BACKGROUND:Environmentally sustainable management of plant fungal diseases requires green agrochemicals that combine high efficacy with novel modes-of-action. Rice blast caused by Magnaporthe oryzae is a destructive crop fungal disease, and developing botanical fungicides with new antifungal mechanisms is urgently needed for its green control. RESULTS:In this study, we report that an ethanol-petroleum ether crude extract of Hydnocarpus hainanensis seeds (designated DF2) strongly suppresses infection of the rice blast fungus M. oryzae. Bioactivity-guided fractionation of DF2 identified hydnocarpic acid and chaulmoogric acid as the principal antifungal constituents, which inhibit conidial germination, appressorium formation and plant infection by M. oryzae. Furthermore, treatment with DF2 significantly perturbed carnitine-related metabolites and severely attenuated fatty acid β-oxidation in M. oryzae conidia. Interestingly, exogenous addition of methylenecyclopropyl acetic acid to the M. oryzae conidial inoculum mimicked these effects, whereas supplementation with l-carnitine reversed them. CONCLUSION:Taken together, our findings indicate that hydnocarpic acid and chaulmoogric acid disrupt fatty acid β-oxidation and impede infection of M. oryzae, and suggest that DF2 could serve as a potential botanical fungicide to control fungal diseases of crops. © 2026 Society of Chemical Industry.

INTRODUCTION:The fungal cell wall, a dynamic structure critical for pathogenesis, is composed of polysaccharides and proteins. UDP-glucose 4-epimerases (UGEs) play a pivotal role in cell wall synthesis by converting UDP-galactose between UDP-glucose. This study investigates the role of MoUGE1 in Magnaporthe oryzae pathogenesis and explores its potential as an antifungal target for fungal disease control. A lead inhibitor targeting MoUGE1 was identified through virtual screening. OBJECTIVES:The aims of this study were to elucidate the role of MoUGE1 in M. oryzae during rice blast pathogenesis, assess its potential as an antifungal target, and identify potential MoUGE1 inhibitors through virtual screening to control rice blast. METHODS:To analyses the role of MoUGE1 in M. oryzae, we generated Δuge1 mutants via split-PCR-mediated gene knockout. The impact of MoUGE1 on fungal growth, cell wall composition, and plant infection was assessed. Metabolomic analysis revealed the impact of MoUGE1 deletion on metabolic processes of rice blast. Structure-based virtual screening, molecular dynamics (MD) simulation and surface plasmon resonance (SPR) used to find hit compounds inhibiting MoUGE1. RESULTS:The Δuge1 exhibited reduced mycelial growth, altered cell wall and cell membrane composition, and impaired plant infection. Metabolomic analysis and western blot revealed accumulation in UDP-galactose and alteration of N-glycosylation, leading to cell wall instability and increased sensitivity to cell wall stressors. Virtual screening identified lig122132 as a potential MoUGE1 inhibitor, which was further confirmed through MD simulation and SPR, showing stable binding to the MoUGE1. CONCLUSION:Our findings emphasize the significance of MoUGE1 in fungal cell wall integrity, appressorium function, and virulence in M. oryzae. Identifying MoUGE1 as a target for antifungal intervention provides new insights into the molecular mechanisms of rice blast pathogenesis and paves the way for developing innovative strategies against this major agricultural disease. The potential MoUGE1 inhibitor, lig122132, is a promising starting point for the development of novel fungicides to control rice blast.

BACKGROUND:The development of biocontrol agents represents a promising strategy to manage banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Although many traditional approaches have isolated beneficial microorganisms from soil or the rhizosphere, studies seeking biocontrol resources from the perspective of banana root endophytes remain scarce. RESULTS:Endophytic microbiome analysis revealed significant enrichment of Bacillota in the wilt-resistant cultivar. Among the isolated strains, Bacillus velezensis JDB15 exhibited the best inhibitory effect against Foc TR4. The fermentation broth of JDB15 significantly inhibited spore germination and caused hyphal membrane damage in pathogens. Mechanistic studies indicated that the lipopeptide surfactin C is a candidate active antimicrobial metabolite produced by JDB15, which disrupts pathogen cell membrane integrity, increases membrane permeability, and induces electrolyte leakage. Another isolated endophytic fungus, Trichoderma harzianum strain JDL4, also exhibited strong antagonistic activity against Foc TR4 probably through mycoparasitism. Combined application of cell-free filtrate from JDB15 and JDL4 demonstrated effective control against multiple plant diseases including banana Fusarium wilt, tomato Fusarium wilt, corn southern leaf blight, and rice blast under controlled conditions. CONCLUSION:We suggest that the antimicrobial activity of JDB15 and JDL4 is most probably attributable to the metabolite surfactin C and likely mycoparasitism, respectively. Co-application of the fermentation filtrates of these two strains exhibited broad-spectrum disease control efficacy and significantly improved disease suppression compared with either strain alone. These findings provide novel biological agents for the control of banana Fusarium wilt and other plant diseases. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Poly(A) tail shortening by deadenylases is a central checkpoint linking mRNA fate to eukaryotic development, yet its impact on fungal pathogenesis remains unexplored. Here, we uncover that the Pan2-Pan3 deadenylase complex is a master regulator of infection in the rice blast fungus Magnaporthe oryzae. Pan2 and Pan3 form a catalytically active complex that localizes to P-bodies and globally trims poly(A) tails to enforce mRNA quality control. Deletion of either or both subunits abolishes this quality-control checkpoint, causing severe virulence loss due to arrested appressorium maturation, disrupted glycogen/lipid mobilization, and impaired autophagy. Integrating poly(A)-seq and transcriptome profiling reveals 390 mRNAs whose poly(A) tails are ≥ 5 nt longer and whose steady-state levels are elevated in the Δpan2Δpan3 mutant; among them, ATG5, GLS2, and DES1-key genes governing autophagy, ER quality control, and ROS detoxification respectively-are directly deadenylated by Pan2-Pan3. Loss of deadenylation destabilizes these mRNAs and reduces their protein output, thereby crippling infection. Our findings establish Pan2-Pan3 complex-mediated deadenylation as an essential post-transcriptional layer that orchestrates fungal virulence through stringent mRNA quality control, offering a novel target for crop protection.

BACKGROUND:The ascomycete Magnaporthe oryzae is a destructive phytopathogenic fungus that causes rice blast disease and can greatly reduce rice yields. Due to the widespread use of synthesized chemical fungicides and the frequent occurrence of fungicide resistance and environmental pollution as a result, it has become increasingly urgent to develop environmentally friendly biocontrol alternatives, including plant crude extracts. RESULTS:In this study, the crude extracts of the tropical plant Millettia pachyloba were screened out and exhibited excellent preventive effects against M. oryzae at 200 μg mL-1. Bio-guided isolation of M. pachyloba was conducted and three isoflavonoids, rotenolone, durmillone, and durallone, whose chemical structures were determined using spectroscopic and spectrometric analyses, were obtained. The three isoflavonoids exhibited antifungal activities on conidial germination and appressorium formation in M. oryzae. Rotenolone had the strongest effect, with EC50 values of 57.81 μg mL-1 on conidial germination and 19.14 μg mL-1 on appressorium formation. Comparative metabolomics showed that differential metabolites were enriched in ABC transporter pathways and amino acid metabolic pathways when M. oryzae conidia were treated with rotenolone, suggesting that rotenolone interferes with amino acid transportation. Moreover, the M. pachyloba crude extract also effectively inhibited the infection of other fungal pathogens on tomato, apple, wheat, and maize. CONCLUSION:The results suggest that isoflavones extracted from M. pachyloba prevent rice blast by inhibiting the infection-related morphogenesis of M. oryzae and may interfere with amino acid transport, demonstrating that M. pachyloba crude extract exhibits potential as a bio-fungicide for controlling fungal diseases in plants. © 2025 Society of Chemical Industry.

AbstractVascular wilt, a disease caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is highly destructive to bananas. Identifying genes that contribute to the fungus's virulence is crucial for understanding its pathogenesis. In this review, we provide an overview of recent research on genes involved in various aspects of Foc TR4's pathogenic process. These include signal recognition and transduction, the formation of cellular structures, regulation through microRNA and epigenetic mechanisms, effector secretion, and toxin secretion. We place a particular emphasis on discussing effectors that either facilitate virulence or serve as elicitors of host defense responses. Given the limited understanding of the molecular mechanisms underlying Foc TR4 pathogenesis, summarizing the research on these functional genes is necessary and timely. Our integrative information will facilitate research on identification of more key genes involved in the invasiveness of Foc TR4, contributing to more systemic understanding of pathogenesis of this important pathogen. These findings will, in turn, offer potential targets for the development of effective fungicides or soil disinfectants to combat this devastating disease.

Snf5 (sucrose nonfermenting) is a core component of the SWI/SNF complexes and regulates diverse cellular processes in model eukaryotes. In plant pathogenic fungi, its biological function and underlying mechanisms remain unexplored. In this study, we investigated the biological roles of MoSnf5 in plant infection and fungal development in the rice blast pathogen Magnaporthe oryzae. The gene deletion mutants of MoSNF5 exhibited slower vegetative hyphal growth, severe defects in conidiogenesis, and impaired virulence and galactose utilization capacities. Domain dissection assays showed that the Snf5 domain and the N- and C-termini of MoSnf5 were all required for its full functions. Co-immunoprecipitation and yeast two-hybrid assays showed that MoSnf5 physically interacts with four proteins, including a transcription initiation factor MoTaf14. Interestingly, the ∆MoTaf14 mutants showed similar phenotypes as the ∆Mosnf5 mutants on fungal virulence and development. Moreover, assays on GFP-MoAtg8 expression and localization showed that both the ∆Mosnf5 and ∆MoTaf14 mutants were defective in autophagy. Taken together, MoSnf5 regulates fungal virulence, growth, and conidiation, possibly through regulating galactose utilization and autophagy in M. oryzae.

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.

Serine/arginine-rich (SR) proteins are well known as splicing factors in humans, model animals and plants. However, they are largely unknown in regulating pre-mRNA splicing of filamentous fungi. Here we report that the SR protein MoSrp1 enhances and suppresses alternative splicing in a model fungal plant pathogen Magnaporthe oryzae . Deletion of MoSRP1 caused multiple defects, including reduced virulence and thousands of aberrant alternative splicing events in mycelia, most of which were suppressed or enhanced intron splicing. A GUAG consensus bound by MoSrp1 was identified in more than 94% of the intron or/and proximate exons having the aberrant splicing. The dual functions of regulating alternative splicing of MoSrp1 were exemplified in enhancing and suppressing the consensus-mediated efficient splicing of the introns in MoATF1 and MoMTP1 , respectively, which both were important for mycelial growth, conidiation, and virulence. Interestingly, MoSrp1 had a conserved sumoylation site that was essential to nuclear localization and enhancing GUAG binding. Further, we showed that MoSrp1 interacted with a splicing factor and two components of the exon-joining complex via its N-terminal RNA recognition domain, which was required to regulate mycelial growth, development and virulence. In contrast, the C-terminus was important only for virulence and stress responses but not for mycelial growth and development. In addition, only orthologues from Pezizomycotina species could completely rescue defects of the deletion mutants. This study reveals that the fungal conserved SR protein Srp1 regulates alternative splicing in a unique manner.

Abstract Endocytosis plays key roles during infection of plant‐pathogenic fungi, but its regulatory mechanisms are still largely unknown. Here, we identified a putative endocytosis‐related gene, PAL1, which was highly expressed in appressorium of Magnaporthe oryzae, and was found to be important for appressorium formation and maturation. Deletion of PAL1 significantly reduced the virulence of M. oryzae due to defects in appressorial penetration and invasive growth in host cells. The Pal1 protein interacted and colocalized with the endocytosis protein Sla1, suggesting it is involved in endocytosis. The Δpal1 mutant was significantly reduced in appressorium formation, which was recovered by adding exogenous cAMP and 3‐isobutyl‐1‐methylxanthine (IBMX). Moreover, the phosphorylation level of Pmk1 in Δpal1 was also reduced, suggesting Pal1 functions upstream of both the cAMP and Pmk1 signalling pathways. As a consequence, the utilization of glycogen and lipid, appressorial autophagy, actin ring formation, localization of septin proteins, as well as turgor accumulation were all affected in the Δpal1 mutant. Taken together, Pal1 regulates cAMP and the Pmk1 signalling pathway for appressorium formation and maturation to facilitate infection of M. oryzae.

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.

AbstractThe Ubp family of deubiquitinating enzymes has been found to play important roles in plant‐pathogenic fungi, but their regulatory mechanisms are still largely unknown. In this study, we revealed the regulatory mechanism of the deubiquitinating enzyme Ubp3 during the infection process of Magnaporthe oryzae. AUBP3 deletion mutant was severely defective in appressorium turgor accumulation, leading to the impairment of appressorial penetration. During appressorium formation, the mutant was also defective in glycogen and lipid metabolism. Interestingly, we found that nitrogen starvation and rapamycin treatment induced the ribophagy process in M. oryzae, which is closely dependent on Ubp3. In the ∆ubp3 mutant, the ribosome proteins and rRNAs were not well degraded on nitrogen starvation and rapamycin treatment. We also found that Ubp3 interacted with the GTPase‐activating protein Smo1 and regulated its de‐ubiquitination. Ubp3‐dependent de‐ubiquitination of Smo1 may be required for Smo1 to coordinate Ras signalling. Taken together, our results showed at least two roles of Ubp3 in M. oryzae: it regulates the ribophagy process and it regulates de‐ubiquitination of GTPase‐activating protein Smo1 for appressorium‐mediated infection.

Soybean cyst nematode (SCN, Heterodera glycines ) is widely considered as the model plant-parasitic nematode, which secretes effector proteins to manipulate host responses. In this study, we cloned a dorsal gland-expressed effector protein SCN-27D09 that belongs to the same family as Hg10A07 in SCN. We used the model plant pathogen rice blast fungus ( Magnaporthe oryzae ) to quickly predict and characterize the functions of SCN-27D09. By using M. oryzae secretion system in barley, we confirmed that the signal peptide of SCN-27D09 has secretory activity and can guide the protein into the host cells. Heterologous expression of SCN-27D09 in M. oryzae significantly enhanced the susceptibility of barley to M. oryzae . SCN-27D09 can inhibit Bax-triggered cell death when expressed in Nicotiana benthamiana . Overexpression of SCN-27D09 in soybean hairy root also increased the susceptibility of soybean plants to SCN. Moreover, yeast two-hybrid and firefly luciferase complementation imaging assays showed that SCN-27D09 interacts with a soybean plant kinase GmIPK-2. Functional characterization of GmIPK-2 revealed its positive role in soybean resistance, indicating that SCN-27D09 might compromise the function of GmIPK-2 to facilitate nematode infection. Our results not only uncover the biological role of SCN-27D09 in suppressing plant defense responses and therefore promoting nematode parasitism, but also reaffirm the potential application of the model plant pathogenic fungus M. oryzae in investigating the pathogenic roles of candidate effectors of phytonematodes.

Soybean cyst nematode (SCN), Heterodera glycines, is a devastating pathogen in soybean worldwide, causing huge yield losses annually. Host-induced gene silencing (HIGS) has shown potentials to control plant parasitic nematodes by generating transgenic plants carrying hairpin constructs against nematode target genes (Chaudhary et al., 2019; Li et al., 2010; Mani et al., 2020; Shivakumara et al., 2017; Tian et al., 2016). Chitin synthesized by chitin synthase (Chs) is present in fungi and nematodes but absent in plants and vertebrate animals and is a target in Magnaporthe oryzae and Fusarium graminearum for designing new fungicides and developing novel resistant varieties by HIGS (Cheng et al., 2015; Kong et al., 2012). Here, we mainly aimed to develop heritable SCN CHS-HIGS soybeans conferring enhanced SCN resistance. Meanwhile, the developed HIGS soybeans were tested for resistance to fungus Fusarium oxysporum, which causes soybean Fusarium wilt disease. First, the sole CHS gene (SCN-CHS) with 3984 bp (GenBank Acc. No.: OK149168) was cloned from SCN HG Type 1.2.3.5.7 (race 4, SCN4). SCN-Chs contained a typical chitin synthase catalytic domain (Chs) and seven transmembrane domains (TMs) (Figure 1a). qRT-PCR analysis indicated that SCN-CHS was highly expressed at egg stage when compared to other developmental stages (Figure 1b). A 420 bp cDNA fragment of SCN-CHS catalytic region positioned at 1936–2355 bp was used to generate transgenic HIGS soybeans employing 'Jack' as wild-type soybean (Figure 1c), and three homozygous transgenic lines ('48-7-5', '55-8-24', and '57-9-2') with yellow seed-coat identical to that of Jack (Figure 1d) were obtained. The amount of SCN4 cysts per plant (left of Figure 1e) and eggs per cyst (Figure 1f) in T2 lines was all significantly reduced when compared to Jack. The cyst numbers among T2 lines differed dramatically from 18 to 76, while the cyst numbers were about 120 in Jack, on the average, and the T2 line '55-8-24(T2)' showed the most inhibited SCN cyst formation with a 6-fold reduction in cyst numbers (left of Figure 1e). These results indicated that various HIGS lines suppressed SCN cyst formation differently. The average numbers of eggs per cyst were 153 in T2 lines, which were significantly decreased by 37.5% when compared to that in Jack (Figure 1f). qRT-PCR analysis showed that SCN-CHS expression in eggs of SCN parasitizing in T2 lines was significantly reduced (Figure 1g). These results suggested that down-regulation of SCN-CHS expression in eggs was likely associated with suppression of SCN cyst and egg formation in T2 soybeans. Analyses of the time-course developmental progress of SCN4 juveniles in T2 roots showed that all juveniles were decreased, and the developmental transition frequency from low to high molt stages was also delayed in all T2 roots (Figure 1h). What is more, the size of cysts formed in T2 lines ranged from 662.33 to 673.92 μm in diameter and was obviously smaller than that in Jack (703.4–724.24 μm) (Figure 1i). Meanwhile, a similar but stronger effectiveness on the suppression of cyst formation was observed in all the SCN HG Type 0 (race 3, SCN3)-infected T2 lines (right of Figure 1e). These results together with SCN4 infection results implied that T2 HIGS soybean lines expressing dsRNA of SCN-CHS showed enhanced broad-spectrum resistance to different SCN HG types (races). The heredity of HIGS soybeans suppressing SCN was evaluated using three T6 lines, '48-7-5(T6)', '55-8-24(T6)', and '57-9-2 (T6)', with infection of SCN4. The amount of both cysts per plant and eggs per cyst was also significantly decreased in all T6 lines (Figure 1j). Moreover, expression of SCN-CHS in SCN eggs was also obviously suppressed in all T6 lines (Figure 1g). All these results suggested that T6 HIGS lines exhibited strong heredity of the boosted resistance to SCN. Subsequently, the three HIGS soybean lines were tested for their resistance against Fusarium oxysporum f. sp. glycines. The longer the lesions are on the hypocotyls, the more serious the disease is. The results showed that the average hypocotyl lesion length in all HIGS lines was significantly decreased when compared to that in Jack (Figure 1k). Afterwards, F. oxysporum was isolated from each HIGS line and Jack as Fo-48-7-5, Fo-55-8-24, Fo-57-9-2, and Fo-Jack, respectively, and they were then cultured on PDA-medium plates and mycelia growth was observed. About 1 week post-inoculation, the average spread size (diameter) of mycelia isolated from all the HIGS soybeans was remarkably reduced when compared to that isolated from Jack (Figure 1l). Meanwhile, after these isolated fungi inoculating Jack, the lesions of hypocotyls infected by Fo-48-7-5, Fo-55-8-24, or Fo-57-9-2 were all significantly shorter than these infected by Fo-Jack (Figure 1m). These results indicated that HIGS of SCN-CHS significantly enhanced soybean resistance against F. oxysporum. In summary, our study showed that host-induced silencing of chitin synthase gene in SCN (SCN-CHS) broadly and durably enhanced soybean resistance against both different SCN races and F. oxysporum, and the developed SCN-CHS HIGS soybeans will be a potential for controlling SCN and Fusarium wilt diseases. This study was funded by grants from National Natural Science Foundation of China (31872924, 31972248), National Key Research and Development Program of China (2018YFD0201002), and State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF201613). We are grateful to Dr. Zhixiang Zhang at Institute of Plant Protection, Chinese Academy of Agricultural Sciences for discussion and suggestions. The authors declare no conflict of interest. LK, SL, JY, and DP designed the experiments. LK, XS, DC, NY, CY, JY, SL, GW, WH, and HP performed the experiments and data analyses. LK and SL wrote the manuscript. All the authors participated in discussion and revision.

The calcium/calcineurin signaling pathway plays a key role in the development and virulence of plant pathogenic fungi, but the regulation of this signaling pathway is still not clear. In this study, we identified a calcineurin regulator MoRCN1 in the plant pathogenic fungus Magnaporthe oryzae and found it is important for virulence by regulating the calcineurin pathway. MoRCN1 deletion mutants were severely decreased in colony growth and conidia formation. More importantly, the deletion of MoRCN1 led to a significant reduction in virulence due to defects in appressorium formation and invasive growth. The ΔMorcn1 mutants were more sensitive to different stresses and induced host ROS accumulation, suggesting a role of MoRCN1 in stress adaptation. We found that MoRCN1 directly interacted with the calcineurin catalytic subunit MoCNA and affected its protein stability, which was therefore important for regulating the calcineurin pathway. Transcriptome analysis showed that MoRCN1 significantly activated 491 genes and suppressed 337 genes in response to calcium ion, partially overlapped with the MoCRZ1-bound genes. Gene Ontology and KEGG pathway analyses indicated that MoRCN1-regulated genes were enriched in stress adaptation, lipid metabolism, and secondary metabolite biosynthesis, reflecting a function of MoRCN1 in host cell adaptation. Altogether, these results suggest MoRCN1 functions as a regulator of the calcium/calcineurin signaling pathway for fungal development and infection of host cells.

The splicing factor Cwf15 is an essential component of the Prp19-associated component of the spliceosome and regulates intron splicing in several model species, including yeasts and human cells. However, the roles of Cwf15 remain unexplored in plant pathogenic fungi. Here, we report that MoCWF15 in the rice blast fungus Magnaporthe oryzae is non-essential to viability and important to fungal virulence, growth and conidiation. MoCwf15 contains a putative nuclear localization signal (NLS) and is localized into the nucleus. The NLS sequence but not the predicted phosphorylation site or two sumoylation sites was essential for the biological functions of MoCwf15. Importantly, MoCwf15 physically interacted with the Prp19-associated splicing factors MoCwf4, MoSsa1 and MoCyp1, and negatively regulated protein accumulations of MoCyp1 and MoCwf4. Furthermore, with the deletion of MoCWF15, aberrant intron splicing occurred in near 400 genes, 20 of which were important to the fungal development and virulence. Taken together, MoCWF15 regulates fungal growth and infection-related development by modulating the intron splicing efficiency of a subset of genes in the rice blast fungus.

Many of the world’s most serious crop diseases are caused by hemibiotrophic fungi. These pathogens have evolved the ability to colonize living plant cells, suppressing plant immunity responses, before switching to necrotrophic growth, in which host cells die, providing the energy to fuel sporulation and spread of the fungus. How hemibiotrophic pathogens switch between these two lifestyles remains poorly understood. Here, we report that the devastating rice blast fungus, Magnaporthe oryzae , manipulates host cellular pH to regulate hemibiotrophy. During infection by M. oryzae , host plant cells are alkalinized to pH 7.8 during biotrophic growth, but later acidified to pH 6.5 during necrotrophy. Using a forward genetic screen, we identified alkaline-sensitive mutants of M. oryzae that were blocked in biotrophic proliferation and impaired in induction of host cell acidification and necrotrophy. These mutants defined components of the PacC-dependent ambient pH signal transduction pathway in M. oryzae . We report that PacC exists as a full-length repressor, PacC559, and a truncated transcriptional activator, PacC222, which localize to the fungal nucleus during biotrophic growth and to the cytoplasm during necrotrophy. During biotrophy, PacC222 directly activates genes associated with nutrient acquisition and fungal virulence, while PacC559 represses genes associated with saprophytic mycelial growth and sporulation, which are subsequently de-repressed during necrotrophy. When considered together, our results indicate that temporal regulation of hemibiotrophy by M. oryzae requires PacC-dependent sensing and manipulation of host cellular pH.Author Summary Crop diseases caused by fungi represent some of the most serious threats to global food security. Many fungal pathogens have evolved the ability to invade living plant tissue and suppress host immunity, before switching to a completely different mode of growth, in which they are able to kill host plant cells. This lifestyle– called hemibiotrophy –is exemplified by the blast fungus, Magnaporthe oryzae , which causes devastating diseases of rice, wheat and many other grasses. We found that during infection by M. oryzae , host cells initially have an alkaline pH, when the fungus is growing in living tissue, but pH rapidly becomes acidic, as host tissue is killed. We identified mutants of the blast fungus that were sensitive to alkaline pH and this enabled us to identify the signal transduction pathway by which the fungus responds to changes in ambient pH. We found that mutants in the pH response pathway were blocked in invasive fungal growth and could not cause acidification of host tissue. Consequently, they are unable to cause blast disease. We characterized the central regulator of this pathway, the PacC transcription factor, which unusually can act as both a repressor and an activator of fungal gene expression. During biotrophic invasive growth, PacC activates many genes previously reported to be required for virulence, including several associated with nutrient acquisition, and at the same time represses genes associated with vegetative growth and sporulation. The PacC signaling pathway is therefore necessary for regulating the switch in fungal lifestyle associated with causing blast disease.

31 Plant fungal pathogens secrete numerous proteins into the apoplast at the 32 plant–fungus contact sites to facilitate colonization. Only a few secreted proteins were 33 functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice 34 blast disease worldwide. ALG3 is an α-1, 3-mannosyltransferase function in N-glycan 35 synthesis for secreted N-glycosylated proteins, and the ∆alg3 mutants show strong 36 defects in cell wall integrity and fungal virulence, indicating a potential effect on the 37 secretion of multiple proteins. In this study, we compared the secretome of wild type 38 and ∆alg3 mutants, and identified 51 proteins that require ALG3 for proper secretion. 39 These are predicted to be involved in metabolic processes, interspecies interactions, 40 cell wall organization, and response to chemicals. The tested secreted proteins 41 localized at the apoplast region surrounding the fungal infection hyphae. Moreover, 42 the N-glycosylation of candidate proteins was significantly changed in the ∆alg3 43 mutant, leading to the reduction of protein secretion and abnormal protein localization. 44 Furthermore, we tested the function of two genes, one is a previously reported M. 45 oryzae gene Invertase 1 (INV1) encoding a secreted invertase, and the other one is a 46 gene encoding an Acid mammalian chinitase (AMCase). The fungal virulence was 47 significantly reduced and the cell wall integrity was altered in the ∆inv1 and ∆amcase 48 mutant strains. Elucidation of the comparative secretome of M. oryzae improves our 49 understanding of the proteins that require ALG3 for secretion, and of their function in 50 fungal virulence and cell wall integrity. 51 52