Type 2C Protein Phosphatase MoPtc6 Plays Critical Roles in the Development and Virulence of Magnaporthe oryzae

Abstract

Rice blast caused by Magnaporthe oryzae pathotype is the worst disease that leads to serious food insecurity globally. Understanding rice blast disease pathogenesis is therefore essential for the development of a blast disease mitigation strategy. Reverse phosphorylation mediated by phosphatases performs a vital function in the activation of diverse biological mechanisms within eukaryotic. However, little has been reported on the roles of PP2Cs in the virulence of blast fungus. In this current work, we deployed functional genomics and biochemical approaches to characterize type 2C protein phosphatase MoPtc6 in blast fungus. Deletion of MoPTC6 led to a drastic reduction in conidiophore development, conidia production, hyphal growth, and stress tolerance. Western blotting assay demonstrated that the phosphorylation level of MoOsm1 was decreased while MoMps1 was increased in the MoPtc6 deletion mutant, and comparative transcriptome assay revealed a higher number of expressed genes between mutant and wild type. Localization assay confirmed that MoPtc6 is sub-localized in the cytoplasm of mycelia, spores, and in the appressoria of M. oryzae. Furthermore, disruption of MoPTC6 impaired appressoria turgor pressure and glycogen utilization; more findings revealed attenuation of hyphal penetration and virulence upon deletion of MoPTC6. Generally, present findings suggested the role of MoPtc6 in the growth and virulence of M. oryzae.

Keywords: Magnaporthe oryzae; phosphorylation; protein phosphatases; stress tolerance; virulence.

Figure 1

Domain structure and phylogenetics of MoPtc6 and its orthologs. (a) The domain architecture of Ptc6 proteins from different fungi (PP2Cs domain and PP2C_SIG domain). The architecture was built using IBS software (IBS.biocuckoo.org, accessed on 20 November 2024). (b) Analysis of the phylogenetic relationship of Ptc6 proteins among different pathogenic fungi. The tree was produced by using MEGA X software version 10.1. (c) Three-dimensional structure of MoPtc6 generated from Swiss Model Software https://www.genecards.org/, accessed on 20 November 2024. Mo: Magnaporthe oryzae; Fo: Fusarium oxysporum; Fg: Fusarium gramineurum; Sc: Saccharomyces Cerevisiae; Bc: Botrytis Cinerea. Nc: Neurospora crassa.

Figure 2

Expression pattern and localization of MoPTC6 in M. oryzae. (a) Transcription patterns of MoPTC6 during interaction with the host; three-week-old rice seedlings were sprayed with Guy11, and the samples were harvested at different time points (4 h, 8 h, 12 h, 24 h, 48 h, and 72 h). (b) Sub-localization of MoPtc6-GFP in the mycelia, conidia, and appressoria. Images were taken by using confocal scanning microscope. (** p < 0.01).

Figure 3

MoPtc6 is important for growth and conidiation in M. oryzae. (a,b) Growth and colony diameters of the mutant and WT grown on solid complete media at 28 °C for ten days. (c,d) Quantification of conidia and conidiophores development in the ∆Moptc6 and WT. (e) Expression levels of conidiation-related genes in ∆Moptc6 compared to the WT. Asterisks show statistically significant differences analyzed by ANOVA (**, p < 0.01). Scale bar = 20 μm.

Figure 4

Sensitivity of ∆Moptc6 mutant and WT to various stresses. (a) Growth of the strains on CM media with 200 µg/mL CR, 0.01% SDS, 200 µg/mL CFW, 1 M KCl, 1 M NaCl, and 10 mM H₂O₂ at 28 °C for ten days. (b) Inhibition of growth of the strains due to the diverse fungal stressors. (c,d) Osm1 phosphorylation levels; MoOsm1 was bound to P38 Thr180/Tyr182 antibody and actin was used as a control. Quantification of band intensity was performed with ImageJ software. (e,f) Mps1 phosphorylation levels; MoMps1 were detected with antiphospho P44/42 antibody, and actin was used as a control. (f) Displayed bands’ intensity in mutant and WT. Asterisks show statistically significant differences (*, p < 0.05; **, p < 0.01).

Figure 5

∆Moptc6 promotes appressorium turgor pressure and mobilization of glycogen. (a,b) Indicates incipient cytorryhsis conducted to examine appressorium turgor pressure for ∆Moptc6, WT, and complementation strains. Appressoria were incubated for 24 h period and tested with various concentrations of glycerol, 1, 2, and 3 M, 5 min before viewed by using microscope. Scale bar = 20 μm. (c,d) Micrograph displayed glycogen utilization from conidium to appressorium and percentages of glycogen levels in conidium and in appressorium at different time points, 4 h, 8 h, 12 h, and 24 h, in mutant compared to WT. Asterisks displays statistically significant (*, p < 0.05; **, p < 0.01). Scale bar = 10 μm.

Figure 6

Virulence analysis of MoPtc6 in M. oryzae. (a,b) The figures displayed the pathogenicity analysis and lesions quantification assay for ∆Moptc6, WT, and complementation strain. Images were captured one-week post-spraying. (c,d) Indicate the lesion areas on barley leaves induced by ΔMoptc6, ΔMoptc6-com, and the WT strains’ mycelia plugs; lesion areas were calculated by using Image J software. (e) Observation of invasive hyphae in ten-day-old barley leaves infected with 5 × 10⁴ spores/mL and photographed after 24 h, 48 h, and 72 h post-inoculation. Asterisks displays statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Scale bar = 20 μm.

Figure 7

Expression of genes between ∆Moptc6 and WT. (a) Global view of gene expression levels in ∆Moptc6 and WT. This figure was generated by Jvenn software version 2.1.0. (b) Volcano figure plotted by using R software 4.3.3, indicating differentially expressed gene (DEG) distribution patterns in ∆Moptc6 mutant and WT. Red dots demonstrate downregulated genes, and blue dots represent up-regulated genes. (c) Displaying the KEGG pathways enriched by differentially expressed genes (DEGs) (p < 0.01).