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Applied and Environmental Microbiology, February 2006, p. 986-993, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.986-993.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Sheng-Cheng Wu,^* Jeffrey E. Halley, Christopher Luttig, Linda M. Fernekes, Gerardo Gutiérrez-Sanchez, Alan G. Darvill, and Peter Albersheim

Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602-4712

Received 5 August 2005/ Accepted 6 November 2005

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-ß-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 mutant were each fractionated by liquid chromatography, and the collected fractions were assayed for endo-ß-1,4-D-glucanase or endo-ß-1,4-D-xylanase activities. Two protein-containing peaks with endo-ß-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 and XYL-6ß) from the parent were each purified to homogeneity. N-terminal amino acid sequencing indicated that XYL-6 is a fragment of XYL-6ß and that XYL-6ß 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-ß-1,4-D-xylanase.

Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.

Present address: Department of Biochemistry, School of Medicine, Emory University, Atlanta, GA 30322.