RESUMO
Ralstonia solanacearum (R. solanacearum) is one of the most devastating pathogens in terms of losses in agricultural production. Bentonite (Bent) is a promising synergistic agent used in development of effective and environmentally friendly pesticides against plant disease. However, the synergistic mechanism of Bent nanoclays with benzothiazolinone (BIT) against R. solanacearum is unknown. In this work, acid-functionalized porous Bent and cetyltrimethylammonium bromide (CTAB) were employed as the core nanoclays, and BIT was loaded into the clay to form BIT-loaded CT-Bent (BIT@CT-Bent) for the control of bacterial wilt disease. BIT@CT-Bent exhibited pH-responsive release behavior that fit the Fickian diffusion model, rapidly releasing BIT in an acidic environment (pH = 5.5). The antibacterial effect of BIT@CT-Bent was approximately 4 times greater than that of the commercial product BIT, and its biotoxicity was much lower than that of BIT under the same conditions. Interestingly, R. solanacearum attracted BIT@CT-Bent into the nanocomposites and induced cytoplasmic leakage and changes in membrane permeability, indicating an efficient and synergistic bactericidal effect that rapidly reduced bacterial density. In addition, BIT@CT-Bent significantly inhibited R. solanacearum biofilm formation and swimming activity, by suppressing the expression of phcA, solR and vsrC. Indeed, exogenous application of BIT@CT-Bent significantly suppressed the virulence of R. solanacearum on tobacco plants, with control effect of 75.48%, 72.08% and 66.08% at 9, 11 and 13 days after inoculation, respectively. This study highlights the potential of using BIT@CT-Bent as an effective, eco-friendly bactericide to control bacterial wilt diseases and for the development of sustainable crop protection strategies.
Assuntos
Bentonita , Ralstonia solanacearum , Bentonita/farmacologia , Bentonita/metabolismo , Antibacterianos/farmacologia , Virulência , Concentração de Íons de Hidrogênio , Ralstonia solanacearum/metabolismo , Doenças das Plantas/prevenção & controle , Doenças das Plantas/microbiologiaRESUMO
Ralstonia solanacearum, a species complex of bacterial plant pathogens that causes bacterial wilt, comprises four phylotypes that evolved when a founder population was split during the continental drift ~180 million years ago. Each phylotype contains strains with RipTAL proteins structurally related to transcription activator-like (TAL) effectors from the bacterial pathogen Xanthomonas. RipTALs have evolved in geographically separated phylotypes and therefore differ in sequence and potentially functionality. Earlier work has shown that phylotype I RipTAL Brg11 targets a 17-nucleotide effector binding element (EBE) and transcriptionally activates the downstream arginine decarboxylase (ADC) gene. The predicted DNA binding preferences of Brg11 and RipTALs from other phylotypes are similar, suggesting that most, if not all, RipTALs target the Brg11-EBE motif and activate downstream ADC genes. Here we show that not only phylotype I RipTAL Brg11 but also RipTALs from other phylotypes activate host genes when preceded by the Brg11-EBE motif. Furthermore, we show that Brg11 and RipTALs from other phylotypes induce the same quantitative changes of ADC-dependent plant metabolites, suggesting that most, if not all, RipTALs induce functionally equivalent changes in host cells. Finally, we report transgenic tobacco lines in which the RipTAL-binding motif Brg11-EBE mediates RipTAL-dependent transcription of the executor-type resistance (R) gene Bs4C from pepper, thereby conferring resistance to RipTAL-delivering R. solanacearum strains. Our results suggest that cell death-inducing executor-type R genes, preceded by the RipTAL-binding motif Brg11-EBE, could be used to genetically engineer broad-spectrum bacterial wilt resistance in crop plants without any apparent fitness penalty.
Assuntos
Ralstonia solanacearum , Ralstonia solanacearum/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Plantas/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologiaRESUMO
Bacterial wilt disease caused by several Ralstonia species is one of the most destructive diseases in Solanaceae crops. Only a few functional resistance genes against bacterial wilt have been cloned to date. Here, we show that the broadly conserved type III secreted effector RipY is recognized by the Nicotiana benthamiana immune system, leading to cell death induction, induction of defense-related gene expression, and restriction of bacterial pathogen growth. Using a multiplexed virus-induced gene-silencing-based N. benthamiana nucleotide-binding and leucine-rich repeat receptor (NbNLR) library, we identified a coiled-coil (CC) nucleotide-binding and leucine-rich repeat receptor (CNL) required for recognition of RipY, which we named RESISTANCE TO RALSTONIA SOLANACEARUM RIPY (RRS-Y). Genetic complementation assays in RRS-Y-silenced plants and stable rrs-y knockout mutants demonstrated that RRS-Y is sufficient to activate RipY-induced cell death and RipY-induced immunity to Ralstonia pseudosolanacearum. RRS-Y function is dependent on the phosphate-binding loop motif of the nucleotide-binding domain but independent of the characterized signaling components ENHANCED DISEASE SUSCEPTIBILITY 1, ACTIVATED DISEASE RESISTANCE 1, and N REQUIREMENT GENE 1 and the NLR helpers NB-LRR REQUIRED FOR HR-ASSOCIATED CELL DEATH-2, -3, and -4 in N. benthamiana. We further show that RRS-Y localization at the plasma membrane is mediated by two cysteine residues in the CC domain and is required for RipY recognition. RRS-Y also broadly recognizes RipY homologs across Ralstonia species. Lastly, we show that the C-terminal region of RipY is indispensable for RRS-Y activation. Together, our findings provide an additional effector/receptor pair system to deepen our understanding of CNL activation in plants.
Assuntos
Nicotiana , Ralstonia solanacearum , Nicotiana/microbiologia , Proteínas de Plantas/metabolismo , Leucina , Resistência à Doença/genética , Ralstonia solanacearum/metabolismo , Membrana Celular/metabolismo , NucleotídeosRESUMO
Ralstonia solanacearum is a widespread plant bacterial pathogen that can launch a range of type III effectors (T3Es) to cause disease. In this study, we isolate a pathogenic R. solanacearum strain named P380 from tomato rhizosphere. Five out of 12 core T3Es of strain P380 are introduced into Pseudomonas syringae DC3000D36E separately to determine their functions in interacting with plants. DC3000D36E that harbors each effector suppresses FliC-triggered Pti5 and ACRE31 expression, ROS burst, and callose deposition. RipAE, RipU, and RipW elicit cell death as well as upregulate the MAPK cascades in Nicotiana benthamiana. The derivatives RipC1ΔDXDX(T/V) and RipWΔDKXXQ but not RipAEK310R fail to suppress ROS burst. Moreover, RipAEK310R and RipWΔDKXXQ retain the cell death elicitation ability. RipAE and RipW are associated with salicylic acid and jasmonic acid pathways, respectively. RipAE and RipAQ significantly promote the propagation of DC3000D36E in plants. The five core T3Es localize in diverse subcellular organelles of nucleus, plasma membrane, endoplasmic reticulum, and Golgi network. The suppressor of G2 allele of Skp1 is required for RipAE but not RipU-triggered cell death in N. benthamiana. These results indicate that the core T3Es in R. solanacearum play diverse roles in plant-pathogen interactions.
Assuntos
Ralstonia solanacearum , Ralstonia solanacearum/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Bactérias/metabolismo , Plantas/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologia , Doenças das Plantas/microbiologiaRESUMO
BACKGROUND: Tobacco is an important economic crop, but the quality and yield have been severely impaired by bacterial wilt disease (BWD) caused by Ralstonia solanacearum. METHODS AND RESULTS: Here, we describe a transgenic approach to prevent BWD in tobacco plants. A new root-specific promoter of an NtR12 gene was successfully cloned. The NtR12 promoter drove GUS reporter gene expression to a high level in roots but to less extent in stems, and no significant expression was detected in leaves. The Ribosome-inactivating proteins (RIP) gene from Momordica charantia was also cloned, and its ability to inhibit Ralstonia solanacearum was evaluated using RIP protein produced by the prokaryotic expression system. The RIP gene was constructed downstream of the NtR12 promoter and transformed into the tobacco cultivar "Cuibi No. 1" (CB-1), resulting in many descendants. The resistance against BWD was significantly improved in transgenic tobacco lines expressing NtR12::RIP. CONCLUSION: This study confirms that the RIP gene confers resistance to BWD and the NtR12 as a new promoter for its specific expression in root and stem. Our findings pave a novel avenue for transgenic engineering to prevent the harmful impact of diseases and pests in roots and stems.
Assuntos
Nicotiana , Ralstonia solanacearum , Nicotiana/metabolismo , Proteínas Inativadoras de Ribossomos/genética , Proteínas Inativadoras de Ribossomos/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ralstonia solanacearum/genética , Ralstonia solanacearum/metabolismo , Regiões Promotoras Genéticas/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Resistência à Doença/genéticaRESUMO
ROPs (Rho-like GTPases from plants) belong to the Rho-GTPase subfamily and serve as molecular switches for regulating diverse cellular events, including morphogenesis and stress responses. However, the immune functions of ROPs in Solanum lycopersicum Linn. (tomato) is still largely unclear. The tomato genome contains nine genes encoding ROP-type small GTPase family proteins (namely SlRop1-9) that fall into five distinct groups as revealed by phylogenetic tree. We studied the subcellular localization and immune response induction of nine SlRops by using a transient overexpression system in Nicotiana benthamiana Domin. Except for SlRop1 and SlRop3, which are solely localized at the plasma membrane, most of the remaining ROPs have additional nuclear and/or cytoplasmic distributions. We also revealed that the number of basic residues in the polybasic region of ROPs tends to be correlated with their membrane accumulation. Though nine SlRops are highly conserved at the RHO (Ras Homology) domains, only seven constitutively active forms of SlRops were able to trigger hypersensitive responses. Furthermore, we analyzed the tissue-specific expression patterns of nine ROPs and found that the expression levels of SlRop3, 4 and 6 were generally high in different tissues. The expression levels of SlRop1, 2 and 7 significantly decreased in tomato seedlings after infection with Ralstonia solanacearum (E.F. Smith) Yabuuchi et al. (GMI1000); the others did not respond. Infection assays among nine ROPs showed that SlRop3 and SlRop4 might be positive regulators of tomato bacterial wilt disease resistance, whereas the rest of the ROPs may not contribute to defense. Our study provides systematic evidence of tomato Rho-related small GTPases for localization, immune response, and disease resistance.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Ralstonia solanacearum , Solanum lycopersicum , Resistência à Doença/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Filogenia , Ralstonia solanacearum/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
CabZIP63 and CaWRKY40 were previously found to be shared in the pepper defense response to high temperature stress (HTS) and to Ralstonia solanacearum inoculation (RSI), forming a transcriptional cascade. However, how they activate the two distinct defense responses is not fully understood. Herein, using a revised genetic approach, we functionally characterized CabZIP23 in the CabZIP63-CaWRKY40 cascade and its context specific pepper immunity activation against RSI by interaction with CabZIP63. CabZIP23 was originally found by immunoprecipitation-mass spectrometry to be an interacting protein of CabZIP63-GFP; it was upregulated by RSI and acted positively in pepper immunity against RSI by virus induced gene silencing in pepper plants, and transient overexpression in Nicotiana benthamiana plants. By chromatin immunoprecipitation (ChIP)-qPCR and electrophoresis mobility shift assay (EMSA), CabZIP23 was found to be directly regulated by CaWRKY40, and CabZIP63 was directly regulated by CabZIP23, forming a positive feedback loop. CabZIP23-CabZIP63 interaction was confirmed by co-immunoprecipitation (CoIP) and bimolecular fluorescent complimentary (BiFC) assays, which promoted CabZIP63 binding immunity related target genes, including CaPR1, CaNPR1 and CaWRKY40, thereby enhancing pepper immunity against RSI, but not affecting the expression of thermotolerance related CaHSP24. All these data appear to show that CabZIP23 integrates in the CabZIP63-CaWRKY40 cascade and the context specifically turns it on mounting pepper immunity against RSI.
Assuntos
Capsicum , Ralstonia solanacearum , Capsicum/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Doenças das Plantas/genética , Reguladores de Crescimento de Plantas/metabolismo , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ralstonia solanacearum/metabolismoRESUMO
Ralstonia solanacearum is a soil-borne pathogen that causes bacterial wilt in plants. The wild-type strain of R. solanacearum undergoes spontaneous phenotype conversion (PC), from a fluidal to non-fluidal colony morphology. PC mutants are non-pathogenic due to reduced virulence factors, and can control wilt diseases as biological control agents. The induction factors of PC in R. solanacearum are currently unclear. Here, we investigated the effect of iron treatment on bacterial growth of wild-type strain and PC mutant, and PC of the wild-type strain in liquid medium. Interestingly, PC was frequently induced in the single cultured wild-type strain by iron treatment; however, PC was not induced in the co-culture. In a co-culture of both strains, the PC mutant showed increased growth compared to the wild-type strain by iron treatment. Furthermore, we investigated the effects of iron treatment on the bacterial growth and PC of the wild-type strain under different culture conditions of medium type (MM broth, BG broth, and water medium), iron compounds, and pH. In BG broth, PC occurred frequently regardless of iron treatment. In MM broth, the optimal conditions for high frequency induction of PC by iron treatments were treatment of iron (III) EDTA, and under pH 7-8. Conversely, PC was not induced by iron treatment in water medium and in MM broth under pH 5 conditions. Common to the culture conditions wherein PC was not induced by iron treatment, the bacterial density of the wild-type strain was as low as 106 CFU mL-1 or less. Finally, we investigated the effects on bacterial growth and PC of the wild-type strain by the iron treatment and addition of culture filtrate after cultivation of the wild-type strain at high concentration. In medium containing only the culture filtrate, PC did not occur. However, in medium containing the culture filtrate and iron, PC occurred frequently. Our results thus suggest that high-density growth of the wild-type strain as well as the presence of iron are involved in inducing PC in R. solanacearum.
Assuntos
Meios de Cultura/metabolismo , Compostos de Ferro/metabolismo , Ralstonia solanacearum/metabolismo , Meios de Cultura/análise , Concentração de Íons de Hidrogênio , Compostos de Ferro/análise , Fenótipo , Doenças das Plantas/microbiologia , Ralstonia solanacearum/genética , Ralstonia solanacearum/crescimento & desenvolvimentoRESUMO
Ralstonia solanacearum injects type III effectors into host cells to cause bacterial wilt in Solanaceae plants. To identify R. solanacearum effectors that suppress effector-triggered immunity (ETI) in plants, we evaluated R. solanacearum RS1000 effectors for their ability to suppress a hypersensitive response (HR) induced by the avirulence (Avr) effector RipAA in Nicotiana benthamiana. Out of the 11 effectors tested, 4 suppressed RipAA-triggered HR cell death. Among them, RipAC contains tandem repeats of the leucine-rich repeat (LRR) motif, which serves as the structural scaffold for a protein-protein interaction. We found that the LRR domain of RipAC was indispensable for the suppression of HR cell death during the recognition of RipAA and another Avr effector RipP1. By yeast two-hybrid screening, we identified N. benthamiana SGT1, an adaptor protein that forms a molecular chaperone complex with RAR1, as a host factor of the RipAC target. RipAC interacted with NbSGT1 in yeast and plant cells. Upon the formation of the molecular chaperone complex, the presence of RipAC markedly inhibits the interaction between NbSGT1 and NbRAR1. The RipAA- and RipP1-triggered HR cell deaths were not observed in NbSGT1-silenced plants. The introduction of RipAC was complementary to the reduced growth of the R. solanacearum mutant strain in N. benthamiana. These findings indicate that R. solanacearum uses RipAC to subvert the NbSGT1-mediated formation of the molecular chaperone complex and suppress ETI responses during the recognition of Avr effectors.
Assuntos
Proteínas de Bactérias/fisiologia , Glucosiltransferases/metabolismo , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Ralstonia solanacearum/metabolismo , Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno , Nicotiana/metabolismo , Nicotiana/microbiologiaRESUMO
Effector proteins delivered inside plant cells are powerful weapons for bacterial pathogens, but this exposes the pathogen to potential recognition by the plant immune system. Therefore, the effector repertoire of a given pathogen must be balanced for a successful infection. Ralstonia solanacearum is an aggressive pathogen with a large repertoire of secreted effectors. One of these effectors, RipE1, is conserved in most R. solanacearum strains sequenced to date. In this work, we found that RipE1 triggers immunity in N. benthamiana, which requires the immune regulator SGT1, but not EDS1 or NRCs. Interestingly, RipE1-triggered immunity induces the accumulation of salicylic acid (SA) and the overexpression of several genes encoding phenylalanine-ammonia lyases (PALs), suggesting that the unconventional PAL-mediated pathway is responsible for the observed SA biosynthesis. Surprisingly, RipE1 recognition also induces the expression of jasmonic acid (JA)-responsive genes and JA biosynthesis, suggesting that both SA and JA may act cooperatively in response to RipE1. We further found that RipE1 expression leads to the accumulation of glutathione in plant cells, which precedes the activation of immune responses. R. solanacearum secretes another effector, RipAY, which is known to inhibit immune responses by degrading cellular glutathione. Accordingly, RipAY inhibits RipE1-triggered immune responses. This work shows a strategy employed by R. solanacearum to counteract the perception of its effector proteins by plant immune system.
Assuntos
Proteínas Fúngicas/genética , Nicotiana/imunologia , Imunidade Vegetal , Ralstonia solanacearum/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas Fúngicas/metabolismo , Ralstonia solanacearum/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Nicotiana/microbiologiaRESUMO
Nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins function as sensors that perceive pathogen molecules and activate immunity. In plants, the accumulation and activation of NLRs is regulated by SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1). In this work, we found that an effector protein named RipAC, secreted by the plant pathogen Ralstonia solanacearum, associates with SGT1 to suppress NLR-mediated SGT1-dependent immune responses, including those triggered by another R. solanacearum effector, RipE1. RipAC does not affect the accumulation of SGT1 or NLRs, or their interaction. However, RipAC inhibits the interaction between SGT1 and MAP kinases, and the phosphorylation of a MAPK target motif in the C-terminal domain of SGT1. Such phosphorylation is enhanced upon activation of immune signaling and contributes to the activation of immune responses mediated by the NLR RPS2. Additionally, SGT1 phosphorylation contributes to resistance against R. solanacearum. Our results shed light onto the mechanism of activation of NLR-mediated immunity, and suggest a positive feedback loop between MAPK activation and SGT1-dependent NLR activation.
Assuntos
Proteínas de Bactérias/metabolismo , Doenças das Plantas/imunologia , Imunidade Vegetal/imunologia , Proteínas de Plantas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas de Plantas/imunologia , Ralstonia solanacearum/imunologia , Ralstonia solanacearum/metabolismo , Nicotiana/metabolismoRESUMO
Many bacterial plant pathogens employ a type III secretion system to inject effector proteins within plant cells to suppress plant immunity. Whether and how effector proteins also co-opt plant metabolism to support extensive bacterial replication remains an open question. Here, we show that Ralstonia solanacearum, the causal agent of bacterial wilt disease, secretes the effector protein RipI, which interacts with plant glutamate decarboxylases (GADs) to alter plant metabolism and support bacterial growth. GADs are activated by calmodulin and catalyze the biosynthesis of gamma-aminobutyric acid (GABA), an important signaling molecule in plants and animals. RipI promotes the interaction of GADs with calmodulin, enhancing the production of GABA. R. solanacearum is able to replicate efficiently using GABA as a nutrient, and both RipI and plant GABA contribute to a successful infection. This work reveals a pathogenic strategy to hijack plant metabolism for the biosynthesis of nutrients that support microbial growth during plant colonization.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Interações Hospedeiro-Patógeno/fisiologia , Plantas/efeitos dos fármacos , Plantas/metabolismo , Arabidopsis , Solanum lycopersicum , Doenças das Plantas/imunologia , Imunidade Vegetal , Plantas/imunologia , Plantas/microbiologia , Ralstonia solanacearum/crescimento & desenvolvimento , Ralstonia solanacearum/metabolismo , Nicotiana , Sistemas de Secreção Tipo III/metabolismo , Virulência , Ácido gama-Aminobutírico/metabolismoRESUMO
Plant pathogenic bacteria cause high crop and economic losses to human societies1-3. Infections by such pathogens are challenging to control as they often arise through complex interactions between plants, pathogens and the plant microbiome4,5. Experimental studies of this natural ecosystem at the microbiome-wide scale are rare, and consequently we have a poor understanding of how the taxonomic and functional microbiome composition and the resulting ecological interactions affect pathogen growth and disease outbreak. Here, we combine DNA-based soil microbiome analysis with in vitro and in planta bioassays to show that competition for iron via secreted siderophore molecules is a good predictor of microbe-pathogen interactions and plant protection. We examined the ability of 2,150 individual bacterial members of 80 rhizosphere microbiomes, covering all major phylogenetic lineages, to suppress the bacterium Ralstonia solanacearum, a global phytopathogen capable of infecting various crops6,7. We found that secreted siderophores altered microbiome-pathogen interactions from complete pathogen suppression to strong facilitation. Rhizosphere microbiome members with growth-inhibitory siderophores could often suppress the pathogen in vitro as well as in natural and greenhouse soils, and protect tomato plants from infection. Conversely, rhizosphere microbiome members with growth-promotive siderophores were often inferior in competition and facilitated plant infection by the pathogen. Because siderophores are a chemically diverse group of molecules, with each siderophore type relying on a compatible receptor for iron uptake8-12, our results suggest that pathogen-suppressive microbiome members produce siderophores that the pathogen cannot use. Our study establishes a causal mechanistic link between microbiome-level competition for iron and plant protection and opens promising avenues to use siderophore-mediated interactions as a tool for microbiome engineering and pathogen control.
Assuntos
Ferro/metabolismo , Microbiota , Doenças das Plantas/microbiologia , Rizosfera , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Interações Hospedeiro-Patógeno , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiologia , Filogenia , Doenças das Plantas/prevenção & controle , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Ralstonia solanacearum/isolamento & purificação , Ralstonia solanacearum/metabolismo , Análise de Sequência de DNA , Sideróforos , Solo/química , Microbiologia do SoloRESUMO
RipX of Ralstonia solanacearum is translocated into host cells by a type III secretion system and acts as a harpin-like protein to induce a hypersensitive response in tobacco plants. The molecular events in association with RipX-induced signaling transduction have not been fully elucidated. This work reports that transient expression of RipX induced a yellowing phenotype in Nicotiana benthamiana, coupled with activation of the defense reaction. Using yeast two-hybrid and split-luciferase complementation assays, mitochondrial ATP synthase F1 subunit α (ATPA) was identified as an interaction partner of RipX from N. benthamiana. Although a certain proportion was found in mitochondria, the YFP-ATPA fusion was able to localize to the cell membrane, cytoplasm, and nucleus. RFP-RipX fusion was found from the cell membrane and cytoplasm. Moreover, ATPA interacted with RipX at both the cell membrane and cytoplasm in vivo. Silencing of the atpA gene had no effect on the appearance of yellowing phenotype induced by RipX. However, the silenced plants improved the resistance to R. solanacearum. Moreover, qRT-PCR and promoter GUS fusion experiments revealed that the transcript levels of atpA were evidently reduced in response to expression of RipX. These data demonstrated that RipX exerts a suppressive effect on the transcription of atpA gene, to induce defense reaction in N. benthamiana.
Assuntos
Proteínas de Bactérias , Resistência à Doença/genética , Nicotiana , Proteínas de Plantas , ATPases Translocadoras de Prótons , Ralstonia solanacearum , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Ralstonia solanacearum/genética , Ralstonia solanacearum/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologiaRESUMO
Ralstonia solanacearum is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects approximately 70 effector proteins into plant cells via the Hrp type III secretion system in an early stage of infection. To identify an as-yet-unidentified avirulence factor possessed by the Japanese tobacco-avirulent strain RS1000, we transiently expressed RS1000 effectors in Nicotiana benthamiana leaves and monitored their ability to induce effector-triggered immunity (ETI). The expression of RipB strongly induced the production of reactive oxygen species and the expressions of defence-related genes in N. benthamiana. The ripB mutant of RS1002, a nalixidic acid-resistant derivative of RS1000, caused wilting symptoms in N. benthamiana. A pathogenicity test using R. solanacearum mutants revealed that the two already known avirulence factors RipP1 and RipAA contribute in part to the avirulence of RS1002 in N. benthamiana. The Japanese tobacco-virulent strain BK1002 contains mutations in ripB and expresses a C-terminal-truncated RipB that lost the ability to induce ETI in N. benthamiana, indicating a fine-tuning of the pathogen effector repertoire to evade plant recognition. RipB shares homology with Xanthomonas XopQ, which is recognized by the resistance protein Roq1. The RipB-induced resistance against R. solanacearum was abolished in Roq1-silenced plants. These findings indicate that RipB acts as a major avirulence factor in N. benthamiana and that Roq1 is involved in the recognition of RipB.
Assuntos
Nicotiana/microbiologia , Doenças das Plantas/microbiologia , Ralstonia solanacearum/metabolismo , Ralstonia solanacearum/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Folhas de Planta/microbiologia , Ralstonia solanacearum/genéticaRESUMO
Ralstonia solanacearum is the causal agent of bacterial wilt disease of plants. This pathogen injects more than 70 type III effector proteins called Rips (Ralstonia-injected proteins) into plant cells to succeed in infection. One of the Rips, RipAL, contains a putative lipase domain that shared homology with Arabidopsis DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1). RipAL significantly suppressed pattern-triggered immunity in leaves of Nicotiana benthamiana. Subcellular localization analyses suggest that RipAL localizes to chloroplasts and targets chloroplast lipids in plant cells. Notably, the expression of RipAL markedly increased the jasmonic acid (JA) and JA-isoleucine levels, and induced the expressions of JA-signaling marker genes in plant leaves. Simultaneously, RipAL greatly reduced the salicylic acid (SA) level and decreased the expression levels of SA-signaling marker genes. Mutations in two putative catalytic residues in the DAD1-like lipase domain abolished the ability of RipAL to induce JA production and suppress SA signaling. Infection of R. solanacearum also induced JA production and simultaneously decreased the SA level in susceptible pepper leaves in a ripAL-dependent manner. The growth of R. solanacearum enhanced in plants with silenced CaICS1, which encodes the SA synthesis enzyme isochorismate synthase 1. These results indicate that SA signaling is involved in the defense response against R. solanacearum and that R. solanacearum uses RipAL to induce JA production and suppress SA signaling in plant cells.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Cloroplastos/metabolismo , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Plantas/imunologia , Plantas/microbiologia , Ralstonia solanacearum/metabolismo , Ácido Salicílico/metabolismo , Arabidopsis/imunologia , Arabidopsis/microbiologia , Capsicum/imunologia , Capsicum/microbiologia , Suscetibilidade a Doenças , Inativação Gênica , Lipase/metabolismo , Fenótipo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal , Transporte Proteico , Pseudomonas syringae/patogenicidade , Transdução de Sinais , Frações Subcelulares/metabolismo , Nicotiana/imunologia , Nicotiana/microbiologia , VirulênciaRESUMO
The subversion of plant cellular functions is essential for bacterial pathogens to proliferate in host plants and cause disease. Most bacterial plant pathogens employ a type III secretion system to inject type III effector (T3E) proteins inside plant cells, where they contribute to the pathogen-induced alteration of plant physiology. In this work, we found that the Ralstonia solanacearum T3E RipAY suppresses plant immune responses triggered by bacterial elicitors and by the phytohormone salicylic acid. Further biochemical analysis indicated that RipAY associates in planta with thioredoxins from Nicotiana benthamiana and Arabidopsis. Interestingly, RipAY displays γ-glutamyl cyclotransferase (GGCT) activity to degrade glutathione in plant cells, which is required for the reported suppression of immune responses. Given the importance of thioredoxins and glutathione as major redox regulators in eukaryotic cells, RipAY activity may constitute a novel and powerful virulence strategy employed by R. solanacearum to suppress immune responses and potentially alter general redox signalling in host cells.
Assuntos
Arabidopsis/imunologia , Proteínas de Bactérias/metabolismo , Nicotiana/imunologia , Imunidade Vegetal , Ralstonia solanacearum/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Arabidopsis/microbiologia , Núcleo Celular/metabolismo , Cisteína/metabolismo , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Oxirredução , Células Vegetais/metabolismo , Ralstonia solanacearum/patogenicidade , Tiorredoxinas/metabolismo , Nicotiana/citologia , Nicotiana/microbiologia , Virulência , gama-Glutamilciclotransferase/metabolismoRESUMO
Ralstonia solanacearum, the causal agent of bacterial wilt disease, is considered one of the most destructive bacterial pathogens due to its lethality, unusually wide host range, persistence and broad geographical distribution. In spite of the extensive research on plant immunity over the last years, the perception of molecular patterns from R. solanacearum that activate immunity in plants is still poorly understood, which hinders the development of strategies to generate resistance against bacterial wilt disease. The perception of a conserved peptide of bacterial flagellin, flg22, is regarded as paradigm of plant perception of invading bacteria; however, no elicitor activity has been detected for R. solanacearum flg22. Recent reports have shown that other epitopes from flagellin are able to elicit immune responses in specific species from the Solanaceae family, yet our results show that these plants do not perceive any epitope from R. solanacearum flagellin. Searching for elicitor peptides from R. solanacearum, we found several protein sequences similar to the consensus of the elicitor peptide csp22, reported to elicit immunity in specific Solanaceae plants. A R. solanacearum csp22 peptide (csp22Rsol ) was indeed able to trigger immune responses in Nicotiana benthamiana and tomato, but not in Arabidopsis thaliana. Additionally, csp22Rsol treatment conferred increased resistance to R. solanacearum in tomato. Transgenic A. thaliana plants expressing the tomato csp22 receptor (SlCORE) gained the ability to respond to csp22Rsol and became more resistant to R. solanacearum infection. Our results shed light on the mechanisms for perception of R. solanacearum by plants, paving the way for improving current approaches to generate resistance against R. solanacearum.
Assuntos
Peptídeos/imunologia , Doenças das Plantas/imunologia , Imunidade Vegetal , Ralstonia solanacearum/metabolismo , Solanaceae/imunologia , Arabidopsis/imunologia , Arabidopsis/microbiologia , Resistência à Doença , Epitopos/imunologia , Flagelina/imunologia , Solanum lycopersicum/imunologia , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Raízes de Plantas/imunologia , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/imunologia , Solanaceae/microbiologia , Nicotiana/imunologia , Nicotiana/microbiologiaRESUMO
The Yersinia outer protein J (YopJ) family of bacterial effectors depends on a novel acetyltransferase domain to acetylate signalling proteins from plant and animal hosts. However, the underlying mechanism is unclear. Here, we report the crystal structures of PopP2, a YopJ effector produced by the plant pathogen Ralstonia solanacearum, in complex with inositol hexaphosphate (InsP6), acetyl-coenzyme A (AcCoA) and/or substrate Resistance to Ralstonia solanacearum 1 (RRS1-R)WRKY. PopP2 recognizes the WRKYGQK motif of RRS1-RWRKY to position a targeted lysine in the active site for acetylation. Importantly, the PopP2-RRS1-RWRKY association is allosterically regulated by InsP6 binding, suggesting a previously unidentified role of the eukaryote-specific cofactor in substrate interaction. Furthermore, we provide evidence for the reaction intermediate of PopP2-mediated acetylation, an acetyl-cysteine covalent adduct, lending direct support to the 'ping-pong'-like catalytic mechanism proposed for YopJ effectors. Our study provides critical mechanistic insights into the virulence activity of YopJ class of acetyltransferases.
Assuntos
Proteínas de Bactérias/química , Yersinia/metabolismo , Acetilcoenzima A/química , Acetilação , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Ácido Fítico/química , Conformação Proteica , Ralstonia solanacearum/metabolismo , Sistemas de Secreção Tipo IIIRESUMO
Plant pathogenic bacteria exerts their pathogenicity through the injection of large repertoires of type III effectors (T3Es) into plant cells, a mechanism controlled in part by type III chaperones (T3Cs). In Ralstonia solanacearum, the causal agent of bacterial wilt, little is known about the control of type III secretion at the post-translational level. Here, we provide evidence that the HpaB and HpaD proteins do act as bona fide R. solanacearum class IB chaperones that associate with several T3Es. Both proteins can dimerize but do not interact with each other. After screening 38 T3Es for direct interactions, we highlighted specific and common interacting partners, thus revealing the first picture of the R. solanacearum T3C-T3E network. We demonstrated that the function of HpaB is conserved in two phytopathogenic bacteria, R. solanacearum and Xanthomonas campestris pv. vesicatoria (Xcv). HpaB from Xcv is able to functionally complement a R. solanacearum hpaB mutant for hypersensitive response elicitation on tobacco plants. Likewise, Xcv is able to translocate a heterologous T3E from R. solanacearum in an HpaB-dependent manner. This study underlines the central role of the HpaB class IB chaperone family and its potential contribution to the bacterial plasticity to acquire and deliver new virulence factors.