Endo-β-1,4-xylanases (EC 3.2.1.8) are key enzymes in the degradation of xylan, the predominant hemicellulose in the cell walls of plants and the second most abundant polysaccharide on earth. A number of endoxylanases are produced by microbial phytopathogens responsible for severe crop losses. These enzymes are considered to play an important role in phytopathogenesis, as they provide essential means to the attacking organism to break through the plant cell wall. Plants have evolved numerous defense mechanisms to protect themselves against invading pathogens, amongst which are proteinaceous inhibitors of cell wall-degrading enzymes. These defense mechanisms are triggered when a pathogen-derived elicitor is recognized by the plant. In this review, the diverse aspects of endoxylanases in promoting virulence and in eliciting plant defense systems are highlighted. Furthermore, the role of the relatively recently discovered cereal endoxylanase inhibitor families TAXI (Triticum aestivum xylanase inhibitor) and XIP (xylanase inhibitor protein) in plant defense is discussed.

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.

Rational protein engineering was applied to improve the limited stability of the glycosyl hydrolase family 11 (GH11) endo-β-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 ∼30–70% and ∼75–95% of their activity after incubation at pH 3 and 4, respectively, in comparison with only ∼23% and ∼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.