RESUMO
Asteroids with diameters less than about 5 km have complex histories because they are small enough for radiative torques (that is, YORP, short for the Yarkovsky-O'Keefe-Radzievskii-Paddack effect)1 to be a notable factor in their evolution2. (152830) Dinkinesh is a small asteroid orbiting the Sun near the inner edge of the main asteroid belt with a heliocentric semimajor axis of 2.19 AU; its S-type spectrum3,4 is typical of bodies in this part of the main belt5. Here we report observations by the Lucy spacecraft6,7 as it passed within 431 km of Dinkinesh. Lucy revealed Dinkinesh, which has an effective diameter of only 720 m, to be unexpectedly complex. Of particular note is the presence of a prominent longitudinal trough overlain by a substantial equatorial ridge and the discovery of the first confirmed contact binary satellite, now named (152830) Dinkinesh I Selam. Selam consists of two near-equal-sized lobes with diameters of 210 m and 230 m. It orbits Dinkinesh at a distance of 3.1 km with an orbital period of about 52.7 h and is tidally locked. The dynamical state, angular momentum and geomorphologic observations of the system lead us to infer that the ridge and trough of Dinkinesh are probably the result of mass failure resulting from spin-up by YORP followed by the partial reaccretion of the shed material. Selam probably accreted from material shed by this event.
RESUMO
Botrytis cinerea is a major plant pathogen infecting more than 1400 plant species. During invasion, the fungus rapidly kills host cells, which is believed to be supported by induction of programmed plant cell death. To comprehensively evaluate the contributions of most of the currently known plant cell death inducing proteins (CDIPs) and metabolites for necrotrophic infection, an optimized CRISPR/Cas9 protocol was established which allowed to perform serial marker-free mutagenesis to generate multiple deletion mutants lacking up to 12 CDIPs. Whole genome sequencing of a 6x and 12x deletion mutant revealed a low number of off-target mutations which were unrelated to Cas9-mediated cleavage. Secretome analyses confirmed the loss of secreted proteins encoded by the deleted genes. Infection tests with the mutants revealed a successive decrease in virulence with increasing numbers of mutated genes, and varying effects of the knockouts on different host plants. Comparative analysis of mutants confirmed significant roles of two polygalacturonases (PG1, PG2) and the phytotoxic metabolites botrydial and botcinins for infection, but revealed no or only weak effects of deletion of the other CDIPs. Nicotiana benthamiana plants with mutated or silenced coreceptors of pattern recognition receptors, SOBIR1 and BAK1, showed similar susceptibility as control plants to infection by B. cinerea wild type and a 12x deletion mutant. These results raise doubts about a major role of manipulation of these plant defence regulators for B. cinerea infection. Despite the loss of most of the known phytotoxic compounds, the on planta secretomes of the multiple mutants retained substantial phytotoxic activity, proving that further, as yet unknown CDIPs contribute to necrosis and virulence. Our study has addressed for the first time systematically the functional redundancy of fungal virulence factors, and demonstrates that B. cinerea releases a highly redundant cocktail of proteins to achieve necrotrophic infection of a wide variety of host plants.
Assuntos
Botrytis , Nicotiana , Botrytis/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Plantas , Nicotiana/genética , Nicotiana/microbiologia , Virulência/genéticaRESUMO
The devastating pathogen Botrytis cinerea infects a broad spectrum of host plants, causing great socio-economic losses. The necrotrophic fungus rapidly kills plant cells, nourishing their wall and cellular contents. To this end, necrotrophs secrete a cocktail of cell wall degrading enzymes, phytotoxic proteins and metabolites. Additionally, many fungi produce specialized invasion organs that generate high invasive pressures to force their way into the plant cell. However, for most necrotrophs, including Botrytis, the biomechanics of penetration and its contribution to virulence are poorly understood. Here, we use a combination of quantitative micromechanical imaging and CRISPR-Cas-guided mutagenesis to show that Botrytis uses substantial invasive pressure, in combination with strong surface adherence, for penetration. We found that the fungus establishes a unique mechanical geometry of penetration that develops over time during penetration events, and which is actin cytoskeleton dependent. Furthermore, interference of force generation by blocking actin polymerization was found to decrease Botrytis virulence, indicating that also for necrotrophs, mechanical pressure is important in host colonization. Our results demonstrate for the first time mechanistically how a necrotrophic fungus such as Botrytis employs this 'brute force' approach, in addition to the secretion of lytic proteins and phytotoxic metabolites, to overcome plant host resistance.
RESUMO
According to their lifestyle, plant pathogens are divided into biotrophic and necrotrophic organisms. Biotrophic pathogens exclusively nourish living host cells, whereas necrotrophic pathogens rapidly kill host cells and nourish cell walls and cell contents. To this end, the necrotrophic fungus Botrytis cinerea secretes large amounts of phytotoxic proteins and cell wall-degrading enzymes. However, the precise role of these proteins during infection is unknown. Here, we report on the identification and characterization of the previously unknown toxic protein hypersensitive response-inducing protein 1 (Hip1), which induces plant cell death. We found the adoption of a structurally conserved folded Alternaria alternata Alt a 1 protein structure to be a prerequisite for Hip1 to exert its necrosis-inducing activity in a host-specific manner. Localization and the induction of typical plant defense responses by Hip1 indicate recognition as a pathogen-associated molecular pattern at the plant plasma membrane. In contrast to other secreted toxic Botrytis proteins, the activity of Hip1 does not depend on the presence of the receptor-associated kinases BRI1-associated kinase 1 and suppressor of BIR1-1. Our results demonstrate that recognition of Hip1, even in the absence of obvious enzymatic or pore-forming activity, induces strong plant defense reactions eventually leading to plant cell death. Botrytis hip1 overexpression strains generated by CRISPR/Cas9 displayed enhanced infection, indicating the virulence-promoting potential of Hip1. Taken together, Hip1 induces a noncanonical defense response which might be a common feature of structurally conserved fungal proteins from the Alt a 1 family.
Assuntos
Botrytis , Células Vegetais , Botrytis/metabolismo , Morte Celular , Virulência , Membrana Celular , Doenças das Plantas/microbiologia , Regulação da Expressão Gênica de PlantasRESUMO
Barley is a staple crop of major global importance and relatively resilient to a wide range of stress factors in the field. Transgenic reporter lines to investigate physiological parameters during stress treatments remain scarce. We generated and characterized transgenic homozygous barley lines (cv. Golden Promise Fast) expressing the genetically encoded biosensor Grx1-roGFP2, which indicates the redox potential of the major antioxidant glutathione in the cytosol. Our results demonstrated functionality of the sensor in living barley plants. We determined the glutathione redox potential (EGSH) of the cytosol to be in the range of -308 mV to -320 mV. EGSH was robust against a combined NaCl (150 mM) and water deficit treatment (-0.8 MPa) but responded with oxidation to infiltration with the phytotoxic secretome of the necrotrophic fungus Botrytis cinerea. The generated reporter lines are a novel resource to study biotic and abiotic stress resilience in barley, pinpointing that even severe abiotic stress leading to a growth delay does not automatically induce cytosolic EGSH oxidation, while necrotrophic pathogens can undermine this robustness.
Assuntos
Técnicas Biossensoriais , Hordeum , Citosol/metabolismo , Hordeum/genética , Hordeum/metabolismo , Estresse Fisiológico , Oxirredução , Glutationa/metabolismo , Técnicas Biossensoriais/métodosRESUMO
SUMOylation as one of the protein post-translational modifications plays crucial roles in multiple biological processes of eukaryotic organisms. Botrytis cinerea is a devastating fungal pathogen and capable of infecting plant hosts at low temperature. However, the molecular mechanisms of low-temperature adaptation are largely unknown in fungi. Combining with biochemical methods and biological analyses, we report that SUMOylation regulates pathogen survival at low temperature and oxidative DNA damage response during infection in B. cinerea. The heat shock protein (Hsp70) BcSsb and E3 ubiquitin ligase BcRad18 were identified as substrates of SUMOylation; moreover, their SUMOylation both requires a single unique SUMO-interacting motif (SIM). SUMOylated BcSsb regulates ß-tubulin accumulation, thereby affecting the stability of microtubules and consequently mycelial growth at low temperature. On the contrary, SUMOylated BcRad18 modulates mono-ubiquitination of the sliding clamp protein proliferating cell nuclear antigen (PCNA), which is involved in response to oxidative DNA damage during infection. Our study uncovers the molecular mechanisms of SUMOylation-mediated low-temperature survival and oxidative DNA damage tolerance during infection in a devastating fungal pathogen, which provides novel insights into low-temperature adaptation and pathogenesis for postharvest pathogens as well as new targets for inhibitor invention in disease control.
Assuntos
Sumoilação , Ubiquitina-Proteína Ligases , Temperatura , Ubiquitina-Proteína Ligases/metabolismo , Estresse Oxidativo , Dano ao DNARESUMO
Pathogenic fungi are subject to DNA damage stress derived from host immune responses during infection. Small ubiquitin-like modifier (SUMO) modification and precursor (pre)-mRNA splicing are both involved in DNA damage response (DDR). However, the mechanisms of how SUMOylation and splicing coordinated in DDR remain largely unknown. Combining with biochemical analysis, RNA-Seq method, and biological analysis, we report that SUMO pathway participates in DDR and virulence in Fusarium graminearum, a causal agent of Fusarium head blight of cereal crops world-wide. Interestingly, a key transcription factor FgSR is SUMOylated upon DNA damage stress. SUMOylation regulates FgSR nuclear-cytoplasmic partitioning and its phosphorylation by FgMec1, and promotes its interaction with chromatin remodeling complex SWI/SNF for activating the expression of DDR-related genes. Moreover, the SWI/SNF complex was found to further recruit splicing-related NineTeen Complex, subsequently modulates pre-mRNA splicing during DDR. Our findings reveal a novel function of SUMOylation in DDR by regulating a transcription factor to orchestrate gene expression and pre-mRNA splicing to overcome DNA damage during the infection of F. graminearum, which advances the understanding of the delicate regulation of DDR by SUMOylation in pathogenic fungi, and extends the knowledge of cooperation of SUMOylation and pre-mRNA splicing in DDR in eukaryotes.
Assuntos
Precursores de RNA , Sumoilação , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/metabolismo , Dano ao DNARESUMO
CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.
Assuntos
Botrytis/fisiologia , Sistemas CRISPR-Cas , Edição de Genes , Oryza/microbiologia , Doenças das Plantas/microbiologia , Ribonucleoproteínas/antagonistas & inibidores , Telômero/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Oryza/genética , Doenças das Plantas/genética , Ribonucleoproteínas/genéticaRESUMO
Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are found throughout several plant-associated microbial taxa and are typically considered to possess cytolytic activity exclusively on dicot plant species. However, cytolytic NLPs are also produced by pathogens of monocot plants such as the onion (Allium cepa) pathogen Botrytis squamosa. We determined the cytotoxic activity of B. squamosa BsNep1, as well as other previously characterized NLPs, on various monocot plant species and assessed the plant plasma membrane components required for NLP sensitivity. Leaf infiltration of NLPs showed that onion cultivars are differentially sensitive to NLPs, and analysis of their sphingolipid content revealed that the GIPC series A : series B ratio did not correlate to NLP sensitivity. A tri-hybrid population derived from a cross between onion and two wild relatives showed variation in NLP sensitivity within the population. We identified a quantitative trait locus (QTL) for NLP insensitivity that colocalized with a previously identified QTL for B. squamosa resistance and the segregating trait of NLP insensitivity correlated with the sphingolipid content. Our results demonstrate the cytotoxic activity of NLPs on several monocot plant species and legitimize their presence in monocot-specific plant pathogens.
Assuntos
Plantas , Proteínas , Peptídeos , Folhas de Planta , EsfingolipídeosRESUMO
Fusarium graminearum is an important plant pathogen and the causal agent of Fusarium head blight (FHB). At present, the principal method of controlling FHB is through fungicides. Fluazinam is an agent with strong broad-spectrum antifungal activity and has been used to control many diseases. However, there are no reported uses of fluazinam for controlling FHB. This study reports the activity and cell toxicology mechanisms of fluazinam on the filamentous fungus F. graminearum and its effect on fungal growth and development. The activity of fluazinam was tested for 95 wild-type field strains of F. graminearum. The EC50 values (the 50% effective concentration) of fluazinam for inhibition of mycelial growth and spore germination ranged from 0.037 µg/ml to 0.179 µg/ml and from 0.039 µg/ml to 0.506 µg/ml, respectively. The fluazinam sensitivity of these strains varied in 4.9 and 13.0 folds, implying that the target of the fungicide remained unchanged. After treatment with 0.3 µg/ml (≈EC90) fluazinam, the production of conidia was reduced, and the cell wall and cell membrane had shrunked; the cell nucleus and septum morphology, cell membrane permeability, and sexual development were not affected. When treated with 0.1 µg/ml (≈EC50) or 0.3 µg/ml fluazinam, the mycelial respiration and deoxynivalenol (DON) synthesis of F. graminearum were decreased. Confocal images showed that the formation of toxisomes was disturbed after fluazinam treatment, suggesting that fluazinam reduces DON synthesis by inhibiting toxisome formation. Infection of wheat coleoptiles revealed that fluazinam had a strong protective activity against F. graminearum. At 250 µg/ml fluazinam the control efficacy of protective treatments reached 100% and controlled strains resistant to carbendazim. These results contribute to the understanding of the mode of action of fluazinam and its application.
Assuntos
Fungicidas Industriais , Fusarium , Aminopiridinas , Fungicidas Industriais/toxicidadeRESUMO
Removal of the deep fascia is recommended in therapy for dermatofibrosarcoma protuberans, but its necessity in the context of micrographic surgery is unclear. A retrospective clinicopathological analysis of 48 patients with dermatofibrosarcoma protuberans treated by micrographic surgery was performed, to determine in which tumours fascia preservation was feasible and safe. Histologically, 93% of tumours on the trunk and extremities and 14% of tumours in the head and neck region were fully located above the fascia. Localization on the head and neck was the only significant risk factor for tumour extension beyond the subcutis (p<0.001). Overall, 44% of tumours were completely excised above the fascia and 56% with deeper excisions. Two deeply infiltrating tumours (4%) on the head recurred, but in none of these lesions was the fascia spared. These results show that micrographic surgery allows fascia preservation in superficial tumours outside the head and neck region.
Assuntos
Dermatofibrossarcoma , Sarcoma , Neoplasias Cutâneas , Dermatofibrossarcoma/diagnóstico por imagem , Dermatofibrossarcoma/cirurgia , Fáscia , Humanos , Cirurgia de Mohs/efeitos adversos , Recidiva Local de Neoplasia/cirurgia , Estudos Retrospectivos , Neoplasias Cutâneas/cirurgiaRESUMO
CRISPR/Cas is a genome editing technology that has opened new dimensions in functional biology. In a recent publication, we presented a highly efficient CRISPR/Cas technique for Botrytis cinerea, which dramatically increases our options to mutagenize and modify single or multiple genes. In this Perspectives article, we describe the essential features of the method and demonstrate with several examples how it opens new avenues for unraveling the virulence mechanisms of Botrytis and other plant pathogenic fungi and can accelerate research for the identification of new antifungal compounds.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Botrytis/genética , Sistemas CRISPR-Cas/genética , Genoma Fúngico/genética , Genoma de Planta , Doenças das PlantasRESUMO
ABSTRACT: Clinical but not histological changes of congenital melanocytic nevi (CMN) with age are well characterized. Our objective was to analyze histological changes of CMN with age and discuss possible clinical implications of our findings. We investigated serial excisions of 21 patients with CMN and compared histological and immunohistochemical features over time. The median number of serial excisions was 6 [interquartile range (IQR) 5-7], the median age at the first excision was 12 months (IQR 5-98), and the median time between the first and last analyzed excision was 53 months (IQR 45-64). The projected adult size of the excised CMN was "large" or "giant" in 14 of the 21 CMN (67%) and "medium" in the remaining lesions (33%). Nineteen CMN (90%) involved the subcutaneous fat, and 16 of the 21 CMN (76%) reached the lower surgical margin. The histological pattern and depth did not change over time but the cellularity and HMB-45 expression of dermal melanocytes decreased in 16 of the 21 patients (76%) and in 15 of the 21 patients (71%), respectively (both P < 0.001). Patients with decreasing HMB-45 expression were significantly younger at the first excision (median 6 months, IQR 4-28) than patients with unchanged HMB-45 expression (median 176 months, IQR 12-186; P = 0.018). The expression of Ki-67 and p16 did not change significantly with age. Our study demonstrates that (1) the cellularity and pigment production of CMN decreases with age, (2) the histological pattern and extension in depth remain stable, and (3) clear resection margins can rarely be achieved in larger CMN.
Assuntos
Nevo Pigmentado/metabolismo , Nevo Pigmentado/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Adolescente , Fatores Etários , Criança , Pré-Escolar , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Lactente , Antígeno Ki-67/metabolismo , Estudos Longitudinais , Masculino , Melanócitos/metabolismo , Melanócitos/patologia , Nevo Pigmentado/congênito , Nevo Pigmentado/cirurgia , Estudos Retrospectivos , Neoplasias Cutâneas/congênito , Neoplasias Cutâneas/cirurgia , Gordura Subcutânea/patologia , Adulto Jovem , Antígeno gp100 de Melanoma/metabolismoRESUMO
The ectoparasitic nematode Xiphinema index transmits grapevine fanleaf virus (GFLV) during feeding on grapevine roots, causing fanleaf degeneration in the plant. Hence, resistance breeding is a key to develop novel rootstocks to overcome such threats. In past years, various grapevine species were screened, and a few candidates with partial resistance were identified. However, they were hardly sufficient for viticulture because of their many agronomical defects. To develop reliably resistant rootstocks applicable in viticulture, multiple Vitis spp. genotypes were analyzed using root inoculation with nematodes in glass vials as an early and easy evaluation test. Resistance levels were evaluated 35 days after inoculation based on nematode reproduction factors, focusing on juveniles and eggs. Infection of grapevines with GFLV was analyzed after inoculation with viruliferous X. index. With this fast screening system, putative candidates with resistances against X. index have been identified for future breeding programs. Particularly, genotypes with the genetic background of Vitis aestivalis and Vitis labrusca were found to be nematode-resistant.
Assuntos
Nematoides , Vitis , Animais , Patrimônio Genético , Genótipo , Doenças das Plantas/genéticaRESUMO
Cohesin plays an important role in chromatid cohesion and has additional functions in higher-order chromatin organization and in transcriptional regulation. The binding of cohesin to euchromatic regions is largely mediated by CTCF or the mediator complex. However, it is currently unknown how cohesin is recruited to pericentric heterochromatin in mammalian cells. Here we define the histone methyltransferase Suv4-20h2 as a major structural constituent of heterochromatin that mediates chromatin compaction and cohesin recruitment. Suv4-20h2 stably associates with pericentric heterochromatin through synergistic interactions with multiple heterochromatin protein 1 (HP1) molecules, resulting in compaction of heterochromatic regions. Suv4-20h mutant cells display an overall reduced chromatin compaction and an altered chromocenter organization in interphase referred to as "chromocenter scattering." We found that Suv4-20h-deficient cells display chromosome segregation defects during mitosis that coincide with reduced sister chromatid cohesion. Notably, cohesin subunits interact with Suv4-20h2 both in vitro and in vivo. This interaction is necessary for cohesin binding to heterochromatin, as Suv4-20h mutant cells display substantially reduced cohesin levels at pericentric heterochromatin. This defect is most prominent in G0-phase cells, where cohesin is virtually lost from heterochromatin, suggesting that Suv4-20h2 is involved in the initial loading or maintenance of cohesion subunits. In summary, our data provide the first compelling evidence that Suv4-20h2 plays essential roles in regulating nuclear architecture and ensuring proper chromosome segregation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Linhagem Celular , Segregação de Cromossomos/fisiologia , Histona-Lisina N-Metiltransferase/genética , Camundongos , Mutação , Estrutura Terciária de Proteína , Transporte Proteico , CoesinasRESUMO
The mitogen-activated protein kinase (MAPK) cassette of the cell wall integrity (CWI) pathway is primarily responsible for orchestrating changes of cell wall. However, functions of this cassette in other cellular processes are not well understood. Here, we found that the Botrytis cinerea mutant of MAPK kinase (BcMkk1) displays more serious defects in mycelial growth, conidiation, responses to cell wall and oxidative stresses, but possesses less reduced virulence than the mutants of its upstream (BcBck1) and downstream (BcBmp3) kinases. Interestingly, BcMkk1, but not BcBck1 and BcBmp3, negatively regulates production of oxalic acid (OA) and activity of extracellular hydrolases (EHs) that are proposed to be virulence factors of B. cinerea. Moreover, we obtained evidence that BcMkk1 negatively controls OA production via impeding phosphorylation of the Per-Arnt-Sim (PAS) kinase BcRim15 by the Ser/Thr kinase BcSch9. In addition, the fungal Pro40 homolog BcPro40 was found to interact simultaneously with three MAPKs, implying that BcPro40 is a scaffold protein of the CWI pathway in B. cinerea. Taken together, results of this study reveal that BcMkk1 negatively modulates virulence via suppressing OA biosynthesis in B. cinerea, which provides novel insight into conserved and species-specific functions of the MAPK kinase in fungi.
Assuntos
Botrytis/metabolismo , Proteínas Fúngicas/metabolismo , MAP Quinase Quinase 1/metabolismo , Ácido Oxálico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Botrytis/genética , Botrytis/patogenicidade , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Proteínas Fúngicas/genética , Genes Fúngicos , MAP Quinase Quinase 1/genética , Modelos Biológicos , Mutação , Estresse Oxidativo , Fosforilação , Doenças das Plantas/microbiologia , Proteínas Serina-Treonina Quinases/genética , Estresse Fisiológico , Virulência/genética , Virulência/fisiologiaRESUMO
Lipid droplets (LDs) control lipid metabolism in eukaryotic cells in general. However, the biogenesis regulation and biological functions of LDs are largely unknown in pathogenic fungi. Rapamycin treatment results in a significant increase of LD biogenesis in Fusarium graminearum. Molecular mechanisms of the target of rapamycin (TOR) pathway in regulating LD biogenesis and the functions of LD in virulence of F. graminearum were investigated in depth by combining genetic, cytological and phenotypic strategies. TOR in Fusarium graminearum (FgTOR) inhibition by rapamycin induces LD biogenesis through the FgPpg1/Sit4 signaling branch. FgPpg1 promotes phosphorylation of protein phosphatase FgNem1 by the protein kinase FgCak1. The phosphorylated FgNem1 dephosphorylates the phosphatidate phosphatase FgPah1. Dephosphorylated FgPah1 is active and stimulates LD biogenesis. Moreover, deletion of FgNem1/Spo7 or FgPah1 leads to serious defects in vegetative growth, sexual development and virulence. The results of this study provide novel insights into the regulatory mechanism and biological functions of the LDs in the devastating pathogenic fungus F. graminearum.
Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/crescimento & desenvolvimento , Fusarium/patogenicidade , Gotículas Lipídicas/metabolismo , Transdução de Sinais , Fusarium/enzimologia , Fusarium/ultraestrutura , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/ultraestrutura , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Tricotecenos/metabolismo , Virulência/efeitos dos fármacosRESUMO
Cancer control by adaptive immunity involves a number of defined death and clearance mechanisms. However, efficient inhibition of exponential cancer growth by T cells and interferon-γ (IFN-γ) requires additional undefined mechanisms that arrest cancer cell proliferation. Here we show that the combined action of the T-helper-1-cell cytokines IFN-γ and tumour necrosis factor (TNF) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumour immunity from oncogene-induced senescence, we used a mouse model in which the Simian virus 40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumours by attenuating p53- and Rb-mediated cell cycle control. When combined, IFN-γ and TNF drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16INK4a (also known as CDKN2A), and downstream Rb hypophosphorylation at serine 795. This cytokine-induced senescence strictly requires STAT1 and TNFR1 (also known as TNFRSF1A) signalling in addition to p16INK4a. In vivo, Tag-specific T-helper 1 cells permanently arrest Tag-expressing cancers by inducing IFN-γ- and TNFR1-dependent senescence. Conversely, Tnfr1(-/-)Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in TNFR1-expressing hosts. Finally, as IFN-γ and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.
Assuntos
Senescência Celular/imunologia , Citocinas/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Células Th1/imunologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Ciclo Celular , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Interferon gama/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Oncogenes/genética , Fosfosserina/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteína do Retinoblastoma/química , Proteína do Retinoblastoma/metabolismo , Fator de Transcrição STAT1/metabolismo , Fatores de Tempo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/imunologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Grey mould, the most important disease of strawberry worldwide, is caused by Botrytis cinerea and a few additional Botrytis spp. Fungicide resistance is a growing problem and has become a limiting factor in strawberry production. In northern Germany, an annual survey of Botrytis isolates from commercial strawberry fields in 2010 to 2017 has revealed high (> 20%) frequencies of resistance to quinone-outside inhibitors, fenhexamid, boscalid, fludioxonil and cyprodinil, as well as lower (< 10%) shares of resistance to the recently released fluopyram. Iprodione and benzimidazoles have not been used in northern Germany for several years or decades, respectively, yet resistance to them was still detected. These observations are largely representative of the situation in many other strawberry-producing regions worldwide. The spread of strains with multiple resistance to several or even all currently used single-site fungicides is of particular concern and is probably promoted by their excessive use. Contaminated nursery material is a newly detected potential vehicle for the spread of strains with (multiple) fungicide resistance. Several complementary non-chemical measures are available to secure strawberry production in the face of weakening fungicide efficacies, and these are briefly discussed.
Assuntos
Botrytis/efeitos dos fármacos , Botrytis/crescimento & desenvolvimento , Farmacorresistência Fúngica , Fragaria/microbiologia , Fungicidas Industriais/farmacologia , Doenças das Plantas/microbiologia , Agricultura/métodos , AlemanhaRESUMO
The Botrytis cinerea VELVET complex regulates light-dependent development and virulence. The goal of this study was to identify common virulence defects of several VELVET mutants and to reveal their molecular basis. Growth, differentiation, physiology, gene expression and infection of fungal strains were analyzed, and quantitative comparisons of in planta transcriptomes and secretomes were performed. VELVET mutants showed reduced release of citric acid, the major acid secreted by the wild-type, whereas no significant role for oxalic acid was observed. Furthermore, a common set of infection-related and secreted proteins was strongly underexpressed in the mutants. Quantitative secretome analysis with 15 N metabolic labeling revealed a correlation of changes in protein and mRNA levels between wild-type and mutants, indicating that transcript levels determine the abundance of secreted proteins. Infection sites kept at low pH partially restored lesion expansion and expression of virulence genes by the mutants. Drastic downregulation of proteases in the mutants was correlated with incomplete degradation of cellular host proteins at the infection site, but no evidence was obtained that aspartyl proteases are required for lesion formation. The B. cinerea VELVET complex controls pathogenic differentiation by regulating organic acid secretion, host tissue acidification, gene expression and protein secretion.