RESUMO
Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M. oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.
Assuntos
Proteínas Fúngicas , Oryza , Doenças das Plantas , Fosforilação , Oryza/microbiologia , Oryza/metabolismo , Doenças das Plantas/microbiologia , Proteínas Fúngicas/metabolismo , Fosfoproteínas/metabolismo , Ascomicetos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteômica , Transdução de SinaisRESUMO
Pathogens produce diverse effector proteins to manipulate host cellular processes. However, how functional diversity is generated in an effector repertoire is poorly understood. Many effectors in the devastating plant pathogen Phytophthora contain tandem repeats of the "(L)WY" motif, which are structurally conserved but variable in sequences. Here, we discovered a functional module formed by a specific (L)WY-LWY combination in multiple Phytophthora effectors, which efficiently recruits the serine/threonine protein phosphatase 2A (PP2A) core enzyme in plant hosts. Crystal structure of an effector-PP2A complex shows that the (L)WY-LWY module enables hijacking of the host PP2A core enzyme to form functional holoenzymes. While sharing the PP2A-interacting module at the amino terminus, these effectors possess divergent C-terminal LWY units and regulate distinct sets of phosphoproteins in the host. Our results highlight the appropriation of an essential host phosphatase through molecular mimicry by pathogens and diversification promoted by protein modularity in an effector repertoire.
Assuntos
Monoéster Fosfórico Hidrolases , Phytophthora , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas/metabolismo , Phytophthora/química , Phytophthora/metabolismo , Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Proteína Fosfatase 2/metabolismo , Doenças das PlantasRESUMO
The rice blast fungus Magnaporthe oryzae causes a devastating disease that threatens global rice (Oryza sativa) production. Despite intense study, the biology of plant tissue invasion during blast disease remains poorly understood. Here we report a high-resolution transcriptional profiling study of the entire plant-associated development of the blast fungus. Our analysis revealed major temporal changes in fungal gene expression during plant infection. Pathogen gene expression could be classified into 10 modules of temporally co-expressed genes, providing evidence for the induction of pronounced shifts in primary and secondary metabolism, cell signaling, and transcriptional regulation. A set of 863 genes encoding secreted proteins are differentially expressed at specific stages of infection, and 546 genes named MEP (Magnaportheeffector protein) genes were predicted to encode effectors. Computational prediction of structurally related MEPs, including the MAX effector family, revealed their temporal co-regulation in the same co-expression modules. We characterized 32 MEP genes and demonstrate that Mep effectors are predominantly targeted to the cytoplasm of rice cells via the biotrophic interfacial complex and use a common unconventional secretory pathway. Taken together, our study reveals major changes in gene expression associated with blast disease and identifies a diverse repertoire of effectors critical for successful infection.
Assuntos
Ascomicetos , Magnaporthe , Oryza , Magnaporthe/fisiologia , Ascomicetos/metabolismo , Transdução de Sinais , Citoplasma/metabolismo , Oryza/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
To cause rice blast disease, the filamentous fungus Magnaporthe oryzae secretes a battery of effector proteins into host plant tissue to facilitate infection. Effector-encoding genes are expressed only during plant infection and show very low expression during other developmental stages. How effector gene expression is regulated in such a precise manner during invasive growth by M. oryzae is not known. Here, we report a forward-genetic screen to identify regulators of effector gene expression, based on the selection of mutants that show constitutive effector gene expression. Using this simple screen, we identify Rgs1, a regulator of G-protein signaling (RGS) protein that is necessary for appressorium development, as a novel transcriptional regulator of effector gene expression, which acts prior to plant infection. We show that an N-terminal domain of Rgs1, possessing transactivation activity, is required for effector gene regulation and acts in an RGS-independent manner. Rgs1 controls the expression of at least 60 temporally coregulated effector genes, preventing their transcription during the prepenetration stage of development prior to plant infection. A regulator of appressorium morphogenesis is therefore also required for the orchestration of pathogen gene expression required for invasive growth by M. oryzae during plant infection.
Assuntos
Ascomicetos , Magnaporthe , Oryza , Magnaporthe/genética , Ascomicetos/genética , Transdução de Sinais , Expressão Gênica , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Oryza/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
Genetic studies have shown essential functions of N-glycosylation during infection of the plant pathogenic fungi, however, systematic roles of N-glycosylation in fungi is still largely unknown. Biological analysis demonstrated N-glycosylated proteins were widely present at different development stages of Magnaporthe oryzae and especially increased in the appressorium and invasive hyphae. A large-scale quantitative proteomics analysis was then performed to explore the roles of N-glycosylation in M. oryzae. A total of 559 N-glycosites from 355 proteins were identified and quantified at different developmental stages. Functional classification to the N-glycosylated proteins revealed N-glycosylation can coordinate different cellular processes for mycelial growth, conidium formation, and appressorium formation. N-glycosylation can also modify key components in N-glycosylation, O-glycosylation and GPI anchor pathways, indicating intimate crosstalk between these pathways. Interestingly, we found nearly all key components of the endoplasmic reticulum quality control (ERQC) system were highly N-glycosylated in conidium and appressorium. Phenotypic analyses to the gene deletion mutants revealed four ERQC components, Gls1, Gls2, GTB1 and Cnx1, are important for mycelial growth, conidiation, and invasive hyphal growth in host cells. Subsequently, we identified the Gls1 N-glycosite N497 was important for invasive hyphal growth and partially required for conidiation, but didn't affect colony growth. Mutation of N497 resulted in reduction of Gls1 in protein level, and localization from ER into the vacuole, suggesting N497 is important for protein stability of Gls1. Our study showed a snapshot of the N-glycosylation landscape in plant pathogenic fungi, indicating functions of this modification in cellular processes, developments and pathogenesis.
Assuntos
Retículo Endoplasmático/metabolismo , Magnaporthe/genética , Magnaporthe/metabolismo , Estudos de Avaliação como Assunto , Proteínas Fúngicas/metabolismo , Deleção de Genes , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Glicosilação , Hifas/genética , Mutação , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteômica/métodos , Deleção de Sequência , Esporos Fúngicos/crescimento & desenvolvimento , Virulência/genéticaRESUMO
In eukaryotes, N6 -methyladenosine (m6 A) is abundant on mRNA, and plays key roles in the regulation of RNA function. However, the roles and regulatory mechanisms of m6 A in phytopathogenic fungi are still largely unknown. Combined with biochemical analysis, MeRIP-seq and RNA-seq methods, as well as biological analysis, we showed that Magnaporthe oryzae MTA1 gene is an orthologue of human METTL4, which is involved in m6 A modification and plays a critical role in autophagy for fungal infection. The Δmta1 mutant showed reduced virulence due to blockage of appressorial penetration and invasive growth. Moreover, the autophagy process was severely disordered in the mutant. MeRIP-seq identified 659 hypomethylated m6 A peaks covering 595 mRNAs in Δmta1 appressoria, 114 m6 A peaks was negatively related to mRNA abundance, including several ATG gene transcripts. Typically, the mRNA abundance of MoATG8 was also increased in the single m6 A site mutant ∆atg8/MoATG8A982C , leading to an autophagy disorder. Our findings reveal the functional importance of the m6 A methylation in infection of M. oryzae and provide novel insight into the regulatory mechanisms of plant pathogenic fungi.
Assuntos
Magnaporthe , Oryza , Ascomicetos , Autofagia/genética , Proteínas Fúngicas/genética , Oryza/genética , Oryza/microbiologia , Doenças das Plantas/microbiologia , RNA , RNA Mensageiro/genéticaRESUMO
Upon fungal and bacterial pathogen attack, plants launch pattern-triggered immunity (PTI) by recognizing pathogen-associated molecular patterns (PAMPs) to defend against pathogens. Although PTI-mediated response has been widely studied, a systematic understanding of the reprogrammed cellular processes during PTI by multi-omics analysis is lacking. In this study, we generated metabolome, transcriptome, proteome, ubiquitome and acetylome data to investigate rice (Oryza sativa) PTI responses to two PAMPs, the fungi-derived chitin and the bacteria-derived flg22. Integrative multi-omics analysis uncovered convergence and divergence of rice responses to these PAMPs at multiple regulatory layers. Rice responded to chitin and flg22 in a similar manner at the transcriptome and proteome levels, but distinct at the metabolome level. We found that this was probably due to post-translational regulation including ubiquitination and acetylation, which reshaped gene expression by modulating enzymatic activities, and possibly led to distinct metabolite profiles. We constructed regulatory atlas of metabolic pathways, including the defence-related phenylpropanoid and flavonoid biosynthesis and linoleic acid derivative metabolism. The multi-level regulatory network generated in this study sets the foundation for in-depth mechanistic dissection of PTI in rice and potentially in other related poaceous crop species.
Assuntos
Oryza , Quitina/metabolismo , Oryza/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteoma/metabolismoRESUMO
The chromatin modulator Set5 plays important regulatory roles in both cell growth and stress responses of Saccharomyces cerevisiae. However, its function in filamentous fungi remains poorly understood. Here, we report the pathogenicity-related gene CgSET5 discovered in a T-DNA insertional mutant M285 of Colletotrichum gloeosporioides. Bioinformatic analysis revealed that CgSET5 encodes a SET domain-containing protein that is a homolog of the budding yeast S. cerevisiae Set5. CgSET5 is important for hyphae growth and conidiation and is necessary for appressorium formation and pathogenicity. CgSet5 regulates appressorium formation in a mitogen-activated protein kinase-independent manner. Inactivation of CgSET5 resulted in a significant reduction in chitin content within the cell wall, indicating CgSet5 plays a vital role in cell wall integrity. CgSet5 is involved in peroxisome biogenesis. We identified CgSet5 as the histone H4 methyltransferase, which methylates the critical H4 lysine residues 5 and 8 in C. gloeosporioides. We carried out a yeast two-hybrid screen to find CgSet5 interacting partners. We found CgSet5 putatively interacts with an inorganic pyrophosphatase named CgPpa1, which co-localized in the cytoplasm with CgSet5. Finally, CgPpa1 was found to strongly interact with CgSet5 in vivo during appressorium formation by bimolecular fluorescence complementation assays. These data corroborate a complex control function of CgSet5 acting as a core pathogenic regulator, which connects cell wall integrity and peroxisome biogenesis in C. gloeosporioides.
Assuntos
Colletotrichum/genética , Metiltransferases/genética , Morfogênese/genética , Doenças das Plantas/microbiologia , Proteínas de Saccharomyces cerevisiae/genética , Parede Celular/genética , Colletotrichum/patogenicidade , Proteínas Fúngicas/genética , Fungos/genética , Fungos/patogenicidade , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/patogenicidade , Mutagênese Insercional/genética , Biogênese de Organelas , Domínios PR-SET/genética , Peroxissomos/genética , Doenças das Plantas/genética , Saccharomyces cerevisiae/genética , Transdução de Sinais/genéticaRESUMO
Pathogen infection is a dynamic process. Here, we employ single-cell transcriptomics to investigate plant response heterogeneity. By generating an Arabidopsis thaliana leaf atlas encompassing 95,040 cells during infection by a fungal pathogen, Colletotrichum higginsianum, we unveil cell-type-specific gene expression, notably an enrichment of intracellular immune receptors in vasculature cells. Trajectory inference identifies cells that had different interactions with the invading fungus. This analysis divulges transcriptional reprogramming of abscisic acid signaling specifically occurring in guard cells, which is consistent with a stomatal closure dependent on direct contact with the fungus. Furthermore, we investigate the transcriptional plasticity of genes involved in glucosinolate biosynthesis in cells at the fungal infection sites, emphasizing the contribution of the epidermis-expressed MYB122 to disease resistance. This work underscores spatially dynamic, cell-type-specific plant responses to a fungal pathogen and provides a valuable resource that supports in-depth investigations of plant-pathogen interactions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Micoses , Proteínas de Arabidopsis/metabolismo , Transcriptoma , Arabidopsis/microbiologia , Folhas de Planta/microbiologiaRESUMO
Gibberella stalk rot (GSR) caused by Fusarium graminearum is one of the most devastating diseases in maize; however, the regulatory mechanism of resistance to GSR remains largely unknown. We performed a comparative multi-omics analysis to reveal the early-stage resistance of maize to GSR. We inoculated F. graminearum to the roots of susceptible (Y331) and resistant (Y331-ΔTE) near-isogenic lines containing GSR-resistant gene ZmCCT for multi-omics analysis. Transcriptome detected a rapid reaction that confers resistance at 1-3 hpi as pattern-triggered immunity (PTI) response to GSR. Many key properties were involved in GSR resistance, including genes in photoperiod and hormone pathways of salicylic acid and auxin. The activation of programmed cell death-related genes and a number of metabolic pathways at 6 hpi might be important to prevent further colonization. This is consistent with an integrative analysis of transcriptomics and proteomics that resistant-mediated gene expression reprogramming exhibited a dynamic pattern from 3 to 6 hpi. Further metabolomics analysis revealed that the amount of many chemical compounds was altered in pathways associated with the phenylpropanoid biosynthesis and the phenylalanine metabolism, which may play key roles to confer the GSR resistance. Taken together, we generated a valuable resource to interpret the defense mechanism during early GSR resistance.
RESUMO
This introductory chapter describes the life cycle of Magnaporthe oryzae, the causal agent of rice blast disease. During plant infection, M. oryzae forms a specialized infection structure called an appressorium, which generates enormous turgor, applied as a mechanical force to breach the rice cuticle. Appressoria form in response to physical cues from the hydrophobic rice leaf cuticle and nutrient availability. The signaling pathways involved in perception of surface signals are described and the mechanism by which appressoria function is also introduced. Re-polarization of the appressorium requires a septin complex to organize a toroidal F-actin network at the base of the cell. Septin aggregation requires a turgor-dependent sensor kinase, Sln1, necessary for re-polarization of the appressorium and development of a rigid penetration hypha to rupture the leaf cuticle. Once inside the plant, the fungus undergoes secretion of a large set of effector proteins, many of which are directed into plant cells using a specific secretory pathway. Here they suppress plant immunity, but can also be perceived by rice immune receptors, triggering resistances. M. oryzae then manipulates pit field sites, containing plasmodesmata, to facilitate rapid spread from cell to cell in plant tissue, leading to disease symptom development.
Assuntos
Ascomicetos , Oryza , Biologia , Proteínas Fúngicas/metabolismo , Magnaporthe/metabolismo , Oryza/metabolismo , Doenças das Plantas , SeptinasRESUMO
Rice blast is a devastating disease caused by the fungal pathogen Magnaporthe oryzae that threatens rice production around the world. The fungus produces a specialized infection cell, called the appressorium, that enables penetration through the plant cell wall in response to surface signals from the rice leaf. The underlying biology of plant infection, including the regulation of appressorium formation, is not completely understood. Here we report the identification of a network of temporally coregulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which regulates genes associated with induction of major physiological changes required for appressorium development-including cell-cycle control, autophagic cell death, turgor generation and melanin biosynthesis-as well as controlling a additional set of virulence-associated transcription factor-encoding genes. Pmk1-dependent phosphorylation of Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organization, polarized exocytosis and effector gene expression, which are necessary for plant tissue invasion. Identification of this regulatory cascade provides new potential targets for disease intervention.
Assuntos
Ascomicetos/enzimologia , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Esporos Fúngicos/enzimologia , Ascomicetos/genética , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/patogenicidade , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Fosforilação , Esporos Fúngicos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , VirulênciaRESUMO
Many pathogenic fungi depend on the development of specialized infection structures called appressoria to invade their hosts and cause disease. Impairing the function of fungal infection structures therefore provides a potential means by which diseases could be prevented. In spite of this extraordinary potential, however, relatively few anti-penetrant drugs have been developed to control fungal diseases, of either plants or animals. In the present study, we report the identification of compounds that act specifically to prevent fungal infection. We found that the organization of septin GTPases, which are essential for appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae, requires very-long-chain fatty acids (VLCFAs), which act as mediators of septin organization at membrane interfaces. VLCFAs promote septin recruitment to curved plasma membranes and depletion of VLCFAs prevents septin assembly and host penetration by M. oryzae. We observed that VLCFA biosynthesis inhibitors not only prevent rice blast disease, but also show effective, broad-spectrum fungicidal activity against a wide range of fungal pathogens of maize, wheat and locusts, without affecting their respective hosts. Our findings reveal a mechanism underlying septin-mediated infection structure formation in fungi and provide a class of fungicides to control diverse diseases of plants and animals.