Genome-Wide Identification and Characterization of Effector Candidates with Conserved Motif in Falciphora oryzae.

Microbes employ effectors to disrupt immune responses and promote host colonization. Conserved motifs including RXLR, LFLAK-HVLVxxP (CRN), Y/F/WxC, CFEM, LysM, Chitin-bind, DPBB_1 (PNPi), and Cutinase have been discovered to play crucial roles in the functioning of effectors in filamentous fungi. Nevertheless, little is known about effectors with conserved motifs in endophytes. This research aims to discover the effector genes with conserved motifs in the genome of rice endophyte Falciphora oryzae. SignalP identified a total of 622 secreted proteins, out of which 227 were predicted as effector candidates by EffectorP. By utilizing HMM features, we discovered a total of 169 effector candidates with conserved motifs and three novel motifs. Effector candidates containing LysM, CFEM, DPBB_1, Cutinase, and Chitin_bind domains were conserved across species. In the transient expression assay, it was observed that one CFEM and one LysM activated cell death in tobacco leaves. Moreover, two CFEM and one Chitin_bind inhibited cell death induced by Bax protein. At various points during the infection, the genes' expression levels were increased. These results will help to identify functional effector proteins involving omics methods using new bioinformatics tools, thus providing a basis for the study of symbiosis mechanisms.

An efficient genetic manipulation protocol for dark septate endophyte Falciphora oryzae.

OBJECTIVE: To investigate the protoplast preparation and transformation system of endophytic fungus Falciphora oryzae. RESULTS: F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9 M KCl solution and 10 mg mL-1 glucanase at 30 °C with shaking at 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar medium containing 1 M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired green fluorescent protein-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots. CONCLUSIONS: This study provided a protoplast transformation system of F. oryzae, creating opportunities for future genetic research in other endophytic fungi.

PoMet3 and PoMet14 associated with sulfate assimilation are essential for conidiogenesis and pathogenicity in Pyricularia oryzae.

Pyricularia oryzae is the causal agent of blast disease on staple gramineous crops. Sulphur is an essential element for the biosynthesis of cysteine and methionine in fungi. Here, we targeted the P. oryzae PoMET3 encoding the enzyme ATP sulfurylase, and PoMET14 encoding the APS (adenosine-5'-phosphosulphate) kinase that are involved in sulfate assimilation and sulphur-containing amino acids biosynthesis. In P. oryzae, deletion of PoMET3 or PoMET14 separately results in defects of conidiophore formation, significant impairments in conidiation, methionine and cysteine auxotrophy, limited invasive hypha extension, and remarkably reduced virulence on rice and barley. Furthermore, the defects of the null mutants could be restored by supplementing with exogenous cysteine or methionine. Our study explored the biological functions of sulfur assimilation and sulphur-containing amino acids biosynthesis in P. oryzae.

Endophytic fungus Falciphora oryzae promotes lateral root growth by producing indole derivatives after sensing plant signals.

The endophytic fungus Falciphora oryzae was initially isolated from wild rice (Oryza granulata) and colonizes many crop species and promotes plant growth. However, the molecular mechanisms underlying F. oryzae-mediated growth promotion are still unknown. We found that F. oryzae was able to colonize Arabidopsis thaliana. The most dramatic change after F. oryzae inoculation was observed in the root architecture, as evidenced by increased lateral root growth but reduced primary root length, similar to the effect of auxin, a significant plant growth hormone. The expression of genes responsible for auxin biosynthesis, transport, and signalling was regulated in Arabidopsis roots after F. oryzae cocultivation. Indole derivatives were detected at significantly higher levels in liquid media after cocultivation compared with separate cultivation of Arabidopsis and F. oryzae. Consistently, the expression of indole biosynthetic genes was highly upregulated in F. oryzae upon treatment with Arabidopsis exudates. Global analysis of Arabidopsis gene expression at the early stage after F. oryzae cocultivation suggested that signals were exchanged to initiate Arabidopsis-F. oryzae interactions. All these results suggest that signalling molecules from Arabidopsis roots are perceived by F. oryzae and induce the biosynthesis of indole derivatives in F. oryzae, consequently stimulating Arabidopsis lateral root growth.

Detection of Oil Chestnuts Infected by Blue Mold Using Near-Infrared Hyperspectral Imaging Combined with Artificial Neural Networks.

Mildew damage is a major reason for chestnut poor quality and yield loss. In this study, a near-infrared hyperspectral imaging system in the 874⁻1734 nm spectral range was applied to detect the mildew damage to chestnuts caused by blue mold. Principal component analysis (PCA) scored images were firstly employed to qualitatively and intuitively distinguish moldy chestnuts from healthy chestnuts. Spectral data were extracted from the hyperspectral images. A successive projections algorithm (SPA) was used to select 12 optimal wavelengths. Artificial neural networks, including back propagation neural network (BPNN), evolutionary neural network (ENN), extreme learning machine (ELM), general regression neural network (GRNN) and radial basis neural network (RBNN) were used to build models using the full spectra and optimal wavelengths to distinguish moldy chestnuts. BPNN and ENN models using full spectra and optimal wavelengths obtained satisfactory performances, with classification accuracies all surpassing 99%. The results indicate the potential for the rapid and non-destructive detection of moldy chestnuts by hyperspectral imaging, which would help to develop online detection system for healthy and blue mold infected chestnuts.

Genome-wide comparative analysis of putative Pth11-related G protein-coupled receptors in fungi belonging to Pezizomycotina.

BACKGROUND: G-protein coupled receptors (GPCRs) are the largest family of transmembrane receptors in fungi, where they play important roles in signal transduction. Among them, the Pth11-related GPCRs form a large and divergent protein family, and are only found in fungi in Pezizomycotina. However, the evolutionary process and potential functions of Pth11-related GPCRs remain largely unknown. RESULTS: Twenty genomes of fungi in Pezizomycotina covering different nutritional strategies were mined for putative Pth11-related GPCRs. Phytopathogens encode much more putative Pth11-related GPCRs than symbionts, saprophytes, or entomopathogens. Based on the phylogenetic tree, these GPCRs can be divided into nine clades, with each clade containing fungi in different taxonomic orders. Instead of fungi from the same order, those fungi with similar nutritional strategies were inclined to share orthologs of putative Pth11-related GPCRs. Most of the CFEM domain-containing Pth11-related GPCRs, which were only included in two clades, were detected in phytopathogens. Furthermore, many putative Pth11-related GPCR genes of phytopathogens were upregulated during invasive plant infection, but downregulated under biotic stress. The expressions of putative Pth11-related GPCR genes of saprophytes and entomopathogens could be affected by nutrient conditions, especially the carbon source. The gene expressions revealed that Pth11-related GPCRs could respond to biotic/abiotic stress and invasive plant infection with different expression patterns. CONCLUSION: Our results indicated that the Pth11-related GPCRs existed before the diversification of Pezizomycotina and have been gained and/or lost several times during the evolutionary process. Tandem duplications and trophic variations have been important factors in this evolution.

Interconversion between Methoxylated and Hydroxylated Polychlorinated Biphenyls in Rice Plants: An Important but Overlooked Metabolic Pathway.

To date, there is limited knowledge on the methoxylation of polychlorinated biphenyls (PCBs) and the relationship between hydroxylated polychlorinated biphenyls (OH-PCBs) and methoxylated polychlorinated biphenyls (MeO-PCBs) in organisms. In this study, rice (Oryza sativa L.) was chosen as the model organism to determine the metabolism of PCBs in plants. Limited para-substituted 4'-OH-CB-61 (major metabolite) and 4'-MeO-CB-61 (minor metabolite) were found after a 5-day exposure to CB-61, while ortho- and meta-substituted products were not detected. Interconversion between OH-PCBs and MeO-PCBs in organisms was observed for the first time. The demethylation ratio of 4'-MeO-CB-61 was 18 times higher than the methylation ratio of 4'-OH-CB-61, indicating that formation of OH-PCBs was easier than formation of MeO-PCBs. The transformation products were generated in the roots after 24 h of exposure. The results of in vivo and in vitro exposure studies show that the rice itself played a key role in the whole transformation processes, while endophytes were jointly responsible for hydroxylation of PCBs and demethylation of MeO-PCBs. Metabolic pathways of PCBs, OH-PCBs, and MeO-PCBs in intact rice plants are proposed. The findings are important in understanding the fate of PCBs and the source of OH-PCBs in the environment.

Exploring dynamic changes of fungal cellular components during nanoemulsion treatment by multivariate microRaman imaging.

Recently, essential oils (EO) have gained a lot of interest for use as antifungal agent in food and agricultural industry and extensive research is ongoing to understand their mode of action. However, the exact mechanism is not yet elucidated. Here, we integrated spectral unmixing and Raman microspectroscopy imaging to unveil the antifungal mechanism of green tea EO based nanoemulsion (NE) against Magnaporthe oryzae. The dramatic change in protein, lipid, adenine, and guanine bands indicate that NE has a significant impact on the protein, lipid and metabolic processes of purine. The results also demonstrated that the NE treatment caused damage to fungal hyphae by inducing a physical injury leading to cell wall damage and loss of integrity. Our study shows that MCR-ALS (Multivariate Curve Resolution-Alternating Least Squares) and N-FINDR (N-finder algorithm) Raman imaging could serve as a suitable complementary package to the traditional methods, for revealing the antifungal mechanism of action of EO/NE.

Bioactive metabolites from Phoma species, an endophytic fungus from the Chinese medicinal plant Arisaema erubescens.

Through bioassay-guided fractionation, the EtOAc extract of a culture broth of the endophytic fungus Phoma species ZJWCF006 in Arisaema erubescens afforded a new α-tetralone derivative, (3S)-3,6,7-trihydroxy-α-tetralone (1), together with cercosporamide (2), ß-sitosterol (3), and trichodermin (4). The structures of compounds were established on the basis of spectroscopic analyses. Compounds 1, 2, and 3 were obtained from Phoma species for the first time. Additionally, the compounds were subjected to bioactivity assays, including antimicrobial activity, against four plant pathogenic fungi (Fusarium oxysporium, Rhizoctonia solani, Colletotrichum gloeosporioides, and Magnaporthe oryzae) and two plant pathogenic bacteria (Xanthomonas campestris and Xanthomonas oryzae), as well as in vitro antitumor activities against HT-29, SMMC-772, MCF-7, HL-60, MGC80-3, and P388 cell lines. Compound 1 showed growth inhibition against F. oxysporium and R. solani with EC50 values of 413.22 and 48.5 µg/mL, respectively. Additionally, compound 1 showed no cytotoxicity, whereas compound 2 exhibited cytotoxic activity against the six tumor cell lines tested, with IC50 values of 9.3 ± 2.8, 27.87 ± 1.78, 48.79 ± 2.56, 37.57 ± 1.65, 27.83 ± 0.48, and 30.37 ± 0.28 µM, respectively. We conclude that endophytic Phoma are promising sources of natural bioactive and novel metabolites.

The COPII subunit MoSec24B is involved in development, pathogenicity and autophagy in the rice blast fungus.

The endoplasmic reticulum (ER) acts as the starting point of the secretory pathway, where approximately one-third of the proteins are correctly folded and modified, loaded into vesicles, and transported to the Golgi for further processing and modification. In this process, COPII vesicles are responsible for transporting cargo proteins from the ER to the Golgi. Here, we identified the inner shell subunit of COPII vesicles (MoSec24B) and explored the importance of MoSec24B in the rice blast fungus. The targeted disruption of MoSec24B led to decreased growth, reduced conidiation, restricted glycogen and lipids utilization, sensitivity to the cell wall and hypertonic stress, the failure of septin-mediated repolarization of appressorium, impaired appressorium turgor pressure, and decreased ability to infect, which resulted in reduced pathogenicity to the host plant. Furthermore, MoSec24B functions in the three mitogen-activated protein kinase (MAPK) signaling pathways by acting with MoMst50. Deletion of MoSec24B caused reduced lipidation of MoAtg8, accelerated degradation of exogenously introduced GFP-MoAtg8, and increased lipidation of MoAtg8 upon treatment with a late inhibitor of autophagy (BafA1), suggesting that MoSec24B regulates the fusion of late autophagosomes with vacuoles. Together, these results suggest that MoSec24B exerts a significant role in fungal development, the pathogenesis of filamentous fungi and autophagy.

A New Species in Pseudophialophora From Wild Rice and Beneficial Potential.

Wild rice (Oryza granulata) is a natural resource pool containing abundant unknown endophytic fungi species. There are few reports on the endophytic fungi in wild rice. Here, one isolate recovered from wild rice roots was identified as a new species Pseudophialophora oryzae sp. nov based on the molecular phylogeny and morphological characteristics. Fluorescent protein-expressing P. oryzae was used to monitor the fungal colonization pattern. Hyphae invaded the epidermis to the inner cortex but not into the root stele. The inoculation of P. oryzae promoted the rice growth, with the growth parameters of chlorophyll content, shoot height, root length, fresh shoot weight, fresh root weight and dry weight increasing by 24.10, 35.32, 19.35, 90.00, 33.3, and 79.17%, respectively. P. oryzae induced up-regulation of nitrate transporter OsPTR9 and potassium transporter OsHAK16 by 7.28 ± 0.84 and 2.57 ± 0.80 folds, promoting nitrogen and potassium elements absorption. In addition, P. oryzae also conferred a systemic resistance against rice blast, showing a 72.65 and 75.63% control rate in sterile plates and potting conditions. This systemic resistance was mediated by the strongly up-regulated expression of resistance-related genes NAC, OsSAUR2, OsWRKY71, EL5, and PR1α. Since P. oryzae can promote rice growth, biomass and induce systemic disease resistance, it can be further developed as a new biogenic agent for agricultural production, providing a new approach for biocontrol of rice blast.

Preparation and characterization of a novel green tea essential oil nanoemulsion and its antifungal mechanism of action against Magnaporthae oryzae.

Blast is one of the most devastating fungal diseases of rice caused by Magnaporthe oryzae. Plant essential oil (EO) can function as antifungal agents and are regarded as a safe and acceptable method for plant disease control. However, EOs are unstable and hydrophobic, which limits its use. In the present study, we aimed for the preparation and characterization of a nanoemulsion (NE) from green tea essential oil (GTO) by ultrasonication method and determined the antifungal activity of NE onM. oryzae. The particle size and zeta potential of the NE were 86.98 nm and -15.1 mV, respectively. The chemical composition and functional groups of GTO and NE were studied by using GC-MS analysis, portable Raman spectroscopy, and FTIR coupled with chemometric analysis. GC-MS analysis showed the major components in GTO and NE were n-Hexyl cinnamaldehyde and L-α-Terpineol. Both GTO and NE showed good antioxidant activity and total phenol content. Moreover, the NE showed good antifungal activity againstM. oryzae which was further confirmed by scanning electron microscopy (SEM) examination. Also, confocal Raman micro-spectroscopy (CRM) revealed the antifungal mechanism of GTO and NE on M. oryzae which proves the cell damage. To the best of our knowledge, this is the first study on the antifungal activity of GTO and NE against M. oryzae and also the use of CRM for the evaluation of the chemical changes in single fungal hyphae in a holistic approach. This study suggests that the prepared NE could be a potential candidate for use as a substitute for synthetic fungicides.

The Endophytic Fungus Piriformospora Indica-Assisted Alleviation of Cadmium in Tobacco.

Increasing evidence suggests that the endophytic fungus Piriformospora indica helps plants overcome various abiotic stresses, especially heavy metals. However, the mechanism of heavy metal tolerance has not yet been elucidated. Here, the role of P. indica in alleviating cadmium (Cd) toxicities in tobacco was investigated. It was found that P. indica improved Cd tolerance to tobacco, increasing Cd accumulation in roots but decreasing Cd accumulation in leaves. The colonization of P. indica altered the subcellular repartition of Cd, increasing the Cd proportion in cell walls while reducing the Cd proportion in membrane/organelle and soluble fractions. During Cd stress, P. indica significantly enhanced the peroxidase (POD) activity and glutathione (GSH) content in tobacco. The spatial distribution of GSH was further visualized by Raman spectroscopy, showing that GSH was distributed in the cortex of P. indica-inoculated roots while in the epidermis of the control roots. A LC-MS/MS-based label-free quantitative technique evaluated the differential proteomics of P. indica treatment vs. control plants under Cd stress. The expressions of peroxidase, glutathione synthase, and photosynthesis-related proteins were significantly upregulated. This study provided extensive evidence for how P. indica enhances Cd tolerance in tobacco at physiological, cytological, and protein levels.

Application of Hyperspectral Imaging for Maturity and Soluble Solids Content Determination of Strawberry With Deep Learning Approaches.

Maturity degree and quality evaluation are important for strawberry harvest, trade, and consumption. Deep learning has been an efficient artificial intelligence tool for food and agro-products. Hyperspectral imaging coupled with deep learning was applied to determine the maturity degree and soluble solids content (SSC) of strawberries with four maturity degrees. Hyperspectral image of each strawberry was obtained and preprocessed, and the spectra were extracted from the images. One-dimension residual neural network (1D ResNet) and three-dimension (3D) ResNet were built using 1D spectra and 3D hyperspectral image as inputs for maturity degree evaluation. Good performances were obtained for maturity identification, with the classification accuracy over 84% for both 1D ResNet and 3D ResNet. The corresponding saliency maps showed that the pigments related wavelengths and image regions contributed more to the maturity identification. For SSC determination, 1D ResNet model was also built, with the determination of coefficient (R 2) over 0.55 of the training, validation, and testing sets. The saliency maps of 1D ResNet for the SSC determination were also explored. The overall results showed that deep learning could be used to identify strawberry maturity degree and determine SSC. More efforts were needed to explore the use of 3D deep learning methods for the SSC determination. The close results of 1D ResNet and 3D ResNet for classification indicated that more samples might be used to improve the performances of 3D ResNet. The results in this study would help to develop 1D and 3D deep learning models for fruit quality inspection and other researches using hyperspectral imaging, providing efficient analysis approaches of fruit quality inspection using hyperspectral imaging.

Insights of roles played by septins in pathogenic fungi.

Septins, a conserved family of GTP-binding proteins, are widely recognized as an essential cytoskeletal component, playing important roles in a variety of biological processes, including division, polarity, and membrane remodeling, in different eukaryotes. Although the roles played by septins were identified in the model organism Saccharomyces cerevisiae, their importance in other fungi, especially pathogenic fungi, have recently been determined. In this review, we summarize the functions of septins in pathogenic fungi in the cell cycle, autophagy, endocytosis and invasion host-microbe interactions that were reported in the last two years in the field of septin cell biology. These new discoveries may be expanded to investigate the functions of septin proteins in fungal pathogenesis and may be of wide interest to the readers of Microbiology and Molecular Pathology.

Vacuolar Protein-Sorting Receptor MoVps13 Regulates Conidiation and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae.

Magnaporthe oryzae (synonym Pyricularia oryzae) is a filamentous fungal pathogen that causes major yield losses in cultivated rice worldwide. However, the mechanisms of infection of M. oryzae are not well characterized. The VPS13 proteins play vital roles in various biological processes in many eukaryotic organisms, including in the organization of actin cytoskeleton, vesicle trafficking, mitochondrial fusion, and phagocytosis. Nevertheless, the function of the Vps13 protein in plant pathogenic fungi has not been explored. Here, we analysed the biological functions of the Vps13 protein in the development and pathogenicity of M. oryzae. Deletion mutants of MoVps13 significantly reduced the conidiation and decreased the rate of fungal infection on hosts. Moreover, the loss of MoVps13 resulted in defective cell wall integrity (CWI) and plasma membrane (PM) homeostasis when treated with chemicals for inducing cell wall stress (200 mg/mL Congo Red or 0.005% SDS) and sphingolipid synthesis inhibitors (2 µM myriocin or 2 µM amphotericin B). This indicated that MoVps13 is also involved in cell wall synthesis and sphingolipid synthesis. Through immunoblotting, autophagic flux detection, co-localization, and chemical drug sensitivity assays, we confirmed the involvement of Movps13 in ER-phagy and the response to ER stress. Additionally, we generated the C-terminal structure of MoVps13 with high accuracy using the alphaflod2 database. Our experimental evidence indicates that MoVps13 is an important virulence factor that regulates the pathogenicity of M. oryzae by controlling CWI, lipid metabolism and the ER-phagy pathway. These results have expanded our knowledge about pathogenic fungi and will help exploration for novel therapeutic strategies against the rice blast fungus.

The chitin deacetylase PoCda7 is involved in the pathogenicity of Pyricularia oryzae.

The fungal cell wall plays an essential role in maintaining cellular integrity and facing complex and changing environmental conditions. Whether a fungus successfully invades a host depends on whether it evades the plant's innate immune system, which recognizes the conserved components of the fungal cell wall, such as chitin. Fungi developed infection-related changes in cell wall composition in co-evolution with nature to solve this problem. One of the changes is the deacetylation of chitin by chitin deacetylase (CDA) to produce a polysaccharide that influences the infection of pathogenic fungi. The present study revealed the functions of PoCda7, a chitin deacetylase in Pyricularia oryzae. Phenotype analysis revealed that the knockout mutant of ΔPocda7 had no significant effect on fungal morphogenic development, including conidiation, germination, appressorial formation and cell wall of conidium and hyphae but was sensitive to reactive oxygen species. Glycerols are necessary to generate sufficient turgor in appressoria for invading the host surface. As a result of the decreased appressorium turgor pressure and decreased appressorium-mediated invasion, the fungal virulence of ΔPocda7 was significantly reduced in host plants. PoCda7 inhibited the cell death of leaves in Nicotiana benthamiana. Additionally, the expression of PoCDA7 was repressed in the early stage of infection. Subcellular localization experiments showed that PoCda7 was localized in the cell wall, and its fluorescence transferred to the EIHM and BIC when the rice blast fungus infected the rice leaf sheath, which was referred to as a candidate apoplastic effector in P. oryzae.

Dark septate endophyte Falciphora oryzae-assisted alleviation of cadmium in rice.

Dark septate endophytes (DSEs) are the typical representatives of root endophytic fungi in heavy metal (HM)-contaminated environments. However, little is known about their roles in the HMs tolerance of hosts and the underlying mechanism. Here, we investigated the biological roles and molecular mechanisms of a DSE strain Falciphora oryzae in alleviating cadmium (Cd) toxicities in rice. It was found that F. oryzae possessed a capacity of accumulating Cd in its vacuoles and chlamydospores. During symbiosis, F. oryzae conferred improved Cd tolerance to rice, decreasing Cd accumulation in roots and translocation to shoots. F. oryzae alleviated Cd toxicity to rice by sequestering Cd in its vacuoles. Further application of F. oryzae as fertilizer in the field could reduce Cd content in rice grains. We identified a SNARE Syntaxin 1 gene through proteomics, which participated in Cd tolerance of F. oryzae by regulating chlamydospore formation and vacuole enlargement. This study provided novel insights into how the DSEs and their host plants combat Cd stress.

Investigation on Data Fusion of Multisource Spectral Data for Rice Leaf Diseases Identification Using Machine Learning Methods.

Rice diseases are major threats to rice yield and quality. Rapid and accurate detection of rice diseases is of great importance for precise disease prevention and treatment. Various spectroscopic techniques have been used to detect plant diseases. To rapidly and accurately detect three different rice diseases [leaf blight (Xanthomonas oryzae pv. Oryzae), rice blast (Pyricularia oryzae), and rice sheath blight (Rhizoctonia solani)], three spectroscopic techniques were applied, including visible/near-infrared hyperspectral imaging (HSI) spectra, mid-infrared spectroscopy (MIR), and laser-induced breakdown spectroscopy (LIBS). Three different levels of data fusion (raw data fusion, feature fusion, and decision fusion) fusing three different types of spectral features were adopted to categorize the diseases of rice. Principal component analysis (PCA) and autoencoder (AE) were used to extract features. Identification models based on each technique and different fusion levels were built using support vector machine (SVM), logistic regression (LR), and convolution neural network (CNN) models. Models based on HSI performed better than those based on MIR and LIBS, with the accuracy over 93% for the test set based on PCA features of HSI spectra. The performance of rice disease identification varied with different levels of fusion. The results showed that feature fusion and decision fusion could enhance identification performance. The overall results illustrated that the three techniques could be used to identify rice diseases, and data fusion strategies have great potential to be used for rice disease detection.

The casein kinase MoYck1 regulates development, autophagy, and virulence in the rice blast fungus.

Casein kinases are serine/threonine protein kinases that are evolutionarily conserved in yeast and humans and are involved in a range of important cellular processes. However, the biological functions of casein kinases in the fungus Magnaporthe oryzae, the causal agent of destructive rice blast disease, are not characterized. Here, two casein kinases, MoYCK1 and MoHRR25, were identified and targeted for replacement, but only MoYCK1 was further characterized due to the possible nonviability of the MoHRR25 deletion mutant. Disruption of MoYCK1 caused pleiotropic defects in growth, conidiation, conidial germination, and appressorium formation and penetration, therefore resulting in reduced virulence in rice seedlings and barley leaves. Notably, the MoYCK1 deletion triggered quick lipidation of MoAtg8 and degradation of the autophagic marker protein GFP-MoAtg8 under nitrogen starvation conditions, in contrast to the wild type, indicating that autophagy activity was negatively regulated by MoYck1. Furthermore, we found that HOPS (homotypic fusion and vacuolar protein sorting) subunit MoVps41, a putative substrate of MoYck1, was co-located with MoAtg8 and positively required for the degradation of MoAtg8-PE and GFP-MoAtg8. In addition, MoYCK1 is also involved in the response to ionic hyperosmotic and heavy metal cation stresses. Taken together, our results revealed crucial roles of the casein kinase MoYck1 in regulating development, autophagy and virulence in M. oryzae.