Vegetative Hyphal Growth Research Articles

Streptomyces umbrella toxin particles block hyphal growth of competing species

Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.

MoSnf5 Regulates Fungal Virulence, Growth, and Conidiation in Magnaporthe oryzae.

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.

Respiratory Quinone Switches from Menaquinone to Polyketide Quinone during the Development Cycle in Streptomyces sp. Strain MNU77.

Type III polyketide synthases (PKSs) found across Streptomyces species are primarily known for synthesis of a vast repertoire of clinically and industrially relevant secondary metabolites. However, our understanding of the functional relevance of these bioactive metabolites in Streptomyces physiology is still limited. Recently, a role of type III PKS harboring gene cluster in producing alternate electron carrier, polyketide quinone (PkQ) was established in a related member of the Actinobacteria, Mycobacteria, highlighting the critical role these secondary metabolites play in primary cellular metabolism of the producer organism. Here, we report the developmental stage-specific transcriptional regulation of homologous type III PKS containing gene cluster in freshwater Streptomyces sp. strain MNU77. Gene expression analysis revealed the type III PKS gene cluster to be stringently regulated, with significant upregulation observed during the dormant sporulation stage of Streptomyces sp. MNU77. In contrast, the expression levels of only known electron carrier, menaquinone biosynthetic genes were interestingly found to be downregulated. Our liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of a metabolite extract from the Streptomyces sp. MNU77 spores also showed 10 times more metabolic abundance of PkQs than menaquinones. Furthermore, through heterologous complementation studies, we demonstrate that Streptomyces sp. MNU77 type III PKS rescues a respiratory defect of the Mycobacterium smegmatis type III PKS deletion mutant. Together, our studies reveal that freshwater Streptomyces sp. MNU77 robustly produces novel PkQs during the sporulation stage, suggesting utilization of PkQs as alternate electron carriers across Actinobacteria during dormant hypoxic conditions. IMPORTANCE The complex developmental life cycle of Streptomyces sp. mandates efficient cellular respiratory reconfiguration for a smooth transition from aerated nutrient-rich vegetative hyphal growth to the hypoxic-dormant sporulation stage. Polyketide quinones (PkQs) have recently been identified as a class of alternate electron carriers from a related member of the Actinobacteria, Mycobacteria, that facilitates maintenance of membrane potential in oxygen-deficient niches. Our studies with the newly identified freshwater Streptomyces sp. strain MNU77 show conditional transcriptional upregulation and metabolic abundance of PkQs in the spore state of the Streptomyces life cycle. In parallel, the levels of menaquinones, the only known Streptomyces electron carrier, were downregulated, suggesting deployment of PkQs as universal electron carriers in low-oxygen, unfavorable conditions across the Actinobacteria family.

A cytosolic thiouridylase gene MoCTU2 in Magnaporthe oryzae is important for vegetative hyphal growth, conidiation, and responses to rapamycin and high temperature

Cytoplasmic thiouridylase Ctu1-Ctu2 protein complex is responsible for thiolation on the wobble base U34 of tRNAs and plays an important role in cell development in model organisms. However, the biological function of this protein complex in plant pathogenic fungi remains unknown. In this study, we identified a cytosolic thiouridylase gene MoCTU2 in the rice blast fungus Magnaporthe oryzae. The ΔMoctu2 mutant showed reduced vegetative hyphal growth and conidiation, and increased responses to rapamycin and high temperature. However, MoCTU2 had a minor impact on the ability of the mutant causing blast disease on rice. MoCTU2 was constitutively expressed at various developmental stages, with a peak during appressorium formation; the MoCtu2-GFP fusion was evenly distributed in the cytoplasm. Altogether, our findings indicate that the cytosolic thiouridylase gene MoCTU2 is required for fungal development, conidiation, and responses to rapamycin and high temperature in the blast fungus.

A polyketide synthase from Verticillium dahliae modulates melanin biosynthesis and hyphal growth to promote virulence

BackgroundDuring the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during “overwintering.” Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear.ResultsWe analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type.ConclusionsWe show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.

The Heterotrimeric Transcription Factor CCAAT-Binding Complex and Ca2+-CrzA Signaling Reversely Regulate the Transition between Fungal Hyphal Growth and Asexual Reproduction.

ABSTRACTThe life cycle of filamentous fungi generally comprises hyphal growth and asexual reproduction. Both growth and propagation processes are critical for invasion growth, spore dissemination, and virulence in fungal pathogens and for the production of secondary metabolites or for biomass accumulation in industrial filamentous fungi. The CCAAT-binding complex (CBC) is a heterotrimeric transcription factor comprising three subunits, HapB, HapC, and HapE, and is highly conserved in fungi. Previous studies revealed that CBC regulates sterol metabolism by repressing several genes in the ergosterol biosynthetic pathway in the human fungal pathogen Aspergillus fumigatus. In the present study, we found dysfunction of CBC caused the abnormal asexual reproduction (conidiation) in submerged liquid culture. CBC suppresses the activation of the brlA gene in the central regulatory pathway for conidiation combined with its upstream regulators fluG, flbD, and flbC by binding to the 5′-CCAAT-3′ motif within conidiation gene promoters, and lack of CBC member HapB results in the upregulation of these genes. Furthermore, when the expression of brlA or flbC is repressed, the submerged conidiation does not happen in the hapB mutant. Interestingly, deletion of HapB leads to enhanced transient cytosolic Ca2+ levels and activates conidiation-positive inducer Ca2+-CrzA modules to enhance submerged conidiation, demonstrating that CrzA works with CBC as a reverse regulator of fungal conidiation. To the best of our knowledge, the finding of this study is the first report for the molecular switch mechanism between vegetative hyphal growth and asexual development regulated by CBC, in concert with Ca2+-CrzA signaling in A. fumigatus.

The velvet protein Vel1 controls initial plant root colonization and conidia formation for xylem distribution in Verticillium wilt.

The conserved fungal velvet family regulatory proteins link development and secondary metabolite production. The velvet domain for DNA binding and dimerization is similar to the structure of the Rel homology domain of the mammalian NF-κB transcription factor. A comprehensive study addressed the functions of all four homologs of velvet domain encoding genes in the fungal life cycle of the soil-borne plant pathogenic fungus Verticillium dahliae. Genetic, cell biological, proteomic and metabolomic analyses of Vel1, Vel2, Vel3 and Vos1 were combined with plant pathogenicity experiments. Different phases of fungal growth, development and pathogenicity require V. dahliae velvet proteins, including Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 heterodimers, which are already present during vegetative hyphal growth. The major novel finding of this study is that Vel1 is necessary for initial plant root colonization and together with Vel3 for propagation in planta by conidiation. Vel1 is needed for disease symptom induction in tomato. Vel1, Vel2, and Vel3 control the formation of microsclerotia in senescent plants. Vel1 is the most important among all four V. dahliae velvet proteins with a wide variety of functions during all phases of the fungal life cycle in as well as ex planta.

A CRISPR/Cas9-mediated in situ complementation method for Phytophthora sojae mutants.

Phytophthora sojae is an important model species for oomycete functional genomics research. Recently, a CRISPR/Cas9‐mediated genome‐editing technology has been successfully established in P. sojae, which has been rapidly and widely applied in oomycete research. However, there is an emerging consensus in the biological community that a complete functional gene research system is needed such as developed in the investigations in functional complementation carried out in this study. We report the development of an in situ complementation method for accurate restoration of the mutated gene. We targeted a regulatory B‐subunit of protein phosphatase 2A (PsPP2Ab1) to verify this knockout and subsequent complementation system. We found that the deletion of PsPP2Ab1 in P. sojae leads to severe defects in vegetative hyphal growth, soybean infection, and loss of the ability to produce sporangia. Subsequently, the reintroduction of PsPP2Ab1 into the knockout mutant remedied all of the deficiencies. This study demonstrates the successful implementation of an in situ complementation system by CRISPR/Cas9, which will greatly accelerate functional genomics research of oomycetes in the post‐genomic era.

Genome-wide mRNA and miRNA analysis in the early stages of germ tube outgrowth in Coprinopsis cinerea

Coprinopsis cinerea is a model mushroom-forming basidiomycete which produces basidiospores during sexual reproduction. This fungus is widely used to study fruiting body formation and development. Molecular mechanisms controlling its growth from vegetative mycelium to multicellular mature fruiting body have been studied extensively. However, little is known about the underlying biological processes during germ tube outgrowth or the transition from basidiospores to multinucleate hyphae. To better understand sexual spore germination in fungi, here we examined the time-dependent cellular events at resting, germinating and fully germinated basidiospores of C. cinerea by genome-wide transcriptional and post-transcriptional analyses and by carbohydrate composition analysis. Our results revealed a high demand of protein degradation, and biosynthesis of various compounds at the early stage of basidiospore gemination and dynamic changes of carbohydrate metabolism throughout the germination process. Seven microRNA-like RNAs (milRNAs) were identified in the resting basidiospores of C. cinerea, six of which were basidiospore-specific. Glycogen and trehalose were shown to be the carbon sources supporting the initiation of germ tube outgrowth. One basidiospore-specific milRNA, cci-milR-37, was found to be a potential regulator of glycogen metabolic pathways related to vegetative hyphal growth. Our results demonstrated the mRNA and miRNA-mediated regulation on energy production, protein and carbohydrate metabolisms at the early developmental stages of germ tube to form totipotent hyphae. To our knowledge, this is the first study to show the roles of miRNAs in mushroom basidiospore germination and out-growth.

Improved chlamydospore formation in Tricholoma bakamatsutake with addition of amino acids in vitro

Abstract Tricholoma bakamatsutake, which forms its fruit body in oak forests, is similar to T. matsutake in shape and odor; therefore, it is expected to have a high economic value similar to T. matsutake. Recently, the fruit bodies of T. bakamatsutake were produced after planting of a Quercus phillyraeoides sapling with a pure culture of the fungal mycelia. This fungus forms pigmented thick-walled spores (chlamydospores [CSs]) terminally on the hyphae in culture and in the field settings. Abundant CS production in vitro seems to be advantageous for cultivating mycorrhizal trees inoculated with T. bakamatsutake CSs. We investigated the effects of adding 16 different nitrogen (N) compounds (three inorganic and 13 organic) to a culture medium on vegetative hyphal growth and CS formation. The growth of T. bakamatsutake was reduced by many of the N sources used. Conversely, the rate of CS formation was improved by the addition of valine or glutamine.

Large-scale identification of lysine acetylated proteins in vegetative hyphae of the rice blast fungus

Lysine acetylation is a major post-translational modification that plays important regulatory roles in diverse biological processes to perform various cellular functions in both eukaryotes and prokaryotes. However, roles of lysine acetylation in plant fungal pathogens were less studied. Here, we provided the first lysine acetylome of vegetative hyphae of the rice blast fungus Magnaporthe oryzae through a combination of highly sensitive immune-affinity purification and high-resolution LC-MS/MS. This lysine acetylome had 2,720 acetylation sites in 1,269 proteins. The lysine acetylated proteins were involved indiverse cellular functions, and located in 820 nodes and 7,709 edges among the protein-protein interaction network. Several amino acid residues nearby the lysine acetylation sites were conserved, including KacR, KacK, and KacH. Importantly, dozens of lysine acetylated proteins are found to be important to vegetative hyphal growth and fungal pathogenicity. Taken together, our results provided the first comprehensive view of lysine acetylome of M.oryzae and suggested protein lysine acetylation played important roles to fungal development and pathogenicity.

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae.

SummaryChitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.

Interactions between Melanin Enzymes and Their Atypical Recruitment to the Secretory Pathway by Palmitoylation.

ABSTRACTMelanins are biopolymers that confer coloration and protection to the host organism against biotic or abiotic insults. The level of protection offered by melanin depends on its biosynthesis and its subcellular localization. Previously, we discovered that Aspergillus fumigatus compartmentalizes melanization in endosomes by recruiting all melanin enzymes to the secretory pathway. Surprisingly, although two laccases involved in the late steps of melanization are conventional secretory proteins, the four enzymes involved in the early steps of melanization lack a signal peptide or a transmembrane domain and are thus considered “atypical” secretory proteins. In this work, we found interactions among melanin enzymes and all melanin enzymes formed protein complexes. Surprisingly, the formation of protein complexes by melanin enzymes was not critical for their trafficking to the endosomal system. By palmitoylation profiling and biochemical analyses, we discovered that all four early melanin enzymes were strongly palmitoylated during conidiation. However, only the polyketide synthase (PKS) Alb1 was strongly palmitoylated during both vegetative hyphal growth and conidiation when constitutively expressed alone. This posttranslational lipid modification correlates the endosomal localization of all early melanin enzymes. Intriguingly, bioinformatic analyses predict that palmitoylation is a common mechanism for potential membrane association of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) in A. fumigatus. Our findings indicate that protein-protein interactions facilitate melanization by metabolic channeling, while posttranslational lipid modifications help recruit the atypical enzymes to the secretory pathway, which is critical for compartmentalization of secondary metabolism.

An Ash1-Like Protein MoKMT2H Null Mutant Is Delayed for Conidium Germination and Pathogenesis in Magnaporthe oryzae.

Ash1 is a known H3K36-specific histone demethylase that is required for normal Hox gene expression and fertility in Drosophila and mammals. However, little is known about the expression and function of the fungal ortholog of Ash1 in phytopathogenic fungus Magnaporthe oryzae. Here we report that MoKMT2H, an Ash1-like protein, is required for conidium germination and virulence in rice. We obtained MoKMT2H null mutant (ΔMoKMT2H) using a target gene replacement strategy. In the ΔMoKMT2H null mutants, global histone methyltransferase modifications (H3K4me3, H3K9me3, H3K27me3, and H3K36me2/3) of the genome were unaffected. The ΔMoKMT2H mutants showed no defect in vegetative hyphal growth, conidium morphology, conidiation, or disease lesion formation on rice leaves. However, the MoKMT2H deletion mutants were delayed for conidium germination and consequently had decreased virulence. Taken together, our results indicated that MoKMT2H plays an important role in conidium germination during appressorium formation in the rice blast fungus and perhaps other pathogenic plant fungi.

A spindle pole antigen gene MoSPA2 is important for polar cell growth of vegetative hyphae and conidia, but is dispensable for pathogenicity in Magnaporthe oryzae.

Spa2 is an important component of the multiprotein complex polarisome, which is involved in the establishment, maintenance, termination of polarized cell growth and is important for defining tip growth of filamentous fungi. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that formed smaller colony and conidia compared with the wild type. In the mutant, a spindle pole antigen gene MoSPA2 was disrupted by the integration of an exogenous plasmid. Targeted gene deletion and complementation assays demonstrated the gene disruption was responsible for the defects of the insertional mutant. Interestingly, the MoSpa2-GFP fusion protein was found to accumulate as a spot at hyphal tips, septa of hyphae and conidial tip cells where germ tubes are usually produced, but not in appressoria, infection hyphae or at the septa of conidia. Furthermore, the deletion mutants of MoSPA2 exhibited slower hyphal tip growth, more hyphal branches, and smaller size of conidial tip cells. However, MoSPA2 is not required for plant infection. These results indicate that MoSPA2 is required for vegetative hyphal growth and maintaining conidium morphology and that spotted accumulation of MoSpa2 is important for its functions during cell polar growth.

MoTlg2, a t-SNARE component is important for formation of the Spitzenkörper and polar deposition of chitin in Magnaporthe oryzae

T-SNAREs are a family of conserved proteins involved in intracellular transport of membrane-coated cargo among subcellular compartments. In this study, we identified a putative t-SNARE gene, MoTLG2, in Magnaporthe oryzae via insertion mutagenesis. Deletion of MoTLG2 resulted in slower vegetative growth and less conidiation relative to the wild-type strain, but the ΔMotlg2 null mutant was as virulent as the wild-type strain. MoTlg2 has 30% overall amino acid identity with Saccharomyces cerevisiae Tlg2, and rescued the defect of monensin de-sensitivity in the yeast strain where TLG2 had been deleted. More importantly, apical regions of the hyphae of the ΔMotlg2 null mutant were only weakly stained by FM4-64, which was reported as an excellent vesicle tracer, suggesting that the Spitzenkörper was not well formed in the ΔMotlg2 null mutant. In addition, more uneven lateral deposition of chitin was observed in the cell wall of vegetative hyphae of the ΔMotlg2 null mutant. Taken together, this study shows that the t-SNARE Tlg2 is important for both vegetative hyphal growth and conidiation, but dispensable for plant infection in filamentous fungi, and suggests that Tlg2 is important for formation of the Spitzenkörper and polar distribution of chitin.

Down-regulation of sidB gene by use of RNA interference in Aspergillus nidulans.

Introduction of the RNA interference (RNAi) machinery has guided the researchers to discover the function of essential vital or virulence factor genes in the microorganisms such as fungi. In the filamentous fungus Aspergillus nidulans, the gene sidB plays an essential role in septation, conidiation and vegetative hyphal growth. In the present study, we benefited from the RNAi strategy for down-regulating a vital gene, sidB, in the fungus A. nidulans. The 21-nucleotide small interfering RNA (siRNA) was designed based on the cDNA sequence of the sidB gene in A. nidulans. Transfection was performed through taking up siRNA from medium by 6 hour-germinated spores. To evaluate the morphologic effects of siRNA on the fungus, germ tube elongation was followed. Moreover, total RNA was extracted and quantitative changes in expression of the sidB gene were analyzed by measuring the cognate sidB mRNA level by use of a quantitative real-time RT-PCR assay. Compared to untreated-siRNA samples, a significant inhibition in germ tube elongation was observed in the presence of 25 nM of siRNA (42 VS 21 µM). In addition, at the concentration of 25 nM, a considerable decrease in sidB gene expression was revealed. Usage of RNAi as a kind of post-transcriptional gene silencing methods is a promising approach for designing new antifungal agents and discovering new drug delivery systems.

A two‐component histidine kinase Shk1 controls stress response, sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum

Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high-osmolarity glycerol mitogen-activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H2 O2 -induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real-time polymerase chain reaction (qRT-PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21-activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild-type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two-component HKs in Sclerotinia.

A serine/threonine-protein phosphatase PP2A catalytic subunit is essential for asexual development and plant infection in Magnaporthe oryzae

Protein phosphatase 2A is a subgroup of widely conserved serine/threonine phosphatases and plays diverse roles in transcription, translation, differentiation, cell cycle, and signal transduction in many organisms. However, its roles in biotrophic and hemi-biotrophic phytopathogenic fungi remain to be investigated. In this study, we isolated an insertional mutant of the rice blast fungus Magnaporthe oryzae that was defective in vegetative hyphal growth. In the mutant, the T-DNA fragment was found to be inserted in the promoter region of a putative serine/threonine protein phosphatase 2A catalytic subunit (PP2Ac) gene MoPPG1. Deletion of MoPPG1 leads to severe defects in vegetative hyphal growth and conidiation. Conidia of the ∆Moppg1 null mutants were misshaped, and most of them were two-celled. The deletion mutants of MoPPG1 did not penetrate into host plant cells and failed to cause any disease lesions on rice leaves. Interestingly, significant reduction was found in the ∆Moppg1 null mutants in expression levels of several Rho GTPase family genes including MgCDC42, MgRHO3, and MgRAC1, which were important for pathogenesis of M. oryzae. Taken together, our results indicated that PP2Ac plays vital roles in asexual development and plant infection by regulating Rho GTPases in the rice blast fungus and perhaps other plant pathogenic fungi.

Heterotrimeric G protein β subunit GPB1 and MAP kinase MPK1 regulate hyphal growth and female fertility in Fusarium sacchari

Heterotrimeric GTP-binding proteins (G proteins) and mitogen-activated protein kinase (MAPK) cascades involve vegetative hyphal growth, development of infection-related structure, colonization in host plant and female fertility in phytopathogenic ascomycete fungi. In this study, a heterotrimeric G protein β subunit (Gβ), GPB1, and MAPK, MPK1, were characterized from Fusarium sacchari (=Gibberella sacchari; mating population B of the G. fujikuroi-species complex). GPB1 and MPK1 showed high homology to known Gβ and Fus3/Kss1 MAP kinases of other filamentous ascomycetes, respectively. Disruption (Δ) of gpb1 suppressed hyphal branching and accelerated aerial hyphae formation in F. sacchari. Oppositely, disruption of mpk1 caused delayed aerial hyphae formation. These indicated that GPB1 regulates vegetative hyphal growth negatively, and MPK1 does positively in F. sacchari. Both Δgpb1 and Δmpk1 showed female sterility. Level of intracellular cAMP in Δgpb1 was lower than wild type. Exogenous cyclic AMP (cAMP) partially restored enhanced aerial hyphae formation. These suggested that abnormal hyphal growth was caused by depletion of intracellular cAMP in Δgpb1. cAMP has been reported to suppress development of perithecia in crossing between wild type strains. Thus, precise regulation of intracellular cAMP level via Gβ/MAPK is essential for normal hyphal growth and fertility.