Diaporthe atlantica enhances tomato drought tolerance by improving photosynthesis, nutrient uptake and enzymatic antioxidant response.

Functional symbiosis with fungal endophytes can help plants adapt to environmental stress. Diaporthe atlantica is one of the most abundant fungal taxa associated with roots of Festuca rubra subsp. pruinosa, a grass growing in sea cliffs. This study aimed to investigate the ability of a strain of this fungus to ameliorate the impact of drought stress on tomato plants. In a greenhouse experiment, tomato plants were inoculated with Diaporthe atlantica strain EB4 and exposed to two alternative water regimes: well-watered and drought stress. Several physiological and biochemical plant parameters were evaluated. Inoculation with Diaporthe promoted plant growth in both water treatments. A significant interactive effect of Diaporthe-inoculation and water-regime showed that symbiotic plants had higher photosynthetic capacity, water-use efficiency, nutrient uptake (N, P, K, Fe and Zn), and proline content under drought stress, but not under well-watered conditions. In addition, Diaporthe improved the enzymatic antioxidant response of plants under drought, through an induced mechanism, in which catalase activity was modulated and conferred protection against reactive oxygen species generation during stress. The results support that Diaporthe atlantica plays a positive role in the modulation of tomato plant responses to drought stress by combining various processes such as improving photosynthetic capacity, nutrient uptake, enzymatic antioxidant response and osmo-protectant accumulation. Thus, drought stress in tomato can be enhanced with symbiotic fungi.

Surface Activation of Titanium Dental Implants by Using UVC-LED Irradiation.

Organic contaminants significantly limit the bioactivity of titanium implants, resulting in the degradation known as the ageing of titanium. To reactivate the surfaces, they can be photofunctionalized, i.e., irradiated with C-range ultraviolet (UVC) light. This descriptive in vitro study compares the effectiveness of novel light-emitting diode (LED) technology to remove contaminant hydrocarbons from three different commercially available titanium dental implants: THD, TiUnite, and SLA. The surface topography and morphology were characterized by scanning electron microscopy (SEM). The chemical compositions were analyzed by X-ray photoelectron spectroscopy (XPS), before and after the lighting treatment, by a pair of closely placed UVC (λ = 278 nm) and LED devices for 24 h. SEM analysis showed morphological differences at the macro- and micro-scopic level. XPS analysis showed a remarkable reduction in the carbon contents after the UVC treatment: from 25.6 to 19.5 C at. % (carbon atomic concentration) in the THD; from 30.2 to 20.2 C at. % in the TiUnite; from 26.1 to 19.2 C at. % in the SLA surface. Simultaneously, the concentration of oxygen and titanium increased. Therefore, LED-based UVC irradiation decontaminated titanium surfaces and improved the chemical features of them, regardless of the kind of surface.

Copper Homeostasis in Aspergillus nidulans Involves Coordinated Transporter Function, Expression and Cellular Dynamics.

Copper ion homeostasis involves a finely tuned and complex multi-level response system. This study expands on various aspects of the system in the model filamentous fungus Aspergillus nidulans. An RNA-seq screen in standard growth and copper toxicity conditions revealed expression changes in key copper response elements, providing an insight into their coordinated functions. The same study allowed for the deeper characterization of the two high-affinity copper transporters: AnCtrA and AnCtrC. In mild copper deficiency conditions, the null mutant of AnctrC resulted in secondary level copper limitation effects, while deletion of AnctrA resulted in primary level copper limitation effects under extreme copper scarcity conditions. Each transporter followed a characteristic expression and cellular localization pattern. Although both proteins partially localized at the plasma membrane, AnCtrC was visible at membranes that resembled the ER, whilst a substantial pool of AnCtrA accumulated in vesicular structures resembling endosomes. Altogether, our results support the view that AnCtrC plays a major role in covering the nutritional copper requirements and AnCtrA acts as a specific transporter for extreme copper deficiency scenarios.

Decontamination of Ti Oxide Surfaces by Using Ultraviolet Light: Hg-Vapor vs. LED-Based Irradiation.

C-range Ultraviolet (UVC) mercury (Hg)-vapor lamps have shown the successful decontamination of hydrocarbons and antimicrobial effects from titanium surfaces. This study focused on surface chemistry modifications of titanium dental implants by using two different light sources, Hg-vapor lamps and Light Emitting Diodes (LEDs), so as to compare the effectivity of both photofunctionalization technologies. Two different devices, a small Hg-vapor lamp (λ = 254 nm) and a pair of closely placed LEDs (λ = 278 nm), were used to irradiate the implants for 12 min. X-ray Photoelectron Spectroscopy (XPS) was employed to characterize the chemical composition of the surfaces, analysing the samples before and after the lighting treatment, performing a wide and narrow scan around the energy peaks of carbon, oxygen and titanium. XPS analysis showed a reduction in the concentration of surface hydrocarbons in both UVC technologies from around 26 to 23.4 C at.% (carbon atomic concentration). Besides, simultaneously, an increase in concentration of oxygen and titanium was observed. LED-based UVC photofunctionalization has been suggested to be as effective a method as Hg-vapor lamps to remove the hydrocarbons from the surface of titanium dental implants. Therefore, due to the increase in worldwide mercury limitations, LED-based technology could be a good alternative decontamination source.

Tolerance to alkaline ambient pH in Aspergillus nidulans depends on the activity of ENA proteins.

Tolerance of microorganisms to abiotic stress is enabled by regulatory mechanisms that coordinate the expression and activity of resistance genes. Alkalinity and high salt concentrations are major environmental physicochemical stresses. Here, we analyzed the roles of sodium-extrusion family (ENA) transporters EnaA, EnaB and EnaC in the response to these stress conditions in the filamentous fungus Aspergillus nidulans. While EnaC has a minor role, EnaB is a key element for tolerance to Na+ and Li+ toxicity. Adaptation to alkaline pH requires the concerted action of EnaB with EnaA. Accordingly, expression of enaA and enaB was induced by Na+, Li+ and pH 8. These expression patterns are altered in a sltAΔ background and completely inhibited in a mutant expressing non-functional PacC protein (palH72). However, a constitutively active PacC form was not sufficient to restore maximum enaA expression. In agreement with their predicted role as membrane ATPases, EnaA localized to the plasma membrane while EnaB accumulated at structures resembling the endoplasmic reticulum. Overall, results suggest different PacC- and SltA-dependent roles for EnaB in pH and salt homeostasis, acting in coordination with EnaA at pH 8 but independently under salt stress.

New insights into copper homeostasis in filamentous fungi.

Copper is a metal ion that is required as a micronutrient for growth and proliferation. However, copper accumulation generates toxicity by multiple mechanisms, potentially leading to cell death. Due to its toxic nature at high concentrations, different chemical variants of copper have been extensively used as antifungal agents in agriculture and medicine. Most studies on copper homeostasis have been carried out in bacteria, yeast, and mammalian organisms. However, knowledge on filamentous fungi is less well documented. This review summarizes the knowledge gathered in the last few years about copper homeostasis in the filamentous fungi Aspergillus fumigatus and Aspergillus nidulans: The mechanism of action of copper, the uptake and detoxification systems, their regulation at the transcriptional level, and the role of copper homeostasis in fungal pathogenicity are presented.

The Early Asexual Development Regulator fluG Codes for a Putative Bifunctional Enzyme.

FluG is a long recognized early regulator of asexual development in Aspergillus nidulans. fluG null mutants show profuse aerial growth and no conidial production. Initial studies reported sequence homology of FluG with a prokaryotic type I glutamine synthetase, but catalytic activity has not been demonstrated. In this study, we conducted an in-depth analysis of the FluG sequence, which revealed a single polypeptide containing a putative N-terminal amidohydrolase region linked to a putative C-terminal γ-glutamyl ligase region. Each region corresponded, separately and completely, to respective single function bacterial enzymes. Separate expression of these regions confirmed that the C-terminal region was essential for asexual development. The N-terminal region alone did not support conidial development, but contributed to increased conidial production under high nutrient availability. Point mutations directed at respective key catalytic residues in each region demonstrated that they were essential for biological function. Moreover, the substitution of the N- and C-terminal regions with homologs from Lactobacillus paracasei and Pseudomonas aeruginosa, respectively, maintained functionality, albeit with altered characteristics. Taken together, the results lead us to conclude that FluG is a bifunctional enzyme that participates in an as yet unidentified metabolic or signaling pathway involving a γ-glutamylated intermediate that contributes to developmental fate.

Boron Tolerance in Aspergillus nidulans Is Sustained by the SltA Pathway Through the SLC-Family Transporters SbtA and SbtB.

Microbial cells interact with the environment by adapting to external changes. Signal transduction pathways participate in both sensing and responding in the form of modification of gene expression patterns, enabling cell survival. The filamentous fungal-specific SltA pathway regulates tolerance to alkalinity, elevated cation concentrations and, as shown in this work, also stress conditions induced by borates. Growth of sltA- mutants is inhibited by increasing millimolar concentrations of boric acid or borax (sodium tetraborate). In an attempt to identify genes required for boron-stress response, we determined the boric acid or borax-dependent expression of sbtA and sbtB, orthologs of Saccharomyces cerevisiae bor1, and a reduction in their transcript levels in a ΔsltA mutant. Deletion of sbtA, but mainly that of sbtB, decreased the tolerance to boric acid or borax. In contrast, null mutants of genes coding for additional transporters of the Solute Carrier (SLC) family, sB, sbtD or sbtE, showed an unaltered growth pattern under the same stress conditions. Taken together, our results suggest that the SltA pathway induces, through SbtA and SbtB, the export of toxic concentrations of borates, which have largely recognized antimicrobial properties.

Copper Resistance in Aspergillus nidulans Relies on the PI-Type ATPase CrpA, Regulated by the Transcription Factor AceA.

Copper homeostasis has been extensively studied in mammals, bacteria, and yeast, but it has not been well-documented in filamentous fungi. In this report, we investigated the basis of copper tolerance in the model fungus Aspergillus nidulans. Three genes involved in copper homeostasis have been characterized. First, crpA the A. nidulans ortholog of Candida albicans CaCRP1 gene encoding a PI-type ATPase was identified. The phenotype of crpA deletion led to a severe sensitivity to Cu+2 toxicity and a characteristic morphological growth defect in the presence of high copper concentration. CrpA displayed some promiscuity regarding metal species response. The expression pattern of crpA showed an initial strong elevation of mRNA and a low continuous gene expression in response to long term toxic copper levels. Coinciding with maximum protein expression level, CrpA was localized close to the cellular surface, however protein distribution across diverse organelles suggests a complex regulated trafficking process. Secondly, aceA gene, encoding a transcription factor was identified and deleted, resulting in an even more extreme copper sensitivity than the ΔcrpA mutant. Protein expression assays corroborated that AceA was necessary for metal inducible expression of CrpA, but not CrdA, a putative metallothionein the function of which has yet to be elucidated.

Tip-to-nucleus migration dynamics of the asexual development regulator FlbB in vegetative cells.

In Aspergillus nidulans, asexual differentiation requires the presence of the transcription factor FlbB at the cell tip and apical nuclei. Understanding the relationship between these two pools is crucial for elucidating the biochemical processes mediating conidia production. Tip-to-nucleus communication was demonstrated by photo-convertible FlbB::Dendra2 visualization. Tip localization of FlbB depends on Cys382 in the C-terminus and the bZIP DNA-binding domain in the N-terminus. FlbE, a critical FlbB interactor, binds the bZIP domain. Furthermore, the absence of FlbE results in loss of tip localization but not nuclear accumulation. flbE deletion also abrogates transcriptional activity indicating that FlbB gains transcriptional competence from interactions with FlbE at the tip. Finally, a bipartite nuclear localization signal is required for nuclear localization of FlbB. Those motifs of FlbB may play various roles in the sequence of events necessary for the distribution and activation of this transcriptionally active developmental factor. The tip accumulation, FlbE-dependent activation, transport and nuclear import sketch out a process of relaying an environmentally triggered signal from the tip to the nuclei. As the first known instance of transcription factor-mediated tip-to-nucleus communication in filamentous fungi, this provides a general framework for analyses focused on elucidating the set of molecular mechanisms coupling apical signals to transcriptional events.

Beyond asexual development: modifications in the gene expression profile caused by the absence of the Aspergillus nidulans transcription factor FlbB.

In the model fungus Aspergillus nidulans, asexual development is induced from vegetative hyphae by a set of early regulators including the bZIP-type transcription factor FlbB. To determine the range of genes under the influence of the transcriptional activity of FlbB and to characterize their role in fungal development, we sequenced and compared the transcriptomes of a ΔflbB mutant and its isogenic wild-type strain at different developmental stages. Results confirmed the activating role of FlbB on downstream regulators of conidiation such as flbD and brlA. However, FlbB has additional functions beyond the induction of asexual development. Among the changes observed, absence of a functional FlbB caused induction of the dba cluster and synthesis of a secondary metabolite with bactericidal properties. In addition, a new transcriptional target of FlbB was unveiled, urdA, that codes for a putative transcription factor that represses premature sexual development. Taken together, our results indicate that the activators of asexual development simultaneously exert a role on other cellular functions, including an inhibitory effect on the sexual cycle, and reinforce the hypothesis that mutually exclusive metabolic and cellular patterns are associated with different morphogenetic programs.

The Mycelium Blueprint: insights into the cues that shape the filamentous fungal colony.

The mycelium is an organised cellular network that develops according to a functionally coherent plan. As it expands, the mycelium is capable of modulating the relative abundance of different cell types to suit the prevailing environmental conditions. This versatile pattern of multicellular development involves sophisticated environmental sensing and intercellular communication systems that have barely been recognised. This review describes an insight into our current understanding of the signalling molecules and mechanisms that take part in the ordered and timely emergence of various cell types and their biological significance. The prospects that this emerging knowledge may offer for the sustainable control of fungal colonisation or dispersal will also be considered.

Transcriptional changes in the transition from vegetative cells to asexual development in the model fungus Aspergillus nidulans.

Morphogenesis encompasses programmed changes in gene expression that lead to the development of specialized cell types. In the model fungus Aspergillus nidulans, asexual development involves the formation of characteristic cell types, collectively known as the conidiophore. With the aim of determining the transcriptional changes that occur upon induction of asexual development, we have applied massive mRNA sequencing to compare the expression pattern of 19-h-old submerged vegetative cells (hyphae) with that of similar hyphae after exposure to the air for 5 h. We found that the expression of 2,222 (20.3%) of the predicted 10,943 A. nidulans transcripts was significantly modified after air exposure, 2,035 being downregulated and 187 upregulated. The activation during this transition of genes that belong specifically to the asexual developmental pathway was confirmed. Another remarkable quantitative change occurred in the expression of genes involved in carbon or nitrogen primary metabolism. Genes participating in polar growth or sexual development were transcriptionally repressed, as were those belonging to the HogA/SakA stress response mitogen-activated protein (MAP) kinase pathway. We also identified significant expression changes in several genes purportedly involved in redox balance, transmembrane transport, secondary metabolite production, or transcriptional regulation, mainly binuclear-zinc cluster transcription factors. Genes coding for these four activities were usually grouped in metabolic clusters, which may bring regulatory implications for the induction of asexual development. These results provide a blueprint for further stage-specific gene expression studies during conidiophore development.

GmcA is a putative glucose-methanol-choline oxidoreductase required for the induction of asexual development in Aspergillus nidulans.

Aspergillus nidulans asexual differentiation is induced by Upstream Developmental Activators (UDAs) that include the bZIP-type Transcription Factor (TF) FlbB. A 2D-PAGE/MS-MS-coupled screen for proteins differentially expressed in the presence and absence of FlbB identified 18 candidates. Most candidates belong to GO term classes involved in osmotic and/or oxidative stress response. Among these, we focused on GmcA, a putative glucose-methanol-choline oxidoreductase which is upregulated in a ΔflbB background. GmcA is not required for growth since no differences were detected in the radial extension upon deletion of gmcA. However, its activity is required to induce conidiation under specific culture conditions. A ΔgmcA strain conidiates profusely under acid conditions but displays a characteristic fluffy aconidial phenotype in alkaline medium. The absence of asexual development in a ΔgmcA strain can be suppressed, on one hand, using high concentrations of non-fermentable carbon sources like glycerol, and on the other hand, when the cMyb-type UDA TF flbD is overexpressed. Overall, the results obtained in this work support a role for GmcA at early stages of conidiophore initiation.

Signaling the induction of sporulation involves the interaction of two secondary metabolites in Aspergillus nidulans.

When growing Aspergillus nidulans hyphae encounter the atmosphere, they initiate a morphogenetic program leading to the production of spores. Mutants that are defective in the fluG gene fail to undergo sporulation because they lack an endogenous diffusible factor that purportedly accumulates on aerial hyphae, thus signaling the initiation of development. In this study, the defect could be reversed by adding culture extracts from a wild-type strain onto a mutant colony. Moreover, a bioassay-guided purification of the active culture extract resulted in the identification of the active agent as dehydroaustinol. However, this meroterpenoid was active only when administered in conjunction with the orsellinic acid derivative diorcinol. These two compounds formed an adduct that was detected by HRMS in an LC-MS experiment. The diorcinol-dehydroaustinol adduct prevented crystal formation of the signal on the surface of aerial hyphae and on an artificially prepared aqueous film and also increased the signal lipophilicity.

8-Carbon oxylipins inhibit germination and growth, and stimulate aerial conidiation in Aspergillus nidulans.

Germination of Aspergillus nidulans conidia in liquid cultures was progressively inhibited at inoculum loads above 1×10(5)conidiamL(-1). High conidial densities also inhibited growth of neighbouring mycelia. The eight-carbon oxylipin 1-octen-3-ol was identified as the main inhibitor in a fraction also containing 3-octanone and 3-octanol. These three oxylipins also increased the conidiation rate of dark-grown surface cultures, but had no effect on liquid cultures. 3-octanone was the most conidiogenic compound. The action of 3-octanone required functional forms of developmental activators fluG, flbB-D and brlA, and was not additive to the conidiogenic effect of stress stimuli such as osmotic stress or carbon starvation. Oxylipins were produced shortly after hyphae made contact with the atmosphere and were most effective on aerial mycelia, indicating that they perform their signalling function in the gas phase.

Elucidation of functional markers from Aspergillus nidulans developmental regulator FlbB and their phylogenetic distribution.

Aspergillus nidulans is a filamentous fungus widely used as a model for biotechnological and clinical research. It is also used as a platform for the study of basic eukaryotic developmental processes. Previous studies identified and partially characterized a set of proteins controlling cellular transformations in this ascomycete. Among these proteins, the bZip type transcription factor FlbB is a key regulator of reproduction, stress responses and cell-death. Our aim here was the prediction, through various bioinformatic methods, of key functional residues and motifs within FlbB in order to inform the design of future laboratory experiments and further the understanding of the molecular mechanisms that control fungal development. A dataset of FlbB orthologs and those of its key interaction partner FlbE was assembled from 40 members of the Pezizomycotina. Unique features were identified in each of the three structural domains of FlbB. The N-terminal region encoded a bZip transcription factor domain with a novel histidine-containing DNA binding motif while the dimerization determinants exhibited two distinct profiles that segregated by class. The C-terminal region of FlbB showed high similarity with the AP-1 family of stress response regulators but with variable patterns of conserved cysteines that segregated by class and order. Motif conservation analysis revealed that nine FlbB orthologs belonging to the Eurotiales order contained a motif in the central region that could mediate interaction with FlbE. The key residues and motifs identified here provide a basis for the design of follow-up experimental investigations. Additionally, the presence or absence of these residues and motifs among the FlbB orthologs could help explain the differences in the developmental programs among fungal species as well as define putative complementation groups that could serve to extend known functional characterizations to other species.

Aspergillus nidulans asexual development: making the most of cellular modules.

Asexual development in Aspergillus nidulans begins in superficial hyphae as the programmed emergence of successive pseudohyphal modules, collectively known as the conidiophore, and is completed by a layer of specialized cells (phialides) giving rise to chains of aerial spores. A discrete number of regulatory factors present in hyphae play different stage-specific roles in pseudohyphal modules, depending on their cellular localization and protein-protein interactions. Their multiple roles include the timely activation of a sporulation-specific pathway that governs phialide and spore formation. Such functional versatility provides for a new outlook on morphogenetic change and the ways we should study it.

FlbC is a putative nuclear C2H2 transcription factor regulating development in Aspergillus nidulans.

Asexual development (conidiation) in Aspergillus is governed by multiple regulators. Here, we characterize the upstream developmental activator FlbC in Aspergillus nidulans. flbC mRNA is detectable throughout the life cycle, at relatively high levels during vegetative growth, early asexual and late sexual developmental phases. The deletion of flbC causes a delay/reduction in conidiation, brlA and vosA expression, and conidial germination. While overexpression of flbC (OEflbC) does not elaborate conidiophores, it inhibits hyphal growth and activates expression of brlA, abaA and vosA, but not wetA. FlbC is conserved in filamentous Ascomycetes containing two C(2) H(2) zinc fingers at the C-terminus and a putative activation domain at the N-terminus. FlbC localizes in the nuclei of both hyphae and developmental cells. Localization and expression of FlbC are not affected by the absence of FlbB or FlbE, and vice versa. Importantly, overexpression of flbC causes growth inhibition and activation of abaA and vosA in the absence of brlA and abaA respectively. In vitro DNA-binding assay reveals that FlbC binds to the brlA, abaA and vosA, but not the wetA, promoters. In summary, FlbC is a putative nuclear transcription factor necessary for proper activation of conidiation, and its balanced activity is crucial for governing growth and development in A. nidulans.

The concerted action of bZip and cMyb transcription factors FlbB and FlbD induces brlA expression and asexual development in Aspergillus nidulans.

Fungi are capable of generating diverse cell types through developmental processes that stem from hyphae, acting as pluripotent cells. The formation of mitospores on emergence of hyphae to the air involves the participation of transcription factors, which co-ordinate the genesis of new cell types, eventually leading to spore formation. In this investigation, we show that bZip transcription factor FlbB, which has been attributed to participate in transducing the aerial stimulus signal, activates the expression of c-Myb transcription factor FlbD. Both factors then jointly activate brlA, a C(2)H(2) zinc finger transcription factor, which plays a central role in spore formation. This sequence of regulatory events resembles developmental control mechanisms involving c-Myb and bZip counterparts in metazoans and plants.