RESUMO
MicroRNAs (miRNAs) are small noncoding RNAs in eukaryotes. Plant endogenous miRNAs play pivotal roles in regulating plant development and defense responses. MicroRNA394 (miR394) has been reported to regulate plant development, abiotic stresses and defense responses. Previous reports showed that miR394 responded to P. infestans inoculation in potato, indicating that miR394 may be involved in defense responses. In this study, we further investigated its role in potato defense against P. infestans. Stable expression of miR394 in tobacco and potato enhances the susceptibility to P. infestans, which is accompanied with the reduced accumulation of ROS and down-regulation of the PTI (pattern-triggered immunity) marker genes. Besides well-known target StLCR, miR394 also targets StA/N-INVE, which encodes a chloroplast Alkaline/Neutral Invertases (A/N-INVE). Both StLCR and StA/N-INVE positively regulate late blight resistance, while miR394 degrades them. Interestingly, StA/N-INVE is located in the chloroplast, indicating that miR394 may manipulate chloroplast immunity. Degradation of StA/N-INVE may affect the chloroplast function and hence lead to the compromised ROS (reactive oxygen species) burst and reduced retrograde signaling from the chloroplast to the nucleus and cytoplasm. In summary, this study provides new information that miR394 targets and degrades StA/N-INVE and StLCR, which are positive regulators, to enhance potato susceptibility to P. infestans.
Assuntos
MicroRNAs , Phytophthora infestans , Solanum tuberosum , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Phytophthora infestans/genética , Phytophthora infestans/metabolismo , Plantas/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Doenças das Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
The oomycete pathogen Phytophthora capsici encodes hundreds of RXLR effectors that enter the plant cells and suppress host immunity. Only a few of these genes are conserved across different strains and species. Such core effectors might target hub genes and immune pathways in hosts. Here, we describe the functional characterization of the core P. capsici RXLR effector RXLR242. The expression of RXLR242 was up-regulated during infection, and its ectopic expression in Nicotiana benthamiana, an experimental plant host, further promoted Phytophthora infection. RXLR242 physically interacted with a group of RAB proteins that belong to the small GTPase family and play a role in regulating transport pathways in the intracellular membrane trafficking system. In addition, RXLR242 impeded the secretion of PATHOGENESIS-RELATED 1 (PR1) protein to the apoplast. This phenomenon resulted from the competitive binding of RXLR242 to RABE1-7. We also found that RXLR242 interfered with the association between RABA4-3 and its binding protein, thereby disrupting the trafficking of the membrane receptor FLAGELLIN-SENSING 2. Thus, RXLR242 manipulates plant immunity by targeting RAB proteins and disrupting protein trafficking in the host plants.
Assuntos
Phytophthora infestans , Phytophthora infestans/metabolismo , Imunidade Vegetal/genética , Doenças das Plantas , Proteínas/metabolismo , Plantas/metabolismo , Transporte ProteicoRESUMO
Plants are resistant to most microbial species due to nonhost resistance (NHR), providing broad-spectrum and durable immunity. However, the molecular components contributing to NHR are poorly characterised. We address the question of whether failure of pathogen effectors to manipulate nonhost plants plays a critical role in NHR. RxLR (Arg-any amino acid-Leu-Arg) effectors from two oomycete pathogens, Phytophthora infestans and Hyaloperonospora arabidopsidis, enhanced pathogen infection when expressed in host plants (Nicotiana benthamiana and Arabidopsis, respectively) but the same effectors performed poorly in distantly related nonhost pathosystems. Putative target proteins in the host plant potato were identified for 64 P. infestans RxLR effectors using yeast 2-hybrid (Y2H) screens. Candidate orthologues of these target proteins in the distantly related non-host plant Arabidopsis were identified and screened using matrix Y2H for interaction with RxLR effectors from both P. infestans and H. arabidopsidis. Few P. infestans effector-target protein interactions were conserved from potato to candidate Arabidopsis target orthologues (cAtOrths). However, there was an enrichment of H. arabidopsidis RxLR effectors interacting with cAtOrths. We expressed the cAtOrth AtPUB33, which unlike its potato orthologue did not interact with P. infestans effector PiSFI3, in potato and Nicotiana benthamiana. Expression of AtPUB33 significantly reduced P. infestans colonization in both host plants. Our results provide evidence that failure of pathogen effectors to interact with and/or correctly manipulate target proteins in distantly related non-host plants contributes to NHR. Moreover, exploiting this breakdown in effector-nonhost target interaction, transferring effector target orthologues from non-host to host plants is a strategy to reduce disease.
Assuntos
Arabidopsis , Resistência à Doença , Especificidade de Hospedeiro , Nicotiana , Doenças das Plantas , Proteínas de Plantas , Arabidopsis/metabolismo , Arabidopsis/parasitologia , Oomicetos/metabolismo , Phytophthora infestans/metabolismo , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/metabolismo , Solanum tuberosum/parasitologia , Nicotiana/metabolismo , Nicotiana/parasitologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Species of the genus Phytophthora, the plant killer, cause disease and reduce yields in many crop plants. Although many Resistance to Phytophthora infestans (Rpi) genes effective against potato late blight have been cloned, few have been cloned against other Phytophthora species. Most Rpi genes encode nucleotide-binding domain, leucine-rich repeat-containing (NLR) immune receptor proteins that recognize RXLR (Arg-X-Leu-Arg) effectors. However, whether NLR proteins can recognize RXLR effectors from multiple Phytophthora species has rarely been investigated. Here, we identified a new RXLR-WY effector AVRamr3 from P. infestans that is recognized by Rpi-amr3 from a wild Solanaceae species Solanum americanum. Rpi-amr3 associates with AVRamr3 in planta. AVRamr3 is broadly conserved in many different Phytophthora species, and the recognition of AVRamr3 homologs by Rpi-amr3 activates resistance against multiple Phytophthora pathogens, including the tobacco black shank disease and cacao black pod disease pathogens P. parasitica and P. palmivora. Rpi-amr3 is thus the first characterized resistance gene that acts against P. parasitica or P. palmivora. These findings suggest a novel path to redeploy known R genes against different important plant pathogens.
Assuntos
Phytophthora infestans , Solanum tuberosum , Solanum , Resistência à Doença/genética , Genes de Plantas , Phytophthora infestans/metabolismo , Doenças das Plantas/genética , Solanum/genética , Solanum tuberosum/genéticaRESUMO
Blue-light (BL) phototropin receptors (phot1 and phot2) regulate plant growth by activating NPH3/RPT2-like (NRL) family members. Little is known about roles for BL and phots in regulating plant immunity. We showed previously that Phytophthora infestans RXLR effector Pi02860 targets potato (St)NRL1, promoting its ability to enhance susceptibility by facilitating proteasome-mediated degradation of the immune regulator StSWAP70. This raises the question: do BL and phots negatively regulate immunity? We employed coimmunoprecipitation, virus-induced gene silencing, transient overexpression and targeted mutation to investigate contributions of phots to regulating immunity. Whereas transient overexpression of Stphot1 and Stphot2 enhances P. infestans colonization of Nicotiana benthamiana, silencing endogenous Nbphot1 or Nbphot2 reduces infection. Stphot1, but not Stphot2, suppressed the INF1-triggered cell death (ICD) immune response in a BL- and NRL1-dependent manner. Stphot1, when coexpressed with StNRL1, promotes degradation of StSWAP70, whereas Stphot2 does not. Kinase-dead Stphot1 fails to suppress ICD, enhance P. infestans colonization or promote StSWAP70 degradation. Critically, BL enhances P. infestans infection, which probably involves phots but not other BL receptors such as cryptochromes and F-box proteins ZTL1 and FKF1. We demonstrate that Stphot1 and Stphot2 play different roles in promoting susceptibility, and Stphot1 kinase activity is required for BL- and StNRL1-mediated immune suppression.
Assuntos
Phytophthora infestans , Fototropinas/metabolismo , Phytophthora infestans/metabolismo , Doenças das Plantas , Imunidade Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Nicotiana/metabolismoRESUMO
Heavy metal pollution causes many soils to become a toxic environment not only for plants, but also microorganisms; however, little is known how heavy metal contaminated environment affects metabolism of phytopathogens and their capability of infecting host plants. In this study the oomycete Phytophthora infestans (Mont.) de Bary, the most harmful pathogen of potato, growing under moderate cadmium stress (Cd, 5 mg/L) showed nitro-oxidative imbalance associated with an enhanced antioxidant response. Cadmium notably elevated the level of nitric oxide, superoxide and peroxynitrite that stimulated nitrative modifications within the RNA and DNA pools in the phytopathogen structures. In contrast, the protein pool undergoing nitration was diminished confirming that protein tyrosine nitration is a flexible element of the oomycete adaptive strategy to heavy metal stress. Finally, to verify whether Cd is able to modify P. infestans pathogenicity, a disease index and molecular assessment of disease progress were analysed indicating that Cd stress enhanced aggressiveness of vr P. infestans towards various potato cultivars. Taken together, Cd not only affected hyphal growth rate and caused biochemical changes in P. infestans structures, but accelerated the pathogenicity as well. The nitro-oxidative homeostasis imbalance underlies the phytopathogen adaptive strategy and survival in the heavy metal contaminated environment.
Assuntos
Cádmio/toxicidade , Phytophthora infestans/efeitos dos fármacos , Phytophthora infestans/metabolismo , Antioxidantes/metabolismo , Homeostase/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Phytophthora infestans/patogenicidade , Doenças das Plantas/etiologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Poluentes do Solo/toxicidade , Solanum tuberosum/microbiologia , Estresse Fisiológico , Virulência/efeitos dos fármacosRESUMO
In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune sensors that recognize and eliminate a wide range of invading pathogens. NLR-mediated immunity is known to be modulated by environmental factors. However, how pathogen recognition by NLRs is influenced by environmental factors such as light remains unclear. Here, we show that the agronomically important NLR Rpi-vnt1.1 requires light to confer disease resistance against races of the Irish potato famine pathogen Phytophthora infestans that secrete the effector protein AVRvnt1. The activation of Rpi-vnt1.1 requires a nuclear-encoded chloroplast protein, glycerate 3-kinase (GLYK), implicated in energy production. The pathogen effector AVRvnt1 binds the full-length chloroplast-targeted GLYK isoform leading to activation of Rpi-vnt1.1. In the dark, Rpi-vnt1.1-mediated resistance is compromised because plants produce a shorter GLYK-lacking the intact chloroplast transit peptide-that is not bound by AVRvnt1. The transition between full-length and shorter plant GLYK transcripts is controlled by a light-dependent alternative promoter selection mechanism. In plants that lack Rpi-vnt1.1, the presence of AVRvnt1 reduces GLYK accumulation in chloroplasts counteracting GLYK contribution to basal immunity. Our findings revealed that pathogen manipulation of chloroplast functions has resulted in a light-dependent immune response.
Assuntos
Cloroplastos/microbiologia , Regulação da Expressão Gênica de Plantas/imunologia , Luz , Proteínas NLR/metabolismo , Phytophthora infestans/metabolismo , Proteínas de Plantas/metabolismo , Agrobacterium/metabolismo , Animais , Cloroplastos/metabolismo , Escherichia coli/metabolismo , Proteínas Fúngicas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Inativação Gênica , Microscopia Confocal , Proteínas NLR/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Plantas/genética , Plântula , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiologia , Nicotiana/metabolismo , Nicotiana/microbiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
To be successful plant pathogens, microbes use "effector proteins" to manipulate host functions to their benefit. Identifying host targets of effector proteins and characterizing their role in the infection process allow us to better understand plant-pathogen interactions and the plant immune system. Yeast two-hybrid analysis and coimmunoprecipitation were used to demonstrate that the Phytophthora infestans effector AVIRULENCE 2 (PiAVR2) interacts with all three BRI1-SUPPRESSOR1-like (BSL) family members from potato (Solanum tuberosum). Transient expression of BSL1, BSL2, and BSL3 enhanced P. infestans leaf infection. BSL1 and BSL3 suppressed INFESTIN 1 elicitin-triggered cell death, showing that they negatively regulate immunity. Virus-induced gene silencing studies revealed that BSL2 and BSL3 are required for BSL1 stability and show that basal levels of immunity are increased in BSL-silenced plants. Immune suppression by BSL family members is dependent on the brassinosteroid-responsive host transcription factor CIB1/HBI1-like 1. The P. infestans effector PiAVR2 targets all three BSL family members in the crop plant S. tuberosum These phosphatases, known for their role in growth-promoting brassinosteroid signaling, all support P. infestans virulence and thus can be regarded as susceptibility factors in late blight infection.
Assuntos
Phytophthora infestans/patogenicidade , Imunidade Vegetal , Proteínas de Plantas/imunologia , Fatores de Virulência/metabolismo , Inativação Gênica , Interações Hospedeiro-Patógeno , Phytophthora infestans/metabolismo , Doenças das Plantas/imunologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Solanum tuberosum/metabolismo , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/microbiologia , Fatores de Virulência/genéticaRESUMO
KEY MESSAGE: Oomycetes MAMP Pep-13 can trigger SERK3/BAK1-independent PTI. Silencing of SERK3/BAK1 in solanaceous plants resulted in reduced expression of brassinosteroid marker genes and enhanced PTI transcriptional responses to Pep-13 treatment. To prevent disease, pattern recognition receptors (PRRs) are responsible for detecting microbe-associated molecular patterns (MAMPs) to switch on plant innate immunity. SOMATIC EMBROYOGENESIS KINASE 3 (SERK3)/BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) is a well-characterized receptor-like kinase (RLK) that serves as a pivotal co-receptor with PRRs to activate immunity following recognition of MAMPs including flg22, EF-Tu, INF1 and XEG1. However, the requirement for SERK3/BAK1 in many pattern-triggered immune (PTI) signaling pathways is not yet known. Pep-13 is an oomycete MAMP that consists of a highly conserved motif (an oligopeptide of 13 amino acids) shared in Phytophthora transglutaminases. Quantitative RT-PCR analysis reveals that the transcripts of three PTI marker genes (WRKY7, WRKY8 and ACRE31) rapidly accumulate in response to three different MAMPs: flg22, chitin and Pep-13. Whereas silencing of SERK3/BAK1 in Nicotiana benthamiana or potato compromised transcript accumulation in response to flg22, it did not attenuate WRKY7, WRKY8 and ACRE31 up-regulation in response to chitin or Pep-13. This indicates that Pep-13 triggers immunity in a SERK3/BAK1-independent manner, similar to chitin. Surprisingly, silencing of SERK3/BAK1 led to significantly increased accumulation of PTI marker gene transcripts following Pep-13 or chitin treatment, compared to controls. This was accompanied by reduced expression of brassinosteroid (BR) marker genes StSTDH, StEXP8 and StCAB50 and StCHL1, which is a negative regulator of PTI, supporting previous reports that SERK3/BAK1-dependent BR signaling attenuates plant immunity. We provide Pep-13 as an alternative to chitin as a trigger of SERK3/BAK1-independent immunity.
Assuntos
Alarminas/metabolismo , Nicotiana/imunologia , Phytophthora infestans/metabolismo , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Solanum tuberosum/imunologia , Brassinosteroides/farmacologia , Quitina/farmacologia , Flagelina/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peptídeos/farmacologia , Phytophthora infestans/efeitos dos fármacos , Imunidade Vegetal/efeitos dos fármacos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Solanum tuberosum/genética , Nicotiana/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
The potato blight agent Phytophthora infestans secretes a range of RXLR effectors to promote disease. Recent evidence indicates that some effectors suppress early pattern-triggered immunity (PTI) following perception of microbe-associated molecular patterns (MAMPs). Phytophthora infestans effector PiSFI3/Pi06087/PexRD16 has been previously shown to suppress MAMP-triggered pFRK1-Luciferase reporter gene activity. How PiSFI3 suppresses immunity is unknown. We employed yeast-two-hybrid (Y2H) assays, co-immunoprecipitation, transcriptional silencing by RNA interference and virus-induced gene silencing (VIGS), and X-ray crystallography for structure-guided mutagenesis, to investigate the function of PiSFI3 in targeting a plant U-box-kinase protein (StUBK) to suppress immunity. We discovered that PiSFI3 is active in the host nucleus and interacts in yeast and in planta with StUBK. UBK is a positive regulator of specific PTI pathways in both potato and Nicotiana benthamiana. Importantly, it contributes to early transcriptional responses that are suppressed by PiSFI3. PiSFI3 forms an unusual trans-homodimer. Mutation to disrupt dimerization prevents nucleolar localisation of PiSFI3 and attenuates both its interaction with StUBK and its ability to enhance P. infestans leaf colonisation. PiSFI3 is a 'WY-domain' RXLR effector that forms a novel trans-homodimer which is required for its ability to suppress PTI via interaction with the U-box-kinase protein StUBK.
Assuntos
Phytophthora infestans/metabolismo , Proteínas Quinases/metabolismo , Proteínas/metabolismo , Solanum tuberosum/imunologia , Solanum tuberosum/microbiologia , Transcrição Gênica , Apoptose/efeitos dos fármacos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Flagelina/farmacologia , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Mutação/genética , Phytophthora infestans/patogenicidade , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/microbiologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Proteínas Quinases/química , Multimerização Proteica , Solanum tuberosum/efeitos dos fármacos , Solanum tuberosum/genética , VirulênciaRESUMO
The oomycete potato blight pathogen Phytophthora infestans secretes a diverse set of proteins to manipulate host plant immunity. However, there is limited knowledge about how and where they are secreted during infection. Here we used the endoplasmic reticulum (ER)-to-Golgi secretion pathway inhibitor brefeldin A (BFA) in combination with liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) to identify extracellular proteins from P. infestans that were conventionally secreted from in vitro-cultured hyphae. We identified 19 proteins with predicted signal peptides that potentially influence plant interactions for which secretion was attenuated by BFA. In addition to inhibition by the apoplastic effector EPIC1, a cysteine protease inhibitor, we show that secretion of the cell wall-degrading pectinesterase enzyme PE1 and the microbe-associated molecular pattern (MAMP)-like elicitin INF4 was inhibited by BFA in vitro and in planta, demonstrating that these proteins are secreted by the conventional, Golgi-mediated pathway. For comparison, secretion of a cytoplasmic RXLR (Arg-[any amino acid]-Leu-Arg) effector, Pi22926, was not inhibited by BFA. During infection, whereas INF4 accumulated outside the plant cell, RXLR effector Pi22926 entered the plant cell and accumulated in the nucleus. The P. infestans effectors, the PE1 enzyme, and INF4 were all secreted from haustoria, pathogen structures that penetrate the plant cell wall to form an intimate interaction with the host plasma membrane. Our findings show the haustorium to be a major site of both conventional and nonconventional secretion of proteins with diverse functions during infection.IMPORTANCE There are many different classes of proteins secreted from Phytophthora infestans that may influence or facilitate infection. Elucidating where and how they are secreted during infection is an important step toward developing methods to control their delivery processes. We used an inhibitor of conventional secretion to identify the following different classes of infection-associated extracellular proteins: cell wall-degrading and cell wall-modifying enzymes, microbe-associated molecular pattern-like proteins that may elicit immune responses, and apoplastic effectors that are predicted to suppress immunity. In contrast, secretion of a cytoplasmic effector that is translocated into host cells is nonconventional, as it is insensitive to inhibitor treatment. This evidence further supports the finding that proteins that are active in the apoplast and effector proteins that are active in the host cytoplasm are differentially secreted by P. infestans Critically, it demonstrates that a disease-specific developmental structure, the haustorium, is a major secretion site for diverse protein classes during infection.
Assuntos
Proteínas Fúngicas/metabolismo , Phytophthora infestans/metabolismo , Fatores de Virulência/metabolismo , Antifúngicos/metabolismo , Brefeldina A/metabolismo , Cromatografia Líquida , Hifas/efeitos dos fármacos , Hifas/metabolismo , Phytophthora infestans/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Espectrometria de Massas em TandemRESUMO
Pathogens secrete effector proteins to interfere with plant innate immunity, in which Ca2+ /calmodulin (CaM) signalling plays key roles. Thus far, few effectors have been identified that directly interact with CaM for defence suppression. Here, we report that SFI5, an RXLR effector from Phytophthora infestans, suppresses microbe-associated molecular pattern (MAMP)-triggered immunity (MTI) by interacting with host CaMs. We predicted the CaM-binding site in SFI5 using in silico analysis. The interaction between SFI5 and CaM was tested by both in vitro and in vivo assays. MTI suppression by SFI5 and truncated variants were performed in a tomato protoplast system. We found that both the predicted CaM-binding site and the full-length SFI5 protein interact with CaM in the presence of Ca2+ . MTI responses, such as FRK1 upregulation, reactive oxygen species accumulation, and mitogen-activated protein kinase activation were suppressed by truncated SFI5 proteins containing the C-terminal CaM-binding site but not by those without it. The plasma membrane localization of SFI5 and its ability to enhance infection were also perturbed by loss of the CaM-binding site. We conclude that CaM-binding is required for localization and activity of SFI5. We propose that SFI5 suppresses plant immunity by interfering with immune signalling components after activation by CaMs.
Assuntos
Calmodulina/metabolismo , Moléculas com Motivos Associados a Patógenos/metabolismo , Phytophthora infestans/metabolismo , Imunidade Vegetal , Proteínas/química , Proteínas/metabolismo , Solanum lycopersicum/imunologia , Solanum lycopersicum/microbiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Cálcio/farmacologia , Membrana Celular/metabolismo , Solanum lycopersicum/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Phytophthora infestans/efeitos dos fármacos , Imunidade Vegetal/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacosRESUMO
The oomycete Phytophthora infestans is the most harmful pathogen of potato. It causes the disease late blight, which generates increased yearly costs of up to one billion euro in the EU alone and is difficult to control. We have performed a large-scale quantitative proteomics study of six P. infestans life stages with the aim to identify proteins that change in abundance during development, with a focus on preinfectious life stages. Over 10 000 peptides from 2061 proteins were analyzed. We identified several abundance profiles of proteins that were up- or downregulated in different combinations of life stages. One of these profiles contained 59 proteins that were more abundant in germinated cysts and appressoria. A large majority of these proteins were not previously recognized as being appressorial proteins or involved in the infection process. Among those are proteins with putative roles in transport, amino acid metabolism, pathogenicity (including one RXLR effector) and cell wall structure modification. We analyzed the expression of the genes encoding nine of these proteins using RT-qPCR and found an increase in transcript levels during disease progression, in agreement with the hypothesis that these proteins are important in early infection. Among the nine proteins was a group involved in cell wall structure modification and adhesion, including three closely related, uncharacterized proteins encoded by PITG_01131, PITG_01132, and PITG_16135, here denoted Piacwp1-3 Transient silencing of these genes resulted in reduced severity of infection, indicating that these proteins are important for pathogenicity. Our results contribute to further insight into P. infestans biology, and indicate processes that might be relevant for the pathogen while preparing for host cell penetration and during infection. The mass spectrometry data have been deposited to ProteomeXchange via the PRIDE partner repository with the data set identifier PXD002446.
Assuntos
Phytophthora infestans/patogenicidade , Proteômica/métodos , Solanum tuberosum/parasitologia , Fatores de Virulência/metabolismo , Parede Celular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Espectrometria de Massas , Phytophthora infestans/crescimento & desenvolvimento , Phytophthora infestans/metabolismo , Doenças das Plantas/parasitologia , Fatores de Virulência/genéticaRESUMO
The potato blight pathogen Phytophthora infestans secretes effector proteins that are delivered inside (cytoplasmic) or can act outside (apoplastic) plant cells to neutralize host immunity. Little is known about how and where effectors are secreted during infection, yet such knowledge is essential to understand and combat crop disease. We used transient Agrobacterium tumefaciens-mediated in planta expression, transformation of P. infestans with fluorescent protein fusions and confocal microscopy to investigate delivery of effectors to plant cells during infection. The cytoplasmic effector Pi04314, expressed as a monomeric red fluorescent protein (mRFP) fusion protein with a signal peptide to secrete it from plant cells, did not passively re-enter the cells upon secretion. However, Pi04314-mRFP expressed in P. infestans was translocated from haustoria, which form intimate interactions with plant cells, to accumulate at its sites of action in the host nucleus. The well-characterized apoplastic effector EPIC1, a cysteine protease inhibitor, was also secreted from haustoria. EPIC1 secretion was inhibited by brefeldin A (BFA), demonstrating that it is delivered by conventional Golgi-mediated secretion. By contrast, Pi04314 secretion was insensitive to BFA treatment, indicating that the cytoplasmic effector follows an alternative route for delivery into plant cells. Phytophthora infestans haustoria are thus sites for delivery of both apoplastic and cytoplasmic effectors during infection, following distinct secretion pathways.
Assuntos
Citoplasma/metabolismo , Phytophthora infestans/metabolismo , Via Secretória , Brefeldina A/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Recuperação de Fluorescência Após Fotodegradação , Phytophthora infestans/efeitos dos fármacos , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Células Vegetais/microbiologia , Proteínas Recombinantes de Fusão/metabolismo , Via Secretória/efeitos dos fármacos , Nicotiana/microbiologiaRESUMO
An emerging area in plant research focuses on antagonism between regulatory systems governing growth and immunity. Such cross talk represents a point of vulnerability for pathogens to exploit. AVR2, an RXLR effector secreted by the potato blight pathogen Phytophthora infestans, interacts with potato BSL1, a putative phosphatase implicated in growth-promoting brassinosteroid (BR) hormone signaling. Transgenic potato (Solanum tuberosum) plants expressing the effector exhibit transcriptional and phenotypic hallmarks of overactive BR signaling and show enhanced susceptibility to P. infestans Microarray analysis was used to identify a set of BR-responsive marker genes in potato, all of which are constitutively expressed to BR-induced levels in AVR2 transgenic lines. One of these genes was a bHLH transcription factor, designated StCHL1, homologous to AtCIB1 and AtHBI1, which are known to facilitate antagonism between BR and immune responses. Transient expression of either AVR2 or CHL1 enhanced leaf colonization by P. infestans and compromised immune cell death activated by perception of the elicitin Infestin1 (INF1). Knockdown of CHL1 transcript using Virus-Induced Gene Silencing (VIGS) reduced colonization of P. infestans on Nicotiana benthamiana Moreover, the ability of AVR2 to suppress INF1-triggered cell death was attenuated in NbCHL1-silenced plants, indicating that NbCHL1 was important for this effector activity. Thus, AVR2 exploits cross talk between BR signaling and innate immunity in Solanum species, representing a novel, indirect mode of innate immune suppression by a filamentous pathogen effector.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Phytophthora infestans/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Brassinosteroides/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno/genética , Phytophthora infestans/genética , Phytophthora infestans/patogenicidade , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Homologia de Sequência de Aminoácidos , Solanum tuberosum/genética , Solanum tuberosum/microbiologia , Regulação para Cima , Fatores de Virulência/genéticaRESUMO
The oomycete Phytophthora infestans is the cause of late blight in potato and tomato. It is a devastating pathogen and there is an urgent need to design alternative strategies to control the disease. To find novel potential drug targets, we used Lifeact-eGFP expressing P. infestans for high resolution live cell imaging of the actin cytoskeleton in various developmental stages. Previously, we identified actin plaques as structures that are unique for oomycetes. Here we describe two additional novel actin configurations; one associated with plug deposition in germ tubes and the other with appressoria, infection structures formed prior to host cell penetration. Plugs are composed of cell wall material that is deposited in hyphae emerging from cysts to seal off the cytoplasm-depleted base after cytoplasm retraction towards the growing tip. Preceding plug formation there was a typical local actin accumulation and during plug deposition actin remained associated with the leading edge. In appressoria, formed either on an artificial surface or upon contact with plant cells, we observed a novel aster-like actin configuration that was localized at the contact point with the surface. Our findings strongly suggest a role for the actin cytoskeleton in plug formation and plant cell penetration.
Assuntos
Actinas/metabolismo , Parede Celular/metabolismo , Phytophthora infestans/citologia , Phytophthora infestans/metabolismo , Células Vegetais/metabolismo , Celulose/metabolismo , Meios de Cultura , Hifas/citologia , Hifas/metabolismo , Solanum lycopersicum/citologia , Solanum lycopersicum/microbiologia , Transporte ProteicoRESUMO
Plant pathogens deliver effectors to manipulate host processes. We know little about how fungal and oomycete effectors target host proteins to promote susceptibility, yet such knowledge is vital to understand crop disease. We show that either transient expression in Nicotiana benthamiana, or stable transgenic expression in potato (Solanum tuberosum), of the Phytophthora infestans RXLR effector Pi02860 enhances leaf colonization by the pathogen. Expression of Pi02860 also attenuates cell death triggered by the P. infestans microbe-associated molecular pattern INF1, indicating that the effector suppresses pattern-triggered immunity. However, the effector does not attenuate cell death triggered by Cf4/Avr4 coexpression, showing that it does not suppress all cell death activated by cell surface receptors. Pi02860 interacts in yeast two-hybrid assays with potato NPH3/RPT2-LIKE1 (NRL1), a predicted CULLIN3-associated ubiquitin E3 ligase. Interaction of Pi02860 in planta was confirmed by coimmunoprecipitation and bimolecular fluorescence complementation assays. Virus-induced gene silencing of NRL1 in N. benthamiana resulted in reduced P. infestans colonization and accelerated INF1-mediated cell death, indicating that this host protein acts as a negative regulator of immunity. Moreover, whereas NRL1 virus-induced gene silencing had no effect on the ability of the P. infestans effector Avr3a to suppress INF1-mediated cell death, such suppression by Pi02860 was significantly attenuated, indicating that this activity of Pi02860 is mediated by NRL1. Transient overexpression of NRL1 resulted in the suppression of INF1-mediated cell death and enhanced P. infestans leaf colonization, demonstrating that NRL1 acts as a susceptibility factor to promote late blight disease.
Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Phytophthora infestans/patogenicidade , Proteínas de Plantas/metabolismo , Solanum tuberosum/microbiologia , Morte Celular/genética , Suscetibilidade a Doenças , Regulação da Expressão Gênica de Plantas , Phytophthora infestans/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal , Folhas de Planta/microbiologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Domínios Proteicos , Solanum tuberosum/genética , Solanum tuberosum/imunologia , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Plant pathogens deliver effectors to alter host processes. Knowledge of how effectors target and manipulate host proteins is critical to understand crop disease. Here, we show that in planta expression of the RXLR effector Pi04314 enhances leaf colonization by Phytophthora infestans via activity in the host nucleus and attenuates induction of jasmonic and salicylic acid-responsive genes. Pi04314 interacts with three host protein phosphatase 1 catalytic (PP1c) isoforms, causing their re-localization from the nucleolus to the nucleoplasm. Re-localization of PP1c-1 also occurs during infection and is dependent on an R/KVxF motif in the effector. Silencing the PP1c isoforms or overexpression of a phosphatase-dead PP1c-1 mutant attenuates infection, demonstrating that host PP1c activity is required for disease. Moreover, expression of PP1c-1mut abolishes enhanced leaf colonization mediated by in planta Pi04314 expression. We argue that PP1c isoforms are susceptibility factors forming holoenzymes with Pi04314 to promote late blight disease.
Assuntos
Nicotiana/enzimologia , Phytophthora infestans/metabolismo , Doenças das Plantas/parasitologia , Proteínas de Plantas/metabolismo , Proteína Fosfatase 1/metabolismo , Solanum tuberosum/enzimologia , Interações Hospedeiro-Patógeno , Phytophthora infestans/genética , Doenças das Plantas/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/parasitologia , Proteínas de Plantas/genética , Ligação Proteica , Proteína Fosfatase 1/genética , Solanum tuberosum/genética , Solanum tuberosum/parasitologia , Nicotiana/genética , Nicotiana/parasitologiaRESUMO
Phytophthora infestans secretes numerous RXLR effectors that modulate host defense and thereby pave the way for successful invasion. Here, we show that the RXLR effector AVR1 is a virulence factor that promotes colonization and suppresses callose deposition, a hallmark of basal defense. To identify host targets of AVR1, we performed yeast two-hybrid screens and selected Sec5 as a candidate. Sec5 is a subunit of the exocyst, a protein complex that is involved in vesicle trafficking. AVR1-like (A-L), a close homolog of AVR1, also acts as a virulence factor, but unlike AVR1, A-L does not suppress CRINKLER2 (CRN2)-induced cell death or interact with Sec5. Compared with AVR1, A-L is shorter and lacks the carboxyl-terminal tail, the T-region that is crucial for CRN2-induced cell death suppression and Sec5 interaction. In planta analyses revealed that AVR1 and Sec5 are in close proximity, and coimmunoprecipitation confirmed the interaction. Sec5 is required for secretion of the pathogenesis-related protein PR-1 and callose deposition and also plays a role in CRN2-induced cell death. Our findings show that P. infestans manipulates an exocyst subunit and thereby potentially disturbs vesicle trafficking, a cellular process that is important for basal defense. This is a novel strategy that oomycete pathogens exploit to modulate host defense.
Assuntos
Interações Hospedeiro-Patógeno , Nicotiana/parasitologia , Phytophthora infestans/imunologia , Doenças das Plantas/imunologia , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Fatores de Virulência/metabolismo , Morte Celular , Glucanos/metabolismo , Phytophthora infestans/metabolismo , Phytophthora infestans/patogenicidade , Doenças das Plantas/parasitologia , Folhas de Planta/imunologia , Folhas de Planta/parasitologia , Proteínas de Plantas/genética , Transporte Proteico , Nicotiana/imunologia , Técnicas do Sistema de Duplo-Híbrido , Fatores de Virulência/genéticaRESUMO
Resistance against oomycete pathogens is mainly governed by intracellular nucleotide-binding leucine-rich repeat (NLR) receptors that recognize matching avirulence (AVR) proteins from the pathogen, RXLR effectors that are delivered inside host cells. Detailed molecular understanding of how and where NLR proteins and RXLR effectors interact is essential to inform the deployment of durable resistance (R) genes. Fluorescent tags, nuclear localization signals (NLSs) and nuclear export signals (NESs) were exploited to determine the subcellular localization of the potato late blight protein R1 and the Phytophthora infestans RXLR effector AVR1, and to target these proteins to the nucleus or cytoplasm. Microscopic imaging revealed that both R1 and AVR1 occurred in the nucleus and cytoplasm, and were in close proximity. Transient expression of NLS- or NES-tagged R1 and AVR1 in Nicotiana benthamiana showed that activation of the R1-mediated hypersensitive response and resistance required localization of the R1/AVR1 pair in the nucleus. However, AVR1-mediated suppression of cell death in the absence of R1 was dependent on localization of AVR1 in the cytoplasm. Balanced nucleocytoplasmic partitioning of AVR1 seems to be a prerequisite. Our results show that R1-mediated immunity is activated inside the nucleus with AVR1 in close proximity and suggest that nucleocytoplasmic transport of R1 and AVR1 is tightly regulated.