Fig 1.
Expression and secretion of MoAbfB during host infection by M. oryzae.
(A) Typical infection phenotype of control (C), incompatible (R), and compatible (S) interactions at 72 hr post M. oryzae infection. (B) Transcriptional and (C) translational expression of MoAbfB in rice leaves, as confirmed by semi-quantitative RT-PCR and western blotting analysis, respectively, in R and S combinations after 48 and/or 72 hr. Rubisco large subunit (RbCl) was set as loading control (D) Western blotting analysis of MoAbfB, OsGlu2, and Rubisco expression in total protein extracts (total) and secreted protein fractions (secretome) extracted from M. oryzae-infected rice leaves. Samples were collected at 72 hr. (E) Transient expression of MoAbfB-mCherry and MoAbrB(w/oSS)-mCherry constructs in sheath cells of transgenic rice harbouring a PBZ1 promoter::GFP reporter. Florescent signals were detected at 48 hr post-particle bombardment. (F) Close-up view and quantification of the mCherry and GFP fluorescent signals in the direction indicated by the arrow.
Table 1.
Strains used in this study.
Table 2.
Characterization of ΔMoAbfB mutants and complemented strains.
Fig 2.
Generation of the ΔMoAbfB mutant and complemented strain.
(A) Gene disruption strategy for MoAbfB (MGG_06843T0). HYG, hygromycin B phosphotransferase marker gene cassette. (B) Genomic DNA samples from a wild-type strain and 3 ΔMoAbfB mutants were digested with BamHI and probed with the 5’-flanking 1,284 bp of the MoAbfB gene. (C) RT-PCR to confirm the loss and recovery of the MoAbfB transcript.
Fig 3.
(A) Conidial suspensions (5 × 105 conidia/mL) of the indicated isolates were sprayed on 3–4 leaf-stage rice leaves. Photographs were taken 7 days after inoculation. (B) Percentage of infected areas of seedlings infected with wild-type fungi, ΔMoAbfB mutants, or MoAbfBc complemented strains. Error bars indicate standard deviation from the mean of 3 independent experiments (*, P < 0.05; Tukey’s test).
Fig 4.
Recombinant MoAbfB protein degrades cell wall structures.
(A) Schematic diagram of signal peptide and conserved domain in MoAbfB. SP, signal peptide; GH43, glycosyl hydrolase family 43 domain; AbfB, Alpha-L-arabinofuranosidase B domain. (B) Nonlinear regression plot of time-dependent MoAbfB and denatured MoAbfB (MoAbfB(D)) release of ρNPA (pH 7.0). (C) Enzyme kinetics assay of recombinant MoAbfB protein. A linear regression plot constructed using GraphPad Prism 5.04 (GraphPad Software Inc., 1992–2010) was created using the Michaelis—Menten saturation kinetic constants for the ability of recombinant MoAbfB to produce ρNPA. (D) Morphology of MoAbfB-digested rice leaf surface (with 5 μg of recombinant MoAbfB protein) after 72 hr of treatment. (E–F) SEM images of MoAbfB protein-digested host cell walls. (E) Boiled enzyme-treated rice leaves exhibited a clear and complete surface structure. (F) Damaged cell surfaces and degraded cell wall matrices caused by MoAbfB treatment. (G) Sugar composition of MoAbfB-digested cell wall extracts. ‘Ara’ = arabinose; ‘Rib’ = ribose; ‘Fuc’ = fucose; ‘Xyl’ = xylose; ‘UI’ = unidentified sugar; ‘GalA’ = galacturonic acid; ‘Man’ = mannose; ‘Gal’ = galactose; ‘Glc’ = glucose; ‘GalNAc’ = N-acetylgalactosamine; ‘GlcNAc’ = N-acetylglucosamine; ‘ManNAc’ = N-acetylmannosamine. (H) Activation of cell death by MoAbfB. Boiled MoAbfB protein (5 μg) (1) and native MoAbfB protein (5 μg) (2) were used as negative and positive controls, respectively; 5 μg (3) and 10 μg (4) of oligosaccharides (glucose equivalent) were dropped onto PBZ1 promoter::GFP rice leaves. GFP signals were detected after 72 hr.
Fig 5.
Pre-treatment with MoAbfB suppresses the virulence of M. oryzae and triggers a host immune response.
(A) Fungus-infected lesions on rice leaves pre-treated with 0.1 μM MoAbfB protein 12 hr prior to inoculation and measured at 72 hpi. (B) Lesion areas were quantified using the APS Assess 2.0 program. Error bars indicate the standard deviation from the mean of 3 independent experiments. (C) Expression of ROS and PR genes detected by RT-PCR.