Table 1.
The activities of antioxidative system in H. oryzae-infected rice roots.
Figure 1.
Laser scanning confocal microscopy of the H. oryzae transformants Ho31gfp and Ho19red.
(a) Hyphae and falcate conidiophores of the Ho31gfp transformant showing constitutive eGFP expression. Bar, 20 µm; Bar in inner panel, 5 µm. (b) Hyphae, conidiophores (arrowhead) and flask-shaped phialides (arrow) of the Ho19red transformant showing strong red fluorescence. Bar, 50 µm.
Figure 2.
Fungal structures of H. oryzae during root infestation.
(a) Dark runner hyphae enwrapping the root surface. Bar, 100 µm. (b) Light and fluorescence microscopy of hyphopodia (arrow) formed by runner hyphae as infection structures on the root surface and corresponding penetration peg (arrowhead). Bars, 20 µm. (c) Intracellular hyphae in cortical cells forming narrow neck-like constriction (arrow) where they cross the cell wall. Bar, 20 µm. (d) Intercellular hyphae expanding and branching occasionally in the cortical layer. Bar, 50 µm. (e) Darkly pigmented and thick-walled chlamydospores on the root surface. Bar, 20 µm. (f) Upon germination, chlamydospores producing septate germ tubes and appressorium-like bulges with corresponding infection pegs. Bars, 10 µm. (g) Germination of intracellular chlamydospores within epidermal cells. Bar, 20 µm. (h) Clusters of inflated, rounded, thick-walled cells compacted in the cortical cells before the formation of microsclerotia. Bar, 20 µm. (i) Light and fluorescence microscopy of darkly pigmented, irregularly lobed, thick-walled intracellular microsclerotia in the cortical cells. Bars, 50 µm. (j) Abundant hyphae and chlamydospores assembled at the basal parts of root hair cells. Bars, 500 µm.
Figure 3.
Colonization pattern of H. oryzae in rice roots.
(a) In a root cross-section, eGFP-tagged hyphae gradually extended from the epidermis to the cortex without penetrating the stele. Bar, 200 µm. (c) A gradual increase in fungal colonization was associated with root maturation. Fungal colonization showing heavy colonization in the differentiation zone (d), slight colonization in the elongation zone, and no colonization in the meristematic zone (e). Bars, 500 µm. (b) and (f) Schematic representations of root colonization by H. oryzae. (b) The colonization pattern as seen in a transverse section. (f) Longitudinal section showing the association of fungal colonization with root maturation. Blue and green indicate living and dead cells, respectively. Red lines and dots: hyphae; black dots: chlamydospores; purple patches: microsclerotia.
Figure 4.
Relative amounts of fungal DNA in rice roots at different time points (1, 3, 5, 7, 10, 15, 20, 25 and 30 d.a.i.).
A fungal colonization curve plotted with the means ± SD of six replicates is shown.
Figure 5.
Vitality of H. oryzae-infected root cells as shown by staining with FM4-64 and DAPI, respectively.
(a) The colonized cells became melanized lesions and showed enhanced fluorescence (arrows) from the intracellular hyphae at 10 d.a.i. (upper panels). Bars, 50 µm. Later (≥ 15 d.a.i.), additional hyphae penetrated the root cells and the lesion-like infected area became larger; moreover, the enhanced fluorescence and plant autofluorescence of some primary infected cells disappeared (arrowheads) (lower panels). Bars, 200 µm. (b) The internalization of FM4-64 into endomembrane structures in fungus-infected cells (arrowheads) and non-invaded cells (arrows) during early colonization (≤ 10 d.a.i.). Bars, 100 µm. (c) At 15 d.a.i., endocytosis disappeared in the cells occupied by microsclerotia (arrowhead), but remained in the adjacent non-infected cells (arrow). Bar, 50 µm. (d) Root segments stained with DAPI. A large number of DAPI-positive nuclei in root cells with slight infection at 5 d.a.i. (left panels; Bars, 400 µm). DAPI-stained nuclei in root cells occupied by abundant hyphae and microsclerotia at 15 d.a.i. (middle panels; Bars, 200 µm). DAPI-stained nuclei disappeared in root cells filled with microsclerotia, but remained in the adjacent non-invaded cells after 15 d.a.i. (right panels; Bars, 100 µm).
Figure 6.
Effects of H. oryzae on root infection by M. oryzae.
(a) – (e) In mock-infected roots, eGFP-tagged M. oryzae propagated in the stele (a) and spread systemically from root to leaf through the vascular tissue (b), causing typical blast symptoms in the leaves (c), roots (d) and stem (e). (f) – (i) In H. oryzae-infected roots, no eGFP-tagged M. oryzae hyphae emerged in the roots (f) and leaf vascular tissues (g), with the disappearance of blast disease on the leaves (h) and stem (i). Bars, 200 µm. Arrows indicate eGFP-tagged M. oryzae.
Figure 7.
H2O2 accumulation caused by H. oryzae.
Light and fluorescence microscopy of mock- and H. oryzae-infected rice roots at different time points after inoculation. The brown granules (arrows, DAB staining) indicate H2O2 accumulation. Bars, rightmost panels, 100 µm; others, 30 µm.
Figure 8.
Systemic protection against rice blast by H. oryzae.
(a) The alleviation of devastating symptoms on the leaves of H. oryzae-infected rice in contrast to mock-infected controls was observed. (b) Left panel, lesion severity was judged in four levels ranging from 1 (resistant) to 4 (highly susceptible) (each treatment, n = 50). Bars indicate the percentage of lesions with each severity level. Right panel, the lesion area was assessed by the %DLA using an Axiovision image analyzer. The values are the means ± SD from 50 leaves of H. oryzae-infected or mock-infected rice (***, P<0.001).
Table 2.
Expression of genes representative for plant defense response.
Figure 9.
Temporal expression analysis of defense-related genes after H. oryzae inoculation.
(a) OsWRKY45 expression was markedly upregulated. (b) Analysis of OsWRKY45, JAmyb and NH1 expression, which is indicative of different defense pathways. Bars represent the means (three replicates) ± SD. Significant differences (one-way ANOVA): *, P<0.05; **, P<0.01; ***, P<0.001.