Translation Initiation Factor eIF4E Positively Modulates Conidiogenesis, Appressorium Formation, Host Invasion and Stress Homeostasis in the Filamentous Fungi Magnaporthe oryzae

Abstract

Translation initiation factor eIF4E generally mediates the recognition of the 5'cap structure of mRNA during the recruitment of the ribosomes to capped mRNA. Although the eIF4E has been shown to regulate stress response in Schizosaccharomyces pombe positively, there is no direct experimental evidence for the contributions of eIF4E to both physiological and pathogenic development of filamentous fungi. We generated Magnaporthe oryzae eIF4E (MoeIF4E3) gene deletion strains using homologous recombination strategies. Phenotypic and biochemical analyses of MoeIF4E3 defective strains showed that the deletion of MoeIF4E3 triggered a significant reduction in growth and conidiogenesis. We also showed that disruption of MoeIF4E3 partially impaired conidia germination, appressorium integrity and attenuated the pathogenicity of ΔMoeif4e3 strains. In summary, this study provides experimental insights into the contributions of the eIF4E3 to the development of filamentous fungi. Additionally, these observations underscored the need for a comprehensive evaluation of the translational regulatory machinery in phytopathogenic fungi during pathogen-host interaction progression.

Keywords: Magnaporthe oryzae; eIF4E3; fungal pathogenesis; mRNA; translational regulation.

FIGURE 1

Domain architecture and phylogeny of M. oryzae eIF4E3 containing proteins. (A) Domain architecture of different fungal proteins containing eIF4E3 domains. (B) Shows maximum likelihood phylogeny constructed entirely for eIF4E3 domain sequence (amino acid) across different fungal species (Lp: Lomentospora prolificans, Sa: Scedosporium apiospermum, Tr: Trichoderma reesei, Tv: Trichoderma virens, Fg: Fusarium graminearum, Fo: Fusarium oxysporum, Fv: Fusarium verticillioides, Ff: Fusarium fujikuroi, Fp: Fusarium proliferatum, Bc: Botrytis cinerea, Ss: Sclerotinia sclerotiorum, Nc: Neurospora crassa, Nt: Neurospora tetrasperma, Sm: Sordaria macrospora, Af: Aspergillus fumigatus, Pz: Penicilliopsis zonata, Ab: Aspergillus brasiliensis, Mo: Magnaporthe oryzae, Sb: Sporothrix brasiliensis, Ssc: Sporothrix schenckii. (C) The expression level of MoeIF4E, MoeIF4E1, and MoeIF4E3 genes during different stages of host-pathogen interaction. Vegetative hyphae were used as the control stage and were assumed as unity (the expression level of MoeIF4E, MoeIF4E1, and MoeIF4E3 at hyphal stage = 1). (*) and (**) represent significant differences p < 0.05 and p < 0.01 respectively.

FIGURE 2

Targeted gene deletion of MoeIF4E3 in M. oryzae. (A) Schematic presentation of targeted disruption of MoeIF4E3 using homologs recombination approach. (B) Southern blot results showing successful replacement of MoeIF4E3 by a single insertion of Hygromycin phosphotransferase (hph) ORF at MoeIF4E3 loci.

FIGURE 3

Subcellular localization of MoeIF4E3 in M. oryzae and in planta. (A) The localization pattern of MoeIF4E3 in asexual spore, during spore germination and in invasive hyphae during plant infection. (B) Localization pattern of MoeIF4E3 with DAPI staining in asexual spore and invasive hyphae. Localization of MoeIF4E3-GFP was visualized using Nikon laser confocal microscopy. The scale bar is 20 and 40 μm.

FIGURE 4

MoeIF4E3 contributes significantly to the vegetative growth and conidiogenesis of M. oryzae. (A) Depicts the average colony diameter and morphology of ΔMoeIF4E3 mutants (ΔMoeif-24, ΔMoeif-72), complemented strain ΔMoeif-comp and the wild-type Guy11 strain cultured on CM, RBM, and MM for 10 days. (B) Statistical demonstration of the average growth rate of respective mutant strains and the wild-type Guy11 on CM, RBM, and MM for 10 days. (C) A statistical representation of conidiation capacity of MoeIF4E3 gene deletion strains and their complement relative to the Guy11 wild-type strain. (D) The reduction in conidiophore and conidia-bearing capabilities of conidiophores of the strains on rice bran medium. The statistical data were analyzed with a Microsoft Excel spreadsheet and GraphPad-prism7. Error bars represent the standard deviation from at least three independent replicates, and (*) and (**) represent significant differences (p < 0.05 and p < 0.01, respectively) between the wild type Guy11 and the respective knockout mutants according to ordinary one-way ANOVA. Bar, 10 μm.

FIGURE 5

Contribution of MoeIF4E3 to promote the pathogenicity of M. oryzae. (A) Represent the hyphae mediated blast lesions on intact and injured barley leaves inoculated with mycelial plugs from ΔMoeIF4E3 mutants (ΔMoeif-24, ΔMoeif-72), ΔMoeif-comp, and the wild-type strain. (B) Depicts the blast lesions due to spore inoculation of respective mutant, complemented strain, and Guy11 on intact and injured barley leaves. (C) Portrays disease lesions on 2 weeks old rice leaf from a susceptible rice cultivar CO39 after spraying with conidia suspensions of Guy11, ΔMoeIF4E3 mutants, and the complemented strain. Photographs were taken after 6 days of inoculation. (D) A graph for scoring lesion types per 1.5 cm² (1 to 4) from rice leaves inoculated with a spore suspension of Guy11, mutants, and complementation strains.

FIGURE 6

Contribution of MoeIF4E3 for invasive growth of M. oryzae in host tissues. (A) Development of appressorium-like structures and hyphal mediated penetration by the MoeIF4E3 mutants and wild-type strain inoculated on barley leaves at 48hpi. Scale bar, 20 μm. (B) In vivo penetration and consequent sheath tissue- colonization activity was assessed using fluorescence microscopy at 24- and 48-hpi. (C) Germination and appressorium formation activity by conidia obtained from the respective mutants and the wild type on an artificial hydrophobic surface. Leaf-sheaths of susceptible rice cultivar CO39 were inoculated with conidia suspension of the same strains used for hyphal mediated penetration on barley leaves. Bar 40 μm.

FIGURE 7

MoeIF4E3 deleted strains are sensitive to various stress in M. oryzae. (A) Colony morphology of Guy11 and MoeIF4E3 deletion mutants on CM plates amended with different stress-inducing osmolytes. The colonies were photographed at 10 days post-inoculation. (B) Statistical analysis of growth inhibition rate of respective mutants and the wild-type strains under different stresses. The percent inhibition was obtained using this formula: Inhibition rate = (the diameter of untreated strain – the diameter of treated strain)/ (the diameter of untreated strain × 100%). Statistical evaluation of percent inhibition was done using non-parametric ANOVA, using the GraphPad Prism7 software. Error bars represent standard deviation from three replications. (C) Expression profiling of different cell-wall-related enzymes during vegetative growth of MoeIF4E3 deleted mutant and Guy11 using qRT-PCR. Error bars represent standard deviation (SD). SD was calculated from three independent biological replications and three technical replicates, and significant levels were estimated using t-tests (*p < 0.05; **p < 0.01).

FIGURE 8

Relative fold change in the expression profile of translation initiation factors of M. oryzae in MoeIF4E3 KO mutants. The graph shows expression profiling of (A). eIF1 isomers, (B). eIF2 complex, (C). eIF3 complex, (D). eIF4F complex and eIF4B, (E). eIF5 complex, and (F). eIF6 in vegetative hyphae of MoeIF4E3 KO mutants. Wild type Guy11 vegetative hyphae were used as control and assumed unity = 1. Error bars represent standard deviation (SD). SD was calculated from three independent biological replications along with three technical replicates. (**p < 0.01 and *p < 0.05, t-test).