Appressorium Formation Research Articles

Identifying substrate triggers for appressorium development in Setosphaeria turcica and functional characterization of Zn(II)2Cys6 transcription factors StTF1 and StTF2

Northern corn leaf blight is a devastating disease caused by Setosphaeria turcica (S. turcica), leading to significant yield losses in maize. S. turcica initiates infection through a specialized structure known as the appressorium, which only forms on conducive substrates. In this study, we introduce a semi‑silicone water polyurethane resin (Si-PUD) that induces only germ tube formation from S. turcica conidia. A mixed coating of Si-PUD and polytetrafluoroethylene successfully triggers appressorium formation. Both coatings maintain optical transparency, chemical resistance, and thermal stability, which facilitate microscopic observations and the development of high-throughput systems. These coatings also demonstrate similar effects on Bipolaris maydis (B. maydis), suggesting their potential universal applicability. Utilizing coating-induced synchronous appressorium formation and proteomic analyses, we identified five genes essential for S. turcica appressorium development. Functional analyses of two zinc binuclear cluster domain-containing transcription factors, StTF1 and StTF2, revealed their critical roles in appressorium development and pathogenicity. This study not only develops a novel method for inducing appressorium formation but also lays the groundwork for rapid screening of environmentally-friendly fungicides that inhibit appressorium development.

Unveiling the functions of the Lim-domain binding protein MaPtaB in Metarhizium acridum.

The Lim-domain binding protein PtaB, a homolog of Mfg1, governs conidiation and biofilm formation in several fungi. PtaB includes a conserved Lim-binding domain and two predicted nuclear localization sequences at its C terminus, and is co-regulated with the transcription factor Som1 downstream of the cyclic AMP-dependent protein kinase A (cAMP/PKA) pathway. However, the function of PtaB in entomopathogenic fungi remain poorly understood. Inactivation of PtaB in Metarhizium acridum resulted in delayed conidial germination, reduced conidial yield and increased sensitivities to cell wall disruptors, ultraviolet B irradiation and heat shock. In addition, the fungal virulence was significantly decreased after deletion of MaPtaB because of impairments in appressorium formation, cuticle penetration and evasion of insect immune responses in M. acridum. The MaPtaB-deletion and MaSom1-deletion strains showed similar phenotypes supporting that MaSom1/MaPtaB complex controls M. acridum normal conidiation and pathogenic progress. Upon loss of MaPtaB or MaSom1, the fungal sporulation mode in M. acridium shifted from microcycle conidiation to normal conidiation on SYA, a microcycle conidiation medium. Transcriptional analysis showed that more differentially expression genes were identified in MaSom1 RNA sequencing, and MaSom1 and MaPtaB may regulate the expression of genes for conidiation, nutrient metabolism and the cell cycle to control conidiation pattern shift. These data corroborate a complex control function for MaPtaB as an important central factor interacting with MaSom1 in the cAMP/PKA pathway, which links stress tolerance, conidiation and virulence in the entomopathogenic fungus M. acridum. © 2024 Society of Chemical Industry.

Functional Characterization of the Transcription Factor CgCyp51 in the Poplar Anthracnose-Causing Fungus Colletotrichum gloeosporioides

Poplar is an economically and ecologically valuable tree species. Anthracnose, which severely affects poplar tree growth, is mainly caused by Colletotrichum gloeosporioides. In the infestation cycle of poplar anthracnose, the entry of C. gloeosporioides into the host tissue depends on the formation of an appressorium. The subsequent development of the appressorium determines the pathogenesis of poplar anthracnose and the degree of damage. Previous studies have found that the transcription factor CgSte12 affects appressorium formation and development by regulating the expression of a series of genes, including the sterol-synthesis-related gene CgCYP51, which influences appressorium formation and development. In this study, knockout and functional analyses of CgCYP51 revealed decreases in differentiation, darkening rate, and turgor pressure of appressoria in mutants. Additionally, compared with the wild-type appressorium, mutant appressoria secreted less mucus and exhibited abnormal penetration pore formation, ultimately leading to decreased pathogenicity. Moreover, CgCyp51 affected the sensitivity of C. gloeosporioides to sterol biosynthesis inhibitors. Considered together, the study findings indicate CgCYP51 is a key CgSte12-regulated gene that affects C. gloeosporioides appressorium formation and development. Furthermore, the study data provide new insights into the molecular basis of C. gloeosporioides appressorium formation and development.

The nucleolin MoNsr1 plays pleiotropic roles in the pathogenicity and stress adaptation in the rice blast fungus Magnaporthe oryzae

The rice blast disease, caused by the fungus Magnaporthe oryzae, is a significant agricultural problem that adversely impacts rice production and food security. Understanding the precise molecular pathways involved in the interaction between the pathogen and its host is crucial for developing effective disease management strategies. This study examines the crucial function of the nucleolin MoNsr1 in regulating M. oryzae physiological functions. ΔMoNsr1 deletion mutants showed reduced fungal growth, asexual sporulation, and pathogenicity compared to the wild-type. Mutants exhibited impaired conidial germination and appressoria formation, reducing infection progression. Additionally, ΔMoNsr1 deletion mutant had less turgor pressure, confirming that MoNsr1 is essential for cell wall biogenesis and resistant to external stresses. Furthermore, ΔMoNsr1 deletion mutant showed enhanced sensitivity to oxidative stress, reactive oxygen species, and cold tolerance. Our results offer a thorough understanding of the function of MoNsr1 in the virulence and stress-resilient capability in M. oryzae. These findings provide insights into the novel targets and contribute to the emergence of innovative approaches for managing rice blast disease.

Rhodopseudomonas palustris Atp2 Protein Exerts Antifungal Effects by Targeting the Ribosomal Protein MoRpl12 in Magnaporthe oryzae.

Rice blast is one of the most hazardous diseases affecting rice production. Previously, we discovered that the Atp2 protein of Rhodopseudomonas palustris could significantly inhibit the appressorium formation and pathogenicity of Magnaporthe oryzae. However, the molecular mechanism of this fungus has remained unknown. This study revealed that Atp2 can enter the cell and interact with the ribosomal protein MoRpl12 of M. oryzae, directly affecting the expression of the MoRpl12 protein. Silencing the MoRPL12 gene can affect cell wall integrity, growth, conidiogenesis, and fungal pathogenicity. The quantitative reverse transcription PCR results showed significant changes in the expression of conidiation-related genes in the MoRPL12 gene-silenced mutants or in the Atp2 protein-treated plants. We further found that Atp2 treatment can influence the expression of ribosomal-related genes, such as RPL, in M. oryzae. Our study revealed a novel antifungal mechanism by which the Atp2 protein binds to the ribosomal protein MoRpl12 and inhibits the pathogenicity of rice blast fungus, providing a new potential target for rice blast prevention and control.

Colletotrichum species associated with anthracnose disease on mango (Mangifera indica L.) cv. Azúcar in Colombia

The role of some Colletotrichum species is uncertain in crops of economic importance for Colombia such as mango (Mangifera indica L.) cv. Azúcar. Eight Colletotrichum isolates from asymptomatic plant tissues were characterized by their pathogenicity on fruits and identified by phylogenetic analysis of the sequences of the ITS, GADPH, Tub2, CHs-1 and Act genomic regions. Colletotrichum asianum, C. gloeosporioides and C. tropicale of the C. gloeosporioides species complex and C. karstii of the C. boninense species complex were identified. The highest virulence was observed for isolates of C. asianum, that was the most prevalent species, and for C. tropicale, that showed high and intermediate virulence levels, indicating diversity in pathogenicity within the same species. The isolate of C. karstii, which displayed intermediate virulence, is the first report on mango in Colombia. Colletotrichum gloeosporioides presented intermediate virulence and was isolated at a low frequency in the present research. The mycelial growth rate did not show differences between species and ranged between 6.1 and 8.3 mm per day. Colletotrichum asianum recorded higher values of conidia germination and appressoria formation than other species, at 77% and 79%, respectively (p < 0.001). The shape and size of conidia did not vary between species. Appressoria exhibited different shapes between isolates and the largest average diameter was for C. karstii with 7.6 µm (p < 0.001). There was a diversity of Colletotrichum species associated with mango cv. Azúcar in Colombia, with differential virulence among isolates of the same species.

PARylation of 14-3-3 proteins controls the virulence of Magnaporthe oryzae

Magnaporthe oryzae is a devastating fungal pathogen that causes the rice blast disease worldwide. The post-translational modification of ADP-ribosylation holds significant importance in various fundamental biological processes. However, the specific function of this modification in M. oryzae remains unknown. This study revealed that Poly(ADP-ribosyl)ation (PARylation) executes a critical function in M. oryzae. M. oryzae Poly(ADP-ribose) polymerase 1 (PARP1) exhibits robust PARylation activity. Disruption of PARylation by PARP1 knock-out or chemical inhibition reveals its involvement in M. oryzae virulence, particularly in appressorium formation. Furthermore, we identified two M. oryzae 14-3-3 proteins, GRF1 and GRF2, as substrates of PARP1. Deletion of GRF1 or GRF2 results in delayed and dysfunctional appressorium, diminished plant penetration, and reduced virulence of the fungus. Biochemical and genetic evidence suggest that PARylation of 14-3-3s is essential for its function in M. oryzae virulence. Moreover, PARylation regulates 14-3-3 dimerization and is required for the activation of the mitogen-activated protein kinases (MAPKs), Pmk1 and Mps1. GRF1 interacts with both Mst7 and Pmk1, and bridges their interaction in a PARylation-dependent manner. This study unveils a distinctive mechanism that PARylation of 14-3-3 proteins controls appressorium formation through MAPK activation, and could facilitate the development of new strategies of rice blast disease control.

The Fungal Transcription Factor BcTbs1 from Botrytis cinerea Promotes Pathogenicity via Host Cellulose Degradation.

Zn(II)2Cys6 proteins constitute the largest group of fungal-specific transcription factors. However, little is known about their functions in the crop killer Botrytis cinerea. In this work, a T-DNA insertion strain M13448 was identified which was inserted into the Zn(II)2Cys6 TF-encoding gene BcTBS1. Knockout of BcTBS1 did not affect mycelia growth, appressorium formation, and sclerotium germination, but impaired fungal conidiation, conidial morphogenesis, conidial germination, infection cushion development, and sclerotial formation. Accordingly, ΔBctbs1 mutants showed reduced virulence in its host plants. Further study proved that BcTBS1, BCIN_15g03870, and BCIN_12g06630 were induced by cellulose. Subsequent cellulase activity assays revealed that the loss of BcTBS1 significantly decreased cellulase activity. In addition, we verified that the BCIN_15g03870 and BCIN_12g06630 genes were positive regulated by BcTBS1 by quantitative real-time reverse-transcription-polymerase chain reaction (qRT-PCR). Taken together, these results suggested that BcTBS1 can promote pathogenicity by modulating cellulase-encoding genes that participate in host cellulose degradation.

Novel cellular functions of Cys2-His2 zinc finger proteins in anthracnose development and dissemination on pepper fruits by Colletotrichum scovillei.

Colletotrichum species are notorious for causing anthracnose on many fruits, leading to significant economic losses worldwide. As a model, we functionally characterized cys2-his2 (C2H2) zinc finger proteins (CsCZFs) in Colletotrichum scovillei, a major causal agent of pepper fruit anthracnose in many countries. In all, 62 CsCZFs were identified by in silico genomic analysis. Twelve were selected based on their expression profiles to generate targeted deletion mutants for functional investigation. ΔCsczf1 markedly reduced conidiation and constitutive expression of CsCZF1 partially recovered conidiation in an asexual reproduction-defective mutant, ΔCshox2. Deletion of CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, impaired autophagy in C. scovillei. ΔCsczf9 was defective in surface recognition, appressorium formation, and suppression of host defenses. CsCZF9 was identified as an essential and novel regulator under the control of the mitogen-activated protein kinase (CsPMK1) in an early step of appressorium development in C. scovillei. This study provides novel insights into CsCZF-mediated regulation of differentiation and pathogenicity in C. scovillei, contributing to understanding the regulatory mechanisms governing fruit anthracnose epidemics.IMPORTANCEThe phytopathogenic fungus Colletotrichum scovillei is known to cause serious anthracnose on chili pepper. However, the molecular mechanism underlying anthracnose caused by this fungus remains largely unknown. Here, we systematically analyzed the functional roles of cys2-his2 zinc finger proteins (CsCZFs) in the dissemination and pathogenic development of this fungus. Our results showed that CsCZF1 plays an important role in conidiation and constitutive expression of CsCZF1 restored conidiation in an asexual reproduction-defective mutant, ΔCshox2. The CsCZF9, a novel target of the mitogen-activated protein kinase (CsPMK1), is essential for surface recognition to allow appressorium formation and suppression of host defenses in C. scovillei. The CsCZF12, orthologous to the calcineurin-responsive transcription factor Crz1, is involved in the autophagy of C. scovillei. Our findings reveal a comprehensive mechanism underlying CsCZF-mediated regulation of differentiation and pathogenicity of C. scovillei, which contributes to the understanding of fruit anthracnose epidemics and the development of novel strategies for disease management.

GPI anchoring controls cell wall integrity, immune evasion and surface localization of ChFEM1 for infection of Cochlibolus heterostrophus1

Glycosylphosphatidylinositol (GPI) anchoring is one of the common post-translational modifications in eukaryotic cells. In fungi, it exerts a wide range of biological functions by targeting proteins to the cell wall, but only few studies focus on the roles of GPI anchoring in plant pathogenic fungi. Here, we reveal a role of GPI anchoring in the maize fungal pathogen Cochlibolus heterostrophus. We found that GPI-anchored proteins were widely accumulated in hyphae, appressorium and infection hyphae of C. heterostrophus. Deletion of ChGPI7, which encodes a key enzyme involved in the biosynthesis of GPI anchors, resulted in significant reduction of vegetative growth and conidiation, as well as virulence due to impairment of appressorium formation and invasive growth. The ΔChgpi7 mutants also showed severe defects in cell wall integrity, resulting in a significant reduction of stress resistance. Deletion of ChGPI7 and hydrofluoric acid (HF) pyridine treatment both led to removal of cell wall GPI-anchored proteins and exposure of chitin, the results suggested that GPI anchored proteins could protect chitin from host immune recognition. A total of 124 proteins were predicted to be GPI anchored proteins in C. heterostrophus, including a putative cell wall glycoprotein ChFEM1. Deletion of ChFEM1 also resulted in significant reduction in virulence and defects in infection structures, as well as cell wall integrity. We further found that cell wall localization and protein abundance of ChFEM1 were affected by ChGPI7. Our results showed that GPI anchoring regulates cell wall integrity and immune evasion for infection of C. heterostrophus.

Exploring the efficacy of endophytic Diaporthe caatingaensis as a biocontrol agent targeting Fusarium strains afflicting coffee plants in Saudi Arabia

BackgroundThe coffee plant is a strategic crop in Saudi Arabia that makes a substantial contribution to the country’s economy. Therefore, it is crucial to continuously monitor and control the fungal phytopathogens that affect coffee plants in order to minimize crop losses and ensure sustainable cultivation. This is the first surveillance report of Fusarium phytopathogens associated with coffee plants in Saudi Arabia. Moreover, the antagonistic efficiency of the endophytic fungus, Diaporthe caatingaensis, was evaluated against the isolated Fusarium phytopathogens. MethodsThe isolated strains were preliminary identified using cultural and microscopic methods and the identification was confirmed using internal transcribed spacer (ITS) sequencing technique. The detached leaf assay was conducted to assess the disease severity of Fusarium phytopathogens against detached coffee leaves. Moreover, dual culture assay was utilized to assess the antagonistic activity of D. caatingaensis. Results and conclusionFusarium oxysporum was found to be the most frequent isolated strain followed by F. solani, F. proliferatum and F. verticillioides strains. Fusarium proliferatum strain was found to be the most severe strain whereas F. solani strain showed the lowest disease severity. On the other hand, propiconazole fungicide was tested for its efficiency against Fusarium pathogens using food poisoning technique, showing that F. oxysporum strain 1 of accession number OP955665 was the most sensitive strain. However, F. proliferatum, F. oxysporum st. 2 (OP959851) and F. solani strains showed no significant response when the propiconazole concentration increase from 150 to 200 ppm. Scanning electron microscope proved the potent antagonistic activity of D. caatingaensis against F. proliferatum and F. oxysporum OP959874 strains through mycoparasitic modes of action as coiling, appressorium formation resulting in complete lysis of fungal mycelium. Accordingly, the current investigation provides the first surveillance data about Fusarium strains associated with coffee plants and also the utilization of D. caatingaensis as a potential biocontrol agent for effective management of Fusarium phytopathogens, avoiding the incidence of fungal resistance to fungicides and the harmful effects of commercial fungicides for sustainable cultivation of coffee in Saudi Arabia.

Antifungal characterizations of a novel endo-β-1,6-glucanase from Flavobacterium sp. NAU1659

β-1,6-Glucan plays a crucial role in fungal cell walls by linking the outer layer of mannoproteins and the inner layer of β-1,3-glucan, contributing significantly to the maintenance of cell wall rigidity. Therefore, the hydrolysis of β-1,6-glucan by β-1,6-glucanase directly leads to the disintegration of the fungal cell wall. Here, a novel β-1,6-glucanase FlGlu30 was identified from the endophytic Flavobacterium sp. NAU1659 and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of purified FlGlu30 were 50℃ and pH 6.0, resulting in a specific activity of 173.1 U/mg using pustulan as the substrate. The hydrolyzed products of FlGlu30 to pustulan were mainly gentianose within 1 h of reaction. With the extension of reaction time, gentianose was gradually hydrolyzed to glucose, indicating that FlGlu30 is an endo-β-1,6-glucanase. The germination of Magnaporthe oryzae Guy11 spores could not be inhibited by FlGlu30, but the appressorium formation of spores was completely inhibited under the concentration of 250.0 U/mL FlGlu30. The disruptions of cell wall and accumulation of intracellular reactive oxide species (ROS) were observed in FlGlu30-treated M. oryzae Guy11 cells, suggesting the significant importance of β-1,6-glucan as a potential antifungal target and the potential application of FlGlu30.Key points• β-1,6-Glucan is a key component maintaining the rigid structure of fungal cell wall.• β-1,6-Glucanase is an antifungal protein with significant potential applications.• FlGlu30 is the first reported β-1, 6-glucanase derived from Flavobacterium.

Transcription Factor CgSte12 Regulates Pathogenicity by Affecting Appressorium Structural Development in the Anthracnose-Causing Fungus Colletotrichum gloeosporioides.

Colletotrichum gloeosporioides is the causal agent of poplar anthracnose, which induces major economic losses and adversely affects the ecosystem services of poplar forests. The appressorium serves as a penetration structure for many pathogenic fungi, including C. gloeosporioides. The production of mucilage and the formation of penetration pegs are critically important for the appressorium-mediated penetration of host tissues. We previously found that CgPmk1 is a key protein involved in appressorium formation, penetration, and pathogenicity. Although CgSte12, which is a transcription factor that functions downstream of CgPmk1, regulates the formation of penetration pegs, its role in C. gloeosporioides appressorium development and pathogenicity has not been elucidated. Here, we developed C. gloeosporioides CgSTE12 mutants and characterized the molecular and cellular functions of CgSTE12. The results showed that mycelial growth and morphology were not affected in the CgSTE12 knockout mutants, which produced normal melanized appressoria. However, these mutants had less mucilage secreted around the appressoria, impaired appressorial cone formation, and the inability to form penetration pores and pegs, which ultimately led to a significant loss of pathogenicity. Our comparative transcriptome analysis revealed that CgSte12 controls the expression of genes involved in appressorium development and function, including genes encoding cutinases, NADPH oxidase, spermine biosynthesis-related proteins, ceramide biosynthesis-related proteins, fatty acid metabolism-related proteins, and glycerophospholipid metabolism-related proteins. Overall, our findings indicate that CgSte12 is a critical regulator of appressorium development and affects C. gloeosporioides pathogenicity by modulating the structural integrity of appressoria.

Oleic Acid and Linoleic Acid Enhances the Biocontrol Potential of Metarhizium rileyi.

Metarhizium rileyi is a wide spread insect fungi with a good biocontrol potentiality to various pests, particularly noctuid insects. However, it is characterized by its slow growth, its sensitivity to abiotic stress, and the slow speed of kill to pests, which hinder its use compared with other entomopathogenic fungi. In this study, the responses of M. rileyi to eight types of lipids were observed; among the lipids, oleic acid and linoleic acid significantly promoted the growth and development of M. rileyi and enhanced its stress tolerances and virulence. An additional mechanistic study demonstrated that exogenous oleic acid and linoleic acid significantly improved the conidial germination, appressorium formation, cuticle degradation, and cuticle infection, which appear to be largely dependent on the up-regulation of gene expression in growth, development, protective, and cuticle-degrading enzymes. In conclusion, exogenous oleic acid and linoleic acid enhanced the stress tolerances and virulence of M. rileyi via protecting conidial germination and promoting cuticle infection. These results provide new insights for the biopesticide development of M. rileyi.

Functional Characterization of the Transcription Factor Gene CgHox7 in Colletotrichum gloeosporioides, Which Is Responsible for Poplar Anthracnose.

Colletotrichum gloeosporioides is the main pathogen that causes poplar anthracnose. This hemibiotrophic fungus, which can severely decrease the economic benefits and ecological functions of poplar trees, infects the host by forming an appressorium. Hox7 is an important regulatory factor that functions downstream of the Pmk1 MAPK signaling pathway. In this study, we investigated the effect of deleting CgHox7 on C. gloeosporioides. The conidia of the ΔCgHox7 deletion mutant germinated on a GelBond membrane to form non-melanized hyphal structures, but were unable to form appressoria. The deletion of CgHox7 weakened the ability of hyphae to penetrate a cellophane membrane and resulted in decreased virulence on poplar leaves. Furthermore, deleting CgHox7 affected the oxidative stress response. In the initial stage of appressorium formation, the accumulation of reactive oxygen species differed between the ΔCgHox7 deletion mutant and the wild-type control. Moreover, CgHox7 expression was necessary for maintaining cell wall integrity. Considered together, these results indicate that CgHox7 is a transcription factor with crucial regulatory effects on appressorium formation and the pathogenicity of C. gloeosporioides.

The C2H2 Transcription Factor Con7 Regulates Vegetative Growth, Cell Wall Integrity, Oxidative Stress, Asexual Sporulation, Appressorium and Hyphopodium Formation, and Pathogenicity in Colletotrichum graminicola and Colletotrichum siamense.

The Colletotrichum genus is listed as one of the top 10 important plant pathogens, causing significant economic losses worldwide. The C2H2 zinc finger protein serves as a crucial transcription factor regulating growth and development in fungi. In this study, we identified two C2H2 transcription factors, CgrCon7 and CsCon7, in Colletotrichum graminicola and Colletotrichum siamense, as the orthologs of Con7p in Magnaporthe oryzae. Both CgrCon7 and CsCon7 have a typical C2H2 zinc finger domain and exhibit visible nuclear localization. Disrupting Cgrcon7 or Cscon7 led to a decreased growth rate, changes in cell wall integrity, and low tolerance to H2O2. Moreover, the deletion of Cgrcon7 or Cscon7 dramatically decreased conidial production, and their knockout mutants also lost the ability to produce appressoria and hyphopodia. Pathogenicity assays displayed that deleting Cgrcon7 or Cscon7 resulted in a complete loss of virulence. Transcriptome analysis showed that CgrCon7 and CsCon7 were involved in regulating many genes related to ROS detoxification, chitin synthesis, and cell wall degradation, etc. In conclusion, CgrCon7 and CsCon7 act as master transcription factors coordinating vegetative growth, oxidative stress response, cell wall integrity, asexual sporulation, appressorium formation, and pathogenicity in C. graminicola and C. siamense.

A Promising Biocontrol Agent of Bacillus velezensis VC3 against Magnaporthe oryzae and Colletotrichum gloeosporioides in Plants

Fungal diseases of plants are one of the key factors causing global crop losses. In this study, we isolated a Bacillus velezensis strain VC3, which was found to have a broad-spectrum inhibitory effect on a variety of phytopathogenic fungi through in vitro and in planta experiments, especially on Magnaporthe oryzae and Colletotrichum gloeosporioides. Further genomic and transcriptomic analyses revealed that the B. velezensis VC3 has multiple functional gene clusters encoding for the synthesis of a variety of antifungal secondary metabolites, including antimicrobial peptides (AMPs) and lipopeptides (LPs). In addition, AMPs and LPs were isolated and purified from B. velezensis VC3 fermentation broth and their antifungal activities were verified in this study. AMPs and LPs significantly inhibited spore germination, appressorium formation, and disease development, and AMPs have a better potential for controlling M. oryzae and C. gloeosporioides than LPs. These findings open new avenues for utilizing B. velezensis VC3 as biocontrol agents, providing potential sustainable solutions for agricultural production.

Dihydroorotase MoPyr4 is required for development, pathogenicity, and autophagy in rice blast fungus

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.

MoSey1 regulates the unfolded protein response, appressorium development, and pathogenicity of Magnaporthe oryzae

Endoplasmic reticulum (ER) is an enclosed three-dimensional eukaryotic membrane network composed of flattened sacs. Fusion of homologous membranes to the ER membrane is essential for the maintenance of this network structure. In yeast, ER membrane fusion is mediated by Sey1p, whose paralogues function distinctly in different species. In this study, we investigated the biological functions of MoSEY1 in the devastating rice blast fungus Magnaporthe oryzae by functional genomic approach. Compared to wild type, deletion of MoSEY1 considerably decreased the growth and conidia production of M. oryzae. Additionally, the absence of MoSEY1 delayed appressorium formation and invasive hyphae growth. The appressorium function was also impaired in ΔMosey1 mutant. Subcellular localization analysis revealed that MoSey1 is localized at the ER. The ΔMosey1 mutant showed augmented sensitivity to ER stress. Additionally, we found that MoSey1 regulated the unfolded protein response, autophagy, and protein secretion in M. oryzae. In conclusion, our study unveiled the involvement of MoSey1 in the development, pathogenesis, and ER functions in M. oryzae.

MoRgs3 functions in intracellular reactive oxygen species perception-integrated cAMP signaling to promote appressorium formation in Magnaporthe oryzae.

We report that MoRgs3 becomes phosphorylated in an oxidative intracellular environment during the appressorium formation stage. We found that this phosphorylation is carried out by MoNdk1, a nucleoside diphosphate kinase. In addition, this phosphorylation leads to a higher binding affinity between MoRgs3 and MoCrn1, a coronin-like actin-binding protein that was implicated in the endocytic transport of several other RGS proteins of Magnaporthe oryzae. We further found that the internalization of MoRgs3 is indispensable for its GTPase-activating protein function toward the Gα subunit MoMagA. Importantly, we characterized how such cellular regulatory events coincide with cAMP signaling-regulated appressorium formation and pathogenicity in the blast fungus. Our studies uncovered a novel intracellular reactive oxygen species signal-transducing mechanism in a model pathogenic fungus with important basic and applied implications.