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Dementia with Lewy bodies (DLB) is a common form of dementia and is characterized by cognitive fluctuations, visual hallucinations, and Parkinsonism. The phenotypic expression of the disease may, in part, relate to alterations in functional connectivity within and between brain networks. This resting‐state study sought to clarify this in DLB, how networks differed from Alzheimer's disease (AD), and whether they were related to clinical symptoms in DLB. Resting‐state networks were estimated using independent component analysis. We investigated functional connectivity changes in 31 DLB patients compared to 31 healthy controls and a disease comparator group of 29 AD patients using dual regression and FSLNets. Within‐network connectivity was generally decreased in DLB compared to controls, mainly in motor, temporal, and frontal networks. Between‐network connectivity was mainly intact; only the connection between a frontal and a temporal network showed increased connectivity in DLB. Differences between AD and DLB were subtle and we did not find any significant correlations with the severity of clinical symptoms in DLB. This study emphasizes the importance of reduced connectivity within motor, frontal, and temporal networks in DLB with relative sparing of the default mode network. The lack of significant correlations between connectivity measures and clinical scores indicates that the observed reduced connectivity within these networks might be related to the presence, but not to the severity of motor and cognitive impairment in DLB patients. Furthermore, our results suggest that AD and DLB may show more similarities than differences in patients with mild disease.

STERILE APETALA (SAP) is known to be an essential regulator of flower development for over 20 years. Loss of SAP function in the model plant Arabidopsis thaliana is associated with a reduction of floral organ number, size and fertility. In accordance with the function of SAP during early flower development, its spatial expression in flowers is confined to meristematic stages and to developing ovules. However, to date, despite extensive research, the molecular function of SAP and the regulation of its spatio-temporal expression still remain elusive. In this work, amino acid sequence analysis and homology modeling revealed that SAP belongs to the rare class of plant F-box proteins with C-terminal WD40 repeats. In opisthokonts, this type of F-box proteins constitutes the substrate binding subunit of SCF complexes, which catalyze the ubiquitination of proteins to initiate their proteasomal degradation. With LC-MS/MS-based protein complex isolation, the interaction of SAP with major SCF complex subunits was confirmed. Additionally, candidate substrate proteins, such as the growth repressor PEAPOD 1 and 2 (PPD1/2), could be revealed during early stages of flower development. Also INDOLE-3-BUTYRIC ACID RESPONSE 5 (IBR5) was identified among putative interactors. Genetic analyses indicated that, different from substrate proteins, IBR5 is required for SAP function. Protein complex isolation together with transcriptome profiling emphasized that the SCFSAP complex integrates multiple biological processes, such as proliferative growth, vascular development, hormonal signaling and reproduction. Phenotypic analysis of sap mutant and SAP overexpressing plants positively correlated SAP function with plant growth during reproductive and vegetative development. Furthermore, to elaborate on the transcriptional regulation of SAP, publicly available ChIP-seq data of key floral homeotic proteins were reanalyzed. Here, it was shown that the MADS-domain transcription factors APETALA 1 (AP1), APETALA 3 (AP3), PISTILLATA (PI), AGAMOUS (AG) and SEPALLATA 3 (SEP3) bind to the SAP locus, which indicates that SAP is expressed in a floral organ-specific manner. Reporter gene analyses in combination with CRISPR/Cas9-mediated deletion of putative regulatory regions further demonstrated that the intron contains major regulatory elements of SAP in Arabidopsis thaliana. In conclusion, these data indicate that SAP is a pleiotropic developmental regulator that acts through tissue-specific destabilization of proteins. The presumed transcriptional regulation of SAP by the floral MADS-domain transcription factors could provide a missing link between the specification of floral organ identity and floral organ growth pathways.

See Jeganathan and Breakspear (doi:10.1093/brain/awz132) for a scientific commentary on this article. Schumacher et al. report a marked and generalised slowing of whole-brain EEG dynamics in Lewy body dementia that distinguishes it from Alzheimer’s disease and healthy ageing. The slowing is related to a loss of dynamic interaction between basal ganglia-thalamic networks and large-scale cortical networks, and may contribute to fluctuating cognition., Lewy body dementia includes dementia with Lewy bodies and Parkinson’s disease dementia and is characterized by transient clinical symptoms such as fluctuating cognition, which might be driven by dysfunction of the intrinsic dynamic properties of the brain. In this context we investigated whole-brain dynamics on a subsecond timescale in 42 Lewy body dementia compared to 27 Alzheimer’s disease patients and 18 healthy controls using an EEG microstate analysis in a cross-sectional design. Microstates are transiently stable brain topographies whose temporal characteristics provide insight into the brain’s dynamic repertoire. Our additional aim was to explore what processes in the brain drive microstate dynamics. We therefore studied associations between microstate dynamics and temporal aspects of large-scale cortical-basal ganglia-thalamic interactions using dynamic functional MRI measures given the putative role of these subcortical areas in modulating widespread cortical function and their known vulnerability to Lewy body pathology. Microstate duration was increased in Lewy body dementia for all microstate classes compared to Alzheimer’s disease (P < 0.001) and healthy controls (P < 0.001), while microstate dynamics in Alzheimer’s disease were largely comparable to healthy control levels, albeit with altered microstate topographies. Correspondingly, the number of distinct microstates per second was reduced in Lewy body dementia compared to healthy controls (P < 0.001) and Alzheimer’s disease (P < 0.001). In the dementia with Lewy bodies group, mean microstate duration was related to the severity of cognitive fluctuations (ρ = 0.56, PFDR = 0.038). Additionally, mean microstate duration was negatively correlated with dynamic functional connectivity between the basal ganglia (r = − 0.53, P = 0.003) and thalamic networks (r = − 0.38, P = 0.04) and large-scale cortical networks such as visual and motor networks in Lewy body dementia. The results indicate a slowing of microstate dynamics and disturbances to the precise timing of microstate sequences in Lewy body dementia, which might lead to a breakdown of the intricate dynamic properties of the brain, thereby causing loss of flexibility and adaptability that is crucial for healthy brain functioning. When contrasted with the largely intact microstate dynamics in Alzheimer’s disease, the alterations in dynamic properties in Lewy body dementia indicate a brain state that is less responsive to environmental demands and might give rise to the apparent slowing in thinking and intermittent confusion which typify Lewy body dementia. By using Lewy body dementia as a probe pathology we demonstrate a potential link between dynamic functional MRI fluctuations and microstate dynamics, suggesting that dynamic interactions within the cortical-basal ganglia-thalamic loop might play a role in the modulation of EEG dynamics.

Figure S1, Supplementary Methods, Tables S1-S4. Additional methods and results of additional analyses as specified in the main text. (PDF 429 kb)

Botrytis cinerea is a plant pathogen that exhibits prominent light responses including the formation of the reproduction structures (photomorphogenesis), secondary metabolites/ pigments, and antioxidant enzymes. A complex regulatory network of photoreceptors, transcription factors (TFs) and chromatin modifiers is supposed to initiate, transmit, and fine-tune the responses to different wavelengths of light on the transcriptional level that finally leads to the observable phenotypes. As the formation of the reproduction structures is strictly regulated by light in this fungus - conidia are formed in the light, sclerotia in the dark - the output can be easily monitored. The GATA-type TFs BcWCL1 (as part of the White Collar complex (WCC)) and BcLTF1 are important regulators as their deletions result in light-independent conidiation (“always conidia”) due to the deregulation of BcLTF2 (Schumacher et al. 2014; Canessa et al. 2013; Cohrs et al. 2016). Study of light-induced gene (LIG) expression in both deletion mutants highlighted the role of the TFs in activating EARLY and in repressing LATE LIG expression, including bcltf2 encoding the master regulator of conidiation and further LTFs that may function downstream or in parallel with BcLTF2 in regulating the conidiation process. As the group of LIGs also contained genes that are induced by various stresses in a BcSAK1-dependent manner, the phosphorylation status of the stress-activated MAPK BcSAK1 was studied and shown to increase after exposure to light. Deletion of bcsak1 impairs LIG expression suggesting that BcSAK1 functions as a co-activator of the WCC in inducing EARLY genes, and as a component releasing the LATE promoters (e.g. bcltf2) from repression by BcLTF1 and the WCC resulting in the expression of the conidiation genes. This preliminary model is in agreement with the observations that the deletion of BcSAK1 results in a “never conidia” phenotype (Segmüller et al. 2007), and that the exposure of the wild type to heat and osmotic stress bypasses the requirement of light for conidiation.

Unlike plants that use light also as source of energy, filamentous fungi exclusively use light as signal; here, light may trigger morphogenesis, pigment formation and circadian rhythms. Light plays an important role in the biology of the gray mold fungus by determining the mode of asexual reproduction (conidia in the light, sclerotia in the dark) and by triggering the differentiation of the apothecia. Field populations of B. cinerea are highly diverse with regard to different phenotypic traits and can be generally divided into two groups; strains that undergo photomorphogenesis (considered as the initial situation) and strains that produce the same reproductive structures under different light conditions (considered as loss of light control). However, the inability to form certain reproductive structures such as conidia is expected to decrease the fitness of the pathogen, and thus, it is questionable why “blind” strains are abundant in field populations, especially as this phenotype may be associated with attenuated virulence as shown for the “always conidia” phenotype caused by SNPs in bcvel1. Therefore, we aim to understand the fundamental mechanisms of photoperception and the involved signal transduction pathways. Though overlaps with the well-studied fungal systems i.e. Neurospora crassa and Aspergillus nidulans exist, the light signaling machinery in B. cinerea appears more sophisticated. Thus, the fungus possesses more photoreceptors than saprophytic fungi and responds to different wavelengths of light (near-UV, blue, green, red, far-red). More than 200 genes are responsive to white light, including 24 transcription factor (TF)-encoding genes. BcLTF2 has an outstanding function as its expression is necessary and sufficient to induce conidiation and simultaneously to suppress sclerotial development. Its tight regulation by several signaling pathways governs the balance between vegetative growth that allows for the colonization of the substrate and entering sclerotial development, and reproduction via conidia in the light.

B. cinerea and other filamentous fungi colonize their substrates in the form of vegetative mycelia and produce asexual spores (macroconidia and microconidia) and sexual spores in high quantities for their distribution and survival. As both processes, vegetative growth and reproduction, are competing for the same limiting resources, they should not occur simultaneously and need therefore be tightly coordinated by genetic programming and environmental conditions. For instance, excessive conidiation (e.g. microcycle conidiation) is disadvantageous as it may prevent vegetative growth and entering the sexual cycle. In this context, light serves as an important cue for the gray mold fungus: It induces the formation of conidia and at the same time represses the formation of sclerotia that represent asexual survival structures and are required for sexual recombination. As conidiation is induced by light, we considered the 24 identified light-responsive transcription factors (LTFs) as possible regulators of the conidiation program in B. cinerea. Nine LTFs have been functionally characterized by deletion and overexpression analyses so far: While BcLTF1 has various functions (growth in light, ROS homoeostasis, virulence, differentiation), other LTFs specifically regulate certain developmental stages such as the initiation of conidiophore development (BcATF1, BcLTF2, BcLTF7) or conidiogenesis (BcLTF3, BcREG1), or do not have obvious functions (BcLTF4, BcLTF5, BcLTF6). The C2H2-TF BcLTF2 has an outstanding function as its expression is necessary and sufficient to induce conidiation and simultaneously to suppress sclerotial development. In contrast, expression of BcLTF7 promotes conidiation but is not required for the process. By expression profiling of the remaining LTFs in mutants exhibiting deregulated conidiation, we identified five further candidates (bcltf12, bcltf15, bcltf16, bcltf20, bcltf21) that may act downstream of the major regulator BcLTF2 in the network of LTFs governing the asexual differentiation programs in the gray mold fungus.

National audience; Botrytis cinerea is a necrotrophic plant pathogen that exhibits prominent light responses including the formation of the reproduction structures (photomorphogenesis), secondary metabolites/ pigments, and antioxidant enzymes. A complex regulatory network of photoreceptors, transcription factors (TFs) and chromatin modulates is supposed to initiate, transmit, and fine-tune the responses to different wavelengths of light on the transcriptional level that finally leads to the observable phenotypes. As the formation of the reproduction structures is strictly regulated by light in this fungus - conidia are formed in the light, sclerotia in the dark - the output can be easily monitored. The GATA-type TFs BcLTF1 and BcWCL1 are important regulators as their deletions resulted in conidiation in light and dark («always conidia») (Schumacher et al. 2014; PLoS Genet 10:e1004040, Canessa et al. 2013; PLoS One 8:e84223). Study of gene expression in both deletion mutants by microarray analyses highlighted the role of the TFs in mediating the transcriptional responses of the majority of lightresponsive genes including a number of other transcriptional regulators. As the group of lightresponsive genes also contained genes that are induced by oxidative and osmotic stress in a BcSAK1-dependent manner (Heller et al. 2012; MPMI 25:802-816), their expression levels were studied in the delta-Bcsak1 mutant. Indeed, light induction does not occur in the mutant background indicating that the MAP kinase cascade is another functional unit of the light regulatory network. More detailed expression analyses of chosen LTF-encoding genes showed that three major transcriptional profiles exist: genes that exhibit maximal expression after 15 min (early), 60 min (moderate) or 120 min (late) of light treatment. Expression levels of the majority of genes decrease after prolonged light exposure (photoadaptation); however, this is not observed for bcltf1. Eight LTFs have been functionally characterized by deletion and overexpression analyses so far: While BcLTF1 has various functions (growth in light, ROS homoeostasis, virulence, differentiation), other LTFs specifically regulate certain developmental stages such as the initiation of conidiophore development or conidiogenesis.

International audience; Botrytis cinerea is responsible of the gray mold disease on a wide range of host plants. During the infection process, it produces unspecific phytotoxins (botrydial and botcinic acid) and other secondary metabolites that play a significanT role in plant tissue colonization. Regulation of the biosynthesis of these bioactive compounds is therefore crucial for the outcome of fungus/plant interactions. Combining genetics and transcriptomic approaches, the role of different signaling cascades, transcription factors, and other regulators such as those belonging to the VELVET complex have recently been investigated. Overall, these studies suggest a strong link between the regulation of secondary metabolism and the light-dependent development in B.cinerea. Further knowledge of the complex regulatory network controlling secondary metabolism in B.cinera is crucial to understand the interaction between B.cinerea and its different host plants and may help to define new strategies to control this crop-devastating fungus.

Just accepted; Botrytis cinerea is a plant pathogenic fungus known for its utilization of light as environmental cue to regulateasexual differentiation: conidia are formed in the light, while sclerotia are formed in the dark. As noorthologues of known regulators of conidiation (e.g.,Aspergillus nidulans BrlA, Neurospora crassa FL)exist in the Leotiomycetes, we initiated a de novo approach to identify the functional counterpart in B.cinerea. The search revealed the light-responsive C2H2 transcription factor BcLTF2 whose expression– usually restricted to light conditions – is necessary and sufficient to induce conidiation and simultaneouslyto suppress sclerotial development. Lightinduced expression of bcltf2 is mediated via a so far unknown pathway, and is attenuated in a (blue) lightdependent fashion by the White Collar complex, BcLTF1 and the VELVET complex. Mutation of either component leads to increased bcltf2 expression and causes light-independent conidiation (always conidia phenotype). Hence, the tight regulation of bcltf2 governs the balance between vegetative growth that allows for the colonization of the substrate and subsequent reproduction via conidia in the light. The orthologue ssltf2 in the closely related species Sclerotinia sclerotiorum is not significantly expressed suggesting that its deregulation may cause the lack of the conidiation program in this fungus.

International audience; Botrytis cinerea is the causal agent of gray mold diseases in a range of dicotyledonous plant species. The fungus can reproduce asexually by forming macroconidia for dispersal and sclerotia for survival; the latter also participate in sexual reproduction by bearing the apothecia after fertilization by microconidia. Light induces the differentiation of conidia and apothecia, while sclerotia are exclusively formed in the absence of light. The relevance of light for virulence of the fungus is not obvious; infections are observed under natural illumination as well as in constant darkness. By a random mutagenesis approach, we identified a novel virulence-related gene encoding a GATA transcription factor (BcLTF1 for light-responsive TF1) with characterized homologues in Aspergillus nidulans (NsdD) and Neurospora crassa (SUB- 1). By deletion and overexpression of bcltf1, we confirmed the predicted role of the TF in virulence, and discovered furthermore its functions in regulation of light-dependent differentiation, the equilibrium between production and scavenging of reactive oxygen species (ROS), and secondary metabolism. Microarray analyses revealed 293 lightresponsivegenes in B. cinerea B05.10 including five further TF-encoding genes (BcLTF2-6), and that the expression levels of the majority of these genes (66%) are modulated by BcLTF1. Bcltf2 encodes a C2H2-TF which is the homologue of N. crassa SAH-1 (short aerial-hyphae-1). Expression levels are increased in mutants exhibiting a hyper-conidiation phenotype such as deletion mutants of BcLTF1, the VELVET protein BcVEL1 and the WHITE COLLAR-like TF BcWCL1, suggesting a role of BcLTF2 in regulation of conidiation. Indeed, Δbcltf2 mutants do not produce conidia in axenic culture and in planta. Notably, mutants form sclerotia under all illumination conditions indicating that the suppression of sclerotial development by light is likely due to the induction of conidiation rather than due to a direct suppression of the sclerotium differentiation program.

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Hintergrund: Eine sichere Versorgung von Bewohnern der Alten- und Pflegeheime mit Arzneimitteln ist für alle beteiligten Heilberufe eine große Herausforderung. Aufgrund der vielen unterschiedlichen Arzneimittel, die älteren Menschen verordnet werden, muss mit Wechselwirkungen zwischen[for full text, please go to the a.m. URL], 10. Deutscher Kongress für Versorgungsforschung; 18. GAA-Jahrestagung

Session 3 : Post-Genome Functional Analysis : P3.6; absent

The grey mould fungus Botrytis cinerea produces two major phytotoxins, the sesquiterpene botrydial, for which the biosynthesis gene cluster has been characterized previously, and the polyketide botcinic acid. We have identified two polyketide synthase (PKS) encoding genes, BcPKS6 and BcPKS9, that are up-regulated during tomato leaf infection. Gene inactivation and analysis of the secondary metabolite spectra of several independent mutants demonstrated that both BcPKS6 and BcPKS9 are key enzymes for botcinic acid biosynthesis. We showed that BcPKS6 and BcPKS9 genes, renamed BcBOA6 and BcBO9 (for B. cinerea botcinic acid biosynthesis), are located at different genomic loci, each being adjacent to other putative botcinic acid biosynthetic genes, named BcBOA1 to BcBOA17. Putative orthologues of BcBOA genes are present in the closely related fungus Sclerotinia sclerotiorum, but the cluster organization is not conserved between the two species. As for the botrydial biosynthesis genes, the expression of BcBOA genes is co-regulated by the Gα subunit BCG1 during both in vitro and in planta growth. The loss of botcinic acid production does not affect virulence on bean and tomato leaves. However, double mutants that do not produce botcinic acid or botrydial (bcpks6Δbcbot2Δ) exhibit markedly reduced virulence. Hence, a redundant role of botrydial and botcinic acid in the virulence of B. cinerea has been demonstrated.

International audience

International audience