Type 2C Protein Phosphatases MoPtc5 and MoPtc7 Are Crucial for Multiple Stress Tolerance, Conidiogenesis and Pathogenesis of Magnaporthe oryzae

Abstract

Protein kinases and phosphatases catalyze the phosphorylation and dephosphorylation of their protein substrates, respectively, and these are important mechanisms in cellular signal transduction. The rice blast fungus Magnaporthe oryzae possesses 6 protein phosphatases of type 2C class, including MoPtc1, 2, 5, 6, 7 and 8. However, only very little is known about the roles of these phosphatases in filamentous fungi. Here in, we deployed genetics and molecular biology techniques to identify, characterize and establish the roles of MoPtc5 and MoPtc7 in M. oryzae development and pathogenicity. We found that during pathogen-host interaction, MoPTC7 is differentially expressed. Double deletion of MoPTC7 and MoPTC5 suppressed the fungal vegetative growth, altered its cell wall integrity and reduced its virulence. The two genes were found indispensable for stress tolerance in the phytopathogen. We also demonstrated that disruption of any of the two genes highly affected appressorium turgor generation and Mps1 and Osm1 phosphorylation levels. Lastly, we demonstrated that both MoPtc5 and MoPtc7 are localized to mitochondria of different cellular compartments in the blast fungus. Taken together, our study revealed synergistic coordination of M. oryzae development and pathogenesis by the type 2C protein phosphatases.

Keywords: blast fungus; pathogenicity; phosphorylation; protein phosphatases.

Figure 1

Domain architecture and phylogenetic analysis of Ptc5 and Ptc7 in different fungi. (A) Domain architecture of Ptc5 and Ptc7 orthologs in in different organisms. (B) A phylogenetic tree generated using MEGA X software and a UPGMA test with 1000 Boot straps replications was conducted. Fg: Fusarium gamineum; Fo: Fusarium oxyporum; Nc: Neurospora crassa; Mo: Magnaporthe oryzae; Bc: Botytis cinera; and Sc: Saccharomyces cerevisae.

Figure 2

Transcriptional expression patterns of M. oryzae type 2C genes during interaction with host. Quantitative realtime (q-PCR) was used to check the genes expressions at the indicated time points. The 4 h time point was taken as background control with tubulin gene serving as internal control. The experiments were repeated three times with three technical replicates each. Error bars represent standard deviations while asterisks show significant differences at different p-values (* p < 0.1; ** p < 0.01). The data were analyzed by one-way ANOVA using Tukey’s multiple-comparison test.

Figure 3

Synergistic roles of MoPtc5 and MoPtc7 in M. oryzae vegetative growth of. (A) The growths of the various strains on different media plates. CM, SYM and RBM were used for the growth of the fungal mycelia and incubated in the dark for 10 days at 28 °C. (B) Bar graphs showing the colony diameters of the various strains on the indicated media. Asterisks show significant differences at different p-values (* p < 0.1; ** p < 0.01) while error bars represent standard deviations from three independent replicates. The data were analyzed by one-way ANOVA using Tukey’s multiple-comparison test.

Figure 4

MoPTC5 deletion causes reduction in conidiation and conidiophores formation of M. oryzae. (A) Conidia production of the strains grown on RBM for 10 days period. (B) Conidiophores formation from Guy11, ΔMoptc5, and ΔMoptc7ΔMoptc5 strains on RBM. ΔMoptc7ΔMoptc5 double mutant shows highly reduced conidiophores formation. (C) Relative expressions of genes involved in M. oryzae conidiation. The various strains were inoculated in liquid CM and incubated at 28 °C under constant shaking at 110 rpm for 3 days, after which total RNAs were extracted for qPCR analysis. Tubulin gene was used as a control. Results are means obtained from three independent replicates. Error bars represent standard deviations while asterisks show significant differences at different p-values (*, p < 0.1; **, p < 0.01; ***, p < 0.001)). The data were analyzed by one-way ANOVA using Tukey’s multiple-comparison test.

Figure 5

MoPtc5 and MoPtc7 are involved in stress resistance in the blast fungus. (A) Colony diameters of the strains were measured 10 days post inoculation on CM media containing cell wall-perturbing agents. (B) Growth inhibition rates of the mutants due to the effects of the cell wall stressing substances (Inhibition rate = (the diameter of untreated strain − the diameter of treated strain)/(the diameter of untreated strain) × 100%. Three independent replicates were involved. Error bars represent standard deviations while asterisks show significant differences at different p-values (** p < 0.01). The data were analyzed by one-way ANOVA using Tukey’s multiple-comparison test. (C) MoMps1 phosphorylation level in ΔMoptc5, ΔMoptc7 and ΔMoptc7ΔMoptc5 mutants. (D) Comparison of band intensities for MoMps1 phosphorylation levels in the various strains as calculated using imaging software. Despite the obvious increase in the phosphorylation levels, the differences were not significant compared to the wild type.

Figure 6

Deletion of MoPTC5 and MoPTC7 does not affect cell wall thickness. (A) Transverse sections of hyphae from Guy11, ΔMoptc5, ΔMoptc7 and ΔMoptc5ΔMoptc7 strains following transmission electron microscopy. (B) Graphs indicating differences in hyphal cell wall thickness between the mutants and Guy11. (C) The transcript levels of chitin encoding genes using actin gene as internal control. Three replicates were involved. Error bars represent standard deviations while asterisks show significant differences at different p-values (* p < 0.1; ** p < 0.01). The data were analyzed by one-way ANOVA using Tukey’s multiple-comparison test.

Figure 7

MoPtc5 and ΔMoptc7ΔMoptc5 are indispensable for tolerance to osmotic and oxidative stresses in the blast fungus. (A) Colony diameters of the various strains were measured 10 days after inoculation on media containing 1 M NaCl, 1 M KCl, 1 M Sorbitol, 5 mM, 10 mM H₂O₂. (B) Inhibition rate of the oxidative and osmotic stressors. Inhibition rate = (the diameter of untreated strain − the diameter of treated strain)/(the diameter of untreated strain × 100%). There were three replicates. Error bars represent standard deviations while asterisks show significant differences at different p-values (* p < 0.1; ** p < 0.01). The data were analyzed by one-way ANOVA using Tukey’s multiple-comparison test. (C) Osm1 protein abundance in ΔMoptc5, ΔMoptc7, ΔMoptc7ΔMoptc5 mutants. (D) Comparison of band intensities for Osm1/Actin phosphorylation levels in the various strains as calculated using imaging software.

Figure 8

Deletion of MoPTC5 and MoPTC7 delayed glycogen mobilization from conidia to appressoria and reduced appressorium turgor pressure. (A,B) represent the percentages of appressorium exhibiting incipient cytorryhsis after being treated with glycerol. (C) Mobilization of glycogen in the mutants and Guy11 at different time points. (D,E) Micrographs showing percentage of conidia and appressoria containing glycogen.

Figure 9

MoPtc5 and MoPtc7 play important roles in M. oryzae virulence. (A) Pathogenicity assay for Guy11, ΔMoptc5, ΔMoptc7 and ΔMoptc7ΔMoptc5 on barley leaves using drops of conidia suspension. (B) The pathogenicity of ΔMoptc5, ΔMoptc7 and ΔMoptc7ΔMoptc5 mutants in comparison to Guy11 using conidia sprays on 3 weeks old barley leaves. (C) Extents of lesions development as blast symptoms caused by the indicated strains, Level 1 (uniform dark brown pinpoint lesions without visible centers), Level 2 (small lesions with distinct centers surrounded by a dark brown margin, 1 mm in diameter), Level 3 (small eyespot lesions approximately 2 mm in length with tan centers surrounded by dark brown margins), Level 4 (intermediate size eyespot lesions, approximately 3–4 mm in length), Level 5 (large eyespot lesions approximately 5 mm in length). (D) Cuticle penetration defects of the mutants.

Figure 10

Localization of MoPtc5 and MoPtc7 in rice blast fungus. (A,B) displays co-localization of MoPtc5, MoPtc7 in hyphae, conidia, appressorium and in plantae as it co-localizes with ATP1_RFP (Red Fluorescence Protein) mitochondria marker. (C,D) graphs representing intensity fluorescence and distance for MoPtc5 and MoPtc7 co-localization. The pictures were taken by a NikonA1 confocal microscope (Scale bar = 20 µm).