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

Abstract

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-␤-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-␤-1,4-D-glucanase or endo-␤-1,4-D-xylanase activities. Two protein-containing peaks with endo-␤-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-6␤) from the parent were each purified to homogeneity. N-terminal amino acid sequencing indicated that XYL-6␣ is a fragment of XYL-6␤ and that XYL-6␤ 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-␤-1,4-D-xylanase.

Interaction Transcriptome Analysis Identifies Magnaporthe oryzae BAS1-4 as Biotrophy-Associated Secreted Proteins in Rice Blast Disease

“…One of the >10-fold upregulated secreted protein genes (see Supplemental Table 1B online) encodes a putative cellulase. However, out of five characterized xylanases (Wu et al, 2006) represented in the microarray, none were upregulated in IH. ESTs from cell wallgrown mycelium were well represented among transcripts that were underexpressed in IH ( Figure 1C; see Supplemental Table 1A online) but not among transcripts for the >10-fold induced IH genes (see Supplemental Table 1B online).…”

“…These genes are expressed in planta after macroscopic symptoms develop (Wu et al, 2006), and they are induced in mycelium grown on nutrient medium containing isolated plant walls as the major carbon source (Wu et al, 2006). The sequenced cDNAs from mycelium grown on cell wall medium identified more secreted proteins than cDNAs from other sources (Ebbole et al, 2004).…”

Structures and Functions of Oligosaccharins: The Role of Endoglycanases

“…For example, Magnaporthe oryzae (formerly Magnaporthe grisea), the destructive rice blast fungus [49], secrete various isoforms of endo-β-1,4-xylanases [49][50][51][52][53] that degrade arabinoxylans, the quantitatively major hemicellulose in the cell walls of Poaceae [54,55]. Some of the arabinoxylandegrading enzymes are probably pathogenicity factors that provide ingress to the cell walls, facilitate biotrophic growth, generate metabolizable nutrients, and release oligosaccharins that elicit host defense responses [53].…”

“…These genes are expressed and their enzyme products secreted differentially under various growth conditions [50][51][52][53]. For example, while most xylanase genes are expressed both in infected rice leaves and in culture three days postinoculation/infection, XYL-3 transcripts were detected at a very early stage (24 h post-inoculation) during infection, and XYL-4, as well as XYL-5, are exclusively expressed in infected host tissues.…”

“…(3) In order to effectively produce and purify M. grisea xylanases for biochemical and biological studies, we chose to express the proteins in the heterologous host Pichia pastoris using either a commercial vector, pPic3.5k, or a modified vector, pPicH, that includes DNA sequences encoding a cmyc epitope and a (His) 6 tag [52]. With the exception of XYL-5, all the genes attempted resulted in secretion of the corresponding protein into the Pichia culture media.…”