RESUMO
Regulator of G-protein signaling (RGS) proteins exhibit GTPase-accelerating protein activities to govern G-protein function. In the rice blast fungus Magnaporthe oryzae, there is a family of at least eight RGS and RGS-like proteins (MoRgs1 to MoRgs8), each exhibiting distinct or shared functions in the growth, appressorium formation, and pathogenicity. MoRgs3 recently emerged as one of the crucial regulators that senses intracellular oxidation during appressorium formation. To explore this unique regulatory mechanism of MoRgs3, we identified the nucleoside diphosphate kinase MoNdk1 that interacts with MoRgs3. MoNdk1 phosphorylates MoRgs3 under induced intracellular reactive oxygen species levels, and MoRgs3 phosphorylation is required for appressorium formation and pathogenicity. In addition, we showed that MoRgs3 phosphorylation determines its interaction with MoCrn1, a coronin-like actin-binding protein homolog, which regulates MoRgs3 internalization. Finally, we provided evidence demonstrating that MoRgs3 functions in MoMagA-mediated cAMP signaling to regulate normal appressorium induction. By revealing a novel signal perception mechanism, our studies highlighted the complexity of regulation during the appressorium function and pathogenicity of the blast fungus. IMPORTANCE: We report that MoRgs3 becomes phosphorylated in an oxidative intracellular environment during the appressorium formation stage. We found that this phosphorylation is carried out by MoNdk1, a nucleoside diphosphate kinase. In addition, this phosphorylation leads to a higher binding affinity between MoRgs3 and MoCrn1, a coronin-like actin-binding protein that was implicated in the endocytic transport of several other RGS proteins of Magnaporthe oryzae. We further found that the internalization of MoRgs3 is indispensable for its GTPase-activating protein function toward the Gα subunit MoMagA. Importantly, we characterized how such cellular regulatory events coincide with cAMP signaling-regulated appressorium formation and pathogenicity in the blast fungus. Our studies uncovered a novel intracellular reactive oxygen species signal-transducing mechanism in a model pathogenic fungus with important basic and applied implications.
Assuntos
AMP Cíclico , Proteínas Fúngicas , Doenças das Plantas , Espécies Reativas de Oxigênio , Transdução de Sinais , Espécies Reativas de Oxigênio/metabolismo , AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Fosforilação , Doenças das Plantas/microbiologia , Oryza/microbiologia , Proteínas RGS/metabolismo , Proteínas RGS/genética , Regulação Fúngica da Expressão Gênica , Núcleosídeo-Difosfato Quinase/metabolismo , Núcleosídeo-Difosfato Quinase/genética , Ascomicetos/genética , Ascomicetos/metabolismo , Ascomicetos/patogenicidade , VirulênciaRESUMO
Autophagy is a central biodegradation pathway critical in eliminating intracellular cargo to maintain cellular homeostasis and improve stress resistance. At the same time, the key component of the mitogen-activated protein kinase cascade regulating cell wall integrity signaling MoMkk1 has an essential role in the autophagy of the rice blast fungus Magnaporthe oryzae. Still, the mechanism of how MoMkk1 regulates autophagy is unclear. Interestingly, we found that MoMkk1 regulates the autophagy protein MoAtg9 through phosphorylation. MoAtg9 is a transmembrane protein subjected to phosphorylation by autophagy-related protein kinase MoAtg1. Here, we provide evidence demonstrating that MoMkk1-dependent MoAtg9 phosphorylation is required for phospholipid translocation during isolation membrane stages of autophagosome formation, an autophagic process essential for the development and pathogenicity of the fungus. In contrast, MoAtg1-dependent phosphorylation of MoAtg9 negatively regulates this process, also impacting growth and pathogenicity. Our studies are the first to demonstrate that MoAtg9 is subject to MoMkk1 regulation through protein phosphorylation and that MoMkk1 and MoAtg1 dichotomously regulate autophagy to underlie the growth and pathogenicity of M. oryzae.IMPORTANCEMagnaporthe oryzae utilizes multiple signaling pathways to promote colonization of host plants. MoMkk1, a cell wall integrity signaling kinase, plays an essential role in autophagy governed by a highly conserved autophagy kinase MoAtg1-mediated pathway. How MoMkk1 regulates autophagy in coordination with MoAtg1 remains elusive. Here, we provide evidence that MoMkk1 phosphorylates MoAtg9 to positively regulate phospholipid translocation during the isolation membrane or smaller membrane structures stage of autophagosome formation. This is in contrast to the negative regulation of MoAtg9 by MoAtg1 for the same process. Intriguingly, MoMkk1-mediated MoAtg9 phosphorylation enhances the fungal infection of rice, whereas MoAtg1-dependant MoAtg9 phosphorylation significantly attenuates it. Taken together, we revealed a novel mechanism of autophagy and virulence regulation by demonstrating the dichotomous functions of MoMkk1 and MoAtg1 in the regulation of fungal autophagy and pathogenicity.
Assuntos
Ascomicetos , Proteínas Fúngicas , Magnaporthe , Fosforilação , Virulência , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Autofagia , Fosfolipídeos/metabolismo , Doenças das Plantas/microbiologia , Regulação Fúngica da Expressão Gênica , Esporos Fúngicos/metabolismoRESUMO
Effector proteins secreted by plant pathogenic fungi are important artilleries against host immunity, but there is no precedent of such effectors being explored as antifungal targets. Here we demonstrate that MoErs1, a species-specific effector protein secreted by the rice blast fungus Magnaporthe oryzae, inhibits the function of rice papain-like cysteine protease OsRD21 involved in rice immunity. Disrupting MoErs1-OsRD21 interaction effectively controls rice blast. In addition, we show that FY21001, a structure-function-based designer compound, specifically binds to and inhibits MoErs1 function. FY21001 significantly and effectively controls rice blast in field tests. Our study revealed a novel concept of targeting pathogen-specific effector proteins to prevent and manage crop diseases.
Assuntos
Proteínas Fúngicas , Oryza , Doenças das Plantas , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Interações Hospedeiro-Patógeno , Papaína/metabolismo , Ascomicetos , MagnaportheRESUMO
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
Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum has led to serious losses in the yields of oilseed rape and other crops every year. Here, we designed and synthesized a series of carboxamide derivatives containing a diphenyl ether skeleton by adopting the scaffold splicing strategy. From the results of the mycelium growth inhibition experiment, inhibition rates of compounds 4j and 4i showed more than 80% to control S. sclerotiorum at a dose of 50 µg/mL, which is close to that of the positive control (flubeneteram, 95%). Then, the results of a structure-activity relationship study showed that the benzyl scaffold was very important for antifungal activity and that introducing a halogen atom on the benzyl ring would improve antifungal activity. Furthermore, the results of an in vitro activity test suggested that these novel compounds can inhibit the activity of succinate dehydrogenase (SDH), and the binding mode of 4j with SDH was basically similar to that of the flutolanil derivative. Morphological observation of mycelium revealed that compound 4j could cause a damage on the mycelial morphology and cell structure of S. sclerotiorum, resulting in inhibition of the growth of mycelia. Furthermore, in vivo antifungal activity assessment of 4j displayed a good control of S. sclerotiorum (>97%) with a result similar to that of the positive control at a concentration of 200 mg/L. Thus, the diphenyl ether carboxamide skeleton is a new starting point for the discovery of new SDH inhibitors and is worthy of further development.
Assuntos
Ascomicetos , Brassica napus , Fungicidas Industriais , Antifúngicos/farmacologia , Ascomicetos/metabolismo , Relação Estrutura-Atividade , Brassica napus/metabolismo , Succinato Desidrogenase/metabolismo , Fungicidas Industriais/farmacologia , Fungicidas Industriais/químicaRESUMO
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
IMPORTANCE: The interplay between plant and pathogen is a dynamic process, with the host's innate defense mechanisms serving a crucial role in preventing infection. In response to many plant pathogen infections, host cells generate the key regulatory molecule, reactive oxygen species (ROS), to limit the spread of the invading organism. In this study, we reveal the effects of fungal peroxisome dynamics on host ROS homeostasis, during the rice blast fungus Magnaporthe oryzae infection. The elongation of the peroxisome appears contingent upon ROS and links to the accumulation of ROS within the host and the infectious growth of the pathogen. Importantly, we identify a peroxisomal 3-ketoacyl-CoA thiolase, MoKat2, responsible for the elongation of the peroxisome during the infection. In response to host-derived ROS, the homodimer of MoKat2 undergoes dissociation to bind peroxisome membranes for peroxisome elongation. This process, in turn, inhibits the accumulation of host ROS, which is necessary for successful infection. Overall, our study is the first to highlight the intricate relationship between fungal organelle dynamics and ROS-mediated host immunity, extending the fundamental knowledge of pathogen-host interaction.
RESUMO
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
Fungal cell wall decomposition enzymes exhibit great potential for the development of efficient antifungal agents. However, their practical application is restricted due to incomplete understanding of the action mode. In our previous study, we identified that a novel outer membrane (OM) ß-1,6-glucanase GluM is deployed by predatory myxobacteria to feed on fungi. In this work, we provide deep insights into the antifungal mechanism of ß-1,6-glucanase and its potential in improving plant disease resistance. The fungal cell wall decomposition ability of GluM resulted in irregular hyphae morphology, changed chitin distribution, increased membrane permeability, and leakage of cell constituents in Magnaporthe oryzae Guy11. Under the attack pattern, the cell wall integrity pathway was activated by strain Guy11 for self-protection. GluM exhibited a distinct endo-model toward fungal cell wall; the favorite substrate of GluM toward fungal ß-1,6-glucan may give reason for its efficient antifungal activity compared with Trichoderma ß-1,6-glucanase. Moreover, released glucans from GluM hydrolysis of fungal cell wall functioned as an elicitor and induced rice immunity by means of jasmonic acid pathway. Based on the dual roles of antifungal properties, gluM transgenic plants conferred enhanced resistance against fungal infection.
Assuntos
Antifúngicos , Glucanos , Antifúngicos/farmacologia , Antifúngicos/metabolismo , Glucanos/metabolismo , Parede Celular/química , Hifas , Quitina/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
As social micropredators, myxobacteria are studied for their abilities to prey on bacteria and fungi. However, their predation of oomycetes has received little attention. Here, we show that Archangium sp. AC19 secretes a carbohydrate-active enzyme (CAZyme) cocktail during predation on oomycetes Phytophthora. These enzymes include three specialized ß-1,3-glucanases (AcGlu13.1, -13.2 and -13.3) that act as a cooperative consortium to target ß-1,3-glucans of Phytophthora. However, the CAZymes showed no hydrolytic effects on fungal cells, even though fungi contain ß-1,3-glucans. Heterologous expression of AcGlu13.1, -13.2 or -13.3 enzymes in Myxococcus xanthus DK1622, a model myxobacterium that antagonizes but does not predate on P. sojae, conferred a cooperative and mycophagous ability that stably maintains myxobacteria populations as a mixture of engineered strains. Comparative genomic analyses suggest that these CAZymes arose from adaptive evolution among Cystobacteriaceae myxobacteria for a specific prey killing behavior, whereby the presence of Phytophthora promotes growth of myxobacterial taxa by nutrient release and consumption. Our findings demonstrate that this lethal combination of CAZymes transforms a non-predatory myxobacterium into a predator with the ability to feed on Phytophthora, and provides new insights for understanding predator-prey interactions. In summary, our work extends the repertoire of myxobacteria predatory strategies and their evolution, and suggests that these CAZymes can be engineered as a functional consortium into strains for biocontrol of Phytophothora diseases and hence crop protection.
Assuntos
Myxococcales , Myxococcus xanthus , Phytophthora , Animais , Myxococcales/genética , Comportamento Predatório , Myxococcus xanthus/genética , Glucanos , Phytophthora/genéticaRESUMO
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
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
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
Apoplastic ascorbate oxidases (AOs) play a critical role in reactive oxygen species (ROS)-mediated innate host immunity by regulating the apoplast redox state. To date, little is known about how apoplastic effectors of the rice blast fungus Magnaporthe oryzae modulate the apoplast redox state of rice to subvert plant immunity. In this study, we demonstrated that M. oryzae MoAo1 is an AO that plays a role in virulence by modulating the apoplast redox status of rice cells. We showed that MoAo1 inhibits the activity of rice OsAO3 and OsAO4, which also regulate the apoplast redox status and plant immunity. In addition, we found that MoAo1, OsAO3, and OsAO4 all exhibit polymorphic variations whose varied interactions orchestrate pathogen virulence and rice immunity. Taken together, our results reveal a critical role for extracellular redox enzymes during rice blast infection and shed light on the importance of the apoplast redox state and its regulation in plant-pathogen interactions.
Assuntos
Magnaporthe , Oryza , Ascomicetos , Ascorbato Oxidase , Interações Hospedeiro-Patógeno , Magnaporthe/fisiologia , Oryza/microbiologia , Oxirredução , Doenças das Plantas/microbiologia , Imunidade VegetalRESUMO
Endoplasmic reticulum (ER) homeostasis is essential for plants to manage responses under environmental stress. Plant immune activation requires the ER, but how ER homeostasis is associated with plant immune activation is largely unexplored. Here we find that transcription of an HVA22 family gene, OsHLP1 (HVA22-like protein 1), is induced by Magnaporthe oryzae infection. Overexpression of OsHLP1 significantly enhances blast disease resistance but impairs ER morphology in rice (Oryza sativa), resulting in enhanced sensitivity to ER stress. OsHLP1 interacts with the NAC (NAM, ATAF, and CUC) transcription factor OsNTL6 at the ER. OsNTL6 localizes to the ER and is relocated to the nucleus after cleavage of the transmembrane domain. OsHLP1 suppresses OsNTL6 protein accumulation, whereas OsNTL6 counteracts OsHLP1 by alleviating sensitivity to ER stress and decreasing disease resistance in OsHLP1 overexpression plants. These findings unravel a mechanism whereby OsHLP1 promotes disease resistance by compromising ER homeostasis when plants are infected by pathogens.
Assuntos
Magnaporthe , Oryza , Resistência à Doença , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Homeostase , Magnaporthe/genética , Magnaporthe/metabolismo , Oryza/genética , Oryza/metabolismo , Doenças das Plantas , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Magnaporthe , Oryza , Etilenos/metabolismo , Interações Hospedeiro-Patógeno/genética , Magnaporthe/genética , Oryza/metabolismo , Doenças das Plantas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The initial stage of rice blast fungus, Magnaporthe oryzae, infection, before 36 h postinoculation, is a critical timespan for deploying pathogen effectors to overcome the host's defences and ultimately cause the disease. However, how this process is regulated at the transcription level remains largely unknown. This study functionally characterized two M. oryzae Early Infection-induced Transcription Factor genes (MOEITF1 and MOEITF2) and analysed their roles in this process. Target gene deletion and mutant phenotype analysis showed that the mutants Δmoeitf1 and Δmoeitf2 were only defective for infection growth but not for vegetative growth, asexual/sexual sporulation, conidial germination, and appressoria formation. Gene expression analysis of 30 putative effectors revealed that most effector genes were down-regulated in mutants, implying a potential regulation by the transcription factors. Artificial overexpression of two severely down-regulated effectors, T1REP and T2REP, in the mutants partially restored the pathogenicity of Δmoeitf1 and Δmoeitf2, respectively, indicating that these are directly regulated. Yeast one-hybrid assay and electrophoretic mobility shift assay indicated that Moeitf1 specifically bound the T1REP promoter and Moeitf2 specifically bound the T2REP promoter. Both T1REP and T2REP were predicted to be secreted during infection, and the mutants of T2REP were severely reduced in pathogenicity. Our results indicate crucial roles for the fungal-specific Moeitf1 and Moeitf2 transcription factors in regulating an essential step in M. oryzae early establishment after penetrating rice epidermal cells, highlighting these as possible targets for disease control.
Assuntos
Magnaporthe , Oryza , Ascomicetos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Oryza/microbiologia , Doenças das Plantas/microbiologia , Esporos Fúngicos/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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
The mitotic exit network (MEN) pathway is a vital kinase cascade regulating the timely and correct progress of cell division. In the rice blast fungus Magnaporthe oryzae, the MEN pathway, consisting of conserved protein kinases MoSep1 and MoMob1-MoDbf2, is important in the development and pathogenicity of the fungus. We found that deletion of MoSEP1 affects the phosphorylation of MoMob1, but not MoDbf2, in contrast to what was found in the buddy yeast Saccharomyces cerevisiae, and verified this finding by in vitro phosphorylation assay and mass spectrometry (MS) analysis. We also found that S43 residue is the critical phosphor-site of MoMob1 by MoSep1, and proved that MoSep1-dependent MoMob1 phosphorylation is essential for cell division during the development of M. oryzae. We further provided evidence demonstrating that MoSep1 phosphorylates MoMob1 to maintain the cell cycle during vegetative growth and infection. Taken together, our results revealed that the MEN pathway has both distinct and conservative functions in regulating the cell cycle during the development and pathogenesis of M. oryzae.