RESUMO
Plants recognize a wide variety of microbial molecules to detect and respond to potential invaders. Recognition of Microbe-Associated Molecular Patterns (MAMPs) by cell surface receptors initiate a cascade of biochemical responses that include, among others, ion fluxes across the plasma membrane. A consequence of such event is a decrease in the concentration of extracellular H+ ions, which can be experimentally detected in plant cell suspensions as a shift in the pH of the medium. Thus, similarly to reactive oxygen species (ROS) accumulation, phosphorylation of MAP kinases and induction of defense-related genes, MAMP-induced medium alkalinization can be used as a proxy for the activation of plant immune responses. Here, we describe a detailed protocol for the measurement of medium alkalinization of tobacco BY-2 cell suspensions upon treatment with two different MAMPs: chitohexamers derived from fungal cell walls (NAG6; N-acetylglucosamine) and the flagellin epitope flg22, found in the bacterial flagellum. This method provides a reliable and fast platform to access MAMP-Triggered Immunity (MTI) in tobacco cell suspensions and can be easily adapted to other plant species as well as to other MAMPs.
RESUMO
Plant defensins are small cysteine-rich peptides that inhibit the growth of a broad range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a putative defensin isolated from Nicotiana megalosiphon upon inoculation with the tobacco blue mould pathogen Peronospora hyoscyami f.sp. tabacina. NmDef02 was heterologously expressed in the yeast Pichia pastoris, and the purified recombinant protein was found to display antimicrobial activity in vitro against important plant pathogens. Constitutive expression of NmDef02 gene in transgenic tobacco and potato plants enhanced resistance against various plant microbial pathogens, including the oomycete Phytophthora infestans, causal agent of the economically important potato late blight disease, under greenhouse and field conditions.
Assuntos
Defensinas/genética , Imunidade Inata , Nicotiana/genética , Doenças das Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Peronospora , Phytophthora , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Solanum tuberosum/genética , Solanum tuberosum/imunologia , Nicotiana/imunologiaRESUMO
Blue mould [Peronospora hyoscyami f. sp. tabacina (Adam) Skalicky 1964] is one of the most important foliar diseases of tobacco that causes significant losses in the Americas, south-eastern Europe and the Middle East. This review summarizes the current knowledge of the mechanisms employed by this oomycete pathogen to colonize its host, with emphasis on molecular aspects of pathogenicity. In addition, key biochemical and molecular mechanisms involved in tobacco resistance to blue mould are discussed. TAXONOMY: Kingdom: Chromista (Straminipila); Phylum: Heterokontophyta; Class: Oomycete; Order: Peronosporales; Family: Peronosporaceae; Genus: Peronospora; Species: Peronospora hyoscyami f. sp. tabacina. DISEASE SYMPTOMS: The pathogen typically causes localized lesions on tobacco leaves that appear as single, or groups of, yellow spots that often coalesce to form light-brown necrotic areas. Some of the leaves exhibit grey to bluish downy mould on their lower surfaces. Diseased leaves can become twisted, such that the lower surfaces turn upwards. In such cases, the bluish colour of the diseased plants becomes quite conspicuous, especially under moist conditions when sporulation is abundant. Hence the name of the disease: tobacco blue mould. INFECTION PROCESS: The pathogen develops haustoria within plant cells that are thought to establish the transfer of nutrients from the host cell, and may also act in the delivery of effector proteins during infection. RESISTANCE: Several defence responses have been reported to occur in the Nicotiana tabacum-P. hyoscyami f. sp. tabacina interaction. These include the induction of pathogenesis-related genes, and a correlated increase in the activities of typical pathogenesis-related proteins, such as peroxidases, chitinases, beta-1,3-glucanases and lipoxygenases. Systemic acquired resistance is one of the best characterized tobacco defence responses activated on pathogen infection.
Assuntos
Nicotiana/parasitologia , Peronospora/patogenicidade , Doenças das Plantas , Doenças das Plantas/imunologia , Doenças das Plantas/parasitologia , Nicotiana/imunologiaRESUMO
In order to identify tobacco (Nicotiana megalosiphon) genes involved in broad-spectrum resistance to tobacco blue mold (Peronospora hyoscyami f. sp. tabacina), suppression subtractive hybridization was used to generate cDNA from transcripts that are differentially expressed during an incompatible interaction. After differential screening by membrane-based hybridization, clones corresponding to 182 differentially expressed genes were selected, sequenced, and analyzed. The cDNA collection comprised a broad repertoire of genes associated with various processes. Northern blot analysis of a subset of these genes confirmed the differential expression patterns between the compatible and incompatible interaction. Subsequent virus-induced gene silencing (VIGS) of four genes that were found to be differentially induced was pursued. While VIGS of a lipid transfer protein gene or a glutamate decarboxylase gene in Nicotiana megalosiphon did not affect blue mold resistance, silencing of an EIL2 transcription factor gene and a glutathione synthetase gene was found to compromise the resistance of Nicotiana megalosiphon to P. hyoscyami f. sp. tabacina. Potentially, these genes can be used to engineer resistance in blue mold-susceptible tobacco cultivars.
Assuntos
Glutationa Sintase/metabolismo , Nicotiana/metabolismo , Nicotiana/microbiologia , Peronospora/fisiologia , Doenças das Plantas/microbiologia , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Glutationa Sintase/genética , Dados de Sequência Molecular , Mutação , Folhas de Planta/microbiologia , Nicotiana/enzimologia , Nicotiana/genética , Fatores de Transcrição/genéticaRESUMO
To understand the molecular basis of a specific plant-pathogen interaction, it is important to identify plant genes that respond to the pathogen attack. Amplified fragment length polymorphism (AFLP) analysis of cDNA was used to identify sugarcane genes differentially expressed in disease-resistant but not in susceptible sugarcane somaclones in response to inoculation with either Ustilago scitaminea or Bipolaris sacchari (also known as Helminthosporium sacchari or Drechslera sacchari), causal agents of smut and eyespot respectively. In total 62 differentially regulated genes were identified, of which 10 were down-regulated and 52 were induced. Of these 52, 19 transcript derived fragments showed homology to known plant gene sequences, most of them related to defense or signaling. The total set of differentially expressed sugarcane genes can be an important resource for further studies aimed at understanding sugarcane pathogen defense.