Cloning and expression of an endo-1,4-β-xylanase from the coffee berry borer, Hypothenemus hampei
- Beatriz Padilla-Hurtado†1,
- Claudia Flórez-Ramos†2,
- Carolina Aguilera-Gálvez1,
- Jefferson Medina-Olaya1,
- Andrés Ramírez-Sanjuan1,
- José Rubio-Gómez1 and
- Ricardo Acuña-Zornosa1Email author
© Padilla-Hurtado et al; licensee BioMed Central Ltd. 2011
Received: 6 October 2011
Accepted: 10 January 2012
Published: 10 January 2012
The coffee berry borer, Hypothenemus hampei, reproduces and feeds exclusively on the mature endosperm of the coffee seed, which has a cell wall composed mainly of a heterogeneous mixture of hemicellulose polysaccharides, including arabinoxylans. Xylanases are digestive enzymes responsible for the degradation of xylan based polymers, hydrolyzing them into smaller molecules that are easier to assimilate by insects. We report the cloning, expression and enzymatic characterization of a xylanase gene that was identified in the digestive tract of the coffee berry borer.
The complete DNA sequence encoding a H. hampei xylanase (HhXyl) was obtained using a genome walking technique in a cDNA library derived from the borer digestive tract. The XIP-I gene was amplified from wheat (Triticum aestivum variety Soisson). A Pichia pastoris expression system was used to express the recombinant form of these enzymes. The xylanase activity and XIP-I inhibitory activity was quantified by the 3,5-dinitrosalicylic (DNS). The biological effects of XIP-I on borer individuals were evaluated by providing an artificial diet enriched with the recombinant XIP-I protein to the insects.
The borer xylanase sequence contains a 951 bp open reading frame that is predicted to encode a 317-amino acid protein, with an estimated molecular weight of 34.92 kDa and a pI of 4.84. Bioinformatic analysis revealed that HhXyl exhibits high sequence homology with endo-β-D-xylanases of Streptomyces bingchenggensis from glycosyl hydrolase 10 (GH10). The recombinant xylanase showed maximal activity at pH 5.5 and 37°C. XIP-I expressed as a recombinant protein inhibited HhXyl activity in vitro and caused individual H. hampei mortality in bioassays when included as a supplement in artificial diets.
A xylanase from the digestive tract of the coffee berry borer was identified and functionally characterized. A xylanase inhibitor protein, XIP-I, from wheat was shown to be a potent inhibitor of this xylanase, suggesting that its deployment has potential as a strategy to control coffee berry borer colonization of coffee plants.
The coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae), is the most important pest of coffee crops worldwide. It causes annual economic losses close to $500 million due to loss of seed weight and premature fall of the bean . In Colombia, it is estimated that borer infestation affects 800,000 acres, affecting the assets of more than half a million coffee growers .
The insect feeds exclusively on the fruit and reproduces inside the coffee seed. Carbohydrates, in the form of polysaccharides and free sugars, comprise 50% of the dry weight of the mature seed of the coffee bean. The polysaccharides include galactomannans (25%), arabinogalactans (17%) and cellulose (8%) . However, other polysaccharides are likely present, including xyloglucan and arabinoxylan, and indeed, acid hydrolysis of seed carbohydrates releases small amounts of xylose .
In order to degrade these polysaccharides, the coffee berry borer would need to secrete a range of glycosyl hydrolases (GHs) into its digestive system. Here we report the first characterization of an endo-β-1,4-xylanase (HhXyl) from the digestive tract of the coffee berry borer. Endo-β-1,4-xylanases (EC 126.96.36.199) catalyze the hydrolysis of the β-1,4-xylosidic bonds in the backbone of xylan based polymers . Most of these enzymes are classified in GH families 10 and 11 based on similarities in the amino acid sequences, catalytic domains, protein folds and overall architecture . Recently, the study of xylanolytic enzymes has been an area of great interest due to their numerous industrial applications [7, 8] and their roles as pathogenicity factors in plant pathogenic microbes .
The complete HhXyl transcript sequence was determined from a digestive tract cDNA library, and its homology with endo-β-1,4-xylanase genes from bacteria of the genus Streptomyces was established. Recombinant HhXyl protein was expressed in a Pichia pastoris system to analyze its enzymatic activity. In addition a xylanase inhibitor protein (XIP-I) from wheat (Triticum aestivum variety Soisson) that inhibits endoxylanases, was cloned and similarly expressed . XIP-I inhibits the microbial GH10 and GH11 families of xylanases in a competitive manner  as well as two α-amylases from barley . The inhibitory activity of recombinant XIP-I against the borer xylanase was evaluated in biochemical and biological assays.
Given their functions as digestive enzymes, the identification and biochemical characterization of glycolytic enzymes in H. hampei represents an important step in designing strategies to improve resistance against the insect. Specific protein inhibitors of these enzymes are sought for use in biotechnological strategies and to generate coffee plants that are resistant to this pest.
Results and Discussion
Isolation and cloning of the xylanase gene from the coffee berry borer
Characterization of the HhXyl xylanase from coffee berry borer
Effect of XIP-I on HhXyl
Vasconcelos et al (2011), reported the role of a xylanase inhibitor in the germination of spores of Phakopsora pachyrhizi. The effect was observed in bioassays in the absence of xylan in the reaction. This suggests that such inhibitors can have important biological effects in different processes in which other glycosyl hydrolases are involved, such as cellulases and mannanases . Further analysis is required to elucidate the mechanism of action.
Another possible interpretation of these data is that XIP-I may inhibit glycosyl hydrolases other than xylanases and have consequent effects on the insect. Two plant proteins named XAIP and XAIP-II, with significant sequence identity and considerable structural similarity to XIP-I, have recently been shown to bind specifically to two structurally very different hydrolases from different GH families (GH11 xylanase and GH13 α-amylase), resulting in inhibition of enzymatic activities [20, 21].
XIP-I inhibits xylanases of the GH10 and GH11 families of fungal, but not bacterial origin in a reversible and competitive way. The exact nature of XIP-I biological activity is still unknown, although it is speculated to play an important role in plant defense against pathogens, and particularly those of fungal origin, because it lacks endogenous inhibitory activity against wheat xylanases [9, 10, 22, 23]. Microbial GH enzymes are important for plant tissue invasion and degradation and so inhibitors of these enzymes are thought to play important roles in defense. It has been demonstrated that this type of inhibitor can be induced by pathogens as XIP-I is expressed strongly after inoculation with the fungus Erysiphe graminis and, in contrast to other inhibitors, XIP-I expression varies with the type of pathogen and the infected tissue .
In this study a functional xylanase gene (HhXyl) from the digestive tracts of the coffee berry borer was cloned and expressed as a recombinant protein in a P. pastoris system. The enzyme has a predicted molecular mass of 34.92 kDa and contains a putative domain conserved in GH10 proteins. The enzyme showed optimal activity in similar conditions to those of the physiological medium in the borer's digestive system and was able to effectively depolymerize xylan. The xylanase inhibitor, XIP-I, from wheat was shown to have inhibitory activity against both the recombinant HhXyl enzyme and crude protein extracts from borer digestive tracts. In addition, biological assays using XIP-I supplements to artificial diets induced mortality in borer individuals. Since xylan is not a quantitatively major component of coffee beans, two possible underlying mechanisms are suggested: i) XIP-I inhibition of xylanases may influence the action of other glycosyl hydrolases in the process of digesting the major coffee bean polysaccharides that constitute the diet of the insect. ii) XIP-I may inhibit other non-xylanase proteins that are important for insect metabolism, these results indicate that use of xylanase inhibitors may provide a strategy to reducing losses caused by the coffee berry borer.
Isolation and preservation of coffee berry borer digestive tracts
H. hampei larvae were grown in dry parchment coffee with a grain humidity of 45%, at an average temperature of 27°C and 75% relative humidity in the dark. Prior to extracting the digestive tracts, larvae were incubated for 10 min at 4°C and then placed in a drop of phosphate-buffered saline (PBS), pH 7.2. For proteomic analysis, the tracts were stored in chilled PBS (pH 7.2) containing 5 mM PMSF (phenylmethylsulfonyl fluoride). Tissues used for mRNA isolation were preserved in 0.1% RNALater™ (Qiagen, Valencia, CA, USA) at -80°C.
Complementary DNA (cDNA) library
Total RNA was extracted from 100 mg of H. hampei larvae digestive tracts with the RNeasy™ kit (Qiagen, Valencia, CA, USA). mRNA was isolated using an Oligotex mRNA Mini Kit (Qiagen, Valencia, CA, USA), following the manufacturer's instructions. The directional cDNA library of the borer was created with the Creator™ SMART™ Library Construction Kit (Clontech, Mountain View, CA, USA). BLAST search of the GenBank database was performed using the processed cDNA sequences, which were compared to all available ESTs and genes (http://www.ncbi.nlm.nih.gov/blast). A BLASTX results with a bit scores greater than 80 and an E-values of less than 10-4 was regarded as a significant match.
Amplification and cloning of the coffee berry borer xylanase gene and XIP-I
Primers used in genome walking PCR
Heterologous expression of the P. pastoris system
The coding sequences for the endo-1,4-β-xylanase (HhXyl) and XIP-I proteins were cloned, without their respective signal sequences, into the pPICZαA vector (EasySelect™ Pichia Expression Kit, Invitrogen) generating the constructs constructs pPICZαA-XIL and pPICZαA-XIP-I respectively. The HhXyl gene was amplified with the forward primer XILHH_FW1_PIC 5'CCGCTCGAG AAAAGACACTTCAAAGACCATGCCAA-3' and the reverse primer XILHH_RV1_PIC 5'-TGCTCTAGA CCCATTAATTGCAAGGCAGCTT-3'and the XIP I gene was amplified with the forward primer XIP1_FW_PIC 5'- CCGCTCGAG AAAAGAATGGCGCCGCTCGCAGCCCGGAG-3'and the reverse primer XIP1_RV_PIC 5'TGCTCTAGA CCGGCGTAGTACTTGATCAAGCTG-3'. Forward primers contain the XhoI restriction site (underlined) and reverse primers XbaI. The PCR conditions were 10 cycles at 95°C for 1 min, 95°C for 25 sec, 59°C for 45 sec and a final extension of 72°C for 30 sec and 30 cycles of 95°C for 25 sec, 56°C for 45 sec, 72°C of 30 sec and a final extension of 72°C for 10 min, respectively. E. coli One Shot Top 10 cells (Invitrogen) were transformed with the constructs pPICZαA-XIL and pPICZαA-XIP-I. Positive clones were selected in LB media containing zeocin (25 μg/ml). The constructs were verified by restriction pattern analysis and sequencing. The GS115 strain of P. pastoris was transformed by electroporation at 2.0 kV, 25 μF and 200 Ω using 5 μg of recombinant plasmids that were linearized with SacI. After three days of growth ak 30°C, the positive colonies were selected on YPDS plates (1% yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol, 2% agar and 100 mg/L zeocin). Integration of the HhXyl and XIP-I genes into the P. pastoris genome was confirmed by PCR using the AOX1 forward (5'-GACTGGTTCCAATTGACAAGC-3') and AOX1 reverse primers (5'-GCAAATGGCATTCTGACATCC-3').
Transformed P. pastoris cells were inoculated into 10 ml of buffered minimal glycerol complex media BMGY (1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer [pH 6.0], 1.34% yeast nitrogen base (YNB), 0.0004% of biotin and 1% (v/v) glycerol) at 30°C and were shaken 250 rpm, until an OD600 between 3.0 and 6.0 was achieved. To induce HhXyl and XIP-I production, P. pastoris cell pellets were grown in BMMY (1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer [pH 6.0], 1.34% YNB, 0.0004% biotin and 0.5% methanol) using 1/5 of the original culture media (10 ml). The cultures were incubated at 30°C and with shaking at 250 rpm. Absolute methanol was added to a final concentration of 0.5% every 24 hours for 5 days. The expression of the secreted proteins was evaluated by SDS-PAGE.
Purification of the recombinant proteins
The recombinant proteins expressed in P. pastoris were purified by affinity chromatography using Ni-NTA resin (Qiagen, Valencia, CA, USA). The proteins that bound to the resin due to their tag of six-histidines N-terminal tag end were eluted with imidazole (500 mM). The eluate fractions were concentrated by ultrafiltration using an Amicon ultra-0.5, 3 kDa® centrifugal filter (Millipore, Bedford, MA) and the concentrated samples were analyzed by SDS-PAGE. Protein concentrations were determined using the Bradford method and bovine serum albumin was used as a standard .
Enzymatic activity assay
A total of 10 μl of HhXyl (0.5 μg/μl) was added to 490 μl of 200 mM of acetate buffer, pH 5.0, containing 4% (w/v) xylan (Sigma X0627). In addition, 10 μl of the digestive tracts extracts (0.5 μg/μl) was evaluated for xylanase activity. After incubation at 37°C for 1 hour, the amount of reduced sugars released by the reactions was quantified by the 3,5-dinitrosalicylic (DNS) method. For each 100 μl of reaction mixture, 100 μl of the Bernfeld reagent was added. The mixture was heated to boiling point, was boiled for 5 min, and the absorbance was measured at 540 nm. Xylose was used as a standard . One unit of xylanase activity is defined as the amount of enzyme releasing 1 μmole of xylose equivalents per minute. All measurements were performed in triplicate.
The effects of pH and temperature on enzymatic activity
To evaluate the effect of pH on xylanase activity, 4% (w/v) xylan from oat spelts (Sigma-Aldrich, St. Louis, MO, USA) were prepared in acetate buffer (pH 3.0 to 5.0) and phosphate buffer (pH 6.0 to 8.0). Each substrate solution was incubated with 10 μl HhXyl solution (0.5 μg/μl) at 37°C for one hour. To evaluate the effect of temperature, the activity assay was performed at pH 5.5 and at temperature ranging from 20 to 90°C. The thermostability of the enzyme was determined by measuring xylanase activity at 37°C for one hour after preincubating the enzyme for 10, 20, 30, 60 or 120 min at 30°C, 37°C, 40°C or 50°C. The xylanase activity measured prior to the preincubation at the different temperatures was considered to be 100%.
Zymogram analysis of xylanase activity
Aliquots (2.5 and 5 μg) of HhXyl enzyme were analyzed by native PAGE (12%), according to the method described by Laemmli . The protein bands were visualized with Coomassie stain. For the zymogram analysis, 2% (w/v) xylan was incorporated into the gel and electrophoresis was performed at 4°C. To evaluate the xylanase activity, the gel was incubated at pH 5.0 for one hour at 37°C. The enzymatic activity was visualized by incubating the gel in Congo red dye (0.25%) for 15 min, followed by successive washes with 1 M NaCl. To intensify the color, the gel was placed in 5% acetic acid.
The effect of XIP-I protein on xylanase enzymatic activity
The biological effects of XIP-I on the coffee berry borer were evaluated by providing the insects with a Cenibroca artificial diet , that was enriched with the recombinant XIP-I protein. Multiwell plates were used to provide 2 ml of food that was supplemented with 30 or 60 μg of XIP-I protein or distilled water. The borer eggs were extracted from the parchment coffee beans that were infested for 10 days, and 20 eggs were placed in each well until hatching. During the first instar larval stage (L1), a drop of the XIP-I solution or distilled water (control) was applied to the preoral cavity of each larva. The diet was maintained under controlled conditions at 27°C and 75% humidity. Each evaluation was performed in duplicate. The development of the larvae was followed daily until they reached the pupal stage when the effect of the diet on their mortality was evaluated. The mortality rate was calculated and was corrected relative to the control in accordance with Abbott's formula .
This work was supported by the Ministry of Agriculture and Rural Development of Colombia and National Federation of Coffee Growers of Colombia (contract # 074/2007), the authors thank the Bioinformatics Unit of CENICAFÉ for its contributions to the sequence analysis and Jocelyn Rose from Department of Plant Biology, Cornell University for reviewing the manuscript and for his valuable contributions.
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