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Natural products, known for their environmental safety, are regarded as a significant basis for the modification and advancement of fungicides. Melatonin, as a low‐cost natural indole, exhibits diverse biological functions, including antifungal activity. However, its potential as an antifungal agent has not been fully explored. In this study, a series of melatonin derivatives targeting the mitogen‐activated protein kinase (Mps1) protein of fungal pathogens were synthesized based on properties of melatonin, among which the trifluoromethyl‐substituted derivative Mt‐23 exhibited antifungal activity against seven plant pathogenic fungi, and effectively reduced the severity of crop diseases, including rice blast, Fusarium head blight of wheat and gray mold of tomato. In particular, its EC50 (5.4 µM) against the rice blast fungus Magnaporthe oryzae is only one‐fourth that of isoprothiolane (22 µM), a commercial fungicide. Comparative analyzes revealed that Mt‐23 simultaneously targets the conserved protein kinase Mps1 and lipid protein Cap20. Surface plasmon resonance assays showed that Mt‐23 directly binds to Mps1 and Cap20. In this study, we provide a strategy for developing antifungal agents by modifying melatonin, and the resultant melatonin derivative Mt‐23 is a commercially valuable, eco‐friendly and broad‐spectrum antifungal agent to combat crop disease. [ABSTRACT FROM AUTHOR]

Melatonin is a low‐cost natural small indole molecule with versatile biological functions. However, melatonin's fungicidal potential has not been fully exploited, and the mechanism remains elusive. Here, we report that melatonin broadly inhibited 13 plant pathogens. In the rice blast fungal pathogen Magnaporthe oryzae, melatonin inhibited fungal growth, formation of infection‐specific structures named appressoria, and plant infection, reducing disease severity. Melatonin entered fungal cells efficiently and colocalized with the critical mitogen‐activated protein kinase named Mps1, suppressing phosphorylation of Mps1. Melatonin's affinity for Mps1 via two hydrogen bonds was demonstrated using surface plasmon resonance and chemical modifications. To improve melatonin's efficiency, we obtained 20 melatonin derivatives. Tert‐butyloxycarbonyl melatonin showed a 25‐fold increase in fungicidal activities, demonstrating the feasibility of chemical modifications in melatonin modification. Our study demonstrated the broad‐spectrum fungicidal effect of melatonin by suppressing Mps1 as one of the targets. Through further systematic modifications, developing an eco‐friendly melatonin derivative of commercial values for agricultural applications appears promising. [ABSTRACT FROM AUTHOR]

As with the majority of the hemibiotrophic fungal pathogens, the rice blast fungus Magnaporthe oryzae uses highly specialized infection structures called appressoria for plant penetration. Appressoria differentiated from germ tubes rely on enormous turgor pressure to directly penetrate the plant cell, in which process lipid metabolism plays a critical role. In this study, we characterized the MoPAH1 gene in M. oryzae, encoding a putative highly conserved phosphatidate phosphatase. The expression of MoPAH1 was up‐regulated during plant infection. The MoPah1 protein is expressed at all developmental and infection stages, and is localized to the cytoplasm. Disruption of MoPAH1 causes pleiotropic defects in vegetative growth, sporulation, and heat tolerance. The lipid profile is significantly altered in the Mopah1 mutant. Lipidomics assays showed that the level of phosphatidic acid (PA) was increased in the mutant, which had reduced levels of diacylglycerol and triacylglycerol. Using a PA biosensor, we showed that the increased level of PA in the Mopah1 mutant was primarily accumulated in the vacuole. The Mopah1 mutant was blocked in both conidiation and the formation of appressorium‐like structures at hyphal tips. It was nonpathogenic and failed to cause any blast lesions on rice and barley seedlings. RNA sequencing analysis revealed that MoPah1 regulates the expression of transcription factors critical for various developmental and infection‐related processes. The Mopah1 mutant was reduced in the expression and phosphorylation of Pmk1 MAP kinase and delayed in autophagy. Our study demonstrates that MoPah1 is necessary for lipid metabolism, fungal development, and pathogenicity in M. oryzae. [ABSTRACT FROM AUTHOR]

Ustilaginoidea virens, causing rice false smut (RFS) is an economically important ascomycetous fungal pathogen distributed in rice-growing regions worldwide. Here, we identified a novel transcription factor UvCGBP1 (Cutinase G-box binding protein) from this fungus, which is unique to ascomycetes. Deletion of UvCGBP1 affected development and virulence of U. virens. A total of 865 downstream target genes of UvCGBP1 was identified using ChIP-seq and the most significant KEGG enriched functional pathway was the MAPK signaling pathway. Approximately 36% of target genes contain the AGGGG (G-box) motif in their promoter. Among the targets, deletion of UvCGBP1 affected transcriptional and translational levels of UvPmk1 and UvSlt2, both of which were important in virulence. ChIP-qPCR, yeast one-hybrid and EMSA confirmed that UvCGBP1 can bind the promoter of UvPmk1 or UvSlt2. Overexpression of UvPmk1 in the ∆UvCGBP1-33 mutant restored partially its virulence and hyphae growth, indicating that UvCGBP1 could function via the MAPK pathway to regulate fungal virulence. Taken together, this study uncovered a novel regulatory mechanism of fungal virulence linking the MAPK pathway mediated by a G-box binding transcription factor, UvCGBP1. [ABSTRACT FROM AUTHOR]

The Pmk1 and Mps1 MAP kinases are essential for appressorium formation and plant infection in Magnaporthe oryzae. However, their exact roles during invasive growth are not clear because pmk1 and mps1 mutants are defective in penetration. To further characterize their functions after penetration, in this study we expressed the Pseudomonas syringae effector HopAI known to inactivate plant MAP kinases in M. oryzae. Constitutive expression of HopAI with the RP27 or TrpC promoter resulted in defects in hyphal growth, conidiation, appressorium penetration and pathogenicity, which is similar to the phenotype of the mps1 mutant. HopAI interacted strongly with Mps1 in vivo and expression of dominant active MKK2 partially suppressed the defects of PRP27-HopAI transformants, which were significantly reduced in Mps1 phosphorylation. When the infection-specific MIR1 ( Magnaporthe-infection-related gene-1) promoter was used to express HopAI, PMIR1-HopAI transformants were defective in the spreading of invasive hyphae and elicited strong defense responses in penetrated plant cells. Expression of HopAI in Fusarium graminearum also mainly affected the activation of Mgv1, an Mps1 orthologue. Taken together, our results showed that Mps1 is the major intracellular target of HopAI when it is overexpressed, and MAP kinase signalling is important for cell-to-cell movement of invasive hyphae in M. oryzae. [ABSTRACT FROM AUTHOR]

Appressorium formation plays a critical role in Magnaporthe oryzae. Mst50 is an adapter protein of the Mst11-Mst7-Pmk1 cascade that is essential for appressorium formation. To further characterize its functions, affinity purification was used to identify Mst50-interacting proteins (MIPs) in this study. Two of the MIPs are Mst11 and Mst7 that are known to interact with Mst50 for Pmk1 activation. Surprisingly, two other MIPs are Mck1 and Mkk2 that are the upstream kinases of the Mps1 pathway. Domain deletion analysis showed that the sterile alpha-motif of Mst50 but not the Ras-association domain was important for its interaction with Mck1 and responses to cell wall and oxidative stresses. The mst50 mutant was reduced in Mps1 activation under stress conditions. MIP11 encodes a RACK1 protein that also interacted with Mck1. Deletion of MIP11 resulted in defects in cell wall integrity, Mps1 phosphorylation and plant infection. Furthermore, Mst50 interacted with histidine kinase Hik1, and the mst50 mutant was reduced in Osm1 phosphorylation. These results indicated that Mst50 is involved in all three MAPK pathways in M. oryzae although its functions differ in each pathway. Several MIPs are conserved hypothetical proteins and may be involved in responses to various signals and crosstalk among signaling pathways. [ABSTRACT FROM AUTHOR]

Various surface signals are recognized by M agnaporthe oryzae to activate the Pmk1 MAP kinase that is essential for appressorium formation and invasive growth. One of upstream sensors of the Pmk1 pathway is the MoMsb2 signalling mucin. However, the activation of MoMsb2 and its relationship with other sensors is not clear. In this study, we showed that the cleavage and transmembrane domains are essential for MoMsb2 functions. Cleavage of MoMsb2 was further confirmed by western blot analysis, and five putative cleavage sites were functionally characterized. Expression of the extracellular region alone partially rescued the defects of M omsb2 in appressorium formation and virulence. The cytoplasmic region of MoMsb2, although dispensable for appressorium formation, was more important for penetration and invasive growth. Interestingly, the M omsb2 cbp1 double mutant deleted of both mucin genes was blocked in Pmk1 activation. It failed to form appressoria on artificial surfaces and was non-pathogenic. In addition, we showed that MoMsb2 interacts with Ras2 but not with MoCdc42 in co-immunoprecipitation assays. Overall, results from this study indicated that the extracellular and cytoplasmic regions of MoMsb2 have distinct functions in appressorium formation, penetration and invasive growth, and MoMsb2 has overlapping functions with Cbp1 in recognizing environmental signals for Pmk1 activation. [ABSTRACT FROM AUTHOR]

Diseases caused by various pathogenic fungi pose a serious threat to global food security. Despite their differences in life cycles, fungal pathogens use well-conserved genetic mechanisms to regulate different developmental and infection processes. This review focuses on the key signaling pathways and recent advances in Magnaporthe oryzae, which is a model for studying fungal–plant interactions. In addition to the core components, a number of upstream genes and downstream targets of the cAMP–PKA and mitogen-activated protein (MAP) pathways have been identified. Recent advances in studies with cytoskeleton organization, effector biology, and ROS signaling in M. oryzae and future directions also are discussed. [ABSTRACT FROM AUTHOR]