

Binding competition analysis of proteins

Biotin labeled Cry1Ie2 protein kept its activity after labeling with biotin and was stable as it was not precipitated while stored at -4 ºC. Biotinylated Cry1Ie2 showed strong binding affinity for BBMVs of A. gemmatalis although its binding affinity for BBMVs of C. includens was not strong but was detectable. The results of binding competition assays of biotinylated Cry1Ie2 with the unlabeled competitors are shown (Fig. 43 and 44).

Fig. 43. Biotinylated Cry1Ie2 binding competition assays; A, Western blot showing binding of biotinylated Cry1Ie2 (18 nM) to BBMV of A. gemmatalis in the presence of 0- to 500- fold increasing concentrations of unlabeled Cry1Ie2, Cry1Ac, Cry1Fa and Cry2Ac7 competitors as the lanes labeled. Western blots were probed with streptavidin-Alexa Fluor 594 conjugate and bands were scanned by red fluorescence. B, plot of % bound biotinylated Cry1Ie2 toxin against increasing concentration of competitor (nM), curves show mean total binding calculated from densitometric analysis of bands in panel A using ImageJ software and the error bars show the standard error of the mean.

Fig. 44. Biotinylated Cry1Ie2 binding competition assays; A, Western blot showing binding of biotinylated Cry1Ie2 (18 nM) to BBMV of C. includens in the presence of 0- to 500- fold increasing concentrations of unlabeled Cry1Ie2, Cry1Ac, Cry1Fa and Cry2Ac7 competitors as the lanes labeled. Western blots were probed with streptavidin-Alexa Fluor 594 conjugate and bands were scanned by red fluorescence. B, plot of % bound biotinylated Cry1Ie2 toxin against increasing concentration of competitor (nM), curves show mean total binding calculated from densitometric analysis of bands in panel A using ImageJ software and the error bars show the standard error of the mean.

With the homologous competitor (Cry1Ie2) binding of biotinylated Cry1Ie2 to the BBMVs of A. gemmatalis and C. includens was displaced with increase in the concentration of unlabeled competitor depicting the specific binding of the protein (Fig. 43A and 44A). In 94 the presence of competitor Cry1Ac, binding of biotinylated Cry1Ie2 to the BBMV of A. gemmatalis is displaced partially and at its higher concentrations it did not affect its binding further. Whereas, Cry1Ac did not displace Cry1Ie2 binding to BBMV of C. includens (Fig. 43A and 44A). The densiometric analysis of fluorescent bands of biotinylated Cry1Ie2 generated in the binding competition experiment in the presence of unlabeled Cry1Fa as heterologous competitor suggested that Cry1Fa did not share the binding sites on BBMVs of A. gemmatalis. The biotin-Cry1Ie2 binding curve shows that Cry1Fa at higher concentrations (100- to 300-fold) partially displaced binding of biotinylated Cry1Ie2 to BBMV of C. includens (Fig. 44B). The unlabeled Cry2Ac7 protein did not compete with Cry1Ie2 for the binding sites on BBMVs of both A. gemmatalis and C. includens almost equally (Fig. 43B and 44B). The binding curves in the presence of heterologous competitors (Fig. 43B and 44B) showed that none of the toxin displaced labeled Cry1Ie2 binding to BBMVs completely and only partial displacement occurred for some higher concentrations of the heterologous competitor propose that these toxins preliminarily do not share binding sites with Cry1Ie2 on the BBMVs of both A gemmatalis and C. includens.

Discussion

Bioassay results (Table II, Chapter 3) showed that Cry1Ac, Cry2Ac7 and Cry1Fa toxins were highly active against A. gemmatalis and C. includens whereas, Cry1Ie2 is moderately toxic to A. gemmatalis and less active against C. includens. Cry1Ie2 is found to be active against O. nubilalis and it has been reported that it does not share binding sites with Cry1Ab and Cry1Fa in O. nubilalis BBMVs (Zhao et al., 2015). We are reporting the toxicity of Cry1Ie2 against two economically important lepidopteran pests A. gemmatalis and C. includens. Cry1Ac and Cry2Ab2 transgenic cotton (Bollgard II) and WideStrike (Cry1Ac/Cry1Fa) transgene demonstrated effective for the control of Spodoptera frugiperda and C. includens in the United States (Sorgatto et al., 2015; Stewart et al., 2001). This suggests that Cry1Ac binding sites are not recognized by Cry1Fa or Cry2Ab2 for the target Lepidopteran insects. The idea of lack of cross resistance between Cry1Fa and Cry1Ac for A. gemmatalis, C. includens, H. virescens and Spodoptera cosmioides is also supported by the data in a recent research (Marques et al., 2016). Previously, it was demonstrated that Cry2Ae does not share binding sites with Cry1Ac, Cry1Ab and Cry1Fa in BBMVs of heliothine species (Gouffon et al., 2011). Bt isolates with the presence of Cry1 and Cry2 genes together conferred high toxicity to these isolates against S. frugiperda, P. xylostella and A. gemmatalis (Monnerat et 95 al., 2007) emphasizing the lack of common binding sites for these toxins. Analysis of data from the competition binding assays shows that Cry1Ie2 binds to BBMV of A. gemmatalis and C. includens specifically and implies that the competitors bind to different sites on BBMVs. It is evident from many previous studies that the main determinant of Cry toxin binding specificity are domain II and III (Adang et al., 2014) and the sequence similarities in these domains result in cross resistance between Cry toxins (Carrière et al., 2015). Low sequence similarity of domain II between Cry1Ie2 and Cry1Ab /Cry1Fa supports the absence of shared binding sites on BBMV of O. nubilalis (Zhao et al., 2015) however, specificity is not always predicted by similarity in Cry proteins and specificity is also related to host-dependent factors (reviewed in Jurat-Fuentes and Crickmore, 2016). This is evident from the mean total binding curves in Fig. 43B and 44B as the extent of displacement of biotinylated Cry1Ie2 protein is different for all the competitors in binding the receptors on BBMV of A. gemmatalis and C. includens. These evidences support our hypothesis of lack of shared binding sites between Cry1Ie2 and Cry1Ac or Cry1Fa or Cry2Ac7 for these insect receptors. Further research is needed to identify the binding sites on the BBMVs of these insects for these toxins. Previous research reports that soybean transgene with Cry1Ac provides high level of control against A. gemmatalis and P. includens (Bernardi et al., 2012). In 2015, Bt-soy plants expressing Cry1Ac proteins were registered for cultivation in Latin American countries for the control of lepidopteran pests (Blanco et al., 2016). Expression of Cry1Ab and Cry2Ab in transgenic rice displayed high resistance to lepidopteran insects and no antagonism between these toxins were found (Zhao et al., 2014) proposing for no shared binding sites between these Cry toxins. We are presenting Cry2Ac7 as a new toxin and tnCry2Ac an isolate from Pakistan along with other effective Bt toxins to transgene plants of economic interest for the control of A. gemmatalis and C. includens and other lepidopteran pests. Our results are in accordance with the earlier findings of the difference in susceptibilities of the two target insects for Cy1A toxins and the same behavior for Cry2 type toxin demands to look for the mode of action of the toxins for A. gemmatalis and P. includens for the construction of better biotoxins and integrated pest management (IPM). Present study demonstrates high toxicity of some Cry toxins for A. gemmatalis and C. includens. Proposed lack of shared binding sites between Cry1Ie2 and Cry1Ac or Cry1Fa or Cry2Ac7 qualifies Cry1Ie2 to be used as controlling agent for these insects alone or pyramided with any or all of the tested toxins to increase the number of target insects.

CHAPTER 6

Discussion and Conclusions

Bt three-domain Cry toxins are effective to control the invasions of insect pests on economically important crops like cotton, maize and soybean. The first objective of this study was the production of Cry proteins and analyze their effectiveness to control lepidopteran insect pests. For this Cry1Ac, Cry1Fa, Cry1Ie2 and Cry2ac7 proteins were produced and purified. As per previous knowledge the three domains of the protoxin are involved in specificity and toxicity to insect whereas C-terminal extension is non-essential for insecticidal activity, the C- terminally truncated Cry1Ac protoxin was bio-assayed in this study. Cry2Ac7 is a member of short protoxin group (70 kDa) and is processed at N-terminal end only (reviewed in de Maagd et al., 2001), so it was produced as full length protoxin. Bioassay results of these protoxins with S. litura classify tnCry1Ac as non-toxic and Cry2ac7 as toxic to this insect. As 3d-Cry proteins are generally trypsin digested prior to test for activity finding bioassays, so we trypsin activated these proteins and named tnCry1Ac as Cry1Ac after trypsin digestion. But the trypsin activated toxins could not be assayed for toxicity to S. litura due to the expiry of the whole laboratory culture. So, these toxins along with two other activated toxins, Cry1Fa and Cry1Ie2, were tested for their toxicities to two important crop pests, A. gemmatalis and C. includens.

Considering the LC50 values in the Table II, among all the toxic proteins Cry1Ac has the highest toxicity for these two insect pests.

To address the second objective of this study, hybrid recombinant construct was made and overexpressed. His-tag chromatography technique was used for ease of purification of H1.2Ac, tnCry1Ac and Cry2Ac7 proteins. Insecticidal activity of H1.2Ac was detected against A. gemmatalis and C. includens. The hybrid recombinant protein found to be nontoxic to C. includens. This suggests that the mode of action of the toxins varies from insect to insect and the way of action of binding of the toxin plays a crucial role in its toxicity to insects. Soybean engineered to produce the Cry1Ac toxin (IntactaTM) efficiently controls larvae of A. gemmatalis and C. includens (Walker et al., 2000), yet production of a single toxin greatly increases the probability of resistance evolution in target insects. Novel insecticidal proteins could reduce the risk of resistance evolution and expand the range of activity if pyramided with Cry1Ac in transgenic soybean. Domain III of Cry1Ac contains a lectin fold involved in binding to N- 97 acetylgalactosamine (GalNAc) on putative receptors (Karlova et al., 2005; Jurat-Fuentes and Adang, 2004; Burton et al., 1999). This affinity for glycosylated proteins was proposed in a sequential binding model to promote interactions of toxin oligomers with receptors, leading to toxin insertion in the cell membrane (Pardo-López et al., 2006). Analysis of Cry2Ac7 domain III determined that out of the N506, Q509, and Y513 amino acid residues in Cry1Ac involved in binding to GalNAc (Burton et al., 1999), only Q509 is conserved. Consequently, we designed a toxin hybrid (H1.2Ac) combining domains I and II from Cry1Ac as the most active Bt toxin against A. gemmatalis and C. includens, and domain III from Cry2Ac7 as a novel toxin with toxicity against both species but predicted not to share binding sites with Cry1Ac (Bel et al., 2017).

Previous reports of toxicity enhancement (Shan et al., 2011; Karlova et al., 2005) and reduction (Masson et al., 2002; Lee et al., 1999) after alteration of domain III in Cry1Ac predicted potential effects on H1.2Ac activity. However, we unexpectedly found that while H1.2Ac displayed high activity against A. gemmatalis, it did not kill or delay development in larvae of C. includens. Results from binding assays suggested that this lack of H1.2Ac toxicity against C. includens was due to absence of specific toxin binding to midgut proteins. Since the only region in the H1.2Ac hybrid diverging from Cry1Ac is domain III, these observations identify this domain as the main Cry1Ac binding specificity (and toxicity) determinant in C. includens. In contrast, insecticidal activity and displacement of Cry1Ac binding by H1.2Ac in BBMV from A. gemmatalis support that in this insect Cry1Ac binding specificity mostly resides in domain II, which is shared between both toxins. However, testing of alternative hybrid toxins containing domains I and II of Cry1Ac and domain III from a toxin inactive against A. gemmatalis would be necessary to conclude the importance of domain II for specificity in that insect. Remarkably, the concentration of binding sites in A. gemmatalis BBMV was higher for Cry1Ac than for H1.2Ac, which may suggest the involvement of Cry1Ac domain III in recognizing a population of binding sites. However, the fact that H1.2Ac and Cry1Ac are equally active against A. gemmatalis larvae supports that interactions with the binding sites recognized by domain III of Cry1Ac may not be relevant to toxicity against A. gemmatalis. Dependence on domain III for recognition of this second population of Cry1Ac binding sites in A. gemmatalis BBMV would also help explain why GalNAc significantly inhibited Cry1Ac binding to BBMV from C. includens while the effect was more modest on Cry1Ac binding to A. gemmatalis BBMV (Bel et al., 2017). Interestingly, previous reports suggest the existence of two populations of Cry1Ac binding sites in C. includens, but not in A. 98 gemmatalis (Bel et al., 2017). One possibility to explain this discrepancy is that while there may be multiple populations of Cry1Ac binding sites in A. gemmatalis BBMV, they are recognized with similar affinity by the toxin, preventing their discrimination as distinct sites in competition-binding assays.

As expected from only sharing one of the two putative binding specificity domains with H1.2Ac, neither Cry1Ac nor Cry2Ac7 displaced all H1.2Ac binding to A. gemmatalis BBMV. These results suggest that sites recognized by domain II from Cry1Ac differ from sites recognized by domain III from Cry2Ac7, as predicted from the lack of shared binding sites between Cry1A and Cry2A toxins in A. gemmatalis (Bel et al., 2017). While one needs to keep in mind the limitations associated to ligand blotting (Daniel et al., 2002), comparison of the pattern of A. gemmatalis BBMV proteins recognized by Cry1Ac, H1.2Ac and Cry2Ac7 toxins suggested sharing of some protein bands between H1.2Ac and Cry1Ac or Cry2Ac7. Further research is needed to identify the proteins present in these electrophoretic regions recognized by these toxins, and to elucidate if H1.2Ac recognizes BBMV proteins not bound by Cry1Ac or Cry2Ac7.

Differential toxicity of H1.2Ac to A. gemmatalis and C. includens clearly supports the existence of differences in the way Cry1Ac interacts with the membrane binding sites in these insects. This observation may suggest that integrative models developed using a particular Cry toxin and insect may not be completely applicable to other species. Importantly, A. gemmatalis and C. includens differ in their susceptibility to Cry1Ac, with A. gemmatalis being most susceptible (Table II, Chapter 3). While speculative, recognition of binding sites that are not conducive to toxicity by domain III of Cry1Ac may help explain lower susceptibility in C. includens compared to A. gemmatalis. Thus, while in A. gemmatalis both domain II and III would be involved in binding to receptors, only domain III would be relevant in C. includens, potentially limiting the number of interactions with lethal receptors. While the present study presents evidence for the importance of Cry1Ac domain III for toxicity against C. includens, further research would be needed to identify lethal Cry1Ac receptors and determine their interactions with domains II and/or III of Cry1Ac. This information contributes to the design of more active insecticidal proteins against this pest and our general understanding of the Cry mode of action in Lepidoptera.

Third objective of this study was addressed by performing the binding competition assays taking Cry1Ie2 as the primary protein. Cry1Ie2 was chosen to study binding inhibition 99 by other Cry proteins (Cry1Ac, Cry2Ac7 and Cry1Fa) because it was least toxic to A. gemmatalis and C. includens and it would be useful to employ other more toxic proteins with it to improve the target range and toxicity of the cry1Ie2 trangene. It has been reported by Zhao et al. (2015) that Cry1Ie2 is nontoxic to H. virescens, H. zea and S. frugiperda but it had toxicity to O. nubilalis. Also, it does not share binding sites in O. nubilalis BBMVs with cry1A or cry1Fa (Zhao et al., 2017. We anticipated the idea to transgene cry1Ie2 to control corn pest and broaden the target pest range of the transgene by pyramiding Cry1Ac or Cry2Ac or Cry1Fa with it. For this the presence of probable shared binding site(s) between these toxins was necessary to analyze. This was analyzed by the binding competition assays which suggested that Cry1Ac or Cry2Ac or Cry1Fa does not compete for the binding sites in A. gemmatalis and C. includens as detailed in Chapter 5.

Concluding our discussion, we produced Cry proteins in soluble form, purified them and quantified their toxicities to important lepidopteran pests. The hybrid protein (H1.2Ac) threw light on the different way of binding of Cry1Ac and Cry2ac7 with the receptors on BBMVs of A, gemmatalis and C. includens. This information would be helpful in designing new toxins for the control of these insect pests and to understand the binding mechanisms of the Cry proteins. Further, these genes could be transferred to plants in some combinations or all together to increase the target range of insect pests and to enhance the efficacy of the transgene.

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