One of Three Pex11 Family Members Is Required for Peroxisomal Proliferation and Full Virulence of the Rice Blast Fungus Magnaporthe oryzae

Abstract

Peroxisomes play important roles in metabolisms of eukaryotes and infection of plant fungal pathogens. These organelles proliferate by de novo formation or division in response to environmental stimulation. Although the assembly of peroxisomes was documented in fungal pathogens, their division and its relationship to pathogenicity remain obscure. In present work, we analyzed the roles of three Pex11 family members in peroxisomal division and pathogenicity of the rice blast fungus Magnaporthe oryzae. Deletion of MoPEX11A led to fewer but enlarged peroxisomes, and impaired the separation of Woronin bodies from peroxisomes, while deletion of MoPEX11B or MoPEX11C put no evident impacts to peroxisomal profiles. MoPEX11A mutant exhibited typical peroxisome related defects, delayed conidial germination and appressoria formation, and decreased appressorial turgor and host penetration. As a result, the virulence of MoPEX11A mutant was greatly reduced. Deletion of MoPEX11B and MoPEX11C did not alter the virulence of the fungus. Further, double or triple deletions of the three genes were unable to enhance the virulence decrease in MoPEX11A mutant. Our data indicated that MoPEX11A is the main factor modulating peroxisomal division and is required for full virulence of the fungus.

Fig 1. Sequence similarities of Pex11 family members.

(A) Sequence alignment of Pex11A proteins from various organisms. The alignment was performed with Clustal W module in MEGA software vision 5 and formatted by GeneDoc (http://www.psc.edu/biomed/genedoc). The identical amino acids are highlighted with black background, the conserved residues with dark gray background, and the similar amino acids with light gray background. The putative amphipathic helices are underlined with black arrows, and hydrophobic regions with hollow arrows. (B) The Neighbor-joining phylogenetic tree of Pex11 proteins constructed using MEGA software. The distance scale represents the differences between the sequences, with 0.1 indicating a 10% difference. Af, Aspergillus fumigatus; An, A. nidulans; Ao, A. oryzae; At, Arabidopsis thaliana; Gz, Gibberella zeae; Hs, Homo sapiens; Mo, M. oryzae; Nc, N. crassa; Pc, P. chrysogenum; Sc, S. cerevisiae.

Fig 2. Expression of MoPEX11A was assessed by GFP expression strategy and quantitative PCR.

(A) The GFP cassette was expressed under the MoPEX11A promoter in M. oryzae wild type and detected by CLSM in various development stages, with the GFP expressed under MPG1 promoter as a control. FAM (fatty acid media), minimal medium with 1% olive oil as sole carbon source. Bar = 5 μm. (B) Quantitative PCR analysis of the relative transcription levels of MoPEX11A in the cultures on the minimal media supplemented with 1% Glucose (MM), Triolein, olive oil, tween 80 or sodium acetate as sole carbon source. (C) The relative transcription levels of MoPEX11A during the conidial germination and appressorial formation.

Fig 3. Pathogenicity test of the MoPEX11 deletion mutants.

(A) Spray-inoculation with conidial suspension (1×10⁵ conidia/ml) of the wild type, KO11A, KO11B, KO11C and complemented strains 11A-com on 2-week-old rice cultivar CO39. The symptoms were recorded at 7 days post-inoculation (dpi). (B), Spray-inoculation with conidial suspension (2×10⁴ conidia/ml) on 7-day-old barley cultivar ZJ-8, recorded at 4 dpi. The 20 μl droplets of conidial suspensions in gradient concentrations were inoculated on intact (C) and wounded barley leaves (D) and cultured for 4 days. Droplet-inoculation with conidial suspensions supplied with 2.5% Glucose was performed on intact (E) and wounded barley leaves (F) and cultured for 4 days. (G) Microscopic analysis of the infection process of the mutants. Droplet-inoculated barley leaves were sampled at 18, 30 and 48 h post-inoculation, discolored and examined microscopely. Bar = 40 μm. (H) Statistic analysis of infection rates of the appressoria of the wild type and the mutants. Standard deviations are indicated by the error bars. Asterisks indicate significant differences at p = 0.05.

Fig 4. Peroxisomal profiles in the wild type and the MoPEX11 mutants.

(A) CLSM analysis of peroxisomes visualized with GFP-PTS1 in the wild type Guy11, KO11A, KO11B and KO11C. Bar = 5 μm. The sizes (B) and numbers (C) of the peroxisomes in the cells of the strains were statistically compared. Standard deviations are indicated by the error bars. For each strain, more than 100 cells were counted. Asterisks indicate significant differences at p = 0.05. (D) TEM analysis of the Peroxisomes (indicated by arrow heads) in Guy11 and KO11A.

Fig 5. Analysis of Woronin bodies and peroxisomes in Guy11 and KO11A by dual fluorescence strategy.

Peroxisomes and Woronin bodies were visualized respectively with GFP-PTS1 and mCherry-Hex1 and detected by CLSM. (A) The profiles of peroxisomes and Woronin bodies in hypha cultured on complete media (CM). (B) Magnified image showing the association of the Woronin bodies to the peroxisomes in KO11A. Bars = 2 μm. (C) Statistically analysis of the Woronin bodies separated, overlaid and associated to the peroxisomes.

Fig 6. Lipid utilization and cell wall integrity test of the wild type and MoPEX11 mutants.

The strains were cultured on minimal medium with 1% Glucose, olive oil or Tween 80 as sole carbon source at 28°C for 8 d (A) and the colonial diameters were measured (B). The strains were cultured on CM supplemented with 200 μg/ml Congo red (CR) for 4 d (C), the colonial diameters were measured and the relative inhibition rates were compared (D). Standard deviations are indicated by the error bars. Asterisks indicate significant differences at p = 0.05.

Fig 7. Lipid utilization and pathogenicity test of the single, double and triple mutants of MoPEX11 genes.

The strains were cultured on minimal medium with 1% Tween 80 as sole carbon source at 28°C for 5 d (A) and the colonial diameters were statistically compared (B). (C) Conidial suspension (1×10⁵ conidia/ml) of the strains were sprayed on 2-week-old rice cultivar CO39 and recorded at 7 dpi. (D) The numbers of lesions on 5-mm length leaf were counted and statistically analyzed. Different letters indicate significant differences between groups (p < 0.05).

Fig 8. Impacts of the deletion of MoPEX11 genes to the transcription of related genes.

The relative abundance of the transcripts were detected by quantitative PCR with the total RNA from cultures on CM as template and β-tubulin gene (MGG00604) gene as internal reference. MoPEX3, MGG_06424; DNM1, MGG_06361; FIS1, MGG_06075.