RESUMEN

Pear (Pyrus communis) is an economically important fruit crop. Drops in yield and even losses of whole plantations are caused by diseases, most importantly fire blight which is triggered by the bacterial pathogen Erwinia amylovora. In response to the infection, biphenyls and dibenzofurans are formed as phytoalexins, biosynthesis of which is initiated by biphenyl synthase (BIS). Two PcBIS transcripts were cloned from fire blight-infected leaves and the encoded enzymes were characterized regarding substrate specificities and kinetic parameters. Expression of PcBIS1 and PcBIS2 was studied in three pear cultivars after inoculation with E. amylovora. Both PcBIS1 and PcBIS2 were expressed in 'Harrow Sweet', while only PcBIS2 transcripts were detected in 'Alexander Lucas' and 'Conference'. Expression of the PcBIS genes was observed in both leaves and the transition zone of the stem; however, biphenyls and dibenzofurans were only detected in stems. The maximum phytoalexin level (~110 µg/g dry weight) was observed in the transition zone of 'Harrow Sweet', whereas the concentrations were ten times lower in 'Conference' and not even detectable in 'Alexander Lucas'. In 'Harrow Sweet', the accumulation of the maximum phytoalexin level correlated with the halt of migration of the transition zone, whereby the residual part of the shoot survived. In contrast, the transition zones of 'Alexander Lucas' and 'Conference' advanced down to the rootstock, resulting in necrosis of the entire shoots.

Asunto(s)

Erwinia amylovora/crecimiento & desarrollo , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Proteínas de Plantas/genética , Sintasas Poliquetidas/genética , Pyrus/microbiología , Sesquiterpenos/metabolismo , Compuestos de Bifenilo/química , Clonación Molecular , Dibenzofuranos/química , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/metabolismo , Tallos de la Planta/metabolismo , Tallos de la Planta/microbiología , Sintasas Poliquetidas/biosíntesis , Sintasas Poliquetidas/metabolismo , Pyrus/genética , Pyrus/metabolismo , Sesquiterpenos/química , Fitoalexinas

RESUMEN

MAIN CONCLUSION: The expression of the apple scab resistance gene Rvi6 in different apple cultivars and lines is not modulated by biotic or abiotic factors. All commercially important apple cultivars are susceptible to Venturia inaequalis, the causal organism of apple scab. A limited number of apple cultivars were bred to express the resistance gene Vf from the wild apple genotype Malus floribunda 821. Positional cloning of the Vf locus allowed the identification of the Rvi6 (formerly HcrVf2) scab resistance gene that was subsequently used to generate cisgenic apple lines. It is important to understand and compare how this resistance gene is transcribed and modulated during infection in conventionally bred cultivars and in cisgenic lines. The aim of this work was to study the transcription pattern of Rvi6 in three classically bred apple cultivars and six lines of 'Gala' genetically modified to express Rvi6. Rvi6 transcription was analyzed at two time points using quantitative real-time PCR (RT-qPCR) following inoculation with V. inaequalis conidia or water. Rvi6 transcription was assessed in relation to five reference genes. ß-Actin, RNAPol, and UBC were the most suited to performing RT-qPCR experiments on Malus × domestica. Inoculation with V. inaequalis conidia under conditions conducive to scab infection failed to produce any significant changes to the transcription level of Rvi6. Rvi6 expression levels were inconsistent in response to external treatments in the different apple cultivars, and transgenic, intragenic or cisgenic lines.

Asunto(s)

Ascomicetos/fisiología , Malus/genética , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta/genética , Actinas/genética , Cruzamiento , ARN Polimerasas Dirigidas por ADN/genética , Regulación de la Expresión Génica de las Plantas , Sitios Genéticos/genética , Genotipo , Malus/inmunología , Malus/microbiología , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Esporas Fúngicas

RESUMEN

Upon pathogen attack, fruit trees such as apple (Malus spp.) and pear (Pyrus spp.) accumulate biphenyl and dibenzofuran phytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenyl synthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan (Sorbus aucuparia) was isolated and functionally expressed in yeast (Saccharomyces cerevisiae). SaB4H was named CYP736A107. No catalytic function of CYP736 was known previously. SaB4H exhibited absolute specificity for 3-hydroxy-5-methoxybiphenyl. In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanine ammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter gene construct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembrane domain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (Malus × domestica) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the fire blight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hs were closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehyde and related compounds were not converted by SaB4H.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Malus/enzimología , Proteínas de Plantas/metabolismo , Sesquiterpenos/metabolismo , Sorbus/enzimología , Secuencia de Aminoácidos , Hidrocarburo de Aril Hidroxilasas/química , Células Cultivadas , Clonación Molecular , Sistema Enzimático del Citocromo P-450/química , ADN Complementario/genética , Cromatografía de Gases y Espectrometría de Masas , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Cinética , Malus/genética , Malus/microbiología , Microsomas/metabolismo , Datos de Secuencia Molecular , Filogenia , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/química , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sesquiterpenos/química , Sorbus/genética , Fracciones Subcelulares/enzimología , Especificidad por Sustrato , Nicotiana/metabolismo , Fitoalexinas

RESUMEN

Fire blight is a devastating disease of Rosaceae plants, such as apple and pear trees. It is characterized by necrosis of plant tissue, caused by the phytopathogenic bacterium Erwinia amylovora. The plant pathogen produces the well-known antimetabolite 6-thioguanine (6TG), which plays a key role in fire blight pathogenesis. Here we report that YcfR, a member of the LTTR family, is a major regulator of 6TG biosynthesis in E. amylovora. Inactivation of the regulator gene (ycfR) led to dramatically decreased 6TG production. Infection assays with apple plants (Malus domestica cultivar Holsteiner Cox) and cell cultures of Sorbus aucuparia (mountain ash, rowan) revealed abortive fire blight pathogenesis and reduced plant response (biphenyl and dibenzofuran phytoalexin production). In the presence of the ΔycfR mutant, apple trees were capable of activating the abscission machinery to remove infected tissue. In addition to unveiling the regulation of 6TG biosynthesis in a major plant pathogen, we demonstrate for the first time that this antimetabolite plays a pivotal role in dysregulating the plant response to infection.

Asunto(s)

Erwinia amylovora/química , Tioguanina/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Benzofuranos/química , Benzofuranos/metabolismo , Compuestos de Bifenilo/química , Compuestos de Bifenilo/metabolismo , Erwinia amylovora/genética , Erwinia amylovora/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Células Vegetales/química , Células Vegetales/metabolismo , Enfermedades de las Plantas/microbiología , Rosaceae/crecimiento & desarrollo , Rosaceae/metabolismo , Rosaceae/microbiología , Sesquiterpenos/química , Sesquiterpenos/metabolismo , Tioguanina/química , Fitoalexinas

RESUMEN

Sulfur for fire: The molecular basis for the biosynthesis of the antimetabolite 6-thioguanine (6TG) was unveiled in Erwinia amylovora, the causative agent of fire blight. Bioinformatics, heterologous pathway reconstitution in E. coli, and mutational analyses indicate that the protein YcfA mediates guanine thionation in analogy to 2-thiouridylase. Assays in planta and in cell cultures reveal for the first time a crucial role of 6TG in fire blight pathogenesis.

Asunto(s)

Erwinia amylovora/metabolismo , Enfermedades de las Plantas/microbiología , Tioguanina/metabolismo , Erwinia amylovora/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo

RESUMEN

Biphenyls and dibenzofurans are the phytoalexins of the Pyrinae, a subtribe of the plant family Rosaceae. The Pyrinae correspond to the long-recognized Maloideae. Economically valuable species of the Pyrinae are apples and pears. Biphenyls and dibenzofurans are formed de novo in response to infection by bacterial and fungal pathogens. The inducible defense compounds were also produced in cell suspension cultures after treatment with biotic and abiotic elicitors. The antimicrobial activity of the phytoalexins was demonstrated. To date, 10 biphenyls and 17 dibenzofurans were isolated from 14 of the 30 Pyrinae genera. The most widely distributed compounds are the biphenyl aucuparin and the dibenzofuran γ-cotonefuran. The biosynthesis of the two classes of defense compounds is not well understood, despite the importance of the fruit crops. More recent studies have revealed simultaneous accumulation of biphenyls and dibenzofurans, suggesting sequential, rather than the previously proposed parallel, biosynthetic pathways. Elicitor-treated cell cultures of Sorbus aucuparia served as a model system for studying phytoalexin metabolism. The key enzyme that forms the carbon skeleton is biphenyl synthase. The starter substrate for this type-III polyketide synthase is benzoyl-CoA. In apples, biphenyl synthase is encoded by a gene family, members of which are differentially regulated. Metabolism of the phytoalexins may provide new tools for designing disease control strategies for fruit trees of the Pyrinae subtribe.

RESUMEN

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 , Fitoalexinas

RESUMEN

Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple 'Golden Delicious', nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple 'Holsteiner Cox,' heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple 'Cox Orange,' expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells.

Asunto(s)

Genes de Plantas , Malus/genética , Complejos Multienzimáticos/genética , Clonación Molecular , ADN Complementario , Escherichia coli/genética , Inmunohistoquímica , Malus/enzimología , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/genética