Mitophagy is a selective autophagic process that maintains cellular homeostasis by degrading damaged mitochondria and is a promising antifungal target. However, few inner mitochondrial membrane (IMM) regulators of mitophagy are known. Here we identify cytochrome c oxidase subunit 6 (MoCox6) as an IMM regulator in Magnaporthe oryzae that binds MoAtg5 and MoAtg14 following outer mitochondrial membrane rupture to mediate mitophagy. MoSirt5 regulates this process by desuccinylating MoCox6 at K144. Structural analysis revealed that residue D95 at the MoSirt5-MoCox6 interface mediates the dual role of MoCox6 in mitophagy and mitochondrial metabolic competence. Deletion of the COX6 gene significantly reduced vegetative growth and virulence in both M. oryzae and Alternaria alternata. Through high-throughput screening, we identified a small-molecule compound, Pan-RAS-IN-1, which targets MoCox6 to inhibit mitophagy, thereby suppressing M. oryzae virulence. Pan-RAS-IN-1 exhibits broad-spectrum antifungal activity, and its application to rice plants significantly suppressed rice blast incidence.

The endosomal sorting complex required for transport (ESCRT) is a highly conserved system for membrane remodeling in eukaryotes. The biological functions of ESCRT-Ⅲ accessory proteins, particularly in Magnaporthe oryzae, remain poorly understood, especially as they relate to the final step in cargo sorting. In this study, we identified the proteins Did2 and Vta1 in M. oryzae and elucidated their biological functions. Our findings indicate that MoDid2 and MoVta1 are predominantly localized to endosomes and play significant roles in vegetative growth, conidiation, and pathogenicity. Furthermore, MoDid2 and MoVta1 are involved in abiotic stress responses and influence endocytosis through their participation in the Oms1 MAPK and Pmk1 MAPK pathways, as well as modulating K63-linked ubiquitination levels. Additionally, MoDid2 affect autophagy but not MoVta1. In summary, our findings reveal a correlation between Did2/Vta1 and pathogenicity of M. oryzae, offering new insights for the control of rice blast disease.

Autophagy is crucial for appressorium development and host invasion by phytopathogenic fungi, including Magnaporthe oryzae. During appressorium maturation, many organelles, such as nuclei, in the conidia need to be degraded through autophagy to be recycled in appressorium. However, the interplay between autophagy and nuclear membrane systems remains poorly understood. In this study, we functionally characterized MoNup50, a nuclear pore-associated protein. Despite sharing limited sequence identity with human and yeast Nup proteins, MoNup50 contains conserved domains typical of nuclear pore complex proteins. Observation under fluorescence microscopy revealed that MoNup50 localizes at the nuclear membrane in M. oryzae. Deletion of MoNUP50 resulted in reduced hyphal growth, spore production, appressorium formation, and pathogenicity, while increasing sensitivity to osmotic stress and cell wall disruption. Notably, MoNup50 interacts with the key autophagy protein MoAtg7, which regulates MoAtg8-PE synthesis during autophagy. Moreover, MoNUP50 deletion led to elevated autophagy levels and increased phosphorylation of the MAPKs Osm1 and Mps1. These findings suggest that MoNup50 is involved in appressorium morphogenesis and pathogenicity by modulating autophagy and MAPK pathways, highlighting the critical role of nuclear pore proteins in M. oryzae pathogenicity and their potential cross-talk with autophagic and MAPK signaling.

Ubiquitination is a highly conserved post-translational modification in eukaryotes, and the deubiquitinase associated molecule with the SH3 domain of STAM (AMSH) plays a critical role in reversing this modification, thereby regulating various cellular processes. However, the specific mechanisms by which AMSH influences pathogenicity through autophagy in pathogenic fungi remain poorly understood. In this study, we investigate the multifaceted role of MoAMSH in the development and pathogenicity of Magnaporthe oryzae. We identify MoAMSH and demonstrate that the E346 residue is crucial for its interactions with MoHse1, MoVps24, and MoAtg13. Functional analyses indicate that MoAMSH is essential for growth, conidiation, conidial germination, appressorium formation, and overall pathogenicity. Additionally, MoAMSH plays a significant role in the response to abiotic stress and exhibits K63-linked deubiquitination activity. Our findings further reveal that MoAMSH regulates development and pathogenicity by participating in MAPK pathways. Notably, MoAMSH acts as a negative regulator of autophagy by inhibiting the interaction between MoAtg6 and MoVps34 through the deubiquitination of K63-linked ubiquitinated MoAtg6. In summary, our results uncover a novel regulatory pathway related to autophagy in pathogenic fungi and suggest that AMSH may serve as a potential target for antifungal therapies.

Rice blast, caused by Magnaporthe oryzae, is a major threat to global rice production. This study explores the antifungal potential of the immunosuppressant FK506 and identifies its target protein, MoFpr1 (FK506-binding protein 1B). FK506 inhibited mycelial growth, appressorium formation, and pathogenicity of M. oryzae in an MoFpr1-dependent manner. Mechanistic analyses revealed that FK506 impairs autophagy and ubiquitination, supported by transcriptomic and metabolomic data. Structural studies using X-ray crystallography and site-directed mutagenesis confirmed the direct interaction between FK506 and MoFpr1, highlighting the importance of residues Gly95 and Ile97. Furthermore, FK506 demonstrated broad-spectrum antifungal activity against various plant pathogens and effectively controlled rice blast in laboratory, net-chamber, and field trials with minimal phytotoxicity. These findings position FK506 as a promising antifungal agent and offer insights into its molecular mechanism, suggesting its potential for sustainable plant disease management.

Macroautophagy/autophagy is an evolutionarily conserved cellular degradation process that is crucial for cellular homeostasis in Magnaporthe oryzae. However, the precise regulatory mechanisms governing autophagy in this organism remain unclear. In this study, we found a multiregional localization of MoSec13 to the vesicle membrane, endoplasmic reticulum, nucleus, and perinucleus. MoSec13 negatively regulated autophagy through specific amino acid residues in its own WD40 structural domain by interacting with MoAtg7 and MoAtg8. We also found that the histone acetyltransferase MoGcn5b mediated the acetylation of MoAtg8 and regulated autophagy activity. Subsequently, we further determined that MoSec13 regulated the acetylation status of MoAtg8 by controlling the interaction between MoGcn5b and MoAtg8 in the nucleus. In addition, MoSec13 maintained lipid homeostasis by controlling TORC2 activity. This multilayered integration establishes MoSec13 as an essential node within the autophagic regulatory network. Our findings fill a critical gap in understanding the role of Sec13 in autophagy of filamentous fungi and provide a molecular foundation for developing new therapeutic strategies against rice blast fungus.ABBREVIATIONS BFA: brefeldin A; BiFC: bimolecular fluorescence complementation; CM: complete medium; CMAC: 7-amino-4-chloromethylcoumarin; Co-IP: co-immunoprecipitation; COPII: coat complex II; GFP: green fluorescent protein; HPH: hygromycin phosphotransferase; MM-N: nitrogen-starvation conditions; NPC: nuclear pore complex; PAS: phagophore assembly site; PE: phosphatidylethanolamine; UPR: unfolded protein response.

Carbon catabolite repression (CCR) and de-repression (CCDR) are critical for fungal development and pathogenicity, yet the underlying regulatory mechanisms remain poorly understood in pathogenic fungi. Here, we identify a serine/threonine protein phosphatase catalytic subunit, Pp4c, as essential for growth, conidiation, virulence, and the utilization of carbohydrates and lipids in Magnaporthe oryzae. We demonstrate that the protein phosphatase 4 complex (Pp4c and Smek1 subunits), the AMP-activated protein kinase (AMPK) Snf1, and the transcriptional regulators CreA (repressor) and Crf1 (activator) collaboratively regulate the utilization of non-preferred carbon sources. Protein interaction and phosphorylation analyses reveal that under glucose-rich conditions, Snf1 and Smek1 directly regulate the phosphorylation status of CreA and Crf1. In contrast, under l-arabinose-rich conditions, Snf1 indirectly modulates the dephosphorylation of these transcription factors via Pp4c and Smek1. Phosphorylation-mediated activation or inactivation of CreA and Crf1 drives CCR and CCDR, thereby governing the metabolism of carbon sources derived from plant cell walls and contributing to fungal pathogenicity. These findings provide deep insights into the regulation of CCR and CCDR, emphasizing their significance in carbon metabolism and pathogenicity in phytopathogenic fungi. The protein phosphatase 4 complex, the Snf1 kinase, and the transcriptional regulators CreA and Crf1 collaboratively control carbon catabolite repression and de-repression in the rice blast fungus Magnaporthe oryzae.

Rice blast, caused by the fungal pathogen Magnaporthe oryzae (M. oryzae), leads to substantial global reductions in crop yield. Clotrimazole (CLZ), a synthetic imidazole derivative with broad-spectrum antifungal properties, is widely employed in clinical and veterinary applications for fungal infections treatment. However, limited information exists regarding its antifungal efficacy and mechanisms against phytopathogenic fungi. This study systematically elucidates the antifungal effects and underlying mechanisms of CLZ against M. oryzae. CLZ exhibited potent inhibitory effects on hyphal growth, conidiation, appressorium development, and pathogenicity. CLZ treatment disrupted cellular membrane integrity and significant depletion of ergosterol content. Molecular analysis revealed that the pharmacophore ring of CLZ directly interacts with the key sterol 14α-demethylase enzymes MoCyp51A and MoCyp51B in the ergosterol biosynthetic pathway. Deletion of MoCYP51A or MoCYP51B exhibited enhanced susceptibility to CLZ, confirming that CLZ effectively suppressed ergosterol biosynthesis to impair membrane integrity in M. oryzae. Additionally, CLZ treatment triggered multiple cytological alterations, including enhanced cell wall deposition, mitochondrial ultrastructural defects, dysregulated mitophagy, and excessive reactive oxygen species (ROS) accumulation. These data collectively reveal that CLZ inhibits fungal virulence of M. oryzae by simultaneously targeting structural and functional damage to the cell wall, cell membrane, and organelles. Safety assessments further confirmed that CLZ exhibits minimal toxicity to both insects and crops. This evidence collectively proves the potential application of CLZ as a promising fungicide for the management of rice blast.

In Magnaporthe oryzae, the Pmk1 MAP kinase signaling pathway regulates appressorium formation, plant penetration, effector secretion, and invasive growth. While the Mst11-Mst7-Pmk1 cascade was characterized two decades ago, knowledge of its signaling in the intracellular network remains limited. In this study, we demonstrate that the endosomal surface scaffolds Pmk1 MAPK signaling and Msb2 activates Ras2 on endosomes in M. oryzae. Protein colocalization demonstrated that Msb2, Ras2, Cap1, Mst50, Mst11, Mst7, and Pmk1 attach to late endosomal membranes. Damage to the endosome-vacuole transport system influences Pmk1 phosphorylation. When Msb2 senses a plant signal, it internalizes and activates Ras2 on endosome membrane surfaces, transmitting the signal to Pmk1 via Mst11 and Mst7. Signal-sensing and delivery proteins are ubiquitinated and sorted for degradation in late endosomes and vacuoles, terminating signaling. Plant penetration and lowered intracellular turgor are required for the transition from late endosomes to vacuoles in appressoria. Our findings uncover an effective mechanism that scaffolds and controls Pmk1 MAPK signaling through endosomal-vacuolar transport, offering new knowledge for the cytological and molecular mechanisms by which the Pmk1 MAPK pathway modulates development and pathogenicity in M. oryzae.

Csn5 is subunit 5 of the COP9 signalosome (CSN), but the mechanism by which it strictly controls the pathogenicity of pathogenic fungi through autophagy remains unclear. Here, we found that Csn5 deficiency attenuated pathogenicity and enhanced autophagy in Magnaporthe oryzae. MoCSN5 knockout led to overubiquitination and overdegradation of MoTor (the core protein of the TORC1 complex [target of rapamycin]) thereby promoted autophagy. In addition, we identified MoCsn5 as a new interactor of MoAtg6. Atg6 was found to be ubiquitinated through linkage with lysine 48 (K48) in cells, which is necessary for infection-associated autophagy in pathogenic fungi. K48-ubiquitination of Atg6 enhanced its degradation and thereby inhibited autophagic activity. Our experimental results indicated that MoCsn5 promoted K48-ubiquitination of MoAtg6, which reduced the MoAtg6 protein content and thus inhibited autophagy. Aberrant ubiquitination and autophagy in ΔMocsn5 led to pleiotropic defects in the growth, development, stress resistance, and pathogenicity of M. oryzae. In summary, our study revealed a novel mechanism by which Csn5 regulates autophagy and pathogenicity in rice blast fungus through ubiquitination.

Rice blast is a serious threat to the safe production of grain crops such as rice and wheat. Sporulation, appressorium formation, and invasive growth of Magnaporthe oryzae are the key stages of the development and spread of rice blast epidemics. M. oryzae is a hemibiotrophic fungus that undergoes changes in available carbon sources during the infection cycle. Lipid is a major storage for M. oryzae spores and a major carbon source used in glycerol synthesis and turgor pressure generation in appressoria. The formation of a dense cell wall melanin layer is necessary for an appressorium to produce turgor and to be pathogenic. The plant cell wall is an important carbon source during the infection stage of M. oryzae . Transcription factors regulate gene expression in fungi and are key intermediates between the reception of external environmental signals and the control of development and pathogenicity in M. oryzae . The disease cycle of M. oryzae is controlled by some key transcription factors, such as sporulation by Cos1 and Hox2, appressorium formation by Sfl1, Hox7, and Vrf1, invasive growth by Mst12 and Mig1, and resistance to host basal immunity by Ap1 and Atf1. This review focuses on describing the key transcription factors of M. oryzae that regulate sporulation, appressorium formation, invasive growth, lipid metabolism, carbohydrate metabolism, melanin synthesis, oxidative response, and host basal immunity, as well as the working mechanism of the transcription factors.

Dihydroorotase (DHOase) is the third enzyme in the six enzymatic reaction steps of the endogenous pyrimidine nucleotide de novo biosynthesis pathway, which is a metabolic pathway conserved in both bacteria and eukaryotes. However, research on the biological function of DHOase in plant pathogenic fungi is very limited. In this study, we identified and named MoPyr4, a homologous protein of Saccharomyces cerevisiae DHOase Ura4, in the rice blast fungus Magnaporthe oryzae and investigated its ability to regulate fungal growth, pathogenicity, and autophagy. Deletion of MoPYR4 led to defects in growth, conidiation, appressorium formation, the transfer and degradation of glycogen and lipid droplets, appressorium turgor accumulation, and invasive hypha expansion in M. oryzae, which eventually resulted in weakened fungal pathogenicity. Long-term replenishment of exogenous uridine-5’-phosphate (UMP) can effectively restore the phenotype and virulence of the ΔMopyr4 mutant. Further study revealed that MoPyr4 also participated in the regulation of the Pmk1-MAPK signaling pathway, co-localized with peroxisomes for the oxidative stress response, and was involved in the regulation of the Osm1-MAPK signaling pathway in response to hyperosmotic stress. In addition, MoPyr4 interacted with MoAtg5, the core protein involved in autophagy, and positively regulated autophagic degradation. Taken together, our results suggested that MoPyr4 for UMP biosynthesis was crucial for the development and pathogenicity of M. oryzae. We also revealed that MoPyr4 played an essential role in the external stress response and pathogenic mechanism through participation in the Pmk1-MAPK signaling pathway, peroxisome-related oxidative stress response mechanism, the Osm1-MAPK signaling pathway and the autophagy pathway.

Magnaporthe oryzae is a pathogenic fungus that seriously harms rice production. Phosphatases and carbon metabolism play crucial roles in the growth and development of eukaryotes. However, it remains unclear how serine/threonine phosphatases regulate the catabolism of triglycerides, a major form of stored lipids. In this study, we identified a serine/threonine protein phosphatase regulatory subunit, Smek1, which is required for the growth, conidiation, and virulence of M. oryzae. Deletion of SMEK1 led to defects in the utilization of lipids, arabinose, glycerol, and ethanol. In glucose medium, the expression of genes involved in lipolysis, long-chain fatty acid degradation, beta-oxidation, and the glyoxylate cycle increased in the Delta smek1 mutant, which is consistent with Delta creA in which a carbon catabolite repressor CREA was deleted. In lipid medium, the expression of genes involved in long-chain fatty acid degradation, beta-oxidation, the glyoxylate cycle, and utilization of arabinose, ethanol, or glycerol decreased in the Delta smek1 mutant, which is consistent with Delta crf1 in which a transcription activator CRF1 required for carbon metabolism was deleted. Lipase activity, however, increased in the Delta smek1 mutant in both glucose and lipid media. Moreover, Smek1 directly interacted with CreA and Crf1, and dephosphorylated CreA and Crf1 in vivo. The phosphatase Smek1 is therefore a dual-function regulator of the lipid and carbohydrate metabolism, and controls fungal development and virulence by coordinating the functions of CreA and Crf1 in carbon catabolite repression (CCR) and derepression (CCDR).

Rice blast is a devastating disease worldwide, threatening rice production and food security. The blast fungus Magnaporthe oryzae invades the host via the appressorium, a specialized pressure-generating structure that generates enormous turgor pressure to penetrate the host cuticle. However, owing to ongoing evolution of fungicide resistance, it is vitally important to identify new targets and fungicides. Here, we show that Trs85, a subunit of the transport protein particle III complex, is essential for appressorium-mediated infection in M. oryzae. We explain how Trs85 regulates autophagy through Ypt1 (a small guanosine triphosphatase protein) in M. oryzae. We then identify a key conserved amphipathic α helix within Trs85 that is associated with pathogenicity of M. oryzae. Through computer-aided screening, we identify a lead compound, SP-141, that affects autophagy and the Trs85-Ypt1 interaction. SP-141 demonstrates a substantial capacity to effectively inhibit infection caused by the rice blast fungus while also exhibiting wide-ranging potential as an antifungal agent with broad-spectrum activity. Taken together, our data show that Trs85 is a potential new target and that SP-141 has potential for the control of rice blast. Our findings thus provide a novel strategy that may help in the fight against rice blast.

Calcineurin, a key regulator of the calcium signaling pathway, is involved in calcium signal transduction and calcium ion homeostasis. Magnaporthe oryzae is a devastating filamentous phytopathogenic fungus in rice, yet little is known about the function of the calcium signaling system. Here, we identified a novel calcineurin regulatory-subunit-binding protein, MoCbp7, which is highly conserved in filamentous fungi and was found to localize in the cytoplasm. Phenotypic analysis of the MoCBP7 gene deletion mutant (ΔMocbp7) showed that MoCbp7 influenced the growth, conidiation, appressorium formation, invasive growth, and virulence of M. oryzae. Some calcium-signaling-related genes, such as YVC1, VCX1, and RCN1, are expressed in a calcineurin/MoCbp7-dependent manner. Furthermore, MoCbp7 synergizes with calcineurin to regulate endoplasmic reticulum homeostasis. Our research indicated that M. oryzae may have evolved a new calcium signaling regulatory network to adapt to its environment compared to the fungal model organism Saccharomyces cerevisiae.

In fungi, the methylcitrate cycle converts cytotoxic propionyl-coenzyme A (CoA) to pyruvate, which enters gluconeogenesis. The glyoxylate cycle converts acetyl-CoA to succinate, which enters gluconeogenesis. The tricarboxylic acid cycle is a central carbon metabolic pathway that connects the methylcitrate cycle, the glyoxylate cycle, and other metabolisms for lipids, carbohydrates, and amino acids. Fungal citrate synthase and 2-methylcitrate synthase as well as isocitrate lyase and 2-methylisocitrate lyase, each evolved from a common ancestral protein. Impairment of the methylcitrate cycle leads to the accumulation of toxic intermediates such as propionyl-CoA, 2-methylcitrate, and 2-methylisocitrate in fungal cells, which in turn inhibits the activity of many enzymes such as dehydrogenases and remodels cellular carbon metabolic processes. The methylcitrate cycle and the glyoxylate cycle synergistically regulate carbon source utilization as well as fungal growth, development, and pathogenic process in pathogenic fungi.

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.

The development and pathogenicity of the fungus Magnaporthe oryzae , the causal agent of destructive rice blast disease, require it to perceive external environmental signals. Opy2, an overproduction-induced pheromone-resistant protein 2, is a crucial protein for sensing external signals in Saccharomyces cerevisiae . However, the biological functions of the homologue of Opy2 in M. oryzae are unclear. In this study, we identified that MoOPY2 is involved in fungal development, pathogenicity, and autophagy in M. oryzae . Deletion of MoOPY2 resulted in pleiotropic defects in hyphal growth, conidiation, germ tube extension, appressorium formation, appressorium turgor generation, and invasive growth, therefore leading to attenuated pathogenicity. Furthermore, MoOpy2 participates in the Osm1 MAPK pathway and the Mps1 MAPK pathway by interacting with the adaptor protein Mst50. The interaction sites of Mst50 and MoOpy2 colocalized with the autophagic marker protein MoAtg8 in the preautophagosomal structure sites (PAS). Notably, the Δ Moopy2 mutant caused cumulative MoAtg8 lipidation and rapid GFP-MoAtg8 degradation in response to nitrogen starvation, showing that MoOpy2 is involved in the negative regulation of autophagy activity. Taken together, our study revealed that MoOpy2 of M. oryzae plays an essential role in the orchestration of fungal development, appressorium penetration, autophagy and pathogenesis.

Our identification and analysis of evolutionary and biological relationships provide us with an unprecedented high-resolution view of the flexible and conserved roles of the AGC family in the topmost fungal pathogens that infect rice, wheat, barley, and millet. Guided by these insights, an AGC member, MoFpk1, was found to be indispensable for M. oryzae development.

Magnaporthe oryzae is the causal agent of rice blast outbreaks. L-ascorbic acid (ASC) is a famous antioxidant found in nature. However, while ASC is rare or absent in fungi, a five-carbon analog, D-erythroascorbic acid (EASC), seems to appear to be a substitute for ASC. Although the antioxidant function of ASC has been widely described, the specific properties and physiological functions of EASC remain poorly understood. In this study, we identified a D-arabinono-1,4-lactone oxidase (ALO) domain-containing protein, MoAlo1, and found that MoAlo1 was localized to mitochondria. Disruption of MoALO1 (ΔMoalo1) exhibited defects in vegetative growth as well as conidiogenesis. The ΔMoalo1 mutant was found to be more sensitive to exogenous H2O2. Additionally, the pathogenicity of conidia in the ΔMoalo1 null mutant was reduced deeply in rice, and defective penetration of appressorium-like structures (ALS) formed by the hyphal tips was also observed in the ΔMoalo1 null mutant. When exogenous EASC was added to the conidial suspension, the defective pathogenicity of the ΔMoalo1 mutant was restored. Collectively, MoAlo1 is essential for growth, conidiogenesis, and pathogenicity in M. oryzae.