TRANSGENIC CONTROL OF THE CITRUS WEEVIL,...
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1 TRANSGENIC CONTROL OF THE CITRUS WEEVIL, DIAPREPES ABBREVIATUS: A THREE-PRONGED APPROACH TARGETING DIGESTION, GUT PHYSIOLOGY, AND GENE EXPRESSION By SULLEY KWEKU BEN-MAHMOUD A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013
Transcript of TRANSGENIC CONTROL OF THE CITRUS WEEVIL,...
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TRANSGENIC CONTROL OF THE CITRUS WEEVIL, DIAPREPES ABBREVIATUS: A THREE-PRONGED APPROACH TARGETING DIGESTION, GUT PHYSIOLOGY,
AND GENE EXPRESSION
By
SULLEY KWEKU BEN-MAHMOUD
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2013
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© 2013 Sulley Kweku Ben-Mahmoud
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To the Ben-Mahmoud family, especially my wife, Theodora
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ACKNOWLEDGMENTS
Above all, I would like to thank my dissertation committee Ronald D. Cave,
Robert G. Shatters Jr., Charles A. Powell, Daniel A. Hahn, and James E. Maruniak. I
am most grateful to R. D. Cave for his comments and guidance. For his advice,
encouragement, and intellectual stimulation, I am profoundly grateful to R. G. Shatters
Jr. I thank C. A. Powell for his support. I cannot go on without extending my sincerest
appreciation to Dov Borovsky for being a great teacher and mentor. I feel fortunate to
have had D. G. Hall, S. L. Lapointe, and J. E. Ramos in my camp, particularly for their
expert advice in various aspects of my research.
I am thankful to D. Borovsky, R. G. Shatters Jr., C. A. Powell, D. G. Hall and S. L.
Lapointe for allowing me to build on studies they begun at the University of Florida and
the USDA-USHRL (Fort Pierce). My appreciation goes to H. Van Praet and B. Diego for
their help in organizing the transgenic citrus inventory and to L. Faulkner, J. Smith and
C. Peck for their help in citrus maintenance. The potted-plant bioassay was an arduous
task and I could not have accomplished it without the help of R. G. Shatters Jr., B.
Diego, A. F. Voss, C. A. Malone, S. Clark, R. Jain, and K. Moulton. My special thanks to
A.S. Hill for her help with maintaining the D. abbreviatus colony and to L. Markle for his
help in setting up the larval bioassays. I am also grateful to E. Egan and L. I. Shaffer for
making their help readily available when I needed it. I really appreciate C. L. McKenzie,
W. B. Hunter, G. A. Luzio, R. P. Niedz, J. A. Manthey, R. G. Cameron and M. E. Hilf at
the USDA-USHRL for making their laboratories available to me. I would be remiss if I
forgot to add P. D’Aiuto, S. Kauffman, and E. Branca to my acknowledgments. The
camaraderie and friendship of E. Van Ekert and C. L. Hawkings is appreciated as we
endured the dissertation process.
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To my friends: Sheri Anderson, Adam Searle, Erik Blosser, Irka Bargielowski,
Rodrigo Diaz, Veronica Manrique, Angie Alejandra Niño, Maud Verstraete, Josh Voss,
Rocco Alessandro, Paul Robbins, Kent Morgan, Pasco Avery, John Prokop, Mary
Prokop and others I have failed to mention: “me da mo nyinara ase!”. I immensely
enjoyed your comradeship.
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 8
LIST OF FIGURES .......................................................................................................... 9
LIST OF ABBREVIATIONS ........................................................................................... 11
ABSTRACT ................................................................................................................... 15
CHAPTER
1 INTRODUCTION AND LITERATURE REVIEW ..................................................... 17
Life cycle of Diaprepes abbreviatus in Citrus .......................................................... 17 Current Control of Diaprepes abbreviatus............................................................... 22
Future Control of Diaprepes abbreviatus ................................................................ 24 Transgenic Control of Diaprepes abbreviatus .................................................. 25
Candidate Toxins ............................................................................................. 26 Delivery of Toxins in a Transgenic Strategy ............................................................ 35
2 RECOMBINANT EXPRESSION AND CHARACTERIZATION OF DIAPREPES ABBREVIATUS CATHEPSIN L1 AND THE ROLE OF CATHEPSIN L PROTEASE INHIBITORS IN THE REGULATION OF CATHEPSINS INVOLVED IN DIGESTION. ................................................................................... 37
Materials and Methods............................................................................................ 39 Molecular Modeling and Docking of Cathepsin L Protease Inhibitor to D.
abbreviatus of Cathepsin L1 .......................................................................... 39
Rearing of Diaprepes abbreviatus .................................................................... 41
Bacterial Strains and Plasmids ......................................................................... 41 Primers ............................................................................................................. 41 Cathepsin L 1 and Cathepsin L Protease Inhibitor Constructs ......................... 42
Site-Directed Mutagenesis ............................................................................... 43 Expression of Recombinant Proteins ............................................................... 44 Purification of Recombinant Proteins ............................................................... 46
Enzymatic Assays ............................................................................................ 47 Results .................................................................................................................... 48
Modeling of Cathepsin L1 and CPI1 ................................................................. 48 Synthesis and Purification of DaCatL1 ............................................................. 49 Synthesis and Purification of CPI ..................................................................... 51 Cathepsin L1 Substrate Specificity and pH optimum ........................................ 51 Effect of inhibitors on Cathepsin L1 activity ...................................................... 52
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Sequence alignment of Cathepsin L1 and Human cathepsins ......................... 53 D. abbreviatus Gut in vitro Assays ................................................................... 53
Discussion .............................................................................................................. 54
3 A METHOD FOR CONTROLLING DIAPREPES ABBREVIATUS LARVAL FEEDING ON CITRUS USING A BACILLUS THURINGIENSIS CYTOLYTIC TOXIN ..................................................................................................................... 69
Materials and Methods............................................................................................ 70 Citrus Plants ..................................................................................................... 70
Primers ............................................................................................................. 71 Nucleic Acid Extractions ................................................................................... 71 Quantitative Polymerase Chain Reaction (qPCR) ............................................ 74
Quantitative Reverse Polymerase Chain Reaction (q-RT-PCR) ....................... 74 Potted-Plant Bioassays .................................................................................... 75
Results .................................................................................................................... 78
Potted-Plant Bioassays ........................................................................................... 79 Discussion .............................................................................................................. 81
4 TARGETING THE PERITROPHIC MEMBRANE OF DIAPREPES ABBREVIATUS VIA RNAi INDUCES A DOSE-DEPENDENT MORTALITY RESPONSE ............................................................................................................ 94
Introduction ............................................................................................................. 94 Materials and Methods............................................................................................ 97
Diaprepes abbreviatus Larvae .......................................................................... 97
Primers ............................................................................................................. 97
Double-stranded RNA Synthesis. ..................................................................... 97 Larval Bioassay ................................................................................................ 99
Results .................................................................................................................. 100
Discussion ............................................................................................................ 101
5 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS .................................. 105
LIST OF REFERENCES ............................................................................................. 112
BIOGRAPHICAL SKETCH .......................................................................................... 126
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LIST OF TABLES
Table page 3-1 Primers used for qPCR and q-RT-PCR .............................................................. 71
3-2 Summary statistics of Ct values obtained from q-RT-PCR analysis of Cyt2Ca1 citrus and non-engineered citrus ......................................................... 90
3-3 Statistical analysis of uninfested root ratio .......................................................... 91
3-4 Statistical analysis of infested root ratio .............................................................. 91
3-5 Statistical analysis of root damage index ............................................................ 92
3-6 Statistical analysis of weight gain of surviving larvae fed on citrus ..................... 92
3-7 Statistical analysis of larval mortality .................................................................. 93
4-1 Primers for Diaprepes abbreviatus dsRNA synthesis. ........................................ 98
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LIST OF FIGURES
Figure page 1-1 General plant and root damage as a result of Diaprepes abbreviates feeding
on transgenic versus wild-type alfalfa. ................................................................ 28
1-2 Amino acid sequence alignments of cathepsin L protease inhibitors (CPI 1 and 2) to cathepsin L 1 and 2 in Diaprepes abbreviatus. .................................... 32
2-1 Modelling of the interaction between Diaprepes abbreviatus cathepsin L1 (DaCatL1) and cathepsin L protease inhibitor 1 (CPI1). ................................. 6060
2-2 SDS-PAGE analysis of recombinant cathepsin L1 purification. .......................... 61
2-3 Western blot analysis of His- tagged DaCatL1. .................................................. 62
2-4 Mass spectrometry analysis of DaCatL1. ........................................................... 62
2-5 Size-exclusion purification of DaCatL1. .............................................................. 63
2-6 SDS PAGE (lanes a-d) and western blot analysis (lane e) of cathepsin L protease inhibitor1 mutant (CPI1mut). ................................................................ 63
2-7 Fluorogenic substrate preference of DaCatL1.. .................................................. 64
2-8 pH activity profile of recombinant DaCatL1 using Z-FR-AFC.............................. 64
2-9 Inhibition of DaCatL1 by small-molecule protease inhibitors. ............................. 65
2-10 Inhibition of cathepsin L protease inhibitors on the activity of DaCatL1. . .......... 65
2-11 Alignment of DaCatL1 with human cathepsin sequences. Highlighted in yellow are the active site residues. ..................................................................... 66
2-12 Influence of TritonTM X-100 (1%) on the interaction between D. abbreviatus gut cathepsins and the cathepsin L protease inhibitors CPI1 and CPI1mut. ...... 67
2-13 Inhibition of D. abbreviatus larval cathepsins by cathepsin L protease inhibitors ............................................................................................................. 68
3-1 Experimental design of potted plant bioassay. ................................................... 84
3-2 Relative levels of transcription of Cyt2Ca1 gene in transgenic citrus plants normalized with the 18S RNA levels. .................................................................. 85
3-3 Some transgenic citrus cuttings used for potted plant bioassays. ...................... 86
3-4 Resistance of Cyt2Ca1 engineered citrus versus wild type control. .................. 87
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3-5 Effect of larval root feeding on Cyt2Ca1 citrus plants and the concomitant effect on larval weight and mortality. .................................................................. 88
4-1 Corrected mortality (post 12 days) due to feeding on dsRNA sequences targeting Diaprepes abbreviatus genes. ........................................................... 104
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LIST OF ABBREVIATIONS
Act Actin
AFC 7-Amino-4-trifluoromethylcoumarin
AMC 7-amino-4-methylcoumarin
ANOVA Analysis of Variance
Bt Bacillus thuringiensis
CA074 (L-3-trans-(Propylcarbamyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester
CAL2 Cathepsin L2
CD Citrus dehydrin
CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
CPI Cathepsin L Protease Inhibitor
CPI1 Cathepsin L Protease Inhibitor 1
CPI1mut Cathepsin L Protease Inhibitor 1 mutant
Ct Cycle Threshold
CTV Citrus Tristeza Virus
DaCatL1 Diaprepes abbreviatus Cathepsin L1
DC Diaphorina citri
DEPC Diethylpyrocarbonate
DNA Deoxyribonucleic Acid
dsRNA double-stranded RNA
DTT Dithiothreitol
E. coli Escherichia coli
E-64 L-trans-Epoxysuccinyl-leucyamido (4-guanidino) butane
EDTA Ethylenediaminetetraacetic acid
FOXO forkhead transcription factor
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FR Phenylalanine and Arginine
GPR Glycine, Proline, and Arginine
GST Glutathione S-transferase
HE High Expresser
His6 Polyhistidine Tag
HumCTB Human Cathepsin B
HumCTH Human Cathepsin H
HumCTK Human Cathepsin K
HumCTL1 Human Cathepsin L1
HumCTS Human Cathepsin S
IPM Integrated Pest Management
IPTG Isopropyl β-D-1-thiogalactopyranoside
IRR Infested Root Ratio
LE Low Expresser
MBP Maltose Binding Protein
ME Medium Expresser
MEROPS Peptidase Database
MES 2-(N-morpholino)ethanesulfonic acid
mRNA Messenger RNA
MS Mass Spectrometry
MW Molecular Weight
OGNC Octyl Glucose Neopentyl Glycol
PAGE Polyacrylamide Gel Electrophoresis
PCR Polymerase Chain Reaction
PMCBP Peritrophic Membrane Chitin Binding Protein
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PMP Peritrophic Matrix Protein
pNA p-nitroanilide
PP Propeptide
qPCR Quantitative Polymerase Chain Reaction
q-RT-PCR Quantitative Reverse Transcriptase Polymerase Chain Reaction
RDI Root Damage Inndex
RdRP RNA dependent RNA polymerase
RFU Relative Fluorescent Unit
RISC RNA Interfering Silencing Complex
RNA Ribonucleic Acid
RNAi RNA interference
rRNA Ribosomal RNA
SDS Sodium Dodecyl Sulfate
SDSC San Diego SuperComputer Center
SEC Size Exclusion Chromatography
siRNA Small Interfering RNA
S-S Bond Disulfide Bond
Suc-AAPF N-succinyl-alanine-alanine-proline-phenyalanine-p-nitroanilide
SUMO Small Ubiquitin-like Modifier
TAE Tris, Acetic Acid, EDTA
Tc-ASH Tc-achaete-scute-homolog
TMOF Trypsin Modulating Oostatic Factor
TPCK Tosyl phenylalanyl chloromethyl ketone
Ubi Ubiquitin
UF-ICBR University of Florida-Interdisciplinary Center for Biotechnology Research
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URR Uninfested Root Ration
USDA-USHRL United States Department of Agriculture-United States Horticultural Research Laboratory
VVR Valine, Valine, and Arginine
WT Wild-Type
Z Benzyloxycarbonyl
Z-FY(tBu)DMK (N-benzyloxycarbonyl-phenylalanyl-t-butyl-tryrosyl diazomethylketone
α-Tub Alpha Tubulin
βME Beta-Mercaptoehtanol
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Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
TRANSGENIC CONTROL OF THE CITRUS WEEVIL, DIAPREPES ABBREVIATUS: A THREE-PRONGED APPROACH TARGETING DIGESTION, GUT PHYSIOLOGY,
AND GENE EXPRESSION
By
Sulley Kweku Ben-Mahmoud
December 2013
Chair: Ronald D. Cave. Co-chair: Robert G. Shatters, Jr. Major: Entomology and Nematology
Diaprepes abbreviatus (L.) continues to threaten citriculture in the USA. A
transgenic citrus rootstock approach is proposed to manipulate citrus to synthesize
molecules selectively toxic to D. abbreviatus in a pyramiding/stacking strategy to delay
the onset of resistance. Candidate toxins are sought to complement Cyt2Ca1 transgenic
citrus, with cathepsin L protease inhibitors to inhibit protein digestion, and dsRNA to
interfere with gene expression.
The synthesis, purification, and characterization of a cysteine protease,
cathepsin L1 (DaCatL1) from D. abbreviatus are described, in addition to endogenous
inhibitors of cathepsins, cathepsin L protease inhibitor (CPI1) and a mutant (CPI1mut).
Complementary DNA encoding DaCatL1 was cloned into an expression vector fused
with GST and His6 tags for copious expression in an Escherichia coli strain. CPI1mut
was engineered by site-directed mutagenesis of CPI1 by replacing Lys101 of CPI1 with
a Cys residue. DaCatL1 was stable in solution with 1% TritonTM X-100, whereas CPI1
and CPI1mut were synthesized in soluble forms. Enzymatically active DaCatL1 (23KDa)
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was cleaved from the 60-KDa fusion protein by buffer exchange at alkaline pH using
size exclusion chromatography after initial purification with a Ni-NTA resin. DaCatL1
had optimal activity at pH 8 with the substrate Z-FR-AFC, and enzyme inhibition assays
with protease inhibitors revealed cryptic chemical characteristics. The inhibitors
effectively inhibited cathepsins extracted from the midgut of 6-week-old larvae at acidic
pH but not at alkaline pH. DaCatL1 is not inhibited by either CPI1 or CPI1mut at pH8.
There appear to be two broad classes (acidic and basic) of cathepsins in D.
abbreviatus.
Quantitative PCR of 75 genetically transformed citrus plants identified 31
containing Cyt2Ca1. An ad hoc criterion using q-RT-PCR data was used to select seven
transgenic citrus plants expressing Cyt2Ca1. Three transgenic citrus events expressing
Cyt2Ca1 were significantly more resistant to feeding damage by D. abbreviatus larvae
in no-choice potted-plant bioassays relative to their untransformed wild type cohorts.
Feeding four dsRNA sequences to silence D. abbreviatus genes identified two
possible targets for RNAi. The peritrophic membrane chitin binding protein and
cathepsin L2 dsRNA sequences are possible candidates for a transgenic strategy to
control D. abbreviatus.
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CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW
Life cycle of Diaprepes abbreviatus in Citrus
Since its accidental introduction into the United States in the mid-1960s, the
citrus weevil, Diaprepes abbreviatus (Linnaeus) (Coleoptera: Curculionidae), has been
a constant threat to the cultivation of citrus. The weevil was first reported in a citrus
grove in Apopka, Florida in 1964 (Beavers and Salhime, 1975). Diaprepes abbreviatus
has since exhibited the ability to spread and increase its pest status. It is currently found
in all the major citrus producing states: Florida, Texas, and California (Lapointe et al.,
2007). It originated in the West Indies (Ernst et al., 2006) where it is an economically
important pest of sugar cane, yams, pineapple, and corn (Hall, 1995). The weevil has a
wide host range, in excess of 300 plant species from 59 plant families that include many
commercially cultivated crops, e.g. corn and potatoes (Simpson et al., 1996). In the
United States, it has established itself as a high-impact, economic pest of citrus. As a
result of its polyphagous feeding habit, D. abbreviatus is a problematic pest to the
cultivation of a number of cash crops and ornamentals (Diaz et al., 2006; Martin et al.,
2009).
Diaprepes abbreviatus has numerous other common names from its association
with a variety of host plants and geographic distribution: Apopka weevil, West Indian
weevil, and sugarcane rootstalk borer weevil are examples. Adults are large and grow
to approximately 2 cm in length, and typically have black elytra with brightly colored
streaks of orange, yellow, red, or gray scales in numerous morphotypes. Adults live
mainly in the crown of trees, where they mate and feed predominantly on leaves
(Beavers and Selhime, 1975). Characteristics of a D. abbreviatus infestation include a
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notching pattern on the leaf edges and appearance of significant frass on leaves. Upon
emergence as adults from underground (larval and pupal stages are almost entirely
subterranean), they fly for a short distance, seeking a host tree which they hardly leave
unless disturbed or when food is depleted (Weissling et al., 2009). Adults have an
average life-span of 3-5 months, with females usually out-living males.
Females have high fecundity (Sirot and Lapointe, 2008). Mating occurs in the
early hours of the day or at night when the temperature is cool. This is the time during
which they are also most active. Females lay eggs in clusters of 30-250 eggs arranged
as a single layer in no particular pattern. These clusters are laid between two leaves,
which they hold together at the edges with a gelatinous substance that they secrete.
Eggs are oval and approximately 1 mm in length, off-white in color but darken as they
age, and hatch in about a week (7-10 days). Larvae are white and legless, and almost
immediately drop to the ground after hatching and burrow into the soil to reach the roots
of trees. In the soil, they feed voraciously on roots, which is their only known source of
nutrition in the wild.
Neonate larvae commence feeding on fibrous roots and then move to larger
roots as they mature. Larvae complete 10 or 11 instars, with the third to ninth instars (3-
4 months after hatching) the most aggressive feeders. Feeding damage may be severe
enough to kill a citrus tree as a result of girdling; however, feeding injuries serves as an
entry point for soil-borne plant pathogens (Graham et al., 2003). Although larvae feed
copiously on roots, they can also survive for long periods without food. The entire
larval stage may last from a few months to slightly over a year.
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Pupation occurs in a vertical chamber that the larva (resting with its head up)
compacts by active wriggling movements. Following pupation, there is a two-week
period during which teneral adults remain quiescent, though they may physically appear
to be ready to go above ground. Adults tunnel through soil to the surface by means of a
pair of deciduous mandiblular cusps which often break off. The adults seek mates
almost as soon as they emerge. The developmental period of the citrus root weevil is
highly variable, ranging from six months to as long as two years, depending on nutrition,
temperature, humidity, and soil moisture.
The larva of the weevil is the most damaging stage. It feeds on the roots, is long-
lived, and is almost entirely subterranean. These factors make dependence on
insecticides nearly futile in achieving cost-effective control. Although accurate economic
estimates are hard to come by, annual loss in the citrus industry due to the weevil in
Florida alone in 1997 were estimated to be in excess of $75 million (Stuart et al., 2006).
This is very significant, particularly for an insect with such a wide host range.
No resistant cultivars of citrus are available (Shapiro and Gottwald, 1995). Using
standard plant breeding techniques to breed D. abbreviatus resistance into commercial
citrus varieties has not been viable as yet, though an antixenotic effect has been
observed in two varieties of Poncirus trifoliata L. Raf. in the laboratory (Lapointe and
Bowman, 2002). A need to develop new methods to control the weevil is therefore
relevant. Transgenic citrus strains with specific resistance to D. abbreviatus could play a
key role in protecting citrus. The current literature is replete with numerous examples of
using transgenic techniques to improve crop resistance to pests (Harlander, 2002;
James, 2012); however, almost all transgenic strategies for crop resistance to insects
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rely on the transgenic expression of a variety of Bacillus thuringiensis (Bt) toxins. The Bt
toxins are pore-forming proteins, with specific insecticidal activities, produced during the
sporulation phase of different strains of the bacteria. Commercially, this has produced
significant successes in the management of numerous lepidopteran and coleopteran
crop pests, particularly in reducing costs and improving yields. This has yet to be
deployed as a strategy in citrus.
Scientists at the USDA-USHRL (United States Department of Agriculture-
Horticultural Research Laboratory) in Fort Pierce, FL identified a gene encoding a Bt
toxin (Cyt2Ca1) active against D. abbreviatus and engineered citrus to express it.
Evaluation of Cyt2Ca1expressing citrus is carried out in my study.
Other strategies for insect control through transgenic crop development include
the use of protease inhibitors that disrupt the insect digestive process, and double-
stranded RNA to interfere with gene expression. Protease inhibitors have shown
promise because many plants use naturally produced protease inhibitors as a strategy
to protect themselves from insects. However, in practice, the success of this strategy
has been limited because many insect pests, especially coleopterans and
lepidopterans, have multiple alternative digestive proteases that are over-expressed to
compensate for inhibited proteases. In mosquitoes, blocking digestive proteases was
shown to be a functional strategy when the mechanism that regulates the expression of
digestive trypsin was targeted (Borovsky and Mahmood, 1995; Borovsky and Meola,
2004). Feeding of soybean trypsin inhibitor and Aedes aegypti trypsin modulating
oostatic factor affected trypsin activity and trypsin biosynthesis, respectively in
Diaprepes abbreviatus (Yan et al., 1999). A similar approach is sought to control D.
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abbreviatus with inhibitors of digestive cathepsin L. Since D. abbreviatus, utilizes both
trypsin and cathepsin for digestion (Yan et al., 1999), targeting both enzymes in concert
with their respective inhibitors may be effective at precluding protein digestion in the
weevil.
RNA interference (RNAi) can be harnessed for crop protection against pests.
RNAi is a gene regulatory mechanism at the mRNA level. Therefore, by down-
regulating the expression of essential genes, growth and development of insect pests
can be impaired, possibly leading to death. The adaptation of RNAi as a pest control
tool is nascent, but precocious. The first application of RNAi for pest control was
demonstrated in Coleoptera and Lepidoptera (Baum et al., 2007; Mao et al., 2007). In
the short time since, RNAi applications has expanded exponentially into insect orders of
agricultural and medical importance (Coutinho-Abreu et al., 2010; Huvenne and
Smagghe, 2010). Novel attempts for D. abbreviatus control may benefit from RNAi.
In summary, this thesis presents alternative strategies for D. abbreviatus
management by targeting digestive proteases with inhibitors and the silencing of gene
expression via RNA interference (RNAi) with double-stranded RNA sequences
(dsRNA). An attempt at making a broad spectrum inhibitor of proteases is pursued and
a preliminary study to identify gene targets for silencing are conducted. The Bt toxin,
protease inhibitors and dsRNA for RNAi are explored for a complimentary method, in a
concerted multiple target effort, to produce transgenic citrus rootstock expressing more
than one toxin with different modes of action to offset the selection of resistance in D.
abbreviatus.
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Current Control of Diaprepes abbreviatus
In citriculture, no satisfactory control for D. abbreviatus is available. Heavy
reliance on chemical insecticides is the mainstay of management of D. abbreviatus.
This is also true in the West Indies, where the impact of natural enemies and predators
on populations of D. abbreviatus has not kept the pest below economic thresholds (Hall
et al., 2001). Dependence on chemical insecticides is not only deleterious to the
environment, toxic to beneficial and non-target insects, and harmful to human and
animal health, but the use of ovicides and adulticides is considered ineffective and/or
expensive (Lapointe, 2000).
Control strategies against D. abbreviatus have been deployed target the various
life stages of the insect: eggs that are vulnerable to parasitoids, larvae that dwell below
ground and adults that feed on foliage. Biological and chemical methods are the
contemporary methods for control. However, none of these strategies have proven to be
efficacious or cost-effective.
Cultural Control: Sanitary-based containment and quarantine measures have
been practiced as a means of limiting the spread of D. abbreviatus. Since the insect
prefers to “hitchhike” as adults on plants and larvae in soil transported by people, this
has by some measure limited the spread of D. abbreviatus, though this is quite difficult
to ensure (Weissling et al., 2009).
Biological Control: Several egg parasitoids have been released in the field for
pest suppression: Quadrastichus haitiensis Gahan (Hymenoptera: Eulophidae),
Ceratogramma etiennei Delvare (Hymenoptera: Trichogrammatidae), Aprostocetus
vaquitarum Wolcott (Hymenoptera: Eulophidae), Fidiobia dominica Evans and Peña
(Hymenoptera: Platygastridae), and Haeckeliania sperata Pinto (Hymenoptera:
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Trichogrammatidae), (Hall et al., 2001; Ulmer et al., 2006). These parasitic wasps have
been introduced to Florida from the West Indies. Results of releases of Q. haitiensis and
A. vaquitarum have not been spectacular. The wasps have failed to spread and expand
their range, perhaps as a result of concurrent insecticide use, and therefore have not
had the desired effect. Another possible explanation is the climatic differences between
the geographic origin of these wasps and Florida. Quadrastichus haitiensis, C. etiennei,
and A. vaquitarum are reported to parasitize greater than 35% of eggs in southeastern
Florida, though it is not clear what levels of control they are achieving statewide.
The entomopathogenic nematode Steinernema riobravis Cabanillas has also
been used in the field for D. abbreviatus control (Stuart et al., 2004). The nematode is
applied to the soil through the irrigation system. On reaching the weevil larvae, it
penetrates into the body and releases mutualistic bacteria that multiply and cause
septicemia. The nematode larvae feed on the decomposing cadaver, grow, and as
adults produce offspring that leave the host cadaver and infect other larvae in the
vicinity. Grower acceptance of the product to augment IPM strategies was mixed
(Georgis et al., 2006) and, due to problems with assuring proper quality control
associated with shipment and storage conditions, this product is no longer
recommended (Weissling et al., 2009). The nematodes used as biological control
agents are constrained by their requirements for efficacy that include optimal
temperature, appropriate soil type, and adequate irrigation (Shapiro-Ilan et al., 2006).
Chemical Control: The primary method of D. abbreviatus control by growers
has been insecticides targeted at both larvae and adults. Since the most damaging
stage of the insect is subterranean, this method has also been met with difficulty due to
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the inability to reach the larvae with the insecticides in toxic doses. Insecticides that
have been used recently include bifenthrin and carbaryl to control adults and
imidacloprid against larvae (Jetter and Godfrey, 2009). Chlorinated hydrocarbons -
heptachlor, dieldrin, and chlordane - were the first insecticides used against the pest
(Nigg et al., 1999) but discontinued for environmental impact concerns.
Other methods listed include the use of oils to prevent leaves from sticking
together, thus exposing eggs to desiccation and also making them vulnerable to
predators and parasitoids. Desiccation of eggs reduces the hatchability of eggs and can
help bring down the population of the weevil in the field.
Future Control of Diaprepes abbreviatus
It is evident that no safe and effective control method is available for this pest.
Dependence on insecticides is inefficient because it is difficult to reach the root system
where larvae cause the most damage and parasitoids have failed to establish and
spread. Novel, cost-effective, and efficient control is needed for D. abbreviatus. For
these reasons, the use of transgenic technology is a reasonable and perhaps the only
strategy to provide control of this weevil.
Pests are a major obstacle to large scale agriculture and consequently their
management has always been important. When properly deployed, the transgenic
technology is efficacious. In the 25 years since the first deployment of transgenic crops,
no adverse effects have been realized (Romeis et al., 2013) and with respect to insect
control, reductions in the need for chemical pesticides have been touted (Lu et al.,
2012). Since the initial introduction of commercially available transgenic crops in 1996,
there are now an estimated 170.3 million planted hectares, up from 1.7 million hectares
in 1996 (James, 2012). Prominent among transgenic plants are insect resistant
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varieties: Bt-engineered sweet corn to protect against the corn earworm, Helicoverpa
zea Boddie (Tabashnik et al., 2008), against the pink bollworm (Pectinophora
gossypiella Saunders) in cotton (Kathage and Qaim, 2012; Tabashnik et al., 2012), and
against the Colorado potato beetle (Leptinotarsa decemlineata Say) in potato (Zhou et
al., 2012).
A major challenge in pest management is the development of resistance to
control strategies. Although transgenic crop varieties have surpassed predictions for the
onset of resistance (Bates et al., 2005), reports of field-evolved resistance in insects
(Tabashnik, 2008; Liu et al., 2010; Gassmann et al., 2011) are beginning to emerge
after more than a decade of commercial availability.
The first commercially available transgenic plants were engineered to express
single toxin genes. Insect management strategies, such as the inclusion of refuges
(localized plantings of non-transgenic crops than can function as a location where the
pest insect can reproduce where they are not under selection pressure), have played a
big role in forestalling resistance but there is concern that this practice is only slowing
the development of resistance and not preventing it. Fortunately, the inherent
capabilities of transgenic plants can be enhanced. In a bid to delay - better still,
preclude - the inception of resistance, it has been advocated that future transgenic
plants be developed to express multiple toxins. This is known as the pyramiding or
stacking strategy (Roush, 1997). This is the strategy suggested for transgenic citrus.
Transgenic Control of Diaprepes abbreviatus
Production of transgenic citrus plants expressing multiple deleterious toxins
against D. abbreviatus is an attractive and feasible control strategy. The key is to
identify multiple physiological/biochemical processes within the weevil that can be
26
targeted as interdiction points. The weevil’s digestive process presents an excellent
target for interdiction. This can be achieved by disrupting gut integrity and/or inhibiting
protein digestion, and interfering with the expression and consequent function of
important genes. Toxins with different modes of action will be particularly advantageous
in a multi-pronged attack on the insect to delay the onset of resistance. Several
candidate toxins at various stages of evaluation are reported herein.
Candidate Toxins
This research is based on the anticipation of imminent resistance to a single-
toxin transgenic strategy for D. abbreviatus. The identification and evaluation of toxins
that work in a variety of ways is given priority. Ideally, toxins with unrelated modes of
action are most suitable to reduce the probability of selecting for individuals with a
particular set of resistant genes. Specifically, the strategies to control D. abbreviatus in
a transgenic citrus variety include the Bt toxin Cyt2Ca1 to disrupt gut integrity.
Secondly, cysteine protease inhibitors (CPI) that target an important digestive enzyme,
cathepsin L1, are also investigated. Finally, double-stranded RNA sequences/molecules
to interfere with gene expression via mediated RNA interference are also screened.
Endotoxins from Bacillus thuringiensis. Insecticidal proteins (endotoxins)
produced by Bt, a soil microorganism, have been widely used in genetic engineering of
food crops in the past two decades. However, the bacterium’s use as an insecticidal
agent extends beyond a century (Crickmore, 2006) as foliar sprays. They are target-
specific with activities against different orders of insects - Lepidoptera, Diptera, and
Coleoptera. The insecticidal proteins, Crystal (Cry) and Cytolytic (Cyt) proteins are
produced during the sporulation phase of the bacteria. Bacillus thuringiensis strains
have evolved these toxins to colonize insects because they serve as a ready source of
27
nutrition for germination of the dormant spore, though a small minority of Cry toxins are
nematicidal (Marroquin et al., 2000; De Maagd et al., 2001). The Cry proteins are
secreted as protoxins by the bacterium, and after ingestion, are activated by gut
proteases to active toxins. The activated toxins bind to receptors in the gut epithelium,
resulting in the creation of pores and lyses of midgut epithelial cells (De Maagd et al.,
2001). The Cyt toxins have a different mechanism of action, but are believed to work in
a detergent-like mechanism by interacting directly with membrane lipids to insert into
the membrane (Manceva et al., 2005). In the Cry engineered transgenic plants, they are
engineered to express the active forms of the enzymes (precluding the need for
activation) and optimized with plant codon usage (Vaughn et al., 2005), making them
more robust and versatile.
Bacillus thuringiensis strains were screened to identify toxins active against D.
abbreviatus (Weathersbee et al., 2006). From this work, a candidate Cyt toxin,
Cyt2Ca1, was identified with activity against D. abbreviatus larvae. A synthetic Cyt2Ca1
gene was constructed with codon optimization specific for highly expressed genes in
citrus and this synthetic gene was engineered into alfalfa in a proof of concept approach
(alfalfa supports all life stages of the weevil). Preliminary work with transgenic alfalfa
(Figure 1-1) has shown that this is a promising strategy for D. abbreviatus control, and
therefore, Cyt2Ca1 was subsequently engineered into citrus.
28
Figure 1-1. General plant and root damage as a result of Diaprepes abbreviates feeding on transgenic versus wild-type alfalfa (Shatters, unpublished data). Four weeks after the addition of 10 larvae to individually potted plants, it is evident that transgenic plants had greater resistance to D. abbreviates injury (Photos by R.G. Shatters, Jr.).
Peptide inhibitors against protein digestion. The ability to break down
proteins and complex carbohydrates, by digestive proteases in the gut, into free amino
acids and simple sugars is essential for growth and development of insects (Bown et al.,
2004). Therefore, the inhibition of digestive proteolysis by protease inhibitors has been
suggested as a pest management strategy (Haq et al., 2004; Christou et al., 2006;
Schlüter et al., 2010).
In many coleopteran species such as the cowpea bruchid (Callosobruchus
maculatus Fabricius) and the western corn rootworm (Diabrotica virgifera virgifera
LeConte), cysteine proteases are the major digestive enzymes (Koiwa et al., 2000; Zhu-
Salzman et al., 2003). In D. abbreviatus, cathepsins (cysteine proteases) are one of two
important groups of enzymes responsible for protein digestion and predominantly active
under acidic conditions. The other is a serine protease with trypsin-like activity that
Cyt2Ca1 Alfalfa
Cyt2Ca1 Alfalfa
Wild-type Alfalfa Wild-type Alfalfa
29
functions in the alkaline range (Yan et al., 1999). Targeting the digestive proteases in
the midgut of D. abbreviatus may contribute to a pyramided strategy.
Preliminary studies have been done to determine the effect of an inhibitor, trypsin
modulating oostatic factor (TMOF), on D. abbreviatus trypsin. This inhibitor is a
decapeptide (YDPAPPPPPP) that has been shown to be the physiological signal that
terminates trypsin biosynthesis through translational control in mosquitoes and other
insects (Borovsky et al., 1993, 1995, 1996). In vitro, TMOF has been shown to inhibit
the activity of gut trypsins, and feeding TMOF to larval D. abbreviatus reduces weight
gain and results in reduced gut trypsin activity (Yan et al., 1999). Dov Borovsky and
Robert Shatters (personal communication) have cloned and expressed TMOF in alfalfa
and demonstrated that feeding citrus weevil the transgenic alfalfa leaves inhibited
trypsin activity in the gut. However, because D. abbreviatus also relies on cathepsins for
digestive protein degradation, blocking trypsin alone is not sufficient to induce significant
mortality. Therefore, research was conducted to characterize the nature of D.
abbreviatus digestive tract cathepsins and to test a strategy to block their activity that
could be deployed through transgenic means.
Cathepsins belong to the papain family of proteins, and many are lysosomal
proteins (Cristofoletti et al., 2005) involved in cellular protein catabolism (Kamboj et al.,
1993; Stachowiak et al., 2004). Through numerous studies of cathepsins, several
classes have been identified (Kirschke, et al., 1977; Turk et al., 2000) and classified
based on characteristics such as their activities with unique substrates and specific
inhibitors, and pH optimum for activity (Rawlings et al., 2012). In humans, mutations
affecting cathepsin activity are implicated in serious diseases such as arthritis, muscular
30
dystrophy and tumors (Hasnain et al., 1992; Gewies and Grim, 2003). In insects,
cathepsins are involved in digestion, embryogenesis, molting, tissue remodeling, and
reproduction (Bown et al., 2004; Deraison et al., 2004; Pyati et al., 2009).
In members of Coleoptera and Hemiptera known to utilize digestive cathepsins
(Terra, 1990), the digestive cathepsins are secreted into the gut lumen by the epithelial
cells. Cathepsins are synthesized as preproproteins; a polypeptide with three
identifiable regions: a pre-region which serves as a signal peptide that causes the
enzyme to enter the cellular secretory pathway where it is ultimately secreted into the
gut lumen; a pro-region which is essential for protein folding and regulates enzymatic
activity; and must be cleaved to generate the mature and active protein (third region of
the preproprotein). All cathepsins are characterized by a cysteine residue at the active
site (Turk et al., 2000) with a broad specificity for cleaving peptide bonds. Upon
synthesis, they remain inactive until the prepro- region of the polypeptide is removed.
The N-terminal pre-region is co-translationally removed (Philip et al., 2007), whereas
the pro-region is cleaved via autocatalysis at the optimum pH of the mature protein. The
pro-region, approximately 11-13 KDa in size, regulates enzymatic activity of the
proteinase domain by its interaction with the active site to block proteolytic activity (Tao
et al., 1994 and Philip et al., 2007). The pro-region is cleaved via autocatalysis at the
optimum pH and the molecule is activated.
More recently, genes encoding small peptides (approximately 11 KDa) with
significant homology to the pro-region of cysteine proteases have been characterized in
the digestive tract of insects (Kurata et al., 2001; Deshapriya et al., 2007; Miyaji et al.,
2010). The activities of the digestive cathepsins are modulated by these smaller
31
proteins (Figure 1-2). Two sequences encoding D. abbreviatus cathepsin L protease
inhibitors (CPI 1 and CPI 2) synthesized in the gut epithelial cells have recently been
discovered (Borovsky, unpublished results). CPI 1 is secreted into the gut lumen;
however, CPI 2 lacks a signal peptide and is believed to function in the epithelium
cytoplasm. Thus, it was hypothesized that the CPI1 modulates cathepsin activity in the
gut lumen. My thesis reveals research conducted to characterize the activity of CPI1
and attempts to modulate its inhibitor activity, to be utilized as a toxin expressed in
citrus to confer D. abbreviatus mortality.
32
CPI 2 ---------- -----MSAPT -KAP--SYLS DQEEWEKFKT GFNRNYDSSD
CPI 1 MLVKVFLLVV LAAVAMSAPS DTAPKQKSLS VEEHWNNFKT KFNRNYESPE
Cathepsin L 1 ---------- MKVFIAACLL VAVSATVLEE TGVKFQAFKL KHGKTYKNQV
Cathepsin L 2 -------MYS LVVLLATLVA YSHAISYQVL VQEQWEQFKL EHGKVYESES
CPI 2 EEAKRFNIFQ QNLQSIREHN EKFERGETTF TQGINQFTDL TKEEFKARHT
CPI 1 EESKRFEIFK NNLKDIQAHQ KKYEAGEVSY QQGVNDFTDL THEEFLATHT
Cathepsin L 1 EETARFNIFK DNLRAIEQHN VLYEQGLVSY KKGINRFTDM TQEEFRAFLT
Cathepsin L 2 ENEYRQSVFM ENLFQINEHN KLYEMGLSSY QMAMNHLGDL TKDEFMRIYT
CPI 2 GLLRRPPQE- ---------- ---------- ---------- ----------
CPI 1 MHFNPKPKS- ---------- ---------- ---------- ----------
Cathepsin L 1 LSSSKKP--- HFNTTEHVLT G--------- ---------L AVPDSIDWRT
Cathepsin L 2 VNMPQLPQSE NLSDSEPWLD LPQDLQGFVT YALPTNLDEV DLPTDIDWRQ
CPI 2 ---------- ---------- ---------- ---------- ----------
CPI 1 ---------- ---------- ---------- ---------- ----------
Cathepsin L 1 KGQVTGVKDQ GNCGSCWAFS VTGSTEAAYY RKAGKLVSLS EQQLVDCS-T
Cathepsin L 2 KGAVTPVKNQ RNCGSCWSFS ATGALEAQWF KKTNKLISLS EQQLVDCSGR
CPI 2 ---------- ---------- ---------- ---------- ----------
CPI 1 ---------- ---------- ---------- ---------- ----------
Cathepsin L 1 DINAGCNGGY LDETFTYVKS KG-LEAESTY PYKGTDGSCK YSASKVVTKV
Cathepsin L 2 YGNHGCHGGW MHWAFGYIKE NGGIDTEQSY PYTAKDGRCA YKPGNKAATV
CPI 2 ---------- ---------- ---------- ---------- ----------
CPI 1 ---------- ---------- ---------- ---------- ----------
Cathepsin L 1 SGHKSLKSED ENALLDAVGN VGPVSVAIDA TY-LSSYESG IYEDDWCSPS
Cathepsin L 2 S-QVIMVPRG ENQLAAKVSS VGPISIAAEV SHKFQFYHSG VYDEPQCGHS
CPI 2 ---------- ---------- ---------- ---------- ----------
CPI 1 ---------- ---------- ---------- ---------- ----------
Cathepsin L 1 ELNHGVLVVG YGTSNGKKYW IVKNSWGGSF GESGYFRLLR GKN-ECGVAE
Cathepsin L 2 -LNHAMLAVG YGSMGGKNFW LVKNSWGTGW GDQGYIRMAK DKNNQCGIAL
CPI 2 -------
CPI 1 -------
Cathepsin L 1 DTVYP--
Cathepsin L 2 MASYPGV
Figure 1-2. Amino acid sequence alignments of cathepsin L protease inhibitors (CPI 1 and 2) to cathepsin L 1 and 2 in Diaprepes abbreviatus. The pre-region of the cathepsin L 1 sequence in highlighted in green and the pro-region is highlighted in yellow.
Double-stranded RNA. RNA interference (RNAi) is an innate mechanism of
gene regulation in eukaryotes. It is an evolutionarily conserved gene-regulatory pathway
(Meister and Tuschl, 2004) that follows several different pathways ultimately to the
33
same fate - the silencing of genes. RNAi was known as a process in plants for many
years prior to its discovery in animals about a decade ago in the nematode
Caenorhabditis elegans (Maupas) (Fire et al., 1998). It has since been identified as a
potential mechanism for pest/vector control. By down-regulating essential gene function
(Price and Gatehouse, 2008), growth, development, and reproduction can be
hampered, thus leading to death or less fit individuals.
The underlying theme among all the different pathways is the requirement of a
double-stranded RNA template. Templates may be of varying length that specifically
hybridize to and result in the cleavage of a specific mRNA sequence transcribed from a
gene. Principally, dsRNA templates are processed by specific endonucleases,
Dicer/Drosha, into short RNA duplexes. These duplexes of short-interfering RNA
(siRNA) are typically 20-23 base pairs in length and staggered. They mediate
translational repression and mRNA degradation via the catalytic activity of argonaute
proteins, as part of a silencing complex (RNA-induced silencing complex: RISC) that
contains single-stranded siRNA (Meister and Tuschl, 2004). The single-stranded siRNA
recognizes complementary strands that are subsequently destroyed.
RNAi in insects is believed to constitute a part of an immune response to viral
infection (Keene et al., 2004). It is possible to manipulate an insect’s RNAi machinery to
attack itself, by presenting dsRNA complementary to its genes, such that it down
regulates the expression of these genes. A limitation of the RNAi mechanism exists in
insects, however. Contrary to C. elegans, plants, and fungi, there is no apparent
amplification of the dsRNA template in insects. There is yet no evidence of the presence
of the enzyme RNA-dependent RNA polymerase (RdRP) in insects for amplifying the
34
RNA signal (Tomoyasu et al., 2008). Despite this limitation, it is still possible to induce
an RNAi response in insects because of the systemic spread of dsRNA in insects
(Huvenne and Smagghe, 2010).
Gene silencing via RNAi has therefore been identified as a potential mechanism
for pest/vector control (Gordon and Waterhouse, 2007). Work on RNAi on the western
corn rootworm and the Colorado potato beetle shows that ingestible dsRNA may be
used in the future to control insects as transgenes expressed in transgenic plants
(Baum et al., 2007; Mao et al., 2007). Numerous other examples (Price and Gatehouse,
2008) highlight the potential of RNAi for pest control and would place it on the frontier of
pest management technology.
The Watson-Crick hybridization requirement of the RNAi mechanism makes it a
very valuable tool for future pest control. Thus, RNAi can be designed to be very
specific (Whyard et al., 2009). However, off-target effects can also occur (Nunes et al.,
2013). Additionally, lessons gleaned from the Shatters laboratory at the USDA-USHRL
investigating RNAi for control of the Asian citrus psyllid, Diaphorina citri Kuwayama,
indicate that it is possible to induce mortality with significant doses dsRNA of any nature
or source. Therefore, a dose-response effect is vital in RNAi experiments.
Bacillus thuringiensis Cyt2Ca1, peptide inhibitors, and dsRNA sequences will
provide a three-pronged approach to D. abbreviatus control in a transgenic strategy.
The efficacy of each of these approaches needs to be independently verified before any
future synergistic studies. My thesis sets out to study these approaches separately. A
discussion of delivery modes in a transgenic strategy is necessary, and I express an
opinion about this in the following section.
35
Delivery of Toxins in a Transgenic Strategy
Having identified suitable toxins for control through artificial diet bioassays, it is
important then to consider a variety of delivery methods for a transgenic variety.
Initially, transgenic alfalfa expressing identified D. abbreviatus toxins was promoted as a
shield crop in citrus groves because the weevil also eats alfalfa. In a proof-of-concept
experiment, the laboratories of Dov Borovsky and Charles Powell at the University of
Florida and Robert Shatters in the USDA-USHRL cloned TMOF active against D.
abbreviatus in alfalfa. Feeding citrus weevil the transgenic alfalfa leaves stopped
trypsin activity in the gut of the weevil (Borovsky et al., unpublished data). On the other
hand, avoidance of transgenic alfalfa and a preference for citrus may compromise the
success of such a strategy and possibly exacerbate the pest status of the weevil.
Citrus is attractive for engineering against D. abbreviatus because of the way it is
cultivated. Commercial cultivation of citrus uses trees produced by grafting the desired
fruiting cultivars (scions) onto rootstocks selected for traits such as resistance to insects
and diseases. Citrus rootstocks can thus be engineered for resistance to D.
abbreviatus, but not the scions, leaving fruits without transgenic genes. The fact that
recombinant proteins would not be expressed in the fruits may boost consumer
acceptance, as the ultimate goal of this project is to have rootstock strains that will
provide a commercially viable pest management strategy in citriculture. An engineered
citrus rootstock delivery mechanism has the principal advantage of being target-specific
on two counts. Firstly, the range of insects that could be affected is narrowed down to
only insects that directly feed on citrus, excluding insects that do not feed on citrus.
Secondly, but more importantly, these toxins are sourced directly from D. abbreviatus
specifically.
36
A third delivery mechanism is to use citrus tristeza virus (CTV) as a vector of
toxins. Citrus tristeza virus, an infectious agent commonly present in citrus, also lends
itself as a vector of dsRNA and protein molecules in citrus (Folimonov et al., 2007).
Engineered to be infective but not pathogenic, several CTV-based transient-expression
vectors have been developed for gene expression or silencing studies in, but not limited
to, citrus (Dawson et al., 2010). The CTV vector has been designed for systemic field
infection of trees to express copious amounts of recombinant gene product for up to
several years. As the replicative form of CTV is double-stranded RNA (Tatineni et al.,
2010), it also enables manipulation for the expression of dsRNA molecules, without a
hair-pin loop targeting specific organisms in an RNA-mediated interference mechanism.
Thus it will be possible to employ the CTV-vector to express peptide inhibitors and/or
dsRNA toxins identified for specific D. abbreviatus control under field conditions. The
greatest advantage of using CTV as a delivery tool is that it drastically cuts the time
typical for the development of resistant trees. Importantly, it removes the requirement to
completely re-plant citrus groves with transgenic strains as it is possible to infect
existing trees with the engineered virus.
37
CHAPTER 2 RECOMBINANT EXPRESSION AND CHARACTERIZATION OF DIAPREPES
ABBREVIATUS CATHEPSIN L1 AND THE ROLE OF CATHEPSIN L PROTEASE INHIBITORS IN THE REGULATION OF CATHEPSINS INVOLVED IN DIGESTION
Proteins are an important food source for D. abbreviatus, similar to many
organisms. The literature is replete with many examples where the inhibition of digestive
proteases resulted in death and retardation of growth of many pest insects (Oppert et
al., 1993; Macedo et al., 2003; Vila et al., 2005). Therefore, inhibiting protein digestion
in insects with protease inhibitors (PIs) has been suggested for pest control (Bown et
al., 2004; Christou et al., 2006). The digestive proteases of targeted pest insects require
in depth characterization to find potential inhibitors of their activities. Cathepsins
constitute one component of a dual proteolytic system in D. abbreviatus (Yan et al.,
1999). Diaprepes abbreviatus cathepsins are not as well characterized as the trypsins
(the other component). Due to the role of D. abbreviatus cathepsin in digestion, they
could be an important target for inhibition.
Gut transcriptome studies from larval stages of D. abbreviatus identified
numerous cathepsins that are turned on or off depending on the life-stage and age of
the insect, suggesting their need for growth and development (Shatters, unpublished
data). Additionally, crude D. abbreviatus gut assays indicate the predominance of
cathepsins active in the acidic range between pH 4-6.5 in a midgut that is partitioned
into acidic (anterior) and basic (posterior) regions, similar to the aphid, Aphis gossypii
Glover (Deraison et al., 2004), and the weevil Otiorhynchus sulcatus (Fabricius)
(Edwards et al., 2010). In order to inhibit the enzyme in a possible transgenic control
strategy of D. abbreviatus in citrus, pure recombinant cathepsins will be invaluable for
kinetic studies. Two of the highly expressed cathepsin L homologues were selected for
38
recombinant synthesis however, only cathepsin L1 (DaCatL1) was successfully
expressed and purified. The results of the characterization of DaCatL1 are presented
herein.
In vitro enzymatic assays used to characterize cathepsins feature small peptide
substrates consisting of a sequence of amino acid residues linked to a measurable
leaving group. Also, Positional Scanning Synthetic Combinatorial Libraries, enable the
determination of the preferred amino acid sequences of substrates for various
proteases including cathepsin L (Cotrin et al., 2004; Choe et al., 2006).The leaving
groups of the substrates may be fluorogenic or chromogenic (Tchoupé et al., 1991). The
classes of cathepsins are grouped according to chemical properties available in the
MEROPS database including the sequence of amino acids in suitably cleaved
substrates (Berger and Schechter, 1970; Rawlings et al., 2012). Cathepsin L has been
shown to have substrate tolerance for hydrophobic residues in the P2 and P3 positions
(Otto and Schirmeister, 1997) with a preference for leucine or arginine at the P2 position
(Clara et al., 2011).
The activities of cathepsins are regulated by small peptide inhibitors called
cystatins. Two endogenous regulators of D. abbreviatus digestive cathepsins, cathepsin
L protease inhibitors (CPI 1 and 2), have been identified (Borovsky, Shatters, and
Powell, unpublished data). Their inhibition of cathepsins is competitive and reversible
and thus, it may be possible to modulate this interaction. Amino acids in the CPI
molecule are predicted to be important for their inhibitory activity. Therefore amino acid
substitutions in CPI may lead to a mutant with modified chemical interaction with
cathepsin. Cathepsins catalytic cysteine is hypothesized to form a disulfide bridge with a
39
proximate cysteine on a CPI mutant. The disulfide bridge formed between the mutant
inhibitor, and enzyme would make the mutant a better inhibitor due to the irreversible
interaction. Site-directed mutagenesis is a technique to introduce amino acid
substitutions in proteins. Polymerase chain reaction utilizing primers that incorporate
codon change flanked by long complementary sequences is used to generate mutations
in a cDNA template via the technique.
Although no PIs are available commercially, as ready-to-use formulations or in
transgenic cells, they can still be complementary to conventional approaches using
chemical and biological pesticides in future pest control strategies. They can make
target pests more vulnerable to other toxins, such as Bt toxins or dsRNA, in a stacked
transgenic approach. Thus, DaCatL1-CPI interactions need to be verified and exploited.
Materials and Methods
Molecular Modeling and Docking of Cathepsin L Protease Inhibitor to D. abbreviatus Cathepsin L1
Homology modelling of DaCatL1 was done using the YASARA Structure program
(Krieger et al., 2002). Different models of DaCatL1 were built from the X-ray coordinates
of the procathepsin L1 from Fasciola hepatica Linnaeus (PDB code 2O6X) (Stack et al.,
2008), the larval midgut procathepsin L3 of Tenebrio molitor Linnaeus (PDB code
3QT4) (Beton et al., 2012), the Cys25Ala mutant of human procathepsin S (PDB code
2C0Y) (Kaulmann et al., 2006), the human procathepsin L (PDB Code 1CS8)
(Coulombe et al., 1996), and the activated cathepsin L of Toxoplasma gondii Nicolle
and Manceaux in combination with the propeptide (PDB code 3F75) (Larson et al.,
2009). Finally, a hybrid model of DaCatL1 was built up from the five previous models.
40
The CPI1 was similarly modelled using the X-ray coordinates of the propeptide
from the larval midgut procathepsin L2 from T. molitor (PDB code 3QJ3) (Beton et al.,
2012), the procathepsin L1 from F. hepatica (PDB code 2O6X) (Stack et al., 2008), the
larval midgut procathepsin L3 of T. molitor (PDB code 3QT4) (Beton et al., 2012), the
Cys25Ala mutant of human procathepsin S (PDB code 2C0Y) (Kaulmann et al., 2006),
and the activated cathepsin L of T. gondii in combination with the propeptide (PDB code
3F75) (Larson et al., 2009). A hybrid model for CPI1 was built from the five previous
models.
PROCHECK (Laskowski et al., 1993) was used to assess the geometric quality
of the three-dimensional models. In this respect, all of the residues of DaCatL1 were
correctly assigned in the allowed regions of the Ramachandran plot except for 9 Gly
residues which occur in the non-allowed region of the plot. Using ANOLEA (Melo and
Feytmans, 1998) to evaluate the model, only 12 residues of DaCatL1 out of 306
exhibited energy over the threshold value. All residues are located in the loop region
connecting alpha-helices and beta-sheets. Molecular models were drawn with the
UCSF Chimera package (Pettersen et al., 2004).
The T. gondii cathepsin L (TgCPL) in complex with its propeptide (PDB code
3F75) (Larson et al., 2009) was taken as a model for docking experiments of the
cathepsin L inhibitor CPI1 on activated DaCatL1. Docking pattern was also rendered
with Chimera.
The SDSC Biology Workbench (San Diego, CA) was used for amino acid
sequence comparisons between DaCatL1 precursor (GenBank: ABG73217).1) and the
Homo sapiens cathepsins L1 isoform 1 preproprotein (NCBI Reference Sequence:
41
NP_001903.1), K (GenBank: AAH16058.1), S (GenBank: AAC37592.1), B (GenBank:
AAH95408.1), and H (GenBank: EAW99143.1).
Rearing of Diaprepes abbreviatus
Diaprepes abbreviatus larvae were obtained from a colony maintained at the
USDA-USHRL, Fort Pierce, Florida on an artificial diet described by Lapointe and
Shapiro (1999).
Bacterial Strains and Plasmids
Escherichia coli strains OneShot TOP 10F’ and DH5-alpha (Invitrogen, Grand
Island, NY) were used for sub-cloning, purification, and sequencing of plasmid DNA.
The plasmid used for checking DNA sequences was pCRTM2.1 (Invitrogen). Expression
plasmids used were pET DUET (EMD Millipore, Billerica, MA) for cathepsin L protease
inhibitors (both wild-type and mutant forms) and pET-41a (+) (EMD Millipore) for
DaCatL1. These plasmids permit the expression of proteins as fusion products, with the
incorporation of a polyhistidine tag (both) and a glutathione-S transferase tag (pET-
41a(+)). A plasmid purification kit (Qiagen, Valencia, CA) was used for all purifications
according to manufacturer’s protocol. An IPTG (Isopropyl β-D-1-thiogalactopyranoside
)-inducible derivative of E. coli strain BL21 (DE3), Rosetta™ (DE3) pLysS Competent
Cells, purchased from EMD Millipore, was used for expression of recombinant proteins.
Bacterial strains were grown in Luria broth (LB) media at 37 °C except after inducing
with IPTG for protein synthesis and expression at which time cells were grown at 25 °C
for at least 4 h up to overnight.
Primers
The primer sequences (5’-3’) listed below were obtained from IDT® (San Diego,
CA) and used for the cloning of DaCatL1, GenBank accession number: GenBank:
42
DQ667143.1 (GstproCatL1-f and GsrproCL1-r) and site-directed mutagenesis of CPI1,
GenBank accession number: EU009453.1, to make the mutant CPI (CPI1mut-f and
CPI1mut-r).
GstproCL1-f:
CTCAATCACTAGTACCGTCTTGGAGGAGACAGGTGTCAAAT
GstproCL1-r:
GCAGTCACTCGAGTATAATTGGATATACGGTATCTTCAGCAACTCC
CPI1mut-f:
GAATTTCTTGCTACTCACACGTGTCACTTCAATCCCAAACCCAAG
CPI1mut-r:
CTTGGGTTTGGGATTGAAGTGACACGTGTGAGTAGCAAGAAATTC
Cathepsin L 1 and Cathepsin L Protease Inhibitor Constructs
The DNA sequences of the pro- and active regions of DaCatL1 were amplified
from a cDNA library and cloned into the expression vector pET-41a (+) DNA. The
primers GstproCL1-f (incorporating the cut site for SpeI) and GstproCL1-r (incorporating
the cut site for XhoI) were used to amplify DaCatL1 using standard polymerase chain
reaction (PCR) protocols. The following thermal cycling conditions were used: 95 °C for
10 min, followed by 30 cycles of 95 °C for 15 sec, 57 °C for 30 sec and 72 °C for 30 sec,
and a final extension at 72 °C for 10 min. The PCR product was verified on an agarose
gel via electrophoresis. The band holding the product was cut from the agarose gel. The
DaCatL1 fragment was recovered from the agarose gel band using Wizard® SV Gel and
PCR Clean-Up System (Promega, Madison, WI) following manufacturer’s instructions.
The restriction enzyme ends incorporated into the amplification primers were cut from
the DaCatL1 fragment with restriction enzymes (SpeI and XhoI, obtained from New
43
England Biolabs®, Ipswich, MA), and the fragment was then cloned into the expression
vector pET-41a. One Shot Top 10 (Invitrogen) competent E. coli cells were transformed
with pET-41a (100 ng) and grown on LB media containing kanamycin (50 µg/mL).
Single colonies were selected and grown overnight at 37 °C in LB media containing
antibiotic in a shaking incubator at 225 rpm. Plasmid (pET41-a) was then extracted from
the E. coli cells using a plasmid extraction kit (Qiagen) and digested with SpeI and Xho1
as described for the DaCatL1 fragment. The linearized vector was separated by
agarose gel electrophoresis, cut from the gel and cleaned as previously described.
SpeI/XhoI-digested pET 41a and DaCatL1 were ligated using T4 DNA ligase overnight
at 4 °C. The ligated product was transformed into One Shot Top 10 competent cells,
single colonies were selected and the plasmids extracted and sequenced. Several
colonies contained the DaCatL1 insert, and glycerol stocks were made and stored at
minus 80 °C.
Cathepsin L protease inhibitor (wild type) (CPI 1, GenBank: EU009453.1) was
cloned into the pETDuet-1 (EMD Millipore) at the multiple cloning site-1 between the
restriction sites BamHI and NotI for synthesis of the recombinant protein as a fusion
product with a Histidine tag to enable selective purification based on affinity to a Ni-NTA
resin.
DNA fragments were sequenced by the genomics core facility at the USDA-
USHRL, and University of Florida-Interdisciplinary Center for Biotechnology Research
(UF-ICBR), Gainesville.
Site-Directed Mutagenesis
Primers (CPI1mut-f and CPI1mut-r) were synthesized to replace Lysine 101 with
Cysteine residue in the CPI1 construct (CPI1 fragment cloned into the pETDuet-1
44
Vector (EMD Millipore) using the QuikChange II XL Site-Directed Mutagenesis Kit and
manufacturer protocol (Agilent, Santa Clara, CA). A PCR was set up with the CPI1
construct as a template, according to the manufacturer’s instructions. The amplified
product was digested with DpnI, cleaned using the Wizard® SV Gel and PCR Clean-Up
System (Promega), and cloned into XL10-Gold Ultracompetent Cells (Agilent). Single
colonies were selected from LB plates (100 µg/mL carbenicillin), grown in LB
carbenicillin media, and plasmids extracted using plasmid extraction kit from Qiagen.
Plasmids were sequenced as described below, and verified constructs were
transformed into Rosetta™ (DE3) pLysS Competent Cells (EMD Millipore) for protein
synthesis.
Expression of Recombinant Proteins
Rosetta™ (DE3) pLysS Competent Cells (EMD Millipore) cells were transformed
with pET-41a carrying DaCatL1 sequence. Multiple colonies with the engineered vector
were tested to verify the biosynthesis of the recombinant proteins. Cell cultures (5 mL)
were initially grown at 37 ºC, 225 rpm in LB medium (30 μg/mL chloramphenicol and 50
μg/mL kanamycin) to OD600 of 0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (1
mM) was then added to induce synthesis of recombinant proteins in treatment cultures.
Controls were grown similarly, except the IPTG induction step was omitted. The
temperature during the induction stage was maintained at 25 ºC for 4-6 h. Cells were
harvested by centrifugation at 16,000 rpm for 5 min at 4 ºC (Eppendorf's refrigerated
bench-top 5702 R centrifuge), re-suspended in 1 mL phosphate buffer saline (50 mM
NaH2PO4, 300 mM NaCl, pH 7.0), and added to 2-mL tubes containing 180 μm acid-
washed glass beads Cells were broken in a FastPrep Instrument for 1 min and
centrifuged at 14,000 rpm for 10 min at 4 ºC. The supernatant was collected and
45
aliquots (20 μL each) were run on sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) using the 4-12% NuPAGE® Novex® Bis-Tris Gel (1.0 mm
thick) (Life Technologies, Carlsbad, CA). DaCatL1 fused with the GST tag ran as a
protein band of 60 KDa, whereas the CPI1 and CPI1mut ran as bands of 11 KDa. To
synthesize larger amounts of recombinant proteins, larger cultures of transformed E.
coli (4-6 L) were grown and harvested as before. Harvested E. coli cells were lysed
using a cell disruptor (Branson 450 Sonifier - SmithKline). However, the DaCatL1
quickly precipitated out of solution. To determine if adding surfactants/detergents
(obtained from Affymetrix, Santa Clara, CA) would improve solubility, the following
detergents were tested: Amphipol (20 ng/mL), CHAPS [3-((3-Cholamidopropyl)
dimethylammonium)-1-propanesulfonate] (8 mM), OGNG [Octyl Glucose Neopentyl
Glycol] (1 µM) and TritonTM X-100 (0.25% and 1%). Incorporating 1% TritonTM X-100 in
all buffers used from lysis stage through nickel resin (Ni-NTA, Qiagen) purification, and
size exclusion chromatography maintained the enzyme in solution. SDS-PAGE was
used to identify the recombinant protein followed by western blot analysis.
Protein quantification was done using the Quick Start™ Bradford (Bio-Rad,
Hercules, CA) method in all cases, except when proteins were solubilized in 1%
TritonTM-X100 in which the RC DC™ Protein Assay (Bio-Rad) was used. Synergy™ HT
Multi-Mode Microplate Reader (BioTek, Winooski, VT) was used to read absorbance.
Western blot analysis of recombinant proteins was done using the Xcell II blot
module from Invitrogen to transfer resolved proteins from SDS-PAGE onto a
nitrocellulose membrane. Antibodies raised against histidine tagged proteins- 6x-His
46
Epitope Tag Monoclonal Antibody kit (Thermo Scientific, Waltham, MA), was used to
locate lanes (bands) with DaCatL1, CPI1, and CPI1mut.
Western Lightning Plus ECL kit (PerkinElmer, Waltham, MA) was used to
visualize proteins on the nitrocellulose membrane with the aid of a KODAK imager
(Image Station 440CF).
Purification of Recombinant Proteins
After lyses of cells via sonicator, cell debris was removed by centrifugation
(Beckman XL-I analytical ultracentrifuge - 20,000 rpm at 4 °C for 40 min). Initial
purification of histidine-tagged recombinant proteins from the crude extract was done
using affinity resin (Ni-NTA Superflow -Qiagen). Equilibrated resin (5 mL; 50 mM Tris
buffer, pH 7, 300 mM NaCl, 1 mM BME, 1% TritonTM, 10% glycerol) was added to 50
mL crude extract and gently mixed in the cold for an hour. Unbound proteins were
removed by centrifugation at low speed (2,000 rpm, 5 min, 4 °C), and bound proteins
were washed with buffer (5 times) by mixing in the cold as before with 5 volumes of
buffer. Recombinant proteins were eluted from Ni-NTA resin with 250 mM imidazole in
buffer (above), by mixing as before. The Ni-NTA resin was removed by low speed
centrifugation and purified proteins were analyzed by SDS-PAGE and western blotting.
The recombinant proteins were then purified by size-exclusion chromatography
(HiprepTM 16/60 SephacrylTM S-100 HR) with an ÄKTA™FPLC™ system (GE
HealthCare Life Sciences, Piscataway Township, NJ). Eluted proteins were
concentrated on centrifugal filters (Amicon® Ultra-15 mL with 3000 and 10, 000 MW cut-
off for cysteine protease inhibitors and recombinant cathepsins respectively, EMD
Millipore), to each aliquot glycerol was added to a final concentration of 20%, and the
proteins were flash frozen and stored at -80 °C.
47
Enzymatic Assays
DaCatL1: The activity of DaCatL1 was initially assayed using three small
fluorogenic peptide substrates, Z-GPR-AMC (Z= benzyloxycarbonyl, GPR = glycine,
proline, and arginine, AMC = 7-amino-4-methylcoumarin), Z-FR-AFC (FR=
phenylalanine and arginine, AFC= 7-Amino-4-trifluoromethylcoumarin), and Z-VVR-
AMC (VVR= valine, valine, and arginine), and three small chromogenic peptide
substrates, Suc-AAPF-pNA (N-succinyl-alanine-alanine-proline-phenyalanine-p-
nitroanilide), H-Arg-pNA.2HCl, and Z-FR-pNA.HCl (all obtained from Enzo® Life
Sciences, Farmingdale, NY) using a fluorometer (Wallac 1420, Victor3TM- PerkinElmer).
However, Z-FR-AFC was found to be the best substrate and was used for all
subsequent assays. The 96-well plate format (Corning® - Corning, NY - 96 Well Flat
Clear Bottom Black Polystyrene TC-Treated Microplates, to prevent well-to-well
crosstalk) was used contained a final assay volume of 150 µl per well. The fluorescent
assays were conducted at excitation/emission wavelengths of 355 nm/535 nm, 25 °C,
with a final substrate concentration of 12.5 µM, whereas the chromogenic assays were
conducted at an absorbance of 405 nm with final substrate concentration of 80 µM.
Fluorescence was monitored at 5 min intervals for up to 120 min. To find the optimum
pH, the following buffers were used at the variously stated pH range: 50 mM sodium
acetate buffer (pH 3-4), 50 mM MES buffer (pH 5-6), and 50 mM Tris-HCl (pH 7-9). For
proteinase inhibitor assays, the enzyme-inhibitor mixtures (without substrate) were pre-
incubated at room temperature for at least 10 min prior to the assay. The assays were
performed in duplicates/triplicates and repeated1-3 times. The inhibition of DaCatL1
was assessed using the protease inhibitors within their physiological working
concentrations: E-64 (L-trans-Epoxysuccinyl-leucyamido (4-guanidino) butane), Z-
48
FY(tBu)DMK (N-benzyloxycarbonyl-phenylalanyl-t-butyl-tryrosyl diazomethylketone),
CA074, cystatin and chymostatin at 50 µM each and aprotinin at 50 nM.
D. abbreviatus gut in vitro assays: To find the cysteine protease(s) activity of
D. abbreviatus cathepsins, crude cathepsins from the guts of 5 actively feeding 6-week-
old larvae were obtained by homogenizing surgically removed midguts in deionized
water (1 larval gut per 100 µl of water). Debris was removed by centrifugation (14,000
rpm at 4 °C) and supernatants saved as 25 µl aliquots. Enzymatic assays were
conducted as described with Z-FR-AFC (12.5 µM) in the presence of cathepsin B
specific inhibitor CA074 (50 µM). Single gut aliquots were removed as needed and
diluted 1:100 for enzymatic assays. One microliter was used per well in a total assay
volume of 150 µl.
To investigate the role TritonTM X-100 might have on protein folding and therefore
the activity of cathepsins and the interaction with the peptide inhibitors, an experiment
was performed in which the guts of four six-week-old larvae were extracted into buffer
containing TritonTM X-100 (1%) and another without TritonTM X-100. Enzymatic assays
were performed at acidic (pH 5.5), neutral (pH 7), and basic (pH 8) conditions using the
substrate Z-FR-AFC, with the gut extracts including either of the two inhibitors. The
cathepsin B inhibitor (CA074) was included at 50 µM. To eliminate the possibility that
TritonTM X-100 affected the interaction with the inhibitors, I tried to remove TritonTM X-
100 using HiPPR® Detergent Removal Spin Columns (Thermo Scientific).
Results
Modeling of Cathepsin L1 and CPI1
DaCatL1 consists of a short N-terminal propeptide with three alpha-helices,
linked to a longer C-terminal catalytic domain which possesses two catalytically active
49
residues Cys120 and His255 located at the centre of the catalytic groove (Figure 2-1 A).
Both domains are organized in such a way that the propeptide completely blocks the
catalytic groove of the active domain, inhibiting the proteinase activity of cathepsin L1
(Figure 2-1 B, C). After cleaving the propeptide from the catalytic groove, DaCatL1
recovers its proteolytic activity.
CPI1 comprises three alpha-helices similarly organized as in the propeptide of
DaCatL1. The amino acid sequences of the propeptide and CPI1 share high similarity (~
70%) but moderate percentages of identity (~25%). According to these common
structural organizations, the modelled CPI1 exhibits similar overall three-dimensional
conformation to the natural propeptide of DaCatL1 with a long C-terminal tail at the end
of the alpha-helical core structure (Figure 2-1 D). The C-terminal tail allows CPI1 to bind
in the catalytic groove of DaCatL1 (Figure 2-1 E) and to block its enzymatic activity. The
enzymatic inhibition is due to steric hindrance at the catalytic groove that prevents the
substrate from interacting with the active residues Cys120 and His255 at the active
groove (Figure 2-1 F). CPI1 interacts with the activated DaCatL1 through a network of
hydrogen bonds forming between the core structure of CPI1 and the upper part of
DaCatL1. The endogenous CPI1 offers D. abbreviatus an efficient mechanism to
regulate cathepsin L activity in the gut by mimicking the natural propeptide which
maintains DaCatL1 inactive.
Synthesis and Purification of DaCatL1
Although the E. coli cloning, synthesis, and subsequent purification of CPI1 and
its mutant (CPI1mut) were straight-forward with standard techniques, the production of
DaCatL1 was very challenging. Previously, unsuccessful attempts at synthesizing D.
abbreviatus cathepsin L2 were made using the yeast expression system of Pichia
50
pastoris (pPICz-alpha, Invitrogen), a Human In Vitro Translation Kit (Thermo Scientific),
and DaCatL1 in E. coli (pET200 Directional TOPO® Expression Kit, Life Technologies) .
To overcome these difficulties, DaCatL1 was expressed in an E. coli system fused to a
GST tag. Syntheses of recombinant proteins were induced with IPTG in DaCatL1-
transformed E. coli cells, alongside non-transformed control cells. Crude bacterial
extracts were obtained and run on SDS-PAGE (Figure 2-2, lanes a-b). DaCatL1 fusion
product was affinity purified (Figure 2-2, lane c) followed by size-exclusion
chromatography of the mature DaCatL1 at pH 8 (Figure 2-2, lane d). In the SDS-PAGE
analysis of DaCatL1 (Figure 2-2), the inactive fusion DaCatL1 with GST tag is present
at the molecular weight size of 60KDa and the active DaCatL1 (Mr 23 KDa) is also
present. Monoclonal antibodies against His6-tag proteins identified DaCatL1 from
transformed E. coli cells but not the non-transformed control (Figure 2-3).
Although the GST tag enabled high synthesis of DaCatL1, the recombinant
enzyme was unstable and readily precipitated out of solution, regardless of the different
conditions that were employed, such as low salt LB cultures, induction and expression
at various temperatures (25 – 37 °C), and the incorporation of the surfactants
(amphipol, CHAPS, OGNG and TritonTM X-100 at different concentrations) during lyses.
DaCatL1 only remained soluble in TritonTM X-100 (1%).
The mass spectrometry analysis showed 34 unique peptides (yellow in Figure 2-
4) indicating 100% probability that the expressed purified protein is DaCatL1 fused to
GST and His6 (end of sequence).
Activated DaCatL1 was eluted under the second protein peak (red arrow in
Figure 2-5) using size-exclusion chromatography and Tris-HCl (pH 8) buffer. The eluted
51
protein Mr was found to be 23 kDa by SDS-PAGE and the protein showed Cathepsin L
activity when incubated with its substrate ( Z-FR-AFC) at pH 8 (Figure 2-2, lane d). The
first protein peak (blue arrow in Figure 2-5) is the non-activated moiety of Cathepsin L
(Mr 60 kDa) after analysis by SDS-PAGE (results not shown).
Synthesis and Purification of CPI
Two forms of CPI 1 were expressed and purified. The first form was the wild
type CPI1 that was discovered in D. abbreviatus. The second form (CPI1mut) was a
mutated CPI1 in which Lys101 was replaced by Cys to form an S-S bond between
Cathepsin L Cys120 and CPI1mut Cys101. The inhibitor was extracted from bacterial
lysates, purified by affinity chromatography on Ni-NTA resin, and then purified to
apparent homogeneity by size exclusion chromatography. At each purification step,
CPI1mut was assayed for homogeneity by SDS-PAGE (Figure 2-6a-d). After size
exclusion chromatography, the inhibitor exhibited apparent homogeneity and was also
assayed by monoclonal antibodies to the His6-tag, proving that staining the purified His6-
CPI1mut with coomassie brilliant blue or analyzing with His6 antibodies detect the same
protein of Mr 11 kDa (Figure 2-6 d and e).
Cathepsin L1 Substrate Specificity and pH Optimum
DaCatL1 hydrolyzed the general cathepsin L, B and H substrate, Z-FR-AFC
(Figure 2-7). Thus, all activity assays were carried out in the presence of 50 µM CA074
(cathepsin B inhibitor). The activity assay showed that DaCatL1 is more active at pH 8
than at pH 5.5. DaCatL1 showed optimal activity at pH 8 as determined with fluorogenic
substrate Z-FR-AFC at 25 ºC (Figure 2-8). The fluorogenic substrate is more sensitive
in the bioassay than its chromogenic counterpart, Z-FR-pNA (results not shown) or Z-
GPR-AMC and Z-VVR-AMC (Figure 2-7). These results indicate that DaCatL1 is an
52
alkaline cathepsin in contrast to the majority of cathepsins that show optimal activity at
acidic pH range. The cathepsin G substrate SucAAPF-pNA (also cleaved by
chymotrypsin-like proteases) and HR-pNA (Cathepsin H and other aminopeptidases)
were not cleaved by DaCatL1 (data not shown).
Effect of Inhibitors on Cathepsin L1 Activity
The activity of DaCatL1 was assessed at pH 8 (optimal enzyme activity, Figure 2-
8) in the absence or presence of known protease inhibitors by incubating the enzyme
and the inhibitors together for at least 10 min and conducting the assay for 120 min.
Aprotinin (serine protease inhibitor) at 50 nM weakly inhibited DaCatL1; it inhibited
DaCatL1 activity almost completely at a 1000-fold higher concentration (data not
shown). In the presence of the general cathepsin inhibitor (excluding cathepsin C and
H) E-64 (50 µM), the activity was inhibited by 26.6%, whereas the specific cathepsin L
inhibitor Z-FY(tBu)DMK (50 µM) caused higher inhibition (40.6%). The specific
cathepsin B inhibitor (CA074) at 50 µM inhibited the enzyme by 25.3%. The general
protease inhibitors cystatin and chymostatin (50 µM each), which also inhibit cathepsin
L, were more effective, inhibiting DaCatL1 activity, by 47.5% and 85.6%, respectively
(Figure 2-9).
To determine if DaCatL1 is affected by CPI1 and CPI1mut, the enzyme was
separately incubated with each inhibitor and its activity followed in the presence and
absence of the inhibitors (Figure 2-10). As inhibitor concentration increased from 1
µMto 10 µM, an increase in inhibition was recorded. Both inhibitors were equally
effective. However, the inhibition of the enzyme by both inhibitors did not reach 50%
53
even in the presence of 10 µMof CPI1 or CPI1mut, indicating that DaCatL1 is only
partially inhibited by CPI1 inhibitors at pH 8.0.
Sequence Alignment of Cathepsin L1 and Human Cathepsins
Alignment of amino acid sequences of DaCatL1 and human cathepsins showed
similarities among the different cathepsin sequences (Figure 2-11). Active site residues
of DaCatL1 and human Cathepsin L1 (HumCTL1): C, H, Q and N, are conserved in all
the different cathepsins. Therefore, folding of the protein molecules is important to
achieve proper catalytic activity of these proteases. Thus, substrate specificities and
substrate binding sites (Turk et al., 2000) are important in distinguishing the different
cathepsins even though high conservation at the catalytic amino acids is observed
(Figure 2-11).
D. abbreviatus Gut in vitro Assays
In acidic and neutral conditions, TritonTM X-100 negatively affected the catalytic
activity of the gut cathepsin Ls, whereas the inhibition of the enzymatic activity of the gut
cathepsin Ls with either inhibitor is not affected by TritonTM X-100 (Figure 2-12). The
enzyme-inhibitor interaction is predominantly influenced by the pH of the incubation
mixture. Since the catalytic activity of the enzyme itself is affected by TritonTM X-100, it
is not clear if the surfactant has an effect on the binding of CPIs to the enzyme. At the
basic pH, TritonTM X-100 does not have an effect on the enzymatic activity of the basic
Cathepsin L or the binding of CPI to it.
Removing TritonTM X-100 using HiPPR® Detergent Removal Spin Columns
(Thermo Scientific) had no effect on the enzymatic activity observed earlier (data not
shown). However, protein recovery of the enzyme after the spin column step was low.
This may be because of the instability of DaCatL1 in the absence of TritonTM X-100.
54
Both CPI inhibitors were very effective inhibitors of digestive cysteine proteases
at pH 4-7 as they inhibited 80- 90% of the gut cathepsin L activities (Figure 2-13);
however, at pH 8 the inhibitors inhibited13- 25% of the gut enzymatic activity. This
suggests that DaCatL1 is a basic cathepsin L in the gut of D. abbreviatus.
Discussion
The interaction between cathepsin L 1 and CPI1 were modeled by Pierre Rougé
(Université de Toulouse, Institut de Recherche pour le Développement). Using Rouge’s
3D models, CPI1 was modified by mutating one of the amino acids that is in close
proximity to Cathepsin L unbridged cysteine in order to bind CPI to Cathepsin L using S-
S bonding. This is postulated to make the binding between CPI and Cathepsin L
irreversible in the hope of using this strategy in transgenic citrus. Cathepsin L protease
inhibitors expressed in the roots of citrus will prevent food digestion in the gut of larval
D. abbreviatus.
The working hypothesis was formed from gut transcriptome studies that DaCatL1
is a major digestive enzyme (R. Shatters, unpublished data). Furthermore, based on the
predominance of high activity of cathepsins at acidic pH, DaCatL1 was postulated to be
active at acidic pH, where the pro-segment of the molecule is cleaved. In the posterior
midgut (alkaline pH), DaCatL1 molecules are bound by modulators of their activity,
cystatins – cysteine protease inhibitors (Otto and Schirmeister, 1997), to protect them
from degradation and therefore conserve the insects energies in resynthesizing new
DaCatL1 molecules. Modeling the interaction between DaCatL1 and CPI (Figure 2-1)
indicates a reversible interaction via hydrogen bonding between specific amino acid
residues on the surface CPI and in the active-site pocket of DaCatL1 which is pH
dependent.
55
The sequence of events in the gut of D. abbreviatus was therefore suggested as
follows. As food particles move through the insect midgut, passing from the anterior end
to the posterior end, molecules (including enzymes) move back and forth, across the
peritrophic membrane to improve the energy efficiency of digestion. Cathepsin L1,
postulated to be active in the anterior region, is therefore available to cleave peptide
bonds. However, in the posterior midgut, DaCatL1 is protected from degradation by
CPI1, by sitting in the active site pocket of the enzyme. A proportion of the DaCatL1-
CPI1 complex is found again in the anterior midgut as it cycles through the gut, where
hydrogen bonds, loosely holding the enzyme and the inhibitor together, are broken to
release active DaCatL1 molecules. This is a measure by the insect to conserve its
energy from the need to resynthesize new enzyme.
To verify this hypothesis, it was important to synthesize and purify recombinant
DaCatL1 and inhibitors to characterize their kinetics. Going a step further, a mutant form
of the inhibitor was synthesized by modifying the amino acid residue directly involved in
the interaction with DaCatL1, such that the chemical bond between this mutant peptide
and DaCatL1 is changed from the weak hydrogen bond to a stronger covalent bond that
is not pH-dependent. Success in this approach was postulated to make available a
more potent inhibitor of cathepsin and a better candidate toxin in a transgenic control
strategy.
The copious synthesis of DaCatL1 in an E. coli system was accomplished by
synthesizing it as a fusion product with a GST tag. However, DaCatL1 was very
unstable in solution; the cause of this instability is not clear. Perhaps differences in
protein processing in a prokaryotic system may contribute to this instability. Still, other
56
polypeptide moieties could be employed that may keep DaCatL1 soluble and retain its
activity, such as the maltose binding protein (MBP) and the small ubiquitin-like modifier
(SUMO) (Kapust and Waugh, 1999; Butt et al., 2005). Of the surfactants employed to
improve the solubility of DaCatL1, only TritonTM X-100 (1%) was effective, though it is
not clear if it affected the characterization of DaCatL1. The list of surfactants assayed is
by no means exhaustive, and other options may be more useful. Additionally, numerous
alternative recombinant protein synthesis technologies are available (Hunt, 2005), but
the expression of heterologous proteins in the E. coli system is simple and straight-
forward.
The characterization of DaCatL1 presents confounding data. DaCatL1
preferentially cleaved Z-FR-AFC (Figure 2-7), which has a hydrophobic phenylalanine at
P2. However, DaCatL1 had optimum activity at pH 8 (Figure 2-8). This is unusual of the
cathepsins in the MEROPS database (Rawlings et al., 2012). Reports of basic cysteine
proteases are rare (Otto and Schirmeister, 1997); most have been found to have
optimum activity in the acidic pH range (pH 5-6.5). Though Z-FR-AFC is an established
cathepsin L substrate (Tchoupé et al., 1991), cathepsins B and H have affinities also for
it. Specific small-molecule inhibitors also aid the characterization of cathepsins. The
general cathepsin inhibitor E-64 inhibits the activities of almost all cysteine proteases,
including cathepsins B, H, and L (Barrett et al., 1982; Turk et al., 2012), but not
cathepsin C (Rozman-Pungercar et al., 2003). Meanwhile, the inhibitor CA074 and its
analogs specifically inhibit cathepsin B (Bogyo et al., 2000), whereas Z-Phe-Tyr(tBu)-
diazomethylketone and analogs specifically inhibits cathepsin L (Shaw et al., 1993).
Some of these inhibitors, among others, were used to characterize DaCatL1. To rule out
57
cathepsin B, the specific cathepsin inhibitor, CA074, was assayed against DaCatL1. In
the presence of CA074 (50 µM), DaCatL1 still retained 75% of its activity (Figure 2-9).
However, the general cathepsin inhibitor E-64 and the specific cathepsin L inhibitor Z-
FY-(tBu)DMK only showed limited inhibition of DaCatL1 (Figure 2-9).The general
cathepsin inhibitor chymostatin was effective at inhibiting DaCatL1 (Figure 2-9),
perhaps because it also inhibits serine proteases that are active at a basic pH.
Additionally, aprotinin, a sole serine protease inhibitor, did not inhibit DaCatL1 at the
same concentration range at which it effective against serine proteases. It is worthy to
re-emphasize here that none of the substrates: Z-GPR-AMC, Z-VVR-AMC, Suc-AAPF-
pNA and H-Arg-pNA.2HCl were cleaved by DaCatL1. Crucially, mass spectrometry
analysis (Figure 2-4) of SDS-PAGE bands cut out for analysis (Figure 2-2), put the
synthesis and purification (Figure 2-5) of DaCatL1 beyond any doubt. DaCatL1, as
synthesized by an E. coli strain, is best described as a cryptic cysteine protease.
These data beget the question: Does DaCatL1 have chymotrypsin-like activity?
When the chymotrypsin-specific inhibitor TPCK (tosyl phenylalanyl chloromethyl ketone)
was incubated with DaCatL1 in the presence of Z-FR-AFC, DaCatL1 was not inhibited;
rather it exhibited full cysteine protease activity (data not shown). This was also the
case with a positive control using a commercially recombinant human cathepsin L1. It is
therefore safe to conclude that DaCatL1 does not have chymotrypsin-like activity.
Aligning DaCatL1 with other sequenced cathepsins revealed the same active site
residues: C, H, Q and N (Figure 2-11), yet these cathepsins have different substrate
preferences (Rawlings et al., 2012). Proper protein folding is therefore a prerequisite for
the activity/function of these proteases to determine their kinetic characteristics. Access
58
of substrates to substrate binding sites is important in distinguishing between the
cathepsins (Turk et al., 2000).TritonTM X-100 was the only surfactant that kept the
protein stable and active in solution. Therefore proof that the enzyme is properly folded
in its native form has to wait for future X-ray crystallographic studies.
Cathepsin L protease inhibitor wild type (CPI1) and its mutant (CPI1mut) were
successfully purified and effectively inhibited the activity of gut cathepsins from D.
abbreviatus larvae at acidic to neutral pH. Even though some inhibition of gut
cathepsins was observed at basic pH with either CPI, it was low (25% or less). This
was also true with DaCatL1 and it is likely that the basic cathepsins are uniquely
regulated and may have their own specific modulators/regulators. The inhibitory potency
of Cathepsin L propeptides were found to be pH dependent and were highly potent at
acidic pH (Carmona et al., 1996).
I was unable to show that CPI1mut is a better candidate for inhibition of D.
abbreviatus acidic cathepsins than the wild-type. The cysteine residues (for instance,
active site cysteine of DaCatL1 and the mutated cysteine residue on CPI1mut) are
probably too far apart to establish an effective S-S bond. X-ray crystallographic data
may allow a better prediction of an amino acid to mutate on the C-terminus of CPI1 to
get it close to the active site of D. abbreviatus digestive cathepsins. Possibly, an
opportunity of evaluating a better mutant was missed due the failure to synthesize
active D. abbreviatus cathepsin L2 in E. coli.
A re-evaluation of the process of digestion in D. abbreviatus may also be
necessary. It is apparent, however, that the DaCatL1-CPI interaction obtained in this
study is not the one modeled and hypothesized. DaCatL1 is entirely different from the
59
acidic cathepsins that are modulated by CPI1. This notwithstanding, the strategy of
mutating endogenous regulators of enzymatic activity to make for better inhibitors is a
feasible and attractive possibility to protect crops. Cathepsin L protease inhibitors are a
potential alternative in a transgenic strategy for crop protection in citrus rootstock
against D. abbreviatus as a component of an integrated IPM system.
60
Figure 2-1. Modelling of the interaction between Diaprepes abbreviatus cathepsin L1
(DaCatL1) and cathepsin L protease inhibitor 1 (CPI1). A shows the overall three-dimensional conformation of DaCatL1 and CPI1. DaCatL1 propeptide (PP) and the catalytic domain are colored violet red and yellow, respectively and the catalytic residue of DaCatL1 C120 and H255 red and blue, respectively. B shows the catalytic groove (black dashed lines) of the activated DaCatL1 and its propeptide PP. C is an enlarged view of the catalytic residues C120 and H255 at the C-terminal tail of the propeptide (PP) bound in the catalytic groove of DaCatL1. D is a ribbon diagram showing the docking of cathepsin inhibitor CPI1 from D. abbreviatus to the activated cathepsin DaCatL1. The inhibitor and the activated cathepsin L1 are colored violet red and yellow, respectively. E shows the molecular surfaces and the binding of CPI 1 into the catalytic groove (black dashed lines) of the activated DaCatL1. The catalytically active C120 and H255 residues are also shown. F is an enlarged view of the catalytic residues C120 and H255 and the C-terminal tail of the cathepsin inhibitor CPI11 binding to the catalytic groove of the activated DaCatL1.
A B
C
D E
F
CPI CPI
CPI
PP PP
PP C120
C120
C120
C120 C120
C120
H255 H255
H255
H255 H255
H255
61
Figure 2-2. SDS-PAGE analysis of recombinant cathepsin L1 purification. Lane on far left has molecular weight markers. Lanes a= control, b= crude bacterial extract, c= affinity (Ni-NTA) purified enzyme, d= activated affinity purified enzyme after size exclusion chromatography and concentration. The red box highlights the recombinant fusion protein, Mr 60KDa before activation and the red arrows shows an activated DaCatL1.
250
75
50
25
20
KDa a b c d
62
Figure 2-3. Western blot analysis of His- tagged DaCatL1. Left lane is a control of
protein extract from E.coli cells with a plasmid not carrying DaCatL1. Right lane protein extract from DaCatL1 in transformed E. coli cells showing two expected bands of Mr 60KDa (GSTHis6-DaCatL1) and Mr 23 KDa (activated DaCatL1 without the GST tag). Bacterial cells were induced with IPTG (as above).
Figure 2-4. Mass spectrometry analysis of DaCatL1.
Control DaCatL1
60 KDa
23 KDa
63
Figure 2-5. Size-exclusion purification of DaCatL1. Activated DaCatL1 was eluted in the second peak (red arrow) during size-exclusion chromatography with Tris-HCl (pH 8). The first peak (blue arrow) was resolved on SDS-PAGE as the 60 KDa fusion product indicated in Figure 2-2.
Figure 2-6. SDS PAGE (lanes a-d) and western blot analysis (lane e) of cathepsin L protease inhibitor1 mutant (CPI1mut). Lane a: control lane of crude bacterial extract; Lane b: CPI1mut transformed E.coli crude protein extract showing 11KDa bands in the red box (the calculated size of the His6- tagged protein); Lane c: affinity (Ni-NTA) purified protein; Lane d: size exclusion chromatography purification after affinity chromatograph (lane c); Lane e: Monoclonal antibody identification of CPI1mut band on nitrocellulose membrane was visualized by fluorescence.
25
10 11KDa
a b c d e
64
Figure 2-7. Fluorogenic substrate preference of DaCatL1. Activity is given as RFU = relative fluorescent unit.
Figure 2-8. pH activity profile of recombinant DaCatL1 using Z-FR-AFC
65
Figure 2-9. Inhibition of DaCatL1 by small-molecule protease inhibitors.
Figure 2-10. Inhibition of cathepsin L protease inhibitors on the activity of DaCatL1. Enzyme activity was followed at pH 8, 25 °C using 2.25 µg of synthesized recombinant enzyme per assay.
66
DaCatL1 ----MKVFIAACLLVAVSATVLEETGVK------FQAFKLKHGKTYKNQVEETARFNIFK
HumCTH MWATLPLLCAGAWLLGVPVCGAAELCVNSLEKFHFKSWMSKHRKTYS-TEEYHHRLQTFA
HumCTK ------MWGLKVLLLPVVS---FALYPEEILDTHWELWKKTHRKQYNNKVDEISRRLIWE
HumCTS ------MKRLVCVLLVCSSAV-AQLHKDPTLDHHWHLWKKTYGKQYKEKNEEAVRRLIWE
HumCTL1 ------MNPTLILAAFCLGIASATLTFDHSLEAQWTKWKAMHNRLYG-MNEEGWRRAVWE
HumCTB ---------------------------------MWQLWASLCCLLVLANARSRPSFHPLS
: :
DaCatL1 DNLR-AIEQHNVLYEQGLVSYKKGINRFTDMTQEEFRAFLTLSSSKKPHFN---TTEHVL
HumCTH SNWR-KINAHN----NGNHTFKMALNQFSDMSFAEIKHKYLWSEPQNCSAT---KSNYLR
HumCTK KNLK-YISIHNLEASLGVHTYELAMNHLGDMTSEEVVQKMTGLKVPLSHSRSNDTLYIPE
HumCTS KNLK-FVMLHNLEHSMGMHSYDLGMNHLGDMTSEEVMSLMSSLRVPSQWQR--NITYKSN
HumCTL1 KNMK-MIELHNQEYREGKHSFTMAMNAFGDMTSEEFRQVMNGFQNRKPRKG---KVFQEP
HumCTB DELVNYVNKRNTTWQAGHNFYNVDMSYLKRLCG----TFLGGPKPPQRVMF----TEDLK
.: : :* * : :. : :
DaCatL1 TGLAVPDSIDWRTKG-QVTGVKDQGNCGSCWAFSVTGSTEAAYYRKAGKLVSLSE--QQL
HumCTH GTGPYPPSVDWRKKGNFVSPVKNQGACGSCWTFSTTGALESAIAIATGKMLSLAE--QQL
HumCTK WEGRAPDSVDYRKKG-YVTPVKNQGQCGSCWAFSSVGALEGQLKKKTGKLLNLSP--QNL
HumCTS PNRILPDSVDWREKG-CVTEVKYQGSCGACWAFSAVGALEAQLKLKTGKLVSLSA--QNL
HumCTL1 LFYEAPRSVDWREKG-YVTPVKNQGQCGSCWAFSATGALEGQMFRKTGRLISLSE--QNL
HumCTB LPASFDAREQWPQCP-TIKEIRDQGSCGSCWAFGAVEAISDRICIHTNAHVSVEVSAEDL
:: :. :: ** **:**:*. . : . :. :.: ::*
DaCatL1 VDCSTDIN--AGCNGGYLDETFTYVKS-KGLEA---------------------------
HumCTH VDCAQDFN-NHGCQGGLPSQAFEYILYNKGIMG---------------------------
HumCTK VDCVSE---NDGCGGGYMTNAFQYVQKNRGIDS---------------------------
HumCTS VDCSTEKYGNKGCNGGFMTTAFQYIIDNKGIDS---------------------------
HumCTL1 VDCSGPQ-GNEGCNGGLMDYAFQYVQDNGGLDS---------------------------
HumCTB LTCCGSMC-GDGCNGGYPAEAWNFWTRKGLVSGGLYESHVGCRPYSIPPCEHHVNGSRPP
: * ** ** :: : : .
DaCatL1 ---ESTYPYKGTDGSCKYSASKVVTKVSGHKSLKSE-DENALLDAVGNVGPVSVAIDATY
HumCTH ---EDTYPYQGKDGYCKFQPGKAIGFVKDVANITIY-DEEAMVEAVALYNPVSFAFEVTQ
HumCTK ---EDAYPYVGQEESCMYNPTGKAAKCRGYREIPEG-NEKALKRAVARVGPVSVAIDASL
HumCTS ---DASYPYKAMDLKCQYDSKYRAATCSKYTELPYG-REDVLKEAVANKGPVSVGVDARH
HumCTL1 ---EESYPYEATEESCKYNPKYSVANDTGFVDIPK--QEKALMKAVATVGPISVAIDAGH
HumCTB CTGEGDTPKCSKICEPGYSPTYKQDKHYGYNSYSVSNSEKDIMAEIYKNGPVEGAFSVYS
: * . :.. . *. : : .*:. ....
DaCatL1 --LSSYESGIYEDDWCS--PSELNHGVLVVGYGTS----NGKKYWIVKNSWGGSFGESGY
HumCTH D-FMMYRTGIYSSTSCHKTPDKVNHAVLAVGYGEK----NGIPYWIVKNSWGPQWGMNGY
HumCTK TSFQFYSKGVYYDESCNS--DNLNHAVLAVGYGIQ----KGNKHWIIKNSWGENWGNKGY
HumCTS PSFFLYRSGVYYEPSCT---QNVNHGVLVVGYGDL----NGKEYWLVKNSWGHNFGEEGY
HumCTL1 ESFLFYKEGIYFEPDCSS--EDMDHGVLVVGYGFESTESDNNKYWLVKNSWGEEWGMGGY
HumCTB D-FLLYKSGVYQHVTGEM---MGGHAIRILGWGVE----NGTPYWLVANSWNTDWGDNGF
: * *:* .*.: :*:* .. :*:: ***. .:* *:
DaCatL1 FRLLRGK-NECGVAEDTVYPII--------- 322
HumCTH FLIERGK-NMCGLAACASYPIPLV------- 335
HumCTK ILMARNKNNACGIANLASFPKM--------- 329
HumCTS IRMARNKGNHCGIASFPSYPEI--------- 331
HumCTL1 VKMAKDRRNHCGIASAASYPTV--------- 333
HumCTB FKILRGQ-DHCGIESEVVAGIPRTDQYWEKI 339
. : :.: : **:
Figure 2-11. Alignment of DaCatL1 with human cathepsin sequences. Highlighted in yellow are the active site residues.
67
Figure 2-12. Influence of TritonTM X-100 (1%) on the interaction between D. abbreviatus gut cathepsins and the cathepsin L protease inhibitors CPI1 and CPI1mut.
68
Figure 2-13. Inhibition of D. abbreviatus larval cathepsins by cathepsin L protease inhibitors
69
CHAPTER 3 A METHOD FOR CONTROLLING DIAPREPES ABBREVIATUS LARVAL FEEDING
ON CITRUS USING A BACILLUS THURINGIENSIS CYTOLYTIC TOXIN
Diaprepes abbreviatus (Linnaeus) rapidly established itself as an important pest
of citrus in USA after its accidental introduction from Puerto Rico in the 1960s (Lapointe
et al., 2007). There have been several attempts to manage this pest in citrus groves, but
they have failed for numerous reasons. A natural insect control strategy that has been
widely exploited for insect control in agriculture is based on the production of insecticidal
proteins, known as delta endotoxins, located in parasporal inclusion bodies of the soil
bacterium, Bacillus thuringiensis (Lacey et al., 2001; Crickmore, 2006; Bravo et al.,
2011). The delta-endotoxins are put into two groups: Crystal (Cry) and Cytolitic (Cyt),
and act differently but culminate in the disruption of gut integrity. The Cry toxins bind
specific receptors and results in pores in the gut epithelium, whereas the Cyt toxins act
in a detergent-like manner to destroy the integrity of the gut epithelium (De Maagd et al.,
2001; Manceva et al., 2005). Numerous toxins from several B. thuringiensis strains
show specific activity to members of Diptera, Lepidoptera, and Coleoptera. Whereas the
Cyt toxins predominantly show dipteran toxicity (Bravo et al., 2007), a few Cyt toxins
(Cyt1Aa and Cyt2Ca) have been found to have toxicity to some coleopteran species
(Soberón et al., 2013), including Cyt2Ca1, which is active against D. abbreviatus
(Weathersbee et al., 2006). Formulations of Bt are used effectively as spray
applications in horticulture, but limitations such as rapid environmental degradation and
the subterranean feeding habit of D. abbreviatus larvae, means such a tactic will be
impractical in citriculture.
Since 1996, significant advancement of the use of Bt as bio-control agents
(Crickmore, 2006) has been the commercial deployment insect-resistant transgenic
70
crops expressing a variety of Cry toxins. However, this has yet to be introduced in
commercial citrus fruit trees. The transgenic crop varieties have been rapidly adopted
by farmers providing insurmountable proof of their resilience against destruction from
feeding damage by pests. Because of the way citrus is commercially cultivated, by
grafting fruiting scions onto resistant rootstock cultivars, it lends itself to a transgenic
citrus rootstock strategy to protect against D. abbreviatus larvae. Citrus rootstocks can
thus be engineered for resistance to D. abbreviatus, but not the scions, leaving fruits
without transgenic genes. Evidence that recombinant products are not expressed in
fruits will boost consumer acceptance. Citriculture can therefore benefit from the
transgenic technology. Thus, the citrus variety ‘Carizzo’ (a hybrid of Citrus sinensis × P.
trifoliata), was engineered to express a recombinant gene product, Cyt2Ca1.
This chapter presents results of work done with transgenic Cyt2Ca1 citrus to
identify transformational events carrying and expressing the recombinant gene product.
A transformational event is a genetically engineered organism (citrus in this case) and
all identical clones resulting from a transformation. In addition, transgenic Cyt2Ca1
citrus was challenged with aggressively feeding D. abbreviatus larvae to determine the
plant’s resistance to feeding damage.
Materials and Methods
Citrus Plants
Several batches of transgenic citrus plant cohorts engineered with Cyt2Ca1 were
available from USDA-USHRL. A total of 75 transgenic citrus cohorts (scions were
transgenic and grafted to non-engineered rootstocks), making up batch 1, were
screened by qPCR on leaf specimens for presence of the Cyt2Ca1 insert. Cuttings of
qPCR validated parental transgenic citrus (Batch 2) were made from Batch 1 and used
71
for transcription analysis by q-RT-PCR. Furthermore, cuttings (Batch 3) were made from
selected batch-2 transgenic citrus plants and used for potted-plant bioassays.
Primers
Primers listed in Table 3-1 were designed and purchased from IDT® (San Diego,
CA) and used for q-PCR and q-RT-PCR amplifications. Primers BtCyt-f and BtCyt-r
were used to amplify Cyt2Ca1, CitCD-f and CitCD-r were used to amplify citrus
dehydrogenase (qPCR reference genes), whereas CitUbi-f and CitUbi-r, CitAct-f and
CitAct-r, and Cit18S-f and Cit18S-r were used to amplify ubiquitin (HarvEST:
UCRCP01), actin (HarvEST: C0LQ97), and 18S RNAs (GenBank: AF206997.1),
respectively (q-RT-PCR reference genes).
Table 3-1. Primers used for qPCR and q-RT-PCR
Name Sequence (5’- 3’) Tm (melting temperature)
°C
BtCyt-f ATG TTC TTC AAC CGC GTT ATC 54.0 BtCyt-r TTA GCT GTT GCT GCA GAT TTT 54.9 CitCD-f TGA GTA CGA GCC GAG TGT TG 56.7 CitCD-r CTG GTG GAT CGG TGA AGT TT 55.2 CitUbi-f TCG CCG ATT ACA ACA TTC AA 52.4 CitUbi-r AGA GGA AAT TAG GCC CAA GC 54.9 CitAct-f AAA TCA AAC CCC AGG CTT CT 54.6 CitAct-r ACC CTT GCG TCG TAC AGT TC 57.4 Cit18s-f AAA CGG CTA CCA CAT CCA AG 55.0 Cit18s-r CCT CCA ATG GAT CCT CGT TA 53.2
Nucleic Acid Extractions
DNA Extraction: Two leaf samples were randomly obtained from batch-1
transgenic citrus plants, and cleaned with RNase™ AWAY (Thermo Scientific, Waltham,
MA). Leaves were thoroughly rinsed with de-ionized water, and excess water was
blotted off. Leaf midribs were excised and finely chopped (a new blade was used per
sample to prevent DNA cross-contamination) for genomic DNA extractions using the
72
FastDNA® Green SPIN Kit (mpBIO, Solon, OH) according to the manufacturer’s
protocol. The purity and concentration of DNA was quantified by NanoDrop™ 1000
(Thermo Scientific).
RNA Extraction: Two leaf samples of qPCR-validated batch-2 Cyt2Ca1 citrus
were obtained and processed as described previously but the midrib-excision step was
skipped. Samples were placed in zipper-sealed sample bags and flash frozen with liquid
nitrogen. The samples were stored at -80 °C until they were ready to be used for RNA
extractions. The TRI Reagent® (Sigma-Aldrich, St Louis, MO) extraction protocol (with
modifications) was used for RNA extractions from leaf samples taken from F1 cohorts.
All materials were ensured to be RNase-free with RNAse AWAY (Thermo Scientific).
The leaf samples were ground to a fine powder under liquid nitrogen with a
mortar and pestle. With a spatula, 150 mg of pulverized leaves were placed into 2-mL
screw-cap tubes. One mL of TRI Reagent® was added to the tubes and the mixture
vortexed then, and incubated at room temperature for 5 min. After incubation, 0.2 mL of
chloroform was added to the mixture, and the mixture was vortexed and incubated for
10 min at room temperature. Proteins and DNA were precipitated and pelleted by
centrifugation at high speed (12,000 rpm for 10 min at 4 °C in a refrigerated bench-top
centrifuge- Eppendorf 5702 R). The partitioned aqueous phase was transferred into a
fresh 1.5-mL tube (DNA- and RNA-free). Five hundred µl of isopropanol were added per
1 mL TRI reagent, vortexed briefly, and incubated for 10 min. The solution was
centrifuged at high speed, and the supernatant was discarded. To wash the pellet, 1 mL
of cold 75% ethanol was added to the pellet and the tube inverted gently. The ethanol
was removed after centrifugation at high speed for 5 min and RNA pellet air-dried for
73
approximately 10 min. The RNA pellet was then re-suspended in 80 µl of nuclease-free
water and stored at -80 ºC.
The RNA suspension was treated with RQ1 DNase (Promega, Madison, WI) to
remove contamination as follows. Twenty µl of 1:1 RQ1 DNase and 10X DNase buffer
was added to the RNA suspension. The mixture was incubated at 37 °C for one hr.
Following incubation, 200 µl nuclease-free water and 300 µl phenol (pH 4.3) were
added to the RNA suspension, vortexed, and centrifuged at high speed for 10 min. The
upper phase obtained after centrifugation was collected into a fresh tube. An equal
volume (0.6 mL) of phenol: chloroform was added to the contents in the tube, vortexed,
and centrifuged at high speed for 10 min. The upper phase was collected into a new
clean tube and sodium acetate precipitation was used to collect RNA (present in this
phase) as follows. One-tenth the volume of sodium acetate (3 M, pH 5.2), and 2.5
volumes of 100% ethanol were added to the upper phase collected and briefly inverted
to mix the contents. The RNA was precipitated by chilling overnight at -80 °C. The RNA
precipitate was pelleted by high speed centrifugation for 20 min and the supernatant
discarded. Three hundred µl of cold 70% ethanol were used to wash the pellet by
inverting the tube gently. The pellet was re-suspended in 50 µl of nuclease-free water.
A second clean-up of the RNA suspension was performed by chilling it for 30 min
at -20 °C in LiCl solution at a final concentration of 2.5 M. Stock LiCl solution was
prepared as 7.5 M in DEPC (1% diethylpyrocarbonate)-treated, nuclease-free water
(autoclaved) and filter sterilized with a 0.2 µm filter. The RNA (50 µl) was re-suspended
in nuclease-free water after pelleting by centrifugation at high speed (4 °C) and washing
with 70% ethanol. RNA concentration, purity, and integrity were verified via
74
spectroscopy using the NanoDrop™ 1000 (Thermo Scientific) instrument and running
on 1.2% agarose gel using DEPC-treated TAE (TAE- 40 mM tris, 20 mM acetic acid,
and 1 mM EDTA (pH 8) prepared with DEPC, treated water).
Quantitative Polymerase Chain Reaction (qPCR)
The qPCR reactions were performed using 150 ng of template DNA in 12.75 µl
total reaction volume with the SensiMix HRMTM Kits (Bioline, Taunton, MA). The
primers, BtCyt-f and BtCyt-r (Table 3-1) were used to amplify the Cyt2Ca1 gene, and
CitCD-f and CitCD-r were used to amplify the control gene (CD- citrus dehydrin- used
as a normalization control). The Cyt2Ca1 amplification was done in triplicate and CD in
duplicate for each transgenic citrus. The CAS1200 Liquid Handling System (Corbett
Robotics- now Qiagen, Valencia, CA) was used to aliquot multiple reactions
simultaneously, and the Rotor-Gene 6000 (Corbett Robotics) was used to run qPCR
reactions. The following thermal cycling conditions were used: 95 °C for 10 min,
followed by 40 cycles of 95 °C for 15 sec, 60 °C for 30 sec and 72 °C for 30 sec.
Amplification reactions were immediately followed by a melt curve analysis of the
product between 70-90 °C. The presence of amplified product was assessed based on
the number of amplification cycles needed to reach a common fixed threshold (cycle
threshold - Ct) in the exponential phase of PCR.
Quantitative Reverse Polymerase Chain Reaction (q-RT-PCR)
The QuantiTect® SYBR® Green (Qiagen) kit was used to conduct q-RT-PCR
reactions. The reactions were performed according to the manufacturer’s
recommendations but with half the final total volume (12.5 µl) suggested. To normalize
Cyt2Ca1 expression data, an experiment was carried out to identify a reference gene
that was stably expressed with the least variation across the root tissues. Three
75
reference genes, ubiquitin, actin, and 18S RNA, were assayed using RNA extracted
from 12 Cyt2Ca1 transgenic citrus and 1 non-engineered citrus control plant. An amount
of 100 ng of RNA templates was used per reaction with the primer pairs BtCyt-f and
BtCyt-r, CitUbi-f and CitUbi-r, and CitAct-f and CitAct-r to amplify Cyt2Ca1, ubiquitin,
and actin, respectively. However, 25 pg of RNA template were used in the 18S RNA
amplification reactions (primers: Cit18s-f and Cit18s-r). Primers were used at a final
concentration of 1 µM. Reverse transcription reactions were performed in duplicate
alongside single no RT (reverse transcriptase) controls to check for DNA contamination,
and no template controls with nuclease-free water to check for cross-contamination.
The CAS1200 Liquid Handling System was used to perform multiple reactions
simultaneously and the Rotor-Gene 6000 was used to run the reactions. The following
thermal cycling conditions were used: 50 °C for 30 min, 95 °C for 15 min, followed by 35
cycles of 94 °C for 15 sec, 60 °C for 30 sec and 72 °C for 30 sec. Amplification
reactions were immediately followed by a melt curve analysis from 70-90 °C to verify the
product. Expression levels were assessed based on the number of amplification cycles
needed to reach a common fixed threshold (cycle threshold - Ct) in the exponential
phase of PCR. Relative levels of transcription of Cyt2Ca1 gene in transgenic citrus
plants was normalized with the 18S RNA levels as the difference between the Ct of 18S
rRNA and Ct of Cyt2Ca1.
Potted-Plant Bioassays
An ad hoc criterion was used to classify transgenic citrus as high, medium, or low
expressers: Ct Difference greater than one = high expressers; Ct Difference less than
one, but greater than minus one = medium expressers; and Ct difference less than
minus one = low expressers (Figure 3-2). Cuttings were made from these plants (batch-
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3). Growth of cuttings was observed to be uneven among the cuttings. Thus, plants that
grew to fairly uniform height were used for the potted plant bioassays. Seven Cyt2Ca1
transgenic citrus plants were selected for this experiment: low expressers (L-A3, L-422);
medium expressers (M-413, M-414); and high expressers (H-3A, H-U2, H-417). Using
an experimental design based on Lapointe and Bowman (2002), 10 cohorts of each
selected transgenic citrus (from 8-month-old cuttings) in addition to 10 cohorts of the
wild type (WT) Carrizo control were used for the potted plant bioassays. Figure 3-1
shows the experimental design.
The aim of the experiment was to challenge these transgenic citrus plants with
three-week-old D. abbreviatus larvae to determine their resistance to wounding as a
result of feeding injury and also to assess the effect on weight gain and mortality of
larvae that feed on transgenic plant roots. Citrus plants (10 each of transgenic citrus
and wild type controls) initially grown in yellow cones were transplanted into 2-L pots
(approximately 11 cm diameter, 25 cm tall) with drain holes containing white, sterile
sand. To prevent escape of larvae from infested pots, two pots were nested into each
other with a plastic screen placed between them and covering drain holes. Root-
volumes of each plant were measured by the displacement of water method before
planting into sterile soil as follows. Plant roots were individually placed into a volumetric
cylinder containing a known volume of water at a predetermined point on the stem (the
soil level). The volume of water displaced was recorded as the equivalent of the volume
of plant roots. After transplanting into a pot containing sand, plants were placed on
benches in the greenhouse and maintained on a fertilizer mix (N: P: K, 20:10:20) diluted
in water provided at a weekly rate of 150 mg/L. Temperature during the experimental
77
period fluctuated between a maximum of 35 °C and minimum of 15 °C, and relative
humidity varied between 34% and 93%. No supplemental light was provided.
Three-week-old D. abbreviatus larvae were obtained from the colony maintained
at USDA-USHRL and reared as described by Lapointe and Shapiro (1999). Forty plants
were infested with larvae as well as five pots containing sterile soil only. Non-infested
plant controls as shown in Figure 3-1 were also set up. In the infested treatments, six
larvae were weighed (20-35 mg individual weight), then placed individually in 10-cm
deep holes in the soil at a distance of 2 cm from the stem of the plant. Larvae and roots
were recovered 28 days later. The final root volume for each plant was determined by
the displacement of water method. The change in root-volume for each plant was
calculated as the ratio of the final root volume to the initial root volume. From this, an
average was determined for each uninfested cohort as the uninfested root ratio (URR).
Similarly, the infested root ratio (IRR) was calculated for infested potted plants. A root
damage index (RDI) was calculated for each citrus plant as the difference between URR
and IRR. Mortality and weights of live larvae were measured. Abbott’s formula (1925)
was used to adjust for mortality not associated with treatment (Cyt2Ca1 expressed in
citrus) to obtain the corrected mortality by subtracting mortality of larvae feeding on the
control wild type citrus roots from the mortality of larvae feeding on transgenic roots.
Corrected percentage mortality was transformed with the arcsine transformation to
normalize the data for statistical analysis. Data were subjected to statistical analysis
using GraphPad Prism 5 (La Jolla, CA) with Dunnett’s multiple comparison tests to
compare differences between transgenic citrus plants and wild type citrus control.
78
Results
Cyt2Ca1-Positive Transgenic Plants A total of 75 Cyt2Ca1 citrus plants (batch 1)
were verified for the presence of the Cyt2Ca1 construct. Of these, 31 transgenic citrus
plants were validated positive for the insert from qPCR analysis. To be considered
positive for Cyt2Ca1 construct, two out of three Cyt2Ca1 qPCR reactions had to have
amplified DNA product as determined by their Ct values and results of melt analysis
(data not shown). Trees representing these Cyt2Ca1-positive transgenic citrus plants
were selected for q-RT-PCR studies.
Cyt2Ca1 expression levels were assessed in the 31 transgenic citrus plants from
batch-2 (Table 3-2). The stability in the expression of ubiquitin and actin, and the
proportion of 18S rRNA in total RNA were assessed for suitability to correct for sample
to sample variation and to normalize Cyt2Ca1 expression data. Cycle threshold (Ct) of
Cyt2Ca1, 18S rRNA, ubiquitin, and actin were transformed to concentration and then
their means determined. The Fishers test was then used to compare differences in
variance between Cyt2Ca1 to 18S rRNA, ubiquitin, and actin. The variance between
Cyt2Ca1 was significantly different from ubiquitin, and actin (F=67050, df=11, P< 0.0001
and F=2069, df=11, P<0.001, respectively), but not 18S rRNA (F=2.38, df=11, P>0.05).
Therefore, 18s rRNA was used to normalize Cyt2Ca1 expression levels in transgenic
citrus plants. Ubiquitin, actin, and 18S rRNA are recommended as reference genes in
citrus (Boava et al., 2011; Yan et al., 2012). Even though the 18S RNA is generally not
recommended as a reference gene when examining changes in the expression of a
gene due to a treatment, because of commonly observed imbalances in mRNA and
rRNA proportions (Vandesompele et al., 2002), this was not the case in this study.
Since, the objective in this study was to determine differences in Cyt2Ca1 expression
79
among plants, not changes in Cyt2Ca1 gene expression due to a treatment, a reference
gene in the strictest definition, was not necessary. Therefore, the 18S RNA proportion of
total RNA was sufficient in this case to normalize gene expression.
Potted-Plant Bioassays
The uninfested root ratio (URR) was significantly higher in the high expressers
than in the low and medium expressers (Figure 3-5a). The wild type (WT) URR had the
highest growth rate increasing threefold during the experimental period, but it was not
significantly different (P > 0.05) from the values for transgenic plants H-3A, H-U2 and H-
417 (URR: 2.34, 2.63 and 2.95, respectively) in the Dunnett’s multiple comparison tests
(Table 3-3). The intrinsic growth of L-A3, L-422, M-413, and M-414 were significantly
lower, with URRs of 2.21, 1.60, 1.29, and 1.44, respectively. The root ratio was higher in
the high expressers than the low and medium expressers, contrary to earlier
observations. The observation that the root growth was higher in the high expressers
was the opposite of what was observed when the cuttings were initially growing (Figure
3-3). This suggests that the effect of Cyt2Ca1 on plant growth is influenced by factors
that were not measured in this study.
The impact of root feeding by larvae after 28 days is manifested by the IRR
(Figure 3-5b). The infested root ratio (IRR) of all the transgenic citrus plants did not
statistically vary from the wild type control in the Dunnett’s multiple comparison test
(Table 3-4). This suggests that larval feeding impacted evenly on the roots of transgenic
and non-transgenic citrus. However, the IRR fails to take into account the intrinsic
growth of roots. The URR normalizes the impact of feeding injury.
To account for the intrinsic root growth, a root damage index (RDI) was
calculated based on a simple assumption. If the Cyt2Ca1 toxin totally precludes larval
80
feeding on the roots, then there should be no significant difference between the URR
and the IRR for a treatment plant. The root damage index (RDI) is calculated as the
difference between URR and IRR. Ideally, an RDI close to zero is reflective of a citrus
plant that tolerates damage from larval root feeding (Figure 3-5c). A small RDI is
representative of resistance of roots to larval feeding damage. A large RDI however,
indicates a plant that is severely impacted by root feeding activity of larvae. In Figure 3-
4, it is observed that the damage to the root of the transgenic plant M-414 is less severe
than the wild type citrus plant. The caveat of the assumption in calculating the RDI is
that larval feeding could have been at the upper regions of the roots resulting in greater
root loss.
Results of the Dunnett’s multiple comparison tests of the RDI are shown in Table
3-5. Transgenic citrus event M-414 recorded the lowest root damage index with 0.50 ±
0.20 (Figure 3-5), but this was not significantly different from transgenic citrus events M-
413 (RDI = 0.61 ± 0.16) and L-422 (RDI = 1.06 ± 0.17) whereas the RDI of WT was
significantly higher (RDI of 2.53 ± 0.47). Larval feeding therefore had a greater impact
on the roots of the wild type control than on transgenic citrus events L-422, M-413 and
M-414 which are more resistant of larval feeding damage.
Weight gains (Figure 3-5d) of surviving larvae from all plant treatments were not
significantly different (Table 3-6). The highest larval mortality among the transgenic
citrus plants was recorded with event M-414 (Figure 3-5e), with a corrected mortality of
43.2 ±11.9%, significantly different (P < 0.10) from the wild type mortality of 8.4 ± 5.1%
(Table 3-7).
81
Discussion
Transformations to develop transgenic plants are not always successful (Clough
and Bent, 1998). Hence, methods of identifying successfully transformed plants are
necessary. In this study, quantitative polymerase chain reaction (qPCR) identified
transgenic citrus plants based on the presence of the Cyt2Ca1 synthetic gene in
regenerated plants from transformation experiments. Furthermore, methods of
assessing the levels of expression of the Cyt2Ca1 insert were necessary to identify
transgenic citrus plants for potted-plant bioassays. Polyacrylamide gel electrophoresis
and western blotting were used to assess the translation of Cyt2Ca1 but the data were
inconclusive. However via q-RT-PCR studies, the level of transcription of Cyt2Ca1was
assessed in cuttings from qPCR validated plants. I was able to develop an ad hoc
criterion (Figure 3-2) for the classification of 31 transgenic citrus plants into low,
medium, and high expressers, from analysis of the cycle threshold (Ct) data from q-RT-
PCR. With this criterion and an ability to achieve uniform growth, suitable transgenic
citrus plants were selected to test their root resistance to D. abbreviatus larval feeding
damage.
The expression of Cyt2Ca1 in the transgenic citrus influences root growth, as
indicated by the various uninfested root ratios (Figure 3-5a). The wild type URR was
significantly higher than three out of four of the evaluated transgenic citrus in the
bioassay (Table 3-3). This is contrary to the earlier observation made with the young
cuttings. High expression transgenic citrus plants developed at a slower rate than their
medium and low expressing cohorts (Figure 3-3). Perhaps this is an effect of age; once
there is a significant canopy available to support root growth, the energy redirected to
support high Bt expression may be insignificant to affect growth. Abiotic factors or
82
nutrition may have contributed to the values of URR attained. For ease of recapturing
larvae from the infested pots, and also to deny larvae other sources of nutrition, sterile
sand was used to set up the bioassays with a mixture of nitrogen, phosphorus, and
potassium provided as fertilizer. Perhaps a better nutritional regime can be designed for
future assays.
The larval mortalities in this study were not as high as those observed earlier by
Weathersbee et al. (2006). When neonates of D. abbreviatus were fed 300 µg/ml of
lyophilized, sporulated culture of Bacillus thuringiensis isolate expressing Cyt2Ca,
80.4% mortality was observed (Weathersbee et al., 2006). Perhaps larvae were
exposed to a lower dose of recombinant Cyt2Ca1 expressed in transgenic citrus roots.
Unfortunately, I was not able to quantify the Cyt2Ca1 recombinant protein. There are
possibilities to enhance the insecticidal activity of Cyt2Ca1 citrus. Synergism between
some of the known Cry and Cyt toxins exists (Pérez et al., 2005; Xue et al., 2005;
Sóberon et al., 2013). Therefore, other Cry toxins such as CryET33 and CryET43
(Weathersbee et al., 2006) may enhance the insecticidal activity of Cyt2Ca1. Peptide
moieties may also enhance the toxicity of Cyt2Ca1 expressed in citrus. The addition of
a short peptide to Cyt2Aa enhanced its toxicity to the aphids Acyrthosiphon pisum
Harris and Myzus persicae Sulzer (Chougule et al., 2013). Likewise, TMOF may
enhance the toxicity of Cyt2Ca1 in transformed citrus. The transgenic citrus plant M-414
may be a candidate for the addition of other toxins that work via other modes of action
to elicit a mortality response.
The root damage index (RDI) is useful in a study such as this because of the
intrinsic differences in root growth ratios. The RDI (Figure 3-5b) normalizes the impact
83
of feeding damage (Figure 3-5c) on the transgenic citrus roots and the wild type citrus
controls. The analysis of RDI culminated in the selection of transgenic plants L-422, M-
413, and M-414 for further field studies. Importantly, these transgenic citrus scored
significantly lower root damage indices (Table 3-5), satisfying the criterion set to identify
transgenic citrus plants resistant to root feeding damage.
A high mortality in D. abbreviatus larvae and a low RDI are two important
characteristics of a D. abbreviatus resistant plant. The transgenic citrus event M-414
had the best of traits amongst the transgenic citrus plants tested in the potted plant
bioassays. Plant M-414 was established to be a medium expresser of Cyt2Ca1 though
it is not clear now why it performed better than both low and high expressers in the
study.
84
Figure 3-1. Experimental design of potted plant bioassay. A total of 70 transgenic citrus
cohorts from 7 transgenic citrus plants and 10 wild type cohorts were used for the
bioassay. Data collected to provide evidence of resistance included: larval mortality,
changes in weight of larvae (mg), differences in root volume (mL) remaining on the plant
after the feeding trial.
85
Figure 3-2. Relative levels of transcription of Cyt2Ca1 gene in transgenic citrus plants normalized with the 18S RNA levels. Transgenic citrus were categorized into high expressers (Ct Difference ≥ 1), medium expressers (Ct Difference less than 1, but greater than -1), and low expressers (Ct Difference ≤ -1). Plants with red, green, and yellow columns were used for potted plant bioassays.
86
Figure 3-3. Some transgenic citrus cuttings used for potted plant bioassays. An observation was made that the rate of growth was inversely proportional to the level of transcription of recombinant Cyt2Ca1 gene; H.E.; high expresser, M.E.; medium expresser, L.E.; low expresser. Photo by S.K. Ben-Mahmoud,
L.E. M.E. H.E.
87
Figure 3-4. Resistance of Cyt2Ca1 engineered citrus versus wild type control. Transgenic citrus plant M-414 (a), non-engineered control citrus plants (b).The root mass excised in (b)- arrowed, is considered as damage/loss. Photos by S.K. Ben-Mahmoud.
(a) (b)
88
Figure 3-5. Effect of larval root feeding on Cyt2Ca1 citrus plants and the concomitant effect on larval weight and mortality. L-A3, L-422, M-413, M-414, H-3A, H-U2 and H-417 are transgenic citrus plants. WT is a wild type citrus control and SOIL is the starved control. Uninfested plant cohorts (a) were set up alongside infested plant cohorts (b) in which 6, 3-week old larvae fed on roots for 28 days (5 plants each). Changes in root volume were recorded as the infested root ratio (IRR) (b) and uninfested root ration (URR) (a). A root damage index (URR-IRR) was assessed from the infested and uninfested potted plants (c). Weight changes of surviving larvae (d) and mortality of larvae fed on plant roots in potted-plant bioassays are also shown (e). Bars sharing the same letter in each were significantly different from the wild type.
89
(a) Uninfested Root Ratio
L-A3
L-422
M-4
13
M-4
14
H-3
AH-U
2
H-4
17 WT
0
1
2
3
4
a
aa a
Citrus Plant
RO
OT
VO
LU
ME
RA
TIO
(D
ay28/D
ay0)
(b) Infested Root Ratio
L-A3
L-422
M-4
13
M-4
14
H-3
AH-U
2
H-4
17 WT
0.0
0.5
1.0
1.5
Citrus Plant
RO
OT
VO
LU
ME
RA
TIO
(D
ay28/D
ay0)
(c) Root Damage Index
L-A3
L-422
M-4
13
M-4
14
H-3
AH-U
2
H-4
17 WT
0
1
2
3
4
b
b b
Citrus Plant
UR
R-I
RR
(d) Weight Gain of Surviving Diaprepes fed on Cyt2Ca1 Citrus
L-A3
L-422
M-4
13
M-4
14
H-3
AH-U
2
H-4
17 WT
SOIL
0
50
100
150
200
c
Citrus Plant
AV
. W
EIG
HT
OF
LIV
E L
AR
VA
E (
mg
)
(e) Mortality of Diaprepes larvae fed on Cyt2Ca1 Citrus
L-A3
L-422
M-4
13
M-4
14
H-3
AH-U
2
H-4
17 WT
SOIL
0
20
40
60
80
d
d
d
Citrus Plant
% C
orr
ecte
d M
ort
ali
ty
90
Table 3-2. Summary statistics of Ct values obtained from q-RT-PCR analysis of Cyt2Ca1 citrus and non-engineered citrus
Cycle Threshold (Ct)
Cyt2Ca1 18S
rRNA Ubiquitin Actin
Mean 11.75 12.49 23.11 17.43
Standard Error 0.41 0.15 0.77 0.53
Standard Deviation 2.26 0.86 2.76 1.89
Sample Variance 5.09 0.75 7.63 3.59
Coefficient of Variation-CV (%) 7.28 2.70 21.25 14.57
Range 8.73 3.85 10.53 7.10
Minimum 9.11 9.83 19.39 15.69
Maximum 17.83 13.67 29.92 22.79
Count 31.00 32.00 13.00 13.00
91
Table 3-3. Statistical analysis of uninfested root ratio
Dunnett's Multiple Comparison Test Mean Diff. Q P < 0.05? Summary 95% CI of diff
WT versus L-A3 0.8980 3.006 Yes * 0.07233 to 1.724
WT versus L-422 1.508 5.048 Yes *** 0.6823 to 2.334
WT versus M-413 1.822 6.099 Yes *** 0.9963 to 2.648
WT versus M-414 1.668 5.584 Yes *** 0.8423 to 2.494
WT versus H-3A 0.7660 2.564 No Ns -0.05967 to 1.592
WT versus H-U2 0.4780 1.600 No Ns -0.3477 to 1.304
WT versus H-417 0.1600 0.5356 No Ns -0.6657 to 0.9857
Transgenic citrus plants: L-A3, L-422, M-413, M-414, H-3A, H-U2 and H-417. WT: Wild Type control.
Table 3-4. Statistical analysis of infested root ratio
Dunnett's Multiple Comparison Test Mean Diff. Q P < 0.05? Summary 95% CI of diff
WT versus L-A3 -0.04000 0.1678 No Ns -0.6987 to 0.6187
WT versus L-422 0.04000 0.1678 No Ns -0.6187 to 0.6987
WT versus M-413 -0.09800 0.4112 No Ns -0.7567 to 0.5607
WT versus M-414 -0.3600 1.511 No Ns -1.019 to 0.2987
WT versus H-3A -0.3020 1.267 No Ns -0.9607 to 0.3567
WT versus H-U2 -0.3480 1.460 No Ns -1.007 to 0.3107
WT versus H-417 0.0880 0.3693 No Ns -0.5707 to 0.7467
Transgenic citrus plants: L-A3, L-422, M-413, M-414, H-3A, H-U2 and H-417. WT: Wild Type control.
92
Table 3-5. Statistical analysis of root damage index
Dunnett's Multiple Comparison Test Mean Diff. Q P < 0.05? Summary 95% CI of diff
WT versus L-A3 0.9380 2.317 No Ns -0.1810 to 2.057
WT versus L-422 1.468 3.626 Yes ** 0.3490 to 2.587
WT versus M-413 1.920 4.742 Yes *** 0.8010 to 3.039
WT versus M-414 2.024 4.999 Yes *** 0.9050 to 3.143
WT versus H-3A 1.070 2.643 No Ns -0.04900 to 2.189
WT versus H-U2 0.8280 2.045 No Ns -0.2910 to 1.947
WT versus H-417 0.07000 0.1729 No Ns -1.049 to 1.189
Transgenic citrus plants: L-A3, L-422, M-413, M-414, H-3A, H-U2 and H-417. WT: Wild Type control.
Table 3-6. Statistical analysis of weight gain of surviving larvae fed on citrus
Dunnett's Multiple Comparison Test Mean Diff. Q P < 0.05? Summary 95% CI of diff
WT versus L-A3 -4.630 0.1450 No ns -93.91 to 84.65
WT versus L-422 30.74 1.021 No ns -53.44 to 114.9
WT versus M-413 56.48 1.876 No ns -27.70 to 140.7
WT versus M-414 51.54 1.712 No ns -32.64 to 135.7
WT versus H-3A 61.42 2.040 No ns -22.76 to 145.6
WT versus H-U2 -18.70 0.6210 No ns -102.9 to 65.48
WT versus H-417 -13.26 0.4404 No ns -97.44 to 70.92
WT versus SOIL 111.4 3.700 Yes ** 27.24 to 195.6
Transgenic citrus plants: L-A3, L-422, M-413, M-414, H-3A, H-U2 and H-417. WT: Wild Type control.
93
Table 3-7. Statistical analysis of larval mortality
Dunnett's Multiple Comparison Test Mean Diff. Q P < 0.10? Summary 95% CI of diff
WT versus L-A3 -0.1974 1.365 No Ns -0.6011 to 0.2063
WT versus L-422 -0.2081 1.439 No Ns -0.6118 to 0.1956
WT versus M-413 -0.2219 1.535 Yes * -0.6257 to 0.1818
WT versus M-414 -0.3769 2.606 Yes ** -0.7806 to 0.02681
WT versus H-3A -0.1974 1.365 Yes * -0.6011 to 0.2063
WT versus H-U2 0.04241 0.2932 No Ns -0.3613 to 0.4461
WT versus H-417 0.0000 0.0000 No Ns -0.4037 to 0.4037
WT versus SOIL -0.6096 4.214 Yes ** -1.013 to -0.2058
Transgenic citrus plants: L-A3, L-422, M-413, M-414, H-3A, H-U2 and H-417. WT: Wild Type control.
94
CHAPTER 4 TARGETING THE PERITROPHIC MEMBRANE OF DIAPREPES ABBREVIATUS VIA
RNAi INDUCES A DOSE-DEPENDENT MORTALITY RESPONSE
Introduction
RNA interference (RNAi) is a recently discovered mechanism in eukaryotes
(Shabalina and Koonin, 2008). RNAi is an important defense against viruses and
transposable elements (Obbard et al., 2009), but the ability to manipulate it for pest
control has been shown (Baum et al., 2007; Mao et al., 2007). RNAi is a sequence-
specific, post-transcriptional silencing of genes, as a result of the degradation of target
mRNA, such that the ability of a cell to produce the cognate protein is greatly diminished
(the cognate gene is said to be silenced). The common underlying theme of RNAi is the
requirement of a double-stranded RNA (dsRNA) template molecule (Meister and
Tuschl, 2004). In RNAi for pest control, the template is typically a relatively large dsRNA
(typically between 200 and 600 bps).
Armed with the sequence data of organisms, it is possible to design and
synthesize dsRNA molecules for the purpose of silencing genes important for
physiological functions within a target organism. These genes are essential genes
without which organisms have reduced fitness or cannot survive. In insects, these
genes might be involved in homeostasis: the insulin signaling and forkhead transcription
factor (FOXO) silenced in Culex pipiens Linnaeus (Sim and Denlinger, 2008), in
digestion: the digestive cellulose enzyme in Reticulitermis flavipes (Kollar) (Zhou et al.,
2007), and in metabolism: cytochrome P450 in Helicoverpa armigera (Hübner) (Mao et
al., 2007) and Plutella xylostella (Linnaeus) (Bautista et al., 2009). Other examples
include genes involved in growth and development: Tc-achaete-scute-homolog (Tc-
ASH) in Tribolium castaneum (Herbst) (Tomoyasu and Denell, 2004), in reproduction
95
and caste differentiation in termites: hexamerin genes in termites (Zhou et al., 2006),
and in blueprints for structural proteins, such as α-tubulin in Diabrotica virgifera
(LeConte) (Baum et al., 2007).
RNAi as a pest control tool is novel, but the pace of its development is
precocious. Although not commercially available yet, the feasibility of this tool as a pest
management strategy has already been shown demonstrated and is now hotly debated
(Gatehouse, 2008). The specificity in the design of dsRNA that can be achieved is often
cited as its biggest advantage. However, issues with quality assurance exist as non-
target effects have also been observed that are yet to be fully understood.
In insects, two inherent characteristics are to be considered in the selection of
target genes. First, barriers such as the peritrophic membrane and cuticle, in addition to
potential degradation of dsRNA molecules, may impair the uptake of dsRNA molecules
(Terenius et al., 2011; Bachman et al., 2013). Second, amplification of the dsRNA
template/signal may be absent. As yet, the enzyme RNA-dependent RNA polymerase
(RdRP) which is responsible for RNAi signal amplification, or its homologues, have not
been found in any insect (Tomoyasu et al., 2008; Duan et al., 2013), though systemic
spread of dsRNA has been shown in some insects (Terenius et al., 2011; Knorr et al.,
2013). These two characteristics make the selection of targets important because the
dsRNA molecules must be able to reach their target tissues to elicit a response.
Delivery of dsRNA molecules to insects in laboratory studies has mainly involved
microinjections, oral uptake as part of artificial diets and transgenic plant varieties, and
topical applications in a mixture with cuticular penetrants. In commercial application as a
strategy for pest insect control, the delivery method of choice, for any pest, will depend
96
on factors such as cost of dsRNA synthesis, feeding behavior and life cycle of pest.
Most likely, there will not be a “one-size-fits-all” approach to the adoption of this
technology. For field pest control purposes, the oral uptake route is likely to be the most
pragmatic. It is envisioned that this would be commercialized by expressing double-
stranded RNA in transgenic plants or by expression in crop plants using an attenuated
plant virus delivery strategy.
Larvae of D. abbreviatus are the most damaging stage of the pest as a result of
root feeding injuries to citrus trees. Novel strategies are needed because no effective
pest management options are available. RNAi might be a suitable tactic to complement
peptide inhibitors of digestion and Cyt2Ca1 citrus for D. abbreviatus control in a novel
transgenic strategy. It will not be pragmatic to deliver dsRNA molecules as spray
formulations because D. abbreviatus larvae, the most destructive stage of the weevil
pest, is effectively, entirely subterranean. Additionally, it is not yet economical to
produce dsRNAs in quantities that will allow soil drenching, assuming that the dsRNAs
are totally innocuous to the environment and non-target organisms. Transgenic citrus or
attenuated CTV virus, expressing dsRNA that work to silence D. abbreviatus specific
genes will be a feasible approach in a manner similar to Baum et al. (2007). Transgenic
corn engineered to express D. virgifera α-tubulin V-ATPase A dsRNA were protected
from larval feeding damage. Since larvae feed on the roots of citrus, genes expressed in
the gut might be more appropriate targets due to direct access to potentially vulnerable
tissues. To this end, the objective of this study is to identify targets that will achieve
significant mortality for an RNAi strategy for D. abbreviatus control.
97
Materials and Methods
Diaprepes abbreviatus Larvae
Two- to three-week-old D. abbreviatus larvae maintained on an artificial diet
(Lapointe and Shapiro, 1999) were obtained from a colony at the USDA-USHRL. Larvae
were individually weighed to select those between 15-30 mg for use in larval bioassays.
Larvae were randomly assigned to treatments.
Primers
Primers were designed and purchased from IDT® (San Diego, CA) to amplify
approximately 300 base pair regions within D. abbreviatus for alpha-tubulin, peritrophic
matrix protein, peritrophic membrane chitin binding protein, and cathepsin L2. The
primer sequences are listed in Table 4-1 in pairs (forward and reverse). The T7
promoter sequences (highlighted in Table 4-1) were added to primers that were used for
synthesizing dsRNA molecules. The melting temperatures are also listed as they guided
the cycling conditions of polymerase chain reactions.
Double-stranded RNA Synthesis.
At the outset, cDNA clones of the target genes were made and stored in the
vector, pCR®2.1-TOPO® (Invitrogen, Grand Island, NY). Approximately 300 base pair
regions of the target genes (α-tubulin, peritrophic matrix protein, peritrophic membrane
chitin binding protein, and cathepsin L2) were identified for dsRNA synthesis. For
control treatments, a random dsRNA sequence from the Asian citrus psyllid (Diaphorina
citri) was obtained from Dr. R. Shatters’ laboratory at the USDA-USHRL. Template DNA
for dsRNA synthesis was synthesized with standard PCR reactions using the AmpliTaq
Gold® kit (Roche, Indianapolis, IN) according to the manufacturer’s protocol. Since all
98
the primers had high melting temperatures (greater than 70 °C), the touchdown PCR
cycling conditions were selected.
Table 4-1. Primers for Diaprepes abbreviatus dsRNA synthesis. Target Primer Name
(forward/Reverse) Sequence (5’- 3’) Tm (melting
temperature) °C
α-Tub DB 1108 TAATCAGACTCACTATAGGGGACTCTGAGTAATATAGTCAACTAAAGCAG
73.7
DB 1109 TAATCAGACTCACTATAGGGCCAAGTCTACGAATACCGCTCTGG
79.2
PMP DB 1126 TAATCAGACTCACTATAGGGGAGTCTTCAGTTGAAAAATCTTTGAAA
75.8
DB 1127 TAATCAGACTCACTATAGGGGTAGTGACAGGTGGCCTAACGGTA
78.4
PMCBP DB 1102 TAATCAGACTCACTATAGGGCATGAAGGTTCACGTTATTCTTTG
76.5
DB 1103 TAATCAGACTCACTATAGGGTGATAGTCACTGTATTCATGTGGTACA
75.1
CAL 2 DB 1118 TAATCAGACTCACTATAGGGATGTATAGTTTAGTA
GTGTTGTGGCA 73.6
DB 1119 TAATCAGACTCACTATAGGGAGGTAATTGAGGCATATTTACTGTGTA
74.0
Legend: α-Tub; alpha-tubulin, PMP; peritrophic matrix protein, PMCBP; peritrophic membrane chitin binding protein, CAL2; cathepsin L2. T7 promoter sequences are highlighted.
The PCR product was quantified (NanoDrop™ 1000 –ThermoScientific, Waltham, MA)
and run on 1.2% agarose gels to verify purity of the amplification product. Amplified
products were purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA)
using the recommended protocol. The HiScribeTM in vitro transcription kit (New England
Biolabs®, Ipswich, MA) was used for dsRNA synthesis. Nuclease-free water was used
for all RNA synthesis. Amount of template (PCR product) used per 40-µl reaction was
approximately 1 µg, and reactions were incubated at 42 °C overnight. One percent
agarose gels (TAE- 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA (pH 8) prepared
with DEPC -treated water were used to check for the integrity of the dsRNA product.
Ammonium acetate precipitation was used to clean the transcription product as follows:
One-tenth volume of 5 M ammonium acetate and 2 volumes of cold ethanol (100%)
were added to the dsRNA product, mixed thoroughly by vortexing for 5 sec, and chilled
99
at -80 °C. Centrifugation at high speed (10 min, 4 °C) was used to create a pellet of
dsRNA. The supernatant was decanted and the pellet washed with 70% ethanol, after
which a pellet was collected via centrifugation (as before) and then briefly air-dried.
Double-stranded RNA was reconstituted in 200 µl RNAse-free water and the
concentration and purity were verified via spectroscopy (NanoDrop™ 1000 -Thermo
Scientific). All dsRNA products were stored at -80 °C until ready for use in larval
bioassays.
Larval Bioassay
A bioassay method, developed to test individual neonate larvae (Weathersbee et
al., 2006), was adopted with clear, 0.2-mL polymerase chain reaction (PCR) tubes to
verify tolerance to dsRNA molecules. D. abbreviatus specific dsRNA molecules and a
control D. citri dsRNA sequence were fed to larvae in artificial diet bioassays as
described herein. The D. citri dsRNA sequence served as a positive control for off-target
effect of non-specific sequences on D. abbreviatus RNAi machinery. Stock of each
dsRNA was diluted with nuclease-free water to the following concentrations: 0.05, 0.20,
0.50, and 1.00 µg/µl water. Fifty-µl aliquots of artificial diet (Lapointe and Shapiro, 1999)
were dispensed into the lids of individual PCR tubes and allowed to cool to room
temperature. Ten µl of one of each dsRNA solution concentration or D. citri dsRNA were
evenly applied to the surface of the artificial diet in a lid and allowed to percolate to yield
the following dosage treatments: 0.5, 2.0, 5.0, and 10 µg per 50 µl artificial diet for each
dsRNA. Each treatment was replicated 20 times. Tubes were air-dried for approximately
20 min in a sterile environment, and individual larvae were randomly transferred per
tube. Three control treatments consisted of 1) individually starved larvae and 2) artificial
diet without dsRNA with 10 µl nuclease free water, and 3) artificial diet only (to correct
100
for no treatment mortality). For each control treatment, 30 larvae were assayed. The
tubes were turned upside down in a sterile centrifuge tube box and kept for 12 days in
the dark in an environmentally controlled chamber set at 26 °C. Larval mortality was
assessed on the twelfth day.
Percentage mortality was corrected with the mortality from diet only, using the
Abbott’s (1925) formula. Statistical analysis of mortality data was done with the
GraphPad Prism 5 (La Jolla, CA). Percentage mortality was transformed with the
arcsine transformation to normalize data for statistical analysis. Differences among
treatment level means were determined by Bonferroni’s multiple comparisons tests to
compare D. abbreviatus dsRNA sequence mortality to the diet and water control
mortality.
Results
There was 30% mortality in larvae that fed on diet only 12 days post feeding.
After percentage mortality (± SE) was corrected for no treatment deaths, 4.8 ± 3.0% of
larvae died due to starvation (Figure 4-1), confirming the ability of D. abbreviatus larvae
to survive for long period without food. Mortality due to water added to diet resulted in
two times the mortality in starved larvae, but was not significantly different (t value= 2.2,
P>0.05). It is not unusual that the D. citri dsRNA resulted in non-target mortality in D.
abbreviatus mortality (Kulkarni et al., 2006; Nunes et al., 2013), but it is curious that the
mortality was not dose-dependent. Mortality as a result of D. citri dsRNA was not
significantly different from the diet and water control (t value 3.27, P>0.05). Larval
mortality as a result of dsRNA sequences targeting specific D. abbreviatus genes
caused significantly (P<0.05) higher mortalities than the control, ranging from 14.3 ±
4.5% to 71.4 ± 7%.
101
At the highest dose of dsRNA fed to larvae, the peritrophic membrane chitin
binding protein- PMCBP resulted in the significantly highest mortality (t value= 3.2,
P<0.05), and at 5.0 µl dsRNA/ 50 µl diet the cathepsin L2- CAL2 dsRNA caused the
significantly highest mortalities (t value= 4.9, P<0.01). However, at 2.0 µg dsRNA/ 50 µl
diet none of the D. abbreviatus dsRNA sequences were significantly different from the
D. citri control mortality (P>0.05), although the CAL2 and α-Tub dsRNA sequences
caused the highest mortalities. At 0.5 µg dsRNA/ 50 µl diet, alpha-tubulin caused the
significantly highest mortality (t value= 4.9, P<0.001).
Importantly, two of the D. abbreviatus dsRNA sequences, PMCBP and CAL2
resulted in mortality that was dose-dependent; the higher the dose the greater the
mortality. The PMCBP dsRNA resulted in 71.4 ± 7.0%, 42.9 ± 9.5%, 35.0 ± 4.0% and
14.3 ± 4.5% at 10.0, 5.0, 2.0 and 0.5 µg/ 50 µl diet respectively, whereas CAL2 resulted
in 50.0 ± 7.0%, 57.1 ± 9.5%, 42.9 ± 4.0% and 28.6 ± 4.5% with decreasing dose of
dsRNA. Mortality due to PMCBP was significantly different at 10 and 5 µg dsRNA / 50
µL artificial diet (P<0.01) whereas cathepsin L2 was significantly different at 5 (P<0.01)
and 0.5 µg dsRNA / 50 µL artificial diet (P<0.05). Mortality due to alpha-tubulin and
peritrophic matrix protein dsRNA sequences was not dose-dependent. The highest
mortality for α-tubulin was 57.1 ± 7.0%, whereas that for the peritrophic matrix protein
was 42.9 ± 7.0%.
Discussion
Feeding D. citri dsRNA to D. abbreviatus larvae elicited mortality significantly
higher than what was observed in diet without dsRNA. It is interesting that this non-
specific response was not dose dependent. Perhaps, dsRNA molecules beyond a
certain threshold induce some cellular response, unknown at this point that generally
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affects global gene expression leading to death. In a study by Nunes et al. (2013), a
green fluorescent protein (GFP)-derived dsRNA sequence affected the expression of
about 10% of the genes of the honey bee, Apis mellifera, even though this gene is not
present in the bee. In the processing of dsRNA fragments to siRNA fragments, several
sequences may be generated that share similarities to non-specific genes and therefore
may affect their expression. This is likely the case with the D. citiri dsRNA sequence
used here, however it is not clear why this effect was not dose-dependent.
Two dsRNA sequences, PMBCP and CAL2, targeting the peritrophic membrane
and the digestive enzyme cathepsin L2, respectively, are good candidates for a novel
strategy for D. abbreviatus control by exploiting RNAi. They elicit mortality that is dose-
dependent in larvae (Figure 4-1). The peritrophic membrane plays key roles in insects,
including the compartmentalization of digestion to improve efficiency (Terra, 2001) and
serving as a physical barrier to prevent mechanical damage (Rao et al., 2004) and to
protect against invading and infectious microbial agents such as protozoa (Coutinho-
Abreu et al., 2013). Cathepsins, meanwhile, are one of two, important digestive
enzymes needed for growth and development of D. abbreviatus (Yan et al., 1999).
Significantly higher mortalities elicited as a result of feeding dsRNA sequences targeting
genes such as PMCBP and CAL2 involved in larval physiology and biochemistry of D.
abbreviatus is indicative of the relevance of the peritrophic membrane and cathepsin L2
to D. abbreviatus’ survival.
The RNAi response in insects is typically specific (Baum et al., 2007; Mao et al.,
2007), but non-target and off-target effects also occur (Zhang et al., 2010; Nunes et al.,
2013). These unintended effects are the result of short-interfering RNA (siRNA)
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sequence similarities to non-target mRNAs when it is processed from dsRNA by dicer
(Qiu et al., 2005). Also, the saturation of the RNAi machinery by non-specific sequences
(Nunes et al., 2013) may overwhelm the organism. It is therefore very significant that
the dsRNA sequence targeting the peritrophic membrane elicited larval mortality that
correlates with the dosage administered (Figure 4-1). This consistency is relevant
towards establishing the validity of an RNAi effect (Terenius et al., 2011).
In spite of the mortality effects induced by dsRNA targeting alpha tubulin and
peritrophic matrix protein, they are not useful candidates for RNAi in D. abbreviatus due
to the inconsistencies with dose (Figure 4-1). However, this is not to imply that the
targets selected are poor. Perhaps other sequence regions may be better at silencing
the respective targets. The rational design of effective sequences is currently very
challenging. A number of sequences, though not entirely random, are typically screened
and fortuitously a candidate sequence is obtained. Perhaps new computational
predictors (Sciabola et al., 2013) may improve currently identified effective sequences
to be more viable.
As a result of the limitations of exploiting RNAi in insects, genes relevant for
survival, growth, and development and expressed in the midgut are the choice targets.
So far, the peritrophic membrane chitin binding protein particularly and cathepsin L2
have shown to be amenable targets in a future pest control strategy in D. abbreviatus.
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-10.0
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
10 µg/diet 5 µg/diet 2 µg/diet 0.5 µg/diet
Psyllid dsRNA
α-Tub
PMP
PMCBP
DaCatL
Diet + water
Figure 4-1. Corrected mortality (post 12 days) due to feeding on dsRNA sequences targeting Diaprepes abbreviatus genes. Legend: control dsRNA sequence targeting a Diaphorina citri gene (Psyllid dsRNA); alpha-tubulin (α-Tub), peritrophic matrix protein (PMP), peritrophic membrane chitin binding protein (PMCBP), cathepsin L2 (CAL2), of D. abbreviatus.
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CHAPTER 5 GENERAL CONCLUSIONS AND FUTURE DIRECTIONS
Toxins with different modes of action are particularly desirable for new transgenic
crop varieties. The single-toxin Bt crop varieties have been commercially deployed in
the field for over a decade, and have exhibited remarkable resilience against resistance
selection in insect populations (Bates et al., 2005). However, it has become apparent
that they may not be sustainable any longer. Indeed, there are recent reports of field-
evolved resistance in insects to the single-toxin Bt crops (Liu et al., 2010; Gassmann et
al., 2011). Multiple-toxin transgenic plants are expected to further delay the inception of
resistance. Thus, the ultimate aim for a transgenic strategy in citriculture is to have
citrus express multiple toxins that act through different modes leading to death of larvae
that feed on roots. Candidate toxins were explored in a three-pronged approach aimed
at inhibiting digestive enzymes (cathepsins) with peptide inhibiters in in vitro enzyme
assays, disrupting gut integrity with the Bt toxin Cyt2Ca1 expressed in transgenic citrus,
and interference with gene expression via RNAi by feeding dsRNA molecules in artificial
diet bioassays.
Protease inhibitors originating from plants have been investigated for control
purposes. Some level of protection has been demonstrated in certain instances, but the
majority of results have been mediocre; indeed, in some cases the opposite effect has
been observed (Murdock and Shade, 2002). Insects are highly adaptable and have
circumvented inhibitors simply through an apparent increase in the expression of
digestive enzymes (Adbeen et al., 2005) or switching to a different complement of
proteases or sequestration of the inhibitor or all of the above. In a stacking/pyramiding
strategy, inhibiting the activity of digestive cathepsins with CPIs may be enough to make
106
the insect more vulnerable to additional toxins, such as Cyt2Ca1, despite their best
efforts at detoxifying the inhibitor. The availability of pure recombinant enzyme enables
the specific determination of the binding characteristics of the enzyme and inhibitor. I
set forth to characterize the interaction between DaCatL1 and CPI1.
My attempts at synthesizing and purifying a Diaprepes abbreviatus cathepsin for
inhibitor characterization with CPI resulted in the serendipitous discovery of a basic
cathepsin, DaCatL1, expressed in E. coli. Recombinant DaCatL1 was only stable in
solution with 1% TritonTM X-100; DaCatL1 was therefore folded favorably in TritonTM X-
100. Whether TritonTM X-100 has any effect on the chemical characteristics of DaCatL1
remains to be seen. Sequence comparison of DaCatL1 to other cathepsins revealed the
same amino acid residues at the active sites. Protein folding is apparently important to
their activity and chemical characteristics. The chemical characteristics of DaCatL1 are
cryptic. DaCatL1 has optimum activity at pH 8 with the general cathepsin L substrate Z-
FR-AFC. Whereas DaCatL1 activity was inhibited by chymostatin, a general protease
inhibitor with activity against cathepsins, it was not inhibited by the specific cathepsin L
inhibitors E-64 and Z-FY(tBu)DMK. Failure to inhibit DaCatL1 with the specific cathepsin
L inhibitors may be explained by the pH optima of the enzyme. Importantly, aprotinin, a
serine protease inhibitor, does not inhibit DaCatL1 at the same concentration effective
with the serine proteases which have pH optima in the basic range. X-ray
crystallographic studies may be utilized to resolve this conundrum.
A recombinant CPI1, an endogenous modulator of cathepsin in D. abbreviatus,
and a mutant CPI1 (CPI1mut) were purified to near homogeneity. CPI1 mutant has
Lys101 replaced with a cysteine residue, making it a more robust inhibitor of D.
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abbreviatus cathepsins. The docking models of CPI1 and DaCatL1 reveal proximity of
Lys101 to the active site residue Cys120 of DaCatL1. Thus, mutating Lys101 to a Cys
was hypothesized to result in the formation of a stronger S-S bond which will be
unaffected by pH. In this way, the interaction of CPI1 with cathepsin will be changed
from a reversible interaction to an irreversible one. I was unable to show CPI1mut to be
a better inhibitor of D. abbreviatus gut cathepsins across the acidic to basic range.
However, CPI1 and CPI1mut are both effective inhibitors of acidic cathepsins. Added to
the inability to inhibit the basic gut cathepsins, CPI1 and CPI1mut were weak inhibitors
of DaCatL1. This sheds new insights into the regulation of digestive cathepsins in D.
abbreviatus. Two activity peaks of gut cathepsins were obtained, one acidic and the
other basic. Therefore, the acidic and basic cathepsins appear to be uniquely distinctive
and may have different modulators of their activity.
Transgenic citrus engineered to express Cyt2Ca1 needed to be evaluated.
Although, the exact mode of action of Cyt2Ca1 was not determined in this study, it was
expected to insert into gut epithelial membranes and disrupt gut integrity resulting in
death. Importantly, three transgenic citrus plants resisted feeding injuries by D.
abbreviatus larva. Using a root damage index, these three plants were less impacted by
feeding damage. Additionally, one of the transgenic plants caused significantly high
mortality to D. abbreviatus larvae. Choice tests and/or field studies might further reveal
the resilience of Cyt2Ca1 citrus to D. abbreviatus larval feeding damage. However,
larval mortality, and weight losses, of three-week old D. abbreviatus when fed on
transgenic citrus were low or not substantial. There may be possibilities of enhancing
Cyt2Ca1 insecticidal activity by adding small peptide moieties.
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The interruption of gene expression via RNAi has become a new tool in the
development of pest control tactics. Preliminary studies investigating the expression of
D. abbreviatus genes exposed to dsRNA sequences in artificial diet show the possibility
of exploiting RNAi for citrus protection. Sequences of 300 base pairs designed to
hybridize with the peritrophic membrane chitin binding protein (PMCBP) and cathepsin
L2 (CAL2) mRNAs elicited dose-response mortality when fed to 2-3- week-old D.
abbreviatus larvae. The specificity of this response needs to be validated because non-
target effects cannot be ruled out entirely at this point. The dsRNA sequences could be
engineered directly in transgenic citrus or could be expressed by an attenuated citrus
virus vector. Currently, a CTV vector is being engineered by scientists at the University
of Florida Citrus Research and Education Center (Lake Alfred, FL) to express the
PMCBP sequence.
Whereas transgenic citrus with its own armor against D. abbreviatus represents
a potential pest management tactic, there are potential bottlenecks to its adoption.
Despite the rapid adoption of transgenic crops worldwide and greater public acceptance
of their products, there are still some concerns. From experience in applying the
transgenic technology in other commercial crops, impediments to faster development
and adoption include cost, regulations (Bradford et al., 2005), public perceptions of the
technology (Rommens, 2010), and concerns related to the safety of the foods and the
environment (Shelton et al., 2002). Other issues often raised include the risk of gene-
flow from transgenic plants to non-transgenic plants (DiFazio et al., 2012), biological
fitness of the transgenic plant (Gassmann et al., 2009), allergenicity of the transgenic
109
product (Goodman et al., 2008), and resistance development (Tabashnik, 2008). These
same issues will have to be addressed with transgenic citrus.
While scientists continuously seek better gene targets for suppression, preferably
targets that will elicit a desired response (reduced fitness, low fecundity, mortality) with
a low dose of dsRNA that is not injurious to non-target organisms, the cost of delivery of
these dsRNA molecules will be important. The persistence of the dsRNA molecules in
the plants and/or the environment will also be important in the choice of a delivery
strategy. The application of attenuated viral agents, like CTV that has the dsRNA
replicative form, implies a regular and cost-effective supply of dsRNA sequences will be
available, provided that the viral genomes do not lose the construct and revert to the
wild type (Burand and Hunter, 2013). The alternative of applying dsRNA in insecticidal
sprays is feasible, but the cost of producing large quantities of dsRNA on a commercial
basis is currently a deterrent.
Safety of the transgenes in my experiments will need to be empirically
determined. However, transgenic citrus rootstocks engineered for resistance to D.
abbreviatus and grafted to a non-transgenic fruit-producing scion will bear fruits without
transgenes. Therefore, evidence that recombinant products produced in the roots but
not mobilized through phloem or xylem will boost consumer acceptance. Cathepsins are
ubiquitous enzymes in numerous organisms (Beton et al., 2012), and therefore it is
possible that peptide inhibitors may also affect similar enzymes in non-target organisms
that feed on citrus. However, many similar peptide inhibitors, for example, legume
inhibitors (Ryan, 1973; Mithöfer and Boland, 2012), are present in foods that are safe
for consumption. The more prudent issue will be the levels of accumulation of peptide
110
inhibitors in edible tissues of transgenic citrus. The proposed vector for expression of D.
abbreviatus toxins in citrus, CTV, has co-evolved with citrus (Moreno et al., 2008) and
the distribution of the virus is throughout the plant (Dawson et al., 2013). Fuchs and
Gonsalves (2007) reported the use of mild strains of CTV for cross-protection of citrus in
Brazil for years. This and other virus-based plant protection practices, including
engineered viruses, are commonplace and no known adverse effects on human health
or environmental safety are reported.
The greatest obstacle to the sustainability of a transgenic citrus tactic could be
the development of resistance in populations of D. abbreviatus exposed to the
transgenic variety. Insects are known to be very adept at developing resistance. The
onset of resistance can in the least be delayed with the incorporation of multiple
insecticidal toxins with different modes of action in the transgenic citrus. In cross-
resistance, tolerance to an insecticidal toxin is enough to protect an individual from a
different, but similar acting toxin. Studies of a strain of the cotton bollworm, Helicoverpa
zea, revealed that resistance to the Bt toxin Cry1Ac protein had increased survivorship
of the pest on pyramided cotton plants as a result of cross resistance between the
Cry1A and another Bt toxin, Cry2A (Brévault et al., 2013). Cross-resistance will be
unlikely in the proposed three-pronged transgenic approach in citrus.
Finally, IPM strategies can prolong the sustainability of a transgenic citrus
strategy (Kos et al., 2009). The inclusion of refuges is acclaimed to have helped delay
insect resistance to Bt crops (Tabashnik et al., 2008). Cultivation of citrus using
transgenic strains may require minimal use of insecticides. However, minimal
insecticide use can function in tandem with biological control. Parasitoids and predators
111
would benefit from the reduced use of broad-spectrum insecticides used in transgenic
citrus groves and augment the control of the weevil. Transgenic citrus will therefore be
“IPM-friendly” at the same time as playing a pivotal role in suppressing pest populations
of D. abbreviatus. My studies bring the strategy of controlling D. abbreviatus with a
transgenic technique a step closer.
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BIOGRAPHICAL SKETCH
Sulley K. Ben-Mahmoud was born in Accra, Ghana. In August 2001, he enrolled
at the University of Ghana, and received his B.Sc. in Biochemistry in June of 2005. He
worked as a teaching assistant at the Biochemistry Department for a year and enrolled
into the entomology program at the University of Ghana under the guidance of Dr.
W.S.K. Gbewonyo in August 2006. He graduated with an M.Phil. in Entomology in June
2009, and was admitted in the same year to the University of Florida, initially under the
mentorship of Dr. Dov Borovsky (retired) at the Florida Medical Entomology Laboratory
in Vero Beach, FL. He switched to the guidance of Dr. Ronald D. Cave in May of 2011
at the Indian River Research and Education Center in Fort Pierce, FL. He did the
majority of his research under the supervision of Dr. Robert G. Shatters Jr. at the United
States Department of Agriculture-Horticultural Research Laboratory, Fort Pierce, FL. He
received his Ph.D. from the University of Florida in the fall of 2013.
