RESUMO
The genetic regulation of Colletotrichum (Glomerella) sexual reproduction does not strictly adhere to the Ascomycota paradigm and remains poorly understood. Morphologically different but sexually compatible strain types, termed plus and minus, have been recognized, but the biological and molecular distinctions between these strain types remain elusive. In this study, we characterized the sexual behaviors of a pair of plus and minus strains of C. fructicola with the aid of live-cell nucleus-localized fluorescent protein labeling, gene expression, and gene mutation analyses. We confirmed a genetically stable plus-to-minus switching phenomenon and demonstrated the presence of both cross-fertilized and self-fertilized perithecia within the mating line (perithecia cluster at the line of colony contact) between plus and minus strains. We demonstrated that pheromone signaling genes (a-factor-like and α-factor-like pheromones and their corresponding GPCR receptors) were differently expressed between vegetative hyphae of the two strains. Moreover, deletion of pmk1 (a FUS/KSS1 mitogen-activate protein kinase) in the minus strain severely limited mating line formation, whereas deletion of a GPCR (FGSG_05239 homolog) and two histone modification factors (hos2, snt2) in the minus strain did not affect mating line development but altered the ratio between cross-fertilization and self-fertilization within the mating line. We propose a model in which mating line formation in C. fructicola involves enhanced protoperithecium differentiation and enhanced perithecium maturation of the minus strain mediated by both cross-fertilization and diffusive effectors. This study provides insights into mechanisms underlying the mysterious phenomenon of plus-minus-mediated sexual enhancement being unique to Colletotrichum fungi. IMPORTANCE Plus-minus regulation of Colletotrichum sexual differentiation was reported in the early 1900s. Both plus and minus strains produce fertile perithecia in a homothallic but inefficient manner. However, when the two strain types encounter each other, efficient differentiation of fertile perithecia is triggered. The plus strain, by itself, can also generate minus ascospore progeny at high frequency. This nontypical mating system facilitates sexual reproduction and is Colletotrichum specific; the underlying molecular mechanisms, however, remain elusive. The current study revisits this longstanding mystery using C. fructicola as an experimental system. The presence of both cross-fertilized and self-fertilized perithecia within the mating line was directly evidenced by live-cell imaging with fluorescent markers. Based on further gene expression and gene mutation analysis, a model explaining mating line development (plus-minus-mediated sexual enhancement) is proposed. Data reported here have the potential to allow us to better understand Colletotrichum mating and filamentous ascomycete sexual regulation.
Assuntos
Colletotrichum/genética , Colletotrichum/fisiologia , Reprodução/genética , Proteínas Fúngicas/genética , FenótipoRESUMO
Filamentous ascomycetes produce complex multicellular structures during sexual reproduction. Little is known about the genetic pathways enabling the construction of such structures. Here, with a combination of classical and reverse genetic methods, as well as genetic mosaic and graft analyses, we identify and provide evidence for key roles for two genes during the formation of perithecia, the sexual fruiting bodies, of the filamentous fungus Podospora anserina. Data indicate that the proteins coded by these two genes function cell-non-autonomously and that their activity depends upon conserved cysteines, making them good candidate for being involved in the transmission of a reactive oxygen species (ROS) signal generated by the PaNox1 NADPH oxidase inside the maturing fruiting body towards the PaMpk1 MAP kinase, which is located inside the underlying mycelium, in which nutrients are stored. These data provide important new insights to our understanding of how fungi build multicellular structures.
Assuntos
Carpóforos/crescimento & desenvolvimento , Carpóforos/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Podospora/crescimento & desenvolvimento , Podospora/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Western Blotting , Celulose/farmacologia , Sequência Conservada , Cisteína/metabolismo , Evolução Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Deleção de Genes , Teste de Complementação Genética , Proteínas de Fluorescência Verde/metabolismo , Mosaicismo , Micélio/metabolismo , Fenótipo , Fosforilação/efeitos dos fármacos , Frações Subcelulares/metabolismo , Vacúolos/metabolismoRESUMO
The molecular pathways involved in the development of multicellular fruiting bodies in fungi are still not well known. Especially, the interplay between the mycelium, the female tissues and the zygotic tissues of the fruiting bodies is poorly documented. Here, we describe PM154, a new strain of the model ascomycetes Podospora anserina able to mate with itself and that enabled the easy recovery of new mutants affected in fruiting body development. By complete genome sequencing of spod1, one of the new mutants, we identified an inositol phosphate polykinase gene as essential, especially for fruiting body development. A factor present in the wild type and diffusible in mutant hyphae was able to induce the development of the maternal tissues of the fruiting body in spod1, but failed to promote complete development of the zygotic ones. Addition of myo-inositol in the growth medium was able to increase the number of developing fruiting bodies in the wild type, but not in spod1. Overall, the data indicated that inositol and inositol polyphosphates were involved in promoting fruiting body maturation, but also in regulating the number of fruiting bodies that developed after fertilization. The same effect of inositol was seen in two other fungi, Sordaria macrospora and Chaetomium globosum. Key role of the inositol polyphosphate pathway during fruiting body maturation appears thus conserved during the evolution of Sordariales fungi.
Assuntos
Fosfatos de Inositol/metabolismo , Podospora/crescimento & desenvolvimento , Podospora/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Fertilidade , Carpóforos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Proteínas de Fluorescência Verde/metabolismo , Inositol/metabolismo , Sistema de Sinalização das MAP Quinases , Mosaicismo , Mutação/genética , Fenótipo , Pigmentos Biológicos/metabolismo , Podospora/enzimologia , Podospora/genética , Transporte Proteico , Reprodução , Sordariales/metabolismo , Esporos Fúngicos/metabolismo , Temperatura , Zigoto/metabolismoRESUMO
The plant pathogenic ascomycete Fusarium graminearum produces perithecia on corn and small grain residues. These perithecia forcibly discharge ascospores into the atmosphere. Little is known about the relationship among the strength of the perithecial wall, the age of the perithecium, and the quantity of ascospores produced. We used a mechanical compression testing instrument to examine the structural failure rate of perithecial walls from three different strains of F. graminearum (two wild type strains, and a mutant strain unable to produce asci). The force required to compress a perithecium by one micrometer (the mean perithecium compression constant, MPCC) was used to determine the strength of the perithecial wall. Over the course of perithecial maturation (5-12days after the initiation of perithecial development), the MPCC was compared to the number of ascospores contained inside the perithecia. The MPCC increased as perithecia matured, from 0.06Nµm-1 at 5d to 0.12Nµm-1 at 12d. The highest number of ascospores was found in older perithecia (12d). The results indicated that for every additional day of perithecial aging, the perithecia become more resilient to compression forces. Every additional day of perithecial aging resulted in â¼900 more ascospores. Knowledge of how perithecia respond to external forces may provide insight into the development of ascospores and the accumulation of turgor pressure. In the future, compression testing may provide a unique method of determining perithecial age in the field, which could extend to management practices that are informed by knowledge of ascospore release and dispersal.
Assuntos
Parede Celular/fisiologia , Fusarium/fisiologia , Esporos Fúngicos/fisiologia , Força CompressivaRESUMO
Filamentous ascomycetes have long been known as producers of a variety of secondary metabolites, many of which have toxic effects on other organisms. However, the role of these metabolites in the biology of the fungi that produce them remains in most cases enigmatic. A major group of fungal secondary metabolites are polyketides. They are chemically diverse, but have in common that their chemical scaffolds are synthesized by polyketide synthases (PKSs). In a previous study, we analyzed development-dependent expression of pks genes in the filamentous ascomycete Sordaria macrospora. Here, we show that a deletion mutant of the pks4 gene is sterile, producing only protoperithecia but no mature perithecia, whereas overexpression of pks4 leads to enlarged, malformed fruiting bodies. Thus, correct expression levels of pks4 are essential for wild type-like perithecia formation. The predicted PKS4 protein has a domain structure that is similar to homologs in other fungi, but conserved residues of a methyl transferase domain present in other fungi are mutated in PKS4. Expression of several developmental genes is misregulated in the pks4 mutant. Surprisingly, the development-associated app gene is not downregulated in the mutant, in contrast to all other previously studied mutants with a block at the protoperithecial stage. Our data show that the polyketide synthase gene pks4 is essential for sexual development and plays a role in regulating fruiting body morphology.
Assuntos
Carpóforos/fisiologia , Proteínas Fúngicas/metabolismo , Policetídeo Sintases/metabolismo , Sordariales/fisiologia , Sequência de Bases , Carpóforos/genética , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Mutação , Policetídeo Sintases/genética , Sordariales/genéticaRESUMO
Fusarium graminearum, the causal agent of Fusarium head blight (FHB) in cereal crops, employs the production of sexual fruiting bodies (perithecia) on plant debris as a strategy for overwintering and dissemination. In an artificial condition (e.g., carrot agar medium), the F. graminearum Z3643 strain was capable of producing perithecia predominantly in the central region of the fungal culture where aerial hyphae naturally collapsed. To unravel the intricate relationship between natural aerial hyphae collapse and sexual development in this fungus, we focused on 699 genes differentially expressed during aerial hyphae collapse, with 26 selected for further analysis. Targeted gene deletion and quantitative real-time PCR analyses elucidated the functions of specific genes during natural aerial hyphae collapse and perithecium formation. Furthermore, comparative gene expression analyses between natural collapse and artificial removal conditions reveal distinct temporal profiles, with the latter inducing a more rapid and pronounced response, particularly in MAT gene expression. Notably, FGSG_09210 and FGSG_09896 play crucial roles in sexual development and aerial hyphae growth, respectively. Taken together, it is plausible that if aerial hyphae collapse occurs on plant debris, it may serve as a physical cue for inducing perithecium formation in crop fields, representing a survival strategy for F. graminearum during winter. Insights into the molecular mechanisms underlying aerial hyphae collapse provides offer potential strategies for disease control against FHB caused by F. graminearum.
RESUMO
The Ascomycetes fungus Colletotrichum fructicola causes severe diseases on a wide range of crops, fruits, and vegetables. Its pathogenic mechanisms, however, remain poorly understood. Mitogen-activated protein kinases (MAPKs) are conserved regulators of fungal development and pathogenesis. In this study, a Fus3/Kss1-related MAPK from C. fructicola was functionally characterized via gene deletion. On potato dextrose agar (PDA) and oatmeal agar media, the CfPMK1 gene deletion mutants (ΔCfPMK1) were slightly reduced in radial growth rate, severely limited in aerial hyphal differentiation and hyphal melanization, and formed deformed perithecia that were smaller in size and more compactly organized relative to wild type. When artificially inoculated on plants, conidia of these mutants failed to differentiate appressoria or penetrate cuticle, and their pathogenicity defect could not be rescued by wounding plant tissue prior to inoculation. On PDA, ΔCfPMK1 mutants were hypersensitive to osmotic stresses, but were more tolerant to membrane and cell wall stresses. Genetic complementation rescued all phenotypic changes associated with CfPMK1 gene deletion. Based on GFP fusion expression, CfPMK1 protein accumulation was detected at all life stages, and the accumulation level was higher in nascent appressoria relative to conidia. Overall, this study identified CfPMK1 as a key regulator of appressorium and sexual development, pathogenesis, and stress tolerance in C. fructicola.
RESUMO
Fusarium neocosmosporiellum (formerly Neocosmospora vasinfecta) is a cosmopolitan fungus that has been reported from soil, herbivore dung, and as a fruit- and root-rot pathogen of numerous field crops, although it is not known to cause significant losses on any crop. Taking advantage of the fact that this species produces prolific numbers of perithecia in culture, the genome of F. neocosmosporiellum was sequenced and transcriptomic analysis across five stages of perithecium development was performed to better understand the metabolic potential for sexual development and gain insight into its life history. Perithecium morphology together with the genome and transcriptome were compared with those of the plant pathogen F. graminearum, a model for studying perithecium development. Larger ascospores of F. neocosmosporiellum and their tendency to discharge as a cluster demonstrated a duality of dispersal: the majority are passively dispersed through the formation of cirrhi, while a minority of spores are shot longer distances than those of F. graminearum. The predicted gene number in the F. neocosmosporiellum genome was similar to that in F. graminearum, but F. neocosmosporiellum had more carbohydrate metabolism-related and transmembrane transport genes. Many transporter genes were differentially expressed during perithecium development in F. neocosmosporiellum, which may account for its larger perithecia. Comparative analysis of the secondary metabolite gene clusters identified several polyketide synthase genes that were induced during later stages of perithecium development. Deletion of a polyketide synthase gene in F. neocosmosporiellum resulted in a defective perithecium phenotype, suggesting an important role of the corresponding metabolite, which has yet to be identified, in perithecium development. Results of this study have provided novel insights into the genomic underpinning of development in F. neocosmosporiellum, which may help elucidate its ability to occupy diverse ecological niches.
RESUMO
Filamentous fungi frequently undergo bistable phenotypic switches. Crippled Growth of Podospora anserina is one such bistable switch, which seems to rely upon the mis-activation of a self-regulated PaMpk1 MAP kinase regulatory pathway. Here, we identify two new partners of this pathway: PaPro1, a transcription factor orthologous to Sordaria macrospora pro1 and Neurospora crassa ADV-1, and IDC4, a protein with an AIM24 domain. Both PaPro1 and IDC4 regulate stationary phase features, as described for the other actors of the PaMpk1 signaling pathway. However, PaPro1 is also involved in the control of fertilization by activating the transcription of the HMG8 and the mating type transcription factors, as well as the sexual pheromones and receptor genes. The roles of two components of the STRIPAK complex were also investigated by inactivating their encoding genes: PaPro22 and PaPro45. The mutants of these genes were found to have the same phenotypes as PaPro1 and IDC4 mutants as well as additional phenotypes including slow growth, abnormally shaped hyphae, pigment accumulation and blockage of the zygotic tissue development, indicating that the STRIPAK complex regulates, in addition to the PaMpk1 one, other pathways in P. anserina. Overall, the mutants of these four genes confirm the model by which Crippled Growth is due to the abnormal activation of the PaMpk1 MAP kinase cascade.
RESUMO
Ascospores act as the primary inoculum of Fusarium graminearum, which causes the destructive disease Fusarium head blight (FHB), or scab. MicroRNAs (miRNAs) have been reported in the F. graminearum vegetative stage, and Fgdcl2 is involved in microRNA-like RNA (milRNA) biogenesis but has no major impact on vegetative growth, abiotic stress or pathogenesis. In the present study, we found that ascospore discharge was decreased in the Fgdcl1 deletion mutant, and completely blocked in the double-deletion mutant of Fgdcl1 and Fgdcl2. Besides, more immature asci were observed in the double-deletion mutant. Interestingly, the up-regulated differentially expressed genes (DEGs) common to ΔFgdcl1 and ΔFgdcl1/2 were related to ion transmembrane transporter and membrane components. The combination of small RNA and transcriptome sequencing with bioinformatics analysis predicted 143 novel milRNAs in wild-type perithecia, and 138 of these milRNAs partly or absolutely depended on Fgdcl1, while only 5 novel milRNAs were still obtained in the Fgdcl1 and Fgdcl2 double-deletion mutant. Furthermore, 117 potential target genes were predicted. Overall, Fgdcl1 and Fgdcl2 genes were partly functionally redundant in ascospore discharge and perithecium-specific milRNA generation in F. graminearum, and these perithecium-specific milRNAs play potential roles in sexual development.