scholarly journals δ-Aminolaevulinic acid synthesis is required for virulence of the wheat pathogen Stagonospora nodorum

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1533-1538 ◽  
Author(s):  
Peter S. Solomon ◽  
Cordula I. Jörgens ◽  
Richard P. Oliver
Keyword(s):  
Single Gene ◽  
Acid Synthesis ◽  
Gene Replacement ◽  
Stagonospora Nodorum ◽  
Homologous Gene ◽  
In Planta ◽  
Gene Encoding ◽  
Aminolaevulinic Acid ◽  
Key Enzyme

δ-Aminolaevulinic acid (ALA) is synthesized in fungi by ALA synthase, a key enzyme in the synthesis of haem. The requirement for ALA synthase in Stagonospora nodorum to cause disease in wheat was investigated. The single gene encoding ALA synthase (Als1) was cloned and characterized. Expression analysis determined that Als1 transcription was up-regulated during germination and also towards the latter stages of the infection. The Als1 gene was further characterized by homologous gene replacement. The inactivation of Als1 resulted in strains producing severely stunted germ tubes leading quickly to death. The strains could be recovered by supplementation with 33 μM ALA. Pathogenicity assays revealed the als1 strains were essentially non-pathogenic, inferring a key role for the synthesis of ALA during in planta growth. Supplementing the strains with ALA restored growth in vitro and also pathogenicity for up to 5 days after inoculation. Further examination by inoculating the als1 strains onto wounded leaves found that pathogenicity was only partially restored, suggesting that host-derived in planta levels of ALA are not sufficient to support growth. This study has identified a key role for fungal ALA synthesis during infection and revealed its potential as an antifungal target.

scholarly journals Contributions ofyap1Mutation and SubsequentatrFUpregulation to Voriconazole Resistance inAspergillus flavus

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Yuuta Ukai ◽  
Miho Kuroiwa ◽  
Naoko Kurihara ◽  
Hiroki Naruse ◽  
Tomoyuki Homma ◽  
...  
Keyword(s):  
Point Mutation ◽  
Nuclear Export ◽  
Resistance Mechanism ◽  
Resistant Mutant ◽  
Gene Replacement ◽  
Bzip Transcription Factor ◽  
Homologous Gene ◽  
Sequencing Analysis ◽  
Content Type

ABSTRACTAspergillus flavusis the second most significant pathogenic cause of invasive aspergillosis; however, its emergence risks and mechanisms of voriconazole (VRC) resistance have not yet been elucidated in detail. Here, we demonstrate that repeated exposure ofA. flavusto subinhibitory concentrations of VRCin vitrocauses the emergence of a VRC-resistant mutant with a novel resistance mechanism. The VRC-resistant mutant shows a MIC of 16 μg/ml for VRC and of 0.5 μg/ml for itraconazole (ITC). Whole-genome sequencing analysis showed that the mutant possesses a point mutation inyap1, which encodes a bZIP transcription factor working as the master regulator of the oxidative stress response, but no mutations in thecyp51genes. This point mutation inyap1caused alteration of Leu558 to Trp (Yap1Leu558Trp) in the putative nuclear export sequence in the carboxy-terminal cysteine-rich domain of Yap1. This Yap1Leu558Trpsubstitution was confirmed as being responsible for the VRC-resistant phenotype, but not for that of ITC, by the revertant to Yap1wild typewith homologous gene replacement. Furthermore, Yap1Leu558Trpcaused marked upregulation of theatrFATP-binding cassette transporter, and the deletion ofatrFrestored susceptibility to VRC inA. flavus. These findings provide new insights into VRC resistance mechanisms via a transcriptional factor mutation that is independent of thecyp51gene mutation inA. flavus.

scholarly journals The Multifunctional β-Oxidation Enzyme Is Required for Full Symptom Development by the Biotrophic Maize Pathogen Ustilago maydis

2006 ◽  
Vol 5 (12) ◽  
pp. 2047-2061 ◽  
Author(s):  
Jana Klose ◽  
James W. Kronstad
Keyword(s):  
Fatty Acids ◽  
Ustilago Maydis ◽  
Long Chain Fatty Acids ◽  
In Planta ◽  
Gene Encoding ◽  
Metabolic Role ◽  
Fungal Phytopathogen ◽  
Long Chain ◽  
Chain Fatty Acids

ABSTRACT The transition from yeast-like to filamentous growth in the biotrophic fungal phytopathogen Ustilago maydis is a crucial event for pathogenesis. Previously, we showed that fatty acids induce filamentation in U. maydis and that the resulting hyphal cells resemble the infectious filaments observed in planta. To explore the potential metabolic role of lipids in the morphological transition and in pathogenic development in host tissue, we deleted the mfe2 gene encoding the multifunctional enzyme that catalyzes the second and third reactions in β-oxidation of fatty acids in peroxisomes. The growth of the strains defective in mfe2 was attenuated on long-chain fatty acids and abolished on very-long-chain fatty acids. The mfe2 gene was not generally required for the production of filaments during mating in vitro, but loss of the gene blocked extensive proliferation of fungal filaments in planta. Consistent with this observation, mfe2 mutants exhibited significantly reduced virulence in that only 27% of infected seedlings produced tumors compared to 88% tumor production upon infection by wild-type strains. Similarly, a defect in virulence was observed in developing ears upon infection of mature maize plants. Specifically, the absence of the mfe2 gene delayed the development of teliospores within mature tumor tissue. Overall, these results indicate that the ability to utilize host lipids contributes to the pathogenic development of U. maydis.

scholarly journals The NtcA-Dependent P1 Promoter Is Utilized for glnA Expression in N2-Fixing Heterocysts of Anabaena sp. Strain PCC 7120

2004 ◽  
Vol 186 (21) ◽  
pp. 7337-7343 ◽  
Author(s):  
Ana Valladares ◽  
Alicia M. Muro-Pastor ◽  
Antonia Herrero ◽  
Enrique Flores
Keyword(s):  
Binding Site ◽  
In Vitro Transcription ◽  
Upstream Region ◽  
Gene Encoding ◽  
Differentiated Cells ◽  
Multiple Promoters ◽  
Key Enzyme ◽  
Anabaena Sp ◽  
Pcc 7120

ABSTRACT Expression of the glnA gene encoding glutamine synthetase, a key enzyme in nitrogen metabolism, is subject to a variety of regulatory mechanisms in different organisms. In the filamentous, N2-fixing cyanobacterium Anabaena sp. strain PCC 7120, glnA is expressed from multiple promoters that generate several transcripts whose abundance is influenced by NtcA, the transcription factor exerting global nitrogen control in cyanobacteria. Whereas RNAI originates from a canonical NtcA-dependent promoter (P1) and RNAII originates from a σ70-type promoter (P2), RNAIV is influenced by NtcA but the corresponding promoter (P3) does not have the structure of NtcA-activated promoters. Using RNA isolated from Anabaena filaments grown under different nitrogen regimens, we observed, in addition to these transcripts, RNAV, which has previously been detected only in in vitro transcription assays and should originate from P4. However, in heterocysts, which are differentiated cells specialized in N2 fixation, RNAI was the almost exclusive glnA transcript. Analysis of P glnA ::lacZ fusions containing different fragments of the glnA upstream region confirmed that fragments carrying P1, P2, or P3 and P4 have the ability to promote transcription. Mutation of the NtcA-binding site in P1 eliminated P1-directed transcription and allowed increased use of P2. The NtcA-binding site in the P1 promoter and binding of NtcA to this site appear to be key factors in determining glnA gene expression in vegetative cells and heterocysts.

scholarly journals Characterisation of a novel endophyte NRPS gene and its role in endophyte-grass symbioses

2007 ◽  
Vol 13 ◽  
pp. 491-493
Author(s):  
H. Harzer ◽  
R.D. Johnson ◽  
S. Rasmussen ◽  
C.R. Voisey ◽  
L.J. Johnson
Keyword(s):  
Fungal Endophytes ◽  
Gene Clusters ◽  
Gene Replacement ◽  
In Planta ◽  
Dna Library ◽  
Peptide Synthetase ◽  
Genomic Dna Library ◽  
Targeted Gene Replacement ◽  
Fungal Secondary Metabolite

Symbiotic grass associations with fungal endophytes (genera Neotyphodium and Epichloë) display enhanced fitness as well as prolonged field persistence over their endophyte free equivalents. Perennial ryegrass, an important agronomic grass, is typically associated with the N. lolii endophyte. The endophyte lives within the intercellular spaces without inducing any symptoms in the plant. The aim of this study is to elucidate the biosynthetic function of fungal secondary metabolite gene clusters. Non-ribosomal peptide synthetase genes (NRPSs) of unknown function were targeted, as these genes are commonly associated with the production of bioactive peptides some of which are ecologically important. Some novel endophyte NRPS genes have been identified using a degenerate PCR screen; one of these, NRPS5 will be discussed here. Clones were obtained by screening a fosmid Epichloë festucae genomic DNA library and we are currently determining gene function by using targeted gene replacement followed by an assessment in vitro and in planta using metabolomics and appropriate bioassay screens. Keywords: endophyte, NRPS, secondary metabolism

scholarly journals Endopolygalacturonase Genes from Colletotrichum lindemuthianum: Cloning of CLPG2 and Comparison of Its Expression to That of CLPG1 During Saprophytic and Parasitic Growth of the Fungus

1997 ◽  
Vol 10 (6) ◽  
pp. 769-775 ◽  
Author(s):  
Sylvie Centis ◽  
Isabelle Guillas ◽  
Nathalie Séjalon ◽  
Marie-Thérèse Esquerré-Tugayé ◽  
Bernard Dumas
Keyword(s):  
Synthetic Medium ◽  
Amino Acid Level ◽  
Single Copy ◽  
Colletotrichum Lindemuthianum ◽  
In Planta ◽  
Gene Encoding ◽  
Host Cell Wall ◽  
Extensive Degradation ◽  
Necrotrophic Stage

Following the previous isolation of CLPG1, a gene encoding an endopolygalacturonase (endoPG) secreted into the culture filtrate of Colletotrichum lindemuthianum, we have isolated and sequenced an additional endoPG gene, CLPG2. This gene is present as a single copy in the genome of the fungus. At the amino acid level, CLPG2 shows 61% identity to CLPG1 and between 37 to 59% identity to other fungal endoPGs. RNA blot analyses of endoPG gene expression were followed with specific probes during in vitro culture of the fungus. When conidia were used to inoculate a synthetic medium containing pectin as sole carbon source, only CLPG1 was found to be expressed after 3 days of culture. However, transferring the mycelium grown on glucose for 4 days to a pectin-containing medium allowed the detection of CLPG1 and CLPG2 transcripts as early as 12 h after transfer on this substrate. Expression of CLPG2 was transient while that of CLPG1 was more prolonged. Immunocytological localization of endoPG in C. lindemuthianum-infected bean tissues with antibodies against CLPG1 confirmed that the protein is produced in planta and is associated with extensive degradation of the host cell wall. Detection of endoPG transcripts by reverse transcription-polymerase chain reaction revealed that CLPG1, but not CLPG2, is expressed at the beginning of the necrotrophic stage of infection. These results show that the two endoPG genes are differentially expressed and that CLPG1 encodes the major secreted endoPG both during saprophytic growth and during plant infection.

scholarly journals Mannitol 1-Phosphate Metabolism Is Required for Sporulation in Planta of the Wheat Pathogen Stagonospora nodorum

2005 ◽  
Vol 18 (2) ◽  
pp. 110-115 ◽  
Author(s):  
Peter S. Solomon ◽  
Kar-Chun Tan ◽  
Richard P. Oliver
Keyword(s):  
Insertional Mutagenesis ◽  
Redox Regulation ◽  
Expressed Sequence Tag ◽  
Stagonospora Nodorum ◽  
Resonance Spectroscopy ◽  
Plant Interactions ◽  
Wild Type ◽  
In Planta ◽  
Mutant Strains

An expressed sequence tag encoding a putative mannitol 1-phosphate dehydrogenase (Mpd1) has been characterized from the fungal wheat pathogen Stagonospora nodorum. Mpd1 was disrupted by insertional mutagenesis, and the resulting mpd1 strains lacked all detectable NAD-linked mannitol 1-phosphate dehydrogenase activity (EC 1.1.1.17). The growth rates, sporulation, and spore viability of the mutant strains in vitro were not significantly different from the wild type. The viability of the mpd1 spores when subjected to heat stress was comparable to wild type. Characterization of the sugar alcohol content by nuclear magnetic resonance spectroscopy revealed that, when grown on glucose, the mutant strains contained significantly less mannitol, less arabitol, but more trehalose than the wildtype strains. The mannitol content of fructose-grown cultures was normal. No secreted mannitol could be detected in wild type or mutants. Pathogenicity assays revealed the disruption of Mpd1 did not affect lesion development, however the mutants were unable to sporulate. These results throw new light on the role of mannitol in fungal plant interactions, suggesting a role in metabolic and redox regulation during the critical process of sporulation on senescing leaf material.

Transgenic expression of a gene encoding a synthetic antimicrobial peptide results in inhibition of fungal growth in vitro and in planta

Plant Science ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 171-181 ◽  
Author(s):  
Jeffrey W. Cary ◽  
Kanniah Rajasekaran ◽  
Jesse M. Jaynes ◽  
Thomas E. Cleveland
Keyword(s):  
Antimicrobial Peptide ◽  
Fungal Growth ◽  
In Planta ◽  
Gene Encoding ◽  
Transgenic Expression

scholarly journals Entner–Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1

2015 ◽  
Vol 112 (31) ◽  
pp. E4298-E4305 ◽  
Author(s):  
Ann-Katrin Felux ◽  
Dieter Spiteller ◽  
Janosch Klebensberger ◽  
David Schleheck
Keyword(s):  
Pseudomonas Putida ◽  
Single Gene ◽  
Gene Clusters ◽  
Degradation Pathway ◽  
Head Group ◽  
Homologous Gene ◽  
Polar Head Group ◽  
Differential Proteomics ◽  
K 12

Sulfoquinovose (SQ; 6-deoxy-6-sulfoglucose) is the polar head group of the plant sulfolipid SQ-diacylglycerol, and SQ comprises a major proportion of the organosulfur in nature, where it is degraded by bacteria. A first degradation pathway for SQ has been demonstrated recently, a “sulfoglycolytic” pathway, in addition to the classical glycolytic (Embden–Meyerhof) pathway in Escherichia coli K-12; half of the carbon of SQ is abstracted as dihydroxyacetonephosphate (DHAP) and used for growth, whereas a C3-organosulfonate, 2,3-dihydroxypropane sulfonate (DHPS), is excreted. The environmental isolate Pseudomonas putida SQ1 is also able to use SQ for growth, and excretes a different C3-organosulfonate, 3-sulfolactate (SL). In this study, we revealed the catabolic pathway for SQ in P. putida SQ1 through differential proteomics and transcriptional analyses, by in vitro reconstitution of the complete pathway by five heterologously produced enzymes, and by identification of all four organosulfonate intermediates. The pathway follows a reaction sequence analogous to the Entner–Doudoroff pathway for glucose-6-phosphate: It involves an NAD+-dependent SQ dehydrogenase, 6-deoxy-6-sulfogluconolactone (SGL) lactonase, 6-deoxy-6-sulfogluconate (SG) dehydratase, and 2-keto-3,6-dideoxy-6-sulfogluconate (KDSG) aldolase. The aldolase reaction yields pyruvate, which supports growth of P. putida, and 3-sulfolactaldehyde (SLA), which is oxidized to SL by an NAD(P)+-dependent SLA dehydrogenase. All five enzymes are encoded in a single gene cluster that includes, for example, genes for transport and regulation. Homologous gene clusters were found in genomes of other P. putida strains, in other gamma-Proteobacteria, and in beta- and alpha-Proteobacteria, for example, in genomes of Enterobacteria, Vibrio, and Halomonas species, and in typical soil bacteria, such as Burkholderia, Herbaspirillum, and Rhizobium.

scholarly journals Cloning and Disruption of pgx4 Encoding an In Planta Expressed Exopolygalacturonase from Fusarium oxysporum

2000 ◽  
Vol 13 (4) ◽  
pp. 359-365 ◽  
Author(s):  
F. I. García-Maceira ◽  
Antonio Di Pietro ◽  
M. Isabel G. Roncero
Keyword(s):  
Amino Acid ◽  
Fusarium Oxysporum ◽  
Vascular Tissue ◽  
Single Copy ◽  
Gene Replacement ◽  
Amino Acid Sequences ◽  
In Planta ◽  
Calculated Molecular Mass ◽  
Tomato Plants

Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato vascular wilt, produces an array of pectinolytic enzymes, including at least two exo-α1,4-polygalac-turonases (exoPGs). A gene encoding an exoPG, pgx4, was isolated with degenerate polymerase chain reaction primers derived from amino acid sequences conserved in two fungal exoPGs. pgx4 encodes a 454 amino acid polypeptide with nine potential N-glycosylation sites and a putative 21 amino acid N-terminal signal peptide. The deduced mature protein has a calculated molecular mass of 47.9 kDa, a pI of 8.0, and 51 and 49% identity with the exoPGs of Cochliobolus carbonum and Aspergillus tubingensis, respectively. The gene is present in a single copy in different formae speciales of F. oxysporum. Expression of pgx4 was detected during in vitro growth on pectin, polygalacturonic acid, and tomato vascular tissue and in roots and stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Two mutants of F. oxy-sporum f. sp. lycopersici with a copy of pgx4 inactivated by gene replacement were as virulent on tomato plants as the wild-type strain.

scholarly journals Cytosolic Isocitrate Dehydrogenase from Arabidopsis thaliana Is Regulated by Glutathionylation

Antioxidants ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 16 ◽  
Author(s):  
Adnan Khan Niazi ◽  
Laetitia Bariat ◽  
Christophe Riondet ◽  
Christine Carapito ◽  
Amna Mhamdi ◽  
...  
Keyword(s):  
Active Site ◽  
Nicotinamide Adenine Dinucleotide ◽  
Single Gene ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
In Planta ◽  
Thioredoxin System ◽  
Close Proximity ◽  
Dependent Inhibition

NADP-dependent (Nicotinamide Adénine Dinucléotide Phosphate-dependent) isocitrate dehydrogenases (NADP-ICDH) are metabolic enzymes involved in 2-oxoglutarate biosynthesis, but they also supply cells with NADPH. Different NADP-ICDH genes are found in Arabidopsis among which a single gene encodes for a cytosolic ICDH (cICDH) isoform. Here, we show that cICDH is susceptible to oxidation and that several cysteine (Cys) residues are prone to S-nitrosylation upon nitrosoglutathione (GSNO) treatment. Moreover, we identified a single S-glutathionylated cysteine Cys363 by mass-spectrometry analyses. Modeling analyses suggest that Cys363 is not located in the close proximity of the cICDH active site. In addition, mutation of Cys363 consistently does not modify the activity of cICDH. However, it does affect the sensitivity of the enzyme to GSNO, indicating that S-glutathionylation of Cys363 is involved in the inhibition of cICDH activity upon GSNO treatments. We also show that glutaredoxin are able to rescue the GSNO-dependent inhibition of cICDH activity, suggesting that they act as a deglutathionylation system in vitro. The glutaredoxin system, conversely to the thioredoxin system, is able to remove S-nitrosothiol adducts from cICDH. Finally, NADP-ICDH activities were decreased both in a catalase2 mutant and in mutants affected in thiol reduction systems, suggesting a role of the thiol reduction systems to protect NADP-ICDH activities in planta. In line with our observations in Arabidopsis, we found that the human recombinant NADP-ICDH activity is also sensitive to oxidation in vitro, suggesting that this redox mechanism might be shared by other ICDH isoforms.

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