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Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

We have previously identified and functionally characterized the transcription factor ACE1 (Pc-ACE1) from Phanerochaete chrysosporium. In Saccharomyces cerevisiae, ACE1 activates the copper-dependent transcription of target genes through a DNA sequence element named ACE. However, the possible target gene(s) of Pc-ACE1 were unknown. An in silico search led to the identification of putative ACE elements in the promoter region of mco1, one of the four clustered genes encoding multicopper oxidases (MCOs) in P. chrysosporium. Since copper exerts an effect at the transcriptional level in MCOs from several organisms, in this work we analysed the effect of copper on transcript levels of the clustered MCO genes from P. chrysosporium, with the exception of the transcriptionally inactive mco3. Copper supplementation of cultures produced an increment in transcripts from genes mco1 and mco2, but not from mco4. Electrophoretic mobility-shift assays revealed that Pc-ACE1 binds specifically to a probe containing one of the putative ACE elements found in the promoter of mco1. In addition, using a cell-free transcription system, we showed that in the presence of cuprous ion, Pc-ACE1 induces activation of the promoter of mco1, but not that of mco2.

MCO1, a multicopper oxidase from Phanerochaete chrysosporium exhibiting strong ferroxidase activity, has recently been described. This enzyme shows biochemical and structural similarities with the yeast Fet3p, a type I membrane glycoprotein that efficiently oxidizes Fe(II) to Fe(III) for its subsequent transport to the intracellular compartment by the iron permease Ftr1p. The genome database of P. chrysosporium was searched to verify whether it includes a canonical fet3 in addition to mco1, and single copies of fet3 and ftr1 orthologues were found, separated by a divergent promoter. Pc-fet3 encodes a 628 aa protein that exhibits overall identities of about 40% with other reported Fet3 proteins. In addition to a secretion signal, it has a C-terminal transmembrane domain, characteristic of these cell-surface-attached ferroxidases. Structural modelling of Pc-Fet3 revealed that the active site has all the residues known to be essential for ferroxidase activity. Pc-ftr1 encodes a 393 aa protein that shows about 38% identity with several Ftr1 proteins from ascomycetes. Northern hybridization studies showed that the mRNA levels of both genes are reduced upon supplementation of the growth medium with iron, supporting the functional coupling of Fet3 and Ftr1 proteins in vivo.

Carotenoids are essential components of the photosynthetic antenna and reaction center complexes, being also responsible for antioxidant defense, coloration, and many other functions in multiple plant tissues. In tomato, salinity negatively affects the development of vegetative organs and productivity, but according to previous studies it might also increase fruit color and taste, improving its quality, which is a current agricultural challenge. The fruit quality parameters that are increased by salinity are cultivar-specific and include carotenoid, sugar, and organic acid contents. However, the relationship between vegetative and reproductive organs and response to salinity is still poorly understood. Considering this, Solanum lycopersicum cv. Micro-Tom plants were grown in the absence of salt supplementation as well as with increasing concentrations of NaCl for 14 weeks, evaluating plant performance from vegetative to reproductive stages. In response to salinity, plants showed a significant reduction in net photosynthesis, stomatal conductance, PSII quantum yield, and electron transport rate, in addition to an increase in non-photochemical quenching. In line with these responses the number of tomato clusters decreased, and smaller fruits with higher soluble solids content were obtained. Mature-green fruits also displayed a salt-dependent higher induction in the expression of PSY1, PDS, ZDS, and LYCB, key genes of the carotenoid biosynthesis pathway, in correlation with increased lycopene, lutein, β-carotene, and violaxanthin levels. These results suggest a key relationship between photosynthetic plant response and yield, involving impaired photosynthetic capacity, increased carotenoid-related gene expression, and carotenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Organisms are exposed to a tough environment, where acute daily challenges, like light, can strongly affect several aspects of an individual's physiology, including pathogenesis. While several fungal models have been widely employed to understand the physiological and molecular events associated with light perception, various other agricultural-relevant fungi still remain, in terms of their responsiveness to light, in the dark. The fungus Botrytis cinerea is an aggressive pathogen able to cause disease on a wide range of plant species. Natural B. cinerea isolates exhibit a high degree of diversity in their predominant mode of reproduction. Thus, the majority of naturally occurring strains are known to reproduce asexually via conidia and sclerotia, and sexually via apothecia. Studies from the 1970′s reported on specific developmental responses to treatments with near-UV, blue, red and far-red light. To unravel the signaling machinery triggering development – and possibly also connected with virulence – we initiated the functional characterization of the transcription factor/photoreceptor BcWCL1 and its partner BcWCL2, that form the White Collar Complex (WCC) in B. cinerea. Using mutants either abolished in or exhibiting enhanced WCC signaling (overexpression of both bcwcl1 and bcwcl2), we demonstrate that the WCC is an integral part of the mentioned machinery by mediating transcriptional responses to white light and the inhibition of conidiation in response to this stimulus. Furthermore, the WCC is required for coping with excessive light, oxidative stress and also to achieve full virulence. Although several transcriptional responses are abolished in the absence of bcwcl1, the expression of some genes is still light induced and a distinct conidiation pattern in response to daily light oscillations is enhanced, revealing a complex underlying photobiology. Though overlaps with well-studied fungal systems exist, the light-associated machinery of B. cinerea appears more complex than those of Neurospora crassa and Aspergillus nidulans. [ABSTRACT FROM AUTHOR]

Abstract: The effect of copper on the expression of genes encoding the ligninolytic enzymes laccase (lcs) and manganese peroxidase (mnp) in Ceriporiopsis subvermispora was evaluated. This metal increased transcript levels of lcs, mnp1 and mnp2. This finding was not unexpected in the case of lcs, since its promoter contains a putative ACE element. Originally characterized in the yeast Saccharomyces cerevisiae, ACE is the target sequence of the ACE1 copper-responsive transcription factor in this microorganism. Analysis of the promoter regions of mnp genes revealed the presence of formerly unnoticed ACE elements. Based on the ace1 gene from Phanerochaete chrysosporium, we isolated and characterized an ACE1-like transcription factor from C. subvermispora (Cs-ACE1) through complementation of a S. cerevisiae ace1Δ strain. Surprisingly, ACE1 factors from both basidiomycetes exhibit substantial differences, not only structurally but also in their ability to complement the aforementioned yeast strain. Specific binding of Cs-ACE1 to its cognate DNA sequence was confirmed by electrophoretic mobility-shift assays. [Copyright &y& Elsevier]

The authors have previously identified and characterized lcs, a gene encoding laccase in the white-rot basidiomycete Ceriporiopsis subvermispora. In this work, the effect of Mn2+ in the production of extracellular laccase in liquid cultures of this fungus has been assessed. It was observed that at low (0–10 μM) concentrations of Mn2+, high titers of lcs-mRNA were obtained, whereas at high (160–194 μM) concentrations of this metal ion, transcripts levels decreased markedly. This phenomenon was observed at different days of growth. On the other hand, Cu2+ or Ag+, but not Zn2+ or Cd2+, led to an accumulation of lcs transcripts only in cultures grown in the absence of Mn2+. A dramatic increase in lcs transcript levels was also obtained with syringic acid, a lignin-related aromatic compound. This effect was more pronounced in cultures lacking Mn2+. In the course of these studies it was observed that Mn2+ stimulates mycelium growth. Thus, although extracellular laccase activity appeared higher in cultures containing 160 or 194 μM Mn2+, i.e. when lcs transcripts were lower, a correlation between lcs-mRNA levels and enzymatic activity was observed when values of the latter were corrected by the amount of mycelium present in the cultures. [ABSTRACT FROM AUTHOR]

The article presents microbiological research into the cloning and characterization of genes encoding the iron-uptake high-affinity protein complex Fet3/Ftr1 in the Phanerochaete chrysosporium basidiomycete. MCO1 is a multicopper oxidase from Phanerochaete chrysosporium that exhibits structural and biochemical similarities with the yeast Fet3p.

Gene-deletion mutants represent a powerful tool to study gene function. The filamentous fungus Neurospora crassa is a well-established model organism, and features a comprehensive gene knockout strain collection. While these mutant strains have been used in numerous studies, resulting in the functional annotation of many Neurospora genes, direct confirmation of gene-phenotype relationships is often lacking, which is particularly relevant given the possibility of background mutations, sample contamination, and/or strain mislabeling. Indeed, spontaneous mutations resulting in phenotypes resembling many cell fusion mutants have long been known to occur at relatively high frequency in N. crassa , and these secondary mutations are common in the Neurospora deletion collection. The identity of these mutations, however, is largely unknown. Here, we report that the Δ ada-3 strain from the N. crassa knockout collection, which exhibits a cell fusion defect, harbors a secondary mutation responsible for this phenotype. Through whole-genome sequencing and genetic analyses, we found a ~30-Kb deletion in this strain affecting a known cell fusion-related gene, so / ham-1 , and show that it is the absence of this gene-and not of ada-3 -that underlies its cell fusion defect. We additionally found three other knockout strains harboring the same deletion, suggesting that this mutation may be common in the collection and could have impacted previous studies. Our findings provide a cautionary note and highlight the importance of proper functional validation of strains from mutant collections. We discuss our results in the context of the spread of cell fusion-defective cheater variants in N. crassa cultures. IMPORTANCE This study emphasizes the need for careful and detailed characterization of strains from mutant collections. Specifically, we found a common deletion in various strains from the Neurospora crassa gene knockout collection that results in a cell fusion-defective phenotype. This is noteworthy because this collection is known to contain background mutations-of a largely unclear nature-that produce cell fusion-defective phenotypes. Our results describe an example of such mutations, and highlight how this common genetic defect could have impacted previous studies that have used the affected strains. Furthermore, they provide a cautionary note about the use of Neurospora strains with similar phenotypes. Lastly, these findings offer additional details relevant to our understanding of the origin and spread of cell fusion-defective cheater variants in N. crassa cultures.