Table 1.
The primers used in this study.
Figure 1.
Predicted amino acid sequence and phylogenetic analysis of MoPex5.
(A) Alignment of predicted amino acid sequence of MoPex5 and its homologues. The amino acid sequences of GzPex5 (EAA68640) from Gibberella zeae, NcPex5 (EAA36111) from Neurospora crassa, AnPex5 (CBF85028) from Aspergillus nidulans, PcPex5 (AY366189) from Penicillium chrysogenum and ScPex5 (CAA89730) from Saccharomyces cerevisiae were aligned with Clustal W. Identical amino acids are highlighted against a black background, conserved residues are shown on a dark gray background, and similar amino acids are shown on a light gray background. Four probable TPR domains of MoPex5 were indicated with a line on the top of the sequence. (B) Phylogenetic relationship among MoPex5 and its homologues. The phylogenetic trees of the amino acid sequences were created using the MEGA 5.0 program according to the result of alignment.
Figure 2.
Mutation of MoPEX5 blocked the PTS1 peroxisomal import pathways and affected the lipid utilization.
(A) The peroxisomal localization of the RFP-PTS1 and GFP-PTS2 in the wild type, Δmopex5 and Δmopex7. Conidia of the transformants Guy11/RFP-PTS1::GFP-PTS2, Δmopex5/RFP-PTS1::GFP-PTS2 and Δmopex7/RFP-PTS1::GFP-PTS2 harvested from 8-day-old CM plates were observed under a confocal fluorescence microscopy. (B) Lipid utilization of the wild type, Δmopex5 and Δmopex7. The Strains were cultured on medium with glucose, olive oil or Tween 80 as sole carbon source for 12 d at 28°C.
Figure 3.
Host infection of the wild type, Δmopex5 and Δmopex7.
(A) The symptoms on the rice cultivar CO39, Brachypodium distachyon Bd21 and Barley ZJ-8 after spray inoculation with conidial suspension in 2×104 and 2×105 conidia/ml and the for 5 d. (B) The inoculated barley leaves were discolored, strained with aniline blue and observed under a fluorescent microscope. C, conidia; A, appressoria; IH, infection hyphae. The four-pointed star indicated hyphae that passed through the cell wall and invaded the neighbor cell. (C) The frequencies of penetration pegs formed by the appressoria after 24 h and 36 h on barley leaves were statistically compared.
Figure 4.
Vegetative growth and mycelial melanization of the wild type, Δmopex5 and Δmopex7.
(A) The colonies of the strains cultured on the complete media (CM) plates for 10 d. (B) Colonial diameters of the strains were measured and statistical analyzed after cultured on CM, CM-C and CM-N for 4 d and 10 d. (C) The conidia in 1×104/ml of the strains were cultured in liquid CM shaking at 150 rpm at 28°C with 24 h day length, then the colors of the cultures were observed after 5 d.
Figure 5.
Conidial generation and viability of Δmopex5, Δmopex7 and the wild type.
(A) The development of the conidia and conidiophores of the strains grown on complete medium. (B) Statistical analysis of conidial production. The conidia produced by the strains grown on complete medium for 9 days were collected and counted. (C) Fluorescence staining of the conidia of the strains. The conidia were strained with fluorescein diacetate (FDA) and Nile red then observed under a fluorescent microscope. a, the viable conidia stained with FDA emitted bright green fluorescence while the dead ones did not; b, a viable conidium stained with FDA and Nile red; c and d, nonviable conidia strained with FDA and Nile red. (D) Percentages of nonviable conidia of the strains on complete media cultured for different time (E) Germination rates of conidia of the strains harvested from 8-day- old complete medium and incubated on inducible membrane.
Figure 6.
Appressorial morphology of the wild type, Δmopex5 and Δmopex7.
(A) Under the light microscope, the 24 h appressoria of Δmopex5 were less pigmented than the wild type and Δmopex7, and numerous residual droplets (indicated by the arrows) presented in the conidia and germ tubes Δmopex5 and Δmopex7 the residues. (B) TEM analysis of 24 h appressoria. The melanin layers (indicated by the arrows) were detected in the wild type and Δmopex7 but absent in Δmopex5. Bars = 0.5 µm.
Figure 7.
Cell wall integrity test of the wild type, Δmopex5 and Δmopex7.
The 5-mm mycelia discs of the strains were cultured on CM or CM supplied with 30 ug/ml Calcofluor white (CM+CFW) for 4 d, then the colonial diameters were measured and the relative inhibition rates of CFW to vegetative growth were calculated. (A) The colonies of the strain cultured for 4 d. (B) the relative inhibition rate of Δmopex5 was higher significantly than that of the wild type and Δmopex7. The relative inhibition rate (%) = [the colonial diameter (DIC) on CM – DIC on (CM+CFW)]/(DIC on CM –5).
Figure 8.
The lipids mobilization and turgor genesis of the wild type, Δmopex5 and Δmopex7.
(A) Numerous lipid residuals (indicated by the arrows) in the 24 h appressoria of Δmopex5 and Δmopex7 were visualized by Nile red staining. To improve the staining efficiency, 10 ppm tricyclazole was added to the conidial suspensions [65]. (B) cytorrhysis and plasmolysis test of 24 h appressoria. The appressoria were soaked in gradient concentrations of Glycerol. The percentages of cytorrhysis and plasmolysis occured were counted and statistically compared. The error bars on the top of the white columns represent standard deviations of plasmolysis.
Figure 9.
The virulence of Δmopex5 and Δmopex7 were offset partially by supplement with glucose or cuticle removal.
(A) The intact leaves of barley ZJ-8 were droplet inoculated with conidial suspensions in 1×105 conidia/ml supplemented with 2.5% glucose on intact leaves of Barley ZJ-8. The symptoms were recorded at 5 d post inoculation. (B) Conidial suspensions in 1×105 conidia/ml were droplet inoculated on the intact and wounded leaves of Barley ZJ-8. The symptoms were recorded at 5 d post inoculation.
Figure 10.
Suppression of H2O2 to the conidial germination of Δmopex5, Δmopex7 and the wild type.
(A) the medium discs containing 0.5 M H2O2 formed larger restraining rings on the germinated conidia of Δmopex5 and Δmopex7 than those of the wild type; (B) The diameters of the restraining rings on the strains were measured and statistical compared.
Figure 11.
Complementation of Δmopex5 and Δmopex7 mutants by reintroduction of MoPEX5 and MoPEX7 genes.
(A) The Δmopex5 and Δmopex7 mutants, the wild strain Guy11 and complemented strains of Δmopex5 (P5R) and Δmopex7 (P7R) were cultured on minimal medium with olive oil as sole carbon source. (B) Leaves from rice plant of cultivar CO39 inoculated with conidial suspensions of the wild type, and complemented strains P5R and P7R.
Figure 12.
Expression of MoPEX5 and MoPEX7 during conidial germination and appressorium development.
Promoter regions of MoPEX5, MoPEX7 and were respectively fused to the GFP green fluorescent protein-encoding allele and introduced into M. oryzae strain Guy 11. Transformants carrying single-copy integrations were identified. Fluorescence of vegetative hyphae and during the appressorium development of germinating conidia on a hydrophobic surface of the transformants were analyzed and compared by epifluorescence microscopy. Control experiment in which a transformant expressing GFP under control of MPG1 [34] gene was allowed to undergo appressorium formation for 24 h.