Antifungal plant flavonoids identified in silico with potential to control rice blast disease caused by Magnaporthe oryzae.

Rice blast disease, caused by the fungus Magnaporthe oryzae, poses a severe threat to rice production, particularly in Asia where rice is a staple food. Concerns over fungicide resistance and environmental impact have sparked interest in exploring natural fungicides as potential alternatives. This study aimed to identify highly potent natural fungicides against M. oryzae to combat rice blast disease, using advanced molecular dynamics techniques. Four key proteins (CATALASE PEROXIDASES 2, HYBRID PKS-NRPS SYNTHETASE TAS1, MANGANESE LIPOXYGENASE, and PRE-MRNA-SPLICING FACTOR CEF1) involved in M. oryzae's infection process were identified. A list of 30 plant metabolites with documented antifungal properties was compiled for evaluation as potential fungicides. Molecular docking studies revealed that 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin exhibited superior binding affinities compared to reference fungicides (Azoxystrobin and Tricyclazole). High throughput molecular dynamics simulations were performed, analyzing parameters like RMSD, RMSF, Rg, SASA, hydrogen bonds, contact analysis, Gibbs free energy, and cluster analysis. The results revealed stable interactions between the selected metabolites and the target proteins, involving important hydrogen bonds and contacts. The SwissADME server analysis indicated that the metabolites possess fungicide properties, making them effective and safe fungicides with low toxicity to the environment and living beings. Additionally, bioactivity assays confirmed their biological activity as nuclear receptor ligands and enzyme inhibitors. Overall, this study offers valuable insights into potential natural fungicides for combating rice blast disease, with 2-Coumaroylquinic acid, Myricetin, Rosmarinic Acid, and Quercetin standing out as promising and environmentally friendly alternatives to conventional fungicides. These findings have significant implications for developing crop protection strategies and enhancing global food security, particularly in rice-dependent regions.

MoAti1 mediates mitophagy by facilitating recruitment of MoAtg8 to promote invasive growth in Magnaporthe oryzae.

Mitophagy is a selective autophagy for the degradation of damaged or excessive mitochondria to maintain intracellular homeostasis. In Magnaporthe oryzae, a filamentous ascomycetous fungus that causes rice blast, the most devastating disease of rice, mitophagy occurs in the invasive hyphae to promote infection. To date, only a few proteins are known to participate in mitophagy and the mechanisms of mitophagy are largely unknown in pathogenic fungi. Here, by a yeast two-hybrid screen with the core autophagy-related protein MoAtg8 as a bait, we obtained a MoAtg8 interactor MoAti1 (MoAtg8-interacting protein 1). Fluorescent observations and protease digestion analyses revealed that MoAti1 is primarily localized to the peripheral mitochondrial outer membrane and is responsible for recruiting MoAtg8 to mitochondria under mitophagy induction conditions. MoAti1 is specifically required for mitophagy, but not for macroautophagy and pexophagy. Infection assays suggested that MoAti1 is required for mitophagy in invasive hyphae during pathogenesis. Notably, no homologues of MoAti1 were found in rice and human protein databases, indicating that MoAti1 may be used as a potential target to control rice blast. By the host-induced gene silencing (HIGS) strategy, transgenic rice plants targeted to silencing MoATI1 showed enhanced resistance against M. oryzae with unchanged agronomic traits. Our results suggest that MoATI1 is required for mitophagy and pathogenicity in M. oryzae and can be used as a target for reducing rice blast.

MoNOT3 Subunit Has Important Roles in Infection-Related Development and Stress Responses in Magnaporthe oryzae.

The multifunctional carbon catabolite repression negative on TATA-box-less complex (CCR4-NOT) is a multi-subunit complex present in all eukaryotes, including fungi. This complex plays an essential role in gene expression; however, a functional study of the CCR4-NOT complex in the rice blast fungus Magnaporthe oryzae has not been conducted. Seven genes encoding the putative CCR4-NOT complex were identified in the M. oryzae genome. Among these, a homologous gene, MoNOT3, was overexpressed during appressorium development in a previous study. Deletion of MoNOT3 in M. oryzae resulted in a significant reduction in hyphal growth, conidiation, abnormal septation in conidia, conidial germination, and appressorium formation compared to the wild-type. Transcriptional analyses suggest that the MoNOT3 gene affects conidiation and conidial morphology by regulating COS1 and COM1 in M. oryzae. Furthermore, Δmonot3 exhibited a lack of pathogenicity, both with and without wounding, which is attributable to deficiencies in the development of invasive growth in planta. This result was also observed in onion epidermal cells, which are non-host plants. In addition, the MoNOT3 gene was involved in cell wall stress responses and heat shock. Taken together, these observations suggest that the MoNOT3 gene is required for fungal infection-related cell development and stress responses in M. oryzae.

The MoLfa1 Protein Regulates Fungal Development and Septin Ring Formation in Magnaporthe oryzae.

Septins play a key regulatory role in cell division, cytokinesis, and cell polar growth of the rice blast fungus (Magnaporthe oryzae). We found that the organization of the septin ring, which is essential for appressorium-mediated infection in M. oryzae, requires long-chain fatty acids (LCFAs), which act as mediators of septin organization at membrane interfaces. However, it is unclear how septin ring formation and LCFAs regulate the pathogenicity of the rice blast fungus. In this study, a novel protein was named MoLfa1 because of its role in LCFAs utilization. MoLfa1 affects the utilization of LCFAs, lipid metabolism, and the formation of the septin ring by binding with phosphatidylinositol phosphates (PIPs), thereby participating in the construction of penetration pegs of M. oryzae. In addition, MoLfa1 is localized in the endoplasmic reticulum (ER) and interacts with the ER-related protein MoMip11 to affect the phosphorylation level of Mps1. (Mps1 is the core protein in the MPS1-MAPK pathway.) In conclusion, MoLfa1 affects conidia morphology, appressorium formation, lipid metabolism, LCFAs utilization, septin ring formation, and the Mps1-MAPK pathway of M. oryzae, influencing pathogenicity.

Sirt5-mediated lysine desuccinylation regulates oxidative stress adaptation in Magnaporthe oryzae during host intracellular infection.

Plant pathogenic fungi elaborate numerous detoxification strategies to suppress host reactive oxygen species (ROS), but their coordination is not well-understood. Here, we show that Sirt5-mediated protein desuccinylation in Magnaporthe oryzae is central to host ROS detoxification. SIRT5 encodes a desuccinylase important for virulence via adaptation to host oxidative stress. Quantitative proteomics analysis identified a large number of succinylated proteins targeted by Sirt5, most of which were mitochondrial proteins involved in oxidative phosphorylation, TCA cycle, and fatty acid oxidation. Deletion of SIRT5 resulted in hypersuccinylation of detoxification-related enzymes, and significant reduction in NADPH : NADP+ and GSH : GSSG ratios, disrupting redox balance and impeding invasive growth. Sirt5 desuccinylated thioredoxin Trx2 and glutathione peroxidase Hyr1 to activate their enzyme activity, likely by affecting proper folding. Altogether, this work demonstrates the importance of Sirt5-mediated desuccinylation in controlling fungal process required for detoxifying host ROS during M. oryzae infection.

A fungal core effector exploits the OsPUX8B.2-OsCDC48-6 module to suppress plant immunity.

Proteins containing a ubiquitin regulatory X (UBX) domain are cofactors of Cell Division Cycle 48 (CDC48) and function in protein quality control. However, whether and how UBX-containing proteins participate in host-microbe interactions remain unclear. Here we show that MoNLE1, an effector from the fungal pathogen Magnaporthe oryzae, is a core virulence factor that suppresses rice immunity by specifically interfering with OsPUX8B.2. The UBX domain of OsPUX8B.2 is required for its binding to OsATG8 and OsCDC48-6 and controls its 26 S proteasome-dependent stability. OsPUX8B.2 and OsCDC48-6 positively regulate plant immunity against blast fungus, while the high-temperature tolerance heat-shock protein OsBHT, a putative cytoplasmic substrate of OsPUX8B.2-OsCDC48-6, negatively regulates defense against blast infection. MoNLE1 promotes the nuclear migration and degradation of OsPUX8B.2 and disturbs its association with OsBHT. Given the high conservation of MoNLE1 among fungal isolates, plants with broad and durable blast resistance might be generated by engineering intracellular proteins resistant to MoNLE1.

MoMkk1 and MoAtg1 dichotomously regulating autophagy and pathogenicity through MoAtg9 phosphorylation in Magnaporthe oryzae.

Autophagy is a central biodegradation pathway critical in eliminating intracellular cargo to maintain cellular homeostasis and improve stress resistance. At the same time, the key component of the mitogen-activated protein kinase cascade regulating cell wall integrity signaling MoMkk1 has an essential role in the autophagy of the rice blast fungus Magnaporthe oryzae. Still, the mechanism of how MoMkk1 regulates autophagy is unclear. Interestingly, we found that MoMkk1 regulates the autophagy protein MoAtg9 through phosphorylation. MoAtg9 is a transmembrane protein subjected to phosphorylation by autophagy-related protein kinase MoAtg1. Here, we provide evidence demonstrating that MoMkk1-dependent MoAtg9 phosphorylation is required for phospholipid translocation during isolation membrane stages of autophagosome formation, an autophagic process essential for the development and pathogenicity of the fungus. In contrast, MoAtg1-dependent phosphorylation of MoAtg9 negatively regulates this process, also impacting growth and pathogenicity. Our studies are the first to demonstrate that MoAtg9 is subject to MoMkk1 regulation through protein phosphorylation and that MoMkk1 and MoAtg1 dichotomously regulate autophagy to underlie the growth and pathogenicity of M. oryzae.IMPORTANCEMagnaporthe oryzae utilizes multiple signaling pathways to promote colonization of host plants. MoMkk1, a cell wall integrity signaling kinase, plays an essential role in autophagy governed by a highly conserved autophagy kinase MoAtg1-mediated pathway. How MoMkk1 regulates autophagy in coordination with MoAtg1 remains elusive. Here, we provide evidence that MoMkk1 phosphorylates MoAtg9 to positively regulate phospholipid translocation during the isolation membrane or smaller membrane structures stage of autophagosome formation. This is in contrast to the negative regulation of MoAtg9 by MoAtg1 for the same process. Intriguingly, MoMkk1-mediated MoAtg9 phosphorylation enhances the fungal infection of rice, whereas MoAtg1-dependant MoAtg9 phosphorylation significantly attenuates it. Taken together, we revealed a novel mechanism of autophagy and virulence regulation by demonstrating the dichotomous functions of MoMkk1 and MoAtg1 in the regulation of fungal autophagy and pathogenicity.

In Vitro and Ex Vivo Antifungal Activities of Metconazole against the Rice Blast Fungus Pyricularia oryzae.

Rice blast, caused by the filamentous fungus Pyricularia oryzae, has long been one of the major threats to almost all rice-growing areas worldwide. Metconazole, 5-(4-chlorobenzyl)-2, 2-dimethyl-1-(1H-1, 2, 4-triazol-1-ylmethyl) cyclopentanol, is a lipophilic, highly active triazole fungicide that has been applied in the control of various fungal pathogens of crops (cereals, barley, wheat), such as the Fusarium and Alternaria species. However, the antifungal activity of metconazole against P. oryzae is unknown. In this study, metconazole exhibited broad spectrum antifungal activities against seven P. oryzae strains collected from rice paddy fields and the wild type strain P131. Scanning electron microscopic analysis and fluorescein diacetate staining assays revealed that metconazole treatment damaged the cell wall integrity, cell membrane permeability and even cell viability of P. oryzae, resulting in deformed and shrunken hyphae. The supplementation of metconazole in vitro increased fungal sensitivity to different stresses, such as sodium dodecyl sulfate, congo red, sodium chloride, sorbitol and oxidative stress (H2O2). Metconazole could inhibit key virulence processes of P. oryzae, including conidial germination, germ tube elongation and appressorium formation. Furthermore, this chemical prevented P. oryzae from infecting barley epidermal cells by disturbing appressorium penetration and subsequent invasive hyphae development. Pathogenicity assays indicated a reduction of over 75% in the length of blast lesions in both barley and rice leaves when 10 µg/mL of metconazole was applied. This study provides evidence to understand the antifungal effects of metconazole against P. oryzae and demonstrates its potential in rice blast management.

CRISPR/Cas9-mediated knockout of Bsr-d1 enhances the blast resistance of rice in Northeast China.

KEY MESSAGE: The blast resistance allele of OsBsr-d1 does not exist in most japonica rice varieties of Jilin Province in China. The development of Bsr-d1 knockout mutants via CRISPR/Cas9 enhances broad-spectrum resistance to rice blast in Northeast China. Rice blast is a global disease that has a significant negative impact on rice yield and quality. Due to the complexity and variability of the physiological races of rice blast, controlling rice blast is challenging in agricultural production. Bsr-d1, a negative transcription factor that confers broad-spectrum resistance to rice blast, was identified in the indica rice cultivar Digu; however, its biological function in japonica rice varieties is still unclear. In this study, we analyzed the blast resistance allele of Bsr-d1 in a total of 256 japonica rice varieties from Jilin Province in Northeast China and found that this allele was not present in these varieties. Therefore, we generated Bsr-d1 knockout mutants via the CRISPR/Cas9 system using the japonica rice variety Jigeng88 (JG88) as a recipient variety. Compared with those of the wild-type JG88, the homozygous Bsr-d1 mutant lines KO#1 and KO#2 showed enhanced leaf blast resistance at the seedling stage to several Magnaporthe oryzae (M. oryzae) races collected from Jilin Province in Northeast China. Physiological and biochemical indices revealed that the homozygous mutant lines produced more hydrogen peroxide than did JG88 plants when infected with M. oryzae. Comparative RNA-seq revealed that the DEGs were mainly involved in the synthesis of amide compounds, zinc finger proteins, transmembrane transporters, etc. In summary, our results indicate that the development of Bsr-d1 knockout mutants through CRISPR/Cas9 can enhance the broad-spectrum resistance of rice in Northeast China to rice blast. This study not only provides a theoretical basis for disease resistance breeding involving the Bsr-d1 gene in Northeast China, but also provides new germplasm resources for disease-resistance rice breeding.

Rice-Magnaporthe oryzae interactions in resistant and susceptible rice cultivars under panicle blast infection based on defense-related enzyme activities and metabolomics.

Rice blast, caused by rice blast fungus (Magnaporthe oryzae), is a global threat to food security, with up to 50% yield losses. Panicle blast is a severe form of rice blast, and disease responses vary between cultivars with different genotypes. Reactive oxygen species (ROS)-mediated signaling reactions and the phenylpropanoid pathway are important defense mechanisms involved in recognizing and resisting against fungal infection. To understand rice-M. oryzae interactions in resistant and susceptible cultivars, we determined dynamic changes in the activities of five defense-related enzymes in resistant cultivar jingsui 18 and susceptible cultivar jinyuan 899 infected with M. oryzae from 4 to 25 days after infection. We then performed untargeted metabolomics analyses to profile the metabolomes of the cultivars under infected and non-infected conditions. Dynamic changes in the activities of five defense-related enzymes were closely related to panicle blast resistance in rice. Metabolome data analysis identified 634 differentially accumulated metabolites (DAMs) between resistant and susceptible cultivars following infection, potentially explaining differences in disease response between varieties. The most enriched DAMs were associated with lipids and lipid-like molecules, phenylpropanoids and polyketides, organoheterocyclic compounds, organic acids and derivatives, and lignans, neolignans, and related compounds. Multiple metabolic pathways are involved in resistance to panicle blast in rice, including biosynthesis of other secondary metabolites, amino acid metabolism, lipid metabolism, phenylpropanoid biosynthesis, arachidonic acid metabolism, arginine biosynthesis, tyrosine metabolism, tryptophan metabolism, tyrosine and tryptophan biosynthesis, lysine biosynthesis, and oxidative phosphorylation.

The link between changing in host carbon allocation and resistance to Magnaporthe oryzae: a possible tactic for mitigating the rice blast fungus.

One of the most destructive diseases affecting rice is rice blast, which is brought on by the rice blast fungus Magnaporthe oryzae. The preventive measures, however, are not well established. To effectively reduce the negative effects of rice blasts on crop yields, it is imperative to comprehend the dynamic interactions between pathogen resistance and patterns of host carbon allocation. This review explores the relationship between variations in carbon allocation and rice plants' ability to withstand the damaging effects of M. oryzae. The review highlights potential strategies for altering host carbon allocation including transgenic, selective breeding, crop rotation, and nutrient management practices as a promising avenue for enhancing rice blast resistance. This study advances our knowledge of the interaction between plants' carbon allocation and M. oryzae resistance and provides stakeholders and farmers with practical guidance on mitigating the adverse effects of the rice blast globally. This information may be used in the future to create varieties that are resistant to M. oryzae.

The Microtubule End Binding Protein Mal3 Is Essential for the Dynamic Assembly of Microtubules during Magnaporthe oryzae Growth and Pathogenesis.

Cytoskeletal microtubules (MTs) play crucial roles in many aspects of life processes in eukaryotic organisms. They dynamically assemble physiologically important MT arrays under different cell conditions. Currently, aspects of MT assembly underlying the development and pathogenesis of the model plant pathogenic fungus Magnaporthe oryzae (M. oryzae) are unclear. In this study, we characterized the MT plus end binding protein MoMal3 in M. oryzae. We found that knockout of MoMal3 results in defects in hyphal polar growth, appressorium-mediated host penetration and nucleus division. Using high-resolution live-cell imaging, we further found that the MoMal3 mutant assembled a rigid MT in parallel with the MT during hyphal polar growth, the cage-like network in the appressorium and the stick-like spindle in nuclear division. These aberrant MT organization patterns in the MoMal3 mutant impaired actin-based cell growth and host infection. Taken together, these findings showed that M. oryzae relies on MoMal3 to assemble elaborate MT arrays for growth and infection. The results also revealed the assembly mode of MTs in M. oryzae, indicating that MTs are pivotal for M. oryzae growth and host infection and may be new targets for devastating fungus control.

OsLRR-RLP2 Gene Regulates Immunity to Magnaporthe oryzae in Japonica Rice.

Rice is an important cereal crop worldwide, the growth of which is affected by rice blast disease, caused by the fungal pathogen Magnaporthe oryzae. As climate change increases the diversity of pathogens, the disease resistance genes (R genes) in plants must be identified. The major blast-resistance genes have been identified in indica rice varieties; therefore, japonica rice varieties with R genes now need to be identified. Because leucine-rich repeat (LRR) domain proteins possess R-gene properties, we used bioinformatics analysis to identify the rice candidate LRR domain receptor-like proteins (OsLRR-RLPs). OsLRR-RLP2, which contains six LRR domains, showed differences in the DNA sequence, containing 43 single-nucleotide polymorphisms (SNPs) in indica and japonica subpopulations. The results of the M. oryzae inoculation analysis indicated that indica varieties with partial deletion of OsLRR-RLP2 showed susceptibility, whereas japonica varieties with intact OsLRR-RLP2 showed resistance. The oslrr-rlp2 mutant, generated using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), showed increased pathogen susceptibility, whereas plants overexpressing this gene showed pathogen resistance. These results indicate that OsLRR-RLP2 confers resistance to rice, and OsLRR-RLP2 may be useful for breeding resistant cultivars.

Targeting Magnaporthe oryzae effector MoErs1 and host papain-like protease OsRD21 interaction to combat rice blast.

Effector proteins secreted by plant pathogenic fungi are important artilleries against host immunity, but there is no precedent of such effectors being explored as antifungal targets. Here we demonstrate that MoErs1, a species-specific effector protein secreted by the rice blast fungus Magnaporthe oryzae, inhibits the function of rice papain-like cysteine protease OsRD21 involved in rice immunity. Disrupting MoErs1-OsRD21 interaction effectively controls rice blast. In addition, we show that FY21001, a structure-function-based designer compound, specifically binds to and inhibits MoErs1 function. FY21001 significantly and effectively controls rice blast in field tests. Our study revealed a novel concept of targeting pathogen-specific effector proteins to prevent and manage crop diseases.

Phenazine biosynthesis protein MoPhzF regulates appressorium formation and host infection through canonical metabolic and noncanonical signaling function in Magnaporthe oryzae.

Microbe-produced secondary metabolite phenazine-1-carboxylic acid (PCA) facilitates pathogen virulence and defense mechanisms against competitors. Magnaporthe oryzae, a causal agent of the devastating rice blast disease, needs to compete with other phyllosphere microbes and overcome host immunity for successful colonization and infection. However, whether M. oryzae produces PCA or it has any other functions remains unknown. Here, we found that the MoPHZF gene encodes the phenazine biosynthesis protein MoPhzF, synthesizes PCA in M. oryzae, and regulates appressorium formation and host virulence. MoPhzF is likely acquired through an ancient horizontal gene transfer event and has a canonical function in PCA synthesis. In addition, we found that PCA has a role in suppressing the accumulation of host-derived reactive oxygen species (ROS) during infection. Further examination indicated that MoPhzF recruits both the endoplasmic reticulum membrane protein MoEmc2 and the regulator of G-protein signaling MoRgs1 to the plasma membrane (PM) for MoRgs1 phosphorylation, which is a critical regulatory mechanism in appressorium formation and pathogenicity. Collectively, our studies unveiled a canonical function of MoPhzF in PCA synthesis and a noncanonical signaling function in promoting appressorium formation and host infection.

The synthetic NLR RGA5HMA5 requires multiple interfaces within and outside the integrated domain for effector recognition.

Some plant sensor nucleotide-binding leucine-rich repeat (NLR) receptors detect pathogen effectors through their integrated domains (IDs). Rice RGA5 sensor NLR recognizes its corresponding effectors AVR-Pia and AVR1-CO39 from the blast fungus Magnaporthe oryzae through direct binding to its heavy metal-associated (HMA) ID to trigger the RGA4 helper NLR-dependent resistance in rice. Here, we report a mutant of RGA5 named RGA5HMA5 that confers complete resistance in transgenic rice plants to the M. oryzae strains expressing the noncorresponding effector AVR-PikD. RGA5HMA5 carries three engineered interfaces, two of which lie in the HMA ID and the other in the C-terminal Lys-rich stretch tailing the ID. However, RGA5 variants having one or two of the three interfaces, including replacing all the Lys residues with Glu residues in the Lys-rich stretch, failed to activate RGA4-dependent cell death of rice protoplasts. Altogether, this work demonstrates that sensor NLRs require a concerted action of multiple surfaces within and outside the IDs to both recognize effectors and activate helper NLR-mediated resistance, and has implications in structure-guided designing of sensor NLRs.

Attenuation of phytofungal pathogenicity of Ascomycota by autophagy modulators.

Autophagy in eukaryotes functions to maintain homeostasis by degradation and recycling of long-lived and unwanted cellular materials. Autophagy plays important roles in pathogenicity of various fungal pathogens, suggesting that autophagy is a novel target for development of antifungal compounds. Here, we describe bioluminescence resonance energy transfer (BRET)-based high-throughput screening (HTS) strategy to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 that is critical for autophagosome formation. We identified ebselen (EB) and its analogs ebselen oxide (EO) and 2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PT) as inhibitors of fungal pathogens Botrytis cinerea and Magnaporthe oryzae ATG4-mediated ATG8 processing. The EB and its analogs inhibit spore germination, hyphal development, and appressorium formation in Ascomycota pathogens, B. cinerea, M. oryzae, Sclerotinia sclerotiorum and Monilinia fructicola. Treatment with EB and its analogs significantly reduced fungal pathogenicity. Our findings provide molecular insights to develop the next generation of antifungal compounds by targeting autophagy in important fungal pathogens.

Distinct genomic contexts predict gene presence-absence variation in different pathotypes of Magnaporthe oryzae.

Fungi use the accessory gene content of their pangenomes to adapt to their environments. While gene presence-absence variation contributes to shaping accessory gene reservoirs, the genomic contexts that shape these events remain unclear. Since pangenome studies are typically species-wide and do not analyze different populations separately, it is yet to be uncovered whether presence-absence variation patterns and mechanisms are consistent across populations. Fungal plant pathogens are useful models for studying presence-absence variation because they rely on it to adapt to their hosts, and members of a species often infect distinct hosts. We analyzed gene presence-absence variation in the blast fungus, Magnaporthe oryzae (syn. Pyricularia oryzae), and found that presence-absence variation genes involved in host-pathogen and microbe-microbe interactions may drive the adaptation of the fungus to its environment. We then analyzed genomic and epigenomic features of presence-absence variation and observed that proximity to transposable elements, gene GC content, gene length, expression level in the host, and histone H3K27me3 marks were different between presence-absence variation genes and conserved genes. We used these features to construct a model that was able to predict whether a gene is likely to experience presence-absence variation with high precision (86.06%) and recall (92.88%) in M. oryzae. Finally, we found that presence-absence variation genes in the rice and wheat pathotypes of M. oryzae differed in their number and their genomic context. Our results suggest that genomic and epigenomic features of gene presence-absence variation can be used to better understand and predict fungal pangenome evolution. We also show that substantial intra-species variation can exist in these features.

Autophagy and cell wall integrity pathways coordinately regulate the development and pathogenicity through MoAtg4 phosphorylation in Magnaporthe oryzae.

Autophagy and Cell wall integrity (CWI) signaling are critical stress-responsive processes during fungal infection of host plants. In the rice blast fungus Magnaporthe oryzae, autophagy-related (ATG) proteins phosphorylate CWI kinases to regulate virulence; however, how autophagy interplays with CWI signaling to coordinate such regulation remains unknown. Here, we have identified the phosphorylation of ATG protein MoAtg4 as an important process in the coordination between autophagy and CWI in M. oryzae. The ATG kinase MoAtg1 phosphorylates MoAtg4 to inhibit the deconjugation and recycling of the key ATG protein MoAtg8. At the same time, MoMkk1, a core kinase of CWI, also phosphorylates MoAtg4 to attenuate the C-terminal cleavage of MoAtg8. Significantly, these two phosphorylation events maintain proper autophagy levels to coordinate the development and pathogenicity of the rice blast fungus.

Multi-Omics Approach Reveals OsPIL1 as a Regulator Promotes Rice Growth, Grain Development, and Blast Resistance.

Rice (Oryza sativa) is a crucial crop, achieving high yield concurrent pathogen resistance remains a challenge. Transcription factors play roles in growth and abiotic tolerance. However, rice phytochrome-interacting factor-like 1 (OsPIL1) in pathogen resistance and agronomic traits remains unexplored. We generated OsPIL1 overexpressing (OsPIL1 OE) rice lines and evaluated their impact on growth, grain development, and resistance to Magnaporthe oryzae. Multiomics analysis (RNA-seq, metabolomics, and CUT&Tag) and RT-qPCR validated OsPIL1 target genes and key metabolites. In the results, OsPIL1 OE rice lines exhibited robust growth, longer grains, and enhanced resistance to M. oryzae without compromising growth. Integrative multiomics analysis revealed a coordinated regulatory network centered on OsPIL1, explaining these desirable traits. OsPIL1 likely acts as a positive regulator, targeting transcriptional elements or specific genes with direct functions in several biological programs. In particular, a range of key signaling genes (phosphatases, kinases, plant hormone genes, transcription factors), and metabolites (linolenic acid, vitamin E, trigonelline, d-glucose, serotonin, choline, genistein, riboflavin) contributed to enhanced rice growth, grain size, pathogen resistance, or a combination of these traits. These findings highlight OsPIL1's regulatory role in promoting important traits and provide insights into potential strategies for rice breeding.