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Expression level analysis of genes involved in aerial hyphal differentiation and conidiation in the mutants. (A) Relative expression level of four hydrophobic protein genes (MGG_09134, MPG1, MGG_10105, and MHP1) in the aerial mycelia of ΔPogpd2 cultured on CM medium under a light–dark cycle or a continuous dark condition. (B) Relative expression level of four melanin synthesis genes (4HNR, AIB1, RSY1, and BUF1) and four conidiation-required transcription factor genes (COS1, CONx2, CON7, and GCC1) in the aerial mycelia of ΔPogpd1 and ΔPogpd2 cultured on CM medium under a light–dark cycle. (C) Relative expression level of seven light-sensitive protein genes (PoWC-1, TWL, TIG1, GCN5, YPD1, PoSIR2, and TRX2) in the aerial mycelia of ΔPogpd2 cultured on CM medium under a light–dark cycle and a continuous dark condition. β-TUBULIN and H3 were selected as reference genes. Error bars represent SD. Significant difference compared with the wild type as estimated by Tukey’s HSD test: ∗P < 0.05 and ∗∗P < 0.01.
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The glycerol-3-phosphate (G-3-P) shuttle is an important pathway for delivery of cytosolic reducing equivalents into mitochondrial oxidative phosphorylation, and plays essential physiological roles in yeast, plants, and animals. However, its role has been unclear in filamentous and pathogenic fungi. Here, we characterize the function of the G-3-P s...
Citations
... The glycerol-3-phophate (G-3-P) shuttle cooperatively operated by cytosolic and mitochondrial G-3-P dehydrogenases is one of the most widely studied metabolite shuttles in exporting reducing equivalents to mitochondria and maintaining the cellular redox status across the eukaryotic taxa (Mráček et al. 2013, Rigoulet et al. 2004, Shen et al. 2006. Recently identified yeast mitochondrial G-3-P dehydrogenase homolog has been shown to participate in regulation of NAD + metabolism and ATP production in P. oryzae, implicating a role in regulation of intracellular redox status and energy metabolism (Shi et al. 2018). Interestingly, gene disruption of gpd2 via T-DNA insertion caused the loss of fungal conidiation and pathogenicity on host plants. ...
... oryzae, the cytosolic operating enzyme of G-3-P shuttle is either awaiting to be discovered or metabolic state-dependent in subcellular localization, for both identified yeast Gpd1 and Gpd2 homologs in P. oryzae localizing in mitochondria (Shi et al. 2018). ...
... oryzae (Shi et al. 2018). However, the importance of cytosolic oxidizing and reducing equivalent balance is still unknown due to lack of cytosolic G-3-P dehydrogenase for characterization in P. oryzae. ...
... The study on the effect and utilization of cell wall components is helpful to understand the mechanism of plant cell wall damage caused by pathogens (53,54). Pyricularia oryzae glycerol-3-phosphate dehydrogenase PoGpd, a cellular oxidoreductase, played an important role in carbon source utilization, mycelial growth, and virulence (55). The a-oxoglutarate dehydrogenase VdOGDH deletion mutants grew slowly in the medium with sucrose, pectin, xylan, starch, and galactose as the sole carbon source, indicating that VdOGDH was important for vegetative growth and carbon utilization of V. dahliae (56). ...
This study showed that α-galactosidase VdGAL4 of V. dahliae could activate plant immune response and plays an important role in conidial morphology and yield, formation of microsclerotia, and mycelial penetration. VdGAL4 deletion mutants significantly reduced the pathogenicity of V. dahliae . These findings deepened the understanding of pathogenic virulence factors and how the mechanism of pathogenic fungi infected the host, which may help to seek new strategies for effective control of plant diseases caused by pathogenic fungi.
... Gpd1, a cytosolic form of glycerol-3-phosphate dehydrogenase, is important for glycerol accumulation in the appressorium (Foster et al., 2017). Disruption of GPD1 or GPD2 leads to reduced fungal virulence on rice (Shi et al., 2018). ...
... Data were analysed with Student's t test. Asterisks represent significant differences (****p < 0.0001) thermophila and F. graminearum(Liu, Yun, et al., 2019a; Liu, Liang, et al.,2019b;Pillai et al., 2017;Shi et al., 2018). The KEGG pathway and GO term analyses of the Mopah1 mutant showed that many DEGs are related to lipid and glycolysis/gluconeogenesis metabolism. ...
As with the majority of the hemibiotrophic fungal pathogens, the rice blast fungus Magnaporthe oryzae uses highly specialized infection structures called appressoria for plant penetration. Appressoria differentiated from germ tubes rely on enormous turgor pressure to directly penetrate the plant cell, in which process lipid metabolism plays a critical role. In this study, we characterized the MoPAH1 gene in M. oryzae, encoding a putative highly conserved phosphatidate phosphatase. The expression of MoPAH1 was up‐regulated during plant infection. The MoPah1 protein is expressed at all developmental and infection stages, and is localized to the cytoplasm. Disruption of MoPAH1 causes pleiotropic defects in vegetative growth, sporulation, and heat tolerance. The lipid profile is significantly altered in the Mopah1 mutant. Lipidomics assays showed that the level of phosphatidic acid (PA) was increased in the mutant, which had reduced levels of diacylglycerol and triacylglycerol. Using a PA biosensor, we showed that the increased level of PA in the Mopah1 mutant was primarily accumulated in the vacuole. The Mopah1 mutant was blocked in both conidiation and the formation of appressorium‐like structures at hyphal tips. It was nonpathogenic and failed to cause any blast lesions on rice and barley seedlings. RNA sequencing analysis revealed that MoPah1 regulates the expression of transcription factors critical for various developmental and infection‐related processes. The Mopah1 mutant was reduced in the expression and phosphorylation of Pmk1 MAP kinase and delayed in autophagy. Our study demonstrates that MoPah1 is necessary for lipid metabolism, fungal development, and pathogenicity in M. oryzae. MoPAH1 is involved in lipid metabolism, vegetative growth, development, MAPK signalling pathways, autophagy, and pathogenicity in the rice blast fungus.
... Data are presented as ± SD. p values were determined using Student's t-test between the two groups and listed in the figure for each substrate. Glycerol 3-phosphate shuttle can direct cytosolic reducing equivalents to the mitochondrial oxidative phosphorylation pathway to generate ATP [70]. Furthermore, utilization of TCA cycle intermediates such as isocitric acid, and fumaric acid also generate NADH for driving the synthesis of ATP through oxidative phosphorylation [71]. ...
... Interestingly we found that glycerol 3-phosphate, α-ketoisocaproic acid, pyruvic acid, isocitric acid, fumaric acid, succinic acid were used less by preOCs from the GCR group compared to sham preOCs. All these substrates can help ATP production by generating the reducing equivalents to run oxidative phosphorylation via either TCA cycle activity or directly shuttling them to the electron transport chain [70,71]. Although it initially appears counterintuitive, our results could be explained by the necessity of a healthy crosstalk between redox and energy metabolisms for cellular function. ...
Space is a high-stress environment. One major risk factor for the astronauts when they leave the Earth’s magnetic field is exposure to ionizing radiation from galactic cosmic rays (GCR). Several adverse changes occur in mammalian anatomy and physiology in space, including bone loss. In this study, we assessed the effects of simplified GCR exposure on skeletal health in vivo. Three months following exposure to 0.5 Gy total body simulated GCR, blood, bone marrow and tissue were collected from 9 months old male mice. The key findings from our cell and tissue analysis are (1) GCR induced femoral trabecular bone loss in adult mice but had no effect on spinal trabecular bone. (2) GCR increased circulating osteoclast differentiation markers and osteoclast formation but did not alter new bone formation or osteoblast differentiation. (3) Steady-state levels of mitochondrial reactive oxygen species, mitochondrial and non-mitochondrial respiration were increased without any changes in mitochondrial mass in pre-osteoclasts after GCR exposure. (4) Alterations in substrate utilization following GCR exposure in pre-osteoclasts suggested a metabolic rewiring of mitochondria. Taken together, targeting radiation-mediated mitochondrial metabolic reprogramming of osteoclasts could be speculated as a viable therapeutic strategy for space travel induced bone loss.
... For stress tests, each of the strains was cultured on minimal medium [MM, 10 g/L D-Glucose, 6 g/L NaNO 3 , 1.52 g/L KH 2 PO 4 , 0.52 g/L KCl, 0.52 g/L MgSO 4 •7H 2 O, 0.1% (v/v) trace elements, 0.1% (v/v) vitamin solution] with 0.8 M sucrose, 0.8 M sorbitol, 0.5 M NaCl, 20 μg/ml Congo red, or 50 μg/ml Calcofluor white (CFW) at 25°C . To assay conidial germination and appressorium formation, 20 μl spore suspensions (5 × 10 4 conidia/ml) were dropped onto hydrophobic coverslips and incubated at 22°C for 4 h post-inoculation (hpi; conidial germination) and for 8 and 24 hpi (appressorium formation; Shi et al., 2018). To observe cAMP's role on appressorium formation, 10 mM 8-bromoadenosine 3',5'-cyclic monophosphate sodium salt (8-Br-cAMP; Sigma, Japan) was added to spore suspensions which were dropped on hydrophobic or hydrophilic surfaces (GelBond) for 8 and 24 hpi. ...
Pyricularia oryzae is an important plant pathogenic fungus that can severely damage rice and wheat crops, leading to significant reductions in crop productivity. To penetrate into and invade tissues of its plant host, this fungus relies on an invasive structure known as an appressorium. Appressorium formation is rigorously regulated by the cAMP-PKA and Pmk1 MAPK pathways. Here, we identified PoRal2, a homologous protein of Schizosaccharomyces pombe Ral2, and characterized its roles in fungal development and virulence in P. oryzae. PoRal2 contains N-terminal kelch repeats and C-terminal BTB domains. PoRal2 is involved in sporulation, aerial hypha and conidiophore differentiation, appressorium formation, plant penetration, and virulence. During appressorium formation, ∆Poral2 mutants generate appressoria with long germ tubes on hydrophobic surfaces. ∆Poral2 mutants exhibited a defective response to exogenous cAMP and the activated RAS2G18V on a hydrophilic surface, indicating impairment in the cAMP-PKA or Pmk1 MAPK signaling pathways. Deletion of PoRAL2 leads to lowered Pmk1 phosphorylation level in the mutant. Moreover, PoRal2 is found to interact with Scd1, Smo1, and Mst50, which are involved in activation of Pmk1. In addition, the expression levels of MPG1, WISH, and PDEH in the cAMP-PKA pathway, RAS2 in both the cAMP-PKA and Pmk1 MAPK pathways, and melanin biosynthesis genes (ALB1, BUF1, and RSY1) were significantly down-regulated in the ∆Poral2. Therefore, PoRal2 is involved in fungal development and virulence by its crosstalk in the cAMP-PKA and Pmk1 MAPK signaling pathways.
... Further, G3P dehydrogenase plays an important role in the virulence of Pseudomonas aeruginosa (Daniels et al., 2014). In Magnaporthe oryzae, the deletion of G3P dehydrogenase had negative effects on fungal development and virulence (Shi et al., 2018). The function of G3P dehydrogenase in Colletotrichum gloeosporioides is also related to conidiation (Wei et al., 2004). ...
... In P. aeruginosa, the analogous G3P dehydrogenase plays a role in producing virulence factors (Daniels et al., 2014). In addition, in the rice blast fungus, M. oryzae, conidia formation and disease development were affected by G3P dehydrogenase, and the G3P dehydrogenase knockout mutant was less virulent than the wild-type strain (Shi et al., 2018). Thus, the G3P dehydrogenase that is indispensable for glycerol production from glucose is an important component of virulence. ...
Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB) in watermelon, a disease that poses a serious threat to watermelon production. Because of the lack of resistant cultivars against BFB, virulence factors or mechanisms need to be elucidated to control the disease. Glycerol-3-phosphate dehydrogenase is the enzyme involved in glycerol production from glucose during glycolysis. In this study, we report the functions of a putative glycerol-3-phosphate dehydrogenase in Ac (GlpdAc) using comparative proteomic analysis and phenotypic observation. A glpdAc knockout mutant, AcΔglpdAc(EV), lost virulence against watermelon in two pathogenicity tests. The putative 3D structure and amino acid sequence of GlpdAc showed high similarity with glycerol-3-phosphate dehydrogenases from other bacteria. Comparative proteomic analysis revealed that many proteins related to various metabolic pathways, including carbohydrate metabolism, were affected by GlpdAc. Although AcΔglpdAc(EV) could not use glucose as a sole carbon source, it showed growth in the presence of glycerol, indicating that GlpdAc is involved in glycolysis. AcΔglpdAc(EV) also displayed higher cell-to-cell aggregation than the wild-type bacteria, and tolerance to osmotic stress and ciprofloxacin was reduced and enhanced in the mutant, respectively. These results indicate that GlpdAc is involved in glycerol metabolism and other mechanisms, including virulence, demonstrating that the protein has pleiotropic effects. Our study expands the understanding of the functions of proteins associated with virulence in Ac.
... Aspergillus fungi can convert and utilize different sugar monomers in the plant as carbon sources for a variety of catabolic pathways that all are connected to glycolysis (Khosravi et al. 2015). In Pyricularia oryzae, glycerol-3-phosphate dehydrogenases, PoGpd1 and PoGpd2, important cellular redox enzymes, played essential physiological roles in hyphal differentiation, utilization of carbon sources, and virulence (Shi et al. 2018). OGDC is located in the mitochondrial matrix and catalyzes the decarboxylation of α-ketoglutarate to produce succinyl CoA with the reduction of NAD + to generate NADH, which is critical for energy metabolism (Bunik and Fernie 2009). ...
Verticillium dahliae, a soil-borne fungus, can invade plant vascular tissue and cause Verticillium wilt. The enzyme α-oxoglutarate dehydrogenase (OGDH), catalyzing the oxidation of α-oxoglutarate in the tricarboxylic acid cycle (TCA), is vital for energy metabolism in the fungi. Here, we identified the OGDH gene in V. dahliae (VdOGDH, VDAG_10018) and investigated its function in virulence by generating gene deletion mutants (ΔVdOGDH) and complementary mutants (ΔVdOGDH-C). When the ΔVdOGDH mutants were supplemented with different carbon sources, vegetative growth on Czapek Dox medium was significantly impaired, suggesting that VdOGDH is crucial for vegetative growth and carbon utilization. Conidia of the ΔVdOGDH mutants were atypically rounded or spherical, and hyphae were irregularly branched and lacked typical whorled branches. Mutants ΔVdOGDH-1 and ΔVdOGDH-2 were highly sensitive to H2O2 in the medium plates and had higher intracellular ROS levels. ΔVdOGDH mutants also had elevated expression of oxidative response-related genes, indicating that VdOGDH is involved in response to oxidative stress. In addition, the disruption of VdOGDH caused a significant increase in the expression of energy metabolism-related genes VdICL, VdICDH, VdMDH, and VdPDH and melanin-related genes Vayg1, VdSCD, VdLAC, VT4HR, and VaflM in the ΔVdOGDH mutants; thus, VdOGDH is also important for energy metabolism and melanin accumulation. Cotton plants inoculated with ΔVdOGDH mutants exhibited mild leaf chlorosis and the disease index was lower compared with wild type and ΔVdOGDH-C strains. These results together show that VdOGDH involved in energy metabolism of V. dahliae, is also essential for full virulence by regulating multiple fungal developmental factors.
... The results suggest a role of grg-1 in A. fatua caryopsis colonization. The gpd2 gene has been shown to be necessary for glycerol utilization, and deletion of the gene results in reduced virulence of Pyricularia oryzae (Shi et al., 2018). Several phosphatase genes were induced during caryopsis colonization, which is of relevance because not only is phosphate turnover central to basic biological functions, but phosphatases have also been shown to be crucial to virulence in F. graminearum (Yun et al., 2015). ...
Fusarium avenaceum F.a.1 is a novel strain of a fungal plant pathogen capable of preferentially decaying wild oat (Avena fatua) caryopses compared with those of wheat (Triticum aestivum). Understanding the molecular mechanisms governing weed seed-pathogen interactions is crucial to developing novel weed seed suppression technologies. Additionally, wild oat often competes with wheat in regions undergoing soil acidification, which leads to increases in soluble concentrations of many metals, including aluminum (Al). There is a dearth of information regarding the gene expression responses of Fusarium species to Al toxicity, or how metal toxicity might influence caryopsis colonization. To address this, a transcriptomic approach was used to investigate molecular responses of F.a.1 during wild oat caryopsis colonization in the presence and absence of chronic, sublethal concentrations of Al (400 μM). Caryopsis colonization was associated with induction of genes related to virulence, development, iron metabolism, oxidoreduction, stress, and detoxification, along with repression of genes associated with development, transport, cell-wall turnover, and virulence. Caryopsis colonization during Al exposure resulted in the induction of genes associated with virulence, detoxification, stress, iron metabolism, oxidoreduction, and cell wall turnover, along with repression of genes associated with cell wall metabolism, virulence, development, detoxification, stress, and transcriptional regulation. Aluminum exposure in the absence of caryopses was associated with induction of genes involved in siderophore biosynthesis, secretion, uptake, and utilization, along with several other iron metabolism-related and organic acid metabolism-related genes. The siderophore-related responses associated with Al toxicity occurred concurrently with differential regulation of genes indicating disruption of iron homeostasis. These findings suggest Al toxicity is attenuated by siderophore metabolism in F.a.1. In summary, both caryopsis colonization and Al toxicity uniquely influence transcriptomic responses of F.a.1.
... Mutant Dmcs1 had dense aerial hyphae on MM, similar to the wild type (Fig. 3A). Because hydrophobins are necessary for the formation of aerial hyphae (Elliot and Talbot 2004;Shi et al. 2018), we measured the expression level of hydrophobin genes in mycelia of Dmcs1 cultured on CM or MM. Compared with the wild type, the transcript levels of three hydrophobin genes (MGG_09134, MGG_10105, and MPG1) in Dmcs1 were downregulated on both CM and MM, whereas MHP1 was upregulated on MM (Fig. 5D). ...
The methylcitrate cycle metabolizes propionyl-CoA, a toxic metabolite, into pyruvate. Pyricularia oryzae (syn. Magnaporthe oryzae) is a phytopathogenic fungus that causes a destructive blast disease in rice and wheat. We characterized the essential roles of the methylcitrate cycle in the development and virulence of P. oryzae using functional genomics. In P. oryzae, the transcript levels of MCS1 and MCL1, which encode a 2-methylcitrate synthase and a 2-methylisocitrate lyase, respectively, were upregulated during appressorium formation and when grown on propionyl-CoA-producing carbon sources. We found that deletion of MCS1 and MCL1 inhibited fungal growth on media containing both glucose and propionate, and media using propionate or propionyl-CoA-producing amino acids (valine, isoleucine, methionine, and threonine) as the sole carbon or nitrogen sources. The Δmcs1 mutant formed sparse aerial hyphae and did not produce conidia on complete medium (CM), while the Δmcl1 mutant showed decreased conidiation. The aerial mycelium of Δmcs1 displayed a lowered NAD+/NADH ratio, reduced nitric oxide content, and downregulated transcription of hydrophobin genes. Δmcl1 showed reduced appressorium turgor, severely delayed plant penetration, and weakened virulence. Addition of acetate recovered the growth of the wild type and Δmcs1 on medium containing both glucose and propionate and recovered the conidiation of both Δmcs1 and Δmcl1 on CM by reducing propionyl-CoA formation. Deletion of MCL1 together with ICL1, an isocitrate lyase gene in the glyoxylate cycle, greatly reduced the mutant's virulence as compared with the single-gene deletion mutants (Δicl1 and Δmcl1). This experimental evidence provides important information about the role of the methylcitrate cycle in development and virulence of P. oryzae by detoxification of propionyl-CoA and 2-methylisocitrate.
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.
