Identification of an endo-beta-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-beta-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-6Delta mutant were each fractionated by liquid chromatography, and the collected fractions were assayed for endo-beta-1,4-D-glucanase or endo-beta-1,4-D-xylanase activities. Two protein-containing peaks with endo-beta-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-6alpha and XYL-6beta) from the parent were each purified to homogeneity. N-terminal amino acid sequencing indicated that XYL-6alpha is a fragment of XYL-6beta and that XYL-6beta 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-beta-1,4-D-xylanase.
Wu
Sheng-Cheng
SC
Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Rd., Athens, GA 30602-4712, USA. wusc@ccrc.uga.edu
Halley
Jeffrey E
JE
Luttig
Christopher
C
Fernekes
Linda M
LM
Gutiérrez-Sanchez
Gerardo
G
Darvill
Alan G
AG
Albersheim
Peter
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D020224:Expressed Sequence Tags; D015870:Gene Expression; D018390:Gene Targeting; D005800:Genes, Fungal; D020082:Magnaporthe / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D008969:Molecular Sequence Data; D009154:Mutation; D012275:Oryza / Q000382:microbiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D004271:DNA, Fungal; D011993:Recombinant Fusion Proteins; D043364:Endo-1,4-beta Xylanases