RESUMO
Phytopathogenic oomycetes, such as Phytophthora infestans, secrete an arsenal of effector proteins that modulate plant innate immunity to enable infection. We describe CRN8, a host-translocated effector of P. infestans that has kinase activity in planta. CRN8 is a modular protein of the CRN effector family. The C-terminus of CRN8 localizes to the host nucleus and triggers cell death when the protein is expressed in planta. Cell death induction by CRN8 is dependent on its localization to the plant nucleus, which requires a functional nuclear localization signal (NLS). The C-terminal sequence of CRN8 has similarity to a serine/threonine RD kinase domain. We demonstrated that CRN8 is a functional RD kinase and that its auto-phosphorylation is dependent on an intact catalytic site. Co-immunoprecipitation experiments revealed that CRN8 forms a dimer or multimer. Heterologous expression of CRN8 in planta resulted in enhanced virulence by P. infestans. In contrast, in planta expression of the dominant-negative CRN8(R469A;D470A) resulted in reduced P. infestans infection, further implicating CRN8 in virulence. Overall, our results indicate that similar to animal parasites, plant pathogens also translocate biochemically active kinase effectors inside host cells.
Assuntos
Núcleo Celular/enzimologia , Phytophthora infestans/patogenicidade , Doenças das Plantas/microbiologia , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Solanum tuberosum/microbiologia , Núcleo Celular/genética , Phytophthora infestans/enzimologia , Phytophthora infestans/genética , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Imunidade Vegetal , Proteínas Serina-Treonina Quinases/genética , Solanum tuberosum/imunologia , Nicotiana/metabolismo , Nicotiana/microbiologiaRESUMO
Pathogens use specialized secretion systems and targeting signals to translocate effector proteins inside host cells, a process that is essential for promoting disease and parasitism. However, the amino acid sequences that determine host delivery of eukaryotic pathogen effectors remain mostly unknown. The Crinkler (CRN) proteins of oomycete plant pathogens, such as the Irish potato famine organism Phytophthora infestans, are modular proteins with predicted secretion signals and conserved N-terminal sequence motifs. Here, we provide direct evidence that CRN N termini mediate protein transport into plant cells. CRN host translocation requires a conserved motif that is present in all examined plant pathogenic oomycetes, including the phylogenetically divergent species Aphanomyces euteiches that does not form haustoria, specialized infection structures that have been implicated previously in delivery of effectors. Several distinct CRN C termini localized to plant nuclei and, in the case of CRN8, required nuclear accumulation to induce plant cell death. These results reveal a large family of ubiquitous oomycete effector proteins that target the host nucleus. Oomycetes appear to have acquired the ability to translocate effector proteins inside plant cells relatively early in their evolution and before the emergence of haustoria. Finally, this work further implicates the host nucleus as an important cellular compartment where the fate of plant-microbe interactions is determined.
Assuntos
Proteínas de Algas/metabolismo , Núcleo Celular/metabolismo , Oomicetos/metabolismo , Proteínas de Algas/genética , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Oomicetos/genética , Oomicetos/patogenicidade , Organismos Geneticamente Modificados , Doenças das Plantas/parasitologia , Folhas de Planta/parasitologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The Irish potato famine pathogen Phytophthora infestans is predicted to secrete hundreds of effector proteins. To address the challenge of assigning biological functions to computationally predicted effector genes, we combined allele mining with high-throughput in planta expression. We developed a library of 62 infection-ready P. infestans RXLR effector clones, obtained using primer pairs corresponding to 32 genes and assigned activities to several of these genes. This approach revealed that 16 of the 62 examined effectors cause phenotypes when expressed inside plant cells. Besides the well-studied AVR3a effector, two additional effectors, PexRD8 and PexRD36(45-1), suppressed the hypersensitive cell death triggered by the elicitin INF1, another secreted protein of P. infestans. One effector, PexRD2, promoted cell death in Nicotiana benthamiana and other solanaceous plants. Finally, two families of effectors induced hypersensitive cell death specifically in the presence of the Solanum bulbocastanum late blight resistance genes Rpi-blb1 and Rpi-blb2, thereby exhibiting the activities expected for Avrblb1 and Avrblb2. The AVRblb2 family was then studied in more detail and found to be highly variable and under diversifying selection in P. infestans. Structure-function experiments indicated that a 34-amino acid region in the C-terminal half of AVRblb2 is sufficient for triggering Rpi-blb2 hypersensitivity and that a single positively selected AVRblb2 residue is critical for recognition by Rpi-blb2.
Assuntos
Proteínas de Algas/metabolismo , Phytophthora infestans/patogenicidade , Proteínas de Plantas/metabolismo , Solanum/genética , Alelos , Sequência de Aminoácidos , Morte Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Imunidade Inata , Dados de Sequência Molecular , Phytophthora infestans/metabolismo , Proteínas de Plantas/genética , Polimorfismo Genético , RNA de Plantas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Solanum/imunologia , Solanum/metabolismo , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/metabolismoRESUMO
Filamentous pathogens, such as plant pathogenic fungi and oomycetes, secrete an arsenal of effector molecules that modulate host innate immunity and enable parasitic infection. It is now well accepted that these effectors are key pathogenicity determinants that enable parasitic infection. In this review, we report on the most interesting features of a representative set of filamentous pathogen effectors and highlight recent findings. We also list and describe all the linear motifs reported to date in filamentous pathogen effector proteins. Some of these motifs appear to define domains that mediate translocation inside host cells.
Assuntos
Proteínas Fúngicas/fisiologia , Fungos/patogenicidade , Doenças das Plantas/microbiologia , Fatores de Virulência/fisiologia , Motivos de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Modelos Moleculares , Doenças das Plantas/imunologia , Plantas/imunologia , Plantas/microbiologia , Estrutura Terciária de Proteína , Transporte Proteico , Fatores de Virulência/química , Fatores de Virulência/genéticaRESUMO
The Arabidopsis mutant downy mildew resistant 6 (dmr6) carries a recessive mutation that results in the loss of susceptibility to Hyaloperonospora parasitica. Here we describe the map-based cloning of DMR6 (At5g24530), which was found to encode a 2-oxoglutarate (2OG)-Fe(II) oxygenase of unknown function. DMR6 transcription is locally induced during infections with both compatible and incompatible H. parasitica isolates. High DMR6 transcript levels were also observed in constitutive defense mutants and after treatment with salicylic acid analog BTH, suggesting that DMR6 has a role during plant defense. Expression analysis of dmr6 mutants, using DNA microarrays and quantitative PCR, showed the enhanced expression of a subset of defense-associated genes, including DMR6 itself, suggesting dmr6-mediated resistance results from the activation of plant defense responses. Alternatively, resistance could be caused by the accumulation of a toxic DMR6 substrate, or by the absence of a DMR6 metabolic product that is required for H. parasitica infection.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genes de Plantas , Cetona Oxirredutases/genética , Oomicetos/fisiologia , Arabidopsis/microbiologia , Sequência de Bases , Primers do DNA , Regulação da Expressão Gênica de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
All plant species are subject to disease. Plant diseases are caused by parasites, e.g. viruses, bacteria, oomycetes, parasitic plants, fungi, or nematodes. In all organisms, gene expression is tightly regulated and underpins essential functions and physiology. The coordination and regulation of both host and pathogen gene expression is essential for pathogens to infect and cause disease. One mode of gene regulation is RNA silencing. This biological process is widespread in the natural world, present in plants, animals and several pathogens. In RNA silencing, small (20-40 nucleotides) non-coding RNAs (small-RNAs, sRNAs) accumulate and regulate gene expression transcriptionally or post-transcriptionally in a sequence-specific manner. Regulation of sRNA molecules provides a fast mode to alter gene activity of multiple gene transcripts. RNA silencing is an ancient mechanism that protects the most sensitive part of an organism: its genetic code. sRNA molecules emerged as regulators of plant development, growth and plant immunity. sRNA based RNA silencing functions both within and between organisms. Here we present the described sRNAs from plants and pathogens and discuss how they regulate host immunity and pathogen virulence. We speculate on how sRNA molecules can be exploited to develop disease resistant plants. Finally, the activity of sRNA molecules can be prevented by proteins that suppress RNA silencing. This counter silencing response completes the dialog between plants and pathogens controlling plant disease or resistance outcome on the RNA (controlling gene expression) and protein level.
RESUMO
Plants are susceptible to a limited number of pathogens. Most infections fail due to active defense or absence of compatibility. Many components of the plant's surveillance system and defense arsenal have been identified in the last decades. However, knowledge is limited on compatibility; in particular, the role of plant factors in the infection process. To gain insight into these processes, we have initiated an Arabidopsis thaliana mutant screen for reduced susceptibility to the downy mildew pathogen Hyaloperonospora parasitica. Ethyl methane sulfonate (EMS) mutants were generated in the highly susceptible Arabidopsis line Ler eds1-2. Eight downy mildew-resistant (dmr) mutants were analyzed in detail, corresponding to six different loci. Microscopic analysis showed that, in all mutants, H. parasitica growth was severely reduced. Resistance of dmr3, dmr4, and dmr5 was associated with constitutive expression of PR-1. Furthermore, dmr3 and dmr4, but not dmr5, also were resistant to Pseudomonas syringae and Golovinomyces orontii, respectively. However, enhanced activation of plant defense was not observed in dmr1, dmr2, and dmr6. We postulate that, in these susceptibility mutants, cellular processes are disrupted which are required for H. parasitica infection. This interesting new set of mutants provides a basis to elucidate the molecular processes underlying susceptibility to downy mildew in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Oomicetos/crescimento & desenvolvimento , Doenças das Plantas/genética , Arabidopsis/microbiologia , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Análise Mutacional de DNA , Metanossulfonato de Etila/toxicidade , Teste de Complementação Genética , Imunidade Inata/genética , Solanum lycopersicum/microbiologia , Mutagênese/efeitos dos fármacos , Mutação , Oomicetos/patogenicidade , Fenótipo , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/microbiologia , Pseudomonas/crescimento & desenvolvimentoRESUMO
The recognition of pathogen effectors by plant immune receptors leads to the activation of immune responses that often include a hypersensitive response (HR): rapid and localized host cell death surrounding the site of attempted pathogen ingress. We have demonstrated previously that the recognition of the Verticillium dahliae effector protein Ave1 by the tomato immune receptor Ve1 triggers an HR in tomato and tobacco. Furthermore, we have demonstrated that tomato Ve1 provides Verticillium resistance in Arabidopsis upon Ave1 recognition. In this study, we investigated whether the co-expression of Ve1 and Ave1 in Arabidopsis results in an HR, which could facilitate a forward genetics screen. Surprisingly, we found that the co-expression of Ve1 and Ave1 does not induce an HR in Arabidopsis. These results suggest that an HR may occur as a consequence of Ve1/Ave1-induced immune signalling in tomato and tobacco, but is not absolutely required for Verticillium resistance.
Assuntos
Arabidopsis/imunologia , Arabidopsis/microbiologia , Resistência à Doença/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Verticillium/fisiologia , Agrobacterium/fisiologia , Arabidopsis/genética , Solanum lycopersicum/metabolismo , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Pseudomonas syringae/fisiologia , Plântula/metabolismo , Plântula/microbiologia , Nicotiana/microbiologiaRESUMO
Plant disease resistance is commonly triggered by early pathogen recognition and activation of immunity. An alternative form of resistance is mediated by recessive downy mildew resistant 1 (dmr1) alleles in Arabidopsis thaliana. Map-based cloning revealed that DMR1 encodes homoserine kinase (HSK). Six independent dmr1 mutants each carry a different amino acid substitution in the HSK protein. Amino acid analysis revealed that dmr1 mutants contain high levels of homoserine that is undetectable in wild-type plants. Surprisingly, the level of amino acids downstream in the aspartate (Asp) pathway was not reduced in dmr1 mutants. Exogenous homoserine does not directly affect pathogen growth but induces resistance when infiltrated in Arabidopsis. We provide evidence that homoserine accumulation in the chloroplast triggers a novel form of downy mildew resistance that is independent of known immune responses.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/microbiologia , Imunidade Inata/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Doenças das Plantas/microbiologia , Aminoácidos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Imunidade Inata/genética , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Doenças das Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologiaRESUMO
Long considered intractable organisms by fungal genetic research standards, the oomycetes have recently moved to the centre stage of research on plant-microbe interactions. Recent work on oomycete effector evolution, trafficking and function has led to major conceptual advances in the science of plant pathology. In this review, we provide a historical perspective on oomycete genetic research and summarize the state of the art in effector biology of plant pathogenic oomycetes by describing what we consider to be the 10 most important concepts about oomycete effectors.