Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations
2015 Engineering Cry4Aa for toxicity against the soybean aphid, Aphis glycines Matsumura Benjamin Robert Deist Iowa State University
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Engineering Cry4Aa for toxicity against the soybean aphid, Aphis glycines Matsumura
by
Benjamin Robert Deist
A thesis submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
Major: Microbiology
Program of Study Committee: Bryony C. Bonning, Major Professor Russell A. Jurenka Gwyn A. Beattie
Iowa State University
Ames, Iowa
2015
Copyright © Benjamin Robert Deist, 2015. All rights reserved. ii
TABLE OF CONTENTS
Page ACKNOWLEDGEMENTS ...... iii
CHAPTER 1. INTRODUCTION
Soybean Aphid ...... 1 Management of Soybean Aphid ...... 2 Aphid Resistant Transgenic Plants ...... 3 Bacillus thuringiensis Derived Toxins for Pest Management ...... 6 Insecticidal Bt Proteins ...... 7 Truncated Toxins ...... 11 Cleavage Site Modifications ...... 15 Binding Modifications ...... 21 Concluding Remarks ...... 31 Research Problem ...... 32 Thesis Organization ...... 32 References ...... 32
CHAPTER 2. MODIFICATION OF A CRY TOXIN FOR ENHANCED EFFICACY AGAINST SOYBEAN APHID (Aphis glycines )
Abstract ...... 45 Introduction ...... 46 Materials and Methods ...... 47 Results ...... 57 Discussion ...... 70 Author Contributions ...... 72 Acknowledgements ...... 72 References ...... 73
CHAPTER 3. CONCLUSIONS & DISCUSSION ...... 76
References ...... 79
APPENDIX. NUCLEOTIDE AND AMINO ACID SEQUENCE OF CRY4AA AND MODIFIED TOXINS ...... 81
iii
ACKNOWLEDGEMENTS
First and foremost, I would like to thank my advisor, Bryony Bonning, for allowing me the opportunity to work in her lab. A special thank you to Nanasaheb
Chougule who provided me with my initial training, Teresa Fernandez-Luna for helping troubleshoot, Yuting Chen for assisting with both PCR methodologies and feeding assays, and Mike Rausch for helping with feeding assays. Last but not least, I would like to thank my loving parents, Robert and Terri Deist, for their lifelong encouragement and support. 1
CHAPTER 1. INTRODUCTION
Insects pose a significant threat to modern agriculture, causing damage to cash and food crops primarily through damage by feeding, but also as vectors of plant disease [1]. Every year, insect pests reduce crop production by an estimated 10-16%, representing billions of dollars in losses [2,3]. Adding to the cost is the 1.2 billion dollars spent on pesticides applied by farmers every year in the United States to manage insect pests [3]. These costs could increase further as insect populations increase due to global climate change [2,4].
Soybean Aphid
Soybean aphid ( Aphis glycines Matsumura) is the foremost pest of soybean in
North America. An invasive hemipteran originating from Asia, soybean aphid was first identified in the United States in Wisconsin in 2000. By the end of 2000 it had been identified in a total of ten states; by 2009 soybean aphid had been identified in thirty states and three Canadian provinces [5]. The soybean aphid requires two host plants, a primary host and a secondary host to complete its lifecycle. In North America the role of primary host is fulfilled by common buckthorn ( Rhamnus cathartica ), an invasive species from Europe, as the soybean aphid’s natural primary hosts, Japanese buckthorn
(Rhamnus japonica ) and Dahurian buckthorn ( Rhamnus davurica ), are not present in substantial numbers in North America [5]. Soybean aphids use the primary host for sexual reproduction and overwintering. The secondary host is utilized as the site of asexual reproduction; for soybean aphid the secondary host is soybean ( Glycines max )[5]. Unfortunately, relatively little is known about the molecular biology and 2 physiology of soybean aphid. Information on the gut environment is inferred from work on other aphid species, while information on the molecular biology of soybean aphid is limited to next generation sequencing work [6,7].
Soybean aphid infests soybean during the growing season, causing up to a 40% yield loss [5]. Damage is caused primarily via feeding, but can also occur by transmission of plant viruses, and the deposition of honeydew on the surface of the soybean promoting the growth of saprophytic fungi [5,8]. Soybean aphid feeding, as well as the growth of fungi on the surface of soybean, interferes with the photosynthetic capability of soybean [5,8]. Soybean aphids pose a potential threat as a vector of plant viruses.
Although soybean aphid has not been linked to viral outbreaks in soybean in North
America, they have been shown to be competent vectors for several plant viruses including Bean yellow mosaic virus , Alfalfa mosaic virus , and Soybean mosaic virus [5,9].
Management of Soybean Aphid
A variety of approaches have been taken to manage soybean aphid. Chemical insecticides are both the most traditional approach to soybean aphid management as well as the most prevalent. Foliar application of organophosphates and pyrethroids is a common strategy for soybean aphid management [5,8,10]. Neonicotinoid-based seed treatments are increasing in popularity for soybean, and can provide some early season protection against soybean aphid, although not necessarily the most economical choice
[5,10]. While insecticides are effective against soybean aphids, insecticide application is not without issue. The use of insecticides increases the cost of soybean production per unit area, cutting into the profits of soybean growers [5,10]. Insecticide use can also 3 have an impact on natural enemies of soybean aphid, thereby limiting the beneficial effects derived from natural enemies on suppressing soybean aphid populations [5,10].
The rapid population growth of soybean aphid poses a potential problem for the long- term use of insecticides. Under optimal conditions, soybean aphids can double their population in less than two days, resulting in upwards of 15 asexual generations per season [11]. Rapid population growth increases the risk of soybean aphid developing resistance to currently employed insecticides highlighting the importance of the proper use of insecticides to preserve their long term efficacy [10].
The development of naturally resistant plant cultivars provides another strategy for the management of soybean aphid. Natural plant resistance utilizes two mechanisms, antibiosis, causing the death of the pest insect, and antixenosis, deterring pest feeding [5,10]. A total of four “resistance to Aphis glycines ” (Rag) genes, Rag 1,
Rag 2, Rag3/Rag3 , and Rag4 , are known to function in soybean resistance to soybean aphid, although more are likely to be involved [5,10]. Unfortunately, rag genes do not provide consistent performance against all soybean aphid biotypes, as certain biotypes display a lack of susceptibility to one or more rag genes [5,10]. Natural host plant resistance offers a strategy for the management of soybean aphid on soybean, particularly in combination with other management approaches.
Aphid Resistant Transgenic Plants
While transgenic approaches are not currently in use for suppression of aphid populations, several strategies have been investigated to that end. Transgenic plants for aphid resistance provide a modern approach to the management of aphids on crops. 4
There are a variety of strategies behind transgenic plants, ranging from the heterologous expression of a toxin/resistance gene found elsewhere in nature to the expression of “designer” toxins modified specifically to target aphids.
Plant lectins are naturally occurring carbohydrate-binding proteins associated with plant defense against a variety of threats including pathogens and feeding by insects [12]. More often than not, plant lectins active against aphids preferentially bind to mannose residues present on the gut epithelium, ultimately disrupting gut integrity resulting in a variety of effects ranging from reduced feeding to insect death [12,13].
The heterologous expression of plant lectins has been used with some success to manage a variety of aphid species, although this approach has yet to be employed against soybean aphid [8]. While the heterologous expression of lectins in plants provides a powerful technology to use against a variety of aphids, it is not without issues, notably a higher risk for off-target activity, as well as inconsistent field performance [8,12].
Another viable strategy for the management of aphids is the use of toxins modified with enhanced activity towards aphids. Such toxins may act within the gut
(e.g. toxins derived from Bacillus thuringiensis , Bt) [14], or within the hemocoel (e.g. neurotoxins derived from spider venom, such as Hv1a) [15]. Different strategies exist to engineer toxins, but the central theme is the identification of the bottleneck limiting or inhibiting activity against the desired target pest and modifying the toxin to reduce the impact of said bottleneck. Common bottlenecks for toxin efficacy against aphids include inefficient transcytosis across the gut epithelium for neurotoxins, limited toxin stability 5 in the aphid gut, a lack of binding to the gut epithelium, and limited activation of protoxins (e.g. Bt toxins) [16]. By targeting these bottlenecks, several modified toxins have been made with activity against a variety of aphid species.
An insect specific neurotoxin derived from spider venom, ω-hexatoxin-Hv1a
(Hv1a), with no measurable activity against aphids when ingested, has been fused to two different carrier proteins to promote transcytosis across the aphid gut into the hemocoel. These toxin-carrier fusion proteins exhibited toxicity against several aphid species [17,18]. Fusion of Hv1a to pea enation mosaic virus (PEMV) coat protein resulted in toxicity against several aphid species, including pea aphid (Acyrthosiphon pisum ), soybean aphid (A. glycines ), green peach aphid (Myzus persicae ), and bird- cherry oat aphid ( Rhopalosiphon padi ) [17]. Fusion of the snowdrop lectin (GNA) to
Hv1a, resulted in toxicity towards green peach aphid and grain aphid ( Sitobion avenae ).
While the use of carrier proteins to deliver insect-specific neurotoxins into the aphid hemolymph is an exciting avenue for future research, modification of the gut-active Bt toxins holds considerable promise, particularly considering that use of modified Bt toxins is less emotive than use of toxins derived from spider and scorpion venom.
The remainder of this section has been published in a review paper by Deist B, Rausch
M, Fernandez-Luna M, Adang M, and Bonning B [19].
6
Bacillus thuringiensis Derived Toxins for Pest Management
The ubiquitous Gram-positive spore-forming bacterium Bacillus thuringiensis (Bt) provides a valuable resource due to its ability to synthesize crystal parasporal inclusions during sporulation [20]. These crystals, which include insecticidal proteins called δ- endotoxins have been extensively used as biological insecticides against insect pests of commercial interest [20]. More than 175 million hectares were planted to transgenic crops expressing Bt toxins in 2013, highlighting the practical importance of these toxins for pest suppression [21].
Bt crystal toxins include the Cry proteins (crystal toxins) and Cyt proteins
(cytolytic toxins) [22,23]. While the ETX/MTX-like and the Binary (Bin)-like toxins do have Cry designations, they belong to structurally different classes of Cry toxins; we refer the reader to an updated review on Cry toxins diversity [23]. The Cry toxins are important virulence factors allowing for the development of the bacterium in dead or weakened insect larvae. The largest group of Cry toxins has three distinct structural domains; the so-called three-domain (3D) Cry toxins provide the primary focus for this review. The Cyt toxins have in vitro cytolytic activity , in addition to in vivo activity against various insects including mosquitos [24]. Cyt toxins which are active against certain Diptera, synergize the toxicity of Cry proteins against mosquitoes and delay the expression of resistance to the latter [25]. While most Bt crystal toxins are encoded by large plasmids, with expression under the control of sporulation-dependent promoters, there are also cryptic Cry toxins that are not expressed [20,26]. The high level of crystal 7 protein expression is controlled by a variety of mechanisms occurring at the transcriptional, post-transcriptional, and post-translational levels [24].
The evolution of resistance by targeted pests is the main threat to the usefulness of Bt toxins. We outline Bt toxin mechanism of action to provide the framework for toxin modification to combat resistance, or to broaden host range.
Insecticidal Bt Proteins
Mode of Action of Bt Toxins
The conserved structure of 3D Cry toxins, as well as results from a considerable amount of research, supports a conserved mode of Cry toxin action [24,27]. The first impact of Cry toxins on the insect is cessation of feeding due to paralysis of the gut and mouthparts [28]. In addition to gut paralysis, midgut cells swell leading to an ion imbalance and death [28]. The molecular events leading to Cry toxin-mediated insect death are controversial [27], but the accepted initial steps are as follows: The Bt Cry and
Cyt proteins require solubilization in the insect midgut to produce protoxins that are typically about 130 kDa, 70 kDa or 27 kDa for Cyt. These in turn are proteolytically cleaved at the C-terminus and/or at the N-terminus by midgut proteases, generating the activated core toxin. The toxin then crosses the peritrophic matrix and binds to receptors in the apical membrane of the midgut cells, with receptor binding being an important determinant of toxin specificity. Toxin insertion into the epithelial membrane forms ion channels or pores, leading to lysis of the cells, damage to the midgut epithelial tissue, and death of the larva (Figure 1a) [27,29,30].
8 8
Figure 1. (a) Schematic representation of the major steps involved in Cry toxicity and proposed sites for modification to increase efficacy and/or broaden toxicity. (b) Examples of insect species successfully targeted by modified toxins [19]. 9
Structure of Bt Toxins and Domain Function
Cry protoxins are typically 130 kDa or 70 kDa proteins [24] with the larger protoxins having an additional carboxyl region that is not required for toxicity but that is required for crystal formation [22,24]. When these crystal proteins are exposed to the alkaline environment of the gut of susceptible larvae, the crystals are solubilized and proteolytically processed at the N-terminus and/or the C-terminus by midgut proteases to yield the active protease-resistant toxin [22,24].
Based on their crystal structures, different Cry toxins (Cry1Aa, Cry1Ac, Cry2Aa,
Cry3Aa, Cry3Bb, Cry4Aa, Cry4Ba and Cry8Ea1 [22,31,32]) have similar folding patterns with three distinct domains [22,24]. Domain I is a seven to eight α-helix bundle comprised of amphipathic helices surrounding the central hydrophobic helix α-5.
Domain I is involved in binding and in pore formation [22,24,33]. Domain II consists of three antiparallel ß-sheets with exposed loop regions involved in interaction with receptors, while domain III is a ß-sandwich of two antiparallel ß-sheets involved in receptor binding and possibly membrane insertion. In contrast, Cyt toxins consist of a single domain in which two outer layers of α-helix are wrapped around a ß-sheet
[24,34].
The role of domain I in membrane insertion and pore formation in the midgut epithelium of the target insect was suggested by the presence of its long, hydrophobic and amphipathic helices [33]. The Umbrella Model proposed by those authors depicts the hydrophobic helical hairpin α4 and α5 as inserting into the membrane and initiating pore formation, while the rest of domain I flattens out on the membrane surface in an 10 umbrella-like molten globule state. Mutant Cry proteins having altered amino acid residues on the putative surface residues of domain I and within the α-helices supported the role of domain I in membrane binding, insertion and pore formation [35].
Domain II is implicated in protein-receptor interactions through the surface- exposed loops at the apices of the three ß-sheets. Due to their similarities to immunoglobulin antigen-binding sites, the loops of domain II were suggested to participate in receptor binding. Site-directed mutagenesis and segment swapping analyses provided support for this hypothesis [24,33]. The ß-sandwich structure of domain III is also suggested to function in receptor binding; evidence for this role in toxin action comes from domain III swap experiments discussed below, and in the case of Cry1Ac, the presence of a ‘pocket’ in domain III that binds N-acetyl galactosamine moieties on protein receptors [36,37]. Domain III is also implicated in maintaining the structural integrity of the toxin molecule by protecting it from proteolysis within the gut of the target organism [22,33].
The Cyt toxins characterized to date are also protoxins that undergo N- and C- terminal processing. The predominantly ß-sheet core structure of Cyt2A suggests a mechanism of action through a ß-barrel-pore [34]. It contains three strands long enough to span the hydrophobic lipid membrane, forming a hydrophobic sheet [34, 38].
In addition to forming pores, Cyt toxins may exert their effects via a general, detergent- like perturbation of the membrane [39]; the Cyt toxins were recently reviewed [40,41].
11
Truncated Toxins
A clear and rational approach to Bt toxin action allowed for improvement of toxin efficacy. Through a systematic analysis of Cry toxin deletions, the role of the N- and C- terminal domains in crystal formation and in toxin activity against different insects has been elucidated. Bt 3D crystal pro-toxins form crystalline inclusions that probably provide protection against proteolytic degradation by the host cell or external environment. Crystal solubilization in the gut of the target insect is required, however, for toxin action. This solubilization step occurs in many cases through a series of cleavages of the C-terminal half of the protoxin. The C-terminal half of the protoxin is cysteine-rich with disulfide bonds and salt bridges that allow for crystal formation
[22,33].
The truncation of Bt toxins has proven to be a useful option for enhancing Bt toxin activity. The goal of this strategy is to circumvent the toxin activation step resulting in improved toxicity against target pests. Toxin truncation also improves expression levels in planta and deletion analyses yield important information about the molecular biology and mode of action of these toxins.
Deletion Analyses: Localization of the Toxic Portion of the Protoxin
Cry protoxins are proteolytically activated to produce the mature active toxin.
This activation step restricts the spectrum of toxicity of these toxins to insects with appropriate gut proteases, with activation of the Bt crystal protein being essential for toxin action [28]. Low susceptibility may be due to lack of proper proteolytic activation or over-digestion of toxin to inactive peptides, and thus a basic knowledge of the toxic 12 portion is helpful for design of new toxins to control non-susceptible pests. To identify the minimal gene fragment that encodes the toxic peptide, deletions at the 5’- or the 3’- end of the Cry1Ab gene from Bt Berliner 1715 [42] and Bt kurstaki HD-1-Dipel [43] and Cry1Ac of Bt kurstaki HD-73 [44] protoxins of ~130 kDa (~60 kDa activated) have been made. Analysis of Bt strains expressing the toxin-sized proteins, indicated that the
N-terminal half of the protoxin is sufficient for toxicity [42-44]. Approximately 400 amino acids can be removed from the C-terminus of the toxin without significant loss of larvicidal activity. Several Cry1A toxins are proteolytically cleaved at residue 28 at the N- terminus [45], followed by removal of the α-helix upon contact with the brush border membrane [46]. Notably, the minimal toxic fragment corresponds to the trypsin- resistant polypeptide [42,45,47]. The full-length (72 kDa) and truncated (61 kDa) forms of Cry11A were expressed in cells of the fall armyworm ( Spodoptera frugiperda ) and in larvae of the cabbage looper ( Trichoplusia ni ) by using a baculovirus expression vector.
While the full-length protein was highly toxic to mosquito larvae, the truncated protein with a 9.6 kDa deletion at the N-terminus was non-toxic [48]. This result corroborates the requirement for most of the N-terminal region either directly or indirectly (i.e. required for appropriate folding) for toxicity.
Crystallographic structural studies [49] were of tremendous value for prediction of functions of the various Cry domains. Based on structural predictions , deletion of 42 amino acid residues from the N-terminus of Cry2A domain I resulted in a 4- to 6-fold increase in toxicity against the Egyptian cotton leafworm ( Spodoptera littoralis ), cotton bollworm ( Helicoverpa armigera ),and the black cutworm (Agrotis ipsilon) [50]. It was 13 speculated that the interaction of the N-terminal hydrophilic helix within the putative transmembrane domain I could interfere with toxin insertion into the membrane, which is required for toxicity [50]. The carboxy-terminal extensions of many Cry toxins mediate formation of bipyramidal crystals that are soluble at high pH under reducing conditions, typical of the lepidopteran midgut. Deletion of the Cry15Aa C-terminal sequence showed that this sequence is not required for activity against the codling moth ( Cydia pomonella ) [51]. In contrast, bipyramidal crystal formation precluded activity against the
Colorado potato beetle ( Leptinotarsa decemlineata ). In this case, activity could be rescued by solubilization of the toxin [52].
Knowledge of the toxic portion of a given toxin is also useful for Cry gene shuffling. The Cry1Ia truncated protein lacking the C-terminus was used to generate mutated variants, by combining Cry gene shuffling with phage-display [53]. The screening of this library for variants that bind brush border membrane vesicles (BBMV) of the banana stem borer ( Telchin licus licus ) and for toxicity, revealed four variants with greater activity against this pest compared to the non-toxic wild type Cry1Ia. These variants provide promising candidate toxins for the future development of T. l. licus resistant transgenic plants.
The truncated Bt toxin gene without the protoxin C-terminus was used for expression of the toxin in the early stages of developing Bt transgenic plants. The Cry1A protoxin was toxic to transgenic tobacco plants [54] while the toxin truncated at the C- terminal proteolytic cleavage site that generates the mature toxin, was non-toxic to the plants [54]. The truncated toxin did confer resistance to the tobacco hornworm 14
(Manduca sexta ), however. Furthermore, expression levels in transgenic tobacco sufficient for insecticidal activity were only achieved with the truncated Cry1A, and not with the Cry1A protoxin [55].
The extensive use of Bt toxins has resulted in field-evolved resistance in some insect pests. Toxin modification for greater efficacy against resistant insects has been achieved with the genetically modified Cry1AbMod and Cry1AcMod toxins. Compared to
Cry1Ab and Cry1Ac, both Cry1AbMod and Cry1AcMod lack 56 amino acids at the N- terminus, including the α-1 helix in domain I. Resistant insects with cadherin gene mutations such as pink bollworm ( Pectinophora gossypiella ) [56] were susceptible to
Cry1AMod toxins, supporting the conclusion that Cry1AbMOD bypasses the toxin oligomerization step induced by binding to cadherin. Interestingly, these Cry1AMod toxins were also toxic to strains of the diamondback moth ( Plutella xylostella ) and
European corn borer ( Ostrinia nubilalis ) which are resistant to the native toxins Cry1Ab and Cry1Ac, but in these examples resistance was not linked to mutations in cadherin
[57]. Recently, Cry1AbMod toxin was reported to counter resistance due to low cadherin expression, but not low alkaline phosphatase expression [58].
Improved Expression of Truncated Toxins
In addition to enhanced in planta expression of the truncated toxin compared to expression of the protoxin, Hayakawa et al. [59] designed a gene encoding the truncated
Cry4Aa with the GC content and codon preference of E. coli . These modifications resulted in significant improvements in expression levels in E. coli , with the recombinant 15 toxin exhibiting the same toxicity against larvae of the common house mosquito ( Culex pipiens ) as the native Cry4Aa.
Park et al. [60] obtained inclusions of truncated Cry1C (Cry1C-t) by combining genetic elements from other endotoxin genes and operons that enhance Cry protein synthesis. Increased levels of Cry1C-t synthesis were achieved by using Cyt1A promoters to drive expression of the N-terminal half of Cry1C, shown to be toxic to beet armyworm larvae ( Spodoptera exigua ) when expressed in the HD1 isolate of B. thuringiensis subsp. kurstaki [60].
Cleavage Site Modifications
Processing of the Cry protoxin into its active form is essential for toxin activity.
Processing is mediated by insect proteases that cleave the protoxin polypeptide at specific sequences. Refer to [61] for a detailed review of the role of proteases in Cry toxin action. Processing of protoxins has been best described in the 3D group of Cry toxins, with relatively little information available on the processing of Cyt, Vip, and binary Cry toxins. Cleavage of the 3D Cry toxins results in the removal of N-terminal peptides, and occasionally C-terminal peptides in larger protoxins, producing the active toxin (Cry3) or a toxin intermediate (Cry1, Cry4, Cry11). For the latter, activation is completed by proteolysis at additional intramolecular sites to produce the active toxin fragments [24,62-64]. Proteolytic cleavage at these sites is required for subsequent events involved in toxicity [65-67], and may be exploited in the design and construction of modified toxins [67,68]. Proteolytic cleavage that results in toxicity is referred to as processing or activation. This section describes evidence supporting proteolytic 16 cleavage as an activation step in 3D Cry toxins and how this process can be utilized for toxin modification.
Toxicity and Protoxin Processing
One of the initial observations that suggested proteolytic processing as a toxin activation step is that feeding a susceptible insect species on protoxin or on activated toxin resulted in similar levels of mortality, and that toxicity could be attenuated when protoxin was fed in the presence of protease inhibitors [65-67]. Toxicity can also be achieved in a non-susceptible species by feeding with activated toxin [69,70] suggesting that inefficient processing of protoxin precludes toxicity. Fragments of activated toxin produced by intramolecular cleavage can remain toxic when both fragments are present in the gut lumen. For example, Cry4Aa is processed to 45 and 20 kDa fragments from a
130 kDa protoxin [64]. Yamagiwa et al. [71] produced GST fusions of both the 45 and 20 kDa fragments. Neither GST (glutathione S-transferase)-45 nor GST-20 was toxic on its own when assayed against Culex pipiens larva. However, the presence of both fragments resulted in insecticidal activity [71], indicating that both are needed for toxicity. Activated Cry toxins that exhibit appreciable toxicity against species that lack susceptibility to the protoxin could be modified by insertion of sequences recognized by the proteases of the target insect to facilitate protoxin activation. The following paragraphs discuss the molecular events following processing that may facilitate toxicity.
Toxin binding to insect gut receptors preferentially occurs after appropriate proteolytic cleavage. Greater specific association was noted between processed toxin 17 and brush border membrane vesicles (BBMV) than between protoxin and BBMV [65-
67,72,73]. Carroll et al. [66] found that chymotrypsin treated Cry3A bound specifically to L. decemlineata BBMV at the pH of the L. decemlineata gut (pH 7.4). In contrast, the
Cry3A protoxin did not display specific binding at either the pH found in the insect gut
(pH 7.4) or at a pH at which the Cry3A protoxin is soluble (pH 10). Trypsin or chymotrypsin treatment of Cry3Ba and Cry3Ca also resulted in specific binding to the
BBMV of L. decemlineata , while the protoxin did not exhibit specific binding [66,72,73].
Activated fragments of Cry1Ab following incubation with M. sexta gut juices bound
BBMV [74]. In ligand blotting experiments, processed Cry4Aa bound BBMV of C. pipiens following incubation with gut extracts [74]. These examples demonstrate that processing of the protoxin preceeds toxin binding to epithelial cells. These results suggest that following proteolytic cleavage the core toxin is relatively more accessible than protoxin for binding to epithelial cells.
Effects of Activation on Toxin Solubility
Proteolytic activation of toxins can also enhance solubility. Toxins with limited solubility form precipitates that restrict interaction with the insect gut environment and would be expected to have limited toxicity. Cry protoxin solubility is dependent on pH
[24,75]. Insect gut pH varies across insect orders; Lepidoptera and Diptera have an alkaline gut pH [24,76], and Coleoptera and Hemiptera have a neutral to acidic gut pH
[66,75,77,78]. Limited toxicity against non-susceptible insect species may result from a gut environment that is not conducive to toxin solubilization. However, Cry solubility can change upon proteolytic cleavage. For example, Cry3A protoxin is only soluble under 18 acidic (pH<4) and alkaline (pH>10) conditions. The susceptible insect species L. decemlineata has a neutral gut pH, an environment where Cry3A protoxin forms insoluble precipitates. However, Carroll et al. [66] showed that the solubility of Cry3A changes upon activation with chymotrypsin, increasing its solubility at neutral pH; both
Cry3A protoxin and activated toxin exhibit similar toxicity against L. decemlineata. These findings suggest that the challenge of toxin insolubility, and the resulting limited toxicity, can be resolved in cases where the insect gut environment is not conducive to solubility.
Activation is Facilitated by Insect Gut Proteases
The examples cited provide compelling evidence that proteolytic cleavage of Cry toxins can be crucial to toxicity. Proper activation of Cry toxins is facilitated by proteases present in the insect gut. Among susceptible species in the orders Lepidoptera and
Diptera, the major gut proteases are of the serine type [24,79], while in Coleoptera the major proteases are cysteine and aspartic proteases, although some use cathepsin G serine proteases. Since activation is a crucial step to achieve toxicity, it has been suggested that the type and/or abundance of insect proteases is important in contributing to toxin specificity. As mentioned earlier, Cry toxicity can be achieved in non-target insect species by protoxin activation prior to feeding. Porcar et al. [70] demonstrated that a mixture of Cry4A and Cry4B isolated from a recombinant strain of
Bt subsp. israelensis that was trypsin activated displayed enhanced toxicity against the pea aphid ( Acyrthosiphon pisum ) relative to non-activated Cry4, suggesting proteolytic activation as an important limiting step in Cry toxicity against aphids. The major proteases utilized by A. pisum are cysteine proteases of the cathepsin L and cathepsin B 19 type [77] whereas dipteran species susceptible to Cry4A and Cry4B have serine proteases [79]. Pea aphids therefore appear to lack the appropriate proteases required for, at least, Cry4 toxin activation.
Insect proteases may also be detrimental to toxicity by degrading or inactivating protoxins by cleavage at inappropriate sites. For example, extensive toxin degradation of several Cry1A toxins is implicated in the insensitivity of S. exigua. However, when toxin and protease inhibitors were fed in combination, a synergistic effect was observed against S. exigua larva, which was decreased as inhibitor concentrations were reduced
[80]. Similarly, increasing the stability of Cry1Fa toxin against larval gut proteases by a cadherin fragment, correlated with increased synergy of toxicity to larvae [81]. In addition to lacking the appropriate proteases required for toxin activation, non-target insects may also harbor toxin degrading proteases [61].
Examples of insufficient protoxin activation and degradation can also be found in cases of field-evolved resistance to Bt toxins. Alterations in protease content and/or activity can result in resistance. Cao et al. [82] investigated several stains of H. armigera with resistance to Cry1Ac and found cases of up-regulated and down-regulated protease activity conferring resistance in strains with low to moderate resistance. Decreased total protease activity resulting in insufficient activation of protoxin was found in some strains, while increased esterase, GST, and chymotrypsin activity resulting in toxin degradation was found in others [82]. These results are supported by in vitro work which described a link between insufficient activation of the protoxin and low toxicity 20
[83]. Although protease activity levels were less important in highly resistant strains, modulation of enzyme levels may be important in insects under low selection pressure.
Modification of Cry Protoxin to Facilitate Proteolytic Activation
Our current understanding of how 1) Cry protoxin processing precedes toxicity,
2) insect gut proteases mediate this processing, and 3) insect gut proteases play a role in determining the specificity of Cry toxins by appropriate proteolytic cleavage, suggests a method for broadening the specificity of 3D Cry toxins to include non-target insects as well as combating Cry resistance. Knowledge of the mode of action of the 3D Cry proteins and of insect gut physiology can be combined for the engineering of designer toxins. Walters et al. [67] demonstrated the feasibility of this approach by achieving toxicity with modified Cry3A (mCry3A) against the relatively non-susceptible western corn rootworm ( Diabrotica virgifera virgifera ). A chymotrypsin G cleavage site introduced between α-helices 3 and 4 of domain I was cleaved by western corn rootworm gut proteases. The introduced cleavage site resulted in enhanced activity and the activated mCry3A bound specifically to D.v. virgifera BBMV [67]. Insertion of protease recognition sequences at appropriate sites should result in enhanced processing of the protoxin to its active form. Similarly, in cases of toxin degradation, toxins can be engineered for removal of deleterious protease sites. Bah et al. [68] created Cry1Aa constructs designed to resist degradation in the spruce budworm
(Choristoneura fumiferana ) by mutating potential trypsin and chymotrypsin sites. These modifications resulted in a 2-4 fold increase in toxicity [68]. Understanding of the protease type in the gut of the target insect, as well as the stability and solubility of the 21 candidate Cry toxin will be essential for the success of designer toxins. Fortunately the
Cry gene database is vast, and the structure, stability and solubility of many Cry toxins have been categorized. Although Cry3A is the only toxin engineered using modified cleavage sites to date, the success of this approach, as well as the toxicity of activated toxins against non-susceptible insects suggests that the approach could be more widely applied.
Potential Post Binding Modification
The focus of this section has been on the processing events that precede Cry toxin binding. However, post binding cleavage could be important for pore formation, a process referred to as proteolytic nicking [84,85]. This process adds an additional facet to Cry toxin modification by introducing cleavage sites to promote pre-pore structures and pore formation after binding. However, it is unclear how important post binding cleavage is for toxicity. Pore formation may not be dependent on post binding cleavage, and protease activity at the membrane of epithelial cells may inhibit pore formation [86-
88]. Additional research is needed to clarify post binding events to assess the potential for Cry modification to stimulate pore formation.
Binding Modifications
Proteolytic activation of Bt toxins, and the subsequent binding to the insect gut epithelium, are important steps for toxicity (reviewed in [23]). Modification of either or both of these steps for a particular Bt toxin should therefore result in altered host range and/or altered toxicity. Several approaches have been taken to modify the binding specificity and affinity of Bt toxins with the ultimate goal of producing designer toxins 22 that target new pest species and counter field evolved resistance. The alteration of Bt toxin binding affinity and specificity can be broken down into four categories: domain or loop swapping between Cry toxins, site-directed mutagenesis, incorporation of binding peptides or fragments from non-Bt toxins, and the generation and subsequent display of
Cry toxin mutant libraries on phage.
Domain Swapping
Domain swapping between Cry toxins is perhaps the oldest method used for engineering toxins with novel properties. These large exchanges can also provide information on the function of the exchanged segments. For example, domain III exchanges between Cry toxins have implicated this domain in both toxin binding and host specificity [89-92]. For this reason, domain III of three domain Cry toxins is used extensively in domain swapping experiments. However, at least one domain II exchange has been successful, with movement of domain II of Cry1Ia to Cry1Ba resulting in a toxin with strong activity against L. decemlineata [93].
Exchanging domain III of the 3D Cry1 toxins between Cry1 toxins has been used to enhance toxicity against particular insect pests [89-91,93], and to enhance the toxicity of more distantly related 3D Cry toxins [92]. It is often the case that the substitution of all or part of domain III from one Bt toxin to another confers the specificity of the donor toxin upon the recipient toxin [89-91]. However, there is an instance of a domain III exchange from a Cry1A-active toxin altering the binding specificity of a coleopteran-active Cry3A toxin. Hybrid Bt toxin, eCry3.1Ab, was generated by the incorporation of a large section of the lepidopteran-active domain III 23 of Cry1Ab into the coleopteran-active modified Cry3A (mCry3A) [67,92] and the resulting hybrid toxin displayed significant activity (approximately 94% mortality) against D. v. virgifera [92]. Interestingly, this is the only documented case of a successful domain exchange between more distantly related Cry toxins.
Although there has been success in using domain swapping as a method for modifying Bt toxins for enhanced efficacy, this approach is limited by a lack of knowledge of what domain II and domain III contribute to the activity of each toxin
[89,91]. Until these issues are addressed, the rational design of Bt toxins via domain swapping is hampered.
Site-Directed Mutagenesis
Site-directed mutagenesis of Cry toxins has resulted in impressive enhancements in toxicity against various insect species. Domain II has been heavily targeted for modification as this domain is involved in receptor binding [24]. Modifications have been used to enhance toxicity of Bt toxins that already display activity against the target insect [94], as well as against non-susceptible insects [95-98]. Loop 1, loop 2, and loop 3 of domain II are of particular interest because they interact with receptors in the gut of the target pest. These loops have been successful targets for alteration, although the success of the loop modifications appears to depend on both the toxin and the target insect. Loop 1 of domain II of Cry3A was altered, resulting in mutants displaying significant increases in toxicity against L. decemlineata , the yellow mealworm ( Tenebrio molitor ), and the cottonwood leaf beetle ( Chrysomela scripta ), as well as an increase in binding affinity to L. decemlineata BBMV [94]. Modifications to loop 1 and loop 2 of 24 domain II of Cry19Aa made it more Cry4Ba-like: Deletions in loop 2 of domain II and the substitution of Cry4Ba loop 1 residues into loop 1 of Cry19Aa resulted in the mutant toxin, designated 19AL1L2. This toxin showed a 42,000-fold increase in toxicity to the yellow fever mosquito ( Aedes aegypti ) [97]. In stark contrast, when attempting to introduce activity against the southern house mosquito ( Culex quinquefasciatus ) and C. pipiens to Cry4Ba, any modifications to loop 1 or loop 2 of domain II resulted in a loss of activity against A. aegypti and common malaria mosquito ( Anopheles quadrimaculatus ).
However, activity against C. pipiens and C. quinquefasciatus was successfully introduced to Cry4Ba through modifications to loop 3 of domain II [96]. These results support the variability of Cry toxin interactions with insect gut receptors based on the particular Cry toxin and insect species, an important factor to consider when selecting a toxin to modify for activity against a particular pest. A toxin with a basal level of toxicity against the target pest is optimal for enhancement by toxin modification.
Site-directed mutagenesis was used to dramatically shift the specificity of
Cry1Aa, a lepidopteran-active toxin. This toxin was modified for toxicity against C. pipiens . Cry1Aa did not cause observable mortality in C. pipiens when fed at a concentration of 100 μg/ml. Loop 1 and loop 2 of domain II were modified to mimic loop 1 and loop 2 of Cry4Ba in both amino acid sequence and loop length. The resulting mutant toxin lost activity against the lepidopteran M. sexta but gained activity against C. pipiens , with an LC 50 of 45 μg/ml [98], marking a considerable improvement in toxicity against this insect. This work further supported the importance not only of the amino acid residues involved in binding to the receptors, but of the length and shape of the 25 loops. Loop 1 of Cry1Aa is shorter than that of Cry4Ba, while loop 2 of Cry1Aa is longer than loop 2 of Cry4Ba. When mutant toxins were produced with a longer loop 2, mosquitocidal activity was not observed [98].
Site directed mutagenesis has been used successfully to modify Bt, and offers a powerful tool to combat resistance in Bt susceptible pest species, as well as to broaden the spectrum of insect pests targeted by Bt toxins. In addition, this approach has provided important insight into the specific Cry toxin residues that interact with insect gut receptors.
Use of Synthetic Peptides and Non-Bt Toxin Regions
The use of synthetic gut binding peptides and non-Bt toxin fragments in the engineering of Bt toxins is a relatively new approach, which has the potential to easily enhance the toxicity of Bt toxins against pests which have little to no susceptibility. The first example involved the fusion of the non-toxic lectin, ricin B-chain, to the C-terminus of lepidopteran-active Cry1Ac via a four amino acid linker region, to produce a toxin designated BtRB [99]. BtRB was expressed in rice and maize and subsequently tested against the striped stem borer ( Chilo suppressalis ), cotton leaf worm S. littoralis , and the maize leafhopper ( Cicadulina mbila ). Mortality was increased against the Cry1Ac susceptible C. suppressalis , from approximately 17% to 75% on maize and from 20-30% to 60-90% on rice. BtRB substantially increased the mortality of Cry1Ac-tolerant S. littoralis , from less than 20% on control maize plants to 78% on BtRB-expressing maize plants at 4 days. The mortality of C. mbila fed on control maize plants was about 20%, while the mortality on BtRB-expressing maize plants was about 95%. Although the ricin 26
B-chain did enhance the activity of Cry1Ac, it did not do so indiscriminately as neither
Cry1Ac nor BtRB displayed activity against the bird cherry-oat aphid ( Rhopalosiphum padi ) [99]. This result may reflect the specificity of ricin B binding to galactose residues and the abundance of these residues in the insect gut, however as discussed above, a number of other factors affect Cry host range. The introduction of toxicity to a Bt toxin against a hemipteran, C. mbila , was significant however, as few Bt toxins display toxicity against hemipteran pests [70,100,101]. In a similar study, replacing domain III of Cry1Ac with Allium sativum agglutinin (ASAL; a mannose-binding lectin) resulted in a 30-fold increase in toxicity against H. armigera and an 8-fold increase against P. gossypiella , as well as an expanded binding profile to H. armigera BBMV in a ligand blot [102].
Through the addition of lectins to Cry1Ac, toxicity against susceptible and non- susceptible insect pests was enhanced, albeit in an unpredictable manner. The incorporation of short gut binding peptides, identified via a phage display library, into Bt toxins provides a method to target specific pests that lack susceptibility to Bt toxins. To date, two papers have been published on the use of receptor binding peptides to modify
Bt toxins.
A 12-aa A. pisum gut binding peptide, designated GBP3.1, previously identified by feeding A. pisum on a phage display library [103] and subsequently found to bind to
A. pisum alanyl aminopeptidase-N (APN), was incorporated into different loops of the cytolytic Bt toxin Cyt2Aa through the addition or substitution of amino acids [104]. The incorporation of GBP3.1 into loops 1, 3 and 4 of Cyt2Aa resulted in mutants with significantly higher binding to A. pisum BBMV, as measured by pull-down assays and 27 surface plasmon resonance. The increase in binding was accompanied by a significant improvement in toxicity to A. pisum , lowering the LC 50 from > 150 μg/ml to 10-29 μg/ml.
The mutant Cyt2Aa toxins also displayed improved activity against the green peach aphid ( Myzus persicae ), lowering the LC 50 from >150 μg/ml to 4393 μg/ml [104].
Interestingly, while GBP3.1 bound to APN, a glycosylphosphatidyl inositol (GPI)- anchored membrane bound protein that has been implicated as a receptor for Bt toxins, in the case of Cyt and possibly the 3D Cry toxins, the specific protein which binds the toxin may not matter.
The ‘sequential binding model’ of 3D Cry toxin-receptor interactions, where Cry toxins interact with a number of insect midgut proteins that facilitate formation of oligomeric structures and membrane insertion [105], supports the concept that receptor identity may not be as fundamental as once thought. For example, the highly toxic Cry11Ba toxin binds at least four different GPI-anchored proteins (alkaline phosphatase, aminopeptidase, and two glucosidases), plus two cadherin-like proteins in the midgut of the malarial mosquito Anopheles gambiae [106,107]. It may be advantageous from the perspective of insecticidal potency to have multiple midgut binding proteins acting as ‘accessory’ receptors to gather toxin at the midgut brush border protein level. The concept of increasing toxin retention leading to increased potency is suggested by the following example: an Asian longhorn beetle ( Anoplophora glabripennis Motschulsky) Cx-cellulase binding peptide, designated PCx, was used to modify Cry3Aa. PCx was fused to either the N-terminus (PCx-Cry3Aa) or C-terminus
(Cry3Aa-PCx) of Cry3Aa. PCx-Cry3Aa and Cry3Aa-PCx both showed increased toxicity 28 against the Mulberry longhorn beetle ( Apriona germari Hope), roughly doubling the mortality of A. germari compared to Cry3Aa. In addition the retention of PCx-Cry3Aa in the midgut of A. germari was significantly increased relative to Cry3Aa [108]. This approach suggests that increasing the ‘bindability’ of Cry toxins may augment recognized Bt toxin receptors such as cadherin, APN, and ALP. Alternatively, increased retention of Bt toxin in the insect gut, as well as proximity to the gut epithelium, may in itself be sufficient for increasing toxicity and target range.
Phage Display
A phage display library expresses a diverse array of peptides or proteins on the surface of bacteriophage. Peptides or proteins that have a higher target binding affinity are selected by screening the phage display library against purified receptor or BBMV immobilized on a substrate [53,109-111]. Alternatively, feeding assays can be used to isolate peptides or proteins that bind to the gut epithelium of a targeted insect [103].
Unbound phage are removed by washing. Bound phage are then eluted and amplified in E. coli for subsequent rounds of selection, and selected phage sequenced to identify sequences encoding peptides of interest.
Phage display libraries expressing mutant Cry toxins offer a potentially powerful method for engineering Cry toxins. The most important aspect of this methodology is the successful display of complete Cry toxins on the surface of a phage, followed by the generation of phage display libraries expressing mutant Cry toxins and the subsequent selection of mutant Cry toxins via binding affinity to BBMVs or purified gut receptors. 29
The display of Cry toxins on the surface of phage has been attempted several times over the past twenty years, with initial attempts resulting only in partial success.
The earliest published attempt to display a Cry toxin on a phage succeeded in only displaying domain II or only loop 2 of domain II of Cry1Aa on M13 phage, despite attempts to display the activated toxin. In this case, the E. coli were unable to produce the activated toxin, possibly due to Cry1Aa being directly or indirectly toxic to the cells
[112]. Despite not displaying activated Cry1Aa, the successful display of domain II and loop 2 of domain II still allowed for the generation of combinatorial libraries that could be used to identify sequences with higher binding, and thus potentially enhanced toxicity, once incorporated into the complete toxin. This goal could also be accomplished by incorporating short binding peptide sequences identified via phage display library into any number of Bt toxins [103-108]. A concurrent attempt to display
Cry1Ac on the surface of a phage was more successful in that Cry1Ac was displayed, and was active against M. sexta and H. virescens . However, when exposed to BBMV, Cry1Ac was cleaved from the phage, limiting in vivo and in vitro applications [113].
Recent efforts with phage display of Bt toxins have been more successful. Cry1Ac was successfully displayed on λ phage by fusing the toxin to the structural D protein, an essential protein for phage stability found in the head of λ phage. The fusion protein successfully bound to brush border membrane proteins, purified from a preparation of
M. sexta BBMV. However, the display of Cry1Ac on λ phage did result in a 20-fold decrease in toxicity against M. sexta [114]. The decrease in toxicity may have resulted from the λ phage inhibiting secondary binding events or pore formation. Fusion of 30
Cry1Ac to the T7 phage structural gene 10B provided comparable mortality in M. sexta to Cry1Ac [115]. Despite the decreased toxicity seen in some instances, the successful display of Cry1Ac on the surface of λ phage and interaction of the displayed toxin with gut receptors is a significant step forward for use of phage display libraries for selection of toxins with enhanced binding properties. The screening of libraries displaying complete toxins under in vivo conditions is less practical, as toxins with increased binding are expected to damage the gut epithelium or kill the insect, thereby hampering isolation of bound phage for further enrichment.
There have been a few attempts to generate and screen Cry mutant libraries for enhanced binding and toxicity via phage display [53,109-111]. Two of these attempts involved the generation of domain II mutant libraries [109,111], while the other two mutant libraries were generated by introducing mutations into random locations in Cry genes [53,110]. However, a noted difficulty with this approach is generation of inclusive mutant libraries [111]. This problem could be alleviated by targeting shorter amino acid sequences for mutation [109], decreasing the number of possible permutations of the target sequence, thereby decreasing the number of mutants needed for an inclusive library. As stated previously, an important consideration is the probable difficulty of in vivo biopanning, as toxin-induced mortality could hamper elution of bound phage from the insect gut. This limitation restricts library screening to the use of purified receptors
[109,111], or BBMV [35,92], which do not necessarily represent biologically relevant conditions, and could lead to the selection of toxins that are not active under in vivo conditions. 31
The generation of mutant Cry toxin libraries and their display on various phage provides a powerful approach for the selection of toxins with enhanced efficacy against target insect pests, but with important limitations. Production of a representative mutant library, particularly when altering specific regions of a Cry toxin, is an important but technically limiting step. Phage display library screens conducted in vitro may not result in toxins active under in vivo conditions.
Concluding Remarks
Toxins derived from the bacterium B. thuringiensis have been used extensively to manage insect pest populations, traditionally through foliar sprays and more recently via transgenic crops. A thorough understanding of the mode of action of these toxins is required for full exploitation of their potential. Some insects of agricultural and human health importance lack susceptibility to Cry toxins while other species evolve resistance in response to intense selection pressure. The lack of toxicity in these cases likely involves interference with crucial steps in toxin action: N- or C-terminal cleavage, intramolecular proteolytic cleavage, and / or binding to the insect gut epithelium (Fig
1a). Based on understanding of Cry toxin mode of action and the gut physiology of targeted pests, specific modifications have been made to counter these limitations to toxicity. Toxin modification has resulted in toxins with increased efficacy against various target pests, and increased understanding of toxin mode of action. Incorporation of such modified toxins into current management strategies will maximize the use of Bt as a tool for crop protection and for management of insect borne disease.
32
Research Problem
Of the Bt-derived toxins that have been tested for efficacy against aphids, we selected the 3-domain toxin, Cry4Aa, for genetic enhancement on the basis that low- level toxicity has been recorded [70]. A lack of binding to soybean aphid gut is a potentially important bottleneck in the low toxicity of Cry4Aa against soybean aphid. To improve toxicity of Cry4Aa against soybean aphid, we isolated and incorporated a soybean aphid gut binding peptide into Cry4Aa, to provide for increased binding to the target gut epithelium cells. Increased toxin binding to the soybean aphid gut epithelium is expected to result in increased toxicity against this pest.
Thesis Organization
In Chapter 2, we report on the isolation and use of the soybean aphid gut binding peptide, SG3, in the modification of Cry4Aa for enhanced binding to the soybean aphid gut. Significant findings from this work and potential future research directions are discussed in Chapter 3.
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CHAPTER 2. MODIFICATION OF A CRY TOXIN FOR ENHANCED EFFICACY AGAINST SOYBEAN APHID (Aphis glycines )
Benjamin R. Deist, Nanasaheb Chougule, Biviana Flores-Escobar, Maria Teresa Fernandez-Luna, and Bryony C. Bonning Department of Entomology, Iowa State University, Ames, IA 50011
Abstract
Soybean aphid poses a significant threat to soybean agriculture in North
America. Current management strategies are heavily dependent on chemical insecticides, which while presently effective, may have negative side effects. Bt toxins have been successfully used in various transgenic crops for almost 20 years to manage lepidopteran and coleopteran pests. However, this strategy has not been used in the management of aphids and other hemipterans due to their low susceptibility to Bt toxins, potentially arising from a lack of binding to the hemipteran gut. To circumvent this limitation in soybean aphids, a phage display library was screened in vivo against the soybean aphid gut to identify candidate gut-binding peptides to add to a Bt toxin.
The in vitro binding capacity of candidate gut-binding peptides was characterized. The candidate with the strongest gut binding was used to engineer the Bt toxin Cry4Aa by insertion of the peptide sequence at seven sites in the toxin: N-terminal region (three sites), loop 2 and loop 3 of domain II, and the loops formed between β12-β13 and β15-
β16 of domain III. Four of the seven engineered toxins maintained stability upon activation and native toxicity against Culex pipiens larvae. The four remaining toxins are currently being tested against soybean aphid.
46
Introduction
Soybean aphid ( Aphis glycines Matsumura) is the foremost pest of soybean
(Glycines max ) in North America. Since the first identification of the soybean aphid in
North America in 2000, soybean aphid has been identified in 30 U.S. states and 3
Canadian provinces [1]. The soybean aphid can cause up to 40% yield loss via feeding, the transmission of plant viruses, and promotion of the growth of sooty mold on the surface of the soybean [1–3].
Current management practices for soybean aphid rely heavily on the use of chemical insecticides, [1,4,5]. However, should resistance to chemical insecticides develop, rapid population growth allows soybean aphid to quickly spread resistance, making the development of alternative management strategies imperative [1,4,5].
Bacillus thuringiensis (Bt) is a soil-dwelling pathogen of invertebrates [6,7]. Bt produces a wide array of toxins, most notably the Crystal (Cry) family of toxins. Specific
Cry toxins display toxicity against lepidopteran, coleopteran, and / or dipteran insects, and kill by disrupting gut integrity via a complex and incompletely understood mode of action [8]. The established steps in the mode of action of Cry toxins are: Solubilization in the insect gut, activation, binding to gut receptors, membrane insertion, and pore formation leading to osmotic lysis [8–10].
Cry toxins have been expressed in transgenic crops for the management of coleopteran and lepidopteran pests for close to twenty years [2]. The success of Cry toxins partially stems from their high degree of specificity, in general Cry toxins are primarily active against lepidopterans, coleopterans, and dipterans [8]. Some 47 hemipteran species, including aphids, have limited susceptibility to Cry toxins [11–14].
Low toxicity of Cry toxins against aphid species can be be explained in some cases by inefficient toxin activation, and / or a lack of Cry toxin binding in the aphid gut [11,15].
Cry toxin engineering can be used to enhance toxicity of Cry toxins with limited activity against a particular species by targeting bottlenecks to toxicity [16].
Cry4Aa is a 3-domain Cry toxin that displays a low level of toxicity against the pea aphid ( Acythrosiphon pisum ), making it an ideal candidate for modification to enhance toxicity against not only pea aphid, but other aphid species such as soybean aphid
[11,17]. Bt toxins, primarily Cry toxins, have been engineered for enhanced activity against novel insect targets, largely through increased processing and increased binding of toxins to the insect gut [16]. Here, we modified Cry4Aa to enhance toxicity against the soybean aphid via the addition of a soybean aphid gut binding peptide. In separate modified toxins, the peptide was added to each of five sites within exposed loops or at the N-terminus of the protein. By incorporating a soybean aphid gut-binding peptide our goal was to increase binding to the gut for enhanced toxicity against the soybean aphid.
Materials and Methods
Soybean Aphid Rearing
Soybean aphids were field collected from Iowa. Soybean aphids were reared in a growth chamber on Williams 82 soybean, ( Glycines max ) (Continuous light, 22°C, 70%
RH). 48
Expression and Purification of Soybean Aphid Alkaline Phosphatase 2 and Aminopeptidase N3
We used two approaches for isolation of peptides that bound to the soybean aphid gut. The first approach was to PCR-amplify cDNA for the abundant, membrane- associated proteins alkaline phosphatase 2 (ALP2) and aminopeptidase N3 (APN3) for expression in insect cells using a recombinant baculovirus expression vector. These sequences were identified from the soybean aphid gut transcriptome [18], PCR amplified and cloned into pFastBac™ (Invitrogen™ by Life technologies™) using standard procedures. Sf21 cells (Invitrogen™ by Life technologies™) in T75 flasks were infected with ALP2 or APN3 baculovirus at an MOI of 5 and cells were incubated at 28 oC. Cell culture medium was harvested 96 hours post infection, centrifuged at 3,000g for 10 minutes [19] and recombinant protein expression assessed by SDS-PAGE (12%).
Recombinant, 6X His-tagged, APN was purified by affinity chromatography, using a TALON agarose (Clontech) column equilibrated with 50 mM NaH 2PO 4, 100 mM NaCl pH 8.0 (Buffer A) and washed with 10 volumes of Buffer A containing 5 mM Imidazole.
Protein was eluted with 1.5 ml Buffer A containing 10 mM imidazole. The collected fractions were analyzed by SDS-PAGE (12%), enzymatic activity [19] and the APN- containing fractions were dialyzed.
Recombinant, 6X His-tagged, ALP was purified by anionic exchange chromatography using a Sepharose Q Fast flow column (GE) equilibrated with 20 mM
Tris-HCl pH 8.5, 1 mM MgCl 2. Anionic exchange chromotagraphy was used instead of affinity chromatography due to inadequate purification by use of the TALON agarose column. Protein was eluted with a NaCl gradient (0.5 – 1 M). The collected fractions 49 were analyzed by SDS-PAGE and enzymatic activity [19]. ALP-containing fractions were dialyzed. For both recombinant proteins, concentration was quantified by Bradford assay with BSA standards [20].
Screen for Peptides that Bind ALP and APN
Peptides were selected from the Ph.D-C7C phage display library. The library is comprised of peptides displayed on the surface of the M13 phage. The peptides consist of a seven amino acid variable region flanked by cysteine residues to form a disulphide bridge, resulting in the seven amino acid variable region configured in a loop. The Ph.D.-
C7C library (New England Biolabs; 10 10 PFU) was diluted 100 fold in 100 uL of 50 mM
Tris-HCl, 150 mM NaCl, pH 7.5 (TBS buffer) and 0.1% Tween. Three cycles of biopanning were performed as follows: polystyrene microtiter wells were coated with ALP or APN
(10µg/ml in TBS Buffer – 0.1% Tween) overnight on a rocking platform at 4°C. Excess binding sites were blocked with 0.1M NaHCO 3 (pH 8.6), 5mg BSA at 4°C for 1h. After 6 washes with TBS –Tween 0.1%, the phage library was added. The library was incubated
1 hour at room temperature on a rocking platform. Unbound phages were removed by
10 washes with TBS – Tween 0.1%. The binding interactions with the phages were disrupted with 0.2M Glycine –HCl (pH 2.2), and 1 mg/ml BSA for 20 minutes, and the reaction was neutralized with 1M Tris-HCl (pH 9.1). An aliquot of the eluate was titered and the remaining eluate was amplified in Escherichia coli for subsequent rounds of biopanning. A total of three rounds of biopanning were performed with TBS with 0.5%
Tween. After the third round, peptide sequences encoded by selected phages were determined from 20 plaques. These peptide sequences were compared to the 50 sequences of peptides selected for binding under in vivo conditions (as described below), but the peptides were not used for further experimentation.
Selection of Soybean Aphid Gut-Binding Peptides
The second approach for isolation of peptides that bind the soybean aphid gut was to screen the same phage display for in vivo binding to the gut. Soybean aphid Gut binding (SG) peptides were identified from the Ph.D.-C7C phage display library (New
England Biolabs) following a previously described in vivo selection protocol [21]. After the third round of enrichment for gut binding peptides, the peptide sequences of 27 phages were determined by sequencing phage DNA.
Construction of Gut Binding SG-GFP Fusions
For further characterization of peptide binding to the soybean aphid gut, constructs for expression of soybean aphid gut binding SG-GFP fusions were generated using PCR. The nucleotide sequence of each SG peptide was incorporated at the 5’ end of the forward primer for GFP (5’-GCTGTGAGCAAGGGCGAGG-3’). Each unique forward primer was paired with the reverse GFP primer
(5’-CCGAAGCTTTTAGCCGCTTTACTTGTACAGCTCG -3’). The SG peptides were connected to GFP via a three glycine linker region, which was used to maintain peptide conformation.
A linker control, comprised of three glycines attached to GFP, was synthesized.
Platinum®Taq (Invitrogen™ by Life technologies™) was used to make each fusion construct. The size of each SG-GFP fusion construct was checked via agarose gel electrophoresis and ethidium bromide staining. Bands of the expected size were 51 excised and the DNA purified using a QIAquick gel extraction kit (Qiagen, Venlo,
Limburg, Netherlands).
SG-GFP fusions were cloned into pBAD-HisB using XhoI and HindIII restriction sites, and subsequently transformed into Top10 Z-competent E. coli cells. Expression of
SG-GFP fusions was induced in 500 ml LB cultures with 0.02% arabinose. The induction was allowed to continue overnight at 20°C and shaking at 220 rpm. After induction, the cultures were centrifuged at 3,000 rpm and the pellets stored at -80°C until purification.
SG-GFP fusion proteins were purified using nickel affinity purification. Purified SG-GFP proteins were assessed by SDS-PAGE and purified protein was quantified via Bradford assay [20].
Preparation of Soybean Aphid Brush Border Membrane Vesicles (BBMV)
Soybean aphid brush border membrane vesicles were prepared following a standardized protocol. Differential precipitation was used to prepare BBMV from 1 gram of whole soybean aphids [22]. The enrichment of BBMV was quantified using an aminopeptidase N (APN) activity assay to assess enrichment of membrane proteins from an early homogenate to the final BBMV. BBMV sample protein content was quantified using a Bradford assay [20]. Protease inhibitor cocktail (Sigma; P8340) was added before BBMV samples were stored at -80°C.
Relative Binding of Soybean Aphid Gut Binding Peptides
The relative binding of gut binding peptides to BBMV was assessed by pull-down assay at room temperature as described previously [23]. Binding conditions were: PBS
(137 mM NaCl, 2.7 mM KCl, 10 mM Na 2HPO 4, 1.8 mM KH 2PO 4), pH 7.4, 1% BSA, and 1% 52
Tween20. Pull-down assays were conducted in duplicate. An ANOVA was performed to identify significant differences in binding based on ImageJ quantification of SG-GFP fusions pulled down with BBMV. Based on the results, the peptide SG3, which was among the peptides that bound the most strongly, was selected for further analysis.
Predictive Modeling of Modified Cry4Aa-S1
Four exterior loops, as well as the N-terminus, were identified as potential sites for addition of the gut binding peptide, SG3. Predictive modeling of modified toxins with the peptide sequence incorporated at different sites on each loop was performed using
Local Meta-Threading-Server (LOMETS) [24]. The amino acid sequences of modified
Cry4Aa-S1’s were submitted to LOMETS and the resulting models were visualized using
PyMol ( The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC.)
Particular attention was paid to whether the models predicted exposure of the peptide sequence on the outside of the toxin molecule, rather than the peptide sequence folding inward. Specific sites for insertion of the gut binding peptide were selected based on these predictive models.
Modification of Cry4Aa-S1
Optimized for expression in E. coli, Cry4Aa-S1 shares the same amino acid sequence as Cry4Aa but differs in nucleotide sequence; in addition Cry4Aa-S1 has a partial C-terminal truncation [25]. The soybean aphid gut binding peptide SG3, including the terminal cysteines, was added to seven separate sites in Cry4Aa-S1 via overlap extension PCR (OE-PCR). The sites were: at the N-terminus (three separate sites), loop 2 and loop 3 of domain II, and the loops formed between β12 and β13 and between β15 53 and β16 of domain III [26]. One addition was made at the N-terminus of the N-terminal peptide, one was made at the N-terminus of the activated toxin, and the final addition was made two amino acids (six nucleotides) in from the N-terminal peptide cleavage site. See Table 1 for the primers used. Platinum®Taq Hi-Fidelity (Invitrogen™ by Life technologies™) DNA polymerase was used in the modification of Cry4Aa-S1 . Genes encoding modified toxins, as well as wild-type Cry4Aa-S1 , were visualized using agarose gel electrophoresis with ethidium bromide staining. Bands of the appropriate size were excised and the DNA purified using a Qiaquick gel extraction kit (Qiagen, Venlo, Limburg,
Netherlands) following the provided protocol. Wild-type Cry4Aa-S1 and the modified toxins were cloned into pGEX-2T using the BamHI and EcoRI restriction sites. Plasmid constructs were then transformed into BL21 Z-competent E. coli .
Expression and Purification of Toxins
Expression of toxins, including the wild type Cry4Aa-S1 , was carried out using a standardized protocol. Overnight LB cultures (5 ml) were used to inoculate 500 ml cultures of Terrific Broth grown in 2000 ml baffled Erlenmeyer flasks. The cultures were grown at 37°C with shaking at 240 rpm until the appropriate OD 600 was reached. Once the OD 600 was reached, expression was induced with 0.06 mM isopropyl-β-D-1- thiogalactopyranoside (IPTG). Cultures were allowed to induce in darkness at 20°C for 5 hours at 220 rpm. After induction, cultures were centrifuged at 3000 rpm for 20 minutes and the cell pellet was stored at -80°C. Toxins were purified using Glutathione
Sepharose® (GSH) 4B beads (GE Healthcare Biosciences AB, Uppsala, Sweden). Toxins were eluted from the beads with 50 units of thrombin. The thrombin was allowed to 54
Table 1. Primer pairings to make modified Cry4Aa-S1. Bolded regions denote SG3 nucleotide sequence. Toxin Forward Primer (F) Reverse Primer (R) Cr4N1 5’ATAGGATCC TGTATGGGGCATACTTTGGGGAGGTGC ATGAACCCGTATCAAAAT3’ 5’TATGAATTCTCACACGGTTTC3’
CR4N2 5’ TGTATGGGGCATACTTTGGGGAGGTGC GGTGAACTGTCGGCA3’ 5’ GCACCTCCCCAAAGTATGCCCCATACA TGAATCGATGAACGTTTC3’
Cr4N3 5’ TGTATGGGGCATACTTTGGGGAGGTGC TCGGCATACACCATC3’ 5’ GCACCTCCCCAAAGTATGCCCCATACA CAGTTCACCTGAATCGA3’
Cr4L2 5’ TGTATGGGGCATACTTTGGGGAGGTGC TCGCTGGACAACAAA3’ 5’ GCACCTCCCCAAAGTATGCCCCATACA GATAACGTTCAGCAGAA3’ 54 Cr4L3 5’ TGTATGGGGCATACTTTGGGGAGGTGC ACCCAGGTGTACACG3’ 5’ GCACCTCCCCAAAGTATGCCCCATACA TTTGTACGTTGCCGG3’
Cr4B12 5’ TGTATGGGGCATACTTTGGGGAGGTGC TATACC CAC CTGACC3’ 5’ GCACCTCCCCAAAGTATGCCCCATACA GATCGTGTTTTTCGG3’
Cr4B15 5’ TGTATGGGGCATACTTTGGGGAGGTGC CAGCAATCGTACTTCAT3’ 5’ GCACCTCCCCAAAGTATGCCCCATACA AAAATTTGAGTGCTGGC3’
Cry4Aa 5'ATAGGATCCATGAACCCG3’ 5’TATGAATTCTCACACGGTTTC3’ 55 incubate with the beads overnight at 4°C, followed by a 2 hour incubation at 20°C. Five
500 μl fractions were collected after trypsin incubation. Fractions, along with samples of the cell lysate and flow through were checked using SDS-PAGE with 12% polyacrylamide gels. The buffer was exchanged to 10 mM Tris-HCl pH 7.5 using Amicon
Ultra-0.5 Centrifugal Filter Devices with a 3-kDa cutoff (Merck Millipore Ltd., Co Cork,
IRL). Toxin concentration was quantified via Bradford assay [20].
Structural Stability of Toxins
To assess the stability of the modified toxins, each toxin was activated using a trypsin digest. A 5% trypsin (w/w) digest was performed on 5 ug of each toxin. The reactions were carried out in 20 ul of 50mM Tris-HCl buffer. Reactions were incubated at 37°C for one hour. After incubation, 4 ul of 4X SDS-Loading dye was added to each reaction and samples were then heated for 8 minutes at 100°C. Samples were run on
12% SDS-polyacrylamide gels. Gels were stained in Coomassie blue R250, destained, and scanned using a tabletop scanner.
Bioassay with Mosquito Larvae
To assess whether the modified toxins retained their native toxicity, bioassays were conducted with mosquito larvae, which are susceptible to Cry4Aa. Bioassays were conducted using two-day-old Culex pipiens . Wild-type Cry4Aa-S1, and modified Cry4Aa-
S1 were used as treatments. It is important to note that while mosquito larval bioassays are normally performed using Cry toxin crystal, Cry4Aa-S1 does not form crystals.
Assays were conducted using a 24 well plate (Corning Incorporated, Corning, New York), with six C. pipiens larvae per well, with three wells per treatment. Each well contained a 56
final volume of 1-ml toxin in ddH 2O. Up to six different concentrations were used per treatment (31.25 ng/ml - 2μg/ml). The concentration ranges were selected based on published Cry4Aa LC 50 data for C. pipiens larvae, which are highly variable [27]. In addition to toxin treatments, each assay had a water only control. Mortality was scored every 24 hours for a period of 3 days. Each toxin was categorized as “toxic” or “non- toxic” to C. pipiens larvae based on bioassay data.
Soybean Aphid Bioassays
To assess whether the modified toxins had increased efficacy against soybean aphids, bioassays were conducted using 3 rd instar soybean aphids. Native and modified toxins were used to spike complete aphid diet [28] with several concentrations ranging from 31.25 μg/ml to 500 μg/ml . Two diet controls were used. The first control was 2X complete aphid diet diluted to 1X with 10 mM Tris-HCl pH 7.5, controlling for both diet quality and toxic effect of the buffer. The second control was 500 μg/ml of BSA in complete aphid diet, controlling for any potential toxic effect of high protein concentration.
A 3 cm petri plate with a 1 cm hole in the lid was used for the bioassays. Each complete aphid diet treatment (100 μl) was placed on Parafilm® stretched thinly over the hole in the lid. Another piece of stretched Parafilm® was placed on top to sandwich the droplet of diet over the hole in the lid. Each plate had 15 soybean aphids, and each treatment had 3 plates. Bioassays were performed under the following rearing conditions: continuous light, 22°C, 70% RH. Mortality was scored every 24 hours for 3 days. Statistically significant differences were assessed using a one-way ANOVA. 57
Results
Selection of Binding Peptides
Eight candidate Soybean aphid Gut binding peptides (SG) were identified from the Ph.D.-C7C phage display library. Eight candidate peptides were selected for further analysis from the 15 unique sequences identified from the third round of biopanning.
SG1 and SG2 were represented four times, SG3 was represented twice, and SG4, SG5,
SG6, SG7, and SG8 were represented once each (Table 2).
Screening of the phage display library Ph.D-C7C against purified soybean aphid
APN3 and ALP2 yielded twelve and seven candidate binding peptides, respectively.
Several peptides selected for binding to APN3 (ABP) showed a distinct similarity to several SG peptides. ABP5 and SG6 share four amino acids, ABP11 and SG3 also share four amino acids, while ABP7 and SG8, and ABP10 and SG7, each have 3 amino acids in common (Table 2). Many of the common amino acids are also found in similar positions. This level of similarity was not observed between peptides selected for binding to ALP2 (PBP) and SG peptides. These results suggest that the peptides SG3, 6, 7 and 8 bind to aminopeptidase N in the soybean aphid gut.
Relative Binding of Soybean Aphid Gut Binding Peptides
SG-GFP fusions were successfully purified from E. coli (Figure 1). Assessment of the in vitro binding capability of the SG-GFP fusions to whole soybean aphid BBMV via pull-down assays revealed two strong candidates for use in modifying Cry4Aa-S1 (Figure
2). One-way ANOVA with a post-hoc Tukey’s test (p<0.05) indicated that SG3-GFP and
SG8-GFP consistently performed the best in pull-down assays. Interestingly, SG1-GFP 58
Table 2. Comparison among SG peptides and a comparison between SG peptides and ABP peptides. Bold amino acids are shared. There were no common amino acid motifs between SG and PBP peptides. Common amino acid motifs between SG and ABP peptides suggest that some SG peptides bind APN. Comparison of SG peptides Comparison of SG and ABP peptides Peptide Amino Acid Sequence Peptide Amino Acid Sequence SG1 TPDSNTE SG3 MG HT LGR SG2 SISSLTH ABP11 MG AEI LR SG3 MGHTLGR SG7 DFP SRG Q SG4 LKNSYKI ABP10 MAN SR IG SG5 CLFTTQPS SG8 STWG RYD SG6 LKNQSDQ ABP7 ENKH TWG SG7 DFPSRGQ SG8 STWGRYD 59
Figure 1. Expression of SG-GFP fusion proteins. Fusion proteins were purified from E. coli cell lysate via nickel affinity chromatography and purity was assessed using SDS- PAGE. SGn-GFP fusions run at 37 kDa. The image is representative of the expression of all SGn-GFP fusions. 60 60
Figure 2. SG3-GFP and SG8-GFP bind soybean aphid BBMV more strongly than the other candidate peptides. Peptide-GFP fusions bound to the BBMV were detected via western blot with anti-GFP antisera. Bands (seen along the bottom of the figure) were analyzed in ImageJ to assess relative amounts of peptide-GFP bound. Data shown represent two replicate experiments. Values obtained were normalized to 1 by setting the values obtained for SG8-GFP to 1 and adjusting the values for other peptide-GFP fusions accordingly. Statistical differences were assessed with a one-way ANOVA performed on data from two replicates. 61 and SG2-GFP, despite being selected for in vivo gut binding, did not bind to BBMV in pull-down assays possibly as a result of differences between in vitro conditions used and the in vivo conditions of phage selection.
Predictive Modeling of Modified Cry4Aa-S1
Predictive modeling of the modified Cry4Aa-S1 toxins was carried out using
LOMETS [24] to identify insertion sites predicted to result in display of the peptide sequence on the exterior of the toxin. The models did provide insight on which side, N- or C-terminal, of the loops targeted in domain II and domain III would be the most exposed. According to the models, SG3 was more exposed on the C-terminal side of the respective loops of Cr4L3 and Cr4B15; while N-terminal insertions resulted in more SG3 exposure for Cr4L2 and Cr4B12 (Figure 3).
Modification of Cry4Aa-S1
The soybean aphid gut binding peptide SG3 was added to seven separate addition sites in Cry4Aa, three at the N-terminus, producing a total of seven modified toxins (Figure 4). Each addition was made to a single toxin, i.e. each modified toxin has a single addition of SG3 at a single site. By adding SG3 to three N-terminal locations, we increased the probability that one of the additions would remain exposed on the active toxin, as the exact cleavage site for the N-terminal peptide is unknown. The other additions were made in loop 2 and loop 3 of domain II, and in the loop formed between
β12 and β13 and the loop formed between β15 and β16 of domain III.
62 6 2
Figure 3. The predicted structures of the Cry4Aa (PDB accession number: 2C9K) loop-modified toxins, Cr4L2, Cr4L3, Cr4B12, and Cr4B15 are shown. Structures predicted by LOMETS and visualized with PyMol. SG3 is shown in pink and indicated with an arrow. Predictive models that showed SG3 exposed on the surface of the toxin were used for selection of the site for peptide insertion in most cases. Both N- and C- terminal insertions of SG3 into the Cr4B15 site resulted in an inward facing loop. The C-terminal insertion site was selected as it provided the least inward facing peptide. 63
Figure 4. SG3 was added via OE-PCR to seven distinct sites in Cry4Aa-S1. This schematic depicts the relative position of each insertion. SG3 was inserted with the terminal cysteines to maintain loop conformation. 64
Upon expression, six out of the seven toxins purified well (see Figure 5 for a representative protein gel). Cr4B12 was not purified at a sufficiently high level for use in our experiments, likely due to protein misfolding.
Structural Stability of Toxins
To test the structural stability of the modified Cry4Aa-S1 toxins relative to the wild-type Cry4Aa-S1, toxins were completely activated using trypsin (Figure 6). Prior to activation with trypsin, wild-type Cry4Aa-S1 yields a 65 kDa band on SDS-PAGE. After activation, Cry4Aa-S1 yields two bands, one at 45 kDa and one at 20 kDa, along with a presumed toxin intermediate of 60 kDa [17,25].
Cry4Aa-S1 yielded 3 bands, as expected, after incubation with trypsin, one unprocessed band at 60 kDa, one band at 45 kDa, and one band at 20 kDa. Four of the modified toxins yielded a similar banding pattern, Cr4N1, Cr4N2, Cr4N3, and Cr4B15. As expected, all of the Cry4Aa-S1 toxins modified at the N-terminus were stable. The N- terminal additions of SG3 comprised the least invasive modifications of Cry4Aa-S1. Both domain II modified toxins, Cr4L2 and Cr4L3, proved to be unstable. Cr4L2 and Cr4L3 showed an unexpected cleavage pattern without incubation with trypsin. A different banding pattern was seen after incubation with trypsin, for Cr4L2 and Cr4L3, indicating that at least one trypsin site was functional in these modified toxins.
Bioassay with Mosquito Larvae
Mosquito larval bioassays were conducted to assess the biological activity of the modified toxins relative to wild-type Cry4Aa-S1 to assess whether modification had impacted toxin structure resulting in loss of native toxicity. 65
Figure 5. Expression of (a) Cry4Aa-S1 and (b) Cr4N1. Cry4Aa-S1 was purified from E. coli cell lysate with Glutathione Sepharose 4B. Purity was assessed by SDS-PAGE. The expected size of Cry4Aa-S1 is 65 kDa.
66 66
Figure 6. Cr4N1, Cr4N2, Cr4N3, and Cr4B15 produce stable, active toxins after trypsin digest. Trypsin digests were carried out with 5 μg of each toxin and 5% trypsin (w/w), and then reactions were run on SDS-PAGE. Undigested toxin shows a primary 65 kDa band with an occasional secondary band at 60 kDa. After incubation with trypsin, 3 bands are present. Properly activated toxins display bands at 45 kDa and 20 kDA. A proteolytic resistant band runs at approximately 60 kDa. Boxes indicate faint 20 kDa bands. 67
Due both to the wide range of reported LC 50 values for Cry4Aa against C. pipiens larvae, and the variation in our own bioassays for toxin treatments, each toxin was categorized simply as toxic or non-toxic (Table 3). Control mortality was minimal
(<10%). Cry4Aa-S1 was toxic to C. pipiens larvae at a level comparable to published LC 50 data. In addition to forming a stable active toxin after incubation with trypsin, Cr4B15 maintained toxicity against C. pipiens larvae. As expected, due to their instability prior to activation, the toxins with SG3 inserted in domain II, Cr4L2 and Cr4L3, did not maintain toxicity against C. pipiens larvae, effectively eliminating them from evaluation as viable candidates for use against soybean aphid. Results for bioassays to date using
Cr4N1, Cr4N2, and Cr4N3 were inconclusive due to high control mortality across multiple replicates. However, it is unlikely that Cr4N1, Cr4N2, or Cr4N3 lost activity against C. pipiens larvae as, of the seven constructs, the SG3 insertions into these constructs were predicted to be the least disruptive to toxin structure, and these toxins were stable on treatment with trypsin.
Soybean Aphid Bioassay
Membrane feeding assays were used to assess toxicity of native Cry4Aa-S1 and the candidate modified Cry4Aa-S1 toxins against soybean aphids. Soybean aphids were susceptible to Cry4Aa-S1 when fed on high concentrations of the toxin in complete aphid diet. Analysis by one-way ANOVA with post-hoc Least Square Means (p<0.05) showed a statistically significant difference between mortality of soybean aphids fed on
500 μg/ml of Cry4Aa-S1, and those fed on 125 μg/ml of Cry4Aa-S1 and 500 μg/ml of BSA
(Figure 7). Bioassays for N-terminal constructs and Cr4B15 are pending. 68
Table 3. Summary of the structural stability of modified toxins, activity against C. pipiens larvae, and whether each toxin was used in soybean aphid bioassays. Toxin Stability Toxicity against C.pipiens Used in A. glycines Assay Cry4Aa Yes Yes Yes Cr4N1 Yes ? Yes Cr4L2 No No No Cr4L3 No No No Cr4B15 Yes Yes Yes Cr4N2 Yes ? Yes Cr4N3 Yes ? Yes 69 69
Figure 7. At high concentrations Cry4Aa induces mortality in soybean aphid. Soybean aphids were fed several toxin concentrations of Cry4Aa in complete aphid diet. Two controls were used, complete aphid diet alone, and complete aphid diet with 500 μg/ml of BSA to control for the impact of high protein concentration on aphid survival. Data shown are from two replicate experiments except for the diet control treatment. SE bars are shown. One-way ANOVA with post-hoc Least Square Means was used to assess differences. 70
Discussion
Following the successful use of a pea aphid gut binding peptide for enhanced binding of Cyt2Aa to the pea aphid gut with associated increased toxicity [23], we sought to modify a Cry toxin for use against a major agricultural pest, the soybean aphid. While Cyt toxins have a lipid based mode of action [29], the mode of action of
Cry toxins is more complex involving multiple binding partners, but is incompletely understood [9]. In addition to the potential for toxin modification to enhance toxicity against the soybean aphid, this work - particularly the gut protein bound by SG3 and whether or not increased binding to those proteins results in enhanced toxicity, may also shed light on toxin mode of action.
Modification of Cry toxin binding has been demonstrated to enhance or bring activity against lepidopteran, coleopteran, and dipteran pests [16], as well as against hemipteran pests [23,30]. Gut binding peptides can be used to enhance binding to the insect gut with a concomitant increase in toxicity against the target insect [23,31].
A total of eight candidate soybean aphid gut binding peptides were identified
(SG1-SG8) via an in vivo screen of a phage display library of random peptides against the soybean aphid gut, with three consecutive rounds of enrichment for gut binding peptides. Unexpectedly, two of the selected peptides, SG1 and SG2, did not bind to whole soybean aphid BBMV in pull-down assays. The lack of binding of these peptides in the in vitro assays might be explained by differences between the in vitro binding conditions, and those of the in vivo phage selection conditions. 71
Interestingly, four of the ABP peptides, selected for binding to recombinant soybean aphid APN3 under in vitro conditions, had several amino acids in common with four of the SG peptides. The common amino acids were also in similar positions between each respective peptide pairing. Most notable was the similarity between SG3 and ABP11, with four common amino acids, out of a possible seven, in a similar order.
Such a degree of similarity supports the hypothesis that SG3 binds to APN3, because as few as three amino acids are necessary for protein - protein interaction [32].
Experiments are underway to test the hypothesis that SG3 binds APN3. The uniqueness of these similarities is highlighted by the dissimilarity between SG peptides and the PBP peptides, selected for in vitro binding to recombinant ALP2.
As with other proteins, the structure of Cry4Aa is integral to its biological activity
[17]. Small changes in the structure or sequence of Cry4Aa can result in significant changes in activity [17,33,34]. The addition of SG3 to loop 2 and loop 3 of domain II of
Cry4Aa-S1 resulted in production of unstable toxins. Addition of five or nine amino acid peptides into different loops of domain II of Cry1Ac, either adding to or replacing existing sequence, also resulted in production of unstable modified toxins that showed no aphid toxicity (Huarong Li, unpublished). Cr4B12 may also have been unstable: After purification of Cr4B12 a faint 65 kDa band was present after SDS-PAGE, indicating low expression, a lack of proper folding, or degradation (data not shown). Based on the available evidence, modifications in domain II of Cry toxins by addition or replacement of multiple amino acids are more likely to compromise toxin stability. Delineation of the 72 key amino acids required for receptor binding will allow for more refined toxin modification with potentially less impact on toxin structure.
Increased understanding of Cry toxin structure as it relates to mode of action will facilitate identification of sites for modification that are less likely to result in toxin instability. Modifications to the N- and C-terminus of activated Cry toxins are the least invasive, as peptide insertions, or other modifications, are less likely to impact the core toxin structure. However, to effectively modify Cry toxins at the N- or C-terminus, knowledge of the N- and/or C-terminal peptide cleavage sites is required. While the structure of the active form of Cry4Aa is known, the exact cleavage sites of both the N- and the C-terminus are unknown. [17].
Taken together, our data clearly show that modification of a Cry toxin with a gut binding peptide presents more structure- and activity -related challenges than modification of a Cyt toxin [23]. This approach does however represent an additional potential approach for production of aphid resistant transgenic plants [35].
Author Contributions
Baculovirus expression of soybean aphid ALP and APN was initiated by NC and completed by BFE. BFE and MTFL purified recombinant ALP and APN and screened the phage display library for peptides that bound to these proteins. All other experiments were conducted by BD. BCB contributed to discussion and analysis of results.
Acknowledgements
The authors thank Brendan Dunphy for provision of mosquito larvae. This work was funded by the USDA AFRI 2012-67013-19457, and by Hatch Act and State of Iowa funds. 73
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CHAPTER 3. CONCLUSIONS & DISCUSSION
Insect resistant transgenic crops represent the most rapidly adopted technology in modern agriculture. Since their introduction in the mid-1990s, Bt crops are now grown on over one billion acres worldwide [1]. Despite the success of insect resistant, transgenic Bt crops, a significant drawback is the low susceptibility of some pest species of economic importance to Bt toxins. Notable among these pests are hemipterans, the sap-sucking insects, which include pests such as aphids and stink bugs. Our ability to genetically modify Bt toxins to overcome significant factors inhibiting toxicity against a target pest allows for the creation of designer toxins.
To date, some effort has been made to modify Bt toxins for toxicity against certain hemipterans. Enhanced binding of Bt toxins to the insect gut, with a concomitant increase in toxicity, has been accomplished using the Cyt toxin Cyt2Aa to target pea aphid ( Acythrosiphon pisum ) [2], and the Cry toxin Cry1Ac to target the maize leafhopper ( Cicadulina mbila ) [3]. Both of these approaches resulted in highly active toxins. More moderate success has been observed with the addition of cathepsin cleavage sites into Cry4Aa to enhance processing in the pea aphid gut ( A. pisum) [4], with some evidence for enhanced toxicity against pea aphid.
Following on from the successful modification of Cyt2Aa with a gut binding peptide for targeting of pea aphid [2], the goal of the research described in Chapter 2 was to engineer a crystal (Cry) toxin using a similar approach. Given the more complex and incompletely understood mechanism of action of Cry toxins [5], engineering of
Cry4Aa using this approach proved more challenging than engineering Cyt2Aa. 77
The modification of Cry4Aa-S1 has provided valuable information however on the relationship between the toxin structure and function. Addition of SG3 to loop 2 or loop
3 of domain II resulted in two unstable toxins that lost activity against C. pipiens larvae.
Domain II has proven to be a challenging domain to modify, while examples of unstable domain II modifications do not exist in the literature, successful modifications were conservative, substituting short amino acid sequences or swapping loops between related toxins [6]. More aggressive modifications, like the addition of SG3, in domain II might not be feasible, though more work would be needed to confirm this. However, larger modifications may prove unnecessary, as only three amino acids are needed to mediate protein-protein interactions [7].
In contrast to domain II modifications, one of the domain III modified Cry4Aa-S1 toxins, Cr4B15, maintained structural stability and toxicity against C. pipiens larvae.
However, when tested against soybean aphid, toxicity was not observed. At high concentrations, Cry4Aa-S1 does cause mortality in soybean aphid, yet Cr4B15 had no discernable effect on the health of soybean aphid, in fact, Cr4B15 appears to have lost activity against soybean aphid. This could implicate the importance of domain III to
Cry4Aa-S1 activity against soybean aphid, though further work would need to be done to confirm this.
N- or C- terminal modifications for Cry4Aa are challenged as the cleavage sites for the N- and C-terminal peptides for Cry4Aa-S1 are unknown. Few Cry toxins have cleavage sites identified, in the case of Cry4Aa, the approximate cleavage sites have been inferred from the structure of the active toxin, though actual cleavage sites have 78 not been confirmed [8]. Data for the three toxins modified at the N-terminus will narrow the position of the cleavage site for the N-terminal peptide.
The SG – GFP fusion proteins constructed for analysis of peptide interaction with the soybean aphid gut were also fed to soybean aphids. Following on from previous work with the pea aphid [9], we examined soybean aphids by fluorescence microscopy for the presence of the GFP fusion proteins in the soybean aphid gut. The results of these experiments are not reported in Chapter 2 because strong fluorescence was not observed, and the source of weak fluorescence when observed was ambiguous
Insufficient feeding by the soybean aphids on the peptide-GFP fusion proteins may explain the ambiguous results. Recently, Nakasu et al [10] reported that a triple alanine linker used for a spider-derived neurotoxin – lectin fusion, was unstable in the Myzus persicae gut. In a previous study [11], a proline-rich linker derived from an aphid transmitted plant virus was used, and proved to be stable in the aphid gut. For our constructs, we used a three glycine linker to link GFP to the candidate gut binding peptides. It is possible that this linker was similarly subject to degradation in the soybean aphid gut, which would explain why feeding of soybean aphids on the peptide-
GFP fusion proteins did not result in fluorescent soybean aphid guts.
The introduction of transgenic crops expressing Bt toxins almost 20 years ago marked a shift in insect pest management strategies [12]. It is time for another shift in how we approach insect pest management. Instead of only screening environmental samples for new toxins, we now have the option to modify characterized toxins to both combat the looming specter of field-evolved resistance, and to enhance efficacy against 79 naturally less-susceptible pest species [6,13]. To facilitate Cry toxin engineering, increased understanding of Cry toxin mode of action and Cry toxin molecular interactions with proteins on the surface of the gut epithelium of the target insect is needed so more precise modifications can be made. Overall, Cry toxin engineering provides an adaptive pest management strategy fit for further development.
REFERENCES
1. Tabashnik, B. E.; Brévault, T.; Carrière, Y. Insect resistance to Bt crops: lessons from the first billion acres. Nat. Biotechnol. 2013 , 31 , 510–21.
2. Chougule, N. P.; Li, H.; Liu, S.; Linz, L. B.; Narva, K. E.; Meade, T.; Bonning, B. C. Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests. Proc. Natl. Acad. Sci. U. S. A. 2013 , 110 , 8465–70.
3. Mehlo, L.; Gahakwa, D.; Nghia, P. T.; Loc, N. T.; Capell, T.; Gatehouse, J. a; Gatehouse, A. M. R.; Christou, P. An alternative strategy for sustainable pest resistance in genetically enhanced crops. Proc. Natl. Acad. Sci. U. S. A. 2005 , 102 , 7812–7816.
4. Rausch, M. A. Modification of the Bt toxin Cry4Aa for improved toxin processing in the gut of the pea aphid ( Acyrthrosiphon pisum ), Iowa State University of Science and Technology, 2014.
5. Vachon, V.; Laprade, R.; Schwartz, J.-L. Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: A critical review. J. Invertebr. Pathol. 2012 , 111 , 1–12.
6. Deist, B. R.; Rausch, M. A.; Fernandez-Luna, M. T.; Adang, M. J.; Bonning, B. C. Bt toxin modification for enhanced efficacy. Toxins (Basel). 2014 , 6, 3005–27.
7. Edwards, R. J.; Palopoli, N. Computational prediction of short linear motifs from protein sequences. Methods Mol. Biol. 2015 1268 , 89-141.
8. Boonserm, P.; Mo, M.; Angsuthanasombat, C.; Lescar, J. Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8- angstrom resolution. J. Bacteriol. 2006 , 188 , 3391–401. 80
9. Liu, S.; Sivakumar, S.; Sparks, W. O.; Miller, W. A.; Bonning, B. C. A peptide that binds the pea aphid gut impedes entry of Pea enation mosaic virus into the aphid hemocoel. Virology 2010 , 401 , 107–116.
10. Nakasu, E. Y. T.; Edwards, M. G.; Fitches, E.; Gatehouse, J. a.; Gatehouse, A. M. R. Transgenic plants expressing ω-ACTX-Hv1a and snowdrop lectin (GNA) fusion protein show enhanced resistance to aphids. Front. Plant Sci. 2014 , 5, 1–8.
11. Bonning, B. C.; Pal, N.; Liu, S.; Wang, Z.; Sivakumar, S.; Dixon, P. M.; King, G. F.; Miller, W. A. Toxin delivery by the coat protein of an aphid-vectored plant virus provides plant resistance to aphids. Nat. Biotechnol. 2014 , 32 , 102–5.
12. Yu, X.; Wang, G.; Huang, S.; Ma, Y.; Xia, L. Engineering plants for aphid resistance: current status and future perspectives. Theor. Appl. Genet. 2014.
13. Gassmann, A. J.; Petzold-Maxwell, J. L.; Clifton, E. H.; Dunbar, M. W.; Hoffmann, A. M.; Ingber, D. A.; Keweshan, R. S. Field-evolved resistance by western corn rootworm to multiple Bacillus thuringiensis toxins in transgenic maize. Proc. Natl. Acad. Sci. U. S. A. 2014 , 111 , 5141–6.
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APPENDIX. NUCLEOTIDE AND AMINO ACID SEQUENCE OF CRY4AA AND MODIFIED TOXINS
SG3 denoted by bolded regions in nucleotide and amino acid sequence. Cry4Aa nucleotide and amino acid sequence Nucleotide ATGAACCCGTATCAAAATAAAAACGAATATGAAACGCTGAACGCATCGCAGAAAAAACTGAACATCTCC AACAACTACACCCGTTACCCGATCGAAAATTCACCGAAACAGCTGCTGCAATCGACCAACTACAAAGATT GGCTGAACATGTGCCAGCAAAACCAGCAATACGGTGGTGACTTCGAAACGTTCATCGATTCAGGTGAAC TGTCGGCATACACCATCGTGGTTGGCACCGTTCTGACGGGTTTTGGTTTCACCACGCCGCTGGGTCTGGC ACTGATTGGCTTTGGCACCCTGATCCCGGTCCTGTTCCCGGCTCAGGACCAAAGCAATACCTGGTCTGATT TTATTACCCAGACGAAAAACATCATCAAAAAAGAAATCGCGAGTACCTACATCTCCAACGCCAACAAAAT TCTGAACCGTAGCTTCAACGTTATCTCTACGTATCATAATCACCTGAAAACCTGGGAAAACAACCCGAACC CGCAGAACACGCAAGATGTGCGTACCCAGATCCAACTGGTTCATTACCACTTCCAGAACGTGATCCCGGA ACTGGTTAATAGCTGCCCGCCGAACCCGTCTGATTGTGACTACTACAACATCCTGGTGCTGAGCAGCTAT GCGCAGGCAGCAAATCTGCATCTGACCGTCCTGAACCAAGCAGTGAAATTCGAAGCGTATCTGAAAAAC AATCGTCAGTTCGACTATCTGGAACCGCTGCCGACCGCAATCGATTACTACCCGGTCCTGACGAAAGCTA TCGAAGATTATACCAACTACTGTGTGACCACGTACAAAAAAGGTCTGAACCTGATCAAAACCACGCCGGA CTCCAACCTGGATGGTAACATCAACTGGAACACCTACAACACGTACCGTACCAAAATGACCACGGCAGTT CTGGACCTGGTCGCTCTGTTCCCGAACTACGATGTCGGTAAATACCCGATTGGTGTGCAGAGCGAACTGA CCCGCGAAATCTACCAAGTGCTGAACTTCGAAGAAAGCCCGTACAAATATTACGACTTCCAGTACCAAGA AGATTCTCTGACGCGTCGTCCGCACCTGTTTACCTGGCTGGATAGTCTGAACTTCTACGAAAAAGCACAG ACCACGCCGAACAACTTTTTCACGTCCCATTACAACATGTTCCACTACACCCTGGACAACATTTCACAGAA AAGTTCCGTTTTCGGTAACCACAACGTCACGGATAAACTGAAATCTCTGGGTCTGGCTACCAATATTTATA TCTTTCTGCTGAACGTTATCTCGCTGGACAACAAATACCTGAACGATTACAACAACATCAGCAAAATGGA CTTTTTCATCACGAATGGCACCCGTCTGCTGGAAAAAGAACTGACGGCGGGCAGTGGTCAGATCACCTAT GATGTGAACAAAAACATCTTCGGTCTGCCGATCCTGAAACGTCGCGAAAACCAGGGTAACCCGACGCTG TTCCCGACCTATGATAACTACTCACATATCCTGTCGTTCATCAAAAGCCTGTCTATCCCGGCAACGTACAA AACCCAGGTGTACACGTTCGCATGGACCCATTCATCGGTTGATCCGAAAAACACGATCTATACCCACCTG ACCACGCAGATCCCGGCAGTTAAAGCTAACAGTCTGGGCACCGCGTCCAAAGTCGTGCAAGGTCCGGGC CACACCGGCGGTGATCTGATCGACTTCAAAGATCATTTCAAAATCACCTGCCAGCACTCAAATTTTCAGCA ATCGTACTTCATTCGTATCCGCTATGCAAGTAATGGCTCCGCAAACACCCGTGCCGTTATCAACCTGTCTA TTCCGGGCGTCGCAGAACTGGGTATGGCACTGAACCCGACCTTCAGTGGTACGGACTACACCAACCTGA AATACAAAGATTTCCAGTATCTGGAATTTAGCAATGAAGTGAAATTTGCCCCGAATCAAAACATTAGTCT GGTGTTCAACCGCTCCGACGTTTACACCAACACCACGGTCCTGATTGATAAAATCGAATTTCTGCCGATTA CCCGTAGCATCCGTGAAGACCGTGAAAAACAAAAACTGGAAACCGTGTGA Amino Acid MNPYQNKNEYETLNASQKKLNISNNYTRYPIENSPKQLLQSTNYKDWLNMCQQNQQYGGDFETFIDSGELS AYTIVVGTVLTGFGFTTPLGLALIGFGTLIPVLFPAQDQSNTWSDFITQTKNIIKKEIASTYISNANKILNRSFNVIS TYHNHLKTWENNPNPQNTQDVRTQIQLVHYHFQNVIPELVNSCPPNPSDCDYYNILVLSSYAQAANLHLTVL NQAVKFEAYLKNNRQFDYLEPLPTAIDYYPVLTKAIEDYTNYCVTTYKKGLNLIKTTPDSNLDGNINWNTYNTY RTKMTTAVLDLVALFPNYDVGKYPIGVQSELTREIYQVLNFEESPYKYYDFQYQEDSLTRRPHLFTWLDSLNFY EKAQTTPNNFFTSHYNMFHYTLDNISQKSSVFGNHNVTDKLKSLGLATNIYIFLLNVISLDNKYLNDYNNISKM DFFITNGTRLLEKELTAGSGQITYDVNKNIFGLPILKRRENQGNPTLFPTYDNYSHILSFIKSLSIPATYKTQVYTFA WTHSSVDPKNTIYTHLTTQIPAVKANSLGTASKVVQGPGHTGGDLIDFKDHFKITCQHSNFQQSYFIRIRYASN GSANTRAVINLSIPGVAELGMALNPTFSGTDYTNLKYKDFQYLEFSNEVKFAPNQNISLVFNRSDVYTNTTVLI DKIEFLPITRSIREDREKQKLETV* 82
Cr4N1 nucleotide and amino acid sequence Nucleotide TGTATGGGGCATACTTTGGGGAGGTGC ATGAACCCGTATCAAAATAAAAACGAATATGAAACGCTGAAC GCATCGCAGAAAAAACTGAACATCTCCAACAACTACACCCGTTACCCGATCGAAAATTCACCGAAACAGC TGCTGCAATCGACCAACTACAAAGATTGGCTGAACATGTGCCAGCAAAACCAGCAATACGGTGGTGACT TCGAAACGTTCATCGATTCAGGTGAACTGTCGGCATACACCATCGTGGTTGGCACCGTTCTGACGGGTTT TGGTTTCACCACGCCGCTGGGTCTGGCACTGATTGGCTTTGGCACCCTGATCCCGGTCCTGTTCCCGGCTC AGGACCAAAGCAATACCTGGTCTGATTTTATTACCCAGACGAAAAACATCATCAAAAAAGAAATCGCGAG TACCTACATCTCCAACGCCAACAAAATTCTGAACCGTAGCTTCAACGTTATCTCTACGTATCATAATCACCT GAAAACCTGGGAAAACAACCCGAACCCGCAGAACACGCAAGATGTGCGTACCCAGATCCAACTGGTTCA TTACCACTTCCAGAACGTGATCCCGGAACTGGTTAATAGCTGCCCGCCGAACCCGTCTGATTGTGACTACT ACAACATCCTGGTGCTGAGCAGCTATGCGCAGGCAGCAAATCTGCATCTGACCGTCCTGAACCAAGCAGT GAAATTCGAAGCGTATCTGAAAAACAATCGTCAGTTCGACTATCTGGAACCGCTGCCGACCGCAATCGAT TACTACCCGGTCCTGACGAAAGCTATCGAAGATTATACCAACTACTGTGTGACCACGTACAAAAAAGGTC TGAACCTGATCAAAACCACGCCGGACTCCAACCTGGATGGTAACATCAACTGGAACACCTACAACACGTA CCGTACCAAAATGACCACGGCAGTTCTGGACCTGGTCGCTCTGTTCCCGAACTACGATGTCGGTAAATAC CCGATTGGTGTGCAGAGCGAACTGACCCGCGAAATCTACCAAGTGCTGAACTTCGAAGAAAGCCCGTAC AAATATTACGACTTCCAGTACCAAGAAGATTCTCTGACGCGTCGTCCGCACCTGTTTACCTGGCTGGATAG TCTGAACTTCTACGAAAAAGCACAGACCACGCCGAACAACTTTTTCACGTCCCATTACAACATGTTCCACT ACACCCTGGACAACATTTCACAGAAAAGTTCCGTTTTCGGTAACCACAACGTCACGGATAAACTGAAATC TCTGGGTCTGGCTACCAATATTTATATCTTTCTGCTGAACGTTATCTCGCTGGACAACAAATACCTGAACG ATTACAACAACATCAGCAAAATGGACTTTTTCATCACGAATGGCACCCGTCTGCTGGAAAAAGAACTGAC GGCGGGCAGTGGTCAGATCACCTATGATGTGAACAAAAACATCTTCGGTCTGCCGATCCTGAAACGTCG CGAAAACCAGGGTAACCCGACGCTGTTCCCGACCTATGATAACTACTCACATATCCTGTCGTTCATCAAAA GCCTGTCTATCCCGGCAACGTACAAAACCCAGGTGTACACGTTCGCATGGACCCATTCATCGGTTGATCC GAAAAACACGATCTATACCCACCTGACCACGCAGATCCCGGCAGTTAAAGCTAACAGTCTGGGCACCGC GTCCAAAGTCGTGCAAGGTCCGGGCCACACCGGCGGTGATCTGATCGACTTCAAAGATCATTTCAAAATC ACCTGCCAGCACTCAAATTTTCAGCAATCGTACTTCATTCGTATCCGCTATGCAAGTAATGGCTCCGCAAA CACCCGTGCCGTTATCAACCTGTCTATTCCGGGCGTCGCAGAACTGGGTATGGCACTGAACCCGACCTTC AGTGGTACGGACTACACCAACCTGAAATACAAAGATTTCCAGTATCTGGAATTTAGCAATGAAGTGAAAT TTGCCCCGAATCAAAACATTAGTCTGGTGTTCAACCGCTCCGACGTTTACACCAACACCACGGTCCTGATT GATAAAATCGAATTTCTGCCGATTACCCGTAGCATCCGTGAAGACCGTGAAAAACAAAAACTGGAAACC GTGTGA
Amino Acid CMGHTLGRCMNPYQNKNEYETLNASQKKLNISNNYTRYPIENSPKQLLQSTNYKDWLNMCQQNQQYGGD FETFIDSGELSAYTIVVGTVLTGFGFTTPLGLALIGFGTLIPVLFPAQDQSNTWSDFITQTKNIIKKEIASTYISNAN KILNRSFNVISTYHNHLKTWENNPNPQNTQDVRTQIQLVHYHFQNVIPELVNSCPPNPSDCDYYNILVLSSYA QAANLHLTVLNQAVKFEAYLKNNRQFDYLEPLPTAIDYYPVLTKAIEDYTNYCVTTYKKGLNLIKTTPDSNLDGN INWNTYNTYRTKMTTAVLDLVALFPNYDVGKYPIGVQSELTREIYQVLNFEESPYKYYDFQYQEDSLTRRPHLF TWLDSLNFYEKAQTTPNNFFTSHYNMFHYTLDNISQKSSVFGNHNVTDKLKSLGLATNIYIFLLNVISLDNKYL NDYNNISKMDFFITNGTRLLEKELTAGSGQITYDVNKNIFGLPILKRRENQGNPTLFPTYDNYSHILSFIKSLSIPA TYKTQVYTFAWTHSSVDPKNTIYTHLTTQIPAVKANSLGTASKVVQGPGHTGGDLIDFKDHFKITCQHSNFQQ SYFIRIRYASNGSANTRAVINLSIPGVAELGMALNPTFSGTDYTNLKYKDFQYLEFSNEVKFAPNQNISLVFNRS DVYTNTTVLIDKIEFLPITRSIREDREKQKLETV*
83
Cr4N2 nucleotide and amino acid sequence Nucleotide ATGAACCCGTATCAAAATAAAAACGAATATGAAACGCTGAACGCATCGCAGAAAAAACTGAACATCTCC AACAACTACACCCGTTACCCGATCGAAAATTCACCGAAACAGCTGCTGCAATCGACCAACTACAAAGATT GGCTGAACATGTGCCAGCAAAACCAGCAATACGGTGGTGACTTCGAAACGTTCATCGATTCA TGTATGG GGCATACTTTGGGGAGGTGC GGTGAACTGTCGGCATACACCATCGTGGTTGGCACCGTTCTGACGGGTT TTGGTTTCACCACGCCGCTGGGTCTGGCACTGATTGGCTTTGGCACCCTGATCCCGGTCCTGTTCCCGGCT CAGGACCAAAGCAATACCTGGTCTGATTTTATTACCCAGACGAAAAACATCATCAAAAAAGAAATCGCGA GTACCTACATCTCCAACGCCAACAAAATTCTGAACCGTAGCTTCAACGTTATCTCTACGTATCATAATCACC TGAAAACCTGGGAAAACAACCCGAACCCGCAGAACACGCAAGATGTGCGTACCCAGATCCAACTGGTTC ATTACCACTTCCAGAACGTGATCCCGGAACTGGTTAATAGCTGCCCGCCGAACCCGTCTGATTGTGACTA CTACAACATCCTGGTGCTGAGCAGCTATGCGCAGGCAGCAAATCTGCATCTGACCGTCCTGAACCAAGCA GTGAAATTCGAAGCGTATCTGAAAAACAATCGTCAGTTCGACTATCTGGAACCGCTGCCGACCGCAATCG ATTACTACCCGGTCCTGACGAAAGCTATCGAAGATTATACCAACTACTGTGTGACCACGTACAAAAAAGG TCTGAACCTGATCAAAACCACGCCGGACTCCAACCTGGATGGTAACATCAACTGGAACACCTACAACACG TACCGTACCAAAATGACCACGGCAGTTCTGGACCTGGTCGCTCTGTTCCCGAACTACGATGTCGGTAAAT ACCCGATTGGTGTGCAGAGCGAACTGACCCGCGAAATCTACCAAGTGCTGAACTTCGAAGAAAGCCCGT ACAAATATTACGACTTCCAGTACCAAGAAGATTCTCTGACGCGTCGTCCGCACCTGTTTACCTGGCTGGAT AGTCTGAACTTCTACGAAAAAGCACAGACCACGCCGAACAACTTTTTCACGTCCCATTACAACATGTTCCA CTACACCCTGGACAACATTTCACAGAAAAGTTCCGTTTTCGGTAACCACAACGTCACGGATAAACTGAAA TCTCTGGGTCTGGCTACCAATATTTATATCTTTCTGCTGAACGTTATCTCGCTGGACAACAAATACCTGAA CGATTACAACAACATCAGCAAAATGGACTTTTTCATCACGAATGGCACCCGTCTGCTGGAAAAAGAACTG ACGGCGGGCAGTGGTCAGATCACCTATGATGTGAACAAAAACATCTTCGGTCTGCCGATCCTGAAACGTC GCGAAAACCAGGGTAACCCGACGCTGTTCCCGACCTATGATAACTACTCACATATCCTGTCGTTCATCAAA AGCCTGTCTATCCCGGCAACGTACAAAACCCAGGTGTACACGTTCGCATGGACCCATTCATCGGTTGATC CGAAAAACACGATCTATACCCACCTGACCACGCAGATCCCGGCAGTTAAAGCTAACAGTCTGGGCACCGC GTCCAAAGTCGTGCAAGGTCCGGGCCACACCGGCGGTGATCTGATCGACTTCAAAGATCATTTCAAAATC ACCTGCCAGCACTCAAATTTTCAGCAATCGTACTTCATTCGTATCCGCTATGCAAGTAATGGCTCCGCAAA CACCCGTGCCGTTATCAACCTGTCTATTCCGGGCGTCGCAGAACTGGGTATGGCACTGAACCCGACCTTC AGTGGTACGGACTACACCAACCTGAAATACAAAGATTTCCAGTATCTGGAATTTAGCAATGAAGTGAAAT TTGCCCCGAATCAAAACATTAGTCTGGTGTTCAACCGCTCCGACGTTTACACCAACACCACGGTCCTGATT GATAAAATCGAATTTCTGCCGATTACCCGTAGCATCCGTGAAGACCGTGAAAAACAAAAACTGGAAACC GTGTGA
Amino Acid MNPYQNKNEYETLNASQKKLNISNNYTRYPIENSPKQLLQSTNYKDWLNMCQQNQQYGGDFETFIDS CMG HTLGRCGELSAYTIVVGTVLTGFGFTTPLGLALIGFGTLIPVLFPAQDQSNTWSDFITQTKNIIKKEIASTYISNAN KILNRSFNVISTYHNHLKTWENNPNPQNTQDVRTQIQLVHYHFQNVIPELVNSCPPNPSDCDYYNILVLSSYA QAANLHLTVLNQAVKFEAYLKNNRQFDYLEPLPTAIDYYPVLTKAIEDYTNYCVTTYKKGLNLIKTTPDSNLDGN INWNTYNTYRTKMTTAVLDLVALFPNYDVGKYPIGVQSELTREIYQVLNFEESPYKYYDFQYQEDSLTRRPHLF TWLDSLNFYEKAQTTPNNFFTSHYNMFHYTLDNISQKSSVFGNHNVTDKLKSLGLATNIYIFLLNVISLDNKYL NDYNNISKMDFFITNGTRLLEKELTAGSGQITYDVNKNIFGLPILKRRENQGNPTLFPTYDNYSHILSFIKSLSIPA TYKTQVYTFAWTHSSVDPKNTIYTHLTTQIPAVKANSLGTASKVVQGPGHTGGDLIDFKDHFKITCQHSNFQQ SYFIRIRYASNGSANTRAVINLSIPGVAELGMALNPTFSGTDYTNLKYKDFQYLEFSNEVKFAPNQNISLVFNRS DVYTNTTVLIDKIEFLPITRSIREDREKQKLETV*
84
Cr4N3 nucleotide and amino acid sequence Nucleotide ATGAACCCGTATCAAAATAAAAACGAATATGAAACGCTGAACGCATCGCAGAAAAAACTGAACATCTCC AACAACTACACCCGTTACCCGATCGAAAATTCACCGAAACAGCTGCTGCAATCGACCAACTACAAAGATT GGCTGAACATGTGCCAGCAAAACCAGCAATACGGTGGTGACTTCGAAACGTTCATCGATTCAGGTGAAC TG TGTATGGGGCATACTTTGGGGAGGTGC TCGGCATACACCATCGTGGTTGGCACCGTTCTGACGGGTT TTGGTTTCACCACGCCGCTGGGTCTGGCACTGATTGGCTTTGGCACCCTGATCCCGGTCCTGTTCCCGGCT CAGGACCAAAGCAATACCTGGTCTGATTTTATTACCCAGACGAAAAACATCATCAAAAAAGAAATCGCGA GTACCTACATCTCCAACGCCAACAAAATTCTGAACCGTAGCTTCAACGTTATCTCTACGTATCATAATCACC TGAAAACCTGGGAAAACAACCCGAACCCGCAGAACACGCAAGATGTGCGTACCCAGATCCAACTGGTTC ATTACCACTTCCAGAACGTGATCCCGGAACTGGTTAATAGCTGCCCGCCGAACCCGTCTGATTGTGACTA CTACAACATCCTGGTGCTGAGCAGCTATGCGCAGGCAGCAAATCTGCATCTGACCGTCCTGAACCAAGCA GTGAAATTCGAAGCGTATCTGAAAAACAATCGTCAGTTCGACTATCTGGAACCGCTGCCGACCGCAATCG ATTACTACCCGGTCCTGACGAAAGCTATCGAAGATTATACCAACTACTGTGTGACCACGTACAAAAAAGG TCTGAACCTGATCAAAACCACGCCGGACTCCAACCTGGATGGTAACATCAACTGGAACACCTACAACACG TACCGTACCAAAATGACCACGGCAGTTCTGGACCTGGTCGCTCTGTTCCCGAACTACGATGTCGGTAAAT ACCCGATTGGTGTGCAGAGCGAACTGACCCGCGAAATCTACCAAGTGCTGAACTTCGAAGAAAGCCCGT ACAAATATTACGACTTCCAGTACCAAGAAGATTCTCTGACGCGTCGTCCGCACCTGTTTACCTGGCTGGAT AGTCTGAACTTCTACGAAAAAGCACAGACCACGCCGAACAACTTTTTCACGTCCCATTACAACATGTTCCA CTACACCCTGGACAACATTTCACAGAAAAGTTCCGTTTTCGGTAACCACAACGTCACGGATAAACTGAAA TCTCTGGGTCTGGCTACCAATATTTATATCTTTCTGCTGAACGTTATCTCGCTGGACAACAAATACCTGAA CGATTACAACAACATCAGCAAAATGGACTTTTTCATCACGAATGGCACCCGTCTGCTGGAAAAAGAACTG ACGGCGGGCAGTGGTCAGATCACCTATGATGTGAACAAAAACATCTTCGGTCTGCCGATCCTGAAACGTC GCGAAAACCAGGGTAACCCGACGCTGTTCCCGACCTATGATAACTACTCACATATCCTGTCGTTCATCAAA AGCCTGTCTATCCCGGCAACGTACAAAACCCAGGTGTACACGTTCGCATGGACCCATTCATCGGTTGATC CGAAAAACACGATCTATACCCACCTGACCACGCAGATCCCGGCAGTTAAAGCTAACAGTCTGGGCACCGC GTCCAAAGTCGTGCAAGGTCCGGGCCACACCGGCGGTGATCTGATCGACTTCAAAGATCATTTCAAAATC ACCTGCCAGCACTCAAATTTTCAGCAATCGTACTTCATTCGTATCCGCTATGCAAGTAATGGCTCCGCAAA CACCCGTGCCGTTATCAACCTGTCTATTCCGGGCGTCGCAGAACTGGGTATGGCACTGAACCCGACCTTC AGTGGTACGGACTACACCAACCTGAAATACAAAGATTTCCAGTATCTGGAATTTAGCAATGAAGTGAAAT TTGCCCCGAATCAAAACATTAGTCTGGTGTTCAACCGCTCCGACGTTTACACCAACACCACGGTCCTGATT GATAAAATCGAATTTCTGCCGATTACCCGTAGCATCCGTGAAGACCGTGAAAAACAAAAACTGGAAACC GTGTGA
Amino Acid MNPYQNKNEYETLNASQKKLNISNNYTRYPIENSPKQLLQSTNYKDWLNMCQQNQQYGGDFETFIDSGEL C MGHTLGRCSAYTIVVGTVLTGFGFTTPLGLALIGFGTLIPVLFPAQDQSNTWSDFITQTKNIIKKEIASTYISNAN KILNRSFNVISTYHNHLKTWENNPNPQNTQDVRTQIQLVHYHFQNVIPELVNSCPPNPSDCDYYNILVLSSYA QAANLHLTVLNQAVKFEAYLKNNRQFDYLEPLPTAIDYYPVLTKAIEDYTNYCVTTYKKGLNLIKTTPDSNLDGN INWNTYNTYRTKMTTAVLDLVALFPNYDVGKYPIGVQSELTREIYQVLNFEESPYKYYDFQYQEDSLTRRPHLF TWLDSLNFYEKAQTTPNNFFTSHYNMFHYTLDNISQKSSVFGNHNVTDKLKSLGLATNIYIFLLNVISLDNKYL NDYNNISKMDFFITNGTRLLEKELTAGSGQITYDVNKNIFGLPILKRRENQGNPTLFPTYDNYSHILSFIKSLSIPA TYKTQVYTFAWTHSSVDPKNTIYTHLTTQIPAVKANSLGTASKVVQGPGHTGGDLIDFKDHFKITCQHSNFQQ SYFIRIRYASNGSANTRAVINLSIPGVAELGMALNPTFSGTDYTNLKYKDFQYLEFSNEVKFAPNQNISLVFNRS DVYTNTTVLIDKIEFLPITRSIREDREKQKLETV*
85
Cr4L2 nucleotide and amino acid sequence Nucleotide ATGAACCCGTATCAAAATAAAAACGAATATGAAACGCTGAACGCATCGCAGAAAAAACTGAACATCTCC AACAACTACACCCGTTACCCGATCGAAAATTCACCGAAACAGCTGCTGCAATCGACCAACTACAAAGATT GGCTGAACATGTGCCAGCAAAACCAGCAATACGGTGGTGACTTCGAAACGTTCATCGATTCAGGTGAAC TGTCGGCATACACCATCGTGGTTGGCACCGTTCTGACGGGTTTTGGTTTCACCACGCCGCTGGGTCTGGC ACTGATTGGCTTTGGCACCCTGATCCCGGTCCTGTTCCCGGCTCAGGACCAAAGCAATACCTGGTCTGATT TTATTACCCAGACGAAAAACATCATCAAAAAAGAAATCGCGAGTACCTACATCTCCAACGCCAACAAAAT TCTGAACCGTAGCTTCAACGTTATCTCTACGTATCATAATCACCTGAAAACCTGGGAAAACAACCCGAACC CGCAGAACACGCAAGATGTGCGTACCCAGATCCAACTGGTTCATTACCACTTCCAGAACGTGATCCCGGA ACTGGTTAATAGCTGCCCGCCGAACCCGTCTGATTGTGACTACTACAACATCCTGGTGCTGAGCAGCTAT GCGCAGGCAGCAAATCTGCATCTGACCGTCCTGAACCAAGCAGTGAAATTCGAAGCGTATCTGAAAAAC AATCGTCAGTTCGACTATCTGGAACCGCTGCCGACCGCAATCGATTACTACCCGGTCCTGACGAAAGCTA TCGAAGATTATACCAACTACTGTGTGACCACGTACAAAAAAGGTCTGAACCTGATCAAAACCACGCCGGA CTCCAACCTGGATGGTAACATCAACTGGAACACCTACAACACGTACCGTACCAAAATGACCACGGCAGTT CTGGACCTGGTCGCTCTGTTCCCGAACTACGATGTCGGTAAATACCCGATTGGTGTGCAGAGCGAACTGA CCCGCGAAATCTACCAAGTGCTGAACTTCGAAGAAAGCCCGTACAAATATTACGACTTCCAGTACCAAGA AGATTCTCTGACGCGTCGTCCGCACCTGTTTACCTGGCTGGATAGTCTGAACTTCTACGAAAAAGCACAG ACCACGCCGAACAACTTTTTCACGTCCCATTACAACATGTTCCACTACACCCTGGACAACATTTCACAGAA AAGTTCCGTTTTCGGTAACCACAACGTCACGGATAAACTGAAATCTCTGGGTCTGGCTACCAATATTTATA TCTTTCTGCTGAACGTTATC TGTATGGGGCATACTTTGGGGAGGTGC TCGCTGGACAACAAATACCTGAA CGATTACAACAACATCAGCAAAATGGACTTTTTCATCACGAATGGCACCCGTCTGCTGGAAAAAGAACTG ACGGCGGGCAGTGGTCAGATCACCTATGATGTGAACAAAAACATCTTCGGTCTGCCGATCCTGAAACGTC GCGAAAACCAGGGTAACCCGACGCTGTTCCCGACCTATGATAACTACTCACATATCCTGTCGTTCATCAAA AGCCTGTCTATCCCGGCAACGTACAAAACCCAGGTGTACACGTTCGCATGGACCCATTCATCGGTTGATC CGAAAAACACGATCTATACCCACCTGACCACGCAGATCCCGGCAGTTAAAGCTAACAGTCTGGGCACCGC GTCCAAAGTCGTGCAAGGTCCGGGCCACACCGGCGGTGATCTGATCGACTTCAAAGATCATTTCAAAATC ACCTGCCAGCACTCAAATTTTCAGCAATCGTACTTCATTCGTATCCGCTATGCAAGTAATGGCTCCGCAAA CACCCGTGCCGTTATCAACCTGTCTATTCCGGGCGTCGCAGAACTGGGTATGGCACTGAACCCGACCTTC AGTGGTACGGACTACACCAACCTGAAATACAAAGATTTCCAGTATCTGGAATTTAGCAATGAAGTGAAAT TTGCCCCGAATCAAAACATTAGTCTGGTGTTCAACCGCTCCGACGTTTACACCAACACCACGGTCCTGATT GATAAAATCGAATTTCTGCCGATTACCCGTAGCATCCGTGAAGACCGTGAAAAACAAAAACTGGAAACC GTGTGA
Amino Acid MNPYQNKNEYETLNASQKKLNISNNYTRYPIENSPKQLLQSTNYKDWLNMCQQNQQYGGDFETFIDSGELS AYTIVVGTVLTGFGFTTPLGLALIGFGTLIPVLFPAQDQSNTWSDFITQTKNIIKKEIASTYISNANKILNRSFNVIS TYHNHLKTWENNPNPQNTQDVRTQIQLVHYHFQNVIPELVNSCPPNPSDCDYYNILVLSSYAQAANLHLTVL NQAVKFEAYLKNNRQFDYLEPLPTAIDYYPVLTKAIEDYTNYCVTTYKKGLNLIKTTPDSNLDGNINWNTYNTY RTKMTTAVLDLVALFPNYDVGKYPIGVQSELTREIYQVLNFEESPYKYYDFQYQEDSLTRRPHLFTWLDSLNFY EKAQTTPNNFFTSHYNMFHYTLDNISQKSSVFGNHNVTDKLKSLGLATNIYIFLLNVI CMGHTLGRCSLDNKYL NDYNNISKMDFFITNGTRLLEKELTAGSGQITYDVNKNIFGLPILKRRENQGNPTLFPTYDNYSHILSFIKSLSIPA TYKTQVYTFAWTHSSVDPKNTIYTHLTTQIPAVKANSLGTASKVVQGPGHTGGDLIDFKDHFKITCQHSNFQQ SYFIRIRYASNGSANTRAVINLSIPGVAELGMALNPTFSGTDYTNLKYKDFQYLEFSNEVKFAPNQNISLVFNRS DVYTNTTVLIDKIEFLPITRSIREDREKQKLETV*
86
Cr4L3 nucleotide and amino acid sequence Nucleotide ATGAACCCGTATCAAAATAAAAACGAATATGAAACGCTGAACGCATCGCAGAAAAAACTGAACATCTCC AACAACTACACCCGTTACCCGATCGAAAATTCACCGAAACAGCTGCTGCAATCGACCAACTACAAAGATT GGCTGAACATGTGCCAGCAAAACCAGCAATACGGTGGTGACTTCGAAACGTTCATCGATTCAGGTGAAC TGTCGGCATACACCATCGTGGTTGGCACCGTTCTGACGGGTTTTGGTTTCACCACGCCGCTGGGTCTGGC ACTGATTGGCTTTGGCACCCTGATCCCGGTCCTGTTCCCGGCTCAGGACCAAAGCAATACCTGGTCTGATT TTATTACCCAGACGAAAAACATCATCAAAAAAGAAATCGCGAGTACCTACATCTCCAACGCCAACAAAAT TCTGAACCGTAGCTTCAACGTTATCTCTACGTATCATAATCACCTGAAAACCTGGGAAAACAACCCGAACC CGCAGAACACGCAAGATGTGCGTACCCAGATCCAACTGGTTCATTACCACTTCCAGAACGTGATCCCGGA ACTGGTTAATAGCTGCCCGCCGAACCCGTCTGATTGTGACTACTACAACATCCTGGTGCTGAGCAGCTAT GCGCAGGCAGCAAATCTGCATCTGACCGTCCTGAACCAAGCAGTGAAATTCGAAGCGTATCTGAAAAAC AATCGTCAGTTCGACTATCTGGAACCGCTGCCGACCGCAATCGATTACTACCCGGTCCTGACGAAAGCTA TCGAAGATTATACCAACTACTGTGTGACCACGTACAAAAAAGGTCTGAACCTGATCAAAACCACGCCGGA CTCCAACCTGGATGGTAACATCAACTGGAACACCTACAACACGTACCGTACCAAAATGACCACGGCAGTT CTGGACCTGGTCGCTCTGTTCCCGAACTACGATGTCGGTAAATACCCGATTGGTGTGCAGAGCGAACTGA CCCGCGAAATCTACCAAGTGCTGAACTTCGAAGAAAGCCCGTACAAATATTACGACTTCCAGTACCAAGA AGATTCTCTGACGCGTCGTCCGCACCTGTTTACCTGGCTGGATAGTCTGAACTTCTACGAAAAAGCACAG ACCACGCCGAACAACTTTTTCACGTCCCATTACAACATGTTCCACTACACCCTGGACAACATTTCACAGAA AAGTTCCGTTTTCGGTAACCACAACGTCACGGATAAACTGAAATCTCTGGGTCTGGCTACCAATATTTATA TCTTTCTGCTGAACGTTATCTCGCTGGACAACAAATACCTGAACGATTACAACAACATCAGCAAAATGGA CTTTTTCATCACGAATGGCACCCGTCTGCTGGAAAAAGAACTGACGGCGGGCAGTGGTCAGATCACCTAT GATGTGAACAAAAACATCTTCGGTCTGCCGATCCTGAAACGTCGCGAAAACCAGGGTAACCCGACGCTG TTCCCGACCTATGATAACTACTCACATATCCTGTCGTTCATCAAAAGCCTGTCTATCCCGGCAACGTACAA ATGTATGGGGCATACTTTGGGGAGGTGC ACCCAGGTGTACACGTTCGCATGGACCCATTCATCGGTTGA TCCGAAAAACACGATCTATACCCACCTGACCACGCAGATCCCGGCAGTTAAAGCTAACAGTCTGGGCACC GCGTCCAAAGTCGTGCAAGGTCCGGGCCACACCGGCGGTGATCTGATCGACTTCAAAGATCATTTCAAA ATCACCTGCCAGCACTCAAATTTTCAGCAATCGTACTTCATTCGTATCCGCTATGCAAGTAATGGCTCCGC AAACACCCGTGCCGTTATCAACCTGTCTATTCCGGGCGTCGCAGAACTGGGTATGGCACTGAACCCGACC TTCAGTGGTACGGACTACACCAACCTGAAATACAAAGATTTCCAGTATCTGGAATTTAGCAATGAAGTGA AATTTGCCCCGAATCAAAACATTAGTCTGGTGTTCAACCGCTCCGACGTTTACACCAACACCACGGTCCTG ATTGATAAAATCGAATTTCTGCCGATTACCCGTAGCATCCGTGAAGACCGTGAAAAACAAAAACTGGAAA CCGTGTGA
Amino Acid MNPYQNKNEYETLNASQKKLNISNNYTRYPIENSPKQLLQSTNYKDWLNMCQQNQQYGGDFETFIDSGELS AYTIVVGTVLTGFGFTTPLGLALIGFGTLIPVLFPAQDQSNTWSDFITQTKNIIKKEIASTYISNANKILNRSFNVIS TYHNHLKTWENNPNPQNTQDVRTQIQLVHYHFQNVIPELVNSCPPNPSDCDYYNILVLSSYAQAANLHLTVL NQAVKFEAYLKNNRQFDYLEPLPTAIDYYPVLTKAIEDYTNYCVTTYKKGLNLIKTTPDSNLDGNINWNTYNTY RTKMTTAVLDLVALFPNYDVGKYPIGVQSELTREIYQVLNFEESPYKYYDFQYQEDSLTRRPHLFTWLDSLNFY EKAQTTPNNFFTSHYNMFHYTLDNISQKSSVFGNHNVTDKLKSLGLATNIYIFLLNVISLDNKYLNDYNNISKM DFFITNGTRLLEKELTAGSGQITYDVNKNIFGLPILKRRENQGNPTLFPTYDNYSHILSFIKSLSIPATYK CMGHTL GRCTQVYTFAWTHSSVDPKNTIYTHLTTQIPAVKANSLGTASKVVQGPGHTGGDLIDFKDHFKITCQHSNFQ QSYFIRIRYASNGSANTRAVINLSIPGVAELGMALNPTFSGTDYTNLKYKDFQYLEFSNEVKFAPNQNISLVFNR SDVYTNTTVLIDKIEFLPITRSIREDREKQKLETV*
87
Cr4B12 nucleotide and amino acid sequence Nucleotide ATGAACCCGTATCAAAATAAAAACGAATATGAAACGCTGAACGCATCGCAGAAAAAACTGAACATCTCC AACAACTACACCCGTTACCCGATCGAAAATTCACCGAAACAGCTGCTGCAATCGACCAACTACAAAGATT GGCTGAACATGTGCCAGCAAAACCAGCAATACGGTGGTGACTTCGAAACGTTCATCGATTCAGGTGAAC TGTCGGCATACACCATCGTGGTTGGCACCGTTCTGACGGGTTTTGGTTTCACCACGCCGCTGGGTCTGGC ACTGATTGGCTTTGGCACCCTGATCCCGGTCCTGTTCCCGGCTCAGGACCAAAGCAATACCTGGTCTGATT TTATTACCCAGACGAAAAACATCATCAAAAAAGAAATCGCGAGTACCTACATCTCCAACGCCAACAAAAT TCTGAACCGTAGCTTCAACGTTATCTCTACGTATCATAATCACCTGAAAACCTGGGAAAACAACCCGAACC CGCAGAACACGCAAGATGTGCGTACCCAGATCCAACTGGTTCATTACCACTTCCAGAACGTGATCCCGGA ACTGGTTAATAGCTGCCCGCCGAACCCGTCTGATTGTGACTACTACAACATCCTGGTGCTGAGCAGCTAT GCGCAGGCAGCAAATCTGCATCTGACCGTCCTGAACCAAGCAGTGAAATTCGAAGCGTATCTGAAAAAC AATCGTCAGTTCGACTATCTGGAACCGCTGCCGACCGCAATCGATTACTACCCGGTCCTGACGAAAGCTA TCGAAGATTATACCAACTACTGTGTGACCACGTACAAAAAAGGTCTGAACCTGATCAAAACCACGCCGGA CTCCAACCTGGATGGTAACATCAACTGGAACACCTACAACACGTACCGTACCAAAATGACCACGGCAGTT CTGGACCTGGTCGCTCTGTTCCCGAACTACGATGTCGGTAAATACCCGATTGGTGTGCAGAGCGAACTGA CCCGCGAAATCTACCAAGTGCTGAACTTCGAAGAAAGCCCGTACAAATATTACGACTTCCAGTACCAAGA AGATTCTCTGACGCGTCGTCCGCACCTGTTTACCTGGCTGGATAGTCTGAACTTCTACGAAAAAGCACAG ACCACGCCGAACAACTTTTTCACGTCCCATTACAACATGTTCCACTACACCCTGGACAACATTTCACAGAA AAGTTCCGTTTTCGGTAACCACAACGTCACGGATAAACTGAAATCTCTGGGTCTGGCTACCAATATTTATA TCTTTCTGCTGAACGTTATCTCGCTGGACAACAAATACCTGAACGATTACAACAACATCAGCAAAATGGA CTTTTTCATCACGAATGGCACCCGTCTGCTGGAAAAAGAACTGACGGCGGGCAGTGGTCAGATCACCTAT GATGTGAACAAAAACATCTTCGGTCTGCCGATCCTGAAACGTCGCGAAAACCAGGGTAACCCGACGCTG TTCCCGACCTATGATAACTACTCACATATCCTGTCGTTCATCAAAAGCCTGTCTATCCCGGCAACGTACAA AACCCAGGTGTACACGTTCGCATGGACCCATTCATCGGTTGATCCGAAAAACACGATC TGTATGGGGCAT ACTTTGGGGAGGTGC TATACCCACCTGACCACGCAGATCCCGGCAGTTAAAGCTAACAGTCTGGGCACC GCGTCCAAAGTCGTGCAAGGTCCGGGCCACACCGGCGGTGATCTGATCGACTTCAAAGATCATTTCAAA ATCACCTGCCAGCACTCAAATTTTCAGCAATCGTACTTCATTCGTATCCGCTATGCAAGTAATGGCTCCGC AAACACCCGTGCCGTTATCAACCTGTCTATTCCGGGCGTCGCAGAACTGGGTATGGCACTGAACCCGACC TTCAGTGGTACGGACTACACCAACCTGAAATACAAAGATTTCCAGTATCTGGAATTTAGCAATGAAGTGA AATTTGCCCCGAATCAAAACATTAGTCTGGTGTTCAACCGCTCCGACGTTTACACCAACACCACGGTCCTG ATTGATAAAATCGAATTTCTGCCGATTACCCGTAGCATCCGTGAAGACCGTGAAAAACAAAAACTGGAAA CCGTGTGA
Amino Acid MNPYQNKNEYETLNASQKKLNISNNYTRYPIENSPKQLLQSTNYKDWLNMCQQNQQYGGDFETFIDSGELS AYTIVVGTVLTGFGFTTPLGLALIGFGTLIPVLFPAQDQSNTWSDFITQTKNIIKKEIASTYISNANKILNRSFNVIS TYHNHLKTWENNPNPQNTQDVRTQIQLVHYHFQNVIPELVNSCPPNPSDCDYYNILVLSSYAQAANLHLTVL NQAVKFEAYLKNNRQFDYLEPLPTAIDYYPVLTKAIEDYTNYCVTTYKKGLNLIKTTPDSNLDGNINWNTYNTY RTKMTTAVLDLVALFPNYDVGKYPIGVQSELTREIYQVLNFEESPYKYYDFQYQEDSLTRRPHLFTWLDSLNFY EKAQTTPNNFFTSHYNMFHYTLDNISQKSSVFGNHNVTDKLKSLGLATNIYIFLLNVISLDNKYLNDYNNISKM DFFITNGTRLLEKELTAGSGQITYDVNKNIFGLPILKRRENQGNPTLFPTYDNYSHILSFIKSLSIPATYKTQVYTFA WTHSSVDPKNTI CMGHTLGRCYTHLTTQIPAVKANSLGTASKVVQGPGHTGGDLIDFKDHFKITCQHSNFQQ SYFIRIRYASNGSANTRAVINLSIPGVAELGMALNPTFSGTDYTNLKYKDFQYLEFSNEVKFAPNQNISLVFNRS DVYTNTTVLIDKIEFLPITRSIREDREKQKLETV*
88
Cr4B15 nucleotide and amino acid sequence Nucleotide ATGAACCCGTATCAAAATAAAAACGAATATGAAACGCTGAACGCATCGCAGAAAAAACTGAACATCTCC AACAACTACACCCGTTACCCGATCGAAAATTCACCGAAACAGCTGCTGCAATCGACCAACTACAAAGATT GGCTGAACATGTGCCAGCAAAACCAGCAATACGGTGGTGACTTCGAAACGTTCATCGATTCAGGTGAAC TGTCGGCATACACCATCGTGGTTGGCACCGTTCTGACGGGTTTTGGTTTCACCACGCCGCTGGGTCTGGC ACTGATTGGCTTTGGCACCCTGATCCCGGTCCTGTTCCCGGCTCAGGACCAAAGCAATACCTGGTCTGATT TTATTACCCAGACGAAAAACATCATCAAAAAAGAAATCGCGAGTACCTACATCTCCAACGCCAACAAAAT TCTGAACCGTAGCTTCAACGTTATCTCTACGTATCATAATCACCTGAAAACCTGGGAAAACAACCCGAACC CGCAGAACACGCAAGATGTGCGTACCCAGATCCAACTGGTTCATTACCACTTCCAGAACGTGATCCCGGA ACTGGTTAATAGCTGCCCGCCGAACCCGTCTGATTGTGACTACTACAACATCCTGGTGCTGAGCAGCTAT GCGCAGGCAGCAAATCTGCATCTGACCGTCCTGAACCAAGCAGTGAAATTCGAAGCGTATCTGAAAAAC AATCGTCAGTTCGACTATCTGGAACCGCTGCCGACCGCAATCGATTACTACCCGGTCCTGACGAAAGCTA TCGAAGATTATACCAACTACTGTGTGACCACGTACAAAAAAGGTCTGAACCTGATCAAAACCACGCCGGA CTCCAACCTGGATGGTAACATCAACTGGAACACCTACAACACGTACCGTACCAAAATGACCACGGCAGTT CTGGACCTGGTCGCTCTGTTCCCGAACTACGATGTCGGTAAATACCCGATTGGTGTGCAGAGCGAACTGA CCCGCGAAATCTACCAAGTGCTGAACTTCGAAGAAAGCCCGTACAAATATTACGACTTCCAGTACCAAGA AGATTCTCTGACGCGTCGTCCGCACCTGTTTACCTGGCTGGATAGTCTGAACTTCTACGAAAAAGCACAG ACCACGCCGAACAACTTTTTCACGTCCCATTACAACATGTTCCACTACACCCTGGACAACATTTCACAGAA AAGTTCCGTTTTCGGTAACCACAACGTCACGGATAAACTGAAATCTCTGGGTCTGGCTACCAATATTTATA TCTTTCTGCTGAACGTTATCTCGCTGGACAACAAATACCTGAACGATTACAACAACATCAGCAAAATGGA CTTTTTCATCACGAATGGCACCCGTCTGCTGGAAAAAGAACTGACGGCGGGCAGTGGTCAGATCACCTAT GATGTGAACAAAAACATCTTCGGTCTGCCGATCCTGAAACGTCGCGAAAACCAGGGTAACCCGACGCTG TTCCCGACCTATGATAACTACTCACATATCCTGTCGTTCATCAAAAGCCTGTCTATCCCGGCAACGTACAA AACCCAGGTGTACACGTTCGCATGGACCCATTCATCGGTTGATCCGAAAAACACGATCTATACCCACCTG ACCACGCAGATCCCGGCAGTTAAAGCTAACAGTCTGGGCACCGCGTCCAAAGTCGTGCAAGGTCCGGGC CACACCGGCGGTGATCTGATCGACTTCAAAGATCATTTCAAAATCACCTGCCAGCACTCAAATTTT TGTAT GGGGCATACTTTGGGGAGGTGC CAGCAATCGTACTTCATTCGTATCCGCTATGCAAGTAATGGCTCCGC AAACACCCGTGCCGTTATCAACCTGTCTATTCCGGGCGTCGCAGAACTGGGTATGGCACTGAACCCGACC TTCAGTGGTACGGACTACACCAACCTGAAATACAAAGATTTCCAGTATCTGGAATTTAGCAATGAAGTGA AATTTGCCCCGAATCAAAACATTAGTCTGGTGTTCAACCGCTCCGACGTTTACACCAACACCACGGTCCTG ATTGATAAAATCGAATTTCTGCCGATTACCCGTAGCATCCGTGAAGACCGTGAAAAACAAAAACTGGAAA CCGTGTGA
Amino Acid MNPYQNKNEYETLNASQKKLNISNNYTRYPIENSPKQLLQSTNYKDWLNMCQQNQQYGGDFETFIDSGELS AYTIVVGTVLTGFGFTTPLGLALIGFGTLIPVLFPAQDQSNTWSDFITQTKNIIKKEIASTYISNANKILNRSFNVIS TYHNHLKTWENNPNPQNTQDVRTQIQLVHYHFQNVIPELVNSCPPNPSDCDYYNILVLSSYAQAANLHLTVL NQAVKFEAYLKNNRQFDYLEPLPTAIDYYPVLTKAIEDYTNYCVTTYKKGLNLIKTTPDSNLDGNINWNTYNTY RTKMTTAVLDLVALFPNYDVGKYPIGVQSELTREIYQVLNFEESPYKYYDFQYQEDSLTRRPHLFTWLDSLNFY EKAQTTPNNFFTSHYNMFHYTLDNISQKSSVFGNHNVTDKLKSLGLATNIYIFLLNVISLDNKYLNDYNNISKM DFFITNGTRLLEKELTAGSGQITYDVNKNIFGLPILKRRENQGNPTLFPTYDNYSHILSFIKSLSIPATYKTQVYTFA WTHSSVDPKNTIYTHLTTQIPAVKANSLGTASKVVQGPGHTGGDLIDFKDHFKITCQHSNF CMGHTLGRCQQ SYFIRIRYASNGSANTRAVINLSIPGVAELGMALNPTFSGTDYTNLKYKDFQYLEFSNEVKFAPNQNISLVFNRS DVYTNTTVLIDKIEFLPITRSIREDREKQKLETV*