Optical Identification of Fruitfly Species Based on Their Wingbeats Using Convolutional Neural Networks.

The spotted wing Drosophila (SWD), Drosophila suzukii, is a significant invasive pest of berries and soft-skinned fruits that causes major economic losses in fruit production worldwide. Automatic identification and monitoring strategies would allow to detect the emergence of this pest in an early stage and minimize its impact. The small size of Drosophila suzukii and similar flying insects makes it difficult to identify them using camera systems. Therefore, an optical sensor recording wingbeats was investigated in this study. We trained convolutional neural network (CNN) classifiers to distinguish D. suzukii insects from one of their closest relatives, Drosophila Melanogaster, based on their wingbeat patterns recorded by the optical sensor. Apart from the original wingbeat time signals, we modeled their frequency (power spectral density) and time-frequency (spectrogram) representations. A strict validation procedure was followed to estimate the models' performance in field-conditions. First, we validated each model on wingbeat data that was collected under the same conditions using different insect populations to train and test them. Next, we evaluated their robustness on a second independent dataset which was acquired under more variable environmental conditions. The best performing model, named "InceptionFly," was trained on wingbeat time signals. It was able to discriminate between our two target insects with a balanced accuracy of 92.1% on the test set and 91.7% on the second independent dataset. This paves the way towards early, automated detection of D. suzukii infestation in fruit orchards.

Mass Trapping Drosophila suzukii, What Would It Take? A Two-Year Field Study on Trap Interference.

The invasion of Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) worldwide has disrupted existing or developing integrated pest management (IPM) programs in soft-skinned fruits. Currently, with a reliance on only broad-spectrum insecticides, there is a critical call for alternative control measures. Behavioural control is one of the pillars of IPM, and, in the present study, it is investigated whether mass trapping could be viable for D. suzukii management. By quantifying trap interference in 4 × 4 replicate trapping grids, an estimate of the attraction radius for a certain attractant and context can be obtained. Traps designed for dry trapping (no drowning solution, but a killing agent inside) and synthetic controlled released experimental lures were tested in a two-year field study. Apple cider vinegar (ACV) was included as a reference bait and trials were performed with 5, 10 and 15 m inter-trap spacings at different seasonal timings. Clear trap interference and, hence, overlapping attraction radii were observed both in spring and summer for both the synthetic lures and ACV. In early spring, ACV shows the most potential for mass trapping, however from June onwards, the experimental dry lures show equal or better results than ACV. Based on our findings, workable trap densities are deemed possible, encouraging further development of mass trapping strategies for the control of D. suzukii.

An Advanced One-Step RT-LAMP for Rapid Detection of Little cherry virus 2 Combined with High-Throughput Sequence-Based Phylogenomics Reveal Divergent Flowering Cherry Isolates.

Little cherry virus 2 (LChV-2, genus Ampelovirus) is considered to be the main causal agent of the economically damaging little cherry disease, which can only be controlled by removal of infected trees. The widespread viral disease of sweet cherry (Prunus avium L.) is affecting the survival of long-standing orchards in North America and Europe, hence the dire need for an early and accurate diagnosis to establish a sound disease control strategy. The endemic presence of LChV-2 is mainly confirmed using laborious time-consuming reverse-transcription (RT-PCR). A rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting a conserved region of the coat protein was developed and compared with conventional RT-PCR for the specific detection of LChV-2. This affordable assay, combined with a simple RNA extraction, deploys desirable characteristics such as higher ability for faster (<15 min), more analytically sensitive (100-fold), and robust broad-range diagnosis of LChV-2 isolates from sweet cherry, ornamental flowering cherry displaying heterogenous viral etiology and, for the first time, newly identified potential insect vectors. Moreover, use of Sanger and total RNA high-throughput sequencing as complementary metaviromics approaches confirmed the LChV-2 RT-LAMP detection of divergent LChV-2 isolates in new hosts and the relationship of their whole-genome was exhaustively inferred using maximum-likelihood phylogenomics. This entails unprecedented critical understanding of a novel evolutionary clade further expanding LChV-2 viral diversity. In conclusion, this highly effective diagnostic platform facilitates strategical support for early in-field testing to reliably prevent dissemination of new LChV-2 outbreaks from propagative plant stocks or newly postulated insect vectors. Validated results and major advantages are herein thoroughly discussed, in light of the knowledge required to increase the potential accuracy of future diagnostics and the essential epidemiological considerations to proactively safeguard cherries and Prunus horticultural crop systems from little cherry disease.

Towards Integrated Pest and Pollinator Management in Intensive Pear Cultivation: A Case Study from Belgium.

Recently, the concept of Integrated Pest Management (IPM) was further extended into Integrated Pest and Pollinator Management (IPPM). Implementation of IPPM strategies entails the combination of actions for pest and pollinator management providing complementary or synergistic benefits for yield and/or quality of the harvest. The aim of this study was to examine IPPM elements (i.e., mixed hedgerow, nesting boxes for mason bees, Osmia spp.) and demonstrate their impact in the practical context of modern commercial fruit cultivation in a 4-year case study in an intensive 'Conference' pear orchard. The outcomes of visual observations during transect walks and molecular analysis of pollen collected by mason bees, showed the importance of additional floral resources for the presence of mason bees and other pollinating insects in the orchard environment. Pear quality assessments indicated that insect-mediated pollination had a significant positive impact, with a tendency for higher quality pears in the close vicinity of Osmia nesting boxes. However, despite the fact that pear pollen was also detected in Osmia spp. nest cells, the amount and frequency of pear pollen collection for their nest built-up turned out to be rather low. In the same intensive pear orchard studied for pollination effects, we simultaneously demonstrate the impact of a mixed hedgerow to enhance integrated pest control.

Towards a Knowledge-Based Decision Support System for Integrated Control of Woolly Apple Aphid, Eriosoma lanigerum, with Maximal Biological Suppression by the Parasitoid Aphelinus mali.

The woolly apple aphid Eriosoma lanigerum (Homoptera: Aphidiae) is an important pest in apple orchards worldwide. Since the withdrawal or restricted use of certain broad-spectrum insecticides, E. lanigerum has become one of the most severe pests in apple growing areas across Western Europe. At present, effective limitation of woolly aphid populations relies on a good synergy between chemical control treatments and biological suppression by beneficial arthropods, especially by its main specific natural enemy, the parasitoid Aphelinus mali (Hymenoptera: Aphelinidae). To develop a knowledge-based decision support system, detailed monitoring data of both species were collected in the field (region of Sint-Truiden, Belgium) for a period of ten years (2010-2020). Aphelinus mali flights were monitored in the field, starting before flowering until the end of the second-generation flight at minimum. The seasonal occurrence of the most important management stages of E. lanigerum, e.g., start of wool production or activity on aerial parts in spring and migration of crawlers from colonies towards flower clusters or shoots, were thoroughly monitored. All obtained data were compared with historical and literature data and analysed in a population dynamics phenological model. Our outcomes showed that the emergence of first-generation A. mali adults (critical for the first parasitation activity and the basis for following A. mali generations in the continuation of the season) can be accurately predicted by the developed model. Hence, this information can be utilized to avoid insecticide sprayings with detrimental side effects at this particular moment as demonstrated by the outcomes of a field trial. In addition, the start of migration of E. lanigerum crawlers towards flower clusters or shoots is accurately predicted by the model. In conclusion, our results demonstrate that the model can be used as decision support system for the optimal timing of control treatments in order to achieve effective control of E. lanigerum with maximal biological suppression by its main natural enemy.

Identification of Blackberry (Rubus fruticosus) Volatiles as Drosophila suzukii Attractants.

The spotted wing drosophila, Drosophila suzukii, is an invasive pest species from Southeast Asia that was recently introduced in Europe and North America. As this fruit fly lays its eggs in ripening soft-skinned fruit, it causes great damage to a variety of crops, including cherries, blueberries, blackberries, raspberries, grapes, plums and strawberries. Consequently, there is a great demand for an effective and species-specific lure, which requires the development of successful attractants. Until now, there is no lure available that is species-specific and can detect the presence of D. suzukii before infestation. As blackberry (Rubus fruticosus) is one of the preferred host crops of D. suzukii, the volatile compounds of R. fruticosus berries are here identified and quantified using multiple headspace SPME (solid phase micro extraction) GC-MS (gas chromatography-mass spectrometry). Subsequently, the attractivity of 33 of the identified compounds was tested with a two-choice laboratory bioassay. Acetaldehyde, hexyl acetate, linalool, myrtenol, L-limonene and camphene came out as significantly attractive to D. suzukii. The first four attractive compounds induced the strongest effect and therefore provided the best prospects to be implemented in a potential lure. These findings could contribute towards the development of more effective attractants for monitoring and mass trapping D. suzukii.

Tunnel entries and a killing agent uncover the importance of fly retention in Drosophila suzukii traps.

BACKGROUND: Spotted wing drosophila (SWD), Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is a pest of stone and small fruits causing considerable economic losses. Current management strategies rely primarily on calendar-based spraying, owing to the poor relationship between monitoring data and damage levels, and the lack of success of mass-trapping tools. The aim of this study was to evaluate different trap models for SWD, with an emphasis on their fly-retention capacity. To this end, we examined and quantified the added value of two fly-retaining trap features; tunnel entries to impede escape and an insecticide-coated inner surface as a killing agent. RESULTS: An insecticide-coated inner surface resulted in significantly higher trap retention after 24 h in the laboratory (4.9- to 7.4-fold greater, depending on trap type) compared to a noncoated trap. Trapping efficacy was significantly improved in field trials by such a killing agent in the trap (1.2- to 4.5-fold greater). Tunnel entries significantly improved trap retention in the laboratory and field (by 1.5-fold). CONCLUSION: The outcomes of this study clearly reveal the substantial impact of the fly-retention capacity of SWD traps on their overall capture performances. It was demonstrated for the first time that an insecticide-coated inner surface as a killing agent significantly improves trap efficacy for SWD. This finding can readily be implemented in any trap model to improve monitoring and mass trapping of SWD. Also tunnel entries were shown to have a significant influence on the fly retention and, hence, substantially enhance trapping efficacy.

Marking Drosophila suzukii (Diptera: Drosophilidae) with Fluorescent Dusts.

The marking of Drosophila suzukii can be an important instrument for studying the ecology and behaviour of this economically important fruit pest, aiding the development of new Integrated Pest Management (IPM) tools or strategies. There is, however, a need for a cost-effective methodology that provides an easily detectable and stable mark. Whereas fluorescent pigment powders are often used in entomological research, the pigments (series, dyes), application techniques, or doses need to be evaluated for each studied species in terms of their efficacy and possible adverse effects on the performance of the insect. The effectiveness of different application techniques and dyes (RadGlo® TP-series) and their effect on the survival of adult D. suzukii were investigated in the laboratory. Furthermore, the influence of the marking on the behaviour of the flies was examined in laboratory trap assays (olfaction) and a field recapture study (general orientation). The persistence and detectability of the marks was evaluated both on living flies (for different application techniques) and dead flies under trapping/storage conditions. The use of fluorescent powders to mark D. suzukii flies yielded a clearly detectable and highly persistent mark, without any adverse effects on the survival and behaviour of the flies.

Olfactory Preference of Drosophila suzukii Shifts between Fruit and Fermentation Cues over the Season: Effects of Physiological Status.

Worldwide monitoring programs of the invasive fruit pest Drosophila suzukii Matsumura (Diptera: Drosophilidae), using fermentation baits like apple cider vinegar (ACV), revealed a counterintuitive period of low trap catches during summer, followed by an autumn peak. In this study, we demonstrate that ACV baited traps indeed provide a distorted image of the D. suzukii population dynamics as it is possible to capture higher numbers during this "low capture period" with synthetic lures. It was hypothesised that the preference of D. suzukii populations for fermentation cues like ACV is most pronounced during autumn, winter and spring, while the flies prefer fresh fruit cues during summer and that this seasonal preference is related to the changing physiology of the flies over the season. To test this hypothesis, the preference between fermentation cues (ACV) and host fruits (strawberries) and the effect of physiology (sex, seasonal morphology and feeding, mating and reproductive status) was investigated both in olfactometer laboratory experiments and a year-round field preference experiment. In olfactometer experiments we demonstrated that protein deprived females, virgin females with a full complement of unfertilised eggs and males show a strong preference for fermentation cues while fully fed reproductive summer morph females generally prefer fruit cues. These findings indicate that D. suzukii is attracted to fermentation volatiles in search of (protein-rich) food and to fruit volatiles in search of oviposition substrates. Winter morph and starved females displayed indiscriminating olfactory behaviour. In the field preference experiment, the hypothesised seasonal shift between fermentation and fruit cues was confirmed. This shift appeared to be highly temperature-related and was similarly observed for summer and winter morphs.

High-Throughput Sequencing Assists Studies in Genomic Variability and Epidemiology of Little Cherry Virus 1 and 2 infecting Prunus spp. in Belgium.

Little cherry disease, caused by little cherry virus 1 (LChV-1) and little cherry virus 2 (LChV-2), which are both members of the family Closteroviridae, severely affects sweet (Prunus avium L.) and sour cherry (P. cerasus L.) orchards lifelong production worldwide. An intensive survey was conducted across different geographic regions of Belgium to study the disease presence on these perennial woody plants and related species. Symptomatic as well as non-symptomatic Prunus spp. trees tested positive via RT-PCR for LChV-1 and -2 in single or mixed infections, with a slightly higher incidence for LChV-1. Both viruses were widespread and highly prevalent in nearly all Prunus production areas as well as in private gardens and urban lane trees. The genetic diversity of Belgian LChV-1 and -2 isolates was assessed by Sanger sequencing of partial genomic regions. A total RNA High-Throughput Sequencing (HTS) approach confirmed the presence of both viruses, and revealed the occurrence of other Prunus-associated viruses, namely cherry virus A (CVA), prune dwarf virus (PDV) and prunus virus F (PrVF). The phylogenetic inference from full-length genomes revealed well-defined evolutionary phylogroups with high genetic variability and diversity for LChV-1 and LChV-2 Belgian isolates, yet with little or no correlation with planting area or cultivated varieties. The global diversity and the prevalence in horticultural areas of LChV-1 and -2 variants, in association with other recently described fruit tree viruses, are of particular concern. Future epidemiological implications as well as new investigation avenues are exhaustively discussed.

New sensitive and fast detection of Little cherry virus 1 using loop-mediated isothermal amplification (RT-LAMP).

Little cherry virus 1 (LChV-1) belongs to the genus Velarivirus, family Closteroviridae, is an economically important pathogen affecting mainly cherry around the world emphasizing the impetus for its efficient and accurate on-site detection. This study describes the development of a reliable diagnostic protocol of LChV-1 based on a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The protocol detects LChV-1 isolates in less than 10 min by fluorescence monitoring using a mobile detection device and is most optimal when performed at 67 °C. Sharp melting curves and unique melting temperatures (Tm) were obtained for the positive samples. Both the RT-LAMP and classical RT-PCR methods are capable of specifically detecting LChV-1 in infected leaf tissues. In addition, the RT-LAMP has remarkable advantages in comparison to RT-PCR. It is at least hundred fold more sensitive, significantly faster (allowing on-field leaf-to-result diagnostic) and efficient at minimal cost. In conclusion, this innovative RT-LAMP approach can contribute to the implementation of sustainable integrated management strategies for detection of LChV-1 in commercial orchards or for horticultural research stations. It is also suitable for decision support in phytosanitary epidemiological programs.

Validation of Individual-Based Markov-Like Stochastic Process Model of Insect Behavior and a "Virtual Farm" Concept for Enhancement of Site-Specific IPM.

The paper reports application of a Markov-like stochastic process agent-based model and a "virtual farm" concept for enhancement of site-specific Integrated Pest Management. Conceptually, the model represents a "bottom-up ethological" approach and emulates behavior of the "primary IPM actors"-large cohorts of individual insects-within seasonally changing mosaics of spatiotemporally complex faming landscape, under the challenge of the local IPM actions. Algorithms of the proprietary PESTonFARM model were adjusted to reflect behavior and ecology of R. cerasi. Model parametrization was based on compiled published information about R. cerasi and the results of auxiliary on-farm experiments. The experiments were conducted on sweet cherry farms located in Austria, Germany, and Belgium. For each farm, a customized model-module was prepared, reflecting its spatiotemporal features. Historical data about pest monitoring, IPM treatments and fruit infestation were used to specify the model assumptions and calibrate it further. Finally, for each of the farms, virtual IPM experiments were simulated and the model-generated results were compared with the results of the real experiments conducted on the same farms. Implications of the findings for broader applicability of the model and the "virtual farm" approach-were discussed.

Mutational analysis of wheat (Triticum aestivum L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism.

Nucleotide pyrophosphatases/phosphodiesterases (NPPs, PF01663) release nucleoside 5'-monophosphates from a wide range of nucleotide substrates. Only very recently, the first plant members of the NPP family were characterised (Joye et al. J Cereal Sci 51: 326-336, 2010), and little is known about their substrate-specifying residues. We elucidated the role of six amino acid residues of the recently identified and characterised Triticum aestivum L. NPP (Joye et al. J Cereal Sci 51: 326-336, 2010). Substitution of the highly conserved catalytic Thr132 into Ser or Ala completely abolished enzyme activity. Mutation of a highly conserved His255 residue into an apolar Ala suprisingly increased enzyme activity against most phosphodiester substrates. Four other residues moderately to highly conserved over NPPs of different organisms were studied as well. Mutation of the Asn153, Asn165 and Glu199 into an Arg, Ser and Asp residue, respectively, increased the relative enzyme activity against p-nitrophenyl phosphate. Furthermore, mutation of Phe194 into Ser increased the relative enzyme activity against adenosine 5'-monophosphate-containing substrates, although the overall enzyme activity of this mutant enzyme decreased. We conclude that the structural requirements and the conservation of the amino acids of the catalytic site of TaNPPr and, by extension, probably of all NPPs, are very stringent.

Truncated derivatives of a multidomain thermophilic glycosyl hydrolase family 10 xylanase from Thermotoga maritima reveal structure related activity profiles and substrate hydrolysis patterns.

Efficient heteroxylan degradation in the context of economically feasible lignocellulosic biomass biorefining requires xylanolytic enzymes with optimal thermostability and specificity. Therefore, the structure activity relationship of a modular thermophilic glycoside hydrolase family 10 xylanase (xylanase A from Thermotoga maritima MSB8, rXTMA) was investigated through construction of six truncated derivatives, lacking at least one of the 2 N- and/or 2 C-terminal modules. The temperatures for optimal activity and stability of the xylanases were strongly influenced by the presence of the different modules and ranged from 60 to 80 degrees C and 50 to 80 degrees C, respectively. In contrast, the pH for optimal activity was only slightly affected (pH 6.0 to 7.0). The tested xylanases retained over 80% activity after 2h pre-incubation at 50 degrees C between pH 5.0 and 11.0. Most unexpectedly, changes in the modular structure led to a 26-fold wide range of specific activities of the enzymes towards xylohexaose, while the activity towards insoluble polymeric heteroxylan was comparable for all but one xylanase. rXTMADeltaC, lacking the C-terminal modules, had a 60% higher specific activity towards the latter substrate than the wild type enzyme. These results show that key properties of XTMA can be tuned to allow for optimal performance of the enzyme in biotechnological processes such as in the bioconversion of lignocellulosic biomass.

Plant cell walls: Protecting the barrier from degradation by microbial enzymes.

Plant cell walls are predominantly composed of polysaccharides, which are connected in a strong, yet resilient network. They determine the size and shape of plant cells and form the interface between the cell and its often hostile environment. To penetrate the cell wall and thus infect plants, most phytopathogens secrete numerous cell wall degrading enzymes. Conversely, as a first line of defense, plant cell walls contain an array of inhibitors of these enzymes. Scientific knowledge on these inhibitors significantly progressed in the past years and this review is meant to give a comprehensive overview of plant inhibitors against microbial cell wall degrading enzymes and their role in plant protection.

Computational design-based molecular engineering of the glycosyl hydrolase family 11 B. subtilis XynA endoxylanase improves its acid stability.

Rational protein engineering was applied to improve the limited stability of the glycosyl hydrolase family 11 (GH11) endo-beta-1,4-xylanase from Bacillus subtilis under acidic conditions. Since the pH dependence of protein stability is governed by the ionisation states of the side chains of its titrable amino acid residues, we explored the strategy of changing pH-stability profiles by altering pK(a) values of key residues through in silico designed mutations. To this end, computational predictions and molecular modelling were carried out using the recently developed pKD software package. Four endoxylanase variants, in which the pK(a) values of either Asp4 and Asp11 or His149 were targeted to shift downwards through incorporation of three to five point mutations, were generated and recombinantly expressed in the cytoplasm of Escherichia coli. All four mutants showed considerably increased functional stability at acid pH levels. They retained approximately 30-70% and approximately 75-95% of their activity after incubation at pH 3 and 4, respectively, in comparison with only approximately 23% and approximately 57%, respectively, for the wild-type enzyme under the experimental conditions. No acidophilic adaptation of the catalytic activity had occurred. In addition, their functional stability and catalytic activity profiles under different temperature and ionic strength conditions were significantly altered. These findings contribute to general understanding of the molecular mechanisms governing the pH-dependent stability of GH11 proteins, and hence they can be applied to enhance the stability and effectiveness of many GH11 endoxylanases used in industry today.

General M13 phage display: M13 phage display in identification and characterization of protein-protein interactions.

In M13 phage display, proteins and peptides are exposed on one of the surface proteins of filamentous phage particles and become accessible to affinity enrichment against a bait of interest. We describe the construction of fragmented whole genome and gene fragment phage display libraries and interaction selection by panning. This strategy allows the identification and characterization of interacting proteins on a genomic scale by screening the fragmented "proteome" against protein baits. Gene fragment libraries allow a more in depth characterization of the protein-protein interaction site by identification of the protein region involved in the interaction.

Phage display based identification of novel stabilizing mutations in glycosyl hydrolase family 11 B. subtilis endoxylanase XynA.

Two combinatorial libraries of glycosyl hydrolase family 11 (GH11) Bacillus subtilis endoxylanase XynA were constructed and displayed on phage. Both phage-displayed libraries were subjected to three consecutive biopanning rounds against immobilized endoxylanase inhibitor TAXI, each time preceded by an incubation step at elevated temperature. DNA sequence analysis of enriched phagemid panning isolates allowed identification of mutations conferring enhanced thermal stability. In particular, substitutions T44C, T44Y, F48C, T87D, and Y94C were retained, and their thermostabilizing effect was confirmed by testing site-directed XynA variants. None of these mutations was identified in earlier endoxylanase engineering studies. Each single mutation increased the half-inactivation temperature by 2-3 degrees C over that of the wild-type enzyme. Intriguingly, the three selected cysteine variants generated dimers by formation of intermolecular disulfide bridges.

Mutational analysis of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum reveals comprehensive insights into their inhibitor insensitivity.

Endo-beta-1,4-xylanases (EC 3.2.1.8; endoxylanases), key enzymes in the degradation of xylan, are considered to play an important role in phytopathogenesis, as they occupy a prominent position in the arsenal of hydrolytic enzymes secreted by phytopathogens to breach the cell wall and invade the plant tissue. Plant endoxylanase inhibitors are increasingly being pinpointed as part of a counterattack mechanism. To understand the surprising XIP-type endoxylanase inhibitor insensitivity of endoxylanases XylA and XylB from the phytopathogen Fusarium graminearum, an extensive mutational study of these enzymes was performed. Using combinatorial and site-directed mutagenesis, the XIP insensitivity of XylA as well as XylB was proven to be solely due to amino acid sequence adaptations in the "thumb" structural region. While XylB residues Cys141, Asp148, and Cys149 were shown to prevent XIP interaction, the XIP insensitivity of XylA could be ascribed to the occurrence of only one aberrant residue, i.e., Val151. This study, in addition to providing a thorough explanation for the XIP insensitivity of both F. graminearum endoxylanases at the molecular level, generated XylA and XylB mutants with altered inhibition specificities and pH optima. As this is the first experimental elucidation of the molecular determinants dictating the specificity of the interaction between endoxylanases of phytopathogenic origin and a plant inhibitor, this work sheds more light on the ongoing evolutionary arms race between plants and phytopathogenic fungi involving recognition of endoxylanases.

Targeted molecular engineering of a family 11 endoxylanase to decrease its sensitivity towards Triticum aestivum endoxylanase inhibitor types.

The Bacillus subtilis endoxylanase XynA (BSXY) is frequently used to improve the functionality of arabinoxylan-containing material in cereal based industries. The presence of endogenous Triticum aestivum xylanase inhibitors (TAXI-I and TAXI-II) in wheat is a real concern as they have a direct negative impact on the efficiency of this enzyme. Here, we used the recently determined structure of the complex between TAXI-I and an endoxylanase of Aspergillus niger to develop inhibitor-insensitive BSXY variants by site-directed mutagenesis of strategically chosen amino acids. We either induced steric hindrance to reject the inhibitors or interrupted key interactions with the inhibitors in the endoxylanase substrate-binding groove. The first strategy was successfully applied to position G12 where G12W combined inhibition insensitivity with unharmed catalytic performance. Variants from the second strategy showed altered inhibitor sensitivities concomitant with changes in enzyme activities and allowed to gain insight in the binding-mode of both TAXI-I and TAXI-II with BSXY.