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Find video protocols related to scientific articles indexed in Pubmed.
Specific adaptation of Ustilaginoidea virens in occupying host florets revealed by comparative and functional genomics.
Nat Commun
PUBLISHED: 01-27-2014
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Ustilaginoidea virens (Cooke) Takah is an ascomycetous fungus that causes rice false smut, a devastating emerging disease worldwide. Here we report a 39.4 Mb draft genome sequence of U. virens that encodes 8,426 predicted genes. The genome has ~25% repetitive sequences that have been affected by repeat-induced point mutations. Evolutionarily, U. virens is close to the entomopathogenic Metarhizium spp., suggesting potential host jumping across kingdoms. U. virens possesses reduced gene inventories for polysaccharide degradation, nutrient uptake and secondary metabolism, which may result from adaptations to the specific floret infection and biotrophic lifestyles. Consistent with their potential roles in pathogenicity, genes for secreted proteins and secondary metabolism and the pathogen-host interaction database genes are highly enriched in the transcriptome during early infection. We further show that 18 candidate effectors can suppress plant hypersensitive responses. Together, our analyses offer new insights into molecular mechanisms of evolution, biotrophy and pathogenesis of U. virens.
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Transcription factor CCG-8 as a new regulator in the adaptation to antifungal azole stress.
Antimicrob. Agents Chemother.
PUBLISHED: 12-16-2013
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Antifungal azoles are widely used for controlling fungal infections. Fungi are able to change the expression of many genes when they adapt to azole stress and increased expression of some of these genes can elevate resistance to azoles. However, the regulatory mechanisms behind transcriptional adaption to azoles in filamentous fungi are poorly understood. In this study, we found that deletion of transcription factor gene, ccg-8, which is known to be a clock-controlled gene, made Neurospora crassa hypersensitive to azoles. A comparative analysis of genome-wide transcriptional responses to ketoconazole between the wild type and the ccg-8 mutant revealed that, among the 488 transcriptionally ketoconazole-up-regulated genes and the 427 transcriptionally ketoconazole-down-regulated genes in the wild type, the transcriptional responses of 78 genes to ketoconazole were regulated by CCG-8. Ketoconazole sensitivity testing of all available knockout mutants for CCG-8-regulated genes revealed that CCG-8 contributed to azole adaption by regulating the ketoconazole responses of many genes, including target gene erg11, azole transporter gene cdr4, hexose transporter gene hxt13, stress response gene NCU06317 (named as kts-1), two transcription factor genes: NCU01386 (named as kts-2) and fsd-1/ndt80, four enzyme encoding genes and six unknown-function genes. CCG-8 also regulated phospholipid synthesis in N. crassa in a similar manner to its homolog, Opi1p in Sacchromyces cerevisiae. However, there was no cross-talk between phospholipid synthesis and azole resistance in N. crassa. CCG-8 homologues are conserved and are common in filamentous fungi. Deletion of CCG-8 homologue encoding gene, Fvccg-8 in Fusarium verticillioides, also made this fungus hypersensitive to antifungal azoles.
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PyrG is required for maintaining stable cellular uracil level and normal sporulation pattern under excess uracil stress in Aspergillus nidulans.
Sci China Life Sci
PUBLISHED: 02-25-2013
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Tight control of the intracellular uracil level is believed to be important to reduce the occurrence of uracil incorporation into DNA. The pyrG gene of Aspergillus nidulans encodes orotidine 5-phosphate decarboxylase, which catalyzes the conversion of orotidine monophosphate (OMP) to uridine monophosphate (UMP). In this study, we found that pyrG is critical for maintaining uracil at a low concentration in A. nidulans cells in the presence of exogenous uracil. Excess uracil and its derivatives had a stronger inhibitory effect on the growth of the pyrG89 mutant with defective OMP decarboxylase activity than on the growth of wild type, and induced sexual development in the pyrG89 mutant but not in wild type. Analysis of transcriptomic responses to excess uracil by digital gene expression profiling (DGE) revealed that genes related to sexual development were transcriptionally activated in the pyrG89 mutant but not in wild type. Quantitative analysis by HPLC showed that the cellular uracil level was 6.5 times higher in the pyrG89 mutant than in wild type in the presence of exogenous uracil. This study not only provides new information on uracil recycling and adaptation to excess uracil but also reveals the potential effects of OMP decarboxylase on fungal growth and development.
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Composition and expression of genes encoding carbohydrate-active enzymes in the straw-degrading mushroom Volvariella volvacea.
PLoS ONE
PUBLISHED: 02-08-2013
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Volvariella volvacea is one of a few commercial cultivated mushrooms mainly using straw as carbon source. In this study, the genome of V. volcacea was sequenced and assembled. A total of 285 genes encoding carbohydrate-active enzymes (CAZymes) in V. volvacea were identified and annotated. Among 15 fungi with sequenced genomes, V. volvacea ranks seventh in the number of genes encoding CAZymes. In addition, the composition of glycoside hydrolases in V. volcacea is dramatically different from other basidiomycetes: it is particularly rich in members of the glycoside hydrolase families GH10 (hemicellulose degradation) and GH43 (hemicellulose and pectin degradation), and the lyase families PL1, PL3 and PL4 (pectin degradation) but lacks families GH5b, GH11, GH26, GH62, GH93, GH115, GH105, GH9, GH53, GH32, GH74 and CE12. Analysis of genome-wide gene expression profiles of 3 strains using 3-tag digital gene expression (DGE) reveals that 239 CAZyme genes were expressed even in potato destrose broth medium. Our data also showed that the formation of a heterokaryotic strain could dramatically increase the expression of a number of genes which were poorly expressed in its parental homokaryotic strains.
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Genetic diversity and population structure of rice pathogen Ustilaginoidea virens in China.
PLoS ONE
PUBLISHED: 01-01-2013
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Rice false smut caused by the fungal pathogen Ustilaginoidea virens is becoming a destructive disease throughout major rice-growing countries. Information about its genetic diversity and population structure is essential for rice breeding and efficient control of the disease. This study compared the genome sequences of two U. virens isolates. Three SNP-rich genomic regions were identified as molecular markers that could be used to analyze the genetic diversity and population structure of U. virens in China. A total of 56 multilocus sequence types (haplotypes) were identified out of 162 representative isolates from 15 provinces covering five major rice-growing areas in China. However, the phylogeny, based on sequences at individual SNP-rich regions, strongly conflicted with each other and there were significant genetic differences between different geographical populations. Gene flow between the different geographical populations and genetic differentiation within each geographical population were also detected. In addition, genetic recombination and genetic isolation resulting from geographic separation was also found.
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Sterol C-22 Desaturase ERG5 Mediates the Sensitivity to Antifungal Azoles in Neurospora crassa and Fusarium verticillioides.
Front Microbiol
PUBLISHED: 01-01-2013
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Antifungal azoles inhibit ergosterol biosynthesis by interfering with lanosterol 14?-demethylase. In this study, seven upregulated and four downregulated ergosterol biosynthesis genes in response to ketoconazole treatment were identified in Neurospora crassa. Azole sensitivity test of knockout mutants for six ketoconazole-upregulated genes in ergosterol biosynthesis revealed that deletion of only sterol C-22 desaturase ERG5 altered sensitivity to azoles: the erg5 mutant was hypersensitive to azoles but had no obvious defects in growth and development. The erg5 mutant accumulated higher levels of ergosta 5,7-dienol relative to the wild type but its levels of 14?-methylated sterols were similar to the wild type. ERG5 homologs are highly conserved in fungal kingdom. Deletion of Fusarium verticillioides erg5 also increased ketoconazole sensitivity, suggesting that the roles of ERG5 homologs in azole resistance are highly conserved among different fungal species, and inhibition of ERG5 could reduce the usage of azoles and thus provide a new target for drug design.
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Involvement of a helix-loop-helix transcription factor CHC-1 in CO(2)-mediated conidiation suppression in Neurospora crassa.
Fungal Genet. Biol.
PUBLISHED: 02-01-2011
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The morphological switch from vegetative growth to conidiation in filamentous fungi is highly regulated, but the understanding of the regulatory mechanisms is limited. In this study, by screening a set of knock-out mutants corresponding to 103 transcription factor encoding genes in Neurospora crassa, a mutant was found to produce abundant conidia in race tubes in which conidiation in the wild-type strain was suppressed. The corresponding gene NCU00749 encodes a protein containing a helix-loop-helix DNA binding region. Unlike enhanced conidiation in ras-1 and sod-1 mutants, which was completely suppressed by antioxidant N-acetyl cysteine, enhanced conidiation in the NCU00749 mutant was only slightly affected by N-acetyl cysteine. When grown on slants, the NCU00749 deletion mutant exhibited earlier conidial formation than the wild-type strain, and this was more evident at a higher (5%) CO(2) concentration. Therefore, we named NCU00749 as conidiation at high carbon dioxide-1 (chc-1). Genes that are highly expressed during conidial development, eas, con-6, con-8 and con-10, were transcribed at a higher rate in the chc-1 deletion mutant than the wild-type strain in response to conidiation induction. To determine the mechanisms by which CHC-1 regulates conidiation, we conducted a RNA sequencing analysis and found that 404 genes exhibited ? 2 fold changes in transcription in response to chc-1 deletion. Among them, fluffy and ada-6, two transcription factor genes that positively regulate conidiation in N. crassa, and rca-1, whose homolog flbD in Aspergillus nidulans is essential for conidiation, were upregulated in the chc-1 deletion mutant. Results of RNA sequencing also suggest that signal transduction via the cAMP and the MAK-2 mediated signal pathways, and ROS generation and removal, mechanisms known to regulate conidiation, are not involved in chc-1 mediated control of conidiation. In addition, chc-1 also influences expression of genes involved in other important biological processes besides conidiation such as carbon metabolism, sphingolipid synthesis, cell wall synthesis, and calcium signaling.
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CDR4 is the major contributor to azole resistance among four Pdr5p-like ABC transporters in Neurospora crassa.
Fungal Biol
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Pdr5p-like ABC transporters play a significant role in azole resistance in Saccharomyces cerevisiae and Candida spp. Most of filamentous fungal species have multiple Pdr5p homologues. In this study, phylogenic analysis identified that filamentous fungi have at least two phylogenically distant groups of Pdr5p homologues. One contains PMR1-like Pdr5p homologues while the other contains both AtrF-like and AtrB-like Pdr5p homologues. Neurospora crassa has a total of four genes encoding Pdr5p homologues including CDR4 (PMR1-like), ATRB (AtrB-like), and ATRF (AtrF-like) and ATRF-2 (AtrF-like). By analyzing the susceptibilities of their knockout mutants to azole drugs including ketoconazole, fluconazole, and itraconazole, we found that deletion of cdr4 increased the susceptibility to antifungal azoles. In contrast, neither single-gene nor triple-gene deletion of atrb, atrf, and atrf-2 could not alter the susceptibility to azoles. In addition, cdr4, but not other Pdr5p homologue-coding genes, responded transcriptionally to ketoconazole stress. Together with the previous findings in other fungal species, these results suggest that the PMR1-like but not the AtrF-like or AtrB-like Pdr5p homologues play a key role in antifungal azole resistance in filamentous fungi.
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Analysis of the role of transcription factor VAD-5 in conidiation of Neurospora crassa.
Fungal Genet. Biol.
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Conidiation is the major mode of reproduction in many filamentous fungi. The Neurospora crassa gene vad-5, which encodes a GAL4-like Zn2Cys6 transcription factor, was suggested to contribute to conidiation in a previous study using a knockout mutant. In this study, we confirmed the positive contribution of vad-5 to conidiation by gene complementation. To understand the role of vad-5 in conidiation, transcriptomic profiles generated by digital gene expression profiling from the vad-5 deletion mutant and the wild-type strain were compared. Among 7559 detected genes, 176 genes were found to be transcriptionally down-regulated and 277 genes transcriptionally upregulated in the vad-5 deletion mutant, using ?1-fold change as a cutoff threshold. Among the down-regulated genes, four which were already known to be involved in conidiation -fluffy, ada-6, rca-1, and eas - were examined further in a time course experiment. Transcription of each of the four genes in the vad-5 deletion mutant was lower than in the wild-type strain during conidial development. Phenotypic observation of deletion mutants for 132 genes down-regulated by vad-5 deletion revealed that deletion mutants for 17 genes, including fluffy, ada-6, and eas, produced fewer conidia than the wild type. By phenotypic observation of deletion mutants for 211 genes upregulated in the vad-5 deletion mutant, two types of deletion mutants were found. One type, which produced more conidia than the wild-type strain, includes deletion mutants for previously characterized genes cat-2, cat-3, and sah-1 and for a non-characterized gene NCU07221. Deletion mutants of NCU06302 and NCU11090, representing the second type, produced conidia earlier than the wild-type strain. Based on these conidiation phenotypes, we designated NCU07221 as high conidial production-1 (hcp-1) and named NCU06302 and NCU11090 as early conidial development-1 (ecd-1) and ecd-2, respectively. Given the collective results from this study, we propose that vad-5 exerts an effect on conidiation by activating genes that positively contribute to conidiation as well as by repressing genes that negatively influence conidial development.
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