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Isw2 proteins, ubiquitous across eukaryotes, exhibit a propensity for DNA binding and exert dynamic influences on local chromosome condensation in an ATP-dependent fashion, thereby modulating the accessibility of neighboring genes to transcriptional machinery. Here, we report the deletion of a putative MoISW2 gene, yielding substantial ramifications on plant pathogenicity. Subsequent gene complementation and chromatin immunoprecipitation sequencing (ChIP-seq) analyses were conducted to delineate binding sites. RNA sequencing (RNA-seq) assays revealed discernible impacts on global gene regulation along chromosomes in both mutant and wild-type strains, with comparative analyses against 55 external RNA-seq data sets corroborating these findings. Notably, MoIsw2-mediated binding and activities delineate genomic loci characterized by pronounced gene expression variability proximal to MoIsw2 binding sites, juxtaposed with comparatively stable expression in surrounding regions. The contingent genes influenced by MoIsw2 activity predominantly encompass niche-determinant genes, including those encoding secreted proteins, secondary metabolites, and stress-responsive elements, alongside avirulence genes. Furthermore, our investigations unveil a spatial correlation between MoIsw2 binding motifs and known transposable elements (TEs), suggesting a potential interplay wherein TE transposition at these loci could modulate the transcriptional landscape of Magnaporthe oryzae in a strain-specific manner. Collectively, these findings position MoIsw2 as a plausible master regulator orchestrating the delicate equilibrium between genes vital for biomass proliferation, akin to housekeeping genes, and niche-specific determinants crucial for ecological adaptability. Stress-induced TE transposition, in conjunction with MoIsw2 activity, emerges as a putative mechanism fostering enhanced mutagenesis and accelerated evolution of niche-determinant genes relative to housekeeping counterparts.IMPORTANCEIsw2 proteins are conserved in plants, fungi, animals, and other eukaryotes. We show that a fungal Isw2 protein in the rice pathogen Magnaporthe oryzae binds to retrotransposon (RT) DNA motifs and affects the epigenetic gene expression landscape of the fungal genome. Mainly ecological niche determinant genes close to the binding motifs are affected. RT elements occur frequently in DNA between genes in most organisms. They move place and multiply in the genome, especially under physiological stress. We further discuss the Isw2 and RT combined activities as a possible sought-after mechanism that can cause biased mutation rates and faster evolution of genes necessary for reacting to abiotic and biotic challenges. The most important biotic challenges for plant pathogens are the ones from the host plants' innate immunity. The overall result of these combined activities will be an adaptation-directed evolution of niche-determinant genes., Competing Interests: The authors declare no conflict of interest.

Signal peptidase (SPase) is responsible for cleavage of N-terminal signal peptides in most secretory precursor proteins and many membrane proteins during maturation. In this study, we identified four components of the SPase complex (FoSec11, FoSpc1, FoSpc2, and FoSpc3) in the banana wilt fungal pathogen Fusarium odoratissimum. We proved that interactions exist among the four SPase subunits by bimolecular fluorescence complementation (BiFC) and affinity purification and mass spectrometry (AP-MS) assays. Among the four SPase genes, FoSPC2 was successfully deleted. FoSPC2 deletion caused defects in vegetative growth, conidiation, and virulence. Loss of FoSPC2 also affected the secretion of some pathogenicity-related extracellular enzymes, suggesting that SPase without FoSpc2 may have a lower efficiency in managing the maturation of the extracellular enzymes in F. odoratissimum . In addition, we found that the Δ FoSPC2 mutant had increased sensitivity to light, and the colonies of the mutant grew faster under all-dark conditions than under all-light conditions. We further observed that deletion of FoSPC2 affected expression of the blue light photoreceptor gene FoWC2 , leading to cytoplasmic accumulation of FoWc2 under all-light conditions. Since FoWc2 has signal peptides, FoSpc2 may regulate the expression and subcellular localization of FoWc2 indirectly. Contrary to its response to light, the Δ FoSPC2 mutant displayed a significant decreased sensitivity to osmotic stress, and culturing the mutant under osmotic stress conditions restored both the localization of FoWc2 and light sensitivity of the Δ FoSPC2 , suggesting that a cross talk between osmotic stress and light response pathways in F. odoratissimum and FoSpc2 takes part in these processes. IMPORTANCE In this study, we identified four components of SPase in the banana wilt pathogen Fusarium odoratissimum and characterized the SPase FoSpc2. Loss of FoSPC2 affected the secretion of extracellular enzymes, suggesting that SPase without FoSpc2 may have a lower efficiency in managing the maturation of the extracellular enzymes in F. odoratissimum . In addition, this is the first time that we have found a relationship between the SPase and fungal light response. Deletion of FoSPC2 resulted in decreased sensitivity to the osmotic stresses but with increased sensitivity to light. Continuous light inhibited the growth rate of the Δ FoSPC2 mutant and affected the cellular localization of the blue light photoreceptor FoWc2 in this mutant, but culturing the mutant under osmotic stress both restored the localization of FoWc2 and eliminated the light sensitivity of the Δ FoSPC2 mutant, suggesting that loss of FoSPC2 may affect a cross talk between the osmotic stress and light response pathways in F. odoratissimum ., Competing Interests: The authors declare no conflict of interest.

Hyphal polarized growth in filamentous fungi requires tip-directed secretion, while additional evidence suggests that fungal exocytosis for the hydrolytic enzyme secretion can occur at other sites in hyphae, including the septum. In this study, we analyzed the role of the exocyst complex involved in the secretion in the banana wilt fungal pathogen Fusarium odoratissimum. All eight exocyst components in F. odoratissimum not only localized to the tips ahead of the Spitzenkörper in growing hyphae but also localized to the outer edges of septa in mature hyphae. To further analyze the exocyst in F. odoratissimum , we attempted single gene deletion for all the genes encoding the eight exocyst components and only succeeded in constructing the gene deletion mutants for exo70 and sec5 ; we suspect that the other 6 exocyst components are encoded by essential genes. Deletion of exo70 or sec5 led to defects in vegetative growth, conidiation, and pathogenicity in F. odoratissimum . Notably, the deletion of exo70 resulted in decreased activities for endoglucosidase, filter paper enzymes, and amylase, while the loss of sec5 only led to a slight reduction in amylase activity. Septum-localized α-amylase (AmyB) was identified as the marker for septum-directed secretion, and we found that Exo70 is essential for the localization of AmyB to septa. Meanwhile the loss of Sec5 did not affect AmyB localization to septa but led to a higher accumulation of AmyB in cytoplasm. This suggested that while Exo70 and Sec5 both take part in the septum-directed secretion, the two play different roles in this process. IMPORTANCE The exocyst complex is a multisubunit tethering complex (MTC) for secretory vesicles at the plasma membrane and contains eight subunits, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84. While the exocyst complex is well defined in eukaryotes from yeast ( Saccharomyces cerevisiae ) to humans, the exocyst components in filamentous fungi show different localization patterns in the apical tips of hyphae, which suggests that filamentous fungi have evolved divergent strategies to regulate endomembrane trafficking. In this study, we demonstrated that the exocyst components in Fusarium odoratissimum are localized not only to the tips of growing hyphae but also to the outer edge of the septa in mature hyphae, suggesting that the exocyst complex plays a role in the regulation of septum-directed protein secretion in F. odoratissimum . We further found that Exo70 and Sec5 are required for the septum-directed secretion of α-amylase in F. odoratissimum but with different influences.

The membrane trafficking system is important for compartmentalization of the biosynthesis pathway and secretion of deoxynivalenol (DON) mycotoxin (a virulence factor) in Fusarium graminearum. Flippases are transmembrane lipid transporters and mediate a number of essential physiological steps of membrane trafficking, including vesicle budding, charging, and protein diffusion within the membrane. However, the roles of flippases in secondary metabolism remain unknown in filamentous fungi. Herein, we identified five flippases (FgDnfA, FgDnfB, FgDnfC1, FgDnfC2, and FgDnfD) in F. graminearum and established their specific and redundant functions in the development and pathogenicity of this phytopathogenic fungus. Our results demonstrate that FgDnfA is critical for normal vegetative growth while the other flippases are dispensable. FgDnfA and FgDnfD were found crucial for the fungal pathogenesis, and a remarkable reduction in DON production was observed in ΔFgDNFA and ΔFgDNFD. Deletion of the FgDNFB gene increased DON production to about 30 times that produced by the wild type. Further analysis showed that FgDnfA and FgDnfD have positive roles in the regulation of trichothecene (TRI) genes (TRI1, TRI4, TRI5, TRI6, TRI12, and TRI101) expression and toxisome reorganization, while FgDnfB acts as a negative regulator of DON synthesis. In addition, FgDnfB and FgDnfD have redundant functions in the regulation of phosphatidylcholine transport, and double deletion of FgDNFB and FgDNFD showed serious defects in fungal development, DON synthesis, and virulence. Collectively, our findings reveal the distinct and specific functions of flippase family members in F. graminearum and principally demonstrate that FgDnfA, FgDnfD, and FgDnfB have specific spatiotemporal roles during toxisome biogenesis., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. The vacuolar protein sorting (Vps) protein Vps27 is a component of ESCRT-0 involved in the multivesicular body (MVB) sorting pathway during endocytosis. In this study, we investigated the function of FgVps27 using a gene replacement strategy. The FgVPS27 deletion mutant (ΔFgvps27) exhibited a reduction in growth rate, aerial hyphae formation and hydrophobicity. It also showed increased sensitivity to cell wall-damaging agents and to osmotic stresses. In addition, FgHog1, the critical component of high osmolarity glycerol response pathway, was mis-localized in the ΔFgvps27 mutant upon NaCl treatment. Furthermore, the ΔFgvps27 mutant was defective in conidial production and was unable to generate perithecium in sexual reproduction. The depletion of FgVPS27 also caused a significant reduction in virulence. Further analysis by domain-specific deletion revealed that the FYVE domain was essential for the FgVps27 function and was necessary for the proper localization of FgVps27-GFP and endocytosis. Another component of ESCRT-0, the FgVps27-interacting partner FgHse1, also played an important role in F. graminearum development and pathogenesis. Overall, our results indicate that ESCRT-0 components play critical roles in a variety of cellular and biological processes., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Electron-transferring flavoprotein (ETF) and its dehydrogenase (ETFDH) are highly conserved electron carriers which mainly function in mitochondrial fatty acid β oxidation. Here, we report the identification and characterization of ETF α and β subunit encoding genes (ETFA and ETFB) and ETFDH encoding gene (ETFDH) in the rice blast fungus Magnaporthe oryzae. It was demonstrated that, by impacting fatty acid metabolism, ETF and ETFDH mutations led to severe growth and conidiation defects, which could be largely rescued by exogenous acetate or carbonate. Furthermore, although conidium germination and appressorium formation appeared to be normal in ETF and ETFDH mutants, most appressoria failed to penetrate the host epidermis due to low turgor pressure. The few appressoria that succeeded in penetration were severely restricted in invasive growth and consequently failed to cause disease. Moreover, ETF mutant etfb(-) induced ROS accumulation in infected host cells and exogenous antioxidant GSH accelerated mutant invading growth without increasing the penetration rate. In addition, mutant etfb(-) displayed elevated lipid body accumulation and reduced ATP synthesis. Taken together, ETF and ETFDH play an important role in fungal development and plant infection in M. oryzae by regulation of fatty acid metabolism, turgor establishment and induction of host ROS accumulation.

We identified the Magnaporthe oryzae avirulence effector AvrPi9 cognate to rice blast resistance gene Pi9 by comparative genomics of requisite strains derived from a sequential planting method. AvrPi9 encodes a small secreted protein that appears to localize in the biotrophic interfacial complex and is translocated to the host cell during rice infection. AvrPi9 forms a tandem gene array with its paralogue proximal to centromeric region of chromosome 7. AvrPi9 is expressed highly at early stages during initiation of blast disease. Virulent isolate strains contain Mg-SINE within the AvrPi9 coding sequence. Loss of AvrPi9 did not lead to any discernible defects during growth or pathogenesis in M. oryzae. This study reiterates the role of diverse transposable elements as off-switch agents in acquisition of gain-of-virulence in the rice blast fungus. The prevalence of AvrPi9 correlates well with the avirulence pathotype in diverse blast isolates from the Philippines and China, thus supporting the broad-spectrum resistance conferred by Pi9 in different rice growing areas. Our results revealed that Pi9 and Piz-t at the Pi2/9 locus activate race specific resistance by recognizing sequence-unrelated AvrPi9 and AvrPiz-t genes, respectively., (No claim to original Chinese government works New Phytologist © 2015 New Phytologist Trust.)

Rab GTPases are master regulators of intracellular membrane trafficking along endocytic and exocytic pathways. In this chapter, we began to characterize the exocytic and recycling Rabs from the filamentous fungus Magnaporthe oryzae (M. oryzae) that causes the rice blast disease. Among the 11 putative Rabs identified from the M. oryzae genome database (MoRabs), MoRab1, MoRab8, and MoRab11 appear orthologs of mammalian Rab1, Rab8, and Rab11 and likely function in exocytosis and endosomal recycling. To test this contention, we cloned, expressed, and determined intracellular localization of the three MoRabs in mammalian cells, in comparison to their human counterparts (hRabs). The MoRabs were well expressed as GFP fusion proteins and colocalized with the tdTomato-labeled hRabs on exocytic and recycling organelles, as determined by immunoblot analysis and confocal fluorescence microscopy. The colocalization supports the contention that the MoRabs are indeed Rab orthologs and may play important roles in the development and pathogenicity of M. oryzae., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Rab5 is a key regulator of early endocytosis by promoting early endosomal fusion and motility. In this study, we have unexpectedly found distinct properties of the two Rab5 homologs (MoRab5A and MoRab5B) from Magnaporthe oryzae, a pathogenic fungus in plants whose infection causes rice blast disease. Like mammalian Rab5, MoRab5A and MoRab5B can bind to several Rab5 effectors in a GTP-dependent manner, including EEA1, Rabenosyn-5, and Rabaptin-5. However, MoRab5A shows distinct binding characteristics in the sense that both the wild-type and the GTP hydrolysis-defective constitutively active mutant bind the effectors equally well in GST pull-down assays, suggesting that MoRab5A is defective in GTP hydrolysis and mostly in the GTP-bound conformation in the cell. Indeed, GTP hydrolysis assays indicate that MoRab5A GTPase activity is dramatically lower than MoRab5B and human Rab5 and is insensitive to RabGAP5 stimulation. We have further identified a Pro residue in the switch I region largely responsible for the distinct MoRab5A properties by characterization of MoRab5A and MoRab5B chimeras and mutagenesis. The differences between MoRab5A and MoRab5B extend to their functions in the cell. Although they both target to early endosomes, only MoRab5B closely resembles human Rab5 in promoting early endosome fusion and stimulating fluid phase endocytosis. In contrast, MoRab5A correlates with another related early endosomal Rab, Rab22, in terms of the presence of the switch I Pro residue and the blocked GTPase activity. Our data thus identify MoRab5B as the Rab5 ortholog and suggest that MoRab5A specializes to perform a non-redundant function in endosomal sorting., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rho GTPases, acting as molecular switches, are involved in the regulation of diverse cellular functions. Rho GTPase activating proteins (Rho GAPs) function as negative regulators of Rho GTPases and are required for a variety of signaling processes in cell development. But the mechanisms underlying Rho GAPs in Rho-mediated signaling pathways in fungi are still elusive. There are eight RhoGAP domain-containing genes annotated in the Magnaporthe oryzae genome. To understand the function of these RhoGAP genes, we generated knockout mutants of each of the RhoGAP genes through a homologous recombination-based method. Phenotypic analysis showed that growth rate of aerial hyphae of the Molrg1 deletion mutant decreased dramatically. The ΔMolrg1 mutant showed significantly reduced conidiation and appressorium formation by germ tubes. Moreover, it lost pathogenicity completely. Deletion of another Rho GAP (MoRga1) resulted in high percentage of larger or gherkin-shaped conidia and slight decrease in conidiation. Appressorial formation of the ΔMoRga1 mutant was delayed significantly on hydrophobic surface, while the development of mycelial growth and pathogenicity in plants was not affected. Confocal fluorescence microscopy imaging showed that MoRga1-GFP localizes to septal pore of the conidium, and this localization pattern requires both LIM and RhoGAP domains. Furthermore, either deleting the LIM or RhoGAP domain or introducing an inactivating R1032A mutation in the RhoGAP domain of MoRga1 caused similar defects as the Morga1 deletion mutant in terms of conidial morphology and appressorial formation, suggesting that MoRga1 is a stage-specific regulator of conidial differentiation by regulating some specific Rho GTPases. In this regard, MoRga1 and MoLrg1 physically interacted with both MoRac1-CA and MoCdc42-CA in the yeast two-hybrid and pull-down assays, suggesting that the actions of these two GAPs are involved in MoRac1 and MoCdc42 pathways. On the other hand, six other putative Rho GAPs (MoRga2 to MoRga7) were dispensable for conidiation, vegetative growth, appressorial formation and pathogenicity, suggesting that these Rho GAPs function redundantly during fungal development. Taking together, Rho GAP genes play important roles in M. oryzae development and infectious processes through coordination and modulation of Rho GTPases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Conidia are primary means of asexual reproduction and dispersal in a variety of pathogenic fungi, and it is widely recognized that they play a critical role in animal and plant disease epidemics. However, genetic mechanisms associated with conidiogenesis are complex and remain largely undefined in numerous pathogenic fungi. We previously showed that Htf1, a homeobox transcription factor, is required for conidiogenesis in the rice pathogen Magnaporthe oryzae. In this study, our aim was to characterize how Htf1 homolog regulates common and also distinctive conidiogenesis in three key Fusarium pathogens: F. graminearm, F. verticillioides, and F. oxysporum. When compared to wild-type progenitors, the gene-deletion mutants in Fusarium species failed to form conventional phialides. Rather, they formed clusters of aberrant phialides that resembled elongated hyphae segments, and it is conceivable that this led to the obstruction of conidiation in phialides. We also observed that mutants, as well as wild-type Fusaria, can initiate alternative macroconidia production directly from hyphae through budding-like mechanism albeit at low frequencies. Microscopic observations led us to conclude that proper basal cell division and subsequent foot cell development of macroconidia were negatively impacted in the mutants. In F. verticillioides and F. oxysporum, mutants exhibited a 2- to 5- microconidia complex at the apex of monophialides resulting in a floral petal-like shape. Also, prototypical microconidia chains were absent in F. verticillioides mutants. F. graminearum and F. verticillioides mutants were complemented by introducing its native HTF1 gene or homologs from other Fusarium species. These results suggest that Fusarium Htf1 is functionally conserved homeobox transcription factor that regulates phialide development and conidiogenesis via distinct signaling pathways yet to be characterized in fungi.

Cdc42, a member of the Rho-family small GTP-binding proteins, is a pivotal signaling switch that cycles between active GTP-bound and inactive GDP-bound forms, controlling actin cytoskeleton organization and cell polarity. In this report, we show that MgCdc42, a Cdc42 ortholog in Magnaporthe grisea, is required for its plant penetration. Consequently, the deletion mutants show dramatically decreased virulence to rice due to the arrest of penetration and infectious growth, which may be attributed to the defect of turgor and superoxide generation during the appressorial development in Mgcdc42 deletion mutants. In addition, the mutants also exhibit pleotropic defects including gherkin-shaped conidia, delayed germination as well as decreased sporulation. Furthermore, dominant negative mutation leads to a similar phenotype to that of the deletion mutants, lending further support to the conclusion that MgCdc42 is required for the penetration and virulence of M. grisea.

The Magnaporthe oryzae avirulence gene AvrPiz-t activates immunity in a gene-for-gene fashion to rice mediated by the blast resistance gene Piz-t. To dissect the molecular mechanism underlying their recognition, we initiated the cloning of AvrPiz-t using a map-based cloning strategy. The AvrPiz-t gene was delimited to an approximately 21-kb genomic fragment, in which six genes were predicted. Complementation tests of each of these six candidate genes led to the final identification of AvrPiz-t, which encodes a 108-amino-acid predicted secreted protein with unknown function and no homologues in M. oryzae or in other sequenced fungi. We found that AvrPiz-t is present in the virulent isolate GUY11 but contains a Pot3 insertion at a position 462 bp upstream from the start codon. Complementation tests of AvrPiz-t genes driven by promoters of varying length revealed that a promoter larger than 462 bp is essential to maintain the AvrPiz-t function. These results suggest that a Pot3 insertion in GUY11 might interfere with the proper function of AvrPiz-t. Additionally, we found that AvrPiz-t can suppress the programmed cell death triggered by mouse BAX protein in Nicotiana benthamiana, identifying a mechanism by which AvrPiz-t may contribute virulence of M. oryzae.

The small GTPase Rho3 is conserved in fungi and plays a key role in the control of cell polarity and exocytosis in yeast. In this report, we show that a Rho3 homolog, MgRho3, is dispensable for polarized hyphal growth in the rice blast fungus Magnaporthe grisea. However, MgRho3 is required for plant infection. Appressoria formed by the Mgrho3 deletion mutants are morphologically abnormal and defective in plant penetration. Conidia of the Mgrho3 deletion mutants are narrower than those of the wild-type strain and delayed in germination. Transformants expressing a dominant negative Mgrho3 allele exhibit similar phenotypes as the Mgrho3 deletion mutant, while transformants expressing a constitutively active allele of MgRho3 can produce normal conidia but remain defective in appressorium formation and plant infection. In contrast, overexpression of wild-type MgRho3 increases the infectivity of M. grisea. Our results reveal a new role for the conserved Rho3 as a critical regulator of developmental processes and pathogenicity of M. grisea.

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases and poses a growing threat to food security worldwide. Like many other filamentous pathogens, rice blast fungus releases multiple types of effector proteins to facilitate fungal infection and modulate host defence responses. However, most of the characterized effectors contain an N‐terminal signal peptide. Here, we report the results of the functional characterization of a nonclassically secreted nuclear targeting effector in M. oryzae (MoNte1). MoNte1 has no signal peptide, but can be secreted and translocated into plant nuclei driven by a nuclear targeting peptide. It could also induce hypersensitive cell death when transiently expressed in Nicotiana benthamiana. Deletion of the MoNTE1 gene caused a significant reduction of fungal growth and conidiogenesis, partially impaired appressorium formation and host colonization, and also dramatically attenuated the pathogenicity. Taken together, these findings reveal a novel effector secretion pathway and deepen our understanding of rice–M. oryzae interactions. [ABSTRACT FROM AUTHOR]