Cyclophilin A and calcineurin functions investigated by gene inactivation, cyclosporin A inhibition and cDNA arrays approaches in the phytopathogenic fungus<i>Botrytis cinerea</i>

Viaud, Muriel; Simon, Adeline; Brygoo, Yves; Pradier, Jean‐Marc; Levis, Caroline

doi:10.1046/j.1365-2958.2003.03798.x

Cited by 123 publications

(140 citation statements)

References 91 publications

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“…Starting from two B. cinerea EST sequences (Viaud et al, 2003) showing high similarity to fungal hexo-and glucokinase genes, respectively, gene-specific primers were designed, and used for the amplification of hxt1 and glk1 gene fragments from genomic DNA of B. cinerea strain B05.10. Using these PCR fragments as hybridization probes, genomic clones containing each of the genes were isolated from a B. cinerea genomic bacteriophage library.…”

Section: Cloning Of Glk1 and Hxk1 Genesmentioning

confidence: 99%

The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits

Rui

Hahn

2007

Microbiology

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Hexose kinases play a central role in the initiation of sugar metabolism of living organisms and have also been implicated in carbon catabolite repression in yeasts and plants. In this study, the genes encoding glucokinase (Glk1) and hexokinase (Hxk1) from the plant-pathogenic ascomycete Botrytis cinerea were isolated and functionally characterized. Glk1-deficient mutants were indistinguishable from the wild-type in all growth parameters tested. In contrast, Dhxk1 mutants lacking Hxk1 showed a pleiotropic growth defect. On artificial media, vegetative growth was retarded, and conidia formation strongly reduced. No or only marginal growth of Dhxk1 mutants was observed when fructose, galactose, sucrose or sorbitol were used as carbon sources, and fructose inhibited growth of the mutant in the presence of other carbon sources. B. cinerea mutants containing hxk1 alleles with point mutations leading to enzymically inactive enzymes showed phenotypes similar to the Dhxk1 disruption mutant, indicating that loss of hexose phosphorylation activity of Hxk1 is solely responsible for the pleiotropic growth defect. Virulence of the Dhxk1 mutants was dependent on the plant tissue: on leaves, lesion formation was only slightly retarded compared to the wild-type, whereas only small lesions were formed on apples, strawberries and tomatoes. The low virulence of Dhxk1 mutants on fruits was correlated with their high contents of sugars, in particular fructose. Heterologous expression of Hxk1 and Glk1 in yeast allowed their enzymic characterization, revealing kinetic properties similar to other fungal hexokinases and glucokinases. Both Dglk1 and Dhxk1 mutants showed normal glucose repression of secreted lipase 1 activity, indicating that, in contrast to yeast, B. cinerea hexose kinases are not involved in carbon catabolite repression. INTRODUCTIONHexose kinases are crucial enzymes for most living cells, by catalysing the intracellular trapping and feeding into metabolism of the hexoses glucose and fructose after their uptake. In addition, hexokinases have been shown in yeast, plants and mammals to participate in glucose signalling (De Winde et al., 1996;Moore et al., 2003;Efrat et al., 1994). Saccharomyces cerevisiae has three enzymes catalysing hexose phosphorylation, namely hexokinases (EC 2 . 7 . 1 . 1) Hxk1 and Hxk2, and glucokinase (EC 2 . 7 . 1 . 2) Glk1. Each of these enzymes can support growth on glucose, while either of the two hexokinases is required for growth on fructose (Lobo & Maitra, 1977;Gancedo et al., 1977). Hxk2, in addition, is mainly required for catabolite repression, which prevents the expression of genes involved in catabolism of less preferred carbon sources in the presence of a more favourable nutrient such as glucose (Entian, 1980). In the presence of glucose, Hxk2 interacts with the carbon catabolite repressor Mig1 and thereby is translocated into the nucleus. In the nucleus, the Hxk2-Mig1 complex seems to form a repressor complex that binds to the promoters of carbon-catabolite-repressible genes (Ahuatzi ...

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Section: Cloning Of Glk1 and Hxk1 Genesmentioning

confidence: 99%

The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits

Rui

Hahn

2007

Microbiology

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“…Analysis of similarity by BLAST among important fungi responsible by diseases showed 74% identity (107/144) with B. cinerea (GenBank AL112297), 73% identity (119/162) with C. neoformas (GenBank EAL22572) and 69% identity (112/162) with M. grisea (Genbank AF293848). Cyclophilins from these fungi are involved in virulence [11][12][13]15].…”

Section: Resultsmentioning

confidence: 99%

“…CsA inhibits appressorium development and hyphal growth in a cyclophilin-dependent manner, indicating cyclophilin as a virulence factor in phytopathogenic fungi and provides evidence for a signaling required for infection structure formation by M. grisea [13]. The cyclophilin null mutant in Botrytis cinerea is able to develop infection structures, but it is in the development of symptom-altered-agents in the development of bean and tomato leaves, while calcineurin inhibition using CsA modifies hyphal morphology and prevents infection structure formation [15]. In Saccharomyces cerevisiae, cyclophilin A is localized to the nucleus and governs the meiotic gene expression program to promote efficient sporulation [16].…”

Section: Introductionmentioning

confidence: 87%

“…The coordinates and structural factor files of apo and bound cyclophilin have been deposited into the Protein Data Bank, with the accession codes 3O7T and 3PMP, respectively. Assays for the Activity of Cyclosporin A. Moniliopthora perniciosa' basidiospores were obtained from brooms hanged them in an illuminated (12:12 diurnal light) cabinet in which a mister sprayed brooms with deionized H 2 O for 1 min every 24 h. Standard procedures to test the effect of CsA on basidiospores germination and germ tube development were adapted from previous studies on CsA inhibitory effect [13] and [15]. The effect of CsA on basidiospore germination was tested by adding 0, 1, 10, 20, and 100, and 1000 μg.mL -1 CsA (Sigma) in 1% DMSO to a basidiospore suspension containing 10 6 spores per mL before germination and germ tube were developed.…”

Section: Crystallization Data Collection and Structure Determinationmentioning

confidence: 99%

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Structural Analysis of Cyclophilin from Moniliophthora Perniciosa and Inhibitory Activity of Cyclosporin an on Germination and Growth of Fungi

2013

Pharm Anal Acta

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Moniliophthora perniciosa is the causal agent of witches' broom disease of cacao. Cyclophilins have been implicated in a wide variety of cellular processes, including the response to environmental stresses, cell cycle control, regulation of calcium signaling and transcriptional control. The involvement of cyclophilins in pathogenicity was described both in pathogenic fungi toward animals and plants. In Yeast, calcineurin and cyclophilin are involved in fungal morphogenesis and virulence. There is evidence that cyclophilin interacts with calcineurin in the absence of cyclosporin A (CsA), and it has been proposed that the activity of CsA acts in the regulatory interaction between cyclophilin and calcineurin; also, fungistatic or fungicide actions have been observed in this activity. The M. perniciosa cyclophilin gene from cDNA of the fruiting body in the vector pET28a has been cloned and the protein was expressed, purified and crystallized. The cyclophilin structures in the apo and bound with CsA forms were solved at 1.85 and 1.47 Ǻ, respectively. Comparison of structures from different organisms indicates conserved structures of cyclophilins. Small differences were found in apo and bound forms; hence, it follows that cyclophilin structures do not have accentuated modifications in the presence of the ligand. However, various hydrogen bonds between side chains of amino acids and water molecules are broken in the ligand site when CsA is bound. The germination spore assays using CsA showed a low inhibitory activity in germination, but high inhibition of the germ tube growth. These results show that cyclophilin plays an important role in the growth process, but not in the germination, hence suggesting that cyclophilin is as potential target for the fungistatic action against M. perniciosa.

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“…The family of sesquiterpenes epoxides inhibit eukaryotic protein synthesis and are major contaminants in crops, producing acute toxicity in humans and domesticated animals upon consumption (Hohn et al, 1995). CND genes from B. cinerea (cnd11 and cnd5) have been shown to present similarity with tri4, suggesting that they could be organized in a physical cluster and be involved in secondary metabolism (Viaud et al, 2003).…”

Section: O C H L I O B O L U S H E T E R O S T R O P H U S C O L mentioning

confidence: 99%

Generation and analysis of expressed sequence tags from Botrytis cinerea

et al. 2006

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Botrytis cinerea is a filamentous plant pathogen of a wide range of plant species, and its infection may cause enormous damage both during plant growth and in the post-harvest phase. We have constructed a cDNA library from an isolate of B. cinerea and have sequenced 11,482 expressed sequence tags that were assembled into 1,003 contigs sequences and 3,032 singletons. Approximately 81% of the unigenes showed significant similarity to genes coding for proteins with known functions: more than 50% of the sequences code for genes involved in cellular metabolism, 12% for transport of metabolites, and approximately 10% for cellular organization. Other functional categories include responses to biotic and abiotic stimuli, cell communication, cell homeostasis, and cell development. We carried out pair-wise comparisons with fungal databases to determine the B. cinerea unisequence set with relevant similarity to genes in other fungal pathogenic counterparts. Among the 4,035 non-redundant B. cinerea unigenes, 1,338 (23%) have significant homology with Fusarium verticillioides unigenes. Similar values were obtained for Saccharomyces cerevisiae and Aspergillus nidulans (22% and 24%, respectively). The lower percentages of homology were with Magnaporthe grisae and Neurospora crassa (13% and 19%, respectively). Several genes involved in putative and known fungal virulence and general pathogenicity were identified. The results provide important information for future research on this fungal pathogen.

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Cyclophilin A and calcineurin functions investigated by gene inactivation, cyclosporin A inhibition and cDNA arrays approaches in the phytopathogenic fungusBotrytis cinerea

Cited by 123 publications

References 91 publications

The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits

The Botrytis cinerea hexokinase, Hxk1, but not the glucokinase, Glk1, is required for normal growth and sugar metabolism, and for pathogenicity on fruits

Structural Analysis of Cyclophilin from Moniliophthora Perniciosa and Inhibitory Activity of Cyclosporin an on Germination and Growth of Fungi

Generation and analysis of expressed sequence tags from Botrytis cinerea

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