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

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 cont...

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Published in:PloS one 2015-12, Vol.10 (12), p.e0145132
Main Authors: Ferrara, Taíse Fernanda da Silva, Schneider, Vanessa Karine, Kishi, Luciano Takeshi, Carmona, Adriana Karaoglanovic, Alves, Marcio Fernando Madureira, Belasque-Júnior, Jose, Rosa, José César, Hunter, Wayne Brian, Henrique-Silva, Flávio, Soares-Costa, Andrea
Format: Article
Language:English
Subjects:
Adults
Amino Acid Sequence
Animals
Aphidoidea
Biocatalysis - drug effects
Biophysics
Candidatus Liberibacter asiaticus
Cathepsin B
Cathepsin B - chemistry
Cathepsin B - genetics
Cathepsin B - isolation & purification
Cathepsin B - metabolism
Cathepsins
Chemical properties
Citrus - parasitology
Citrus fruits
Cloning
Cysteine
Cysteine proteinase
Diaphorina citri
Disease
Disease control
Enzymes
Evolution
Gene expression
Gene Expression Regulation, Enzymologic - drug effects
Genomics
Hemiptera
Hemiptera - drug effects
Hemiptera - enzymology
Hemiptera - genetics
Hemiptera - growth & development
Hymenoptera
Insect control
Insects
Laboratories
Larva - genetics
Liviidae
Molecular biology
Molecular Sequence Data
Mortality
Nilaparvata lugens
Peptidase
Peptidases
Peptides - metabolism
Pichia pastoris
Plant diseases
Plant Diseases - parasitology
Plant Diseases - prevention & control
Protease inhibitors
Protease Inhibitors - pharmacology
Proteinase inhibitors
Real-Time Polymerase Chain Reaction
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Sequence Alignment
Sequence Analysis, Protein
Substrates
Sugarcane
Tandem Mass Spectrometry
Thiols
Tissues
Transgenic plants
Trypsin - metabolism
Yeast
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0145132