Transcriptome Analysis Reveals Mycelial and Fruiting Responses to Lithium Chloride in Coprinopsis cinerea.

Lithium chloride (LiCl) has been used in signalling and molecular studies of animals, plants, and yeast. However, information on its roles in basidiomycetous fungi is still limited. In this study, we used RNA-Seq to study the effects of LiCl on Coprinopsis cinerea. LiCl enhanced mycelial growth and inhibited fruiting body formation in C. cinerea. RNA-Seq of the LiCl-treated C. cinerea resulted in a total of 14,128 genes. There were 1199 differentially expressed genes (DEGs) between the LiCl-treated samples and control samples in the mycelium stage (the first time point), and 1391 DEGs were detected when the control samples were forming hyphal knots while the treated samples were still in the mycelium (the second time point). Pathway enrichment analysis of the DEGs revealed a significant association between enhanced mycelium growth in the LiCl-treated C. cinerea and metabolic pathways. In addition, the DEGs involved in cellular process pathways, including "cell cycle-yeast" and "meiosis-yeast", were identified in suppressed C. cinerea fruiting body formation by LiCl under favourable environmental conditions. As LiCl can predominantly inhibit the activity of glycogen synthase kinase3 (GSK3), our findings suggest that LiCl affects the expression of genes involved in fruiting body initiation and cellular processes by inhibiting GSK3 activity which is essential for fruiting body formation.

The Genome-Wide Characterization of Alternative Splicing and RNA Editing in the Development of Coprinopsis cinerea.

Coprinopsis cinerea is one of the model species used in fungal developmental studies. This mushroom-forming Basidiomycetes fungus has several developmental destinies in response to changing environments, with dynamic developmental regulations of the organism. Although the gene expression in C. cinerea development has already been profiled broadly, previous studies have only focused on a specific stage or process of fungal development. A comprehensive perspective across different developmental paths is lacking, and a global view on the dynamic transcriptional regulations in the life cycle and the developmental paths is far from complete. In addition, knowledge on co- and post-transcriptional modifications in this fungus remains rare. In this study, we investigated the transcriptional changes and modifications in C. cinerea during the processes of spore germination, vegetative growth, oidiation, sclerotia formation, and fruiting body formation by inducing different developmental paths of the organism and profiling the transcriptomes using the high-throughput sequencing method. Transition in the identity and abundance of expressed genes drive the physiological and morphological alterations of the organism, including metabolism and multicellularity construction. Moreover, stage- and tissue-specific alternative splicing and RNA editing took place and functioned in C. cinerea. These modifications were negatively correlated to the conservation features of genes and could provide extra plasticity to the transcriptome during fungal development. We suggest that C. cinerea applies different molecular strategies in its developmental regulation, including shifts in expressed gene sets, diversifications of genetic information, and reversible diversifications of RNA molecules. Such features would increase the fungal adaptability in the rapidly changing environment, especially in the transition of developmental programs and the maintenance and balance of genetic and transcriptomic divergence. The multi-layer regulatory network of gene expression serves as the molecular basis of the functioning of developmental regulation.

Pulsed Electric Field Treatments with Nonlethal Field Strength Alter the Properties of Bacterial Spores.

ABSTRACT: Pulsed electric field (PEF) treatment, an alternative to thermal processing in the food industry, is insufficient to inactivate bacterial spores. Although spores that have been treated in this manner remain alive, specific understanding of their physiological properties is limited. The purpose of this study is to describe the morphology, viability, and germination behavior of Bacillus atrophaeus spores treated with PEF. Our findings indicate that nonlethal PEF treatment results in spore deformation, dipicolinic acid (DPA) leakage, and a shorter and more uniform germination lag time (\(\def\upalpha{\unicode[Times]{x3B1}}\)\(\def\upbeta{\unicode[Times]{x3B2}}\)\(\def\upgamma{\unicode[Times]{x3B3}}\)\(\def\updelta{\unicode[Times]{x3B4}}\)\(\def\upvarepsilon{\unicode[Times]{x3B5}}\)\(\def\upzeta{\unicode[Times]{x3B6}}\)\(\def\upeta{\unicode[Times]{x3B7}}\)\(\def\uptheta{\unicode[Times]{x3B8}}\)\(\def\upiota{\unicode[Times]{x3B9}}\)\(\def\upkappa{\unicode[Times]{x3BA}}\)\(\def\uplambda{\unicode[Times]{x3BB}}\)\(\def\upmu{\unicode[Times]{x3BC}}\)\(\def\upnu{\unicode[Times]{x3BD}}\)\(\def\upxi{\unicode[Times]{x3BE}}\)\(\def\upomicron{\unicode[Times]{x3BF}}\)\(\def\uppi{\unicode[Times]{x3C0}}\)\(\def\uprho{\unicode[Times]{x3C1}}\)\(\def\upsigma{\unicode[Times]{x3C3}}\)\(\def\uptau{\unicode[Times]{x3C4}}\)\(\def\upupsilon{\unicode[Times]{x3C5}}\)\(\def\upphi{\unicode[Times]{x3C6}}\)\(\def\upchi{\unicode[Times]{x3C7}}\)\(\def\uppsy{\unicode[Times]{x3C8}}\)\(\def\upomega{\unicode[Times]{x3C9}}\)\(\def\bialpha{\boldsymbol{\alpha}}\)\(\def\bibeta{\boldsymbol{\beta}}\)\(\def\bigamma{\boldsymbol{\gamma}}\)\(\def\bidelta{\boldsymbol{\delta}}\)\(\def\bivarepsilon{\boldsymbol{\varepsilon}}\)\(\def\bizeta{\boldsymbol{\zeta}}\)\(\def\bieta{\boldsymbol{\eta}}\)\(\def\bitheta{\boldsymbol{\theta}}\)\(\def\biiota{\\boldsymbol{\iota}}\)\(\def\bikappa{\boldsymbol{\kappa}}\)\(\def\bilambda{\boldsymbol{\lambda}}\)\(\def\\bimu{\boldsymbol{\mu}}\)\(\def\binu{\boldsymbol{\nu}}\)\(\def\bixi{\boldsymbol{\xi}}\)\(\def\biomicron{\boldsymbol{\micron}}\)\(\def\bipi{\boldsymbol{\pi}}\)\(\def\birho{\boldsymbol{\rho}}\)\(\def\bisigma{\boldsymbol{\sigma}}\)\(\def\bitau{\boldsymbol{\\tau}}\)\(\def\biupsilon{\boldsymbol{\upsilon}}\)\(\def\biphi{\boldsymbol{\phi}}\)\(\def\bichi{\boldsymbol{\chi}}\)\(\def\bipsy{\boldsymbol{\psy}}\)\(\def\biomega{\boldsymbol{\omega}}\)\(\def\bupalpha{\bf{\alpha}}\)\(\def\bupbeta{\bf{\beta}}\)\(\def\bupgamma{\bf{\gamma}}\)\(\def\bupdelta{\bf{\delta}}\)\(\def\bupvarepsilon{\bf{\varepsilon}}\)\(\def\bupzeta{\bf{\zeta}}\)\(\def\bupeta{\bf{\eta}}\)\(\def\buptheta{\bf{\theta}}\)\(\def\bupiota{\bf{\iota}}\)\(\def\bupkappa{\bf{\kappa}}\)\(\def\\buplambda{\bf{\lambda}}\)\(\def\bupmu{\bf{\mu}}\)\(\def\bupnu{\bf{\nu}}\)\(\def\bupxi{\bf{\xi}}\)\(\def\bupomicron{\bf{\micron}}\)\(\def\buppi{\bf{\pi}}\)\(\def\buprho{\bf{\rho}}\)\(\def\bupsigma{\bf{\sigma}}\)\(\def\buptau{\bf{\tau}}\)\(\def\bupupsilon{\bf{\upsilon}}\)\(\def\bupphi{\bf{\phi}}\)\(\def\bupchi{\bf{\chi}}\)\(\def\buppsy{\bf{\psy}}\)\(\def\bupomega{\bf{\omega}}\)\(\def\bGamma{\bf{\Gamma}}\)\(\def\bDelta{\bf{\Delta}}\)\(\def\bTheta{\bf{\Theta}}\)\(\def\bLambda{\bf{\Lambda}}\)\(\def\bXi{\bf{\Xi}}\)\(\def\bPi{\bf{\Pi}}\)\(\def\bSigma{\bf{\Sigma}}\)\(\def\bPhi{\bf{\Phi}}\)\(\def\bPsi{\bf{\Psi}}\)\(\def\bOmega{\bf{\Omega}}\)\({T_{{\rm{lag}}}}\)), but that there is no change in release duration (\(\Delta {T_{{\rm{release}}}}\)), germination ratio, or viability. Based on our findings, we conclude that an intact morphologic state and DPA content are not prerequisites for germination and full viability and that, in contrast to nutrient-induced germination in which initially slowly released DPA triggers subsequent germination events, leaked DPA during PEF treatment does not. Spores that have been subjected to this procedure remain dormant and preserve their full germinability. We found that PEF-treated spores respond to germinants more quickly and with less heterogeneity, possibly because the tiny cracks formed on the spore surface facilitate the germinants' access to the germination receptors situated on the spore's inner membrane. The consensus view that nonlethal PEF has less impact on spores that are still capable of forming CFUs under proper conditions is one-sided. This research advances our understanding of how spores behave following nonlethal PEF treatment and gives information on the topics of nosocomial sterilization, food safety, and public health.

Effect of oxygen on the germination and culturability of Bacillus atrophaeus spores / Efecto del oxígeno sobre la germinación y cultivabilidad de las esporas de Bacillus atrophaeus

The effect of oxygen on the germination and culturability of aerobic Bacillus atrophaeus spores was investigated in this study. Under oxic or anoxic conditions, various nutritional and non-nutritional germinants were utilized to induce germination. Tb3+-dipicolinic acid fluorescence assay and phase-contrast microscopy were used to track the germination process. The final germination level, germination half time, and germination speed were used to define germination kinetics. Colony-forming unit enumeration was used to assess the culturability of germinated spores germinated with or without oxygen. The results show that in the absence of oxygen, the final germination level was unaffected, germination half time decreased by up to 35.0%, germination speed increased by up to 27.4%, and culturability decreased by up to 95.1%. It is suggested that oxygen affects some germinant receptor-dependent germination pathways, implying that biomolecules engaged in these pathways may be oxygen-sensitive. Furthermore, spores that have completed the germination process in either anoxic or oxic conditions may have different culturability. This research contributed to a better understanding of the fundamental mechanism of germination.(AU)

PIN3 positively regulates the late initiation of ovule primordia in Arabidopsis thaliana.

Ovule initiation determines the maximum ovule number and has great impact on seed number and yield. However, the regulation of ovule initiation remains largely elusive. We previously reported that most of the ovule primordia initiate asynchronously at floral stage 9 and PINFORMED1 (PIN1) polarization and auxin distribution contributed to this process. Here, we further demonstrate that a small amount of ovule primordia initiate at floral stage 10 when the existing ovules initiated at floral stage 9 start to differentiate. Genetic analysis revealed that the absence of PIN3 function leads to the reduction in pistil size and the lack of late-initiated ovules, suggesting PIN3 promotes the late ovule initiation process and pistil growth. Physiological analysis illustrated that, unlike picloram, exogenous application of NAA can't restore these defective phenotypes, implying that PIN3-mediated polar auxin transport is required for the late ovule initiation and pistil length. qRT-PCR results indicated that the expression of SEEDSTICK (STK) is up-regulated under auxin analogues treatment while is down-regulated in pin3 mutants. Meanwhile, overexpressing STK rescues pin3 phenotypes, suggesting STK participates in PIN3-mediated late ovule initiation possibly by promoting pistil growth. Furthermore, brassinosteroid influences the late ovule initiation through positively regulating PIN3 expression. Collectively, this study demonstrates that PIN3 promotes the late ovule initiation and contributes to the extra ovule number. Our results give important clues for increasing seed number and yield of cruciferous and leguminous crops.

Effect of oxygen on the germination and culturability of Bacillus atrophaeus spores.

The effect of oxygen on the germination and culturability of aerobic Bacillus atrophaeus spores was investigated in this study. Under oxic or anoxic conditions, various nutritional and non-nutritional germinants were utilized to induce germination. Tb3+-dipicolinic acid fluorescence assay and phase-contrast microscopy were used to track the germination process. The final germination level, germination half time, and germination speed were used to define germination kinetics. Colony-forming unit enumeration was used to assess the culturability of germinated spores germinated with or without oxygen. The results show that in the absence of oxygen, the final germination level was unaffected, germination half time decreased by up to 35.0%, germination speed increased by up to 27.4%, and culturability decreased by up to 95.1%. It is suggested that oxygen affects some germinant receptor-dependent germination pathways, implying that biomolecules engaged in these pathways may be oxygen-sensitive. Furthermore, spores that have completed the germination process in either anoxic or oxic conditions may have different culturability. This research contributed to a better understanding of the fundamental mechanism of germination.

Interaction of brassinosteroid and cytokinin promotes ovule initiation and increases seed number per silique in Arabidopsis.

Ovule initiation is a key step that strongly influences ovule number and seed yield. Notably, mutants with enhanced brassinosteroid (BR) and cytokinin (CK) signaling produce more ovules and have a higher seed number per silique (SNS) than wild-type plants. Here, we crossed BR- and CK-related mutants to test whether these phytohormones function together in ovule initiation. We determined that simultaneously enhancing BR and CK contents led to higher ovule and seed numbers than enhancing BR or CK separately, and BR and CK enhanced each other. Further, the BR-response transcription factor BZR1 directly interacted with the CK-response transcription factor ARABIDOPSIS RESPONSE REGULATOR1 (ARR1). Treatments with BR or BR plus CK strengthened this interaction and subsequent ARR1 targeting and induction of downstream genes to promote ovule initiation. Enhanced CK signaling partially rescued the reduced SNS phenotype of BR-deficient/insensitive mutants whereas enhanced BR signaling failed to rescue the low SNS of CK-deficient mutants, suggesting that BR regulates ovule initiation and SNS through CK-mediated and -independent pathways. Our study thus reveals that interaction between BR and CK promotes ovule initiation and increases seed number, providing important clues for increasing the seed yield of dicot crops.

Chromosome-level de novo assembly of Coprinopsis cinerea A43mut B43mut pab1-1 #326 and genetic variant identification of mutants using Nanopore MinION sequencing.

The homokaryotic Coprinopsis cinerea strain A43mut B43mut pab1-1 #326 is a widely used experimental model for developmental studies in mushroom-forming fungi. It can grow on defined artificial media and complete the whole lifecycle within two weeks. The mutations in mating type factors A and B result in the special feature of clamp formation and fruiting without mating. This feature allows investigations and manipulations with a homokaryotic genetic background. Current genome assembly of strain #326 was based on short-read sequencing data and was highly fragmented, leading to the bias in gene annotation and downstream analyses. Here, we report a chromosome-level genome assembly of strain #326. Oxford Nanopore Technology (ONT) MinION sequencing was used to get long reads. Illumina short reads was used to polish the sequences. A combined assembly yield 13 chromosomes and a mitochondrial genome as individual scaffolds. The assembly has 15,250 annotated genes with a high synteny with the C. cinerea strain Okayama-7 #130. This assembly has great improvement on contiguity and annotations. It is a suitable reference for further genomic studies, especially for the genetic, genomic and transcriptomic analyses in ONT long reads. Single nucleotide variants and structural variants in six mutagenized and cisplatin-screened mutants could be identified and validated. A 66 bp deletion in Ras GTPase-activating protein (RasGAP) was found in all mutants. To make a better use of ONT sequencing platform, we modified a high-molecular-weight genomic DNA isolation protocol based on magnetic beads for filamentous fungi. This study showed the use of MinION to construct a fungal reference genome and to perform downstream studies in an individual laboratory. An experimental workflow was proposed, from DNA isolation and whole genome sequencing, to genome assembly and variant calling. Our results provided solutions and parameters for fungal genomic analysis on MinION sequencing platform.

Carbon metabolism and transcriptome in developmental paths differentiation of a homokaryotic Coprinopsis cinerea strain.

The balance and interplay between sexual and asexual reproduction is one of the most intriguing mysteries in the study of fungi. The choice of developmental strategy reflects the ability of fungi to adapt to the changing environment. However, the evolution of developmental paths and the metabolic regulation during differentiation and morphogenesis are poorly understood. Here, an analysis was performed of carbohydrate metabolism and gene expression regulation during the early differentiation process from the vegetative mycelium, to the differentiated structures, fruiting body, oidia and sclerotia, of a homokaryotic fruiting Coprinopsis cinerea strain A43mutB43mut pab1-1 #326. Changes during morphogenesis and the evolution of developmental strategies were followed. Conversion between glucose and glycogen and between glucose and beta-glucan were the main carbon flows in the differentiation processes. Genes related to carbohydrate transport and metabolism were significantly differentially expressed among paths. Sclerotia displayed a set of specifically up-regulated genes that were enriched in the carbon metabolism and energy production and conversion processes. Evolutionary transcriptomic analysis of four developmental paths showed that all transcriptomes were under the purifying selection, and the more stressful the environment, the younger the transcriptome age. Oidiation has the lowest value of transcriptome age index (TAI) and transcriptome divergence index (TDI), while the fruiting process has the highest of both indexes. These findings provide new insights into the regulations of carbon metabolism and gene expressions during the early stages of fungal developmental paths differentiation, and improve our understanding of the evolutionary process of life history and reproductive strategy in fungi.

Effective Modulating Brassinosteroids Signal to Study Their Specific Regulation of Reproductive Development and Enhance Yield.

Brassinosteroid (BR) is a family of bioactive steroid hormones that plays vital roles in plant growth and development. The BR-mediated regulation of plant growth and architecture has been well studied. However, relatively few studies have investigated the BR-related regulation of reproductive development because of the difficulties in excluding non-specific regulation and secondary responses from severe vegetative phenotypes and poor nutritional status. Furthermore, differentially regulating the BR signal in vegetative and reproductive organs is problematic. Thus, establishing a method for modulating the BR signal only in reproductive organs or during reproductive developmental stages will be beneficial. Additionally, the utility of BR applications for crop production is limited because of deleterious side-effects, including the associated decrease in the planting density and lodging resistance. Moreover, enhancing the BR signal may lead to feedback inhibition. In this study, we developed a transformation system for modulating the BR signal differentially during reproductive and vegetative developmental stages. This system involves transformations with different combinations of a reproductive tissue-specific promoter, coding sequences that increase or decrease the BR signal, and various genotypic backgrounds with enhanced or decreased BR signals. The enhanced BR signal generated in transformants was targeted to reproductive organs without affecting vegetative organs. This system may be useful for studying the BR-specific regulation of plant reproductive development and shows promise for optimizing seed yield.

Modified recipe to inhibit fruiting body formation for living fungal biomaterial manufacture.

Living fungal mycelium with abolished ability to form fruiting bodies is a self-healing substance, which is particularly valuable for further engineering and development as materials sensing environmental changes and secreting signals. Suppression of fruiting body formation is also a useful tool for maintaining the stability of a mycelium-based material with ease and lower cost. The objective of this study was to provide a biochemical solution to regulate the fruiting body formation, which may replace heat killing of mycelium in practice. The concentrations of glycogen synthase kinase-3 (GSK-3) inhibitors, such as lithium chloride or CHIR99021 trihydrochloride, were found to directly correlate with the development of fruiting bodies in the mushroom forming fungi such as Coprinopsis cinerea and Pleurotus djamor. Sensitive windows to these inhibitors throughout the fungal life cycle were also identified. We suggest the inclusion of GSK-3 inhibitors in the cultivation recipes for inhibiting fruiting body formation and regulating mycelium growth. This is the first report of using a GSK-3 inhibitor to suppress fruiting body formation in living fungal mycelium-based materials. It provides an innovative strategy for easy, reliable, and low cost maintenance of materials containing living fungal mycelium.

Human proteins with target sites of multiple post-translational modification types are more prone to be involved in disease.

Many proteins can be modified by multiple types of post-translational modifications (Mtp-proteins). Although some post-translational modifications (PTMs) have recently been found to be associated with life-threatening diseases like cancers and neurodegenerative disorders, the underlying mechanisms remain enigmatic to date. In this study, we examined the relationship of human Mtp-proteins and disease and systematically characterized features of these proteins. Our results indicated that Mtp-proteins are significantly more inclined to participate in disease than proteins carrying no known PTM sites. Mtp-proteins were found significantly enriched in protein complexes, having more protein partners and preferred to act as hubs/superhubs in protein-protein interaction (PPI) networks. They possess a distinct functional focus, such as chromatin assembly or disassembly, and reside in biased, multiple subcellular localizations. Moreover, most Mtp-proteins harbor more intrinsically disordered regions than the others. Mtp-proteins carrying PTM types biased toward locating in the ordered regions were mainly related to protein-DNA complex assembly. Examination of the energetic effects of PTMs on the stability of PPI revealed that only a small fraction of single PTM events influence the binding energy of >2 kcal/mol, whereas the binding energy can change dramatically by combinations of multiple PTM types. Our work not only expands the understanding of Mtp-proteins but also discloses the potential ability of Mtp-proteins to act as key elements in disease development.