Table 1.
The primers used in this study.
Figure 1.
Similarity of MoPEX19 homologs.
(A) Amino acid sequences of HsPex19 (XP_501231.1) from humans, GzPex19 (XP_390112.1) from Gibberella zeae (F. graminearum), NcPex19 (XP_961091.1) from N. crassa, AfPex19 (XP_754525.1) from Aspergillus fumigatus, and ScPex19 (CAA98630) from S. cerevisiae were aligned with ClustalW. Identical amino acids are highlighted against a black background, conserved residues are shown on a dark gray background, and similar residues on a light gray background. (B) Phylogenetic relationship of PEX19 homologs calculated with neighbor-joining method using the MEGA 5.0 program according to alignment.
Figure 2.
Complementation of Δscpex19 mutant by MoPEX19.
S. cerevisiae wild-type (BY4741), Δscpex19, and transformants BY4741+pYES2, BY4741+MoEX19, Δscpex19+pYES2 and Δscpex19+MoPEX19 were cultured on SD plates with glucose (A) or oleic acid (B). The expression of MoPEX19 restored the ability of Δscpex19 to grow on oleic acid.
Figure 3.
Sequential expression and cellular localization of MoPEX19.
(A) Relative transcript abundance of MoPEX19 during appressorial development. Transcript abundance normalized to β-tubulin gene was measured by quantitative PCR at each time point and compared with that in the non-incubated conidia. (B) Fluorescent microscopy of co-transformants with GFP–MoPEX19 and RFP–PTS1 in hyphae, conidia and appressoria of Magnaporthe oryzae. The fluorescence of GFP–MoPEX19 was predominantly in the cytoplasm with some punctate enhancement, which partially overlapped with the red fluorescence of RFP–PTS1. Bar = 10 μm.
Figure 4.
MoPEX19 gene deletion and mutant complementation.
(A) Diagram showing that the 1.22-kb MoPEX19 coding region was replaced by the 1.36-kb HPH cassette. An inner fragment within the deletion region was used as the probe for Southern blotting. Scale bar = 500 bp. (B) Total genomic DNA was isolated from the wild-type (lane 1), ectopic transformants (lanes 2 and 3), and potential Δmopex19 mutants (lanes 4–8, Δmopex19-11, Δmopex19-14, Δmopex19-20, Δmopex19-32 and Δmopex19-44), digested with SacI and subjected to Southern blotting. A 3943-bp hybridization band was detected in the wild-type, whereas 4971-bp bands were present in the five potential mutants, consistent with the gene deletion events. Ectopic transformant generated two bands, one of which was in equal size to the wild-type. (C) Gene transcription analysis of wild-type (Guy-11), Δmopex19-11 and Δmopex19-44, and Δmopex19-com by quantitative PCR. MoPEX19 transcripts were detected in similar abundance in the wild-type and complemented strains, but were completely undetectable in the mutants.
Figure 5.
Distribution of the peroxisomal matrix proteins and PMPs in Δmopex19 mutants.
Wild-type, Δmopex19-44 and complemented strains were transformed with GFP–PTS1, GFP–PTS2 and GFP–PMP47, respectively. Conidia of the transformants were harvested from 5-day-old CM plates and detected using confocal fluorescence microscopy. In the wild-type, the GFP–PTS1, GFP–PTS2 and GFP–PMP47 were all observed in punctate pattern, whereas in Δmopex19, GFP fluorescence was dispersed in the cytoplasm. In the complemented strains, GFP fluorescence was recovered into punctate patterns. Bar = 10 μm.
Figure 6.
Peroxisome and woronin bodies were absent in Δmopex19 mutants.
(A) Ultrastructure of the wild-type and Δmopex19 mutant. Hyphae and conidia from 7-day-old CM plates were analyzed by TEM. Peroxisomes, nuclei and mitochondria were detected in the wild-type (upper left), while the peroxisomes were absent in Δmopex19 (lower left). Typically 3–5 woronin bodies were seen beside the intercellular septum in the wild-type (upper right), but were undetectable in Δmopex19 (lower right). Bar = 0.2 μm. (B) Fluorescent localization of GFP-PTS1 and RFP-HEX1 in the wild-type and Δmopex19 mutant. Bar = 10 μm.
Figure 7.
Growth tests to determine the ability of Δmopex19 to utilize lipids as sole carbon source.
Wild-type, Δmopex19 and complemented strains were inoculated with a plug of mycelium on minimal medium (MM) supplemented with 0.5% (v/v) Tween 80, olive oil or 12 g/l sodium acetate and cultured at 28°C for 12 days.
Figure 8.
Tolerance of Δmopex19 to H2O2 and methyl viologen.
Wild-type, Δmopex19 and complemented strains were cultured on CM and CM supplemented with H2O2 (A) or methyl viologen (B) at 28°C for 5 days.
Figure 9.
Vegetative growth of wild-type, Δmopex19 mutants and complemented strains.
(A) The strains were cultured on CM at 28°C for 10 days. The radical growth, aerial hyphae growth and colonial pigmentation were decreased in Δmopex19 strains. (B) Measurement and statistic comparison of radial growth of the strains. Means and standard errors were calculated from three independent repeats. Single asterisks indicate significant differences to the wild-type (P<0.05).
Figure 10.
MoPEX19 deletion reduced the conidiation and conidial morphology and viability.
(A) Wild-type, Δmopex19 and complemented strains cultured on CM for 7 days were examined by light microscopy. Bar = 50 μm. (B) Statistical analysis of conidia produced on CM culture at 28°C for 10 days. Means and standard errors were calculated from three independent repeats. Double asterisks indicate significant differences to the wild-type (P<0.01). (C) Normal (I) and nonviable conidia (II) of Δmopex19 under light microscopy. Bar = 10 μm. (D) Viable conidia stained with FDA emitted green fluorescence while the dead ones did not. Bar = 10 μm. (E) Statistical analysis of viability of conidia cultured on CM at 28°C for 10 days. Means and standard errors were calculated from three independent repeats (at least 150 conidia of each strain were measured for each repeat). Double asterisks indicate significant differences to the wild-type (P<0.01).
Figure 11.
Conidial germination and appressorial formation of Δmopex19.
The conidial germination rates (A) and appressorial formation rates (B) of Δmopex19, wild-type and complemented strains on hydrophobic surface were calculated. (C) Conidia and appressoria incubated for 12 h. The appressoria of Δmopex19 were less pigmented than those of the control strains, and cellular leakage occurred in the germ tubes and appressoria of Δmopex19. (D) Nile red staining showed that more lipid residues were present in the conidia and germ tubes of Δmopex19 incubated for 24 h. (E) Cytorrhysis assay to compare the appressorial turgor genesis of Δmopex19 and the controls. The 24-h appressoria were soaked in glycerol and the rates of cytorrhysis were calculated. Means and standard errors were calculated from three independent repeats (at least 100 conidia of each strain were measured for each repeat). Single asterisks indicate significant differences at P<0.05 and double asterisks indicate P<0.01.
Figure 12.
Tolerance to CR to compare the integrity of the cell wall.
Wild-type, Δmopex19 and complemented strains were cultured on CM supplemented with 200 μg/ml CR for 5 days.
Figure 13.
Pathogenicity test of Δmopex19.
(A) Spray-inoculation with conidial suspension (1×105 conidia/ml) of Δmopex19, wild-type and complemented strains on 2-week-old rice cultivar CO39. The symptoms were recorded at 7 days post-inoculation. (B) Spray-inoculation with conidial suspension (2×104 conidia/ml) on 7-day-old barley cultivar ZJ-8. The symptoms were recorded at 4 days post-inoculation. Detached barley leaves inoculated with 5-mm mycelial plugs (C) or with 20-μl droplets of conidial suspensions (2×104 conidia/ml) (D) were cultured for 4 days. (E) Wounded barley leaves inoculated with 20-μl conidial suspensions (2×104 conidia/ml) were cultured for 4 days. (F) Droplet-inoculated barley leaves were sampled at 24 and 48 h post-inoculation, discolored, and examined under a light microscope. The conidia (C), appressoria (A) and invasive hyphae (IH) are marked. Bar = 20 μm.