scholarly journals Deletion of Mid1, a putative stretch-activated calcium channel in Claviceps purpurea, affects vegetative growth, cell wall synthesis and virulence

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 3922-3933 ◽  
Author(s):  
Jörg Bormann ◽  
Paul Tudzynski
Keyword(s):  
Vegetative Growth ◽  
Pathogenic Fungus ◽  
Axenic Culture ◽  
Gene Replacement ◽  
Major Effect ◽  
Growth Defect ◽  
Claviceps Purpurea ◽  
Disease Symptoms

The putative Claviceps purpurea homologue of the Saccharomyces cerevisiae stretch-activated calcium ion channel Mid1 was investigated for its role in vegetative growth, differentiation and pathogenicity on rye (Secale cereale). Gene replacement mutants of Cl. purpurea mid1 were not affected in polar growth and branching in axenic culture but showed a significantly reduced growth rate. The growth defect could not be complemented by Ca2+ supplementation, in contrast to mid1 mutants in yeast, but the altered sensitivity of the mutants to changes in external and internal Ca2+ concentrations indicates some role of Mid1 in Ca2+ homeostasis. The major effect of mid1 deletion, however, was the complete loss of virulence: infected rye plants showed no disease symptoms at all. Detailed analyses of in vitro-infected rye ovaries demonstrated that the Δmid1 mutants had multiple apical branches and were unable to infect the host tissue, suggesting that Mid1 is essential for generating the necessary mechanical force for penetration. This is believed to be the first report of an essential role for a Mid1 homologue in the virulence of a plant-pathogenic fungus.

scholarly journals Structure and inhibition of Cryptococcus neoformans sterylglucosidase to develop antifungal agents

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nivea Pereira de Sa ◽  
Adam Taouil ◽  
Jinwoo Kim ◽  
Timothy Clement ◽  
Reece M. Hoffmann ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Rational Design ◽  
Antifungal Agents ◽  
Pathogenic Fungus ◽  
Pathogenic Fungi ◽  
Growth Defect ◽  
Wild Type ◽  
Secondary Infections ◽  
Heavy Burden

AbstractPathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3β-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3β-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1’s substrate specificity and enable the rational design of antifungal agents targeting Sgl1.

scholarly journals Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 118 (4) ◽  
pp. 609-617
Author(s):  
M Winey ◽  
M R Culbertson
Keyword(s):  
Growth Defect ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Endonuclease Activity ◽  
Temperature Dependent ◽  
Direct Role ◽  
Trna Splicing

Abstract Two unlinked mutations that alter the enzyme activity of tRNA-splicing endonuclease have been identified in yeast. The sen1-1 mutation, which maps on chromosome 12, causes temperature-sensitive growth, reduced in vitro endonuclease activity, and in vivo accumulation of unspliced pre-tRNAs. The sen2-1 mutation does not confer a detectable growth defect, but causes a temperature-dependent reduction of in vitro endonuclease activity. Pre-tRNAs do not accumulate in sen2-1 strains. The in vitro enzyme activities of sen1-1 and sen2-1 complement in extracts from a heterozygous diploid, but fail to complement in mixed extracts from separate sen1-1 and sen2-1 haploid strains. These results suggest a direct role for SEN gene products in the enzymatic removal of introns from tRNA that is distinct from the role of other products known to affect tRNA splicing.

scholarly journals Transcript analysis reveals an extended regulon and the importance of protein–protein co-operativity for the Escherichia coli methionine repressor

2006 ◽  
Vol 396 (2) ◽  
pp. 227-234 ◽  
Author(s):  
Ferenc Marincs ◽  
Iain W. Manfield ◽  
Jonathan A. Stead ◽  
Kenneth J. Mcdowall ◽  
Peter G. Stockley
Keyword(s):  
Escherichia Coli ◽  
Gene Replacement ◽  
Dna Arrays ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Metabolic Role ◽  
Mobility Shift Assay

We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5′ promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein–protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein–protein and protein–DNA affinity.

scholarly journals The INFLUENCE OF TEMPERATURE AND FUNGAL COMMUNITY ON GROWTH AND SPORULATION OF Diaporthe FROM FRUIT PLANTS

2020 ◽  
Vol 19 (5) ◽  
pp. 71-79
Author(s):  
Barbara Anna Abramczyk ◽  
Ewa Dorota Król ◽  
Ewa Dorota Zalewska ◽  
Beata Zimowska
Keyword(s):  
Vegetative Growth ◽  
Growth And Development ◽  
Pathogenic Fungus ◽  
Extreme Temperature ◽  
Fruit Trees ◽  
Climatic Conditions ◽  
Effect Of Temperature ◽  
Influence Of Temperature ◽  
Fruit Plants

The purpose of this experiment was to determine the influence of temperature and fungi colonizing fruit plants on Diaporthe, a pathogenic fungus in Poland. The biotic series method was used to test in vitro the effects of the fungi colonizing fruit trees on isolates of Diaporthe. Among the 13 fungal species tested, six inhibited the growth and development of Diaporthe; while another two species were neutral and the rest showed the lack of limiting impact in relation to the pathogen. Fungi present in the shoots of fruit plants was able to limit the growth and development of Diaporthe spp., both in chemically-protected and non-protected orchards. Studies on the effect of temperature indicated that the optimum temperature for vegetative growth of isolates of Diaporthe ranged from 20°C to 30°C, and for conidial sporulation from 25°C to 30°C. However, the ability of Diaporthe sp. to survive at extreme temperature (–20°C and +35°C) enables their vegetative growth in climatic conditions worldwide.

scholarly journals Efficacy of resistance selection to Verticillium wilt in strawberry (Fragaria x ananassa Duch.) tissue culture

2012 ◽  
Vol 64 (3) ◽  
pp. 3-12 ◽  
Author(s):  
Jadwiga I. Żebrowska
Keyword(s):  
Tissue Culture ◽  
Verticillium Wilt ◽  
Verticillium Dahliae ◽  
Pathogenic Fungus ◽  
Genetic Resistance ◽  
Fragaria X Ananassa ◽  
Post Inoculation ◽  
Disease Symptoms ◽  
Resistance Selection

The soil-borne pathogenic fungus <i>Verticillium dahliae</i> Kleb. causes economic losses in crops in temperate regions of the world and hence is the most studied species. Strawberry (<i>Fragaria x ananassa</i> Duch.) belongs to plant species susceptible to <i>Verticillium dahliae</i>, although the response to infection caused by this pathogen is varied and depends on the cultivar. Due to a lack of efficient methods in Verticillium wilt elimination, the selection of genetically resistant plant material is a priority direction in breeding programs. Efficacy of resistance selection to <i>Verticillium dahliae</i> Kleb. in strawberry tissue culture was examined on the basis of response to <i>in vitro</i> infection by this pathogenic fungus in two tissue cultured strawberry cultivars, i.e. 'Filon' and 'Teresa'. Culture was conducted for 16 months in an environmentally controlled growth room at 18-20°C, 60-70% relative humidity and light intensity of 100 µm E × m<sup>-2</sup> × s<sup>-1</sup> on a 16h light / 8h dark cycle. Subcultures were proliferated every 6 weeks on modified Murashige and Skoog medium. Four hundred microplants from each tissue cultured cultivar were inoculated under <i>in vitro</i> conditions at the 4-leaf stage with a homogenate of liquid mycelium of <i>Verticillium dahliae</i> serving as the selecting agent. Disease symptoms were observed at 15, 30, 45, 60, and 75th days post inoculation. The extent of leaf chlorosis was rated on a scale of 0-4. At day 75<sup>th</sup> post inoculation, the percentage of totally chlorotic plants in micropropagated cv. Teresa reached the value of 76.27%, whereas the proportion of such plants in inoculated tissue cultured cv. Filon reached the value of 89.40%. Also, the index of infection calculated for very severe disease symptoms in the subclone 'Teresa' reached the mean value lower when compared with that calculated for subclone 'Filon' (0.0962 and 0.1150, respectively). These results suggested that the micropropagated cv. Teresa exhibited higher genetic resistance to the selecting agent in comparison with the tissue cultured cv. Filon, and it was consistent with field resistance of both cultivars to this pathogen. Therefore, the procedure of <i>in vitro</i> selection used in this study was quite efficient to distinguish varying genetic resistance to <i>Verticillium dahliae</i> in the two examined strawberry subclones, and can be recommended as a suitable method for the estimation of susceptibility to Verticillium wilt in different strawberry genotypes.

scholarly journals The role of Dicer-dependent RNA interference in regulating cross-species communication during fungus-fungus interactions

2021 ◽  
Author(s):  
Edoardo Piombo ◽  
Ramesh Raju Vetukuri ◽  
Anders Broberg ◽  
Pruthvi B Kalyandurg ◽  
Sandeep Kushwaha ◽  
...  
Keyword(s):  
Rna Interference ◽  
Pathogenic Fungus ◽  
Hydrolytic Enzymes ◽  
Plant Diseases ◽  
Parasitic Fungus ◽  
Transcriptional Gene Silencing ◽  
Clonostachys Rosea ◽  
Stem Loop

Dicer-like (DCL) proteins play a vital role in transcriptional and post-transcriptional gene silencing, also known as RNA interference (RNAi), by cleaving double-stranded RNAs or single-stranded RNAs with stem-loop structures into small RNAs . Although DCL-mediated RNAi can regulate interspecific communication between pathogenic/mutualistic organisms and their hosts, its role in parasitic fungus-fungus interactions is yet to be investigated . In this study, we deleted dcl genes in the mycoparasitic fungus Clonostachys rosea and analyzed the transcriptome and secondary metabolome to characterize the regulatory functions of DCL-dependent RNAi in mycoparasitism. Deletion of dcl2 resulted in a mutant with reduced growth rate, pigment production and antagonism towards the plant pathogenic fungus Botrytis cinerea . Moreover, the Δ dcl2 mutant displayed a reduced ability to control fusarium foot rot disease on wheat, caused by Fusarium graminearum , and reduced production of 62 secondary metabolites (SM) including yellow‐coloured sorbicillinoids. Transcriptome sequencing of the in vitro interaction between the C. rosea Δ dcl2 strain and B. cinerea or F. graminearum identified downregulation of genes coding for transcription factors, membrane transporters, hydrolytic enzymes and SM biosynthesis enzymes putatively involved in antagonistic interactions, in comparison with the C. rosea wild type interaction. Sixty-one putative novel microRNA-like RNAs (milRNAs) were identified in C. rosea , and 11 was upregulated in the Δ dcl2 mutant. In addition to putative endogenous gene targets, these DCL2-dependent milRNAs were predicted to target B . cinerea and F. graminearum virulence factor genes, which showed an increased expression during interaction with the Δ dcl2 mutant incapable of producing the targeting milRNAs. This paper constitutes the first step in elucidating the role of RNAi in mycoparasitism, with important implications for biological control of plant diseases. This study further indicates a possible cross-species regulatory activity of fungal milRNAs, emphasizing a novel role of RNAi in fungal interactions and ecology.

scholarly journals Tetracycline-Regulatable System To Tightly Control Gene Expression in the Pathogenic Fungus Candida albicans

2000 ◽  
Vol 68 (12) ◽  
pp. 6712-6719 ◽  
Author(s):  
Hironobu Nakayama ◽  
Toshiyuki Mio ◽  
Shigehisa Nagahashi ◽  
Michiko Kokado ◽  
Mikio Arisawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Culture Media ◽  
Pathogenic Fungus ◽  
Elongation Factor ◽  
Growth Defect ◽  
Control Gene ◽  
Control Gene Expression

ABSTRACT Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformisluciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement ofN-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.

scholarly journals CPTF1, a CREB-Like Transcription Factor, Is Involved in the Oxidative Stress Response in the Phytopathogen Claviceps purpurea and Modulates ROS Level in Its Host Secale cereale

2004 ◽  
Vol 17 (4) ◽  
pp. 383-393 ◽  
Author(s):  
Eva Nathues ◽  
Suchitra Joshi ◽  
Klaus B. Tenberge ◽  
Marcell von den Driesch ◽  
Birgitt Oeser ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Stress Response ◽  
Expressed Sequence Tag ◽  
Axenic Culture ◽  
Gene Replacement ◽  
Oxidative Stress Response ◽  
Claviceps Purpurea ◽  
In Planta ◽  
Est Analysis

CPTF1, a transcription factor with significant homology to ATF/CREB bZIP factors, was identified during an expressed sequence tag (EST) analysis of in planta-expressed genes of the phytopathogen Claviceps purpurea. Using a gene-replacement approach, deletion mutants of cptf1 were created. Expression studies in axenic culture showed that the H2O2-inducible gene cpcat1 (encoding a secreted catalase) had a reduced basal expression level and no longer responded to oxidative stress in the Δcptf1 mutant. Biochemical analyses indicated that CPTF1 is a general regulator of catalase activity. Δcptf1 mutants showed significantly reduced virulence on rye. Electron microscopical in situ localization revealed significant amounts of H2O2 in Δcptf1-infected rye epidermal tissues, indicating that the plant tissue displayed an oxidative burst-like reaction, an event not detected in wild-type infections. These data indicate that CPTF1 is involved not only in oxidative stress response in the fungus but also in modulation of the plant's defense reactions.

scholarly journals A Single Nucleotide Mutation in Adenylate Cyclase Affects Vegetative Growth, Sclerotial Formation and Virulence of Botrytis cinerea

2020 ◽  
Vol 21 (8) ◽  
pp. 2912
Author(s):  
Xue Chen ◽  
Xiaohong Zhang ◽  
Pinkuan Zhu ◽  
Yiwen Wang ◽  
Yantao Na ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Botrytis Cinerea ◽  
Vegetative Growth ◽  
Pathogenic Fungus ◽  
Site Directed Mutagenesis ◽  
Growth Defect ◽  
Single Nucleotide ◽  
Single Nucleotide Mutation ◽  
Nucleotide Mutation ◽  
Sclerotial Formation

Botrytis cinerea is a pathogenic fungus that causes gray mold disease in a broad range of crops. The high intraspecific variability of B. cinerea makes control of this fungus very difficult. Here, we isolated a variant B05.10M strain from wild-type B05.10. The B05.10M strain showed serious defects in mycelial growth, spore and sclerotia production, and virulence. Using whole-genome resequencing and site-directed mutagenesis, a single nucleotide mutation in the adenylate cyclase (BAC) gene that results in an amino acid residue (from serine to proline, S1407P) was shown to be the cause of various defects in the B05.10M strain. When we further investigated the effect of S1407 on BAC function, the S1407P mutation in bac showed decreased accumulation of intracellular cyclic AMP (cAMP), and the growth defect could be partially restored by exogenous cAMP, indicating that the S1407P mutation reduced the enzyme activity of BAC. Moreover, the S1407P mutation exhibited decreased spore germination rate and infection cushion formation, and increased sensitivity to cell wall stress, which closely related to fungal development and virulence. Taken together, our study indicates that the S1407 site of bac plays an important role in vegetative growth, sclerotial formation, conidiation and virulence in B. cinerea.

scholarly journals The potential of silver nanoparticles to control Rhizoctonia solani (AG3-PT) growth in vitro

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
HARTATI OKTARINA ◽  
JAMES WOODHALL ◽  
IAN SINGLETON
Keyword(s):  
Silver Nanoparticles ◽  
Rhizoctonia Solani ◽  
Plant Pathogens ◽  
Pathogenic Fungus ◽  
Subsequent Development ◽  
Hyphal Growth ◽  
Disease Symptoms ◽  
Fungal Plant Pathogens ◽  
Sclerotium Production

Silver nanoparticles (AgNPs) have known anti-microbial properties and therefore have the potential to be used to control fungal plant pathogens. In this study we investigated the growth of a plant pathogenic fungus, Rhizoctonia solani (AG3-PT) in the presence of AgNPs. The effect of AgNPs at two different levels (20 and 50 mg L-1) on hyphal growth and sclerotium production and viability in R. solani was investigated. The results showed that at 20 mg AgNPs L-1 R. solani hyphal growth was reduced along with the production of sclerotia. The results indicate that AgNPs have the potential to control R. solani growth and subsequent development of plant disease symptoms.

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