Deletion of Two Endo-β-1,4-Xylanase Genes Reveals Additional Isozymes Secreted by the Rice Blast Fungus

Abstract

Fungal pathogens secrete hydrolases during infection of plant tissues capable of fragmenting the primary cell wall polysaccharides of the host. Magnaporthe grisea, the fungal pathogen that causes blast disease of graminaceous monocots, secretes two distinct endo-β-1,4-D-xylanases when grown on xylan-rich rice cell walls as the carbon source. We have previously reported the cloning of the genes encoding these two xylanases, XYL1 and XYL2 (formerly XYN22 and XYN33, respectively; see S.-C. Wu, S. Kauffmann, A. G. Darvill, and P. Albersheim, Mol. Plant-Microbe Interact. 8:506–514, 1995). We now present three M. grisea mutants created by selective deletion of XYL1 and/or XYL2. The xyl1 mutant grows as well as the parent in culture medium when rice cell walls or xylan is the sole carbon source. Under the same conditions, the xyl2 mutant grows slightly slower than the parent, whereas the xyl1/xyl2 double mutant exhibits a 50% reduction in accumulation of total mycelial mass. Under conditions idealized for infection, all three mutants infect host plants as efficiently as the parent, indicating neither XYL1 nor XYL2 is required by M. grisea for infection. Endoxylanase assays showed that, at the stationary stage of growth in culture when the accumulation of total xylanase activity is at its maximum, the xyl1 mutant retains ≈ 88%, xyl2, 39%, and xyl1/xyl2, 19% of the endoxylanase activity of the parent. Partial protein purification of xylanases secreted by the xyl1/xyl2 double mutant revealed four distinct endoxylanase activities. One of the xylanases, XYL3, is present in the culture filtrate of both the parent and the mutant strains, and, like XYL1, has been identified as a member of the Family G xylanases. The other three xylanases are not found in the culture filtrates of the parent or of the xyl1 mutant. Thus, M. grisea appears capable of secreting additional xylanases and does so when XYL2, a member of the Family F glycanases, has been eliminated.

Keywords

• electroporation
• fungal pathogenesis
• gene disruption
• hypersensitive response
• selection markers