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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.
Assuntos
Ascomicetos , Magnaporthe , Oryza , Ascomicetos/metabolismo , Núcleo Celular/metabolismo , Transporte Biológico , Sinais Direcionadores de Proteínas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Esporos Fúngicos/metabolismoRESUMO
Dihydroxynapthalene-(DHN) and L-dihydroxyphenylalanine (L-DOPA) are two types of dominant melanin in fungi. Fungal melanins with versatile functions are frequently associated with pathogenicity and stress tolerance. In rice blast fungus, Magnaporthe oryzae, DHN melanin is essential to maintain the integrity of the infectious structure, appressoria; but the role of the tyrosinase-derived L-DOPA melanin is still unknown. Here, we have genetically and biologically characterized a tyrosinase gene (MoTyr) in M. oryzae. MoTyr encodes a protein of 719 amino acids that contains the typical CuA and CuB domains of tyrosinase. The deletion mutant of MoTyr (ΔMoTyr) was obtained by using a homologous recombination approach. Phenotypic analysis showed that conidiophore stalks and conidia formation was significantly reduced in ΔMoTyr. Under different concentrations of glycerol and PEG, more appressoria collapsed in the mutant strains than in the wild type, suggesting MoTyr is associated with the integrity of the appressorium wall. Melanin measurement confirmed that MoTyr loss resulted in a significant decrease in melanin synthesis. Accordingly, the loss of MoTyr stunted the conidia germination under stress conditions. Importantly, the MoTyr deletion affected both infection and pathogenesis stages. These results suggest that MoTyr, like DHN pigment synthase, plays a key role in conidiophore stalks formation, appressorium integrity, and pathogenesis of M. oryzae, revealing a potential drug target for blast disease control.
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Primary inoculum that survives overwintering is one of the key factors that determine the outbreak of plant disease. Pathogenic resting structures, such as chlamydospores, are an ideal inoculum for plant disease. Puzzlingly, Magnaporthe oryzae, a devastating fungal pathogen responsible for blast disease in rice, hardly form any morphologically changed resting structures, and we hypothesize that M. oryzae mainly relies on its physiological alteration to survive overwintering or other harsh environments. However, little progress on research into regulatory genes that facilitate the overwintering of rice blast pathogens has been made so far. Serine threonine protein kinase AGC/AKT, MoSch9, plays an important role in the spore-mediated pathogenesis of M. oryzae. Building on this finding, we discovered that in genetic and biological terms, MoSch9 plays a critical role in conidiophore stalk formation, hyphal-mediated pathogenesis, cold stress tolerance, and overwintering survival of M. oryzae. We discovered that the formation of conidiophore stalks and disease propagation using spores was severely compromised in the mutant strains, whereas hyphal-mediated pathogenesis and the root infection capability of M. oryzae were completely eradicated due to MoSch9 deleted mutants' inability to form an appressorium-like structure. Most importantly, the functional and transcriptomic study of wild-type and MoSch9 mutant strains showed that MoSch9 plays a regulatory role in cold stress tolerance of M. oryzae through the transcription regulation of secondary metabolite synthesis, ATP hydrolyzing, and cell wall integrity proteins during osmotic stress and cold temperatures. From these results, we conclude that MoSch9 is essential for fungal infection-related morphogenesis and overwintering of M. oryzae.
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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.
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The interconversion of CO2 and HCO3 - catalyzed by carbonic anhydrases (CAs) is a fundamental biochemical process in organisms. During mammalian-pathogen interaction, both host and pathogen CAs play vital roles in resistance and pathogenesis; during planta-pathogen interaction, however, plant CAs function in host resistance but whether pathogen CAs are involved in pathogenesis is unknown. Here, we biologically characterized the Magnaporthe oryzae CA (MoCA1). Through detecting the DsRED-tagged proteins, we observed the fusion MoCA1 in the mitochondria of M. oryzae. Together with the measurement of CA activity, we confirmed that MoCA1 is a mitochondrial zinc-binding CA. MoCA1 expression, upregulated with H2O2 or NaHCO3 treatment, also showed a drastic upregulation during conidiogenesis and pathogenesis. When MoCA1 was deleted, the mutant ΔMoCA1 was defective in conidiophore development and pathogenicity. 3,3'-Diaminobenzidine (DAB) staining indicated that more H2O2 accumulated in ΔMoCA1; accordingly, ATPase genes were downregulated and ATP content decreased in ΔMoCA1. Summarily, our data proved the involvement of the mitochondrial MoCA1 in conidiogenesis and pathogenesis in the rice blast fungus. Considering the previously reported HCO3 - transporter MoAE4, we propose that MoCA1 in cooperation with MoAE4 constitutes a HCO3 - homeostasis-mediated disease pathway, in which MoCA1 and MoAE4 can be a drug target for disease control.
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[This corrects the article DOI: 10.3389/fpls.2021.748120.].
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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.
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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.
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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.
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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.
Assuntos
Ascomicetos , Magnaporthe , Oryza , Doenças das PlantasRESUMO
Flowering is the first committed step of plant sexual reproduction. While the developing flower is a strong sink requiring large quantity of sugars from photosynthetic source tissues, this process is under-temper-spatially controlled via hormone signaling pathway and nutrient availability. Sugar transporters SUT/SUC and SWEET mediate sugars movement across membranes and play a significant role in various physiological processes, including reproductive organ development. In Petunia axillaris, a model ornamental plant, 5 SUT/SUC and 36 SWEET genes are identified in the current version of the genome. Analysis of their gene structure and chromosomal locations reveal that SWEET family is moderately expanded. Most of the transporter genes are abundantly expressed in the flower than in other organs. During the five flower developmental stages, transcript levels of PaSUT1, PaSUT3, PaSWEET13c, PaSWEET9a, PaSWEET1d, PaSWEET5a and PaSWEET14a increase with the maturation of the flower and reach their maximum in the fully open flowers. PaSWEET9c, the nectar-specific PhNEC1 orthologous, is expressed in matured and fully opened flowers. Moreover, determination of sugar concentrations and phytohormone dynamics in flowers at the five developmental stages shows that glucose is the predominant form of sugar in young flowers at the early stage but depletes at the later stage, whereas sucrose accumulates only in maturated flowers prior to the corolla opening. On the other hand, GA3 content and to a less extent IAA and zeatin decreases with the flower development; however, JA, SA and ABA display a remarkable peak at mid- or later flower developmental stage.
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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.