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Lichens are of great ecological importance but mechanisms regulating lichen symbiosis are not clear. Umbilicaria muhlenbergii is a lichen-forming fungus amenable to molecular manipulations and dimorphic. Here, we established conditions conducive to symbiotic interactions and lichen differentiation and showed the importance of UMP1 MAP kinase in lichen development. In the initial biofilm-like symbiotic complexes, algal cells were interwoven with pseudohyphae covered with extracellular matrix. After longer incubation, fungal-algal complexes further differentiated into primitive lichen thalli with a melanized cortex-like and pseudoparenchyma-like tissues containing photoactive algal cells. Mutants deleted of UMP1 were blocked in pseudohyphal growth and development of biofilm-like complexes and primitive lichens. Invasion of dividing mother cells that contributes to algal layer organization in lichens was not observed in the ump1 mutant. Overall, these results showed regulatory roles of UMP1 in symbiotic interactions and lichen development and suitability of U. muhlenbergii as a model for studying lichen symbiosis. The mechanisms regulating fungal-algal interactions during the formation of lichen symbioses are not clear. Here, Wang et al. establish conditions conducive to symbiotic interactions and lichen differentiation using a fungus amenable to genetic manipulation, showing the importance of a MAP kinase in lichen development.

The multiprotein-bridging factor 1 (MBF1) gene family is well known in archaea, non-lichenized fungi, plants, and animals, and contains stress tolerance-related genes. Here, we identified four unique mbf1 genes in the lichenized fungi Endocarpon spp. A phylogenetic analysis based on protein sequences showed the translated MBF1 proteins of the newly isolated mbf1 genes formed a monophyletic clade different from other lichen-forming fungi and Ascomycota groups in general, which may reflect the evolution of the biological functions of MBF1s. In contrast to the lack of function reported in yeast, we determined that lysine 114 in the deduced Endocarpon pusillum MBF1 protein (EpMBF1) had a specific function that was triggered by environmental stress. Further, the Endocarpon -specific C-terminus of EpMBF1 was found to participate in stress tolerance. Epmbf1 was induced by a number of abiotic stresses in E. pusillum and transgenic yeast, and its stress-resistant ability was stronger than that of the yeast mbf1 . These findings highlight the evolution and function of EpMBF1 and provide new insights into the co-evolution hypothesis of MBF1 and TATA-box-binding proteins.

Herba Siegesbeckiae (HS), derived from the aerial parts of three plants, Siegesbeckia orientalis (SO), S. glabrescens (SG), and S. pubescens (SP), has been used for the treatment of inflammatory diseases in China for centuries. In the present study, hydrodistillation was applied to extract essential oils from dried SO, SG, and SP aerial parts, and chemical composition analysis by gas chromatography–mass spectrometry (GC-MS) led to the identification of a total of 148 compounds (56 in SO, 62 in SG, and 59 in SP). The main components in the essential oils of SO, SG, and SP differed significantly. In vitro anti-inflammatory activity assays showed that SP essential oils (IC50, 0.97 μg/mL) significantly reduced the ability of lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages to release NO, and the SO essential oil (IC50, 14.99 μg/mL) was better than the others at inhibiting the LPS-induced release of cytokine IL-6. Furthermore, the essential oils exhibited antitumor activities (IC50, 37.72–123.16 μg/mL) against Hep3B (liver) and Hela (cervical) cells. Linear regression analysis showed that, caryophyllene oxide peak area percentages showed remarkably high negative correlation coefficients with IC50 values of Hep3B and Hela cytotoxicity, which suggested the contribution of this compound on the cancer cell cytotoxicity of three essential oils. Finally, the ITS1-5.8S-ITS2 region was amplified and sequenced in order to generate genomic reference sequences for each plant. These can be used to identify the origins of the plants, and will assist other research studies related to these three plants.

Delimiting species boundaries among closely related lineages often requires a range of independent data sets and analytical approaches. Similar to other organismal groups, robust species circumscriptions in fungi are increasingly investigated within an empirical framework. Here we attempt to delimit species boundaries in a closely related clade of lichen-forming fungi endemic to Asia, the Hypogymnia hypotrypa group (Parmeliaceae). In the current classification, the Hypogymnia hypotrypa group includes two species: H. hypotrypa and H. flavida, which are separated based on distinctive reproductive modes, the former producing soredia but absent in the latter. We reexamined the relationship between these two species using phenotypic characters and molecular sequence data (ITS, GPD, and MCM7 sequences) to address species boundaries in this group. In addition to morphological investigations, we used Bayesian clustering to identify potential genetic groups in the H. hypotrypa/H. flavida clade. We also used a variety of empirical, sequence-based species delimitation approaches, including: the \"Automatic Barcode Gap Discovery\" (ABGD), the Poisson tree process model (PTP), the General Mixed Yule Coalescent (GMYC), and the multispecies coalescent approach BPP. Different species delimitation scenarios were compared using Bayes factors delimitation analysis, in addition to comparisons of pairwise genetic distances, pairwise fixation indices (FST). The majority of the species delimitation analyses implemented in this study failed to support H. hypotrypa and H. flavida as distinct lineages, as did the Bayesian clustering analysis. However, strong support for the evolutionary independence of H. hypotrypa and H. flavida was inferred using BPP and further supported by Bayes factor delimitation. In spite of rigorous morphological comparisons and a wide range of sequence-based approaches to delimit species, species boundaries in the H. hypotrypa group remain uncertain. This study reveals the potential limitations of relying on distinct reproductive strategies as diagnostic taxonomic characters for Hypogymnia and also the challenges of using popular sequence-based species delimitation methods in groups with recent diversification histories.

Acute lung injury (ALI) is an inflammatory condition and there are no effective treatments. A novel new compound---colchicine-myricetin hybrid (CMyrH) was herein designed and synthesized. To evaluate the activity of CMyrH in ALI, we used a bleomycin (BLM) induced BEAS-2B injury model in vitro and established a well-recognized rat model of BLM-induced lung injury in vivo . The results demonstrated that colchicine-myricetin hybrid protected BEAS-2B cells against BLM-induced cell injury in an increased dose manner, and reduced wet/dry weight ratio, histological scoring, and inflammation cytokines IL-1β, IL-6, IL-18, and TNF-α levels of lung tissue of the rats. Furthermore, we found colchicine-myricetin hybrid inhibited caspase-1, ASC, GSDMD, and NLRP-3 expression in vivo . Meanwhile, we used molecular docking to analyze the binding mode of colchicine-myricetin hybrid and human neutrophil elastase (HNE), it revealed that colchicine-myricetin hybrid showed strong binding affinity toward human neutrophil elastase when compared to its parent molecules. In conclusion, It is suggested that colchicine-myricetin hybrid antagonized acute lung injury by focusing on multi-targets via multi-mechanisms, and might be served as a potential therapeutic agent for acute lung injury.

The lichen-forming fungus was isolated from the desert lichen Endocarpon pusillum that is extremely drought resistant.To understand the molecular mechanisms of drought resistance in the fungus,we employed RNA-seq and quantitative real-time PCR to compare and characterize the differentially expressed genes in pure culture at two different water levels and with that in desiccated lichen.The comparative transcriptome analysis indicated that a total of 1781 genes were differentially expressed between samples cultured under normal and PEG-induced drought stress conditions.Similar to those in drought resistance plants and non-lichenized fungi,the common drought-resistant mechanisms were differentially expressed in E.pusillum.However,the expression change of genes involved in osmotic regulation in E.pusillum is different,which might be the evidence for the feature of drought adaptation.Interestingly,different from other organisms,some genes involved in drought adaption mechanisms showed significantly different expression patterns between the presence and absence of drought stress in E.pusillum.The expression of 23 candidate stress responsive genes was further confirmed by quantitative real-time PCR using dehydrated E.pusillum lichen thalli.This study provides a valuable resource for future research on lichen-forming fungi and shall facilitate future functional studies of the specific genes related to drought resistance.

Nitric oxide (NO) has been evidenced to mediate biosynthesis of polyphenols in Inonotus obliquus. However, it remains unknown how NO regulates their biosynthesis. Here we show that higher cellular NO levels coincided with higher accumulation of S-nitrosothiols (SNO; the products of NO combined with a specific residue in glutathione or proteins) and polyphenols, and higher activity of denitrosylated S-nitrosoglutathione reductase (GSNOR) and thioredoxin reductase (TrxR). This homeostasis was breached by GSNOR or TrxR inhibitors. Inhibiting GSNOR boosted TrxR activity, but reduced SNO formation, coinciding with an enhanced production of polyphenols. Likewise, inhibiting TrxR increased GSNOR activity and SNO production, but downregulated accumulation of polyphenols. Inhibiting GSNOR or TrxR also modified the polyphenolic profiles of I. obliquus. Suppressing GSNOR-enhanced biosynthesis of phelligridins C and H, inoscavin C and methyl inoscavin B, but reduced that of phelligridin D, methyl inoscavin A, davallialactone and methyl davallialactone, the typical polyphenols in I. obliquus. Similarly, downregulating TrxR increased production of phelligridin D, methyl inoscavin A, davallialactone, and methyl davallialactone, but shrinking that of phelligridins C and H, methyl inoscavin B and inoscavin C. Thus, in I. obliquus, the state of S-nitrosylation and denitrosylation affects not only the accumulation of polyphenols, but also their metabolic profiles. [PUBLICATION ABSTRACT]

This study investigated the photosynthetic rate of the lichen Endocarpon pusillum at the Chinese Academy of Sciences Shapotou Desert Research Station and estimated its annual contribution to the carbon budget in the ecosystem. The software SigmaPlot 10.0 with ＂Macro-Area below curves＂ was used to calculate the carbon fixation capacity of the lichen. The total carbon budget （ΣC） of the lichen was obtained by subtracting the respiratory carbon loss （ΣDR） from the photosynthetic carbon gain （ΣNP）. Because water from precipitation plays an important role in photosynthesis in this ecosystem, the annual carbon budget of E. pusillum at the station was estimated based on the three-year average precipitation data from 2009 to 2011. Our results indicate that the lichen fixes 14.6 g Cm-2 annually. The results suggest that artificial inoculation of the crust lichen in the Tengger Desert could not only help reduce the sand and dust storms but also offer a significant carbon sink, fixing a total of 438000 t of carbon over the 30000 km2 of the Tengger Desert. The carbon sink could potentially help mitigate the atmospheric greenhouse effect. Our study suggests that the carpet-like lichen E. pusillum is an excellent candidate for ＂Bio-carpet Engineering＂ of arid and semi-arid regions.

This work deals with the survival analyses of the symbionts isolated from the lichen E. pusillum under desiccation and starvation stress. The mycobiont of the symbionts was under the desiccation in combination with starvation stress, and under starvation stress alone as well. The phycobiont of the symbionts was under desiccation stress alone. The experiments were detected by means of the biomass size, weight and cell density, deformity of the hyphae and cells, and metabolic activity through SEM (scanning electron microscopy), TEM (transmission electron microscopy), FM (fluorescence microscopy), spectrophotometry, and FCM (flow cytometry). The results show that the mycobiont can survive for seven months under desiccation stress in combination with starvation stress, and for eight months under starvation stress alone. The phycobiont can survive for two months under desiccation stress. It can provide a scientific basis for further research of the reproduction biology of lichens and arid desert biocarpet engineering to fix sand and carbon.

Umbilicaria muhlenbergii is the only known dimorphic lichenized fungus that grows in the hyphal form in lichen thalli but as yeast cells in axenic cultures. However, the regulation of yeast-to-hypha transition and its relationship to the establishment of symbiosis are not clear. In this study, we show that nutrient limitation and hyperosmotic stress trigger the dimorphic change in U. muhlenbergii. Contact with algal cells of its photobiont Trebouxia jamesii induced pseudohyphal growth. Treatments with the cAMP diphosphoesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) induced pseudohyphal/hyphal growth and resulted in the differentiation of heavily melanized, lichen cortex-like structures in culture, indicating the role of cAMP signaling in regulating dimorphism. To confirm this observation, we identified and characterized two Gα subunits UmGPA2 and UmGPA3. Whereas deletion of UmGPA2 had only a minor effect on pseudohyphal growth, the ΔUmgpa3 mutant was defective in yeast-to-pseudohypha transition induced by hyperosmotic stress or T. jamesii cells. IBMX treatment suppressed the defect of ΔUmgpa3 in pseudohyphal growth. Transformants expressing the UmGPA3 G45V or UmGPA3 Q208L dominant active allele were enhanced in the yeast-to-pseudohypha transition and developed pseudohyphae under conditions noninducible to the wild type. Interestingly, T. jamesii cells in close contact with pseudohyphae of UmGPA3 G45V and UmGPA3 Q208L transformants often collapsed and died after coincubation for over 72 h, indicating that improperly regulated pseudohyphal growth due to dominant active mutations may disrupt the initial establishment of symbiotic interaction between the photobiont and mycobiont. Taken together, these results show that the cAMP-PKA pathway plays a critical role in regulating dimorphism and symbiosis in U. muhlenbergii.