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| The pathogenicity assay of the moivd mutants. (A) Pathogenicity test of the moivd mutants on excised barley leaves. The conidium was set at a concentration of 1 × 10 5 spores/ml. (B) Spray inoculation assay of the moivd mutants on rice seedlings. The conidium was set at a concentration of 5 × 10 4 spores/ml. (C) Quantitative analysis of lesion numbers in 10 cm 2 leaf size. The data was calculated from three independent replicates. The same lowercases 'a' or 'b' on the sample bar indicate no significant differences between samples. The different lowercases indicate significant differences (P < 0.05; t test). (D) Statistic analysis of the invasive hyphae growth rate. The hyphae are divided into four types according to the growth speed. Type 1 indicates no penetration; Type 2 indicates penetration with a small peg; Type 3 represents the invasive hyphae in extension. Type 4 indicates the hyphae crossing the second host cell. Different color asterisk indicates the different hyphae type. Bar = 10 µm. (E) DAB staining of barley leaves infected by moivd-3after inoculation for 24 h. The asterisk indicates the stained infected host cell by DAB (1 mg/ml). Bar = 50 µm.
Source publication
Isovaleryl-CoA dehydrogenase (IVD), a member of the acyl-CoA dehydrogenase (ACAD) family, is a key enzyme catalyzing the conversion of isovaleryl-CoA to β-methylcrotonyl-CoA in the third reaction of the leucine catabolism pathway and simultaneously transfers electrons to the electron-transferring flavoprotein (ETF) for ATP synthesis. We previously...
Contexts in source publication
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... investigate the role of MoIVD in M. oryzae pathogenicity, we evaluated the mutant phenotype in infection-related development including conidial germination, appressorium formation and host infection. Although we did not find any significant difference between mutant and wild type strains in conidial germination and appressorium formation ( Supplementary Table S2), their virulence on excised barley leaves showed differences, and moivd-3 and moivd-6 produced smaller disease lesions than Guy11 and moivd- 3/MoIVD (Figure 4A). Likewise, the mutants also produced much smaller and fewer lesions on rice seedlings than Guy11 and moivd-3/MoIVD (Figures 4B,C). ...
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... we did not find any significant difference between mutant and wild type strains in conidial germination and appressorium formation ( Supplementary Table S2), their virulence on excised barley leaves showed differences, and moivd-3 and moivd-6 produced smaller disease lesions than Guy11 and moivd- 3/MoIVD (Figure 4A). Likewise, the mutants also produced much smaller and fewer lesions on rice seedlings than Guy11 and moivd-3/MoIVD (Figures 4B,C). Furthermore, sheath infection assay was used to examine infectious growth in host cells, and moivd-3 and moivd-6 displayed lower penetration frequency and delayed hyphal growth after penetration in comparison to Guy11 and moivd-3/MoIVD at 24 h post- inoculation/infection ( Figure 4D). ...
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... the mutants also produced much smaller and fewer lesions on rice seedlings than Guy11 and moivd-3/MoIVD (Figures 4B,C). Furthermore, sheath infection assay was used to examine infectious growth in host cells, and moivd-3 and moivd-6 displayed lower penetration frequency and delayed hyphal growth after penetration in comparison to Guy11 and moivd-3/MoIVD at 24 h post- inoculation/infection ( Figure 4D). These results suggest MoIVD is required for full pathogenicity of M. oryzae. ...
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... accumulation is a symbol of host defense against pathogen attack and could be tested by DAB staining assay. Using this method, we found that the barley cells infected by the mutant exhibited a darker brown color than the ones infected by Guy11 and complemented strain moivd-3/MoIVD (Figure 4E), suggesting more ROS accumulation in the mutant-infected cells. ...
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... this end, we performed GC-MS to test the content of 6 short chain fatty acids in moivd-3. Our results showed a very high concentration of isovaleric acid (54.06 ± 5.55 µg/g) in moivd-3, which was about 450-fold that in Guy11 (0.12 ± 0.03 µg/g) ( Figure 6A and Supplementary Figure S4). As a control, we also examined the content of 35 medium and long chain fatty acids by GC- MS and found no significant difference between moivd-3 and Guy11 except several fatty acids with slight changes (Supplementary Figure S5). ...
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... IVD mutation destroys leucine catabolism at the third step, involving the conversion of isovaleryl-CoA to β-methylcrotonyl-CoA. An accumulation of leucine has not been observed in IVD mutants ( Mohsen et al., 1998;Mohsen and Vockley, 2015) and we also did not find an obvious high level of leucine in moivd-3 (Supplementary Figure S9), but rather a high level of isovaleric acid was present ( Figure 6A and Supplementary Figure S4). These findings suggest that the IVD mutation led to a block of leucine catabolism at the third step, and that accumulated isovaleryl-CoA is converted into isovaleric acid that is not further degraded. ...
Citations
... The constructed vector DNA was introduced into the knockout mutant protoplast for gene complementation, and resulting transformants were screened using 50 µg/mL chlorimuron-ethyl to select successful complementary strains. The detailed fungal protoplast preparation and transformation methods have been described previously [55]. The sub-cellular localization of MoMyb13 was observed by confocal microscopy (Nikon A1, Tokyo, Japan). ...
The Myb family of transcription factors (TFs) is a large and functionally diverse group found in all eukaryotes. Its role in fungi remains poorly studied, despite the fact that it is thought to play a role in the pathogenicity of fungal pathogens. In this study, we have characterized the functional role of a Myb family TF called MoMyb13 in the rice blast fungus, Magnaporthe oryzae. MoMyb13 has orthologues only in ascomycete fungi, making it of special interest. Localization experiments confirmed that MoMyb13 is located in the nuclei, as expected for a TF. Phenotypic analysis showed that MoMyb13 mutants exhibited reduced growth, white instead of dark colonies, formed no conidia and, consequently, no conidial appressoria. The mutants completely lost pathogenicity, despite being able to form dark hyphal appressoria at their hyphae ends. Furthermore, the mutant colonies lost hydrophobicity and had significantly reduced expression of the hydrophobin MPG1 that MoMyb13 appears to regulate. However, overexpression of MPG1 in the mutants restored hydrophobicity, but not pathogenicity. Stress assay showed that the mutants were more sensitive to SDS, CR, and H2O2, but more tolerant to NaCl and SOR. In summary, our study revealed the crucial function of MoMyb13 in the growth, conidiation, hydrophobicity, stress response, and pathogenicity of M. oryzae. MoMyb13 is thus needed in the late and very early stages of infection for the spreading of the fungus to other plants and the early establishment of infection in other plants.
... Studies in isovaleryl-CoA dehydrogenase of Magnaporthe oryzae (plant fungal pathogen) demonstrate that this enzyme participates in Leu catabolism and is necessary for vegetative growth, conidiation, and pathogenicity (Li et al., 2019). ...
Leucine, isoleucine, and valine, collectively termed Branched Chain Amino Acids (BCAA), are hydrophobic amino acids (AAs) and are essential for most eukaryotes since in these organisms they cannot be biosynthesized and must be supplied by the diet. These AAs are structurally relevant for muscle cells and, of course, important for the protein synthesis process. The metabolism of BCAA and its participation in different biological processes in mammals have been relatively well described. However, for other organisms as pathogenic parasites, the literature is really scarce. Here we review the BCAA catabolism, compile evidence on their relevance for pathogenic eukaryotes with special emphasis on kinetoplastids and highlight unique aspects of this underrated pathway.
... Interestingly, the localization of DNM1 can be observed in both peroxisomes and mitochondria, and it plays a critical role in both peroxisomal and mitochondrial fission [230]. In addition, the isovaleryl-CoA dehydrogenase IVD, another mitochondrial-localized enzyme, is involved in leucine catabolism and contributes to fungal vegetative growth, conidiation, and pathogenicity [231]. The acetoacetyl-CoA acetyltransferase ACAT1 is also mitochondria localized, whereas ACAT2 is localized to the cytoplasm [232]. ...
Magnaporthe oryzae is one of the most devastating pathogenic fungi that affects a wide range of cereal plants, especially rice. Rice blast disease causes substantial economic losses around the globe. The M. oryzae genome was first sequenced at the beginning of this century and was recently updated with improved annotation and completeness. In this review, key molecular findings on the fungal development and pathogenicity mechanisms of M. oryzae are summarized, focusing on fully characterized genes based on mutant analysis. These include genes involved in the various biological processes of this pathogen, such as vegetative growth, conidia development, appressoria formation and penetration, and pathogenicity. In addition, our syntheses also highlight gaps in our current understanding of M. oryzae development and virulence. We hope this review will serve to improve a comprehensive understanding of M. oryzae and assist disease control strategy designs in the future.
... One of the immune responses by the host is a burst of reactive oxygen species (ROS) triggered by the innate immune system recognizing the pathogen (Jwa & Hwang, 2017;Smirnoff & Arnaud, 2019). The ROS at the penetration site can be detected by staining cells with 3,3′-diaminobenzidine (DAB) (Li et al., 2019). The pathogen-host struggle at this early biotrophic stage directly determines the outcome, whether the subsequent infection hyphae can survive and disease occurs (Vargas et al., 2012). ...
... The protoplast cells were prepared as described previously (Li et al., 2019), then the DNA was introduced to the protoplasts. For gene deletion assay, at least 2 μg of gene deletion cassette DNA was transformed into the wild-type strain 98-06, and the transformants were screened on TB3 medium (6 g/L casamino acids, 6 g/L yeast extract, 200 g/L sucrose, 15 g/L agar) with 250 μg/ml hygromycin. ...
... The DAB staining to indicate host ROS formed during M. oryzae infection was performed as described previously (Li et al., 2019). A conidial suspension of 5 × 10 4 spores/ml was sprayed onto 2-week-old barley and incubated for 24 h. ...
The initial stage of rice blast fungus, Magnaporthe oryzae, infection, before 36 h postinoculation, is a critical timespan for deploying pathogen effectors to overcome the host's defences and ultimately cause the disease. However, how this process is regulated at the transcription level remains largely unknown. This study functionally characterized two M. oryzae Early Infection‐induced Transcription Factor genes (MOEITF1 and MOEITF2) and analysed their roles in this process. Target gene deletion and mutant phenotype analysis showed that the mutants Δmoeitf1 and Δmoeitf2 were only defective for infection growth but not for vegetative growth, asexual/sexual sporulation, conidial germination, and appressoria formation. Gene expression analysis of 30 putative effectors revealed that most effector genes were down‐regulated in mutants, implying a potential regulation by the transcription factors. Artificial overexpression of two severely down‐regulated effectors, T1REP and T2REP, in the mutants partially restored the pathogenicity of Δmoeitf1 and Δmoeitf2, respectively, indicating that these are directly regulated. Yeast one‐hybrid assay and electrophoretic mobility shift assay indicated that Moeitf1 specifically bound the T1REP promoter and Moeitf2 specifically bound the T2REP promoter. Both T1REP and T2REP were predicted to be secreted during infection, and the mutants of T2REP were severely reduced in pathogenicity. Our results indicate crucial roles for the fungal‐specific Moeitf1 and Moeitf2 transcription factors in regulating an essential step in M. oryzae early establishment after penetrating rice epidermal cells, highlighting these as possible targets for disease control.
... Propionyl-CoA is a toxic metabolite that leads to sparse aerial hyphae and less conidiation and in M. oryzae is immediately converted to pyruvate by the methylcitrate cycle (Yan et al., 2019). βmethylcrotonyl-CoA is the intermediary product of the leucine catabolism pathway, which ultimately degrades to form acetyl-CoA. Reports have shown defects in leucine metabolism or accumulation of the intermediary products lead to less conidiation and pigmentation (Li et al., 2019). Similarly, l-aspartate was found to be overproduced in the knockdown mutant compared with the wild type. ...
Aims:
To study the altered metabolic pathways and metabolites produced in overexpression and knockdown mutants of a global regulator named MoLAEA, which was recently found to regulate the expression of the genes involved in secondary metabolism in one of the most destructive plant pathogens, Magnaporthe oryzae.
Methods and results:
Mass spectrometry-based global untargeted metabolomic profiling was used to identify altered metabolites. Metabolites were extracted from the mutant strains of MoLAEA using two extraction methods viz., aqueous and organic extraction and data acquired using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive and negative polarities. Levels of metabolites involved in various biological pathways such as amino acid as well as polyamine biosynthesis, fatty acid and pyrimidine metabolism showed remarkable change in the mutant strains. Interestingly, metabolites involved in stress responses were produced in higher quantities in the overexpression strain whereas, certain overproduced metabolites were associated with distinctive phenotypic changes in the overexpression strain compared to the wild-type. Further, the expression of several genes involved in the stress responses was found to have higher expression in the overexpression strain.
Conclusions:
The global regulator MoLAEA is involved in secondary metabolism in the plant pathogen M. oryzae such that the mutant strains showed altered level of several metabolites involved in the biosynthesis pathways compared to the wild-type. Also, metabolites involved in stress responses were overproduced in the overexpression strain and this can be seen in the higher growth in media amended with stress-inducing agents or higher expression of genes involved in stress response in the overexpression strain compared to the wild-type.
Significance and impact:
This is the first report of metabolite profiling relative to the global regulation of secondary metabolism in M. oryzae, where secondary metabolism is poorly understood. It opens up avenues for more relevant investigations on the genetic regulation of several of the metabolites found in the analysis, which have not been previously characterized in M. oryzae.
... Gene clusters of tIpks and nrps were less in the four main clinical species (S. brasiliensis, S. schenckii, S. globosa, and S. luriei) than those of the environmental and other clinical species. (Li Y. et al., 2019). Hsp70 functioned as a stress-related transcriptional co-activator is required for fungal virulence (Weissman et al., 2020). ...
Sporothrix species are commonly isolated from environmental and clinical samples. As common causes of zoonotic mycosis, Sporothrix species may result in localized or disseminated infections, posing considerable threat to animal and human health. However, the pathogenic profiles of different Sporothrix species varied, in virulence, geographic location and host ranges, which have yet to be explored. Analysing the genomes of Sporothrix species are useful for understanding their pathogenicity. In this study, we analyzed the whole genome of 12 Sporothrix species and six S. globosa isolates from different clinical samples in China. By combining comparative analyses with Kyoto Encyclopedia of Genes and Genomes (KEGG), Carbohydrate-Active enZymes (CAZy), antiSMASH, Pfam, and PHI annotations, Sporothrix species showed exuberant primary and secondary metabolism processes. The genome sizes of four main clinical species, i.e., S. brasiliensis, S. schenckii, S. globosa, and S. luriei were significantly smaller than other environmental and clinical Sporothrix species. The contracted genes included mostly CAZymes and peptidases genes that were usually associated with the decay of plants, as well as the genes that were associated with the loss of pathogenicity and the reduced virulence. Our results could, to some extent, explain a habitat shift of Sporothrix species from a saprobic life in plant materials to a pathogenic life in mammals and the increased pathogenicity during the evolution. Gene clusters of melanin and clavaric acid were identified in this study, which improved our understanding on their pathogenicity and possible antitumor effects. Moreover, our analyses revealed no significant genomic variations among different clinical isolates of S. globosa from different regions in China.
... By contrast, hFH[N80][3R/3A] delivered proteins into chloroplasts ( Figure 1B), indicating that the last three arginines are crucial for mitochondrial specificity. Next, using the mitochondrial presequence of Isovaleryl-CoA dehydrogenase of Magnaporthe oryzae (mIVD), a blast fungus (Li et al., 2019), we found that mIVD[N80] delivered proteins into both chloroplasts and mitochondria in Arabidopsis protoplasts ( Figures 1C, D) Figures 1C, D), indicating that the arginine residues of mIVD[N80] are critical for protein import into mitochondria in plants. Taken together, these results indicate that presequences of animal and fungal proteins behave more or less the same way in plants. ...
Plants have two endosymbiotic organelles, chloroplast and mitochondrion. Although they have their own genomes, proteome assembly in these organelles depends on the import of proteins encoded by the nuclear genome. Previously, we elucidated the general design principles of chloroplast and mitochondrial targeting signals, transit peptide, and presequence, respectively, which are highly diverse in primary structure. Both targeting signals are composed of N-terminal specificity domain and C-terminal translocation domain. Especially, the N-terminal specificity domain of mitochondrial presequences contains multiple arginine residues and hydrophobic sequence motif. In this study we investigated whether the design principles of plant mitochondrial presequences can be applied to those in other eukaryotic species. We provide evidence that both presequences and import mechanisms are remarkably conserved throughout the species. In addition, we present evidence that the N-terminal specificity domain of presequence might have evolved from the bacterial TAT (twin-arginine translocation) signal sequence.
In plant-pathogen interactions, Magnaporthe oryzae causes blast disease on more than 50 species of 14 monocot plants, including important crops such as rice, millet, and most 15 recently wheat. M. oryzae is a model fungus for studying plant-microbe interaction, and the main source for fungal pathogenesis in the field. Here we report that MoJMJD6 is required for conidium germination and appressorium formation in M. oryzae. We obtained MoJMJD6 mutants (ΔMojmjd6) using a target gene replacement strategy. The MoJMD6 deletion mutants were delayed for conidium germination, glycogen, and lipid droplets utilization and consequently had decreased virulence. In the ΔMojmjd6 null mutants, global histone methyltransferase modifications (H3K4me3, H3K9me3, H3K27me3, and H3K36me2/3) of the genome were unaffected. Taken together, our results indicated that MoJMJD6 function as a nuclear protein which plays an important role in conidium germination and appressorium formation in the M. oryzae. Our work provides insights into MoJMJD6-mediated regulation in the early stage of pathogenesis in plant fungi.
Cadmium (Cd) contamination is becoming a widespread environmental problem. However, the differential responsive mechanisms of Cd hyperaccumulator Solanum nigrum to low or high dose of Cd are not well documented. In this study, phenotypic and physiological analysis firstly suggested that the seedlings of S. nigrum showed slight leaf chlorosis symptoms under 25 μM Cd and severe inhibition on growth and photosynthesis under 100 μM Cd. Further proteomic analysis identified 105 differentially expressed proteins (DEPs) in the Cd-treated leaves. Under low dose of Cd stress, 47 DEPs are mainly involved in primary metabolic processes, while under high dose of Cd stress, 92 DEPs are mainly involved in photosynthesis, energy metabolism, production of phytochelatin and reactive oxygen species (ROS). Protein-protein interaction (PPI) network analysis of DEPs support above differential responses in the leaves of S. nigrum to low and high dose of Cd treatments. This work provides the differential responsive mechanisms in S. nigrum to low and high dose of Cd, and the theoretical foundation for the application of hyperaccumulating plants in the phytoremediation of Cd-contaminated soils.
