A family 11 xylanase from the pathogen Botrytis cinerea is inhibited by plant endoxylanase inhibitors XIP-I and TAXI-I

Abstract

The phytopathogen fungus Botrytis cinerea produces various glycosidases which are secreted during plant infection. In this study, the XynBc1 cDNA that encodes a xylanase from family 11 glycoside hydrolase from B. cinerea was identified by homology-based analysis, cloned by reverse transcription RT-PCR, fully sequenced, and heterologously expressed in Pichia pastoris X-33. The purified recombinant protein obtained by chelating-affinity chromatography demonstrated high catalytic activity (180 ± 23 U/mg) and efficiently degraded low viscosity xylan [ K m = 10 ± 3 g L −1, V max = 0.50 ± 0.04 μmol xylose min −1, and k cat = 136 ± 11.5 s −1 at pH 4.5 and 25 °C]. XynBc1 was further tested for its ability to interact with wheat XIP and TAXI type xylanase inhibitors which have been implicated in plant defence. The xylanase activity of XynBc1 produced in P. pastoris was strongly inhibited by both XIP-I and TAXI-I in a competitive manner, with a K i of 2.1 ± 0.1 and 6.0 ± 0.2 nM, respectively, whereas no inhibition was detected with TAXI-II. We also showed that XynBc1 mRNAs accumulated during early stages of plant tissue infection.