RESUMEN
Plant pathogenic bacteria are responsible for the loss of hundreds of millions of dollars each year, impacting a wide range of economically relevant agricultural crops. The plant immune system detects conserved bacterial molecules and deploys an arsenal of effective defense measures at different levels; however, during compatible interactions, some pathogenic bacteria suppress and manipulate the host immunity and colonize and infect the plant host. Different bacteria employ similar strategies to circumvent plant innate immunity, while other tactics are specific to certain bacterial species. Recent studies have highlighted the secondary messenger c-di-GMP as a key molecule in the transmission of environmental cues in an intracellular regulatory network that controls virulence traits in many plant pathogenic bacteria. In this review, we focus on the recent knowledge of the molecular basis of c-di-GMP signaling mechanisms that promote or prevent the evasion of bacterial phytopathogens from the plant immune system. This review will highlight the considerable diversity of mechanisms evolved in plant-associated bacteria to elude plant immunity.
Asunto(s)
Productos Agrícolas/microbiología , GMP Cíclico/análogos & derivados , Evasión Inmune , Oryza/microbiología , Inmunidad de la Planta/genética , Productos Agrícolas/genética , Productos Agrícolas/inmunología , GMP Cíclico/biosíntesis , GMP Cíclico/inmunología , Defensinas/biosíntesis , Defensinas/inmunología , Erwinia amylovora/genética , Erwinia amylovora/inmunología , Erwinia amylovora/patogenicidad , Regulación de la Expresión Génica , Oryza/genética , Oryza/inmunología , Oxilipinas/inmunología , Oxilipinas/metabolismo , Pseudomonas syringae/genética , Pseudomonas syringae/inmunología , Pseudomonas syringae/patogenicidad , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/inmunología , Sesquiterpenos/inmunología , Sesquiterpenos/metabolismo , Transducción de Señal , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/inmunología , Virulencia , Xanthomonas/genética , Xanthomonas/inmunología , Xanthomonas/patogenicidad , Xylella/genética , Xylella/inmunología , Xylella/patogenicidad , FitoalexinasRESUMEN
Microbe-associated molecular patterns (MAMPs) are molecules, or domains within molecules, that are conserved across microbial taxa and can be recognized by a plant or animal immune system. Although MAMP receptors have evolved to recognize conserved epitopes, the MAMPs in some microbial species or strains have diverged sufficiently to render them unrecognizable by some host immune systems. In this study, we carried out in vitro evolution of the Arabidopsis thaliana flagellin receptor FLAGELLIN-SENSING 2 (FLS2) to isolate derivatives that recognize one or more flagellin peptides from bacteria for which the wild-type Arabidopsis FLS2 confers little or no response. A targeted approach generated amino acid variation at FLS2 residues in a region previously implicated in flagellin recognition. The primary screen tested for elevated response to the canonical flagellin peptide from Pseudomonas aeruginosa, flg22. From this pool, we then identified five alleles of FLS2 that confer modest (quantitatively partial) recognition of an Erwinia amylovora flagellin peptide. Use of this Erwinia-based flagellin peptide to stimulate Arabidopsis plants expressing the resulting FLS2 alleles did not lead to a detectable reduction of virulent P. syringae pv. tomato growth. However, combination of two identified mutations into a single allele further increased FLS2-mediated responses to the E. amylovora flagellin peptide. These studies demonstrate the potential to raise the sensitivity of MAMP receptors toward particular targets.
Asunto(s)
Presentación de Antígeno/genética , Proteínas de Arabidopsis/fisiología , Arabidopsis , Flagelina/inmunología , Fragmentos de Péptidos/inmunología , Proteínas Quinasas/fisiología , Alelos , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Erwinia amylovora/inmunología , Erwinia amylovora/patogenicidad , Evolución Molecular , Flagelina/química , Inmunidad Innata/genética , Mutación/fisiología , Fragmentos de Péptidos/química , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Proteínas Quinasas/genéticaRESUMEN
The molecular basis of resistance and susceptibility of host plants to fire blight, a major disease threat to pome fruit production globally, is largely unknown. RNA-sequencing data from challenged and mock-inoculated flowers were analyzed to assess the susceptible response of apple to the fire blight pathogen Erwinia amylovora. In presence of the pathogen 1,080 transcripts were differentially expressed at 48 h post inoculation. These included putative disease resistance, stress, pathogen related, general metabolic, and phytohormone related genes. Reads, mapped to regions on the apple genome where no genes were assigned, were used to identify potential novel genes and open reading frames. To identify transcripts specifically expressed in response to E. amylovora, RT-PCRs were conducted and compared to the expression patterns of the fire blight biocontrol agent Pantoea vagans strain C9-1, another apple pathogen Pseudomonas syringae pv. papulans, and mock inoculated apple flowers. This led to the identification of a peroxidase superfamily gene that was lower expressed in response to E. amylovora suggesting a potential role in the susceptibility response. Overall, this study provides the first transcriptional profile by RNA-seq of the host plant during fire blight disease and insights into the response of susceptible apple plants to E. amylovora.
Asunto(s)
Erwinia amylovora/inmunología , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Malus/inmunología , Enfermedades de las Plantas/inmunología , Erwinia amylovora/crecimiento & desarrollo , Malus/microbiología , Enfermedades de las Plantas/microbiologíaRESUMEN
In the rosaceous subtribe Pyrinae (formerly subfamily Maloideae), pathogen attack leads to formation of biphenyls and dibenzofurans. Accumulation of these phytoalexins was studied in greenhouse-grown grafted shoots of Malus domestica cv. 'Holsteiner Cox' and Pyrus communis cv. 'Conference' after inoculation with the fire blight bacterium, Erwinia amylovora. No phytoalexins were found in leaves. However, both classes of defence compounds were detected in the transition zone of stems. The flanking stem segments above and below this zone, which were necrotic and healthy, respectively, were devoid of detectable phytoalexins. The transition zone of apple stems contained the biphenyls 3-hydroxy-5-methoxyaucuparin, aucuparin, noraucuparin and 2'-hydroxyaucuparin and the dibenzofurans eriobofuran and noreriobofuran. In pear, aucuparin, 2'-hydroxyaucuparin, noreriobofuran and in addition 3,4,5-trimethoxybiphenyl were detected. The total phytoalexin content in the transition zone of pear was 25 times lower than that in apple. Leaves and stems of mock-inoculated apple and pear shoots lacked phytoalexins. A number of biphenyls and dibenzofurans were tested for their in vitro antibacterial activity against some Erwinia amylovora strains. The most efficient compound was 3,5-dihydroxybiphenyl (MIC=115 µg/ml), the immediate product of biphenyl synthase which initiates phytoalexin biosynthesis.
Asunto(s)
Benzofuranos/metabolismo , Malus/metabolismo , Enfermedades de las Plantas/microbiología , Pyrus/metabolismo , Sesquiterpenos/metabolismo , Benzofuranos/química , Benzofuranos/farmacología , Erwinia amylovora/efectos de los fármacos , Erwinia amylovora/inmunología , Malus/inmunología , Malus/microbiología , Pruebas de Sensibilidad Microbiana , Inmunidad de la Planta , Hojas de la Planta/metabolismo , Tallos de la Planta/inmunología , Tallos de la Planta/metabolismo , Tallos de la Planta/microbiología , Pyrus/microbiología , Sesquiterpenos/química , Sesquiterpenos/farmacología , FitoalexinasRESUMEN
Transgenic antisense flavanone-3-hydroxylase apple plants were produced to mimic the effect of the agrochemical prohexadione-Ca on apple leaves. This enzyme inhibitor for 2-oxoglutarate dependent dioxygenases is used as a growth retardant and for control of secondary fire blight of leaves. Like using the agent, silencing of flavanone-3-hydroxylase leads to an accumulation of flavanones in leaves, but in contrast not to the formation of 3-deoxyflavonoids. In prohexadione-Ca treated leaves the 3-deoxyflavonoid luteoforol is formed from accumulating flavanones, acting as an antimicrobial compound against the fire blight pathogen Erwinia amylovora. Seemingly, the silencing of just one of the 2-oxoglutarate dependent dioxygenases (in apple also flavonol synthase and anthocyanidin synthase take part downstream in the pathway) does not provide a sufficiently high ratio of flavanones to dihydroflavonols. This seems to be needed to let the dihydroflavonol-4-reductase/flavanone-4-reductase enzyme reduce flavanones to luteoforol, and to let this be reduced by the leucoanthocyanidin-4-reductase/3-deoxyleucoanthocyanidin-4-reductase, each acting with their respective weak secondary activities. Accordingly, also the intended inducible resistance to fire blight by prohexadione-Ca is not observed with the antisense flavanone-3-hydroxylase apple plants. On the other hand, for most transgenic lines with strong flavanone-4-reductase down-regulation, up-regulation of gene expression for the other flavonoid genes was found. This provides further evidence for the feedback regulation of flavonoid gene expression having been previously reported for the prohexadione-Ca inhibited apple plants.
Asunto(s)
Flavanonas/biosíntesis , Silenciador del Gen , Malus/genética , Oxigenasas de Función Mixta/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacología , Benzopiranos/metabolismo , Benzopiranos/farmacología , Clonación Molecular , Medios de Cultivo/metabolismo , Susceptibilidad a Enfermedades/inmunología , Susceptibilidad a Enfermedades/microbiología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Erwinia amylovora/efectos de los fármacos , Erwinia amylovora/inmunología , Erwinia amylovora/patogenicidad , Flavanonas/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Ácidos Cetoglutáricos/farmacología , Malus/enzimología , Malus/inmunología , Malus/microbiología , Oxigenasas de Función Mixta/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacología , Brotes de la Planta/enzimología , Brotes de la Planta/microbiología , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/inmunología , Plantas Modificadas Genéticamente/microbiología , Plásmidos/genética , Plásmidos/metabolismo , Transcripción GenéticaRESUMEN
Fire blight is an invasive disease caused by Erwinia amylovora that threatens pome fruit production globally. Effective implementation of phytosanitary control measures depends upon rapid, reliable pathogen detection and disease diagnosis. We developed a lateral-flow immunoassay specific for E. amylovora with a detection limit of log 5.7 CFU/ml, typical of pathogen concentrations in symptomatic plant material. The simple assay had comparable sensitivity to standard culture plating, serum agglutination and nested PCR when validated for application in a phytosanitary laboratory as a confirmatory test of cultured isolates and for first-line diagnosis of phytosanitary samples that represent the full range of commercial, ornamental and forestry host species. On-site validation in ring-trials with local plant inspectors demonstrated robust and reliable detection (compared to subsequent plating and PCR analysis). The simplicity, inspector acceptance and facilitation of expedited diagnosis (from 2 days for laboratory submitted samples to 15 min with the immunoassay), offers a valuable tool for improved phytosanitary control of fire blight.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Cromatografía de Afinidad/métodos , Erwinia amylovora/química , Enfermedades de las Plantas/microbiología , Animales , Anticuerpos Inmovilizados/química , Erwinia amylovora/inmunología , Sueros Inmunes/química , Límite de Detección , Conejos , Rosaceae/microbiologíaRESUMEN
Harpin elicits rapid and localized programmed cell death in plants, also known as the hypersensitive response (HR). Here we report that HrpN from Erwinia amylovora led to rapid cell death in maize leaves within 24 h and also induced the expression of systemic acquired resistance genes, such as ZmPR1 and ZmPR5. Surprisingly, the results of DAB staining showed that there was no H(2)O(2) accumulation in maize leaves during the HR process, and semi-quantitative RT-PCR revealed that there was also no difference in the expression of the ZmRboh genes. These results suggest that HrpN-induced cell death may be independent of H(2)O(2) accumulation in maize leaves.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Muerte Celular , Peróxido de Hidrógeno/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Erwinia amylovora/genética , Erwinia amylovora/inmunología , Regulación de la Expresión Génica de las Plantas , Inmunidad Innata , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Enfermedades de las Plantas/inmunología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Proteínas de Plantas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ácido Salicílico/análisis , Zea mays/genética , Zea mays/metabolismo , Zea mays/microbiologíaRESUMEN
Fire blight is a devastating bacterial disease of rosaceous plants. Its damage to apple production is a major concern, since no existing control option has proven to be completely effective. Some commercial apple varieties, such as 'Florina' and 'Nova Easygro', exhibit a consistent level of resistance to fire blight. In this study, we used an F1 progeny of 'Florina' × 'Nova Easygro' to build parental genetic maps and identify quantitative trait loci (QTLs) related to fire blight resistance. Linkage maps were constructed using a set of microsatellites and enriched with amplified fragment length polymorphism (AFLP) markers. In parallel, progeny plants were artificially inoculated with Erwinia amylovora strain CFBP 1430 in a quarantine glasshouse. Shoot length measured 7 days after inoculation (DAI) and lesion length measured 7 and 14 DAI were used to calculate the lesion length as a percentage of the shoot length (PLL1 and PLL2, respectively). Percent lesion length data were log10-transformed (log10(PLL)) and used to perform the Kruskal-Wallis test, interval mapping (IM), and multiple QTL mapping (MQM). Two significant fire blight resistance QTLs were detected in 'Florina'. One QTL was mapped on linkage group 10 by IM and MQM; it explained 17.9% and 15.3% of the phenotypic variation by MQM with log10(PLL1) and log10(PLL2) data, respectively. A second QTL was identified on linkage group 5 by MQM with log10(PLL2) data; it explained 10.1% of the phenotypic variation. Genotyping the plants of 'Florina' pedigree with the microsatellites flanking the QTLs showed that the QTLs on linkage groups 5 and 10 were inherited from 'Jonathan' and 'Starking' (a 'Red Delicious' sport mutation), respectively. Other putative QTLs (defined as QTLs with LOD scores above the chromosomal threshold and below the genome-wide threshold) were detected by IM on linkage groups 5 and 9 of 'Nova Easygro'.
Asunto(s)
Erwinia amylovora , Malus/genética , Malus/microbiología , Enfermedades de las Plantas/genética , Sitios de Carácter Cuantitativo , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Cruzamiento , Mapeo Cromosómico , Cruzamientos Genéticos , Erwinia amylovora/inmunología , Erwinia amylovora/patogenicidad , Estudios de Asociación Genética , Ligamiento Genético , Sitios Genéticos , Marcadores Genéticos , Genotipo , Inmunidad Innata/genética , Malus/clasificación , Malus/inmunología , Repeticiones de Microsatélite , Fenotipo , Enfermedades de las Plantas/microbiología , Carácter Cuantitativo HeredableRESUMEN
Erwinia amylovora [(BURRILL) WINSLOW et al.] (Ea), the causal agent of fire blight, was detected in plant samples and pure bacterial cultures by means of PCR, IFAS and ELISA. Polyclonal antibodies of Neogen Europe Ltd. were used for IFAS and PTA-ELISA and laboratory-generated primers EaF72 and EaR560 for PCR. Using the BIOLOG system and an immature pear fruit assay, identities of all Ea strains were confirmed as the fire blight bacterium. In assays of pure Ea cultures, PTA-ELISA, and both IFAS and PCR were sensitive to concentrations 10(6)-10(5) and 10(5)-10(4) CFU/mL, respectively. When saprophytic bacteria associated with Ea in plant samples were tested as potentially cross-reacting bacteria, PTA-ELISA and IFAS gave 20 and 14 % cross-reactions, respectively. In plant samples, the presence of Ea was more reliably detected by IFAS (at a dilution of 1 : 1000) than by PTA-ELISA (to dilution 1 : 100). The capacity to detect Ea might be increased using an optimized PCR, but for PCR prepared from infected plant samples it was necessary to use the bacterial DNA isolated with a DNeasy Plant Mini Kit (Qiagen). In this case the PCR was sensitive to a concentration of 10(5) CFU/mL. PCR was much more specific than either immunochemical technique, because no false positives were observed when primers EaF72 and EaR560 were used.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Erwinia amylovora/aislamiento & purificación , Técnica del Anticuerpo Fluorescente Indirecta , Enfermedades de las Plantas/microbiología , Reacción en Cadena de la Polimerasa , Anticuerpos Antibacterianos , Ensayo de Inmunoadsorción Enzimática/métodos , Erwinia amylovora/genética , Erwinia amylovora/inmunología , Juego de Reactivos para Diagnóstico , Rosaceae/microbiología , Sensibilidad y EspecificidadRESUMEN
A total of 20 putative strains of Erwinia amylovora originating from 11 samples of host plants with symptoms of fire blight were analyzed in detail using commercial polyclonal antibodies in immunochemical tests. Fourteen strains reacted negatively in all tests; 6 strains reacted positively with a polyclonal antibody for PTA-ELISA (plate-trapped antigen-enzyme linked immunosorbent assay) at a concentration corresponding to A620 = 0.1, while at A620 readings of 0.01 and 0.001 the results were negative. Five strains reacted positively with a polyclonal antibody for indirect immunofluorescence test at all tested concentrations. Three of those strains were positive in the PCR test with AMSbL and AMSbR primers designed for detection of E. amylovora. In hypersensitivity test in tobacco and in immature pear fruit assay, all putative strains were negative while a known reference strain of E. amylovora gave a typical hypersensitive-reaction response. On a medium with 5% sucrose the reference strain of E. amylovora produced levan while putative strains did not. After modification of the PCR protocol, 3 putative strains reacted as negatives. Optimization of PCR test was achieved by finding the optimum annealing temperature and time for primers. The recommended annealing temperature (49 degrees C) for these primers was increased to 55 degrees C and the annealing time was reduced from 2 min to 30 s. Using the microbial identification system Biolog those 3 strains were identified as Pantoea dispersa (1 strain) and Pantoea agglomerans (2 strains). The strains are supposed to be white variants of the species P. dispersa and P. agglomerans occurring less frequently than the yellow variants. Since there were positive reactions in our immunochemical tests these strains could cause false positives in routine screening of plant samples.