Eukaryotic Cell Differentiation Research Articles

The evolution of multicellularity and cell differentiation symposium: bridging evolutionary cell biology and computational modelling using emerging model systems.

'The evolution of multicellularity and cell differentiation' symposium, organized as part of the EuroEvoDevo 2024 meeting on June 25-28th in Helsinki (Finland), addressed recent advances on the molecular and mechanistic basis for the evolution of multicellularity and cell differentiation in eukaryotes. The symposium involved over 100 participants and brought together 10 speakers at diverse career stages. Talks covered various topics at the interface of developmental biology, evolutionary cell biology, comparative genomics, computational biology, and ecology using animal, protist, algal and mathematical models. This symposium offered a unique opportunity for interdisciplinary dialog among researchers working on different systems, especially in promoting collaborations and aligning strategies for studying emerging model species. Moreover, it fostered opportunities to promote early career researchers in the field and opened discussions of ongoing work and unpublished results. In this Meeting Review, we aim to promote the research, capture the spirit of the meeting, and present key topics discussed within this dynamic, growing and open community.

HDACs MADS-domain protein interaction: a case study of HDA15 and XAL1 in Arabidopsis thaliana

ABSTRACT Cellular behavior, cell differentiation and ontogenetic development in eukaryotes result from complex interactions between epigenetic and classic molecular genetic mechanisms, with many of these interactions still to be elucidated. Histone deacetylase enzymes (HDACs) promote the interaction of histones with DNA by compacting the nucleosome, thus causing transcriptional repression. MADS-domain transcription factors are highly conserved in eukaryotes and participate in controlling diverse developmental processes in animals and plants, as well as regulating stress responses in plants. In this work, we focused on finding out putative interactions of Arabidopsis thaliana HDACs and MADS-domain proteins using an evolutionary perspective combined with bioinformatics analyses and testing the more promising predicted interactions through classic molecular biology tools. Through bioinformatic analyses, we found similarities between HDACs proteins from different organisms, which allowed us to predict a putative protein-protein interaction between the Arabidopsis thaliana deacetylase HDA15 and the MADS-domain protein XAANTAL1 (XAL1). The results of two-hybrid and Bimolecular Fluorescence Complementation analysis demonstrated in vitro and in vivo HDA15-XAL1 interaction in the nucleus. Likely, this interaction might regulate developmental processes in plants as is the case for this type of interaction in animals.

Time-resolved small RNA transcriptomics of the ichthyosporean Sphaeroforma arctica.

Ichthyosporea, a clade of holozoans, represent a clade closely related to animals, and thus hold a key phylogenetic position for understanding the origin of animals. We have previously discovered that an ichthyosporean, Sphaeroforma arctica, contains microRNAs (miRNAs) as well as the miRNA processing machinery. This was the first discovery of miRNAs among the closest single-celled relatives of animals and raised intriguing questions about the roles of regulatory small RNAs in cell development and differentiation in unicellular eukaryotes. Like many ichthyosporeans, S. arctica also undergoes a transient multicellular developmental life cycle. As miRNAs are, among other roles, key regulators of gene expression during development in animals, we wanted to investigate the dynamics of miRNAs during the developmental cycle in S. arctica. Here we have therefore collected a comprehensive time-resolved small RNA transcriptome linked to specific life stages with a substantially higher sequencing depth than before, which can enable further discovery of functionally relevant small RNAs. The data consists of Illumina-sequenced small RNA libraries from two independent biological replicates of the entire life cycle of S. arctica with high temporal resolution. The dataset is directly linked and comes from the same samples as a previously published mRNA-seq dataset, thus enabling direct cross-functional analyses.

Genome-Wide Identification of G Protein-Coupled Receptors in Ciliated Eukaryotes

G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and play important roles in many physiological processes. As a representative group of protozoa, ciliates represent the highest stage of eukaryotic cell differentiation and evolution in terms of their reproductive mode, two-state karyotype, and extremely diverse cytogenesis patterns. GPCRs have been poorly reported in ciliates. In this study, we identified 492 GPCRs in 24 ciliates. Using the existing classification system for animals, GPCRs in ciliates can be assigned to four families, including families A, B, E, and F. Most (377 members) belong to family A. The number of GPCRs is extremely different in different ciliates; the Heterotrichea ciliates usually have more GPCRs than other ciliates. Parasitic or symbiotic ciliates usually have only a few GPCRs. Gene/genome duplication events seem to play important roles in the expansion of the GPCR superfamily in ciliates. GPCRs in ciliates displayed seven typical domain organizations. GPCRs in an ortholog group are common and conserved in all ciliates. The gene expression analysis of the members in this conserved ortholog group in the model ciliate, Tetrahymena thermophila, suggested that these GPCRs play important roles in the life cycle of ciliates. In summary, this study provides the first comprehensive genome-wide identification of GPCRs in ciliates, improving our understanding of the evolution and function of GPCR in ciliates.

Transcript profiling of Polycomb gene family in Oryza sativa indicates their abiotic stress-specific response.

The precise regulation of gene expression is required for the determination of cell fate, differentiation, and developmental programs in eukaryotes. The Polycomb Group (PcG) genes are the key transcriptional regulators that constitute the repressive system, with two major protein complexes, Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2). Previous studies have demonstrated the significance of these proteins in regulation of normal growth and development processes. However, the role of PcG in adaptation of crops to abiotic stress is still not well understood. The present study aimed to a comprehensive genome-wide identification of the PcG gene family in one of the economically important staple crops, Oryza sativa. Here, a total of 14 PcG genes have been identified, which were distributed over eight chromosomes. Protein structure analysis revealed that both the complexes have distinct domain and motifs that are conserved within the complexes. In silico promoter analysis showed that PcG gene promoters have abundance of abiotic stress-responsive elements. RNA-seq based expression analysis revealed that PcG genes are differentially expressed in different tissues and responded variably in different environmental stress. Validation of gene expression by qRT-PCR showed that most of the genes were upregulated at 1-h time point in shoot tissue and at 24-h time point in root tissue under the drought and salinity stress conditions. These findings provide important and extensive information on the PcG family of O. sativa, which will pave the path for understanding their role in stress signaling in plants.

Role of Caspase Family in Intervertebral Disc Degeneration and Its Therapeutic Prospects.

Intervertebral disc degeneration (IVDD) is a common musculoskeletal degenerative disease worldwide, of which the main clinical manifestation is low back pain (LBP); approximately, 80% of people suffer from it in their lifetime. Currently, the pathogenesis of IVDD is unclear, and modern treatments can only alleviate its symptoms but cannot inhibit or reverse its progression. However, in recent years, targeted therapy has led to new therapeutic strategies. Cysteine-containing aspartate proteolytic enzymes (caspases) are a family of proteases present in the cytoplasm. They are evolutionarily conserved and are involved in cell growth, differentiation, and apoptotic death of eukaryotic cells. In recent years, it has been confirmed to be involved in the pathogenesis of various diseases, mainly by regulating cell apoptosis and inflammatory response. With continuous research on the pathogenesis and pathological process of IVDD, an increasing number of studies have shown that caspases are closely related to the IVDD process, especially in the intervertebral disc (IVD) cell apoptosis and inflammatory response. Therefore, herein we study the role of caspases in IVDD with respect to the structure of caspases and the related signaling pathways involved. This would help explore the strategy of regulating the activity of the caspases involved and develop caspase inhibitors to prevent and treat IVDD. The aim of this review was to identify the caspases involved in IVDD which could be potential targets for the treatment of IVDD.

A long noncoding RNA promotes parasite differentiation in African trypanosomes.

The parasite Trypanosoma brucei causes African sleeping sickness that is fatal to patients if untreated. Parasite differentiation from a replicative slender form into a quiescent stumpy form promotes host survival and parasite transmission. Long noncoding RNAs (lncRNAs) are known to regulate cell differentiation in other eukaryotes. To determine whether lncRNAs are also involved in parasite differentiation, we used RNA sequencing to survey the T. brucei genome, identifying 1428 previously uncharacterized lncRNA genes. We find that grumpy lncRNA is a key regulator that promotes parasite differentiation into the quiescent stumpy form. This function is promoted by a small nucleolar RNA encoded within the grumpy lncRNA. snoGRUMPY binds to messenger RNAs of at least two stumpy regulatory genes, promoting their expression. grumpy overexpression reduces parasitemia in infected mice. Our analyses suggest that T. brucei lncRNAs modulate parasite-host interactions and provide a mechanism by which grumpy regulates cell differentiation in trypanosomes.

Research Progress in Predicting DNA Methylation Modifications and the Relation with Human Diseases.

DNA methylation is an important mode of regulation in epigenetic mechanisms, and it is one of the research foci in the field of epigenetics. DNA methylation modification affects a series of biological processes, such as eukaryotic cell growth, differentiation, and transformation mechanisms, by regulating gene expression. In this review, we systematically summarized the DNA methylation databases, prediction tools for DNA methylation modification, machine learning algorithms for predicting DNA methylation modification, and the relationship between DNA methylation modification and diseases such as hypertension, Alzheimer's disease, diabetic nephropathy, and cancer. An in-depth understanding of DNA methylation mechanisms can promote accurate prediction of DNA methylation modifications and the treatment and diagnosis of related diseases.

Flexible, Biocompatible PET Sheets: A Platform for Attachment, Proliferation and Differentiation of Eukaryotic Cells

Transparent, flexible, biaxially oriented polyethylene terephthalate (PET) sheets were modified by bioactive polymer-fibronectin top layers for the attachment of cells and growth of muscle fibers. Towards this end, PET sheets were grafted with 4-(dimethylamino)phenyl (DMA) groups from the in situ generated diazonium cation precursor. The arylated sheets served as macro-hydrogen donors for benzophenone and the growth of poly(2-hydroxy ethyl methacrylate) (PHEMA) top layer by surface-confined free radical photopolymerization. The PET-PHEMA sheets were further grafted with fibronectin (FBN) through the 1,1-carbonyldiimidazole coupling procedure. The bioactive PET-PHEMA-I-FBN was then employed as a platform for the attachment, proliferation and differentiation of eukaryotic cells which after a few days gave remarkable muscle fibers, of ~120 µm length and ~45 µm thickness. We demonstrate that PET-PHEMA yields a fast growth of cells followed by muscle fibers of excellent levels of differentiation compared to pristine PET or standard microscope glass slides. The positive effect is exacerbated by crosslinking PHEMA chains with ethylene glycol dimethacrylate at initial HEMA/EGDA concentration ratio = 9/1. This works conclusively shows that in situ generated diazonium salts provide aryl layers for the efficient UV-induced grafting of biocompatible coating that beneficially serve as platform for cell attachment and growth of muscle fibers.

Notch Signaling and MicroRNA: The Dynamic Duo Steering Between Neurogenesis and Glioblastomas.

Notch signaling is an evolutionary conserved pathway that plays a central role in development and differentiation of eukaryotic cells. It has been well documented that Notch signaling is inevitable for neuronal cell growth and homeostasis. It regulates process of differentiation from early embryonic stages to fully developed brain. To achieve this streamlined development of neuronal cells, a number of cellular processes are being orchestrated by the Notch signaling. Abrogated Notch signaling is related to several brain tumors, including glioblastomas. On the other hand, microRNAs are small molecules that play decisive role in mediating and modulating Notch signaling. This review discusses the crucial role of Notch signaling in development of nervous system and how this versatile pathway interplay with microRNAs in glioblastoma. This review sheds light on interplay between abrogated Notch signaling and miRNAs in the regulation of neuronal differentiation with special focus on miRNAs mediated regulation of tumorigenesis in glioblastoma. Furthermore, it discusses different aspects of neurogenesis modulated by the Notch signaling that could be exploited for the identification of new diagnostic tools and therapies for the treatment of glioblastoma.

Nanomaterials for Autophagy-Related miRNA-34a Delivery in Cancer Treatment.

Autophagy is an evolutionary conserved physiological process with a fundamental role during development, differentiation, and survival of eukaryotic cells. On the other hand, autophagy dysregulation is observed in many pathological conditions, including cancer. In particular, tumor growth and progression are accompanied and promoted by increased autophagy that allows cancer cells to escape apoptosis and to proliferate also in harsh microenvironments. It is, therefore, clear that the impairment of the autophagic process may represent a valid strategy to inhibit or reduce cancer growth and progression. Among the plethora of molecular players controlling cancer growth, a group of small endogenous noncoding RNAs called microRNAs (miRNAs) has recently emerged. In fact, miRNAs can act as either oncogenes or oncosuppressors depending on their target genes. Moreover, among miRNAs, miRNA-34a has been connected with both tumor repression and autophagy regulation, and its expression is frequently lost in many cancers. Therefore, enforced expression of miRNA-34a in cancer cells may represent a valid strategy to reduce cancer growth. However, such strategy is limited by the fast biodegradation and short half-life of miRNA-34a and by the lack of an efficient intracellular delivery system. The following review describes the autophagic process and its role in cancer as well as the role of miRNAs in general and miRNA-34a in particular in regulating tumor growth by modulating autophagy. Finally, we describe the use of nanoparticles as a promising strategy to selectively deliver miRNA-34a to tumor cells for therapeutic and diagnostic purposes.

The Legionella kinase LegK7 exploits the Hippo pathway scaffold protein MOB1A for allostery and substrate phosphorylation

During infection, the bacterial pathogen Legionella pneumophila manipulates a variety of host cell signaling pathways, including the Hippo pathway which controls cell proliferation and differentiation in eukaryotes. Our previous studies revealed that L. pneumophila encodes the effector kinase LegK7 which phosphorylates MOB1A, a highly conserved scaffold protein of the Hippo pathway. Here, we show that MOB1A, in addition to being a substrate of LegK7, also functions as an allosteric activator of its kinase activity. A crystallographic analysis of the LegK7-MOB1A complex revealed that the N-terminal half of LegK7 is structurally similar to eukaryotic protein kinases, and that MOB1A directly binds to the LegK7 kinase domain. Substitution of interface residues critical for complex formation abrogated allosteric activation of LegK7 both in vitro and within cells and diminished MOB1A phosphorylation. Importantly, the N-terminal extension (NTE) of MOB1A not only regulated complex formation with LegK7 but also served as a docking site for downstream substrates such as the transcriptional coregulator YAP1. Deletion of the NTE from MOB1A or addition of NTE peptides as binding competitors attenuated YAP1 recruitment to and phosphorylation by LegK7. By providing mechanistic insight into the formation and regulation of the LegK7-MOB1A complex, our study unravels a sophisticated molecular mimicry strategy that is used by L. pneumophila to take control of the host cell Hippo pathway.

Cyclic di-GMP in Streptomycetes: A New Conformation, New Binding Mode, New Receptor, and a New Mechanism to Control Cell Development.

A recent paper by Gallagher etal. (2020) demonstrates that c-di-GMP controls spore formation in Streptomyces venezuelae through sequestering the sporulation sigma factor σWhiG and presents the crystal structure of a ternary complex between c-di-GMP, σWhiG, and its anti-sigma factor, RsiG.

A Mathematical Model of the Cell Differentiation in Multicellular Eukaryotes

The cell differentiation in multicellular eukaryotes is one of the most curious phenomena. The recent gene and genome sequencing reveals that most of differentiated cells in a multicellular eukaryote carry a common genome and that such a genome contains the expanded repertoire of genes of proteins associated with the cell-cell adhesion, intercellular and intracellular signal transduction and transcriptional regulation. The cell differentiation occurs in the assembly consisting of a large number of cells after the cell proliferation, and this process is regarded as a stochastic process. Its formulation starts with the master equation in the present paper. The cell differentiation is reproduced in the equation of the most probable path derived from the master equation, when the short-range and long-range interactions between the cells as well as the transition probability between the proliferation and differentiation modes are considered. Moreover, the equation of the most probable path explains the experimental results such as the “memory”, tissue culture and the preparation of induced pluripotent stem (iPS) cells in embryology, if the long-range interaction is considered to be the regulation of gene transcription under the influence of intracellular signal transduction from the receptor accepting the ligand secreted by other types of cells and the short-range interaction is considered to stabilize the intracellular signal transduction by the contact between the same type of cells. The “organizer” found in the initial development of embryo is also explained as the cells that preferentially express the specific gene of a ligand to rouse the long-range interaction. In conclusion, the present study proposes that the complicated intercellular and intracellular signal transduction causing the cell differentiation is ascribed to the long-range interaction between distinctive types of cells and the short-range interaction between the same type of cells.

Contact Sites of rDNA Clusters with FANK1 Gene Correspond to Repressed Chromatin

rDNA genes play an important role in epigenetic regulation and in differentiation of eukaryotic cells. Using the 4C (circular chromosome conformation capture) approach and model HEK293T cells, we analyzed the rDNA-contacting gene FANK1, using anchor located inside rDNA genes. At the 5' end of the FANK1 gene we detected frequent contacts with rDNA clusters. The contact sites coincide with the border where chromatin state changes and nucleosome positioning. The adjacent genes DHX32, BCCIP and UROS are located in the active chromatin and are transcribed, but do not contact with rDNA genes, while FANK1 gene is silenced, and is located in repressed chromatin. Heat shock treatment dramatically changes the pattern of rDNA contacts in the region and induces about 4-fold increase in activation of the FANK1 gene. We conclude that rDNA contacts may be involved in repression of the FANK1 gene.

The putative deubiquitinating enzyme MoUbp4 is required for infection-related morphogenesis and pathogenicity in the rice blast fungus Magnaporthe oryzae.

Ubiquitination is a key regulatory mechanism that affects numerous important biological processes, including cellular differentiation and pathogenesis in eukaryotic cells. Attachment of proteins to ubiquitin is reversed by specialized proteases, deubiquitinating enzymes (DUBs), which are essential for precursor processing, maintaining ubiquitin homeostasis and promoting protein degradation by recycling ubiquitins. Here, we report the identification of a novel non-pathogenic T-DNA-tagged mutant T612 of Magnaporthe oryzae with a single insertion in the second exon of MoUBP4, which encodes a putative ubiquitin carboxyl-terminal hydrolase. Targeted gene deletion mutants of MoUBP4 are significantly reduced in mycelial growth, conidiation, and increased in tolerance to SDS and CR (Congo red) cell-wall damage. The ΔMoubp4 mutants are blocked in penetration and invasive growth, which results in the loss of pathogenicity. Many conidia produced by the ΔMoubp4 mutants are unable to form appressoria and mobilization and degradation of glycogen and lipid droplets are significantly delayed. Moreover, immunohybridization analysis revealed that total protein ubiquitination levels of the null mutants were significantly increased, indicating that MoUbp4 functions as a deubiquitination enzyme. Taken together, we conclude that MoUbp4 is required for deubiquitination, infection-related morphogenesis and pathogenicity in M. oryzae.

Role of Folate Receptor 3 in Tyrosine Kinase Inhibitor Discontinuation in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by Philadelphia chromosome and its molecular counterpart, the BCR-ABL1 fusion gene. Discontinuation of tyrosine kinase inhibitors (TKIs) therapy after achieving a persistent deep molecular response (DMR) is an urgently-needed treatment goal for patients and it was written in NCCN guideline Version 2.2017 for CML. Indeed, various studies have confirmed the feasibility of stopping TKIs therapy in many regions. However, increasing evidence demonstrated that TKIs are unable to eliminate quiescent leukemic stem cells (LSCs), which lead to treatment resistance or relapse after discontinuation of TKIs treatment. Multiple works have confirmed that approximately 50-60% of DMR patients stopping TKIs will lose their response and require retreatment. The patients who will lose the treatment-free remission (TFR) remains unknown currently.We selected bone marrow of 14 patients (matched 7 relapse and 7 non-relapse patients) who discontinued TKIs therapy in our center for transcriptome analysis to discover the differences between relapse and non-relapse patients. Bioinformatics indicated that folate receptor 3 (FOLR3) was highly expressed in non-relapse patients and only in the non-relapsers (Figure 1A). Further work found these samples all carried a common SNP mutation. Then we respectively transferred newly diagnosed chronic phase (CP) CML patients derived bone marrow CD34+ cells and CML K562 cells with lentiviral vectors containing FOLR3 SNP, FOLR3 SNP shRNA, and wild type FOLR3. The result indicated that FOLR3 SNP significantly promoted the clonogenicity of CD34+/K562 cells, while loss of FOLR3 SNP hindered cell differentiation (Figure 1B and D are data of CD34, Figure 1C and E are data of K562). K562 subcutaneous tumor formation in balb/c node mice confirmed that tumors weight and volume of FOLR3 SNP shRNA group were higher than control. While, small animal PET scanning showed that maximum standardized uptake value (SUVmax) of 18F-FDG of FOLR3 SNP+ group was higher than the rest. Experiments in vivo and vitro synergistically proved FOLR3 SNP promoted tumor cell differentiation, delayed tumor growth.To better understand how FOLR3 SNP promote tumor cell differentiation and delay tumor growth, we performed second transcriptome analysis. Consistently, both in FOLR3 SNP+ CD34+ and K562 cells, enrichment analysis revealed that differentially expressed genes were enriched in mitochondria associated Gene Ontology (GO) biological process (Figure F), in which mitochondria complex V matched genes were most significant, such as ATP5 family. In vitro, we demonstrated that ATP syntheses, maximal respiration and spare respiratory capacity of FOLR3 SNP+ CD34+/K562 cells were significantly higher than control and shFOLR3 counterpart through seahorse XF cell mito stress test (Figure G and H refer to CD34 and K562, separately). Electron microscope also exhibited an increase of mitochondrial in FOLR3 SNP+ cells.Mitochondria play an essential role in energy generation, cell signalling, differentiation, death and senescence in eukaryotic cells. We detected a series of genes related to aging, cell cycle and mitochondria unfolded protein response. The results showed that cell cycle kinase such as CDK4 decreased in FOLR3 SNP+ CD34+/K562 cells. On the other hand, senescence associated genes seemed increased. In conclusion, we highlighted the connection of FOLR3 and post-cessation relapse. FOLR3 SNP could be an indicator of TFR. Its internal mechanism might be the mitochondrion activation induced aging of residual leukemia cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Involvement of the cysteine protease BcAtg4 in development and virulence of Botrytis cinerea.

Autophagy serves as a survival mechanism against starvation and has been reported to be important for cell growth and differentiation in eukaryotes. Here, we investigated the function of a cysteine protease BcAtg4 in the gray mold fungus Botrytis cinerea. Yeast complementation experiments revealed that Bcatg4 can functionally replace the counterpart of yeast. Subcellular localization exhibited that BcAtg4 diffused in cytoplasm at different developmental stages. Targeted gene deletion of Bcatg4 (ΔBcatg4) led to autophagy blocking and a significant retardation in growth and conidiation. In addition, ΔBcatg4 failed to form sclerotia. Infection tests demonstrated that ΔBcatg4 was severely attenuated in virulence on different host plant tissues. All of the phenotypic defects were restored by reintroducing an intact copy of Bcatg4 into ΔBcatg4. These results indicate that Bcatg4 plays multiple roles in the developmental processes and pathogenesis of B. cinerea.

Dual reporter genetic mouse models of pancreatic cancer identify an epithelial-to-mesenchymal transition-independent metastasis program.

Epithelial‐to‐mesenchymal transition (EMT) is a recognized eukaryotic cell differentiation program that is also observed in association with invasive tumors. Partial EMT program in carcinomas imparts cancer cells with mesenchymal‐like features and is proposed as essential for metastasis. Precise determination of the frequency of partial EMT program in cancer cells in tumors and its functional role in metastases needs unraveling. Here, we employed mesenchymal cell reporter mice driven by αSMA‐Cre and Fsp1‐Cre with genetically engineered mice that develop spontaneous pancreatic ductal adenocarcinoma (PDAC) to monitor partial EMT program. Both αSMA‐ and Fsp1‐Cre‐mediated partial EMT programs were observed in the primary tumors. The established metastases were primarily composed of cancer cells without evidence for a partial EMT program, as assessed by our fate mapping approach. In contrast, metastatic cancer cells exhibiting a partial EMT program were restricted to isolated single cancer cells or micrometastases (3–5 cancer cells). Collectively, our studies identify large metastatic nodules with preserved epithelial phenotype and potentially unravel a novel metastasis program in PDAC.

The Autophagy Gene BcATG8 Regulates the Vegetative Differentiation and Pathogenicity of Botrytis cinerea.

Autophagy is a conserved degradation process that maintains intracellular homeostasis to ensure normal cell differentiation and development in eukaryotes. ATG8 is one of the key molecular components of the autophagy pathway. In this study, we identified and characterized BcATG8, a homologue of Saccharomyces cerevisiae (yeast) ATG8 in the necrotrophic plant pathogen Botrytis cinerea Yeast complementation experiments demonstrated that BcATG8 can functionally complement the defects of the yeast ATG8 null mutant. Direct physical interaction between BcAtg8 and BcAtg4 was detected in the yeast two-hybrid system. Subcellular localization assays showed that green fluorescent protein-tagged BcAtg8 (GFP-BcAtg8) localized in the cytoplasm as preautophagosomal structures (PAS) under general conditions but mainly accumulated in the lumen of vacuoles in the case of autophagy induction. Deletion of BcATG8 (ΔBcAtg8 mutant) blocked autophagy and significantly impaired mycelial growth, conidiation, sclerotial formation, and virulence. In addition, the conidia of the ΔBcAtg8 mutant contained fewer lipid droplets (LDs), and quantitative real-time PCR (qRT-PCR) assays revealed that the basal expression levels of the LD metabolism-related genes in the mutant were significantly different from those in the wild-type (WT) strain. All of these phenotypic defects were restored by gene complementation. These results indicate that BcATG8 is essential for autophagy to regulate fungal development, pathogenesis, and lipid metabolism in B. cinereaIMPORTANCE The gray mold fungus Botrytis cinerea is an economically important plant pathogen with a broad host range. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. Exploring the fundamental biology of B. cinerea can provide the theoretical basis for sustainable and long-term disease management. Autophagy is an intracellular process for degradation and recycling of cytosolic materials in eukaryotes and is now known to be vital for fungal life. Here, we report studies of the biological role of the autophagy gene BcATG8 in B. cinerea The results suggest that autophagy plays a crucial role in vegetative differentiation and virulence of B. cinerea.