Figure 1.
Isolation of ATS from abc3Δ appressoria in M. oryzae.
(A) Wild-type S. pombe cells were treated with extracellular fluid (E/F) or appressorial extract (A/E) from the wild-type or abc3Δ M. oryzae strain for 6 h and stained with calcofluor white (CFW). Arrowheads indicate aberrant deposition of septal/cell wall material at the cell tip(s). Bars = 10 µm. (B) Schematic representation of the S. pombe cell-based assay used to guide the purification of ATS and to confirm ATS as an efflux substrate of the Abc3 transporter. MoABC3 refers to M. oryzae ABC3.
Figure 2.
ATS shares structural and functional properties with digoxin.
Molecular mass of ATS (A) or digoxin (B) identified by APCI method. Molecular masses shown are sodium adducts of ATS or digoxin (both, m/z 780). Insets depict the predominant peaks of ATS or digoxin with their respective retention times. (C) and (D) Tandem mass spectra of ATS and digoxin, respectively. The ionization products characteristic of the steroidal nucleus (m/z 390), mono- and bi-sugar (m/z 520 and 650, respectively) molecules are highlighted. (E) Wild-type S. pombe cells were treated with residual solvent, ATS, or digoxin for 6 h and stained with CFW. Arrowheads show aberrant septal/cell wall biogenesis. Bars = 5 µm. (F) Conidia from wild-type M. oryzae were germinated on agarose in the presence of residual solvent, ATS, or digoxin and stained with CFW after 4 h. Excess cell wall deposits are indicated with arrowheads. Bar = 10 µm.
Figure 3.
ATS associates with Tef2 in S. pombe and M. oryzae.
(A) Loss of SpTef2-function simulates ATS effect in S. pombe. Cell wall staining of the wild-type or tef2Δ S. pombe cells using CFW. Red arrowheads depict defective septal/cell wall deposition. Scale bar equals 10 micron. (B) Effect of digoxin on subcellular localization of SpTef2-RFP or Swo1-GFP in S. pombe cells. The strains expressing the indicated fusion proteins were stained with CFW and analysed by epifluorescence microscopy. Arrowheads show distinct aggregates of SpTef2-RFP. Bar = 10 µm. (C) Effect of ATS on localization of RFP-Tef2 in M. oryzae vegetative hyphae (upper panels; Scale Bar = 5 µm) and conidia (middle and lower panels; Bar represents 10 µm) co-stained with DAPI to aid visualization of nuclei. Arrowheads denote aberrant perinuclear aggregates and/or patches of RFP-Tef2. BF, Bright Field.
Figure 4.
ATS plays a role in ion homeostasis during pathogenesis in M. oryzae.
(A) ATS increases sensitivity of wild-type M. oryzae towards specific cations. Excess or permissive concentrations of Ca+2, Na+, or Mg+2 ion were added to the germinating wild-type conidia in the presence or absence of ATS. Arrows show delayed appressorial development (longer germ tubes) in the presence of ATS, which was otherwise seen only in the presence of excess concentration of the ions under control condition. Bar = 10 µm. (B) Sensitivity of the abc3Δ towards permissive concentratios of indicated cations. Arrows indicate delayed response in terms of longer germ tubes. Bars = 10 µm. (C) Effect of excess Ca+2 on appressorial function/host penetration efficiency in M. oryzae. Penetration efficiency was evaluated at 28 hpi by staining callose deposits with Aniline Blue. Arrowheads depict appressoria successful in host penetration. Bar = 10 µm. (D) Penetration efficiency of the appressoria was calculated as % appressorial function at 28 hpi. Data represent mean ± SEM from 3 individual experiments (n = 100 each per replicate). (E) Rice leaf sheaths were inoculated with wild-type M. oryzae in the presence of residual solvent or ATS for 24 h, and stained with aniline blue (right panels) for induced callose deposits (arrow) underneath the sites of host penetration (appressorial function). Asterisk shows occasional callose deposition. Bars = 10 µm. (F) Quantification of appressorial function at 30 hpi. The data represents mean ± SEM from 3 individual assays.
Figure 5.
SpTef2 function and the F-actin cytoskeleton in S. pombe.
(A) Sensitivity of tef2Δ S. pombe cells towards Ca+2 in the growth medium. Serial dilutions of the wild-type or tef2Δ cells were inoculated under indicated growth conditions. (B) Morphology and dynamics of GFP-labelled F-actin cytoskeleton in wild-type S. pombe treated with ATS, digoxin or Ca+2. The tef2Δ strain was analyzed in parallel. Arrowheads show excess accumulation of F-actin patches and/or short, spooling cables at the cell end(s). The maximum projection images shown here represent the compressed z-stack sections. Bar equals 10 µm.
Figure 6.
Exogenous ATS or digoxin alters the F-actin cytoskeleton in M. oryzae.
Morphology (A) and dynamics (B) of the F-actin patches in wild type M. oryzae expressing Abp1-RFP and treated with ATS, digoxin, or 0.1 M CaCl2. Arrowheads depict developing appressoria. Bars = 10 µm.
Figure 7.
Excess ATS or digoxin induces cell death in the host plants and reduces blast disease severity.
(A) Barley leaf explants were treated with residual solvent or ATS for 72 h, stained with trypan blue and observed using bright field optics. Arrowhead and arrows show visible (inset) and localized cell death, respectively, in the inoculation zone. (B) Transmission electron micrographs of residual solvent-, ATS- or digoxin-treated rice leaf explant stained with CeCl3 after 48 h of treatment. Arrowheads depict cerium perhydroxide granules and/or plasmolysis after ATS or digoxin treatment for 48 h. CW, cell wall; M, mitochondrion; and V, vacuole. Bars = 1 µm. (C) Transcript levels of Pathogenesis Related genes tested by real-time qRT-PCR in rice after 24 h of treatment. Data represent mean ± SEM of two independent experiments with three replicates each. Perox, peroxidase; Tub, tubulin. (D) Detached barley leaf pieces were inoculated with wild-type conidia in the absence or presence of 200 µM digoxin (DG). The disease symptoms were evaluated at 6 dpi. Arrowhead denotes disease lesion. The data represents observations from 3 independent experiments.
Figure 8.
Working model for the role of ATS in M. oryzae pathogenesis.
(A) Schematic representation of accumulation of ATS, in the wild type or abc3Δ appressoria, affecting host entry. (B) The figure illustrates a proposed crosstalk/mechanistic link between ATS accumulation and ion homeostasis, Tef2-function, and F-actin dynamics during M. oryzae pathogenesis.