Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing

  • Taíse Fernanda da Silva Ferrara ,

    Contributed equally to this work with: Taíse Fernanda da Silva Ferrara, Vanessa Karine Schneider

    Affiliation Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil

  • Vanessa Karine Schneider ,

    Contributed equally to this work with: Taíse Fernanda da Silva Ferrara, Vanessa Karine Schneider

    Affiliation Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil

  • Luciano Takeshi Kishi,

    Affiliation Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil

  • Adriana Karaoglanovic Carmona,

    Affiliation Department of Biophysics, Federal University of São Paulo, São Paulo, SP, Brazil

  • Marcio Fernando Madureira Alves,

    Affiliation Department of Biophysics, Federal University of São Paulo, São Paulo, SP, Brazil

  • Jose Belasque-Júnior,

    Affiliation Department of Phytopathology and Nematology, University of São Paulo, Piracicaba, São Paulo, SP, Brazil

  • José César Rosa,

    Affiliation USDA, ARS, 2001 South Rock Road, Fort Pierce, Florida, United States of America

  • Wayne Brian Hunter,

    Affiliation Protein Chemistry Center and Department of Molecular and Cellular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil

  • Flávio Henrique-Silva,

    Affiliation Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil

  • Andrea Soares-Costa

    andreasc@ufscar.br

    Affiliation Laboratory of Molecular Biology, Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, SP, Brazil

Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing

  • Taíse Fernanda da Silva Ferrara, 
  • Vanessa Karine Schneider, 
  • Luciano Takeshi Kishi, 
  • Adriana Karaoglanovic Carmona, 
  • Marcio Fernando Madureira Alves, 
  • Jose Belasque-Júnior, 
  • José César Rosa, 
  • Wayne Brian Hunter, 
  • Flávio Henrique-Silva, 
  • Andrea Soares-Costa
PLOS
x

Abstract

Huanglonbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB). DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM) and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM). The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM) and CaneCPI-4 (Ki = 0.05 nM) and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM). RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.

Introduction

Citrus cultivation has considerable worldwide economic importance. Citrus fruits are currently produced in 140 countries, with an annual production of more than 122 million tons. According to the Food and Agriculture Organization of the United Nations, the main citrus producers are China, Brazil, USA, India and Mexico [1]. However, losses occur due to agricultural pests and diseases. Huanglongbing (HLB), also known as citrus greening disease [2, 3], is considered the most serious disease of citrus [4]. HLB has been known in China for nearly hundred years, having first been reported in 1919 [5, 6]. In Brazil (represented by the state of São Paulo) and the United States (represented by the state of Florida), HLB was first reported in 2004 [7, 8, 9] and 2005 [10], respectively. The occurrence of HLB was also confirmed in other countries in North, Central, and South America after the year of 2007 [11, 12, 13, 14].

In Africa, HLB is associated with the bacterium Candidatus Liberibacter africanus and the vector is the psyllid Trioza erytreae (Del Guercio) (Hemiptera: Triozidae). In Asian and American countries HLB is associated with Ca. L. asiaticus and the vector is Diaphorina citri Kuwayama (Hemiptera: Liviidae). In Brazil and southern Texas, there is a third variant denominated Ca. L. americanus [8, 15], which is less heat tolerant and less prevalent than Ca. L. asiaticus [16, 17].

Candidatus Liberibacter spp. colonize the conducting vessels of the plant, blocking the phloem and triggering the disease development process. The most common symptoms are blotchy leaf mottle, defoliation, yellow shoots and aborted seeds. The fruit exhibits irregular maturation, inverted coloration, a reduction in size, deformation and frequent dropping [4]. The acquisition of Ca. L. asiaticus can occur through either D. citri nymphs (4th and 5th instars) or adults [18].

If HLB control actions are not adopted, an orchard can become economically unviable in seven to ten years after the onset of symptoms, whereas younger orchards can become economically unviable within five years [19]. Among the control strategies for HLB disease, one of the widely used consists in controlling the disease vector, Diaphorina citri Kuwayama through chemical control [20, 21]. The biological control also has been studied. There are two known parasitoids for the control of D. citri: Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae) and Tamarixia radiata Waterston (Hymenoptera: Eulophidae) [22].

Alternative strategies for insect control have been developed to reduce the dependence on chemical pesticides. There are many reports of transgenic plants overexpressing peptidase inhibitors for insect control, such as sugarcane expressing the soybean Kunitz trypsin inhibitor (SKTI) and soybean Bowman-Birk inhibitor (SBBI), which retard the growth of Diatraea saccharalis larvae feeding on the leaves of transformed plants [23]. A 53% mortality rate was found for Leptinotarsa decemlineata larvae reared with transgenic potato leaves overexpressing oryzacystatin I [24]. The work of [25] demonstrated that Myzus persicae and Acyrthosiphon pisum nymphs feeding on Arabidopsis plants overexpressing a barley-cystatin presented a significant delay to reach the adult phase, demonstrating the interference of the cystatin in the development of these insects. Another alternative is the development of plants that overexpress double-stranded RNA (dsRNA) to inhibit gene expression on the RNA level. [26] reported the development of transgenic plants overexpressing dsRNA for insect control, describing the expression of 246 bp dsRNA for V-ATPase A in transgenic maize. This strategy led to a significant reduction in the attack of the roots by Diabrotica virgifera virgifera LeConte. [27] reported the expression of dsRNA in rice for the midgut genes hexose transporter (NlHT1), carboxypeptidase (Nlcar) and trypsin-like serine peptidase (Nltry) of the hemipteran Nilaparvata lugens. The nymphs of this insect that fed on the transgenic rice plants did not exhibit the lethal phenotype, but the transcript level of the target genes was reduced.

D. citri studies involving RNA interference have been performed to evaluate the effect of gene silencing in the development of the insect, aiming HLB control. Application of a dsRNA specific for five CYP4 genes caused a significant higher mortality in D. citri adults compared to a control group [28]. [29] analyzed EST sequences of D. citri to identify potential targets for RNA interference in Bactericerca cockerelli and suggested that RNAi targets have a potential application against D. citri, because there is a phylogenetic conservation between the two psyllids. [30] evaluated the effect of topical application of specific dsRNAs for D. citri awd gene to nymphs and [31] performed the transient expression dsRNA and siRNA for the same gene in the phloem and associated cells of Citrus macrophylla and evaluated the effect on insects that fed on the plants. Both works related malformed-wing and reduced survival in adults.

There are many reports about transgenic citrus plants with desirable characteristics [32]. However, few studies based on development of transgenic citrus plants against HLB were performed. [33] reported resistance against HLB of two transgenic sweet orange cultivars ‘Hamlin’ and ‘Valencia’ overexpressing an Arabidopsis thaliana NPR1 gene constitutively and driven by a phloem specific promoter of Arabidopsis. Until the present moment, there is no report of a transgenic citrus plant overexpressing peptidase inhibitors.

For the application of such alternative strategies, targets must been identified and characterized in Diaphorina citri. [34] report the identification of cysteine peptidases in the gut of insects belonging to the orders Hemiptera, Diptera and Coleoptera. As D. citri belongs to the order Hemiptera, it likely has cysteine peptidases in its digestive tract. Given the severity of HLB and the importance of the citrus industry to the global economy, the aims of the present study were to perform the recombinant expression and kinetic characterization of a cathepsin B-like cysteine peptidase (DCcathB) from Diaphorina citri and analyze the gene expression of DCcathB in different insect developmental phases and tissues. These findings can contribute to the validation and possible use of this enzyme as a target for the development of a HLB control strategy.

Materials and Methods

In silico analysis of DCcathB sequence

The search for a gene that encodes a cysteine peptidase in Diaphorina citri was performed from transcriptome data developed by the United States Department of Agriculture (USDA) [35]. A partial sequence of a cathepsin B (gi 110456453) was used to search for similarities in the local database through the transcriptome assembly of D. citri using the BLAST program [36].

In silico analysis was performed with a complete sequence of a cathepsin B-like cysteine peptidase using the InterPro database [37] to check the presence of domains for cysteine peptidase and the position of the pro-region. The signal peptide prediction was performed using the SignalP program [38] and potential N-glycosylation sites present in the sequence were analyzed using the NetNGlyc 1.0 Server program [39]. For an accurate analysis of the similarity of DCcathB with cathepsin B-like cysteine peptidases from other hemipterans, an alignment was performed using the Multalin program [40].

RNA isolation and cDNA synthesis

Adults, nymphs and eggs of D. citri were collected in Fundo de Defesa da Citricultura–Fundecitrus, Araraquara, São Paulo–Brazil, under the responsibility of the Dr. José Belasque Jr. No field studies were performed in the present work. No specific permits were required for the described studies.

Total RNA was extracted separately from each insect phase following the methods described by [41]. cDNA synthesis was performed using the Improm II Reverse Transcription System Kit (Promega) with 2 μg of RNA, 0.5 μg of Oligo dT, 1 U of ImProm IITM reverse transcriptase, in a final volume of 15 μL, following the manufacturer’s instructions. The cycle utilized for reaction was 25°C for 5 min, 42°C for 60 min and 70°C, 15 min. The experiment was performed in thermal cycler (Eppendorf Mastercycler Gradient Thermocycler).

Construction of expression vector

Full-length cDNA coding for a proenzyme form of a cathepsin B-like cysteine peptidase of Diaphorina citri was cloned in the vector pPICZαC in fusion with the secretion α-factor for protein secretion into the medium. The open reading frame coding for the cysteine peptidase protein was obtained through polymerase chain reaction (PCR) amplification using cDNA from adult insects as the template and the specific primers CatBPpicZ-F and CatBPpicZ-R (Table 1) with restriction sites for the enzymes EcoRI and XbaI. Briefly, 1 μL of template DNA, 200 μM of dNTP (Invitrogen), 2.5 μL of Taq Buffer [100 mM Tris-HCl (pH 8.8 at 25°C), 500 mM of KCl and 0.8% (v/v) of Nonidet P40 (Thermo Scientific)], 1.25 mM of MgCl2, 10 pmol of each primer and 1 U of Taq DNA polymerase (Thermo Scientific) were used in a 25 μL reaction. The PCR protocol began at 94°C for 3 min, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C and 1 min at 72°C and final extension for 7 min at 72°C in a thermal cycler (Eppendorf Mastercycler Gradient Thermocycler). The amplification product was purified, digested with EcoRI and XbaI and inserted into a pPICZαC vector (Invitrogen) previously digested with the same enzymes. The ligation reaction between the amplicon and the pPICZαC vector was performed in a standard reaction using T4 DNA Ligase (Invitrogen) and transformed in E. coli DH5α competent cells using the calcium chloride method [42].

Colonies were screened on LB low salt agar medium (Sigma Aldrich) plates with the addition of 25 μg/mL of Zeocin™. The recombinant clone pPICZαC_DCcathB was selected and sequenced using the dideoxy method [43] in the MegaBaceTM1000 DNA sequencer using the DYEnamic ET Terminator kit (GE Healthcare). The sequencing primers were CatB_Int-F, CatB_Int-R and the universal primers were α-Factor (forward) and AOX1 (reverse) (Table 1).

Recombinant expression of DCcathB in Pichia pastoris

The recombinant plasmid was linearized with the enzyme PmeI (New England Biolabs) and transformed into Pichia pastoris KM71H strain competent cells through electroporation. The competent yeast cells were prepared following the method described [44]. The transformation was performed using the Gene Pulser Unit (BIO-RAD) in a cuvette (0.2 cm, 25 uF, 200 Ω), following the instructions of the EasySelect Pichia Expression Kit (Invitrogen). The culture was plated on YPDS medium (1% yeast extract, 2% peptone, 2% dextrose, 1 M of sorbitol and 1.5% bacteriological agar) containing 100 μg/mL of Zeocin™ and additional plates with 200 μg/mL of Zeocin™ for the selection of multi-copy clones. The plates were incubated for 48 h at 30°C. PCR was performed with transformed yeast colonies to screen the recombinant clones [45].

The colonies selected by PCR were screened for protein expression in 24-well plates with 3 mL of BMGY medium (1% yeast extract, 2% peptone, 100 mM of potassium phosphate buffer (pH 7.0), 1.34% yeast nitrogen base, 4 x 10−5% biotin and 1% glycerol) for 48 h at 30°C and 250 rpm. The cells were then centrifuged for 5 min at 1700 x g and re-suspended in 2 mL of BMMY medium (same composition as the BMGY medium, but the replacement of glycerol with 0.5% methanol) for expression for 144 h at 30°C and 250 rpm, with the daily addition of methanol (0.75%). Aliquots of 100 μL were removed every 24 h and 10 μL of the culture supernatant was analyzed using SDS-PAGE 12% [46] stained with Coomassie Blue R-250. For large scale expression, a colony with the highest level of expression was selected and inoculated in 10 mL of BMGY for growth for 24 h at 30°C and 250 rpm. The culture was transferred to a flask with 500 mL of BMGY and incubated at 30°C and 250 rpm until OD600 4–5. The cells were centrifuged for 5 min at 1500 x g and homogenized in 100 mL of BMMY for 24 h at 30°C and 250 rpm. The culture was centrifuged and the supernatant containing the secreted recombinant protein was vacuum filtered through a 0.44 μm PVDF membrane.

Purification of recombinant protein DCcathB by affinity chromatography

The yeast medium containing the secreted DCcathB protein was purified through an affinity chromatography in a Ni-NTA superflow nickel column (Qiagen). The column was previously equilibrated with buffer containing 10 mM Tris-HCl, 100 mM NaCl and 50 mM NaH2PO4, pH 8.0. All purification steps were performed at 4°C. The protein was eluted with the same buffer with increasing imidazole concentrations (10, 25, 50, 75, 100 and 250 mM). The purified protein was analyzed in 12% SDS-PAGE. Fractions containing the purified protein were dialyzed using membranes of 3500 MW (Pierce) for 2 hours at 4°C in buffer containing 10 mM Tris-HCl, 100 mM NaCl and 50 mM NaH2PO4, pH 8.0. The protein was then concentrated in the SPD1010 SpeedVac® System (ThermoSavant) for 3 hours and in Vivaspin™ 3000 MWCO (GE Healthcare) for 1.5 h. The protein concentration was determined using Bradford’s method [47] in Hitachi U-5100 spectrophotometer.

Mass spectrometry analysis of DCcathB

Protein samples were separated by SDS-PAGE 12% and analyzed in a MALDI TOF/TOF mass spectrometer after in-gel trypsin digestion. Briefly, selected gel bands were excised and combined. SDS and CBB were removed by washing the gels three times with 50% ACN in 0.1M ammonium bicarbonate (pH 7.8), followed by dehydration in neat acetonitrile. Gel bands were dried in a Speed Vac instrument (Savant, New York, NY) and were swollen in 20 μL of 0.1 M (pH 7.8) ammonium bicarbonate containing 0.5 μg trypsin (Promega, Madison, USA), followed by the addition of 50 μL of 0.1 M ammonium bicarbonate to cover the entire gel piece. Trypsin hydrolysis was carried out at 37°C for 24 h and the reaction was stopped by the addition of 5 μL of formic acid (98%). Peptides were extracted from gel pieces and desalted in microtips filled with POROS R2 (PerSeptive Biosystems, Foster City, CA) previously equilibrated in in 0.2% formic acid. After loading, the sample was desalted by washing two times with 150 μL of 0.2% formic acid. The peptides were eluted from the microtips with 30 μL of 60% methanol/5% formic acid. The sample was dried down in a speed vac and mixed with matrix solution (5 mg/mL of α-cyano-4 hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid) and applied to the MALDI target plate as air dried drops at room temperature. The MALDI TOF/TOF mass spectrometer (Axima Performance, Kratos-Shimadzu, Manchester, UK) was calibrated with a mixture of bradykynin fragment (1–7), angiotensin II, renin and ACTH (mass accuracy < 50 ppm). The CID-MS/MS spectra of each detected ion were obtained in data-dependent acquisition mode. The peak list was obtained from CID-MS/MS spectra using Launchpad v. 2.8 (Kratos-Shimadzu, Manchester, UK) and submitted to a database search using the MASCOT program version 2.2.04 (Matrixscience, Manchester, UK) directly against the ORF sequence. The database search parameters accepted one missing trypsin cleavage and methionine oxidation. Mass tolerance was 1.2 Da for precursor ions and 0.8 Da for product ions. Protein was considered to be identified by MASCOT score > 56.5% level of significance (p < 0.05) and FDR less than 1%. The amino acid sequences of the tryptic peptides were compared with the amino acid sequence of the cysteine peptidase in the USDA database of the D. citri transcriptome and the amino acid sequence obtained by sequencing performed at the Molecular Biology Laboratory of the Federal University of São Carlos, SP, Brazil.

Kinetic characterization of DCcathB

According to [48], different recombinant systems have been used for cathepsin B expression but in all of these the peptidases were activated following processing. Furthermore, according to [49], to generate active peptidases it is necessary to remove the propeptide region and this can be removed autocatalytically by incubating the protein in acetate buffer at pH 4–4.5. The activation of the recombinant enzyme was performed using the method described by [49] with modifications. The enzyme in buffer containing 10 mM Tris HCl, 50 mM NaH2PO3 and 100 mM NaCl at pH 8.0 was activated with the addition of 50% acetic acid until reaching pH 5.0 and incubation for 60 min at 37°C.

Assays for determination of the catalytic activity of DCcathB cysteine peptidase (2.14 nM) were performed in 100 mM sodium acetate buffer (pH 5.5) containing 2.5 mM DTT (dithiotreitol) in final volume of 500 μL. The enzyme was pre-activated for 3 min at 30°C before the addition of the substrate. The fluorogenic substrates Benzyloxycarbonyl-L-phenylalanyl-L-arginine-4-methylcoumaryl-7-amide (Z-F-R-AMC) (Calbiochem) and Benzyloxycarbonyl-L-arginyl-L-arginine-4-methylcoumaryl-7-amide (Z-R-R-AMC) (Calbiochem) were added at concentrations ranging from 1 to 75 mM and the enzyme activity was continuously monitored in a Hitachi F-2500 spectrofluorometer with fluorescence measured at λex = 380 and λem = 460 nm following the procedure described by [50]. The Michaelis-Menten constant (Km) was determined using the GraFit program [51]. Tests were carried out in triplicate.

Inhibition assays of DCcathB activity

The inhibition DCcathB activity was determined spectrofluorometrically using the substrate Z-F-R-AMC. Fluorescence was measured in a Hitachi F-2500 spectrofluorometer at λex = 380 and λem = 460 nm. Inhibitory activity was determined by measuring the residual hydrolytic activity of the cysteine peptidase. The enzyme (2,14 μM) was added to 100 mM sodium acetate buffer (pH 5.5) containing 2.5 mM DTT, in a final volume of 500 μL, and pre-incubated for 3 minutes at 30°C. Then, the substrate Z-F-R-AMC (37.5 mM) was added and the DCcathB activity was measured in the presence of the following inhibitors: CaneCPI-4 (0,04; 0,08; 0,12; 0,16; 0,20 nM) [52], synthetic epoxide peptide E-64 (L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane) (Calbiochem) (0,004, 0,008; 0,012; 0,016; 0,020 μM) [53] and synthetic inhibitor CA-074 [N-(l-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline] (Calbiochem) (0,026; 0,052; 0,078; 0,104; 0,13 nM) [54]. The residual DCcathB activity was measured. The inhibition constant (Ki) was calculated following Morrison’s procedure using the GraFit program. All experiments were carried out in triplicate. The results were used to determine the IC50 [55] for E-64 and CA-074.

Gene expression analysis of DCcathB in insect developmental phases and tissues

The developmental phases of egg, nymph (5th instar) and adult and the tissues of head, gut and remaining tissues were used for DCcathB gene expression analysis by reverse transcription quantitative PCR (RT-qPCR). Egg, gut, head and remaining tissues were collected with a needle, placed in a 1.5-mL tube with 500 μL of Trizol reagent (Invitrogen) and incubated on ice to maintain the integrity of the material. The RNA isolation was performed according to [41]. RNA was quantified in a Nanodrop Spectrophotometer ND-1000 (Thermo Scientific) and integrity was checked in 1% agarose gel stained with ethidium bromide.

Each RNA sample was individually treated with 1U/μL of DNase I Amplification Grade (Invitrogen) and 10x DNase I Reaction Buffer (final concentration 1x). The samples were incubated for 15 min at room temperature to remove traces of genomic DNA. DNase I was inactivated by adding of 1 μL of 25mM EDTA/μL of DNase and incubation at 65°C for 10 min.

The primers CathBD.citriRT_F, CathBD.citriRT_R, ActinRT_F, ActinRT_R, 18S_Foward and 18S_Reverse (Table 1) were designed with the Primer 3 program, version 4.0 [56]. To ensure that the DCcathB primers designed to RT-qPCR will be specific and amplify only the fragment of interest of this enzyme, we used the Primer-BLAST Program [57] and an alignment with the primers with cathepsins B-like identified in the D. citri transcriptome [35] was performed.

The amplification reactions were conducted using 5 μL of 1x Platinum SYBR Green qPCR Supermix UDG (Invitrogen), 1 μL of cDNA (100 ng) and 0.4 μM of each primer, in a final volume of 10 μL. Due to the constitutive expression, the reference gene chosen for analysis of DCcathB gene expression in developmental phases was the actin gene (genbank accession number: 110456519) and for DCcathB gene expression analysis in tissues the reference gene was 18S rRNA (genebank accession number: 110671475). Both genes are cited as reference in studies involving expression analyses in D. citri [28, 30, 58, 59]. The reactions were conducted in triplicate in the Eco Real-time System (Illumina) using the following reaction cycle: 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 30 s, 54°C for 30 s and 72°C for 40 s. The melt curve was performed using the following cycle: 95°C for 15 s, 54°C for 15 s and 95°C for 15 s, to analyze the absence of non-specific amplification products.

The data were analyzed using the 2-ΔΔCt method [60]. The standard curve was performed using a serial dilution of cDNA with the initial concentration of 100 ng. No template controls (NTC) were performed to ensure the absence of sample contamination in every run. The following parameters were established for the reactions: efficiency > 90%, slops > -3.25 < -3.6 and R2 > 98%. The Relative Expression Software Tool (REST) 2005 version 1.9.12 from Qiagen [61] was used to determine the significance of the values obtained in the gene expression analysis and the difference in gene expression among phases and tissues was considered significant when p-value was less than 0.05 with a 95% confidence interval.

Results and Discussion

DCcathB sequence analysis

A complete cDNA sequence encoding the D. citri cathepsin B-like cysteine peptidase, DCcathB, with open reading frame of 1125 bp (genbank accession number (KT835051) was identified using the databank of the Diaphorina citri transcriptome [35]. The protein has a predicted sequence of 374 amino acids and a relative molecular mass of 41.9 kDa. According to the analysis performed in the InterPro database, DCcathB presented characteristics of a cysteine peptidase with regions that potentially corresponds to an active site of cathepsins B represented by the amino acid residues at the positions 142–153 and 315–325. The DCcathB pro-region of 38 amino acids (DIVDQVNNNVTSTWQARHNFHPDTPVSYLSSLAGTRPL) extends from residues 32 to 69 and the mature peptide extends from residues 70 to 374. The signal peptide of 17 amino acids, (MWAVGVLVLIATQSLAI) as predicted by the Signal P Program and four potential N-glycosylation sites were predicted using the NetNGlyc 1.0 Server.

The features of DCcathB were displays in Fig 1. The alignment shows the presence of the region corresponding to the occlusion loop, which is characteristic of cathepsin B-like enzymes [62]. The cleavage site between the pro-peptide and mature protein is predicted to occur in the sequence PL^DESD…, resulting in D70 as the N-terminal amino acid of the mature enzyme. The sequence of pro-peptide region contains a unique N-glycosylation site at N40 and the sequence correspondent to the mature protein contains three N-glycosylation sites at N102, N235 and N253. The papain cysteine peptidase family has a pro-region that performs functions such as transport to endosomal or lysosomal compartments for the correct folding of the active mature protein and pro-enzyme activity inhibition [49].

thumbnail
Fig 1. Alignment of DCcathB with similar cathepsin B-like cysteine peptidases from hemipterans.

Diaphorina citri (genbank accession number: 110456454), Nilaparvata lugens (genbank accession number: 22535408), Aphis citricidus (genbank accession number: 161343879), Riptortus pedestris (genbank accession number: 501291537), Acyrthosiphon pisum (genbank accession number: 209863079). Conserved identical residues are marked in black boxes and white boxes show conserved residues with more than 50% identity. The DCcathB predicted peptide signal of seventeen residues is underlined in black. The probable occlusion loop characteristic of cathepsin B-like cysteine peptidases is represented by the dashed box. The potential cleavage site between the propeptide (residues 32 and 69) and mature DCcathB is indicated by an arrow. The predicted conserved catalytic triad C-H-N is indicated by asterisks. Alignment was generated using the Multalign program with default parameters.

https://doi.org/10.1371/journal.pone.0145132.g001

DCcathB expression and purification

The SDS-PAGE 12% analysis revealed the expression of the recombinant protein at 24 hours of induction (Fig 2A) and the purified DCcathB as a single and intact band of approximately 50 kDa corresponding to the protein eluted in imidazole fractions of 10 and 25 mM (Fig 2B). The yield of purified DCcathB protein was 2 mg/L of P. pastoris cell culture.

thumbnail
Fig 2. DCcathB expression.

A–SDS-PAGE 12% stained with Coomassie blue showing the induction of pPICZαC_DCcathB supplemented with 0.75% methanol. M: molecular weight BenchMark™ Protein Ladder (Invitrogen). Induction Times: 1–0 h; 2–24 h; 3–48 h; 4–72 h; 5–96 h; 6–120 h; 7–144 h. B–Purification of DCcathB. M: molecular weight marker (Invitrogen). 1—Eluate. 2—Wash buffer without imidazole. 3—Protein eluted in 10 mM imidazole. 4—Protein eluted in 10 mM imidazole. 5—Protein eluted in 25 mM imidazole. 6—Protein eluted in 25 mM imidazole.

https://doi.org/10.1371/journal.pone.0145132.g002

The predicted molecular mass of DCcathB is 41.9 kDa. However, due to the fusion with α-factor, c-myc epitope and C-terminal histidine tag, the expected size for the recombinant protein is approximately 46 kDa. SDS-PAGE analysis revealed a band of 50 kDa probably corresponding to the glycosylated protein.

Mass spectrometry analysis

For the verification of the DCcathB amino acid sequence, the purified protein was subjected to sequencing by mass spectrometry. The MALDI TOF/TOF data obtained by CID-MS/MS provided the amino acid sequences of two peptides: QNCYNPSYESTYR (m/z 1625.07 ± 0.41 at residue position 249 to 261) (Fig 3A) and SGVYQHNFGDSIGLHAVR (m/z 1957.32 ± 0.38 at residue position 303 to 320) (Fig 3B). These tryptic peptides correspond to 8.3% sequence coverage and a total MASCOT score of 185. The results obtained by mass spectrometry identified two peptides present in the amino acid sequence of the predicted protein obtained in the USDA database of the D. citri transcriptome (lcl|Sequence 1 ORF: 50.1174 Frame +2 Cathepsin B) as well in the sequence generated by the sequencing of the pPICZαC_DCcathB expression plasmid. The results obtained by mass spectrometry confirm the identity of the purified protein as a cathepsin B-like cysteine peptidase.

thumbnail
Fig 3. CID-MS/MS spectra of tryptic peptides from DCCathB.

Amino acid sequences were deduced from product ions and identified (bold) in the cysteine peptidase sequence deduced from D. citri ESTs.

https://doi.org/10.1371/journal.pone.0145132.g003

Determination of enzyme activity

As the purified DCcathB was produced in its pro-enzyme form, a strategy was adopted to remove the pro-region for subsequent enzyme activation. Once activated, autocatalytic cleavage occurs and the mature protein can process other precursor molecules, with the occurrence of a chain reaction that allows the removal of the pro-region [63].

The DCcathB enzyme was able to hydrolyze the substrates Z-F-R-AMC and Z-R-R-AMC substrates and Km values were determined. For Z-F-R-AMC the Km value was 23.5 μM. This value is very similar to the Km of the cathepsin B-like cysteine peptidases yEmCBP1 and yEmCBP2 from Echinococcus multilocularis (Km = 20.45 and 27.17 μM, respectively) [64]. For Z-R-R-AMC the Km value was 6.13 μM, demonstrating greater affinity. Specificity analysis of cathepsin L midgut Tenebrio molitor was performed using different substrates, demonstrating capability to hydrolyze the substrate Z-R-R-AMC, but a greater preference for substrates with hydrophobic residues at the P2 position substrate [65]. However, cathepsin B unlike other cysteine peptidases from papain family, can accept substrates with arginine residue at the P2 position due to the location of an E245 residue in the subsite S2. This residue is important for the specificity of cathepsin B [66]. This result is important for the characterization of DCcathB therefore demonstrated the enzyme is capable of hydrolyzing the Z-R-R-AMC substrate, a selective substrate for cathepsin B.

DCcathB catalytic activity inhibition

The inhibitory activity of DCcathB was evaluated using the sugarcane recombinant cystatin CaneCPI-4 [52] and the synthetic inhibitors CA-074 and E-64. CaneCPI-4 efficiently inhibited DCcathB catalytic activity, with a Ki value of 0.05 nM, which demonstrated a strong interaction between the D. citri cathepsin B-like cysteine peptidase and the cystatin. This value is consistent with data described by [67], who report that the recombinant human cystatin C inhibits human cathepsin B activity, with a Ki value of 0.38 nM. Furthermore, [52, 68] respectively obtained Ki values of 0.5 nM and 0.83 nM for CaneCPI-4 against recombinant human cathepsin B. Cystatins are competitive and reversible inhibitors of cysteine peptidases [69]. The structure of cathepsin B-like cysteine peptidases has an occluding loop that blocks the cleft of the enzyme active site [62] and hinders the access of cystatin to this site, resulting in high Ki values for most cystatins against cathepsin B when compared to other cysteine peptidases that lack this loop [70, 71, 72].

Structural differences between the cystatin CaneCPI-4 and other cystatins may be the key to the inhibitory profile against cathepsin B. This cystatin is included in group III of the cystatin classification and lacks the N-terminal exclusive phytocystatin consensus sequence LAR-[FY]-N-[VI]-x(3)-N motif. Moreover, the conserved motif is represented by QVVAG in the cystatin superfamily, but is represented by QVVSG in CaneCPI-4 [52, 73]. [68] conducted modeling studies by homology to determine regions of phytocystatins responsible for cathepsin B catalytic activity inhibition. Analyzing CaneCPI-4, variations were found in some hydrophobic amino acids between the α-helix and the β sheet, including a glutamine residue at position 30 and glycine at positions 47 and 56, which accounted for a decrease in hydrophobic interactions in the hydrophobic core. These changes contribute to flexibility and, consequently, inhibitory activity against human cathepsin B. The inhibition assays performed with CaneCPI-4 and DCcathB clearly demonstrate the high affinity of this inhibitor for the DCcathB enzyme.

Analyzes of DCcathB inhibition activity using the inhibitors E-64 and CA-074 were also performed and demonstrated an interaction between the enzyme and both inhibitors. The IC50 was 0.095 nM for CA-074 and 0.014 μM for E-64. CA-074 is a selective inhibitor of cathepsin B [54]. The interaction between CA-074 and DCcathB enzyme is evidence that DCcathB is a cathepsin B like enzyme.

DCcathB gene expression in insect developmental phases and tissues

Studies with cathepsin B-like cysteine peptidases in insects indicated that these enzymes have a role in several biological processes as digestion of food proteins in midgut [34, 74, 75]; degradation and mobilization of yolk proteins during embryogenesis [76, 77, 78, 79]; programmed cell death; metamorphosis [80, 81, 82] and defense against enemies [83, 84]. A detailed study about the differential expression between infected and non-infected D.citri nymphs and adults demonstrated that a cathepsin B-like is down-regulated and categorized in defense/immune response-related contigs [84].

For better understanding the role of DCcathB in D. citri, RT-qPCR was performed to analyze the gene expression of the DCcathB in developmental phases of D.citri: egg, nymphs (5th instar) and adults and in the following tissues: head, gut and remaining tissues. Fig 4A shows the gene expression pattern of DCcathB in egg, nymph and adult phases. DCcathB presented lower expression in egg, suggesting that DCcathB has a basal level of expression in this phase. [85] performed the gene expression analysis of a digestive cathepsin L-like of Sphenophorus levis and described a basal expression of the enzyme at the egg. However there was a notable increase in the production of mRNA encoding DCcathB in D. citri nymphs 9-fold and adults 9.3-fold in comparison to the egg phase. This result is interesting because the nymph phase is very important for the acquisition and transmission of the CLas. Moreover, when nymphs fed on CLas-infected plants, the concentration of the bacterium increased significantly and the transmission to the plants is more effective. The adults infected in the nymph phase are able to transmit the bacterium immediately after emergence. In contrast, adults that fed on infected plants did not presented significantly increase of the concentration and are poor transmitters of the bacterium [84, 86, 87]. Thus, a strategy that uses DCcathB as target for D. citri nymph control may be effective.

thumbnail
Fig 4. DCcathB expression analysis by RT-qPCR in D. citri egg, nymph and adult (A) and head, gut and remaining tissues (B).

The quantification calibrator in developmental phases was egg and for the tissues was gut. Error bars were calculated according [60]. The difference was significant when the p-value was lower than 0.05.

https://doi.org/10.1371/journal.pone.0145132.g004

Gene expression analysis of DCcathB in D. citri head, gut and remaining tissues showed notable increase in the production of mRNA encoding DCcathB in the gut, being 3333-fold and 175-fold, respectively, relative to the head and remaining tissues. Fig 4B shows gene expression pattern of DCcathB in each tissue analyzed. This result suggests that this enzyme has a digestive function in D. citri. Several studies reported the presence of cathepsins B in the gut of Hemipterans, demonstrating the importance of these enzymes in digestion process of this insect class. In other Hemiptera insect, the aphid Acyrthosiphon pisum, 28 cathepsins B-like were identified and five cathepsins B-like were preferentially expressed in the gut [88]. The presence of cathepsins B-like that are specifically expressed in the gut of Tuberaphis styraci, Nilaparvata lugens, Rhodnius prolixus and Triatoma infestans was reported by [83, 89, 90, 91].

Considering that DCcathB is highly and preferentially expressed in the gut and nymph phase, our results contribute to the establishment of future strategies of control possibly based on the overexpression of peptidase inhibitors in citrus plants for HLB control.

Acknowledgments

Research supported by the Brazilian fostering agencies Fapesp (Proc. 2012/24278-5) and Fundo de Defesa da Citricultura. T.F.S.F.–received a grant from Capes, V.K.S.–received a grant from Fapesp.

Author Contributions

Conceived and designed the experiments: TFSF ASC FHS. Performed the experiments: TFSF VKS LTK MFMA. Analyzed the data: TFSF VKS LTK MFMA JCR JBJ WBH ASC. Contributed reagents/materials/analysis tools: LTK JCR ASC FHS. Wrote the paper: TFSF VKS LTK MFMA AKC JBJ JCR WBH FHS ASC.

References

  1. 1. Fao. Statistical Yearbook. Food and Agriculture Organization of the United Nations. 2013. Available: http://www.fao.org/home/en/.
  2. 2. Tsai JH, Liu YH. Biology of Diaphorina citri (Homoptera: Psyllidae) on four host plants. J Econ Entomol. 2000; 93(6): 1721–1725. pmid:11142304.
  3. 3. Boina DR, Onagbola EO, Salyani M, Stelinski LL. Antifeedant and sublethal effects of imidacloprid on Asian citrus psyllid, Diaphorina citri. Pest Manag Sci. 2009; 65(8): 870–877. pmid:19431217.
  4. 4. Bové JM. Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology. 2006; 88: 7–37.
  5. 5. Reinking OA. Diseases of economic plants in South China. Philippine Agriculturist. 1919; 8: 109–135.
  6. 6. Lin KH. Observation on yellow shoot of citrus. Etiological study of yellow shoot of citrus. Acta Phytoph Sinica. 1956; 2: 1–42.
  7. 7. Coletta-Filho HD, Targon MLPN, Takita MA, De Negri JD, Pompeu J, Machado MA, et al. First Report of the Causal Agent of Huanglongbing (“Candidatus Liberibacter asiaticus”) in Brazil. Plant Dis. 2004; 88(12): 1382.
  8. 8. Texeira DC, Ayres J, Kitajima EW, Danet L, Jagoueix-Eveillard S, Saillard C, et al. First Report of a Huanglongbing-Like Disease of Citrus in Sao Paulo State, Brazil and Association of a New Liberibacter Species, “Candidatus Liberibacter americanus”, with the Disease. Plant Dis. 2005; 89: 107.
  9. 9. Teixeira DC, Saillard C, Eveillard S, Danet JL, Costa PI, Ayres AJ, et al. ‘Candidatus Liberibacter americanus’, associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil. International journal of systematic and evolutionary microbiology. 2005; 55: 1857–1862. pmid:16166678
  10. 10. Halbert SE. The discovery of huanglongbing in Florida. In: Proceedings of the 2nd International Citrus Canker and Huanglongbing Research Workshop, Florida Citrus Mutual, Orlando, FL. 2005. Available: http://freshfromflorida.s3.amazonaws.com/2nd_International_Canker_Huanglongbing_Research_Workshop_2005.pdf. Accessed 05 January 2015.
  11. 11. Martínez Y, Llauger R, Batista L, Luis M, Iglesia A, Collazo C, et al. First report of ‘Candidatus Liberibacter asiaticus’ associated with Huanglongbing in Cuba. Plant Pathol. 2009; 58(2): 389.
  12. 12. Manjunath KL, Ramadugu C, Majil VM, Williams S, Irey M, Lee RF. First Report of the Citrus Huanglongbing Associated Bacterium ‘Candidatus Liberibacter asiaticus’ from Sweet Orange, Mexican Lime, and Asian Citrus Psyllid in Belize. Plant Dis. 2010; 94(6): 781.
  13. 13. Luis M, Collazo C, Llauger R, Blanco E, Peña I, et al. Occurrence of citrus huanglongbing in Cuba and association of the disease with Candidatus Liberibacter asiaticus. J Plant Pathol. 2009; 91: 709–712.
  14. 14. Matos L, Hilf ME, Camejo J. First Report of ‘Candidatus Liberibacter asiaticus’ Associated with Citrus Huanglongbing in the Dominican Republic. Plant Dis. 2009; 93(6): 668.
  15. 15. Wulff NA, Zhang S, Setubal JC, Almeida NF, Martins EC, Harakava R, et al. The Complete Genome Sequence of ‘Candidatus Liberibacter americanus’, Associated with Citrus Huanglongbing. Molecular Plant-Microbe Interactions. 2013; 27(2): 163–176.
  16. 16. Lopes SA, Bertolini E, Frare GF, Martins EC, Wulff NA, Teixeira DC, et al. Graft Transmission Efficiencies and Multiplication of ‘Candidatus Liberibacter americanus’ and ‘Ca. Liberibacter asiaticus’ in Citrus Plants. Phytopathology. 2009; 99(3): 301–306. pmid:19203283
  17. 17. Lopes SA, Frare GF, Bertolini E, Cambra M, Fernandes NG, Ayres AJ, et al. Liberibacters Associated with Citrus Huanglongbing in Brazil: ‘Candidatus Liberibacter asiaticus’ Is Heat Tolerant, ‘Ca. L. americanus’ Is Heat Sensitive. Plant Dis. 2009; 93(3): 257–262.
  18. 18. Xu CF, Xia YH, Li KB, Ke C. Further study of the transmission of citrus huanglongbing by a psyllid, Diaphorina citri Kuwayama. In: Timmer LW, Garnsey SM, Navarro L (eds) Proc of the 10th Conf Int Organ Citrus Virol Riverside, CA, 1998. Available: http://www.imok.ufl.edu/hlb/database/pdf/00000029.pdf. Accessed 02 September 2015.
  19. 19. Gottwald TR, Da Graça JV, Bassanezi RB. Citrus huanglongbing: the pathogen and its impact. Plant Health Progress. 6 Sep 2007. Available: http://www.apsnet.org/publications/apsnetfeatures/Pages/HuanglongbingImpact.aspx. Accessed 10 September 2015.
  20. 20. Cocco A, Hoy MA. Toxicity of Organosilicone Adjuvants and Selected Pesticides to the Asian Citrus Psyllid (Hemiptera: Psyllidae) and Its Parasitoid Tamarixia radiata (Hymenoptera: Eulophidae). Florida Entomologist. 2008; 91(4): 610–620.
  21. 21. Avery PB, Hunter WB, Hall DG, Jackson MA, Powell CA, Rogers ME. Diaphorina citri (Hemiptera: Psyllidae) Infection and Dissemination of the Entomopathogenic FungusIsaria fumosorosea (Hypocreales: Cordycipitaceae) Under Laboratory Conditions. Florida Entomologist. 2009; 92(4): 608–618.
  22. 22. Halbert SE, Manjunath KL. Asian Citrus Psyllids (Sternorrhyncha: Psyllidae) and Greening Disease of Citrus: A Literature Review and Assessment of Risk in Florida. Florida Entomologist. 2004; 87(3): 330–353.
  23. 23. Falco MC, Silva-Filho MC. Expression of soybean proteinase inhibitors in transgenic sugarcane plants: effects on natural defense against Diatraea saccharalis. Plant Physiology and Biochemistry. 2003; 41(8): 761–766.
  24. 24. Lecardonnel A, Chauvin L, Jouanin L, Beaujean A, Prévost G, Sangwan-Norreel B. Effects of rice cystatin I expression in transgenic potato on Colorado potato beetle larvae. Plant Science. 1999; 140(1): 71–79.
  25. 25. Carrillo L, Martinez M, Álvarez-Alfageme F, Castañera P, Smagghe G, Diaz I, et al. A barley cysteine-proteinase inhibitor reduces the performance of two aphid species in artificial diets and transgenic Arabidopsis plants. Transgenic Research. 2011; 20(2): 305–319. pmid:20567901
  26. 26. Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O, et al. Control of coleopteran insect pests through RNA interference. Nat Biotech. 2007; 25(11): 1322–1326.
  27. 27. Zha W, Peng X, Chen R, Du B, Zhu L, He G. Knockdown of midgut genes by dsRNA-transgenic plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens. Plos One. 2011; 6(5): e20504. pmid:21655219
  28. 28. Killiny N, Hajeri S, Tiwari S, Gowda S, Stelinski LL. Double-stranded RNA uptake through topical application, mediates silencing of five CYP4 genes and suppresses insecticide resistance in Diaphorina citri. Plos One. 2014; 9(10): e110536. pmid:25330026
  29. 29. Wuriyanghan H, Rosa C, Falk BW. Oral delivery of double-stranded RNAs and siRNAs induces RNAi effects in the potato/tomato psyllid, Bactericerca cockerelli. Plos One. 2011; 6(11): e27736. pmid:22110747.
  30. 30. El-Shesheny I, Hajeri S, El-Hawary I, Gowda S, Killiny N. Silencing Abnormal Wing Disc Gene of the Asian Citrus Psyllid, Diaphorina citri Disrupts Adult Wing Development and Increases Nymph Mortality. Plos One. 2013; 8(5): e65392. pmid:23734251
  31. 31. Hajeri S, Killiny N, El-Mohtar C, Dawson WO, Gowda S. Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease (Huanglongbing). J Biotechnol. 2014; 176: 42–49. pmid:24572372.
  32. 32. Donmez D, Simsek O, Izgu T, Kacar YA, Mendi YY. Genetic transformation in citrus. TheScientificWorldJournal. 2013; 2013: 491207. pmid:23983635.
  33. 33. Dutt M, Barthe G, Irey M, Grosser J. Transgenic Citrus Expressing an Arabidopsis NPR1 Gene Exhibit Enhanced Resistance against Huanglongbing (HLB; Citrus Greening). Plos One. 2015; 10(9): e0137134. pmid:26398891.
  34. 34. Terra WR, Ferreira C. Insect digestive enzymes: properties, compartmentalization and function. Comparative biochemistry and physiology / B. 1994; 109(1): 1–62.
  35. 35. Reese J, Christenson MK, Leng N, Saha S, Cantarel B, Lindeberg M, et al. Characterization of the Asian Citrus Psyllid Transcriptome. Journal of genomics. 2014; 2: 54–58. pmid:24511328
  36. 36. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389–3402. pmid:PMC146917.
  37. 37. Hunter S, Jones P, Mitchell A, Apweiler R, Attwood TK, Bateman A, et al. InterPro in 2011: new developments in the family and domain prediction database. Nucleic Acids Res. 2012; 40:D306–D312. pmid:22096229
  38. 38. Bendtsen JD, Nielsen H, Von-Heijne G, Brunak S. Improved Prediction of Signal Peptides: SignalP 3.0. Journal of Molecular Biology. 2004; 340(4): 783–795. pmid:15223320
  39. 39. Gupta R, Jung E, Brunak S. Prediction of N-glycosylation sites in human proteins. NetNGlyc 1.0. (NetNGlyc website) 2004. Available: http://www.cbs.dtu.dk/services/NetNGlyc. Accessed 07 February 2012.
  40. 40. Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988; 16(22): 10881–10890. pmid:2849754
  41. 41. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987; 162(1): 156–159. pmid:2440339.
  42. 42. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A laboratory Manual 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989.
  43. 43. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. P Natl Acad Sci USA. 1977; 74(12): 5463–5467. pmid:PMC431765.
  44. 44. Cregg JM. Pichia protocols. Totowa, NJ: Humana Press; 2007.
  45. 45. Akada R, Murakane T, Nishizawa Y. DNA extraction method for screening yeast clones by PCR. Biotechniques. 2000; 28(4): 668–70, 672, 674.pmid:10769744.
  46. 46. Laemmli UK. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature. 1970; 227(5259): 680–685. pmid:5432063
  47. 47. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248–254. pmid:942051.
  48. 48. Barrett AJ, Rawlings ND, Woessner JF. Handbook of proteolytic enzymes 2nd ed. London: Academic Press, 1998.
  49. 49. Bromme D, Nallaseth FS, Turk B. Production and activation of recombinant papain-like cysteine proteases. Methods. 2004; 32(2): 199–206. pmid:14698633.
  50. 50. Anastasi A, Brown MA, Kembhavi AA, Nicklin MJ, Sayers CA, Sunter DC, et al. Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. Biochemical Journal. 1983; 211(1): 129–138. pmid:PMC1154336.
  51. 51. Leatherbarrow RJ. GraFit Version 5. Erithacus Software Ltd. ed. Horley: UK, 2001. Available: http://www.erithacus.com/grafit/binary/GraFit%20version%205.pdf. Accessed 10 February 2015.
  52. 52. Gianotti A, Sommer CA, Carmona AK, Henrique-Silva F. Inhibitory effect of the sugarcane cystatin CaneCPI-4 on cathepsins B and L human breast cancer cell invasion. Biol Chem. 2008; 389: 447–453. pmid:18208350
  53. 53. Barrett AJ, Kembhavi AA, Hanada K. E-64 [L-trans-epoxysuccinyl-leucyl-amido(4-guanidino)butane] and related epoxides as inhibitors of cysteine proteinases. Acta Biol Med Ger. 1981; 40(10–11): 1513–1517. pmid:7044005.
  54. 54. Murata M, Miyashita S, Yokoo C, Tamai M, Hanada K, Hatayama K, et al. Novel epoxysuccinyl peptides. Selective inhibitors of cathepsin B, in vitro. Febs Lett. 1991; 280(2): 307–310. pmid:2013328.
  55. 55. Nagase and Salvesen . Proteolytic enzymes. A practical approach. In: Benyon RJ and Bond JS, editors. New York: Oxford University Press; 2001. pp. 131–146.
  56. 56. Rozen S, Skaletsky H. Primer3 on the WWW for General Users and for Biologist Programmers. In: Misener S, Krawetz S, editors. Bioinformatics Methods and Protocols. Totowa: Humana Press; 1999. pp. 365–386.
  57. 57. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012; 13: 134. pmid:22708584.
  58. 58. Tiwari S, Gondhalekar AD, Mann RS, Scharf ME, Stelinski LL. Characterization of five CYP4 genes from Asian citrus psyllid and their expression levels in Candidatus Liberibacter asiaticus-infected and uninfected psyllids. Insect molecular biology. 2011; 20(6): 733–744. pmid:21919983
  59. 59. Marutani-Hert M, Hunter WB, Hall DG. Gene Response to Stress in the Asian Citrus Psyllid (Hemiptera: Psyllidae). Florida Entomologist. 2010; 93(4): 519–525.
  60. 60. Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods. 2001; 25(4): 402–408. pmid:11846609
  61. 61. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002; 30(9): e36. pmid:11972351
  62. 62. Musil D, Zucic D, Turk D, Engh RA, Mayr I, Huber R, et al. The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity. The EMBO Journal. 1991;10(9): 2321–2330. pmid:PMC452927.
  63. 63. Vernet T, Khouri HE, Laflamme P, Tessier DC, Musil R, Gour-Salin BJ, et al. Processing of the papain precursor. Purification of the zymogen and characterization of its mechanism of processing. J Biol Chem. 1991; 266(32): 21451–21457. pmid:1939177
  64. 64. Sako Y, Nakaya K, Ito A. Echinococcus multilocularis: Identification and functional characterization of cathepsin B-like peptidases from metacestode. Experimental Parasitology. 2011; 127(3): 693–701. pmid:21095185
  65. 65. Cristofoletti PT, Ribeiro AF, Terra WR. The cathepsin L-like proteinases from the midgut of Tenebrio molitor larvae: Sequence, properties, immunocytochemical localization and function. Insect Biochemistry and Molecular Biology. 2005; 35(8): 883–901. pmid:15944084
  66. 66. Hasnain S, Hirama T, Huber CP, Mason P, Mort JS. Characterization of cathepsin B specificity by site-directed mutagenesis. Importance of Glu245 in the S2-P2 specificity for arginine and its role in transition state stabilization. J Biol Chem. 1993; 268(1): 235–240. pmid:8093241
  67. 67. Cimerman N, Prebanda MT, Turk B, Popovic T, Dolenc I et al. Interaction of cystatin C variants with papain and human cathepsins B, H and L. J Enzyme Inhib. 1999; 14: 167–174. pmid:10445041
  68. 68. Valadares N, Dellamano M, Soares-Costa A, Henrique-Silva F, Garratt R. Molecular determinants of improved cathepsin B inhibition by new cystatins obtained by DNA shuffling. BMC Structural Biology. 2010; 10(1): 30.
  69. 69. Barrett AJ. The cystatins: a new class of peptidase inhibitors. Trends in Biochemical Sciences. 1987; 12: 193–196.
  70. 70. Abrahamson M. [49] Cystatins. Methods in Enzymology. Volume 244: Academic Press; 1994. p. 685–700. pmid:7845245
  71. 71. Ohtsubo S, Kobayashi H, Noro W, Taniguchi M, Saitoh E. Molecular Cloning and Characterization of Oryzacystatin-III, a Novel Member of Phytocystatin in Rice (Oryza sativa L. japonica). Journal of Agricultural and Food Chemistry. 2005; 53(13): 5218–5224. pmid:15969500
  72. 72. Martinez M, Abraham Z, Gambardella M, Echaide M, Carbonero P, Diaz I. The strawberry gene Cyf1 encodes a phytocystatin with antifungal properties. Journal of Experimental Botany. 2005; 56(417): 1821–1829. pmid:15897228
  73. 73. Reis EM, Margis R. Sugarcane phytocystatins: Identification, classification and expression pattern analysis. Genetics and molecular biology. 2001; 24: 291–296.
  74. 74. Houseman JG, MacNaughton WK and Downe AER. Cathepsin B and aminopeptidase in the posterior midgut of Euschistus euschistoides (Hemiptera: Pentatomidae). Can Entomol. 1984; 116: 1393–1396.
  75. 75. Terra WR, Ferreira C, Garcia ES. Origin, distribution, properties and functions of the major Rhodnius prolixus midgut hydrolases. Insect Biochemistry. 1988; 18(5): 423–434.
  76. 76. Cho WL, Tsao SM, Hays AR, Walter R, Chen JS, Snigirevskaya ES, et al. Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor. J Biol Chem. 1999; 274(19): 13311–13321. pmid:10224092.
  77. 77. Izumi S, Yano K, Yamamoto Y, Takahashi SY. Yolk proteins from insect eggs: Structure, biosynthesis and programmed degradation during embryogenesis. Journal of insect physiology. 1994; 40(9): 735–746.
  78. 78. Medina M, León P, Vallejo CG. Drosophila cathepsin B-like proteinase: A suggested role in yolk degradation. Archives of Biochemistry and Biophysics. 1988; 263: 355–363. pmid:3132106
  79. 79. Zhao XF, Wang JX, Xu XL, Schmid R, Wieczorek H. Molecular cloning and characterization of the cathepsin B-like proteinase from the cotton boll worm, Helicoverpa armigera. Insect molecular biology. 2002; 11(6): 567–575. pmid:12421414.
  80. 80. Takahashi N, Kurata S, Natori S. Molecular cloning of cDNA for the 29 kDa proteinase participating in decomposition of the larval fat body during metamorphosis of Sarcophaga peregrina (flesh fly). Febs Lett. 1993; 334(2): 153–157. pmid:8224239.
  81. 81. Shiba H, Uchida D, Kobayashi H, Natori M. Involvement of cathepsin B- and L-like proteinases in silk gland histolysis during metamorphosis of Bombyx mori. Arch Biochem Biophys. 2001; 390(1): 28–34. pmid:11368511.
  82. 82. Lee KS, Kim BY, Choo YM, Yoon HJ, Kang PD, Woo SD, et al. Expression profile of cathepsin B in the fat body of Bombyx mori during metamorphosis. Comp Biochem Physiol B Biochem Mol Biol. 2009; 154(2): 188–194. pmid:19539774.
  83. 83. Kutsukake M, Shibao H, Nikoh N, Morioka M, Tamura T, Hoshino T, et al. Venomous protease of aphid soldier for colony defense. Proc Natl Acad Sci U S A. 2004; 101(31): 11338–11343. pmid:15277678.
  84. 84. Vyas M, Fisher TW, He R, Nelson W, Yin G, Cicero JM, et al. Asian Citrus Psyllid Expression Profiles Suggest Candidatus Liberibacter Asiaticus-Mediated Alteration of Adult Nutrition and Metabolism, and of Nymphal Development and Immunity. Plos One. 2015; 10(6): e0130328. pmid:26091106.
  85. 85. Fonseca FP, Soares-Costa A, Ribeiro AF, Rosa JC, Terra WR, Henrique-Silva F. Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis. Insect Biochem Mol Biol. 2012; 42(1): 58–69. pmid:22100428.
  86. 86. Pelz-Stelinski KS, Brlansky RH, Ebert TA, Rogers ME. Transmission parameters for Candidatus liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae). J Econ Entomol. 2010; 103(5): 1531–1541. pmid:21061950.
  87. 87. Inoue H, Ohnishi J, Ito T, Tomimura K, Miyata S, Iwanami T, et al. Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage. Annals of Applied Biology. 2009; 155(1): 29–36.
  88. 88. Rispe C, Kutsukake M, Doublet V, Hudaverdian S, Legeai F, Simon JC, et al. Large gene family expansion and variable selective pressures for cathepsin B in aphids. Mol Biol Evol. 2008; 25(1): 5–17. pmid:17934209.
  89. 89. Noda H, Kawai S, Koizumi Y, Matsui K, Zhang Q, Furukawa S, et al. Annotated ESTs from various tissues of the brown planthopper Nilaparvata lugens: A genomic resource for studying agricultural pests. Bmc Genomics. 2008; 9: 117. pmid:PMC2311293.
  90. 90. Liu S, Ding Z, Zhang C, Yang B, Liu Z. Gene knockdown by intro-thoracic injection of double-stranded RNA in the brown planthopper, Nilaparvata lugens. Insect Biochem Mol Biol. 2010; 40(9): 666–671. pmid:20599616.
  91. 91. Ribeiro JMC, Genta FA, Sorgine MHF, Logullo R, Mesquita RD, Paiva-Silva GO, et al. An Insight into the Transcriptome of the Digestive Tract of the Bloodsucking Bug, Rhodnius prolixus. PLoS Neglected Tropical Diseases. 2014; 8(1): e2594. pmid:PMC3886914.