RESUMO
Autophagy and Cell wall integrity (CWI) signaling are critical stress-responsive processes during fungal infection of host plants. In the rice blast fungus Magnaporthe oryzae, autophagy-related (ATG) proteins phosphorylate CWI kinases to regulate virulence; however, how autophagy interplays with CWI signaling to coordinate such regulation remains unknown. Here, we have identified the phosphorylation of ATG protein MoAtg4 as an important process in the coordination between autophagy and CWI in M. oryzae. The ATG kinase MoAtg1 phosphorylates MoAtg4 to inhibit the deconjugation and recycling of the key ATG protein MoAtg8. At the same time, MoMkk1, a core kinase of CWI, also phosphorylates MoAtg4 to attenuate the C-terminal cleavage of MoAtg8. Significantly, these two phosphorylation events maintain proper autophagy levels to coordinate the development and pathogenicity of the rice blast fungus.
Assuntos
Ascomicetos , Magnaporthe , Oryza , Fosforilação , Virulência , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Magnaporthe/metabolismo , Autofagia , Parede Celular/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Regulação Fúngica da Expressão GênicaRESUMO
The emergence of fungicide resistance severely threatens crop production by limiting the availability and application of established fungicides. Therefore, it is urgent to identify new fungicidal targets for controlling plant diseases. Here, we characterized the function of a conserved homoserine O-acetyltransferase (HOA) from the rice blast fungus Magnaporthe oryzae that could serve as the candidate antifungal target. Deletion of the MoMET2 and MoCYS2 genes encoding HOAs perturbed the biosynthesis of methionine and S-adenyl methionine, a methyl group donor for epigenetic modifications, and severely attenuated the development and virulence of M. oryzae. The ∆Momet2 mutant is significantly increased in 5-methylcytosine (5mC) modification that represses the expression of genes required for pathogenicity, including MoGLIK and MoCDH-CYT. We further showed that host-induced gene silencing (HIGS) targeting MoMET2 and MoCYS2 effectively controls rice blasts. Our studies revealed the importance of HOA in the development and virulence of M. oryzae, which suggests the potential feasibility of HOA as new targets for novel anti-rice blast measurements.
Assuntos
Magnaporthe , Oryza , Virulência/genética , Oryza/metabolismo , Metionina/genética , Expressão Gênica , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
The rice blast fungus Magnaporthe oryzae forms specialized infectious structures called appressoria that breach host cells to initiate infection. Previous studies demonstrated that the regulator of G-protein signaling (RGS)-like protein MoRgs7 undergoes endocytosis upon fungal sensing of hydrophobic environmental cues to activate cAMP signaling required for appressorium formation. However, the mechanism by which MoRgs7 internalizes and its fate remains undetermined. We here show that MoSep1, a conserved protein kinase of Mitotic Exit Network (MEN), phosphorylates MoRgs7 to regulate its function. MoRgs7 phosphorylation determines its interaction with MoCrn1, a coronin-like actin-binding protein homolog that also modulates the internalization of MoRgs7. Importantly, the endocytic transport of MoRgs7 is critical for its GTPase-activating protein (GAP) function important in cAMP signaling. Together, our findings revealed a novel mechanism by which M. oryzae activates MoRgs7-mediated hydrophobic cue-sensing signal transduction involving protein phosphorylation and endocytic transport to govern appressorium formation and fungal pathogenicity.
Assuntos
Magnaporthe , Oryza , Humanos , Fosforilação , Sinais (Psicologia) , Magnaporthe/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Oryza/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Regulação Fúngica da Expressão GênicaRESUMO
Magnaporthe oryzae causes rice blasts posing serious threats to food security worldwide. During infection, M. oryzae utilizes several transmembrane receptor proteins that sense cell surface cues to induce highly specialized infectious structures called appressoria. However, little is known about the mechanisms of intracellular receptor tracking and their function. Here, we described that disrupting the coat protein complex II (COPII) cargo protein MoErv14 severely affects appressorium formation and pathogenicity as the ΔMoerv14 mutant is defective not only in cAMP production but also in the phosphorylation of the mitogen-activated protein kinase (MAPK) MoPmk1. Studies also showed that either externally supplementing cAMP or maintaining MoPmk1 phosphorylation suppresses the observed defects in the ΔMoerv14 strain. Importantly, MoErv14 is found to regulate the transport of MoPth11, a membrane receptor functioning upstream of G-protein/cAMP signaling, and MoWish and MoSho1 function upstream of the Pmk1-MAPK pathway. In summary, our studies elucidate the mechanism by which the COPII protein MoErv14 plays an important function in regulating the transport of receptors involved in the appressorium formation and virulence of the blast fungus.
Assuntos
Magnaporthe , Oryza , Virulência , Magnaporthe/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Membrana Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Esporos Fúngicos/metabolismoRESUMO
Mitochondria are highly dynamic organelles that constantly change their morphology to adapt to the cellular environment through fission and fusion, which is critical for a cell to maintain normal cellular functions. Despite the significance of this process in the development and pathogenicity of the rice blast fungus Magnaporthe oryzae, the underlying mechanism remains largely elusive. Here, we identified and characterized a mitochondrial outer membrane translocase, MoTom20, in M. oryzae. Targeted gene deletion revealed that MoTom20 plays an important role in vegetative growth, conidiogenesis, penetration, and infectious growth of M. oryzae. The growth rate, conidial production, appressorium turgor, and pathogenicity are decreased in the ΔMotom20 mutant compared with the wild-type and complemented strains. Further analysis revealed that MoTom20 localizes in mitochondrion and plays a key role in regulating mitochondrial fission and fusion balance, which is critical for infectious growth. Finally, we found that MoTom20 is involved in fatty-acid utilization, and its yeast homolog ScTom20 is able to rescue the defects of ΔMotom20 in mitochondrial morphology and pathogenicity. Overall, our data demonstrate that MoTom20 is a key regulator for mitochondrial morphology maintenance, which is important for infectious growth of the rice blast fungus M. oryzae. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Assuntos
Proteínas Fúngicas , Mitocôndrias , Oryza , Doenças das Plantas , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Mitocôndrias/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Ascomicetos/genética , Ascomicetos/patogenicidade , Regulação Fúngica da Expressão Gênica , Membranas Mitocondriais/metabolismo , Virulência , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Dinâmica Mitocondrial , Deleção de GenesRESUMO
The inhibition of BRD4 bromodomain is an effective therapeutic strategy for a variety of diseases in which BRD4 are implicated. Herein, we identified a small-molecule BRD4 inhibitor hit named compound 3 using high-throughput screening. The 1.6 Å resolution co-crystal structure confirmed that the compound occupies the KAc recognition pockets of BRD4 by forming key hydrogen bonds with Asn140 and engaging in hydrophobic interactions, thus impedes the binding of acetylated lysine to BRD4. These findings suggest compound 3 can be a lead compound to develop a structurally novel BRD4 inhibitors.
Assuntos
Proteínas de Ciclo Celular , Proteínas Nucleares , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição/metabolismo , Ensaios de Triagem em Larga Escala , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
Microbe-produced secondary metabolite phenazine-1-carboxylic acid (PCA) facilitates pathogen virulence and defense mechanisms against competitors. Magnaporthe oryzae, a causal agent of the devastating rice blast disease, needs to compete with other phyllosphere microbes and overcome host immunity for successful colonization and infection. However, whether M. oryzae produces PCA or it has any other functions remains unknown. Here, we found that the MoPHZF gene encodes the phenazine biosynthesis protein MoPhzF, synthesizes PCA in M. oryzae, and regulates appressorium formation and host virulence. MoPhzF is likely acquired through an ancient horizontal gene transfer event and has a canonical function in PCA synthesis. In addition, we found that PCA has a role in suppressing the accumulation of host-derived reactive oxygen species (ROS) during infection. Further examination indicated that MoPhzF recruits both the endoplasmic reticulum membrane protein MoEmc2 and the regulator of G-protein signaling MoRgs1 to the plasma membrane (PM) for MoRgs1 phosphorylation, which is a critical regulatory mechanism in appressorium formation and pathogenicity. Collectively, our studies unveiled a canonical function of MoPhzF in PCA synthesis and a noncanonical signaling function in promoting appressorium formation and host infection.
Assuntos
Ascomicetos , Magnaporthe , Oryza , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Oryza/metabolismo , Fenazinas/metabolismo , Doenças das Plantas/genéticaRESUMO
Lesion mimic mutants (LMMs) are valuable genetic resources for unraveling plant defense responses including programmed cell death. Here, we identified a rice (Oryza sativa) LMM, spotted leaf 38 (spl38), and demonstrated that spl38 is essential for the formation of hypersensitive response-like lesions and innate immunity. Map-based cloning revealed that SPL38 encodes MEDIATOR SUBUNIT 16 (OsMED16). The spl38 mutant showed enhanced resistance to rice pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo) and exhibited delayed flowering, while OsMED16-overexpressing plants showed increased rice susceptibility to M. oryzae. The OsMED16-edited rice lines were phenotypically similar to the spl38 mutant but were extremely weak, exhibited growth retardation, and eventually died. The C-terminus of OsMED16 showed interaction with the positive immune regulator PATHOGENESIS RELATED 3 (OsPR3), resulting in the competitive repression of its chitinase and chitin-binding activities. Furthermore, the ospr3 osmed16 double mutants did not exhibit the lesion mimic phenotype of the spl38 mutant. Strikingly, OsMED16 exhibited an opposite function in plant defense relative to that of Arabidopsis (Arabidopsis thaliana) AtMED16, most likely because of 2 amino acid substitutions between the monocot and dicot MED16s tested. Collectively, our findings suggest that OsMED16 negatively regulates cell death and immunity in rice, probably via the OsPR3-mediated chitin signaling pathway.
Assuntos
Oryza , Xanthomonas , Proteínas de Plantas/metabolismo , Imunidade Inata , Morte Celular/genética , Apoptose , Xanthomonas/fisiologia , Doenças das Plantas/genética , Regulação da Expressão Gênica de Plantas , Resistência à Doença/genéticaRESUMO
Inflammatory breast cancer (IBC) is a rare and highly invasive form of breast cancer, renowned for its aggressive behavior, malignant capacity, and unfavorable prognosis. Despite considerable advancements in comprehending the underlying biology of IBC, the immune cell infiltration (ICI) profile in IBC remains inadequately elucidated. The current work endeavors to investigate the ICI characteristics of IBC and ascertain the pivotal immune cell subtypes and genes that impact its prognosis. The present study employed microarray data from the GEO database to demonstrate that IBC exhibited a lower abundance of activated mast cells (AMC) in comparison to non-inflammatory breast cancer (nIBC) or normal breast tissue (NBT). Additionally, the mRNA expression level of the gene polo-like kinase 5 (PLK5), which was correlated with AMC, was found to be lower in IBC relative to nIBC or NBT. Furthermore, this investigation provided compelling evidence indicating a potential association between a decreased mRNA expression level of PLK5 and a shorter progression-free survival in patients with breast cancer. The gene set enrichment analysis performed on PLK5 revealed that the gene expression in IBC was closely associated with diverse immune functions and pathways. Besides, a negative correlation has been established between PLK5 mRNA expression level and a majority of immune checkpoint-related genes, thereby suggesting the potential suitability of immunotherapy treatment for IBC. In summary, this study offers valuable insights into the ICI profile of IBC and identifies potential target PLK5 for improving its clinical management.
RESUMO
Clathrin-mediated endocytosis (CME) is imperative for physiological processes in eukaryotic cells. In fungi, the Pan1/End3/Sla1 complex controls the transition between early and late stages of CME. Although it is acknowledged that End3 uses its N-terminal to interact with the C-terminal of Sla1, detailed mechanism remains obscure. Magnaporthe oryzae, the pathogenic fungus of rice, cause blast disease that threatens rice production worldwide. Here we report the detailed interaction mechanism between End3 and Sla1 of M. oryzae, i.e. MoEnd3 and MoSla1. The two EH domains of MoEnd3 (MoEnd3-EH1 and MoEnd3-EH2) is different both in evolution and calcium binding, but are indispensable for conformational stability of each other, an unreported effect of tandem-arranged EH domains. MoEnd3-EH1 and MoEnd3-EH2 interact with peptide MoSla11145-1155 containing a NPF motif with a conserved mode, and MoEnd3-EHs (containing both EH1 and EH2 domains) binds MoSla11145-1155 with a higher affinity, supporting the synergetic effect of EH domains. In addition, MoEnd3-EHs also recognize peptide MoSla1971-981 with a new MPF motif that has not been reported before, while Sla1 of yeast contains a DPF motif that bears EH domain interaction ability. Collectively, our research shows that the two EH domains of End3 synergize to interact with dual XPF motifs of Sla1, which conforms to a bivalent receptor-bivalent ligand model to improve both affinity and specificity.
Assuntos
Peptídeos , Saccharomyces cerevisiae , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Peptídeos/metabolismo , Endocitose/fisiologiaRESUMO
GTP-binding protein (G-protein) and regulator of G-protein signaling (RGS) mediated signal transduction are critical in the growth and virulence of the rice blast pathogen Magnaporthe oryzae. We have previously reported that there are eight RGS and RGS-like proteins named MoRgs1 to MoRgs8 playing distinct and shared regulatory functions in M. oryzae and that MoRgs1 has a more prominent role compared to others in the fungus. To further explore the unique regulatory mechanism of MoRgs1, we screened a M. oryzae cDNA library for genes encoding MoRgs1-interacting proteins and identified MoCkb2, one of the two regulatory subunits of the casein kinase (CK) 2 MoCk2. We found that MoCkb2 and the sole catalytic subunit MoCka1 are required for the phosphorylation of MoRgs1 at the plasma membrane (PM) and late endosome (LE). We further found that an endoplasmic reticulum (ER) membrane protein complex (EMC) subunit, MoEmc2, modulates the phosphorylation of MoRgs1 by MoCk2. Interestingly, this phosphorylation is also essential for the GTPase-activating protein (GAP) function of MoRgs1. The balance among MoRgs1, MoCk2, and MoEmc2 ensures normal operation of the G-protein MoMagA-cAMP signaling required for appressorium formation and pathogenicity of the fungus. This has been the first report that an EMC subunit is directly linked to G-protein signaling through modulation of an RGS-casein kinase interaction.
Assuntos
Ascomicetos/metabolismo , Ascomicetos/patogenicidade , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Virulência/fisiologia , Caseína Quinases/metabolismo , Fosforilação , Transdução de Sinais/fisiologiaRESUMO
The fungal cell wall plays an essential role in maintaining cell morphology, transmitting external signals, controlling cell growth, and even virulence. Relaxation and irreversible stretching of the cell wall are the prerequisites of cell division and development, but they also inevitably cause cell wall stress. Both Mitotic Exit Network (MEN) and Cell Wall Integrity (CWI) are signaling pathways that govern cell division and cell stress response, respectively, how these pathways cross talk to govern and coordinate cellular growth, development, and pathogenicity remains not fully understood. We have identified MoSep1, MoDbf2, and MoMob1 as the conserved components of MEN from the rice blast fungus Magnaporthe oryzae. We have found that blocking cell division results in abnormal CWI signaling. In addition, we discovered that MoSep1 targets MoMkk1, a conserved key MAP kinase of the CWI pathway, through protein phosphorylation that promotes CWI signaling. Moreover, we provided evidence demonstrating that MoSep1-dependent MoMkk1 phosphorylation is essential for balancing cell division with CWI that maintains the dynamic stability required for virulence of the blast fungus.
Assuntos
Parede Celular/fisiologia , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Magnaporthe/patogenicidade , Mitose , Oryza/microbiologia , Doenças das Plantas/microbiologia , Parede Celular/microbiologia , Proteínas Fúngicas/genética , Fosforilação , Transdução de SinaisRESUMO
The ß-1,6-glucan is the key linker between mannoproteins in the outermost part of the cell wall and ß-1,3-glucan/chitin polysaccharide to maintain the rigid structure of the cell wall. The ß-1,6-glucanase GluM, which was purified from the fermentation supernatant of Corallococcus sp. EGB, was able to inhibit the germination of Fusarium oxysporum f. sp. cucumerinum conidia at a minimum concentration of 2.0 U/mL (0.08 µg/mL). The survival rates of GluM-treated conidia and monohyphae were 10.4% and 30.7%, respectively, which were significantly lower than that of ß-1,3-glucanase treatment (Zymolyase, 20.0 U/mL; equate to 1.0 mg/mL) (72.9% and 73.9%). In contrast to ß-1,3-glucanase treatment, the high-osmolarity glycerol (HOG) pathway of F. oxysporum f. sp. cucumerinum cells was activated after GluM treatment, and the intracellular glycerol content was increased by 2.6-fold. Moreover, the accumulation of reactive oxygen species (ROS) in F. oxysporum f. sp. cucumerinum cells after GluM treatment induced apoptosis, but it was not associated with the increased intracellular glycerol content. Together, the results indicate that ß-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents. IMPORTANCE Phytopathogenic fungi are the most devastating plant pathogens in agriculture, causing enormous economic losses to global crop production. Biocontrol agents have been promoted as replacements to synthetic chemical pesticides for sustainable agriculture development. Cell wall-degrading enzymes (CWDEs), including chitinases and ß-1,3-glucanases, have been considered as important armaments to damage the cell wall. Here, we found that F. oxysporum f. sp. cucumerinum is more sensitive to ß-1,6-glucanase GluM treatment (0.08 µg/mL) than ß-1,3-glucanase Zymolyase (1.0 mg/mL). The HOG pathway was activated in F. oxysporum f. sp. cucumerinum cells after GluM treatment, and the intracellular glycerol content was significantly increased. Moreover, the decomposition of F. oxysporum f. sp. cucumerinum cell wall by GluM induced the burst of intracellular ROS and apoptosis, which eventually leads to cell death. Therefore, we suggest that the ß-1,6-glucan of the fungal cell wall may be a better antifungal target compared to the ß-1,3-glucan.
Assuntos
Fusarium , Glicerol , Espécies Reativas de Oxigênio/metabolismo , Glicerol/metabolismo , Parede Celular , Antifúngicos/farmacologia , Esporos Fúngicos , Morte Celular , Doenças das Plantas/prevenção & controle , Doenças das Plantas/microbiologiaRESUMO
The plasma membrane (PM) functions as a physical border between the extracellular and cytoplasmic environments that contribute to the interaction between host plants and pathogenic fungi. As a specific sterol constituent in the cell membrane, ergosterol plays a significant role in fungal development. However, the role of ergosterol in the infection of the rice blast fungus Magnaporthe oryzae remains unclear. In this study, we found that a sterol reductase, MoErg4, is involved in ergosterol biosynthesis and the regulation of plasma membrane integrity in M. oryzae. We found that defects in ergosterol biosynthesis disrupt lipid raft formation in the PM and cause an abnormal distribution of the t-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein MoSso1, inhibiting its interaction with the v-SNARE protein MoSnc1. In addition, we found that MoSso1-MoSnc1 interaction is important for biotrophic interface complex development and cytoplasmic effector protein secretion. Our findings suggested that ergosterol-enriched lipid rafts constitute a platform for interactions among various SNARE proteins that are required for the development and pathogenicity of M. oryzae.
Assuntos
Ascomicetos , Magnaporthe , Oryza , Virulência , Proteínas Fúngicas/metabolismo , Ascomicetos/metabolismo , Proteínas SNARE/metabolismo , Oryza/metabolismo , Doenças das Plantas/microbiologiaRESUMO
Plant amino acid transporters regulate not only long-distance transport and reallocation of nitrogen (N) from source to sink organs, but also the amount of amino acids in leaves hijacked by invading pathogens. However, the function of amino acid transporters in plant defense responses to pathogen infection remains unknown. In this study, we found that the rice amino acid transporter gene OsLHT1 was expressed in leaves and up-regulated by maturation, N starvation, and inoculation of the blast fungus Magnaporthe oryzae. Knock out of OsLHT1 resulted in development stage- and N supply-dependent premature senescence of leaves at the vegetative growth stage. In comparison with the wild type, Oslht1 mutant lines showed sustained rusty red spots on fully mature leaf blades irrespective of N supply levels. Notably, no relationship between the severity of leaf rusty red spots and concentration of total N or amino acids was found in Oslht1 mutants at different developmental stages. Disruption of OsLHT1 altered transport and metabolism of amino acids and biosynthesis of flavones and flavonoids, enhanced expression of jasmonic acid- and salicylic acid-related defense genes, production of jasmonic acid and salicylic acid, and accumulation of reactive oxygen species. OsLHT1 inactivation dramatically prevented the leaf invasion by M. oryzae, a hemi-biotrophic ascomycete fungus. Overall, these results establish a link connecting the activity of an amino acid transporter with leaf metabolism and defense against rice blast fungus.
Assuntos
Ascomicetos , Magnaporthe , Oryza , Magnaporthe/fisiologia , Senescência Vegetal , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Salicilatos/metabolismo , Oryza/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismoRESUMO
Virtually all proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. The current work reports the 3.6-Å-resolution X-ray structure of the cytochrome aa3 -600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which have been soaked with the quinol-analog inhibitor HQNO (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.
Assuntos
Bacillus subtilis/metabolismo , Cobre/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Hidroquinonas/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Grupo dos Citocromos b/química , Transporte de Elétrons , Ligação de Hidrogênio , Modelos Moleculares , Naftóis/metabolismo , Oxirredutases , Conformação Proteica , Subunidades Proteicas/química , Bombas de Próton/química , Bombas de Próton/metabolismo , Terpenos/metabolismo , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
Oxaliplatin (OXA) has been recognized as a third-generation platinum-based chemotherapeutic agent with stellar therapeutic efficacy in managing colorectal cancer (CRC). Nevertheless, resistance to OXA in CRC patients hinders its effectiveness. Shikonin (SHI), a natural naphthoquinone derived from Arnebia euchroma (Royle) Johnst., features a broad pharmacological profile and minimal toxicities. To assess the synergism of SHI and OXA towards OXA-resistant CRC cells (HCT116R ), we employed in vitro and in vivo pharmacological assays. Our experiments provided evidence that SHI, either alone or in combination with OXA, considerably reduced cell proliferation, triggered apoptosis, and induced the generation of reactive oxygen species (ROS) in HCT116R cells. Furthermore, the combination of SHI and OXA dramatically curbed the extent of HCT116R -initiated xenograft growth in mouse models. Bioinformatics, western blot, and ROS assays highlighted that the mechanisms of SHI against OXA-resistant CRC cells may involve the induction of cellular responses to chemical stress, intrinsic apoptosis, as well as endoplasmic reticulum stress pathways mediated by ROS. Notably, the synergism of SHI+OXA was partially abrogated by an ROS inhibitor N-acetyl cysteine. Our findings imply the potential of SHI to boost the sensitivity of OXA to CRC, offering promising benefits for clinical strategies to combat OXA resistance.
Assuntos
Neoplasias Colorretais , Naftoquinonas , Animais , Camundongos , Humanos , Oxaliplatina/farmacologia , Oxaliplatina/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Apoptose , Naftoquinonas/farmacologia , Naftoquinonas/uso terapêutico , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Estresse do Retículo EndoplasmáticoRESUMO
Arms race co-evolution of plant-pathogen interactions evolved sophisticated recognition mechanisms between host immune receptors and pathogen effectors. Different allelic haplotypes of an immune receptor in the host mount distinct recognition against sequence or non-sequence related effectors in pathogens. We report the molecular characterization of the Piks allele of the rice immune receptor Pik against rice blast pathogen, which requires two head-to-head arrayed nucleotide-binding sites and leucine-rich repeat proteins. Like other Pik alleles, both Piks-1 and Piks-2 are necessary and sufficient for mediating resistance. However, unlike other Pik alleles, Piks does not recognize any known AvrPik variants of Magnaporthe oryzae. Sequence analysis of the genome of an avirulent isolate V86010 further revealed that its cognate avirulence (Avr) gene most likely has no significant sequence similarity to known AvrPik variants. Piks-1 and Pikm-1 have only two amino acid differences within the integrated heavy metal-associated (HMA) domain. Pikm-HMA interacts with AvrPik-A, -D, and -E in vitro and in vivo, whereas Piks-HMA does not bind any AvrPik variants. Characterization of two amino acid residues differing Piks-1 from Pikm-1 reveal that Piks-E229Q derived from the exchange of Glu229 to Gln229 in Piks-1 gains recognition specificity against AvrPik-D but not AvrPik-A or -E, indicating that Piks-E229Q partially restores the Pikm spectrum. By contrast, Piks-A261V derived from the exchange of Ala261 to Val261 in Piks-1 retains Piks recognition specificity. We conclude that Glu229 in Piks-1 is critical for Piks breaking the canonical Pik/AvrPik recognition pattern. Intriguingly, binding activity and ectopic cell death induction is maintained between Piks-A261V and AvrPik-D, implying that positive outcomes from ectopic assays might be insufficient to deduce its immune activity against the relevant effectors in rice and rice blast interaction.
Assuntos
Ascomicetos , Magnaporthe , Oryza , Alelos , Magnaporthe/fisiologia , Receptores Imunológicos/metabolismo , Oryza/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Interações Hospedeiro-PatógenoRESUMO
During plant-pathogenic fungi and host plants interactions, numerous pathogen-derived proteins are secreted resulting in the activation of the unfolded protein response (UPR) pathway. For efficient trafficking of secretory proteins, including those important in disease progression, the cytoplasmic coat protein complex II (COPII) exhibits a multifunctional role whose elucidation remains limited. Here, we discovered that the COPII cargo receptor MoErv29 functions as a target of MoHac1, a previously identified transcription factor of the UPR pathway. In Magnaporthe oryzae, deletion of MoERV29 severely affected the vegetative growth, conidiation and biotrophic invasion of the fungus in susceptible rice hosts. We demonstrated that MoErv29 is required for the delivery of secreted proteins through recognition and binding of the amino-terminal tripeptide motifs following the signal peptide. By using bioinformatics analysis, we predicted a cargo spectrum of MoErv29 and found that MoErv29 is required for the secretion of many proteins, including extracellular laccases and apoplastic effectors. This secretion is mediated through the conventional endoplasmic reticulum-Golgi secretion pathway and is important for conferring host recognition and disease resistance. Taken together, our results revealed how MoErv29 operates on effector secretion, and our findings provided a critical link between COPII vesicle trafficking and the UPR pathway.
Assuntos
Magnaporthe , Oryza , Ascomicetos , Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Oryza/metabolismo , Doenças das Plantas/microbiologia , VirulênciaRESUMO
Magnaporthe oryzae secretes several effectors that modulate and hijack rice processes to colonize host cells, but the underlying mechanisms remain unclear. We report on a novel cytoplasmic effector MoIug4 that targets the rice ethylene pathway as a transcription repressor to subvert host immunity. We found that MoIug4 binds to the promoter of the host OsEIN2 gene that encodes a central signal transducer in the ethylene-signaling pathway. We also identified a MoIug4 interacting protein, OsAHL1, which acts as an AT-hook motif-containing protein binding to the A/T-rich promoter regions. Our knockout and overexpression studies showed that OsAHL1 positively regulates plant immunity in response to M. oryzae infection. OsAHL1 exhibits transcriptional regulatory activities by binding the OsEIN2 promoter region, similar to MoIug4. Intriguingly, we found that MoIug4 exhibits a higher binding affinity than OsAHL1 to the OsEIN2 promoter, suggesting differential regulatory specificities. These results revealed a counter-defense strategy by which the pathogen effector suppresses the activation of host defense genes by interfering with host transcription activator functions.