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1.  Cloning and expression of an endo-1,4-β-xylanase from the coffee berry borer, Hypothenemus hampei 
BMC Research Notes  2012;5:23.
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1756-0500-5-23
PMCID: PMC3283504  PMID: 22233686
2.  Cloning and biochemical characterization of an endo-1,4-β-mannanase from the coffee berry borer hypothenemus hampei 
BMC Research Notes  2013;6:333.
Background
The study of coffee polysaccharides-degrading enzymes from the coffee berry borer Hypothenemus hampei, has become an important alternative in the identification for enzymatic inhibitors that can be used as an alternative control of this dangerous insect. We report the cloning, expression and biochemical characterization of a mannanase gene that was identified in the midgut of the coffee berry borer and is responsible for the degradation of the most abundant polysaccharide in the coffee bean.
Methods
The amino acid sequence of HhMan was analyzed by multiple sequence alignment comparisons with BLAST (Basic Local Alignment Search Tool) and CLUSTALW. A Pichia pastoris expression system was used to express the recombinant form of the enzyme. The mannanase activity was quantified by the 3,5-dinitrosalicylic (DNS) and the hydrolitic properties were detected by TLC.
Results
An endo-1,4-β-mannanase from the digestive tract of the insect Hypothenemus hampei was cloned and expressed as a recombinant protein in the Pichia pastoris system. This enzyme is 56% identical to the sequence of an endo-β-mannanase from Bacillus circulans that belongs to the glycosyl hydrolase 5 (GH5) family. The purified recombinant protein (rHhMan) exhibited a single band (35.5 kDa) by SDS-PAGE, and its activity was confirmed by zymography. rHhMan displays optimal activity levels at pH 5.5 and 30°C and can hydrolyze galactomannans of varying mannose:galactose ratios, suggesting that the enzymatic activity is independent of the presence of side chains such as galactose residues. The enzyme cannot hydrolyze manno-oligosaccharides such as mannobiose and mannotriose; however, it can degrade mannotetraose, likely through a transglycosylation reaction. The Km and kcat values of this enzyme on guar gum were 2.074 mg ml-1 and 50.87 s-1, respectively, which is similar to other mannanases.
Conclusion
This work is the first study of an endo-1,4-β-mannanase from an insect using this expression system. Due to this enzyme’s importance in the digestive processes of the coffee berry borer, this study may enable the design of inhibitors against endo-1,4-β-mannanase to decrease the economic losses stemming from this insect.
doi:10.1186/1756-0500-6-333
PMCID: PMC3765340  PMID: 23965285
Endo-mannanase; Coffee berry borer; Hypotenemus hampei; Glycosyl hydrolase
3.  α-Amylase inhibitor-1 gene from Phaseolus vulgaris expressed in Coffea arabica plants inhibits α-amylases from the coffee berry borer pest 
BMC Biotechnology  2010;10:44.
Background
Coffee is an important crop and is crucial to the economy of many developing countries, generating around US$70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US$500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an α-amylase inhibitor gene (α-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.
Results
We transformed C. arabica with the α-amylase inhibitor-1 gene (α-AI1) from the common bean, Phaseolus vulgaris, under control of the seed-specific phytohemagglutinin promoter (PHA-L). The presence of the α-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting. Immunoblotting and ELISA experiments using antibodies against α-AI1 inhibitor showed a maximum α-AI1 concentration of 0.29% in crude seed extracts. Inhibitory in vitro assays of the α-AI1 protein against H. hampei α-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity.
Conclusions
This is the first report showing the production of transgenic coffee plants with the biotechnological potential to control the coffee berry borer, the most important insect-pest of crop coffee.
doi:10.1186/1472-6750-10-44
PMCID: PMC2914071  PMID: 20565807
4.  Spiroacetals in the Colonization Behaviour of the Coffee Berry Borer: A ‘Push-Pull’ System 
PLoS ONE  2014;9(11):e111316.
Coffee berries are known to release several volatile organic compounds, among which is the spiroacetal, conophthorin, an attractant for the coffee berry borer Hypothenemus hampei. Elucidating the effects of other spiroacetals released by coffee berries is critical to understanding their chemo-ecological roles in the host discrimination and colonization process of the coffee berry borer, and also for their potential use in the management of this pest. Here, we show that the coffee berry spiroacetals frontalin and 1,6-dioxaspiro [4.5] decane (referred thereafter as brocain), are also used as semiochemicals by the coffee berry borer for host colonization. Bioassays and chemical analyses showed that crowding coffee berry borers from 2 to 6 females per berry, reduced borer fecundity, which appeared to correlate with a decrease in the emission rates of conophthorin and frontalin over time. In contrast, the level of brocain did not vary significantly between borer- uninfested and infested berries. Brocain was attractive at lower doses, but repellent at higher doses while frontalin alone or in a blend was critical for avoidance. Field assays with a commercial attractant comprising a mixture of ethanol and methanol (1∶1), combined with frontalin, confirmed the repellent effect of this compound by disrupting capture rates of H. hampei females by 77% in a coffee plantation. Overall, our results suggest that the levels of frontalin and conophthorin released by coffee berries determine the host colonization behaviour of H. hampei, possibly through a ‘push-pull’ system, whereby frontalin acts as the ‘push’ (repellent) and conophthorin acting as the ‘pull’ (attractant). Furthermore, our results reveal the potential use of frontalin as a repellent for management of this coffee pest.
doi:10.1371/journal.pone.0111316
PMCID: PMC4224388  PMID: 25380135
5.  Annotation of a hybrid partial genome of the coffee rust (Hemileia vastatrix) contributes to the gene repertoire catalog of the Pucciniales 
Coffee leaf rust caused by the fungus Hemileia vastatrix is the most damaging disease to coffee worldwide. The pathogen has recently appeared in multiple outbreaks in coffee producing countries resulting in significant yield losses and increases in costs related to its control. New races/isolates are constantly emerging as evidenced by the presence of the fungus in plants that were previously resistant. Genomic studies are opening new avenues for the study of the evolution of pathogens, the detailed description of plant-pathogen interactions and the development of molecular techniques for the identification of individual isolates. For this purpose we sequenced 8 different H. vastatrix isolates using NGS technologies and gathered partial genome assemblies due to the large repetitive content in the coffee rust hybrid genome; 74.4% of the assembled contigs harbor repetitive sequences. A hybrid assembly of 333 Mb was built based on the 8 isolates; this assembly was used for subsequent analyses. Analysis of the conserved gene space showed that the hybrid H. vastatrix genome, though highly fragmented, had a satisfactory level of completion with 91.94% of core protein-coding orthologous genes present. RNA-Seq from urediniospores was used to guide the de novo annotation of the H. vastatrix gene complement. In total, 14,445 genes organized in 3921 families were uncovered; a considerable proportion of the predicted proteins (73.8%) were homologous to other Pucciniales species genomes. Several gene families related to the fungal lifestyle were identified, particularly 483 predicted secreted proteins that represent candidate effector genes and will provide interesting hints to decipher virulence in the coffee rust fungus. The genome sequence of Hva will serve as a template to understand the molecular mechanisms used by this fungus to attack the coffee plant, to study the diversity of this species and for the development of molecular markers to distinguish races/isolates.
doi:10.3389/fpls.2014.00594
PMCID: PMC4215621  PMID: 25400655
genome; coffee rust; coffee; plant pathogens diversity; RNA-seq; genetic variants
6.  New Record for the Coffee Berry Borer, Hypothenemus hampei, in Hawaii 
The coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) is endemic to Africa and is the most devastating pest of coffee worldwide. The female bores a hole in the coffee berry and deposits her eggs inside. Upon hatching, larvae feed on the seeds, thus reducing both quality and yields of the marketable product. The coffee berry borer was found in the district of Kona on the island of Hawaii in August 2010 and appears to be restricted to that area.
doi:10.1673/031.011.11701
PMCID: PMC3281356  PMID: 22225430
bark beetle; broca; Scolytinae
7.  Some Like It Hot: The Influence and Implications of Climate Change on Coffee Berry Borer (Hypothenemus hampei) and Coffee Production in East Africa 
PLoS ONE  2011;6(9):e24528.
The negative effects of climate change are already evident for many of the 25 million coffee farmers across the tropics and the 90 billion dollar (US) coffee industry. The coffee berry borer (Hypothenemus hampei), the most important pest of coffee worldwide, has already benefited from the temperature rise in East Africa: increased damage to coffee crops and expansion in its distribution range have been reported. In order to anticipate threats and prioritize management actions for H. hampei we present here, maps on future distributions of H. hampei in coffee producing areas of East Africa. Using the CLIMEX model we relate present-day insect distributions to current climate and then project the fitted climatic envelopes under future scenarios A2A and B2B (for HADCM3 model). In both scenarios, the situation with H. hampei is forecasted to worsen in the current Coffea arabica producing areas of Ethiopia, the Ugandan part of the Lake Victoria and Mt. Elgon regions, Mt. Kenya and the Kenyan side of Mt. Elgon, and most of Rwanda and Burundi. The calculated hypothetical number of generations per year of H. hampei is predicted to increase in all C. arabica-producing areas from five to ten. These outcomes will have serious implications for C. arabica production and livelihoods in East Africa. We suggest that the best way to adapt to a rise of temperatures in coffee plantations could be via the introduction of shade trees in sun grown plantations. The aims of this study are to fill knowledge gaps existing in the coffee industry, and to draft an outline for the development of an adaptation strategy package for climate change on coffee production. An abstract in Spanish is provided as Abstract S1.
doi:10.1371/journal.pone.0024528
PMCID: PMC3173381  PMID: 21935419
8.  On the Eyes of Male Coffee Berry Borers as Rudimentary Organs 
PLoS ONE  2014;9(1):e85860.
The coffee berry borer, Hypothenemus hampei, is the most damaging insect pest of coffee worldwide. Like males in other species in the genus, male coffee berry borers have a lower number of facets in the compound eyes than females. The rudimentary eyes in male coffee berry borers could be an evolutionary response to their cryptic life habit, whereby they are born inside a coffee berry and never leave the berry. The main objective of the study was to determine if the differences in the number of facets translates into differences in visual acuity. We used low-temperature scanning electron microscopy to visualize and quantify the number of facets in the compound eyes. There was a significantly lower (p<0.0001) number of facets in males (19.1±4.10) than in females (127.5±3.88). To assess visual acuity, we conducted optomotor response experiments, which indicate that females respond to movement, while males did not respond under the conditions tested. The coffee berry borer is an example of an insect whereby disuse of an organ has led to a rudimentary compound eye. This is the first study that has experimentally tested responses to movement in bark beetles.
doi:10.1371/journal.pone.0085860
PMCID: PMC3896430  PMID: 24465752
9.  Overview of the functional virulent genome of the coffee leaf rust pathogen Hemileia vastatrix with an emphasis on early stages of infection 
Hemileia vastatrix is the causal agent of coffee leaf rust, the most important disease of coffee Arabica. In this work, a 454-pyrosequencing transcriptome analysis of H. vastatrix germinating urediniospores (gU) and appressoria (Ap) was performed and compared to previously published in planta haustoria-rich (H) data. A total of 9234 transcripts were identified and annotated. Ca. 50% of these transcripts showed no significant homology to international databases. Only 784 sequences were shared by the three conditions, and 75% were exclusive of either gU (2146), Ap (1479) or H (3270). Relative transcript abundance and RT-qPCR analyses for a selection of genes indicated a particularly active metabolism, translational activity and production of new structures in the appressoria and intense signaling, transport, secretory activity and cellular multiplication in the germinating urediniospores, suggesting the onset of a plant-fungus dialogue as early as at the germ tube stage. Gene expression related to the production of carbohydrate-active enzymes and accumulation of glycerol in germinating urediniospores and appressoria suggests that combined lytic and physical mechanisms are involved in appressoria-mediated penetration. Besides contributing to the characterization of molecular processes leading to appressoria-mediated infection by rust fungi, these results point toward the identification of new H. vastatrix candidate virulence factors, with 516 genes predicted to encode secreted proteins.
doi:10.3389/fpls.2014.00088
PMCID: PMC3953675  PMID: 24672531
appressorium; coffee leaf rust; germinating urediniospore; haustorium; pyrosequencing; transcriptome
10.  Identification and characterization of the Non-race specific Disease Resistance 1 (NDR1) orthologous protein in coffee 
BMC Plant Biology  2011;11:144.
Background
Leaf rust, which is caused by the fungus Hemileia vastatrix (Pucciniales), is a devastating disease that affects coffee plants (Coffea arabica L.). Disadvantages that are associated with currently developed phytoprotection approaches have recently led to the search for alternative strategies. These include genetic manipulations that constitutively activate disease resistance signaling pathways. However, molecular actors of such pathways still remain unknown in C. arabica. In this study, we have isolated and characterized the coffee NDR1 gene, whose Arabidopsis ortholog is a well-known master regulator of the hypersensitive response that is dependent on coiled-coil type R-proteins.
Results
Two highly homologous cDNAs coding for putative NDR1 proteins were identified and cloned from leaves of coffee plants. One of the candidate coding sequences was then expressed in the Arabidopsis knock-out null mutant ndr1-1. Upon a challenge with a specific strain of the bacterium Pseudomonas syringae (DC3000::AvrRpt2), analysis of both macroscopic symptoms and in planta microbial growth showed that the coffee cDNA was able to restore the resistance phenotype in the mutant genetic background. Thus, the cDNA was dubbed CaNDR1a (standing for Coffea arabica Non-race specific Disease Resistance 1a). Finally, biochemical and microscopy data were obtained that strongly suggest the mechanistic conservation of the NDR1-driven function within coffee and Arabidopsis plants. Using a transient expression system, it was indeed shown that the CaNDR1a protein, like its Arabidopsis counterpart, is localized to the plasma membrane, where it is possibly tethered by means of a GPI anchor.
Conclusions
Our data provide molecular and genetic evidence for the identification of a novel functional NDR1 homolog in plants. As a key regulator initiating hypersensitive signalling pathways, CaNDR1 gene(s) might be target(s) of choice for manipulating the coffee innate immune system and achieving broad spectrum resistance to pathogens. Given the potential conservation of NDR1-dependent defense mechanisms between Arabidopsis and coffee plants, our work also suggests new ways to isolate the as-yet-unidentified R-gene(s) responsible for resistance to H. vastatrix.
doi:10.1186/1471-2229-11-144
PMCID: PMC3212813  PMID: 22023696
11.  Thermal Tolerance of the Coffee Berry Borer Hypothenemus hampei: Predictions of Climate Change Impact on a Tropical Insect Pest 
PLoS ONE  2009;4(8):e6487.
Coffee is predicted to be severely affected by climate change. We determined the thermal tolerance of the coffee berry borer , Hypothenemus hampei, the most devastating pest of coffee worldwide, and make inferences on the possible effects of climate change using climatic data from Colombia, Kenya, Tanzania, and Ethiopia. For this, the effect of eight temperature regimes (15, 20, 23, 25, 27, 30, 33 and 35°C) on the bionomics of H. hampei was studied. Successful egg to adult development occurred between 20–30°C. Using linear regression and a modified Logan model, the lower and upper thresholds for development were estimated at 14.9 and 32°C, respectively. In Kenya and Colombia, the number of pest generations per year was considerably and positively correlated with the warming tolerance. Analysing 32 years of climatic data from Jimma (Ethiopia) revealed that before 1984 it was too cold for H. hampei to complete even one generation per year, but thereafter, because of rising temperatures in the area, 1–2 generations per year/coffee season could be completed. Calculated data on warming tolerance and thermal safety margins of H. hampei for the three East African locations showed considerably high variability compared to the Colombian site. The model indicates that for every 1°C rise in thermal optimum (Topt.), the maximum intrinsic rate of increase (rmax) will increase by an average of 8.5%. The effects of climate change on the further range of H. hampei distribution and possible adaption strategies are discussed. Abstracts in Spanish and French are provided as supplementary material Abstract S1 and Abstract S2.
doi:10.1371/journal.pone.0006487
PMCID: PMC2715104  PMID: 19649255
12.  Cryptosexuality and the Genetic Diversity Paradox in Coffee Rust, Hemileia vastatrix 
PLoS ONE  2011;6(11):e26387.
Background
Despite the fact that coffee rust was first investigated scientifically more than a century ago, and that the disease is one of the major constraints to coffee production - constantly changing the socio-economic and historical landscape of the crop - critical aspects of the life cycle of the pathogen, Hemileia vastatrix, remain unclear. The asexual urediniospores are regarded as the only functional propagule: theoretically, making H. vastatrix a clonal species. However, the well-documented emergence of new rust pathotypes and the breakdown in genetic resistance of coffee cultivars, present a paradox.
Methods and Results
Here, using computer-assisted DNA image cytometry, following a modified nuclear stoichiometric staining technique with Feulgen, we show that meiosis occurs within the urediniospores. Stages of spore development were categorised based on morphology, from the spore-mother cell through to the germinating spore, and the relative nuclear DNA content was quantified statistically at each stage.
Conclusions
Hidden sexual reproduction disguised within the asexual spore (cryptosexuality) could explain why new physiological races have arisen so often and so quickly in Hemileia vastatrix. This could have considerable implications for coffee breeding strategies and may be a common event in rust fungi, especially in related genera occupying the same basal phylogenetic lineages.
doi:10.1371/journal.pone.0026387
PMCID: PMC3216932  PMID: 22102860
13.  Production of Beauveria bassiana Fungal Spores on Rice to Control the Coffee Berry Borer, Hypothenemus hampei, in Colombia 
Two isolates of fungal entomopathogen Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) were grown on cooked rice using diphasic liquid-solid fermentation in plastic bags to produce and harvest spore powder. The cultures were dried and significant differences were found for isolates and time of harvest. The spores were harvested manually and mechanically and after the cultures were dried for nine days, when moisture content was near 10%. After harvesting, spores were submitted to quality control to assess concentration, germination, purity, moisture content, particle size and pathogenicity to the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae). Spore productivity on cooked rice was less than 1×1010 spores/g using both manually and mechanically harvesting methodologies. Germination at 24 hours was over 75% and pathogenicity against H. hampei was over 92.5%. This methodology is suitable for laboratory and field studies, but not for industrial production when a high concentration of spores are required for formulation and field applications.
doi:10.1673/031.008.4101
PMCID: PMC3127422
spore harvesting; germination; pathogenicity; broca
14.  Molecular diagnosis of bird-mediated pest consumption in tropical farmland 
SpringerPlus  2014;3:630.
Biodiversity loss will likely have surprising and dramatic consequences for human wellbeing. Identifying species that benefit society represents a critical first step towards predicting the consequences of biodiversity loss. Though natural predators prevent billions of dollars in agricultural pest damage annually, characterizing which predators consume pests has proven challenging. Emerging molecular techniques may illuminate these interactions. In the countryside of Costa Rica, we identified avian predators of coffee’s most damaging insect pest, the coffee berry borer beetle (Coleoptera:Scolytidae Hypothenemus hampeii), by assaying 1430 fecal samples of 108 bird species for borer DNA. While feeding trials confirmed the efficacy of our approach, detection rates were low. Nevertheless, we identified six species that consume the borer. These species had narrow diet breadths, thin bills, and short wings; traits shared with borer predators in other systems. Borer predators were not threatened; therefore, safeguarding pest control necessitates managing species beyond those at risk of regional extinction by maintaining populations in farmland habitats. Generally, our results demonstrate potential for pairing molecular methods with ecological analyses to yield novel insights into species interactions.
Electronic supplementary material
The online version of this article (doi:10.1186/2193-1801-3-630) contains supplementary material, which is available to authorized users.
doi:10.1186/2193-1801-3-630
PMCID: PMC4216319  PMID: 25392800
Agriculture; Avian; Coffee berry borer beetle; DNA barcoding; Molecular scatology
15.  Climate Change or Urbanization? Impacts on a Traditional Coffee Production System in East Africa over the Last 80 Years 
PLoS ONE  2013;8(1):e51815.
Global environmental changes (GEC) such as climate change (CC) and climate variability have serious impacts in the tropics, particularly in Africa. These are compounded by changes in land use/land cover, which in turn are driven mainly by economic and population growth, and urbanization. These factors create a feedback loop, which affects ecosystems and particularly ecosystem services, for example plant-insect interactions, and by consequence agricultural productivity. We studied effects of GEC at a local level, using a traditional coffee production area in greater Nairobi, Kenya. We chose coffee, the most valuable agricultural commodity worldwide, as it generates income for 100 million people, mainly in the developing world. Using the coffee berry borer, the most serious biotic threat to global coffee production, we show how environmental changes and different production systems (shaded and sun-grown coffee) can affect the crop. We combined detailed entomological assessments with historic climate records (from 1929–2011), and spatial and demographic data, to assess GEC's impact on coffee at a local scale. Additionally, we tested the utility of an adaptation strategy that is simple and easy to implement. Our results show that while interactions between CC and migration/urbanization, with its resultant landscape modifications, create a feedback loop whereby agroecosystems such as coffee are adversely affected, bio-diverse shaded coffee proved far more resilient and productive than coffee grown in monoculture, and was significantly less harmed by its insect pest. Thus, a relatively simple strategy such as shading coffee can tremendously improve resilience of agro-ecosystems, providing small-scale farmers in Africa with an easily implemented tool to safeguard their livelihoods in a changing climate.
doi:10.1371/journal.pone.0051815
PMCID: PMC3544928  PMID: 23341884
16.  Differential expression of maize chitinases in the presence or absence of Trichoderma harzianum strain T22 and indications of a novel exo- endo-heterodimeric chitinase activity 
BMC Plant Biology  2010;10:136.
Background
The interaction of plants with endophytic symbiotic fungi in the genus Trichoderma alters the plant proteome and transcriptome and results in enhanced plant growth and resistance to diseases. In a previous study, we identified the numerous chitinolytic enzyme families and individual enzymes in maize which are implicated in plant disease resistance and other plant responses.
Results
We examined the differential expression of the entire suite of chitinolytic enzymes in maize plants in the presence and absence of T. harzianum. Expression of these enzymes revealed a band of chitinolytic enzyme activity that had greater mass than any known chitinase. This study reports the characterization of this large protein. It was found to be a heretofore undiscovered heterodimer between an exo- and an endo-enzyme, and the endo portion differed between plants colonized with T. harzianum and those grown in its absence and between shoots and roots. The heterodimeric enzymes from shoots in the presence and absence of T. harzianum were purified and characterized. The dimeric enzyme from Trichoderma-inoculated plants had higher specific activity and greater ability to inhibit fungal growth than those from control plants. The activity of specific chitinolytic enzymes was higher in plants grown from Trichoderma treated seeds than in control plants.
Conclusions
This is the first report of a dimer between endo- and exochitinase. The endochitinase component of the dimer changed post Trichoderma inoculation. The dimer originating from Trichoderma inoculated plants had a higher antifungal activity than the comparable enzyme from control plants.
doi:10.1186/1471-2229-10-136
PMCID: PMC3017806  PMID: 20594307
17.  A new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) affects Soybean Asian rust (Phakopsora pachyrhizi) spore germination 
BMC Biotechnology  2011;11:14.
Background
Asian rust (Phakopsora pachyrhizi) is a common disease in Brazilian soybean fields and it is difficult to control. To identify a biochemical candidate with potential to combat this disease, a new chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP) leaves was cloned into the pGAPZα-B vector for expression in Pichia pastoris.
Results
A cDNA encoding a chitinase-like xylanase inhibitor protein (XIP) from coffee (Coffea arabica) (CaclXIP), was isolated from leaves. The amino acid sequence predicts a (β/α)8 topology common to Class III Chitinases (glycoside hydrolase family 18 proteins; GH18), and shares similarity with other GH18 members, although it lacks the glutamic acid residue essential for catalysis, which is replaced by glutamine. CaclXIP was expressed as a recombinant protein in Pichia pastoris. Enzymatic assay showed that purified recombinant CaclXIP had only residual chitinolytic activity. However, it inhibited xylanases from Acrophialophora nainiana by approx. 60% when present at 12:1 (w/w) enzyme:inhibitor ratio. Additionally, CaclXIP at 1.5 μg/μL inhibited the germination of spores of Phakopsora pachyrhizi by 45%.
Conclusions
Our data suggests that CaclXIP belongs to a class of naturally inactive chitinases that have evolved to act in plant cell defence as xylanase inhibitors. Its role on inhibiting germination of fungal spores makes it an eligible candidate gene for the control of Asian rust.
doi:10.1186/1472-6750-11-14
PMCID: PMC3045311  PMID: 21299880
18.  Cuticle-degrading proteases produced by the entomopathogenic fungus Beauveria bassiana in the presence of coffee berry borer cuticle 
Brazilian Journal of Microbiology  2008;39(2):301-306.
A Brazilian isolate of Beauveria bassiana (CG425) that shows high virulence against the coffee berry borer (CBB) was examined for the production of subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases. Fungal growth was either in nitrate-medium or in CBB cuticle-containing medium under both buffered and unbuffered conditions. In unbuffered medium supplemented with cuticle, the pH of cultures dropped and Pr1 and Pr2 activities were detected in high amounts only at a pH of 5.5 or higher. In buffered cultures, Pr1 and Pr2 activities were higher in medium supplemented with cuticle compared to activities with nitrate-medium. The Pr1 and Pr2 activities detected were mostly in the culture supernatant. These data suggest that Pr1 and Pr2 proteases produced by strain CG425 are induced by components of CBB cuticle, and that the culture pH influences the expression of these proteases, indicating the occurrence of an efficient mechanism of protein secretion in this fungus. The results obtained in this study extend the knowledge about protease production in B. bassiana CG425, opening new avenues for studying the role of secreted proteases in virulence against the coffee berry borer during the infection process.
doi:10.1590/S1517-838220080002000019
PMCID: PMC3768393  PMID: 24031220
subtilisin-like protease; trypsin-like protease; Hypothenemus hampei
19.  Chitinolytic enzymes of the rumen ciliate Eudiplodinium maggii 
Folia Microbiologica  2012;57(4):317-319.
The ability of rumen ciliates to digest chitin is clearly recognized. We investigated the chitinolytic system of the rumen ciliate Eudiplodinium maggii. The ciliates were grown in a selectively faunated sheep. They were isolated from the rumen and purified by sedimentation. A crude enzyme preparation was prepared following incubation of ciliates with antibiotics. This was done in order to reduce their contamination with intracellular bacteria. The activity of particular enzymes was examined by quantification of the products released from specific substrates. It was stated that the optimum conditions for the detected activities varied between 4.5 and 5.5 pH, and 45 and 55 °C. β-N-Acetylglucosaminidase was found as an enzyme of the highest activity (4.2 μmol/l released product per mg protein per h). The activities of endochitinase and exochitinase were almost two times lower than that of β-N-acetylglucosaminidase. Zymographic studies revealed the presence of two endochitinases, two exochitinases and two β-N-acetylglucosaminidases in the examined preparation.
doi:10.1007/s12223-012-0133-6
PMCID: PMC3389239  PMID: 22528307
20.  Studies on the Cloning and Expression of Bbchit1 Gene of Beauveria bassiana NCIM 1216 
Indian Journal of Microbiology  2011;51(3):396-402.
Beauveria bassiana is a biocontrol agent which shows entomopathogenecity on insect pests especially the Lepidopterons invading the agriculturally important crops. The mode of infection is through the cuticle by degrading the chitin present on it which is enabled by the exochitinase enzyme of Bbchit1 gene. A good quality genomic DNA was isolated from Beauveria bassiana NCIM 1216 and amplified with specific primers to isolate the gene corresponding to Bbchit1 which codes for the exochitinase enzyme that is responsible for pathogenesis. The Bbchit1 gene of B. bassiana was transformed with the binary plasmid pBANF-bar-pAN-Bbchit1, in which the Bbchit1 gene was placed downstream of the constitutive gpd promoter, which was mediated by A. tumefaciens, and transformants were selected on the basis of herbicide resistance. Fifty herbicide resistant colonies were obtained and analyzed. The exochitinase produced by these transformants was observed maximum on the 7th day of inoculation in both which was 0.09 μmol/ml/min for the purified fraction and 0.06 μmol/ml/min for the crude extract. The chitinolytic activity was observed maximum at pH 5 and at temperature of 40°C. The genetically modified pure form can be used in the production of transgenic plants and in bringing out commercial formulation for the control of Lepidopteran pests.
doi:10.1007/s12088-011-0116-3
PMCID: PMC3209929  PMID: 22754023
Beauveria bassiana; Bbchit1; Exochitinase
21.  A new method to evaluate the biocontrol potential of single spore isolates of fungal entomopathogens 
Fifty Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) strains isolated from the coffee berry borer were used to develop a novel screening method aimed at selecting strains with the highest biocontrol potential. The screening method is based on percent insect mortality, average survival time, mortality distribution, percent spore germination, fungal life cycle duration, and spore production on the insect. Based on these parameters, only 11 strains merited further study. The use of a sound scientific protocol for the selection of promising fungal entomopathogens should lead to more efficient use of time, labor, and financial resources in biological control programs.
PMCID: PMC1615244  PMID: 17119619
Hypothenemus hampei; coffee berry borer; broca; Beauveria; bioassays
22.  An acidic, thermostable exochitinase with β-N-acetylglucosaminidase activity from Paenibacillus barengoltzii converting chitin to N-acetyl glucosamine 
Biotechnology for Biofuels  2014;7(1):174.
Background
N-acetyl-β-D-glucosamine (GlcNAc) is widely used as a valuable pharmacological agent and a functional food additive. The traditional chemical process for GlcNAc production has some problems such as high production cost, low yield, and acidic pollution. Hence, to identify a novel chitinase that is suitable for bioconversion of chitin to GlcNAc is of great value.
Results
A novel chitinase gene (PbChi74) from Paenibacillus barengoltzii was cloned and heterologously expressed in Escherichia coli as an intracellular soluble protein. The gene has an open reading frame (ORF) of 2,163 bp encoding 720 amino acids. The recombinant chitinase (PbChi74) was purified to apparent homogeneity with a purification fold of 2.2 and a recovery yield of 57.9%. The molecular mass of the purified enzyme was estimated to be 74.6 kDa and 74.3 kDa by SDS-PAGE and gel filtration, respectively. PbChi74 displayed an acidic pH optimum of 4.5 and a temperature optimum of 65°C. The enzyme showed high activity toward colloidal chitin, glycol chitin, N-acetyl chitooligosaccharides, and p-nitrophenyl N-acetyl β-glucosaminide. PbChi74 hydrolyzed colloidal chitin to yield N-acetyl chitobiose [(GlcNAc)2] at the initial stage, which was further converted to its monomer N-acetyl glucosamine (GlcNAc), suggesting that it is an exochitinase with β-N-acetylglucosaminidase activity. The purified PbChi74 coupled with RmNAG (β-N-acetylglucosaminidase from Rhizomucor miehei) was used to convert colloidal chitin to GlcNAc, and GlcNAc was the sole end product at a concentration of 27.8 mg mL-1 with a conversion yield of 92.6%. These results suggest that PbChi74 may have great potential in chitin conversion.
Conclusions
The excellent thermostability and hydrolytic properties may give the exochitinase great potential in GlcNAc production from chitin. This is the first report on an exochitinase with N-acetyl-β-D-glucosaminidase activity from Paenibacillus species.
doi:10.1186/s13068-014-0174-y
PMCID: PMC4280004  PMID: 25550712
Chitinase; Exochitinase; Paenibacillus barengoltzii; Chitin conversion; N-acetyl-β-glucosamine
23.  Coffee Berry Borer Joins Bark Beetles in Coffee Klatch 
PLoS ONE  2013;8(9):e74277.
Unanswered key questions in bark beetle-plant interactions concern host finding in species attacking angiosperms in tropical zones and whether management strategies based on chemical signaling used for their conifer-attacking temperate relatives may also be applied in the tropics. We hypothesized that there should be a common link in chemical signaling mediating host location by these Scolytids. Using laboratory behavioral assays and chemical analysis we demonstrate that the yellow-orange exocarp stage of coffee berries, which attracts the coffee berry borer, releases relatively high amounts of volatiles including conophthorin, chalcogran, frontalin and sulcatone that are typically associated with Scolytinae chemical ecology. The green stage of the berry produces a much less complex bouquet containing small amounts of conophthorin but no other compounds known as bark beetle semiochemicals. In behavioral assays, the coffee berry borer was attracted to the spiroacetals conophthorin and chalcogran, but avoided the monoterpenes verbenone and α-pinene, demonstrating that, as in their conifer-attacking relatives in temperate zones, the use of host and non-host volatiles is also critical in host finding by tropical species. We speculate that microorganisms formed a common basis for the establishment of crucial chemical signals comprising inter- and intraspecific communication systems in both temperate- and tropical-occurring bark beetles attacking gymnosperms and angiosperms.
doi:10.1371/journal.pone.0074277
PMCID: PMC3779205  PMID: 24073204
24.  The genome sequence and effector complement of the flax rust pathogen Melampsora lini 
Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their hosts.
doi:10.3389/fpls.2014.00098
PMCID: PMC3970004  PMID: 24715894
rust; flax; Melampsora; effector; virulence; avirulence
25.  Validation of reference genes for normalization of qPCR gene expression data from Coffea spp. hypocotyls inoculated with Colletotrichum kahawae 
BMC Research Notes  2013;6:388.
Background
Coffee production in Africa represents a significant share of the total export revenues and influences the lives of millions of people, yet severe socio-economic repercussions are annually felt in result of the overall losses caused by the coffee berry disease (CBD). This quarantine disease is caused by the fungus Colletotrichum kahawae Waller and Bridge, which remains one of the most devastating threats to Coffea arabica production in Africa at high altitude, and its dispersal to Latin America and Asia represents a serious concern. Understanding the molecular genetic basis of coffee resistance to this disease is of high priority to support breeding strategies. Selection and validation of suitable reference genes presenting stable expression in the system studied is the first step to engage studies of gene expression profiling.
Results
In this study, a set of ten genes (S24, 14-3-3, RPL7, GAPDH, UBQ9, VATP16, SAND, UQCC, IDE and β-Tub9) was evaluated to identify reference genes during the first hours of interaction (12, 48 and 72 hpi) between resistant and susceptible coffee genotypes and C. kahawae. Three analyses were done for the selection of these genes considering the entire dataset and the two genotypes (resistant and susceptible), separately. The three statistical methods applied GeNorm, NormFinder, and BestKeeper, allowed identifying IDE as one of the most stable genes for all datasets analysed, and in contrast GADPH and UBQ9 as the least stable ones. In addition, the expression of two defense-related transcripts, encoding for a receptor like kinase and a pathogenesis related protein 10, were used to validate the reference genes selected.
Conclusion
Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the interaction between Coffea spp. and C. kahawae.
doi:10.1186/1756-0500-6-388
PMCID: PMC3849654  PMID: 24073624

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