Identification of an endo-{szligbeta}-1,4-D-Xylanase from Magnaporthe grisea by Gene Knockout Analysis, Purification, and Heterologous Expression

Magnaporthe grisea, a destructive ascomycetous pathogen of rice, secretes cell wall-degrading enzymes into a culture medium containing purified rice cell walls as the sole carbon source. From M. grisea grown under the culture conditions described here, we have identified an expressed sequenced tag, XYL-6, a gene that is also expressed in M. grisea-infected rice leaves 24 h postinoculation with conidia. This gene encodes a protein about 65% similar to endo-{szligbeta}-1,4-D-glycanases within glycoside hydrolase family GH10. A M. grisea knockout mutant for XYL-6 was created, and it was shown to be as virulent as the parent strain in infecting the rice host. The proteins secreted by the parent strain and by the xyl-6[Delta] mutant were each fractionated by liquid chromatography, and the collected fractions were assayed for endo-{szligbeta}-1,4-D-glucanase or endo-{szligbeta}-1,4-D-xylanase activities. Two protein-containing peaks with endo-{szligbeta}-1,4-D-xylanase activity secreted by the parent strain are not detectable in the column eluant of the proteins secreted by the mutant. The two endoxylanases (XYL-6[alpha] and XYL-6{szligbeta}) from the parent were each purified to homogeneity. N-terminal amino acid sequencing indicated that XYL-6[alpha] is a fragment of XYL-6{szligbeta} and that XYL-6{szligbeta} is identical to the deduced protein sequence encoded by the XYL-6 gene. Finally, XYL-6 was introduced into Pichia pastoris for heterologous expression, which resulted in the purification of a fusion protein, XYL-6H, from the Pichia pastoris culture filtrate. XYL-6H is active in cleaving arabinoxylan. These experiments unequivocally established that the XYL-6 gene encodes a secreted endo-{szligbeta}-1,4-D-xylanase.