A nonclassically secreted effector of Magnaporthe oryzae targets host nuclei and plays important roles in fungal growth and plant infection.

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases and poses a growing threat to food security worldwide. Like many other filamentous pathogens, rice blast fungus releases multiple types of effector proteins to facilitate fungal infection and modulate host defence responses. However, most of the characterized effectors contain an N-terminal signal peptide. Here, we report the results of the functional characterization of a nonclassically secreted nuclear targeting effector in M. oryzae (MoNte1). MoNte1 has no signal peptide, but can be secreted and translocated into plant nuclei driven by a nuclear targeting peptide. It could also induce hypersensitive cell death when transiently expressed in Nicotiana benthamiana. Deletion of the MoNTE1 gene caused a significant reduction of fungal growth and conidiogenesis, partially impaired appressorium formation and host colonization, and also dramatically attenuated the pathogenicity. Taken together, these findings reveal a novel effector secretion pathway and deepen our understanding of rice-M. oryzae interactions.

Magnaporthe oryzae Chloroplast Targeting Endo-ß-1,4-Xylanase I MoXYL1A Regulates Conidiation, Appressorium Maturation and Virulence of the Rice Blast Fungus.

Endo-ß-1,4-Xylanases are a group of extracellular enzymes that catalyze the hydrolysis of xylan, a principal constituent of the plant primary cell wall. The contribution of Endo-ß-1,4-Xylanase I to both physiology and pathogenesis of the rice blast fungus M. oryzae is unknown. Here, we characterized the biological function of two endoxylanase I (MoXYL1A and MoXYL1B) genes in the development of M. oryzae using targeted gene deletion, biochemical analysis, and fluorescence microscopy. Phenotypic analysis of ∆Moxyl1A strains showed that MoXYL1A is required for the full virulence of M. oryzae but is dispensable for the vegetative growth of the rice blast fungus. MoXYL1B, in contrast, did not have a clear role in the infectious cycle but has a critical function in asexual reproduction of the fungus. The double deletion mutant was severely impaired in pathogenicity and virulence as well as asexual development. We found that MoXYL1A deletion compromised appressorium morphogenesis and function, leading to failure to penetrate host cells. Fluorescently tagged MoXYL1A and MoXYL1B displayed cytoplasmic localization in M. oryzae, while analysis of MoXYL1A-GFP and MoXYL1B-GFP in-planta revealed translocation and accumulation of these effector proteins into host cells. Meanwhile, sequence feature analysis showed that MoXYL1A possesses a transient chloroplast targeting signal peptide, and results from an Agrobacterium infiltration assay confirmed co-localization of MoXYL1A-GFP with ChCPN10C-RFP in the chloroplasts of host cells. MoXYL1B, accumulated to the cytoplasm of the host. Taken together, we conclude that MoXYL1A is a secreted effector protein that likely promotes the virulence of M. oryzae by interfering in the proper functioning of the host chloroplast, while the related xylanase MoXYL1B does not have a major role in virulence of M. oryzae.

Corrigendum: eIF3k Domain-Containing Protein Regulates Conidiogenesis, Appressorium Turgor, Virulence, Stress Tolerance, and Physiological and Pathogenic Development of Magnaporthe oryzae Oryzae.

[This corrects the article DOI: 10.3389/fpls.2021.748120.].

eIF3k Domain-Containing Protein Regulates Conidiogenesis, Appressorium Turgor, Virulence, Stress Tolerance, and Physiological and Pathogenic Development of Magnaporthe oryzae Oryzae.

The eukaryotic translation initiation factor 3 (eIF3) complex consists of essential and non-essential sub-complexes. Non-essential eIF3 complex subunits, such as eIF3e, eIF3j, eIF3k, and eIF3l, modulate stress tolerance and enhance the lifespan of Neurospora crassa and Caenorhabditis elegans. However, there is limited knowledge of the role of the non-essential eIF3 sub-complex in the pathophysiological development of plant fungal pathogens. Here, we deployed genetic and biochemical techniques to explore the influence of a hypothetical protein containing eIF3k domain in Magnaporthe oryzae Oryzae (MoOeIF3k) on reproduction, hyphae morphogenesis, stress tolerance, and pathogenesis. Also, the targeted disruption of MoOeIF3k suppressed vegetative growth and asexual sporulation in ΔMoOeif3k strains significantly. We demonstrated that MoOeIF3k promotes the initiation and development of the rice blast disease by positively regulating the mobilization and degradation of glycogen, appressorium integrity, host penetration, and colonization during host-pathogen interaction. For the first time, we demonstrated that the eIF3k subunit supports the survival of the blast fungus by suppressing vegetative growth and possibly regulating the conversions and utilization of stored cellular energy reserves under starvation conditions. We also observed that the deletion of MoOeIF3k accelerated ribosomal RNA (rRNA) generation in the ΔMoOeif3k strains with a corresponding increase in total protein output. In summary, this study unravels the pathophysiological significance of eIF3k filamentous fungi. The findings also underscored the need to systematically evaluate the individual subunits of the non-essential eIF3 sub-complex during host-pathogen interaction. Further studies are required to unravel the influence of synergetic coordination between translation and transcriptional regulatory machinery on the pathogenesis of filamentous fungi pathogens.

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

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.

MoGLN2 Is Important for Vegetative Growth, Conidiogenesis, Maintenance of Cell Wall Integrity and Pathogenesis of Magnaporthe oryzae.

Glutamine is a non-essential amino acid that acts as a principal source of nitrogen and nucleic acid biosynthesis in living organisms. In Saccharomyces cerevisiae, glutamine synthetase catalyzes the synthesis of glutamine. To determine the role of glutamine synthetase in the development and pathogenicity of plant fungal pathogens, we used S. cerevisiae Gln1 amino acid sequence to identify its orthologs in Magnaporthe oryzae and named them MoGln1, MoGln2, and MoGln3. Deletion of MoGLN1 and MoGLN3 showed that they are not involved in the development and pathogenesis of M. oryzae. Conversely, ΔMogln2 was reduced in vegetative growth, experienced attenuated growth on Minimal Medium (MM), and exhibited hyphal autolysis on oatmeal and straw decoction and corn media. Exogenous l-glutamine rescued the growth of ΔMogln2 on MM. The ΔMogln2 mutant failed to produce spores and was nonpathogenic on barley leaves, as it was unable to form an appressorium-like structure from its hyphal tips. Furthermore, deletion of MoGLN2 altered the fungal cell wall integrity, with the ΔMogln2 mutant being hypersensitive to H2O2. MoGln1, MoGln2, and MoGln3 are located in the cytoplasm. Taken together, our results shows that MoGLN2 is important for vegetative growth, conidiation, appressorium formation, maintenance of cell wall integrity, oxidative stress tolerance and pathogenesis of M. oryzae.

Bayogenin 3-O-cellobioside confers non-cultivar-specific defence against the rice blast fungus Pyricularia oryzae.

Rice cultivars from japonica and indica lineage possess differential resistance against blast fungus as a result of genetic divergence. Whether different rice cultivars also show distinct metabolomic changes in response to P. oryzae, and their role in host resistance, are poorly understood. Here, we examine the responses of six different rice cultivars from japonica and indica lineage challenged with P. oryzae. Both susceptible and resistant rice cultivars expressed several metabolites exclusively during P. oryzae infection, including the saponin Bayogenin 3-O-cellobioside. Bayogenin 3-O-cellobioside level in infected rice directly correlated with their resistant attributes. These findings reveal, for the first time to our knowledge that besides oat, other grass plants including rice produces protective saponins. Our study provides insight into the role of pathogen-mediated metabolomics reprogramming in host immunity. The correlation between Bayogenin 3-O-Cellobioside levels and blast resistance suggests that engineering saponin expression in cereal crops represents attractive and sustainable disease management.

Disruption of putative short-chain acyl-CoA dehydrogenases compromised free radical scavenging, conidiogenesis, and pathogenesis of Magnaporthe oryzae.

Short-chain acyl-CoA dehydrogenase (Scad) mediated ß-oxidation serves as the fastest route for generating essential energies required to support the survival of organisms under stress or starvation. In this study, we identified three putative SCAD genes in the genome of the globally destructive rice blast pathogen Magnaporthe oryzae, named as MoSCAD1, MoSCAD2, and MoSCAD3. To elucidate their function, we deployed targeted gene deletion strategy to investigate individual and the combined influence of MoSCAD genes on growth, stress tolerance, conidiation and pathogenicity of the rice blast fungus. First, localization and co-localization results obtained from this study showed that MoScad1 localizes to the endoplasmic reticulum (ER), MoScad2 localizes exclusively to the mitochondria while MoScad3 partially localizes to the mitochondria and peroxisome at all developmental stages of M. oryzae. Results obtained from this investigation showed that the deletion of MoSCAD1 and MoSCAD2 caused a minimal but significant reduction in the growth of ΔMoscad1 and ΔMoscad2 strains, while, growth characteristics exhibited by the ΔMoscad3 strain was similar to the wild-type strain. Furthermore, we observed that deletion of MoSCAD2 resulted in drastic reduction in conidiation, delayed conidia germination, triggered the development of abnormal appressorium and suppressed host penetration and colonization efficiencies of the ΔMoscad1 strain. This study provides first material evidence confirming the possible existence of ER ß-oxidation pathway in M. oryzae. We also infer that mitochondria ß-oxidation rather than peroxisomal and ER ß-oxidation play an essential role in the vegetative growth, conidiation, appressorial morphogenesis and progression of pathogenesis in M. oryzae.