Currently, the control of phytopathogenic fungi faces challenges arising from chemical resistance and biosafety concerns. Triazole fungicides (TFs), as ergosterol biosynthesis inhibitors, possess broad‑spectrum and high‑efficacy characteristics. However, their single target site has led to increasing field resistance, highlighting the urgent need to discover molecular targets with novel mechanisms of action. Although P‑type ATPases drive fungal pathogenicity, their potential as pesticide targets remains unexplored. Here, we identified APT5 as a broad-spectrum target protein commonly recognized by multiple TFs and elucidated its interaction mechanism. Using Magnaporthe oryzae (M. oryzae) as a model, in vivo experiments revealed that knockout of MoAPT5 inhibited mycelial growth and reduced the sensitivity of M. oryzae to TFs, whereas complementation restored both growth and sensitivity. Further pathogenicity assays showed that the ΔMoapt5 mutant exhibited reduced conidial production, abnormal conidial morphology, delayed germ tube elongation, and decreased pathogenicity on rice leaves, as well as markedly reduced sensitivity to the protective and curative effects of TFs. In vitro binding assays demonstrated that TFs exhibited strong affinity for MoAPT5 and inhibited its ATPase activity by approximately 50%. Molecular docking and dynamics simulations further revealed that TFs bind primarily to a hydrophobic pocket formed by transmembrane helices TM2, TM4, TM5, and TM6 of MoAPT5, with the complex remaining conformationally stable. These findings provide new insights into the multi-target mechanism of TFs and offer a potential target for overcoming existing resistance issues and developing novel fungicides.

Currently, the control of phytopathogenic fungi faces challenges arising from chemical resistance and biosafety concerns. Triazole fungicides (TFs), as ergosterol biosynthesis inhibitors, possess broad-spectrum and high-efficacy characteristics. However, their single target site has led to increasing field resistance, highlighting the urgent need to discover molecular targets with novel mechanisms of action. Although P-type ATPases drive fungal pathogenicity, their potential as pesticide targets remains unexplored. Here, we identified MoAPT5 as a potential target protein commonly recognized by multiple TFs and elucidated its interaction mechanism. Using Magnaporthe oryzae (M. oryzae) as a model, in vivo experiments revealed that knockout of MoAPT5 inhibited mycelial growth and reduced the sensitivity of M. oryzae to TFs, whereas complementation restored both growth and sensitivity. Further pathogenicity assays showed that the ΔMoapt5 mutant exhibited reduced conidial production, abnormal conidial morphology, delayed germ tube elongation, and decreased pathogenicity on rice leaves, as well as markedly reduced sensitivity to the protective and curative effects of TFs. In vitro binding assays demonstrated that TFs exhibited strong affinity for MoAPT5 and inhibited its ATPase activity by approximately 50%. Molecular docking and dynamics simulations further revealed that TFs bind primarily to a hydrophobic pocket formed by transmembrane helices TM2, TM4, TM5, and TM6 of MoAPT5, with the complex remaining conformationally stable. These findings provide new insights into the multi-target mechanism of TFs and offer a potential target for overcoming existing resistance issues and developing novel fungicides.

Rice blast disease, caused by Magnaporthe oryzae, significantly threatens global rice yields. The Pmk1-MAPK signaling pathway is crucial for the infection process, but the precise regulatory mechanisms of Pmk1 remain unclear. Our research reveals that sumoylation of Pmk1 is vital for its infectious function. A sumoylation site at K347 and two small ubiquitin-related modifier (SUMO)-interacting motifs (SIMs) in Pmk1 are highly conserved across fungi. This sumoylation, orchestrated by Smt3 and Siz1, reduces the phosphorylation of Pmk1 by tuning its interaction with Mst7. The Pmk1 sumoylation is high in hyphae and less in conidia, and it intensifies during appressorium maturation. It may act as a molecular brake to prevent excessive Pmk1 phosphorylation during appressorium formation, without affecting phosphatase Pmp1 or the localization of Pmk1. Mutations at K347 lead to hyperphosphorylation of Pmk1 and Mst12, and overexpression of appressorium-related genes. The Δpmk1/Pmk1K347R mutant shows deficiencies in storage utilization, turgor accumulation, and septin ring formation. Our study highlights the critical role of sumoylation dynamically balancing Pmk1 function via phosphorylation crosstalk, crucial for infection of M. oryzae, thus proposing a conserved target for antifungal strategies across fungal pathogens.

Hexose transporters (HXT) play a crucial role in the pathogenicity of Magnaporthe oryzae, serving not only as key facilitators for acquiring and transporting sugar nutrients to support pathogen development, but also as sugar sensors which receive transduction signals. The objective of this study is to investigate the impact of MoHXT1-3 on rice pathogenicity and hexose affinity. MoHXT1-3 deletion mutants were generated using CRISPR/Cas9 technology, and their affinity for hexose was evaluated through yeast complementation assays and electrophysiological experiments in Xenopus oocytes. The results suggest that MoHXT1 does not contribute to melanin formation or hexose transportation processes. Conversely, MoHXT2, despite displaying lower affinity towards the hexoses tested in comparison to MoHXT3, is likely to have a more substantial impact on pathogenicity. The analysis of the transcription profiles demonstrated that the deletion of MoHXT2 caused a decrease in the expression of MoHXT3, whereas the knockout of MoHXT3 resulted in an upregulation of MoHXT2 transcription. It is noteworthy that the MoHXT2M145K variant displayed an incapacity to transport hexoses. This investigation into the functional differences in hexose transporters in Magnaporthe oryzae provides insights into potential advances in new strategies to target hexose transporters to combat rice blast by blocking carbon nutrient supply.

The RNA polymerase II degradation factor Degradation Factor 1 (Def1) is important for DNA damage repair and plays various roles in eukaryotes; however, the biological role in plant pathogenic fungi is still unknown. In this study, we investigated the role of Def1 during the development and infection of the rice blast fungus Magnaporthe oryzae. The deletion mutant of Def1 displayed slower mycelial growth, less conidial production, and abnormal conidial morphology. The appressoria of Δdef1 was impaired in the penetration into host cells, mainly due to blocking in the utilization of conidial storages, such as glycogen and lipid droplets. The invasive growth of the Δdef1 mutant was also retarded and accompanied with the accumulation of reactive oxygen species (ROS) inside the host cells. Furthermore, compared with the wild type, Δdef1 was more sensitive to multiple stresses, such as oxidative stress, high osmotic pressure, and alkaline/acidic pH. Interestingly, we found that Def1 was modified by O-GlcNAcylation at Ser232, which was required for the stability of Def1 and its function in pathogenicity. Taken together, the O-GlcNAc modified Def1 is required for hyphae growth, conidiation, pathogenicity, and stress response in M. oryzae. This study reveals a novel regulatory mechanism of O-GlcNAc-mediated Def1 in plant pathogenic fungi.

Cell wall polysaccharides play key roles in fungal development, virulence, and resistance to the plant immune system, and are synthesized from many nucleotide sugars in the endoplasmic reticulum (ER)-Golgi secretory system. Nucleotide sugar transporters (NSTs) are responsible for transporting cytosolic-derived nucleotide sugars to the ER lumen for processing, but their roles in plant-pathogenic fungi remain to be revealed. Here, we identified two important NSTs, NST1 and NST2, in the rice blast fungus Magnaporthe oryzae. Both NSTs were localized in the ER, which was consistent with a function in transporting nucleotide sugar for processing in the ER. Sugar transport property analysis suggested that NST1 is involved in transportation of mannose and glucose, while NST2 is only responsible for mannose transportation. Accordingly, deletion of NSTs resulted in a significant decrease in corresponding soluble saccharides abundance and defect in sugar utilization. Moreover, both NSTs played important roles in cell wall integrity, were involved in asexual development, and were required for full virulence. The NST mutants exhibited decreasing external glycoproteins and exposure of inner chitin, which resulted in activation of the host defence response. Altogether, our results revealed that two sugar transporters are required for fungal cell wall polysaccharides accumulation and full virulence of M. oryzae.

Lipid droplets are important storages in fungal conidia and can be used by plant pathogenic fungi for infection. However, the regulatory mechanism of lipid droplets formation and the utilization during fungal development and infection are largely unknown. Here, in Magnaporthe oryzae, we identified a lipid droplet-associated protein Nem1 that played a key role in lipid droplets biogenesis and utilization. Nem1 was highly expressed in conidia, but lowly expressed in appressoria, and its encoded protein was localized to lipid droplets. Deletion of NEM1 resulted in reduced numbers of lipid droplets and decreased content of diacylglycerol (DAG) or triacylglycerol (TAG). NEM1 was required for asexual development especially conidia production. The Δnem1 mutant was nearly loss of virulence to host plants due to defects in appressorial penetration and invasive growth. Remarkably, Nem1 was regulated by the TOR signaling pathway and involved in the autophagy process. The Ser303 residue of Nem1 could be phosphorylated by the cAMP-PKA signaling pathway and was important for biological function of Nem1. Together, our study revealed a regulatory mechanism of lipid biogenesis and metabolism during the conidium and appressorium formation of the rice blast fungus.

BACKGROUND:The rice (Oryza sativa) gene Xa7 has been hypothesized to be a typical executor resistance gene against Xanthomonas oryzae pv. oryzae (Xoo), and has conferred durable resistance in the field for decades. Its identity and the molecular mechanisms underlying this resistance remain elusive.RESULTS:Here, we filled in gaps of genome in Xa7 mapping locus via BAC library construction, revealing the presence of a 100-kb non-collinear sequence in the line IRBB7 compared with Nipponbare reference genomes. Complementary transformation with sequentially overlapping subclones of the BACs demonstrated that Xa7 is an orphan gene, encoding a small novel protein distinct from any other resistance proteins reported. A 27-bp effector binding element (EBE) in the Xa7 promoter is essential for AvrXa7-inducing expression model. XA7 is anchored in the endoplasmic reticulum membrane and triggers programmed cell death in rice and tobacco (Nicotiana benthamiana). The Xa7 gene is absent in most cultivars, landraces, and wild rice accessions, but highly homologs of XA7 were identified in Leersia perrieri, the nearest outgroup of the genus Oryza.CONCLUSIONS:Xa7 acts as a trap to perceive AvrXa7 via EBEAvrXa7 in its promoter, leading to the initiation of resistant reaction. Since EBEAvrXa7 is ubiquitous in promoter of rice susceptible gene SWEET14, the elevated expression of which is conducive to the proliferation of Xoo, that lends a great benefit for the Xoo strains retaining AvrXa7. As a result, varieties harboring Xa7 would show more durable resistance in the field. Xa7 alleles analysis suggests that the discovery of new resistance genes could be extended beyond wild rice, to include wild grasses such as Leersia species.