MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Plant Sciences' source. Thu, 09 Feb 2012 14:32:16 +0100 http://www.medworm.com/index.php?rid=5533156&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_32 Secreted proteins from plants and phytopathogens play important roles in their interactions and contribute to elaborate mechanisms of attack, defense, and counter-defense, as well as surveillance and signaling. There is therefore considerable interest in developing techniques to characterize “secretomes.” Here, we describe the use of the yeast secretion trap (YST) functional screen to isolate and identify secreted proteins that are accumulated and detected in the extracellular matrix of eukaryotes. This method involves fusing cDNAs generated or derived from plants, pathogens, or infected tissue to a yeast (Saccharomyces cerevisiae) invertase (suc2) reporter gene lacking its signal peptide, transforming the resulting fusion library into an invertase-deficient yeast strain, and p... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533156 Fri, 23 Dec 2011 08:23:42 +0100 5533156 http://www.medworm.com/index.php?rid=5533155&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_31 Agrobacterium tumefaciens-mediated transformation (ATMT) has become an important tool for functional genomics in fungi. ATMT-based approaches such as random insertional mutagenesis and targeted knockout are widely used for gene functional analysis in plant-pathogen interactions. Here, we describe a protocol for the identification of pathogenicity and virulence genes through random insertional mutagenesis using the fungal wilt pathogen Verticillium dahliae as an example for the protocol. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533155 Fri, 23 Dec 2011 08:23:42 +0100 5533155 http://www.medworm.com/index.php?rid=5533154&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_30 PCR-based DNA array technology is one of the most suitable techniques to detect and identify multiple pathogens in a single assay. Out of the different array platforms that currently exist, membrane-based DNA macroarrays are the most convenient for plant disease diagnosis because of low costs, great sensitivity, and modest equipment requirements. Here we describe a protocol for routine detection of plant pathogens using DNA macroarrays, i.e., from sampling to analysis of hybridization results. Diagnosis can be completed within 36 h after sample collection. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533154 Fri, 23 Dec 2011 08:23:42 +0100 5533154 http://www.medworm.com/index.php?rid=5533153&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_2 Genome sequence data on fungal pathogens provide the opportunity to carry out a reverse genetics approach to uncover gene function. Efficient methods for targeted genome modifications such as knockout and in locus over-expression are in high demand. Here we describe two efficient single-step cloning strategies for construction of vectors for Agrobacterium tumefaciens-mediated transformation (ATMT). Targeted genome modifications require integration by a homologous double crossover event, which is achieved by placing target sequences on either side of a selection marker gene in the vector. Protocols are given for two single-step vector construction techniques. The In-Fusion cloning technique is independent of compatible restriction enzyme sites in the vector and the fragment to be cloned. Th... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533153 Fri, 23 Dec 2011 08:23:42 +0100 5533153 http://www.medworm.com/index.php?rid=5533152&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_29 Autophagy is a complex degradative process by which cytosolic material, including organelles, is randomly sequestered within double-membrane bound vesicles termed autophagosomes and targeted for degradation. Initially described as a nutrient stress adaptation response, the process of autophagy is now recognized as a central mechanism involved in many developmental processes. In this chapter, we provide guidelines to assess the initial steps of autophagy by monitoring autophagic body vacuolar accumulation. We employed a standard electron microscopy approach to observe the vacuoles of nutrient stressed fungal cells. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533152 Fri, 23 Dec 2011 08:23:42 +0100 5533152 http://www.medworm.com/index.php?rid=5533151&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_28 The increasing availability of sequenced genomes for plant pathogenic fungi has revolutionized molecular plant pathology in recent years. However, the genetic regulatory networks underlying many important components of pathogenesis remain poorly defined. Although the protocols outlined in this chapter can be utilized to identify genes regulating a wide range of biological processes in many filamentous fungi, we focus on describing how to identify genes through forward and reverse genetics, using the plant pathogenic fungus Fusarium verticillioides as a model for the protocol. Specifically, this chapter explains how to create a collection of insertional mutants via Restriction Enzyme Mediated Integration (REMI) and how to screen mutants with a high-throughput method to visualize defects in ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533151 Fri, 23 Dec 2011 08:23:42 +0100 5533151 http://www.medworm.com/index.php?rid=5533150&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_27 This article focuses specifically on the induction and production of the type B sesquiterpenoid trichothecene mycotoxins. Methods are described which permit in liquid culture the small or large scale production and detection of deoxynivalenol (DON) and its various acetylated derivatives. A wheat (Triticum aestivum L.) ear inoculation assay is also explained which allows the direct comparison of mycotoxin production by species, chemotypes and strains with different growth rates and/or disease-causing abilities. Each of these methods is robust and can be used for either detailed time-course studies or end-point analyses. Various analytical methods are available to quantify the levels of DON, 3A-DON and 15A-DON. Some criteria to be considered when making selections between the different analy... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533150 Fri, 23 Dec 2011 08:23:42 +0100 5533150 http://www.medworm.com/index.php?rid=5533149&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_26 Several species of filamentous fungi contain so-called dispensable or supernumerary chromosomes. These chromosomes are dispensable for the fungus to survive, but may carry genes required for specialized functions, such as infection of a host plant. It has been shown that at least some dispensable chromosomes are able to transfer horizontally (i.e., in the absence of a sexual cycle) from one fungal strain to another. In this paper, we describe a method by which this can be shown. Horizontal chromosome transfer (HCT) occurs during co-incubation of two strains. To document the actual occurrence of HCT, it is necessary to select for HCT progeny. This is accomplished by transforming two different drug-resistance genes into the two parent strains before their co-incubation. In one of the strains... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533149 Fri, 23 Dec 2011 08:23:42 +0100 5533149 http://www.medworm.com/index.php?rid=5533148&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_25 Many characterized fungal effector proteins are small secreted proteins. Effectors are defined as those proteins that alter host cell structure and/or function by facilitating pathogen infection. The identification of effectors by molecular and cell biology techniques is a difficult task. However, with the availability of whole-genome sequences, these proteins can now be predicted in silico. Here, we describe in detail how to identify and characterize effectors from a defined fungal proteome using in silico techniques. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533148 Fri, 23 Dec 2011 08:23:42 +0100 5533148 http://www.medworm.com/index.php?rid=5533147&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_24 We describe in detail how to perform lipid filter and liposome-binding assays and provide suggestions for troubleshooting potential problems with these assays. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533147 Fri, 23 Dec 2011 08:23:42 +0100 5533147 http://www.medworm.com/index.php?rid=5533146&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_23 Fungicide resistance assays are useful to determine if a fungal pathogen has developed resistance to a fungicide used to manage the disease it causes. Laboratory assays are used to determine loss of sensitivity, or resistance, to a fungicide and can explain fungicide failures and for developing successful fungicide recommendations in the field. Laboratory assays for fungicide resistance are conducted by measuring reductions in growth or spore germination of fungi in the presence of fungicide, or by molecular procedures. This chapter describes two techniques for measuring fungicide resistance, using the sugarbeet leaf spot fungus Cercospora beticola as a model for the protocol. Two procedures are described for fungicides from two different classes; growth reduction for triazole (sterol deme... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533146 Fri, 23 Dec 2011 08:23:42 +0100 5533146 http://www.medworm.com/index.php?rid=5533145&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_22 In recent years, a voluminous amount of genomic data has been generated for several plant pathogenic fungi. Multiple studies have utilized these genomic data to advance our knowledge about the molecular mechanisms of plant pathogenesis. However, not all plant pathogenic fungi share the same infection strategies, and several genes have been identified that are crucial for plant pathogenesis in one fungus, but dispensable in others. In order for data on biological relevance to keep pace with accumulating genomic data, new biological assays need to be developed for several pathogenic fungi. Accordingly, we have developed an in vitro assay that allows us to monitor morphological changes in hyphal development as the head blight pathogen Fusarium graminearum infects wheat. Using previously froze... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533145 Fri, 23 Dec 2011 08:23:42 +0100 5533145 http://www.medworm.com/index.php?rid=5533144&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_21 Genetic transformation is an essential tool for the modern study of gene function and the genetic improvement of an organism. The genetic transformation of many fungal species is well established and can be carried out by utilizing different transformation methods including electroporation, Agrobacterium, biolistics, or polyethylene glycol (PEG)-mediated transformation. Due to its technical simplicity and common equipment requirements, PEG-mediated transformation is still the most commonly used method for genetic transformation in filamentous fungi. Here, we describe a PEG-based protocol developed for genetic transformation of Stagonospora nodorum, a fungal pathogen of wheat. This protocol is directly applicable to other fungi especially those in the Dothideomycete class of fungi. (Source:... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533144 Fri, 23 Dec 2011 08:23:42 +0100 5533144 http://www.medworm.com/index.php?rid=5533143&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_20 Studies of the population genetics of fungal and oomycetous phytopathogens are essential to clarifying the disease epidemiology and devising management strategies. Factors commonly associated with higher organisms such as migration, natural selection, or recombination, are critical for the building of a clearer picture of the pathogen in the landscape. In this chapter, we focus on a limited number of experimental and analytical methods that are commonly applied in population genetics. At first, we present different types of qualitative and quantitative traits that could be identified morphologically (phenotype). Subsequently, we describe several molecular methods based on dominant and codominant markers, and we provide our assessment of the advantages and shortfalls of these methods. Third... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533143 Fri, 23 Dec 2011 08:23:42 +0100 5533143 http://www.medworm.com/index.php?rid=5533142&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_1 Fungal cells are highly complex as their metabolism is compartmentalized harboring various types of subcellular organelles that are bordered by one or more membranes. Knowledge about the intracellular localization of transporter proteins is often required for the understanding of their biological function. Among different approaches available, the localization analysis based on the expression of GFP fusions is commonly used as a relatively fast and cost-efficient method that allows visualization of proteins of interest in both live and fixed cells. In addition, inactivation of transporter genes is an important tool to resolve their specific function. Here we provide a detailed protocol for the deletion and localization analysis of ABC transporters in the filamentous fungus Penicillium chry... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533142 Fri, 23 Dec 2011 08:23:42 +0100 5533142 http://www.medworm.com/index.php?rid=5533141&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_19 Microarrays are a valuable technology to study fungal physiology on a transcriptomic level. Various microarray platforms are available comprising both single and two channel arrays. Despite different technologies, preprocessing of microarray data generally includes quality control, background correction, normalization, and summarization of probe level data. Subsequently, depending on the experimental design, diverse statistical analysis can be performed, including the identification of differentially expressed genes and the construction of gene coexpression networks. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533141 Fri, 23 Dec 2011 08:23:42 +0100 5533141 http://www.medworm.com/index.php?rid=5533140&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_18 The development of confocal microscopy and its application to studies of plant–pathogen interactions have revolutionised research into the role of selected molecules and cell components in pathogen infection strategies and plant defence responses. Confocal microscopy allows high-resolution visualisation of a variety of fluorescent and fluorescently tagged molecules in both fixed and living cells, not only in single cells but also in intact tissues. Confocal microscopes greatly improve image quality by reducing interference by out-of-focus light and can capture high-resolution serial optical sections through samples in the z-axis. In combination with a range of computational image analysis techniques, confocal microscopy provides a powerful tool by which molecules, molecular interacti... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533140 Fri, 23 Dec 2011 08:23:42 +0100 5533140 http://www.medworm.com/index.php?rid=5533139&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_17 Next-Generation Sequencing (NGS) methods have revolutionized various aspects of genomics including transcriptome analysis. Digital expression analysis is all set to replace analog expression analysis that uses microarray chips through their cost-effectiveness, reproducibility, accuracy, and speed. The last 2 years have seen a surge in the development of statistical methods and software tools for analysis and visualization of NGS data. Large amounts of NGS data are available for pathogenic fungi and oomycetes. As the analysis results start pouring in, it brings about a paradigm shift in the understanding of host pathogen interactions with discovery of new transcripts, splice variants, mutations, regulatory elements, and epigenetic controls. Here we describe the core technology of the new se... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533139 Fri, 23 Dec 2011 08:23:42 +0100 5533139 http://www.medworm.com/index.php?rid=5533138&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_16 Targeted gene replacement is one of the primary strategies for functional characterization of fungal genes and several methods have been developed for this purpose over the years. The increased availability of genome sequence information in the present times has enabled wider adoption of protocols based on the knowledge of the gene sequence and its surrounding region. Among such targeted gene replacement approaches, the spilt-marker method has gained popularity in filamentous fungi. This method involves only two rounds of PCR and does not require any subcloning. It is based on the availability of a marker gene (e.g., the hygromycin gene) and sequences of the gene of interest, as well as around 1 kb long regions flanking the gene on either side. The technique includes PCR amplification of t... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533138 Fri, 23 Dec 2011 08:23:42 +0100 5533138 http://www.medworm.com/index.php?rid=5533137&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_15 Proteomics and transcriptomics are established functional genomics tools commonly used to study filamentous fungi. Metabolomics has recently emerged as another option to complement existing techniques and provide detailed information on metabolic regulation and secondary metabolism. Here, we describe broad generic protocols that can be used to undertake metabolomics studies in filamentous fungi. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533137 Fri, 23 Dec 2011 08:23:42 +0100 5533137 http://www.medworm.com/index.php?rid=5533136&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_14 Laser microdissection (LM) of plant tissues infected with a fluorescent protein-tagged fungus is a useful method for obtaining samples highly enriched in fungal RNA for downstream analysis such as hybridization to a microarray. This paper outlines the requirements for successful LM of infected tissues and details a set of protocols for (1) preparing and sectioning infected tissue samples under conditions that preserve both RNA integrity and cytological features; (2) capturing fungal structures via LM; and (3) extraction and amplification of transcripts for further analysis. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533136 Fri, 23 Dec 2011 08:23:42 +0100 5533136 http://www.medworm.com/index.php?rid=5533135&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_13 In this chapter a method for the heterologous production of fungal proteins in the yeast Pichia pastoris is described. Starting with cloning of the sequence encoding the gene of interest into the expression vector, this protocol describes P. pastoris transformation, production of the protein in a fermentor, and purification of the protein. This method has successfully been used for the production of a number of fungal effector proteins. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533135 Fri, 23 Dec 2011 08:23:42 +0100 5533135 http://www.medworm.com/index.php?rid=5533134&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_12 Recently, great strides have been made in the area of host-pathogen interactions involving necrotrophic fungi. In this article we describe a method to identify, produce, and characterize effectors that are important in host–necrotrophic fungal pathogen interactions, and to genetically characterize the interactions. The main strength of this method is the combined use of pathogen inoculation, a pathogen culture filtrate bioassay, and genetic analysis of susceptibility and sensitivity in segregating host-mapping populations. These methods have been successfully used to identify several Stagonospora nodorum necrotrophic effectors and to characterize the genetic and phenotypic effects of individual host–effector interactions in the wheat-S. nodorum system. S. nodorum isolates that ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533134 Fri, 23 Dec 2011 08:23:42 +0100 5533134 http://www.medworm.com/index.php?rid=5533133&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_11 The yeast two-hybrid (Y2H) system is a binary method widely used to determine direct interactions between paired proteins. Although having certain limitations, this method has become one of the two main systemic tools (along with affinity purification/mass spectrometry) for interactome mapping in model organisms including yeast, Arabidopsis, and humans. It has also become the method of choice for investigating host–pathogen interactions in fungal pathosystems involving crop plants. This chapter describes general procedures to use the GAL4-based Y2H system for identification of host proteins that directly interact with proteinaceous fungal effectors, thus being their potential targets. The procedures described include cDNA library construction through in vivo recombination, library sc... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533133 Fri, 23 Dec 2011 08:23:42 +0100 5533133 http://www.medworm.com/index.php?rid=5533132&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-501-5_10 One of the primary roles of the cell surface is to provide an effective barrier to various external environmental factors. Specifically, the surface properties of organisms serve as a critical obstacle to pathogen attack. Since its inception, Atomic Force Microscopy (AFM) has enabled nanoscale imaging of cell surfaces in their native state. However AFM has yet to be systematically applied toward resolving surface features and the forces underpinning plant-fungal interactions. In an effort to understand the physical forces involved at the plant-microbe interface, we describe a method for the attachment of fungal spores to AFM tips and the subsequent measurement of unbinding forces between spores with a range of substrates and plant surfaces under physiologically relevant conditions. Investi... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5533132 Fri, 23 Dec 2011 08:23:42 +0100 5533132 http://www.medworm.com/index.php?rid=5204027&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_23 Analysis of gene expression data sets is a potent tool for gene function prediction, cis-element discovery, and hypothesis generation for the model plant Arabidopsis thaliana, and more recently for other agriculturally relevant species. In the case of Arabidopsis thaliana, experiments conducted by individual researchers to document its transcriptome have led to large numbers of data sets being made publicly available for data mining by the so-called “electronic northerns,” co-expression analysis and other methods. Given that approximately 50% of the genes in Arabidopsis have no function ascribed to them by “conventional” homology searches, and that only around 10% of the genes have had their function experimentally determined in the laboratory, these analyses can ac... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204027 Sun, 11 Sep 2011 03:35:58 +0100 5204027 http://www.medworm.com/index.php?rid=5204026&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_22 The ubiquitous signaling molecule nitric oxide (NO) plays an important role in seed biology. Experiments with this biologically important gas require special provisions because NO in aerobic environments is readily converted into other oxides of nitrogen. In this chapter, we describe methods for the application of NO as a gas, and through the use of NO-donor compounds. We included information on the removal or reduction of NO with NO scavengers. Methods for detecting NO using NO-reactive fluorescent probes, and an apparatus incorporating an oxidizer column are also described. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204026 Sun, 11 Sep 2011 03:35:58 +0100 5204026 http://www.medworm.com/index.php?rid=5204025&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_21 The formation of abnormal isoaspartyl residues derived from aspartyl or asparaginyl residues is a major source of spontaneous protein misfolding in cells. The repair enzyme protein l-isoaspartyl methyltransferase (PIMT) counteracts such damage by catalyzing the conversion of abnormal isoaspartyl residues to their normal aspartyl forms. Thus, this enzyme contributes to the survival of many organisms, including plants. Analysis of the accumulation of isoaspartyl-containing proteins and its modulation by the PIMT repair pathway, using germination tests, immunodetection, enzymatic assays, and HPLC analysis, gives new insights in understanding controlling mechanisms of seed longevity and vigor. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204025 Sun, 11 Sep 2011 03:35:58 +0100 5204025 http://www.medworm.com/index.php?rid=5204024&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_20 Evidence is emerging that reactive oxygen species (ROS) and antioxidants, together with plant hormones and other reactive species, such as reactive nitrogen species, are part of signalling networks pertinent to plant stress responses, cell division, and cell death. Consequently, they play pivotal roles in the regulation of seed development and maturation, germination and dormancy, seedling establishment, and seed ageing. Importantly, ROS, although essentially required at low concentrations, must be kept under stringent control by antioxidants. If the balance between pro- and antioxidative processes is lost and ROS production prevails, oxidative stress is the result, which can induce cell death and ultimately seed death. This chapter offers a variety of protocols for the determination of RO... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204024 Sun, 11 Sep 2011 03:35:58 +0100 5204024 http://www.medworm.com/index.php?rid=5204023&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_19 A number of genes and proteins are expressed in a tissue- or cell layer-specific manner. Spatial patterns of gene expression are critical to understanding gene function. Tissue printing provides a simple and rapid method to analyze localization of mRNA and protein at the tissue and cellular levels. This is especially convenient for gene expression analysis in hard tissues, such as seeds that are often difficult to section. Seed RNA or protein can be transferred onto a suitable membrane by printing the cut surface of a bisected seed. This method has been used successfully to determine mRNA and protein localization in seed research. The resolution of printed seed images and RNA and protein signals in tissue printing is sufficient to identify embryo- or endosperm-specific expression of variou... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204023 Sun, 11 Sep 2011 03:35:58 +0100 5204023 http://www.medworm.com/index.php?rid=5204022&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_18 Magnetic resonance imaging (MRI) is a superior noninvasive diagnostic tool widely used in clinical medicine, with more than 60 million MRI tests performed each year worldwide. More specialized high-resolution MRI systems capable of a resolution that is 100–1,000 times higher than standard MRI instruments are used primarily in materials science, but are used with increasing frequency in plant physiology. We have shown that high-resolution 1H-nuclear magnetic resonance (NMR) microimaging can provide a wealth of information about the internal anatomy of plant seeds as small as 1 mm or even smaller. This chapter covers the methods associated with these imaging techniques in detail. We also discuss the application of 1H-NMR microimaging to study in vivo seed imbibition, germination, and e... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204022 Sun, 11 Sep 2011 03:35:58 +0100 5204022 http://www.medworm.com/index.php?rid=5204021&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_17 Nuclear magnetic resonance (NMR) spectroscopy has been successfully applied to profile a variety of primary and secondary metabolites in whole intact plant seeds in vivo. The nondestructive nature of NMR spectroscopy allows direct metabolic studies to be performed on the same seed throughout a given physio­logical process or key lifecycle transition, such as dormancy breakage, germination, and early postgerminative growth. Multinuclear NMR is capable of evaluating seed quality by assessing nondestructively nutrient reserves and seed protectants at seed maturity and to further monitor reserve mobilization following germination, which is critical for seedling emergence. In this chapter, we illustrate the use of several in vivo NMR techniques for metabolite profiling in seeds. Importantly... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204021 Sun, 11 Sep 2011 03:35:58 +0100 5204021 http://www.medworm.com/index.php?rid=5204020&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_16 Mass spectrometry (MS) is rapidly becoming an indispensable tool for the analysis of posttranslational modifications (PTMs) of proteins, and particularly histone PTMs that regulate physiological processes. The more traditional bottom-up approach of searching for modifications on peptides rather than intact proteins (top-down) has proven useful for finding phosphorylation, acetylation, and ubiquitination sites. With the use of modern instrumentation and various MS-based techniques, peptides and their PTMs can be characterized in a high-throughput manner while still maintaining high sensitivity and specificity. In complement to bottom-up MS, recent advances in MS technology, such as high-field Fourier transform ion cyclotron resonance (FTICR)-mass spectrometry, have permitted the study of in... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204020 Sun, 11 Sep 2011 03:35:58 +0100 5204020 http://www.medworm.com/index.php?rid=5204019&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_15 The chromatin structure determines gene expression and thereby regulates developmental processes in the plant. The molecular mechanisms regulating the induction and release of seed dormancy are still largely unknown and the underlying changes in chromatin organization have hardly been analyzed. Most chromatin studies in plants have been performed on vegetative tissues and have focused on seedlings. The composition of seeds hampers molecular analyses and requires adaptation of the methods that are used for other tissues. Here, we give an overview of the current methods that are used to study different aspects of chromatin organization in seeds. Cytogenetic methods, like fluorescence in situ hybridization and immunolocalization, are used to study chromatin at the microscopic level. Changes i... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204019 Sun, 11 Sep 2011 03:35:58 +0100 5204019 http://www.medworm.com/index.php?rid=5204018&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_14 The seed is the dispersal unit of plants and must survive the vagaries of the environment. It is the object of intense genetic and genomic studies because processes related to seed quality affect crop yield and the seed itself provides food for humans and animals. Presently, the general aim of postgenomics analyses is to understand the complex biochemical and molecular processes underlying seed quality, longevity, dormancy, and vigor. Due to advances in functional genomics, the recent past years have seen a tremendous progress in our understanding of several aspects of seed development and germination. Here, we describe the proteomics protocols (from protein extraction to mass spectrometry) that can be used to investigate several aspects of seed physiology, including germination and its ho... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204018 Sun, 11 Sep 2011 03:35:58 +0100 5204018 http://www.medworm.com/index.php?rid=5204017&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_13 MicroRNAs (miRNAs) play an important role in gene regulation in many plant tissues and organs during various developmental stages. Previous studies have suggested the importance of gene regulation by miRNA in seeds. Characterizing the expression of miRNAs and their target genes in dormant and germinating seeds helps to gain a better understanding of the regulatory role of miRNAs during seed dormancy and germination. This can be achieved by implementing a simple miRNA extraction method using fractionation with isopropanol and Northern blot analysis using nonradioactive miRNA probes. Functional analysis of miRNA target genes potentially associated with seed dormancy and germination can be examined using mutant seeds in which specific miRNAs are deregulated by introducing silent mutations in ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204017 Sun, 11 Sep 2011 03:35:58 +0100 5204017 http://www.medworm.com/index.php?rid=5204016&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_12 Activation tagging is an important tool for gene discovery in plants. This method utilizes a T-DNA sequence that contains four tandem copies of the cauliflower mosaic virus 35S enhancer sequence or promoters oriented outward to the T-DNA border sequences. These elements enhance the expression of genes neighboring on either side of the randomly integrated T-DNA, resulting in gain-of-function phenotypes. Activation tagging has identified a number of genes, including those fundamental to plant development, such as the floral inducer gene, FLOWERING LOCUS T  (FT  ). The methods surrounding activation-tagging approaches are described in this chapter. While seeds have generally not been the targets of these methods in the past, activation tagging provides a powerful approach to unc... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204016 Sun, 11 Sep 2011 03:35:58 +0100 5204016 http://www.medworm.com/index.php?rid=5204015&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_11 Seed dormancy is a trait that is under multigenic control and affected strongly by environmental factors. Thus, seed dormancy is a typical quantitative trait. Natural accessions of Arabidopsis thaliana exhibit a great deal of genetic variation for seed dormancy. This natural variation can be used to identify genes controlling this trait by means of quantitative trait loci (QTL) mapping. In this chapter, we describe how QTL mapping for seed dormancy in Arabidopsis thaliana can be performed and how QTL analyses can be used to eventually identify the causal gene. Methods and recourses available specifically for Arabidopsis are described or referred to. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204015 Sun, 11 Sep 2011 03:35:58 +0100 5204015 http://www.medworm.com/index.php?rid=5204014&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_10 Here, we describe a series of methods suitable for the reproducible and abundant isolation of total RNA, genomic DNA, and total protein from dry or imbibed Arabidopsis seeds. The resulting material is suitable for most standard molecular biology procedures. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204014 Sun, 11 Sep 2011 03:35:58 +0100 5204014 http://www.medworm.com/index.php?rid=5204013&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_9 Abscisic acid (ABA) plays an important role in the control of seed dormancy and germination. Identification of hormone metabolism genes from a particular plant species of interest is an essential step in hormone research. The function of these gene products is validated by biochemical analysis using heterologous expression systems, such as E. coli and yeast. ABA 8′-hydroxylase is a subfamily of P450 monooxygenases and is encoded by CYP707A genes. CYP707A catalyzes the committed step in the major ABA catabolic pathway. In this chapter, we describe the methods for RNA extraction from seeds, cloning the CYP707A cDNAs, protein expression in yeast, and biochemical analysis of their gene products. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204013 Sun, 11 Sep 2011 03:35:58 +0100 5204013 http://www.medworm.com/index.php?rid=5204012&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_8 We describe here an in vitro assay for abscisic acid (ABA) 8′-hydroxylase that was developed using microsomes extracted from (+)-ABA-induced corn suspension cultures. This assay may be useful for further characterization and monitoring of ABA 8′-hydroxylase activities in germinating seeds, seedlings, and other tissues. Additionally, the optimization protocols provided here may be adapted towards improving in vitro enzyme assays for other cytochrome P450 enzymes expressed in plants. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204012 Sun, 11 Sep 2011 03:35:58 +0100 5204012 http://www.medworm.com/index.php?rid=5204011&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_7 Seed dormancy and germination are regulated by several plant hormones, such as abscisic acid, gibberellin, auxin (indole-3-acetic acid), ethylene, and brassinosteroid. Endogenous concentrations of a hormone are determined by the balance between biosynthesis and deactivation, and contribute to the regulation of physiological responses. Therefore, profiling of all hormones and their metabolites (hormonome) is a powerful approach to elucidate the regulatory networks of hormone metabolism. The methods involved in the use of liquid chromatography–electrospray ionization–tandem mass spectrometry to develop a high-sensitive and high-throughput hormonome platform are described in this chapter. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204011 Sun, 11 Sep 2011 03:35:58 +0100 5204011 http://www.medworm.com/index.php?rid=5204010&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_6 Abscisic acid (ABA) is a vital phytohormone that regulates seed maturation and germination, seedling growth, and adaptation to environmental stresses. ABA functions through a complex network of signaling pathways, where the cell response is initiated by an ABA receptor which triggers downstream signaling cascades to induce the final physiological effects. Two classes of technologies may be used for the isolation of ABA receptors. One is the genetic screening for ABA receptor mutants, and another is the biochemical isolation of ABA-binding proteins that are putative ABA receptors. We implemented biochemical approaches, namely, the purification of ABA-binding proteins to identify a putative ABA receptor; this protein was further characterized by a combination of biochemical and reverse genet... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204010 Sun, 11 Sep 2011 03:35:58 +0100 5204010 http://www.medworm.com/index.php?rid=5204009&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_5 Most often, the samples used for molecular analysis of dormancy are populations of seeds. An essential survival characteristic of seed populations inhabiting the variable surface layers of the soil is that individuals in the population do not behave uniformly. In addition, seed dormancy (SD) status of the whole population constantly changes even in the dry state. For these and other reasons, production of appropriate and adequately characterized seed samples is the key to the correct and most informative interpretation of molecular studies. This is particularly important when the aim is to describe and explain seed behaviour in the natural environment. Molecular studies of seed dormancy, and especially ecologically relevant behaviour, such as dormancy cycling, should therefore involve char... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204009 Sun, 11 Sep 2011 03:35:58 +0100 5204009 http://www.medworm.com/index.php?rid=5204008&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_4 Many seeds of coniferous species display a deep primary dormancy at maturity and require several weeks of pretreatment to produce seed populations that germinate in a vigorous and timely manner. Facilitating an efficient transition from dormancy to germination by devising improved protocols for dormancy breakage is not only important to conifer seed research, aiding in the study of the dormancy process itself, but is also of interest and applicability to commercial forest nursery operations. In the forests of British Columbia, Canada, several conifer species are well-adapted to their environment, with seeds needing to experience long durations in the moist state at cool or fluctuating temperatures. These include yellow-cedar (Callitropsis nootkatensis), western white pine (Pinus monticola)... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204008 Sun, 11 Sep 2011 03:35:58 +0100 5204008 http://www.medworm.com/index.php?rid=5204007&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_3 Seeds are very attractive and convenient for molecular genetic studies that challenge principal biological phenomena related to the initiation and suppression of growth (e.g., germination and dormancy, respectively). The number of reports in this field is rapidly expanding. Seed dormancy is a widely misinterpreted biological attribute. One of the main reasons is the general neglect of reliable dormancy assays; often, the sole criterion of current dormancy assays is the total germination of a seed population after a defined period of time. This is a very insensitive and inaccurate method, particularly when comparing dormancy levels of seeds from different genotypes, seeds subjected to different treatments, or seeds originating from different environments. Other seed parameters are far more ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204007 Sun, 11 Sep 2011 03:35:58 +0100 5204007 http://www.medworm.com/index.php?rid=5204006&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_2 Seeds form a convenient vehicle for storage of germplasm, both for agricultural purposes and conservation of wild species. When required, seeds can be taken from storage and germinated, and plants can be propagated for the desired purpose, e.g., crop production or biome restoration. However, seed dormancy often interferes with stand establishment or industrial utilization in crops and germination of wild species. An anticipated termination of dormancy (i.e., before crop harvest) also occurs, with preharvest sprouting as a consequence. In order to overcome these problems, a better understanding of dormancy is required. This chapter is devoted to discuss the achievement of such understanding in problematic species. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204006 Sun, 11 Sep 2011 03:35:58 +0100 5204006 http://www.medworm.com/index.php?rid=5204005&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-231-1_1 Artificial regeneration of forests through planting requires high quantities of quality seeds for growing vigorous seedlings. These seedlings are raised in nurseries, where germination capacity (GC) and speed are the most important germination parameters. Germination performance is enhanced by prescribing species-specific dormancy-breaking treatments to individual seedlots in bare-root and container nurseries. For most conifer species in British Columbia, the dormancy-breaking treatments and germination conditions have been worked out, but fine-tuning and optimization could improve germination capacity and speed of germination. Implications of inter- and intra-species variations in germination behaviour and seed quality and their influence on the development of unintentional directional se... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5204005 Sun, 11 Sep 2011 03:35:58 +0100 5204005 http://www.medworm.com/index.php?rid=5137753&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_17 The identification of phosphorylation on proteins has become practicable for many laboratories in recent years, largely due to improvements in mass spectrometry (MS) and the development of methods to selectively enrich for phosphorylated peptides and proteins. However, phosphorylation is a dynamic and reversible modification which plays a central role in many biological processes including intracellular signalling. Therefore, the quantitative analysis of phosphorylated proteins and peptides is a subject of intense interest. We discuss three applications of isobaric tags for relative and absolute quantitation (iTRAQ) to the analysis of phosphopeptides from a variety of sample materials. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137753 Wed, 17 Aug 2011 16:49:26 +0100 5137753 http://www.medworm.com/index.php?rid=5137752&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_16 Protein phosphorylation is a dynamic process of widespread regulatory significance. Phosphoproteomics attempts to provide a global view of this process during biological processes, but the approach is generally limited by the low relative amounts of phosphoproteins in biological samples. Although mass spectrometry (MS)-based technologies exist for the in-depth characterization of protein phosphorylation, these techniques are typically highly focused, have low throughput, and generally require special equipment and expertise. These specialized techniques are best used to support hypotheses generated by an initial broad-based survey, like the one described here. In this chapter, we outline a 2D gel-based phosphoproteomic methodology based on relatively inexpensive materials and basic, widely... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137752 Wed, 17 Aug 2011 16:49:26 +0100 5137752 http://www.medworm.com/index.php?rid=5137751&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_15 Identification of protein kinase targets and specific inhibition of individual kinase isoforms on the protein level in planta are important techniques to elucidate signal transduction pathways. The use of ATP-binding pocket mutants, the so-called gatekeeper mutants, that accommodate N6-enlarged nucleotides and kinase inhibitors has allowed a dramatic increase in kinase isoform selectivity. In this chapter, we describe protocols for the identification and mutation of the gatekeeper residue, radiolabeling of N6-modified nucleotides, analysis of protein targets by using [32P]-labeled N6-modified nucleotides, and in vivo inhibition of kinase activity combined with subsequent molecular readouts. The chapter includes alternative approaches for the described techniques, considerations for other k... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137751 Wed, 17 Aug 2011 16:49:26 +0100 5137751 http://www.medworm.com/index.php?rid=5137750&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_14 Enzyme–substrate interactions are weak and occur only transiently and thus, a faithful analysis of these interactions typically requires elaborated biochemical methodology. The bimolecular-fluorescence complementation (BiFC) assay, also referred to as split YFP assay, is a powerful and straightforward tool to test protein–protein interactions. This system is commonly used due to many advantages and especially due to its simple ease of use. BIFC relies on the reconstitution of an N-terminal and C-terminal half of YFP into a functional, i.e., fluorescent protein. Noteworthy, the dissociation constant of the two YFP halves is much lower than the association constant leading to a stabilization of the protein–protein interaction to be monitored. Whereas this property is someti... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137750 Wed, 17 Aug 2011 16:49:25 +0100 5137750 http://www.medworm.com/index.php?rid=5137749&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_9 Signal transduction through mitogen-activated protein kinase (MAPK) cascades regulates many cellular responses. One example of a stimulus-mediated MAPK signaling network in plants is the self-incompatibility (SI) response in Papaver rhoeas, which represents an important mechanism to prevent self-fertilization. This involves interaction of pistil S-locus determinants with a pollen receptor in an incompatible interaction, resulting in a Ca2+-dependent signaling network involving activation of a MAPK, p56, and stimulation of several caspase-like activities, resulting in programmed cell death (PCD). MAPK inhibitors provide a useful tool to dissect these mechanisms and distinguish their regulation by different signaling pathways. U0126 is a potent, noncompetitive, and specific inhibitor of MAPK... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137749 Wed, 17 Aug 2011 16:49:25 +0100 5137749 http://www.medworm.com/index.php?rid=5137748&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_13 Plasma-membrane-localized receptor kinases are essential for cell–cell communication and as sensors for the extracellular environment. Receptor function is dependent on their distribution in the membrane and interaction with other proteins that are either membrane-localized, present in the cytoplasm, or in the extracellular space. The organized distribution and mobility of receptor kinases is, therefore, thought to regulate the efficiency of downstream signaling. This chapter describes two methods to study receptor mobility in the plasma membrane. Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). Especially, the combination of FRAP and FCS provides a better insight into plasma membrane receptor mobility. (Source: Springer protocols fee... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137748 Wed, 17 Aug 2011 16:49:25 +0100 5137748 http://www.medworm.com/index.php?rid=5137747&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_12 Transmembrane receptor-kinases are widespread throughout eukaryotes and their activities are known to regulate all kinds of cellular responses in diverse organs and cell types. In order to guarantee the correct amplitude and duration of signals, receptor levels at the cellular surface need to be tightly controlled. The regulation of receptor degradation is the most direct way to achieve this and elaborate mechanisms are in place to control this process. Therefore, the rate of receptor degradation is a parameter of central importance for understanding the dynamics of a signal transduction cascade. Unfortunately, degradation of transmembrane receptors is a complicated multistep process that involves internalization from the plasma membrane, invagination into the lumen of endosomal compartmen... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137747 Wed, 17 Aug 2011 16:49:25 +0100 5137747 http://www.medworm.com/index.php?rid=5137746&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_11 Receptor kinases are essential for the cellular perception of signals. The classical model for activation of the receptor kinase involves dimerization, induced by the binding of the ligand. The mechanisms by which plant receptors transduce signals across the cell surface are largely unknown but plant receptors seem to dimerize as well. In this chapter, we describe two fluorescence fluctuation techniques, fluorescence cross-correlation spectroscopy and photon counting histogram analysis, to study the oligomerization state of receptor kinases in living plant cells in a quantitative manner. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137746 Wed, 17 Aug 2011 16:49:25 +0100 5137746 http://www.medworm.com/index.php?rid=5137745&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_10 The highly conserved nature of the protein kinase catalytic domain and the low permeability of plant cell membranes pose a challenge to the development of specific inhibitors that target individual protein kinases in vivo. Here, we describe a chemical-genetic approach to specifically sensitize individual plant kinases to cell-permeable small molecules that do not inhibit wild-type kinases. In this approach, a single amino-acid substitution is introduced in the ATP-binding site of the enzyme enabling specific binding of ATP-competitive molecules. Cell-permeable molecules can then be used to specifically target the sensitized allele in transgenic Arabidopsis thaliana plants that do not express the wild-type form of the kinase. This strategy provides a useful tool for the functional character... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137745 Wed, 17 Aug 2011 16:49:25 +0100 5137745 http://www.medworm.com/index.php?rid=5137744&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_8 Protein phosphorylation by protein kinases can be reversed by the action of protein phosphatases. In plants, the Ser/Thr-specific phosphatases dominate among the protein phosphatase families with the type 2C protein phosphatases (PP2Cs) being the most abundant among them. PP2Cs are monomeric enzymes that require metal cations for their activity and are insensitive to known phosphatase inhibitors. PP2Cs were shown to counteract the mitogen-activated protein kinase (MAP kinase/MAPK) activities in plants and to regulate developmental and stress signaling pathways. Studies of PP2C activities can be performed in vitro using recombinant proteins. The potential substrates of PP2Cs can be tested for dephosphorylation by the phosphatase in vitro. We have found that the stress-induced PP2Cs from alf... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137744 Wed, 17 Aug 2011 16:49:24 +0100 5137744 http://www.medworm.com/index.php?rid=5137743&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_7 Cytokinins, like other phytohormones, act in plants as signaling molecules at very low concentrations. The system that mediates between their chemical recognition and the responses that they induce requires a hormone receptor that, together with down-stream located elements, forms a signaling network, converting the signal into a specific response. Identification of the cytokinin-binding histidine kinases CRE1/AHK4, AHK3, and AHK2 as cytokinin receptors in Arabidopsis was an important milestone in the elucidation of cytokinin signal transduction pathways. Their molecular characterization through the use of transgenic E. coli strains revealed that a variety of cytokinin compounds may have signaling functions, but only with specific receptors. This indicates that differential ligand specific... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137743 Wed, 17 Aug 2011 16:49:24 +0100 5137743 http://www.medworm.com/index.php?rid=5137742&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_6 We describe in detail complementation experiments of a mutant in CDKA;1, the major cell cycle kinase in Arabidopsis, with phosphorylation-site variants of CDKA;1. CDKA;1 versions were generated either by mimicking a phosphorylated amino acid by replacing the respective residue with a negatively charged amino acid, e.g., aspartate or glutamate, or by mutating it to a non-phoshorylatable amino acid, such as alanine, valine, or phenylalanine. The genetic complementation studies were accompanied by the isolation of these kinase variants from plant extract and subsequent kinase assays to determine changes in their activity levels. This work allowed us to judge the importance of ­posttranslational regulation of CDKA;1 in plants and has shown that the molecular mechanistics of CDK function ar... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137742 Wed, 17 Aug 2011 16:49:24 +0100 5137742 http://www.medworm.com/index.php?rid=5137741&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_5 Mitogen-activated protein (MAP) kinase pathways are conserved in eukaryotes and transmit a plethora of stimuli. MAP kinases (MAPKs) are part of signalling modules that consist of three to four tiers of protein kinases in a phosphorylation cascade. MAPKs are known to phosphorylate specific substrates at specific sites at a ­threonine or serine residue followed by proline, but the surrounding amino acids of the phosphorylation site and docking interactions are also important for substrate recognition. MAPK activity can be assayed by detecting their phosphotransferase activity, their activation state, or detecting the switching on or off reaction of specific genes, or cellular responses. Prior to the kinase assay, specific MAPK proteins can be immunoprecipitated either by MAPK-specific an... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137741 Wed, 17 Aug 2011 16:49:24 +0100 5137741 http://www.medworm.com/index.php?rid=5137740&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_4 Orderly progression of the eukaryotic cell cycle is governed by a coordinated response to intrinsic and extracellular cues through activation of cyclin-dependent kinases (CDKs). It is therefore important to verify the kinase activity of distinct types of CDKs during the cell cycle. The immunoprecipitation-coupled kinase assay is a useful procedure to evaluate CDK activity in vivo. Although a specific antibody is usually required for immunoprecipitation, transgenic plant cells expressing tag- or marker protein-fused CDKs are also suitable for this purpose. In addition, the baculovirus expression system is a valuable tool for analyzing CDK activity in vitro, because activation of CDKs is regulated by posttranscriptional modification systems that are active in the insect host cells. (Source: ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137740 Wed, 17 Aug 2011 16:49:24 +0100 5137740 http://www.medworm.com/index.php?rid=5137739&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_3 In vitro functional studies of eukaryotic kinases are often constrained by the availability of pure and ­enzymatically active kinase of interest. Though numerous proteins have been synthesized by cell-based systems, in vivo production of properly folded, eukaryotic proteins remains a challenging task. Current wheat-germ-based cell-free in vitro translation systems present a plausible alternative for protein synthesis since majority of eukaryotic proteins could be obtained in their native folded form with general protocols. The use of special in vitro translation vectors with ligation-independent cloning sites and cleavable affinity tags eliminates further bottlenecks of the protein producing procedure and makes this system a reasonable method for simultaneous generation of active kinas... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137739 Wed, 17 Aug 2011 16:49:24 +0100 5137739 http://www.medworm.com/index.php?rid=5137738&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_2 Major progress has been made in unravelling of regulatory mechanisms in eukaryotic cells. Modification of target protein properties by reversible phosphorylation events has been found to be one of the most prominent cellular control processes in all organisms. The phospho-status of a protein is dynamically controlled by protein kinases and counteracting phosphatases. Therefore, monitoring of kinase and phosphatase activities, identification of specific phosphorylation sites, and assessment of their functional significance are of crucial importance to understand development and homeostasis. Recent advances in the area of molecular biology and biochemistry, for instance, mass spectrometry-based phosphoproteomics or fluorescence spectroscopical methods, open new possibilities to reach an unpr... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137738 Wed, 17 Aug 2011 16:49:24 +0100 5137738 http://www.medworm.com/index.php?rid=5137737&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-264-9_1 The growing number of fully annotated genomes of model and nonmodel plant species, such as Arabidopsis, Brachypodium, ­grapevine, maize, rice, rape seed, soybean, tomato, and others, has led to a tremendous increase in sequence information. The novel genome information has been translated into the putative proteomes and set the ground for a wealth of yet to be explored kinomes. At the same time, cellular pathways are now analyzed in model plants with an unprecedented depth revealing new ­mechanisms of regulation and integration of individual modules into regulatory networks. These studies are promoted by new developments in biochemical, molecular, and cell biological techniques of which we provide a comprehensive and state-of-the-art overview in the present methods book (Fig. 1). (... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5137737 Wed, 17 Aug 2011 16:49:23 +0100 5137737 http://www.medworm.com/index.php?rid=5106037&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_21 Chloroplasts are metabolically important organelles that perform many essential functions within plant cells. The chloroplasts can be subdivided into six distinct sub-compartments to which a protein may be ultimately targeted. These sub-compartments are defined as the outer envelope membrane (OEM), the inner envelope membrane (IEM), the thylakoid membrane, and three aqueous sub-compartments – the intermembrane space (IMS), the stroma, and the thylakoid lumen. The process by which proteins are targeted to the chloroplastic envelope membrane remains a challenging question in cell biology. Our understanding of protein targeting to the OEM is very limited, whereas targeting of membrane proteins to the IEM appears to utilize at least two targeting pathways called the stop-transfer and the... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106037 Mon, 08 Aug 2011 15:18:43 +0100 5106037 http://www.medworm.com/index.php?rid=5106036&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_20 Chloroplasts have one of the most complicated structures among organelles. They have three membrane systems, the outer and inner envelope membranes and the thylakoid membrane, which enclose three aqueous spaces: the intermembrane space between the two envelope membranes, the stroma, and the thylakoid lumen. Each of the chloroplast’s sub-organellar compartments houses a distinct set of proteins that perform distinct functions. Determining the sub-organellar location of a protein in the chloroplast is vital for understanding or verifying the function of the protein. Here, we present protocols for determining the sub-organellar location of a chloroplast protein. The protein of interest is synthesized and labeled with [35S]methionine by an in vitro translation system, and imported into i... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106036 Mon, 08 Aug 2011 15:18:42 +0100 5106036 http://www.medworm.com/index.php?rid=5106035&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_19 This chapter focuses on the techniques of chloroplast isolation; their fractionation into envelopes, stroma, and thylakoids; and their further use for in vitro protein transport assays. In addition to the isolation of thylakoids, this chapter also describes the experimental steps of both protein translocation across the thylakoid membrane and protein integration into the membrane. Protein translocation and integration can be analysed by the radioactive labelling of substrate proteins using an in vitro transcription and translation system. The translocated or integrated proteins can then be detected by autoradiography. Our protocol allows the analysis of these transport systems in wild-type Arabidopsis or mutants that lack or overexpress soluble or membrane transport factors that could be o... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106035 Mon, 08 Aug 2011 15:18:42 +0100 5106035 http://www.medworm.com/index.php?rid=5106034&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_18 Most chloroplast proteins are synthesized in the cytosol as preproteins with N-terminal cleavable transit peptides and are imported into the organelle through the TOC–TIC translocon system. Import involves a complex set of recognition and membrane translocation steps that ensure the fidelity and unidirectional transport of the polypeptide across the double-membrane chloroplast envelope. To understand the mechanism of import, the molecular interactions and energetics of each step must be defined. Here, we describe the methods for capturing intermediates in the import process through the manipulation of the energy state of chloroplasts, and the use of two different chemical cross-linking approaches to examine the molecular interactions that mediate the import process and to assess the ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106034 Mon, 08 Aug 2011 15:18:42 +0100 5106034 http://www.medworm.com/index.php?rid=5106033&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_17 In vitro chloroplast protein import assays have been performed since the late 1970s, initially with plant species (e.g., pea and spinach) that readily provide an abundant source of starting material and also, subsequently, a good yield of chloroplasts for import assays. However, the sequencing of the Arabidopsis genome paved the way for an additional model system that is more amenable to genetic analysis, as a complement to the more biochemically orientated models such as pea and spinach. A prerequisite for this change was an efficient and reliable protocol for the isolation of chloroplasts for use in protein import assays, enabling biochemical approaches to be combined with the genetic potential of the plant. The method described here was developed as a rapid and low-cost procedure that c... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106033 Mon, 08 Aug 2011 15:18:41 +0100 5106033 http://www.medworm.com/index.php?rid=5106032&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_16 This article reviews the various prediction methods that identify plastid targeting sequences, and those that can help estimate location and topology within the plastid or plastid membranes. The most successful approaches are described in detail, with detailed notes to help avoid common pitfalls and advice on interpreting conflicting or ambiguous results. In most cases, it is best to try multiple approaches, and we also cover the powerful new integrated databases that provide a selected blend of experimental data and predictions. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106032 Mon, 08 Aug 2011 15:18:41 +0100 5106032 http://www.medworm.com/index.php?rid=5106031&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_15 Proteolysis is a key process for maintaining homeostasis in all living cells. The ability to degrade specific metabolic enzymes and regulatory proteins is essential for both cellular integrity and function. Equally important is the efficient removal of damaged or otherwise inactive polypeptides, especially during periods of developmental change or stress adaptation. Being one of the most metabolically active plant organelles, chloroplasts require various proteases to control overall protein quality. Much has been revealed about these chloroplast proteases over the last decade, and yet the identity of their native protein substrates remains elusive. In this chapter, we describe a variation upon a classic genetic approach to identify protease substrates based on the comparative protein degra... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106031 Mon, 08 Aug 2011 15:18:40 +0100 5106031 http://www.medworm.com/index.php?rid=5106030&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_14 Chloroplasts as descendents of a cyanobacterial endosymbiont have retained, during evolution, their own genome together with the gene expression machinery, including the translation apparatus. Therefore, chloroplast protein synthesis is not only a key process in organello biogenesis and maintenance, but it also represents the major regulatory step in chloroplast gene expression. In fact, several independent evidences have shown that the accumulation of template messengers is not limiting in the expression of chloroplast genes. On the contrary, translation regulatory processes based on selection of translatable mRNA by either nucleus-encoded activation factors or sensors of the assembly status of chloroplast multiprotein complexes have been reported. Additionally, we have shown that organel... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106030 Mon, 08 Aug 2011 15:18:40 +0100 5106030 http://www.medworm.com/index.php?rid=5106029&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_13 In plant organelles, specific C residues are modified to U by RNA editing. Short RNA sequences surrounding the target site (i.e., cis-elements) are recognized by trans-factors, which were recently shown to be pentatricopeptide repeat (PPR) proteins. PPR proteins consist of tandem arrays of a highly degenerate unit of 35 (pentatrico) amino acids, and PPR motifs are believed to recognize specific RNA sequences. In Arabidopsis thaliana, more than 450 sites are edited in mitochondria and plastids, and a similar number of PPR proteins are encoded in the nuclear genome. To study how the tandem array of a PPR motif facilitates the recognition of RNA sequences, an efficient biochemical strategy is an in vitro binding assay of recombinant PPR proteins with target RNA. This analysis is especially po... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106029 Mon, 08 Aug 2011 15:18:40 +0100 5106029 http://www.medworm.com/index.php?rid=5106028&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_12 Most chloroplast genes in land plants are represented by multiple transcript isoforms that arise via differential splicing, endo- and exo-nucleolytic processing, and/or RNA editing. Exploration of the functional significance and mechanisms of these processing events is an active area of current research. This chapter focuses on methods that can be used to define the termini of chloroplast RNAs, quantify the relative levels of alternative processed RNA isoforms, and identify the binding sites of proteins that mediate chloroplast RNA processing. Various approaches for defining the sequence specificity of chloroplast RNA binding proteins are discussed, as are the parameters to consider in designing in vitro assays for RNA binding activities. A protocol is provided for a poisoned-primer extens... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106028 Mon, 08 Aug 2011 15:18:39 +0100 5106028 http://www.medworm.com/index.php?rid=5106027&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_11 The regulation of gene expression is still one of the major issues in modern plant molecular biology. The amount of RNA in a cell is regulated by both transcriptional and posttranscriptional events. Methods to determine these steady-state levels of RNAs, such as Northern analysis, ribonuclease protection assay (RPA), and quantitative real-time PCR, do not discriminate between regulation by de novo RNA synthesis and the influence by degradation or stabilization. To assess the rate of transcription of individual genes, run-on transcription is utilized. To this end, isolated chloroplasts are used in brief in vitro transcription reactions in the presence of radiolabeled nucleotides, with a subsequent hybridization of the isolated RNA with DNA fragments spotted on membranes. Here, we describe a... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106027 Mon, 08 Aug 2011 15:18:39 +0100 5106027 http://www.medworm.com/index.php?rid=5106026&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_10 Many areas of chloroplast research require methods that can assess the quality and quantity of chloroplast DNA (cpDNA). The study of chloroplast functions that depend on the proper maintenance and expression of the chloroplast genome, understanding cpDNA replication and repair, and the development of technologies for chloroplast transformation are just some of the disciplines that require the isolation of high-quality cpDNA. Arabidopsis thaliana offers several advantages for studying these processes because of the sizeable collection of mutants and natural varieties (accessions) available from stock centers and a broad community of researchers that has developed many other genetic resources. Several approaches for the isolation and quantification of cpDNA have been developed, but little co... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106026 Mon, 08 Aug 2011 15:18:39 +0100 5106026 http://www.medworm.com/index.php?rid=5106025&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_9 Protocols developed for plastome engineering in Nicotiana tabacum rely on biolistic delivery of the transforming DNA to chloroplasts in intact leaf tissue; integration of the foreign DNA into the plastid genome by homologous recombination via flanking plastid DNA (ptDNA) targeting regions; and gradual dilution of non-transformed ptDNA during cultivation in vitro. Plastid transformation in Arabidopsis was obtained by combining the tobacco leaf transformation protocol with Arabidopsis-specific tissue culture and plant regeneration protocols. Because the leaf cells in Arabidopsis are polyploid, this protocol yielded sterile plants. Meristematic cells in a shoot apex or cells of a developing embryo are diploid. Therefore, we developed a regulated embryogenic root culture system that will gener... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106025 Mon, 08 Aug 2011 15:18:38 +0100 5106025 http://www.medworm.com/index.php?rid=5106024&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_8 We describe a method for the analysis of Arabidopsis plant material by TEM, primarily for the assessment of plastid ultrastructure. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106024 Mon, 08 Aug 2011 15:18:38 +0100 5106024 http://www.medworm.com/index.php?rid=5106023&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_7 Amyloplasts, organelles responsible for the synthesis and storage of starch, are of critical importance to gravitropism in higher plants. We discuss two methods that are useful for describing the histology and behavior of amyloplasts. First, because mutants with little or no plastidic starch accumulation are defective in their gravitropic response, we review a method to observe starch accumulation quickly in plant tissue. Second, we discuss a method for measuring amyloplast sedimentation in the dynamic environment of Arabidopsis root columella cells, which is thought to provide a directional cue to a reoriented plant. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106023 Mon, 08 Aug 2011 15:18:38 +0100 5106023 http://www.medworm.com/index.php?rid=5106022&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_6 Chloroplast photorelocation movement is essential for the sessile plant survival and plays a role for efficient photosynthesis and avoiding photodamage of chloroplasts. There are several ways to observe or detect chloroplast movement directly or indirectly. Here, techniques for the induction of chloroplast movement and how to detect the responses, as well as various points of attention and advice for the experiments, are described. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106022 Mon, 08 Aug 2011 15:18:37 +0100 5106022 http://www.medworm.com/index.php?rid=5106021&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_5 Stromules are thin stroma-filled tubules that extend from all plastid types in all multicellular plants examined. They are most easily visualised by epifluorescence or confocal microscopy of plastids containing green fluorescent protein (GFP) or other fluorescent proteins. Transient expression of gene constructs encoding plastid-targeted GFP following bombardment of whole plants or organs of Arabidopsis with gold or tungsten particles coated with plasmid DNA is a relatively rapid and simple means of producing material for observation of stromules. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106021 Mon, 08 Aug 2011 15:18:37 +0100 5106021 http://www.medworm.com/index.php?rid=5106020&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_4 Although chloroplasts have their own genome, most chloroplast proteins are encoded in the nuclear genome and are targeted to chloroplasts posttranslationally. In vitro import studies with isolated chloroplasts have been widely used and have helped to elucidate the complex mechanisms involved in protein targeting to chloroplasts. Recently, an in vivo targeting method using protoplasts emerged as an alternative method to investigate protein targeting into chloroplasts. The present study describes a set of principles and methods, including polyethylene glycol-mediated reporter plasmid transformation, fluorescence microscopy, immunocytochemistry, and Western blotting, for studying chloroplast interior and envelope membrane protein targeting using protoplasts isolated from Arabidopsis thaliana ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106020 Mon, 08 Aug 2011 15:18:37 +0100 5106020 http://www.medworm.com/index.php?rid=5106019&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_3 Immunofluorescence microscopy reveals localization of proteins in cells and tissues by means of highly specific, fluorescently labeled antibodies. This technique is an important complement to localization methods that use genetically encoded fluorescent tags. This chapter describes the five stages of immunofluorescence localization of proteins in plant chloroplasts in sectioned leaf tissue: (1) fixation, (2) tissue embedding and sectioning, (3) treatment of sections prior to immunolabeling, (4) immunostaining, and (5) fluorescence microscopy and image capture. Protocols for both cryosectioning and sectioning of low-melting-point wax-embedded samples are described. Immunofluorescence localization in chloroplasts is complicated by their intense autofluorescence background. Measures to suppre... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106019 Mon, 08 Aug 2011 15:18:36 +0100 5106019 http://www.medworm.com/index.php?rid=5106018&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_2 Methods are described which allow one to observe chloroplasts in mesophyll cells from leaves of Arabidopsis, determine their number per cell, measure their area, and determine a value for chloroplast coverage inside mesophyll cells. Non-green plastids can also be imaged either by using staining, or by exploiting fluorescent proteins targeted to the plastid in non-green parts of the plant, such as the roots, in transgenic Arabidopsis. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106018 Mon, 08 Aug 2011 15:18:36 +0100 5106018 http://www.medworm.com/index.php?rid=5106017&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-234-2_1 The power of Arabidopsis as a model organism lies in the depth and breadth of genetic tools available for its study. This also applies to the study of chloroplast biology. Although vast numbers of mutants have been identified in Arabidopsis, the continued use of forward-genetic screening approaches remains valuable for the isolation and study of previously overlooked mutants and novel mutations in sensitised backgrounds (i.e., suppressors or enhancers of previously known mutants). In addition, reverse-genetic collections of insertional mutants are now extensive and provide unique opportunities for gene function discovery. Here, we describe methods for the chemical mutagenesis of Arabidopsis, the screening of mutants visually, on the basis of gene-expression phenotypes (scored as reduced or... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=5106017 Mon, 08 Aug 2011 15:18:35 +0100 5106017 http://www.medworm.com/index.php?rid=4991700&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_19 Unlike rational protein engineering, directed evolution provides an a priori approach toward the engineering of improved proteins and novel promoters. This minimally recursive technique builds upon small improvements by selecting and combining the best changes. Protein–protein/DNA interactions, catalytic efficiency, or resilience to inhibitors can be improved by thousands of times. By working within a subspace of homologous sequences, DNA shuffling recombines that subspace. Individuals are screened for a particular trait or two and selected for when they meet a set threshold. Here we explain basic principles to follow and provide procedures for the preparation, fragmentation, efficient size fractionation, and purification of parental material, as well as for the reassembly and rescue... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991700 Sat, 02 Jul 2011 14:08:51 +0100 4991700 http://www.medworm.com/index.php?rid=4991699&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_18 DamID (DNA adenine methylation identification) is an adenine methylation-based tagging method designed to map protein–DNA interactions in vivo. DamID, an alternative method to chromatin immunoprecipitation (ChIP), is based on the covalent linking of a “fingerprint” in the vicinity of the DNA-binding sites of the protein of interest. The fingerprints can be further mapped by simple molecular approaches. First developed by van Steensel’s group in Drosophila melanogaster (1), DamID was successfully adapted to Arabidopsis thaliana, and its feasibility demonstrated by using the well-known yeast GAL4 transcription factor (2). The method was further used to establish a genome-wide map of the target sites of LHP1, a regulatory chromatin protein in A. thaliana (3). (Source: ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991699 Sat, 02 Jul 2011 14:08:51 +0100 4991699 http://www.medworm.com/index.php?rid=4991698&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_17 Physical interactions between transcription factors and specific DNA sites are essential for gene regulation. Recent progress in genome-wide in vivo techniques, like chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-SEQ), enables plant researchers to generate genome-wide, high-resolution DNA-binding maps of transcription factors. These new types of data require the use of advanced bioinformatic tools in order to understand the molecular mechanisms of functional specificity and target gene regulation by transcription factors. Here, we will review the use of a genome browser to visualize genome-wide DNA-binding maps of plant transcription factors along with other publicly available data and the program MEME to determine DNA sequence motifs in the bound regions. We al... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991698 Sat, 02 Jul 2011 14:08:51 +0100 4991698 http://www.medworm.com/index.php?rid=4991697&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_16 Chromatin immunoprecipitation (ChIP) is a valuable tool to detect the interaction in vivo between a DNA-associated protein and DNA fragments. Combined with approaches to assess gene expression in response to accumulation of a transcription factor, it is possible to identify direct responsive targets from targets that are indirectly responsive to accumulation of the transcription factor. ChIP may be used to confirm in vivo association of a transcriptional regulator with suspected target DNA fragments. ChIP may also be used to discover new targets, and when combined with high-throughput approaches to identify DNA fragments associated with a transcription factor, it may provide a tool to study the gene regulatory networks active during plant development and/or response to the environment. Fur... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991697 Sat, 02 Jul 2011 14:08:51 +0100 4991697 http://www.medworm.com/index.php?rid=4991696&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_15 In the following chapter we describe methods and protocols to analyze the interaction of proteins with DNA using footprinting and related techniques based on the modification of DNA with either hydroxyl radicals or methylating agents. Footprinting, based on the protection from chemical modification of DNA through the specific binding of a protein, gives information about the nucleotides that are in close contact with the protein upon binding. The derived missing nucleoside and interference techniques identify nucleotides that are energetically important for protein binding. These methods are highly valuable to study in detail the interaction of a transcription factor with nucleotides on both strands of its target DNA sequence. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991696 Sat, 02 Jul 2011 14:08:50 +0100 4991696 http://www.medworm.com/index.php?rid=4991695&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_14 DNA-binding proteins, including transcription factors, play essential roles in many biological processes. The identification of the DNA sequences to which these proteins bind is a first, yet still challenging, step for determining their functions. SELEX provides an excellent tool for deciphering protein DNA-binding sequence specificity, and it has been widely adopted for addressing fundamental biological questions (1, 2). SELEX is an experimental procedure that involves the progressive selection, from a large combinatorial double-stranded oligonucleotide library, of DNA ligands with variable DNA-binding affinities and specificities by repeated rounds of partition and amplification. In this chapter, we describe a SELEX protocol that we have successfully applied to both plant and animal MYB ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991695 Sat, 02 Jul 2011 14:08:50 +0100 4991695 http://www.medworm.com/index.php?rid=4991694&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_9 Transcription factors are modular in nature in all organisms. In general, they have a DNA binding domain, one or more transcription activation and/or repressor domain, and often a dimerization domain. In many cases, transcription factors also have other protein–protein interaction domain(s). Mapping these functional domains in transcription factors is critical in understanding their molecular function. In this chapter, protocols for mapping the DNA binding domain and the transcription activation domain of a bHLH class of transcription factor are described. In principle, these protocols can be applied to other classes of transcription factors for mapping their functional domains. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991694 Sat, 02 Jul 2011 14:08:50 +0100 4991694 http://www.medworm.com/index.php?rid=4991693&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_8 Most transcription factors fulfill their role in protein complexes. As a consequence, information about their interaction capacity sheds light on a protein’s function and the molecular mechanism underlying this activity. The yeast two-hybrid GAL4 (Y2H) assay is a powerful method to unravel and identify the composition of protein complexes. This in vivo based system makes use of two functional protein domains of the GAL4 transcription factor, each fused to a protein of interest. Upon interaction between the two proteins under study, a transcriptional activator gets reconstituted and reporter genes get activated, allowing the yeast to grow on selective medium. In this chapter protocols are given for Y2H library screening, directed Y2H screening, Y2H matrix screening, and YnH screening ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991693 Sat, 02 Jul 2011 14:08:50 +0100 4991693 http://www.medworm.com/index.php?rid=4991692&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_13 Non-cell-autonomous (NCA) control of plant development is an emerging field. Transcription factors (TFs) are the most important plant proteins involved in development and cell fate determination. In plants specialized intercellular symplastic channels, called plasmodesmata (PD), facilitate and regulate the NCA action of TFs. NCA-TFs move from cell to cell either selectively or non-selectively depending upon the specific interactions with PD or the pathway proteins. Here we describe different approaches to establish the role of TFs in NCA control of its function and the characteristic movement behavior. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991692 Sat, 02 Jul 2011 14:08:50 +0100 4991692 http://www.medworm.com/index.php?rid=4991691&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_12 Similar to the activities of transcription factors (TFs) in other eukaryotes, activities of many plant TFs are determined via regulated proteolysis by the ubiquitin/26S proteasome system. Thus, to fully understand the function of a TF, it is important to determine the fate of the active TF protein and unravel the environmental and intrinsic signals that control its total cellular level. Here we describe how to determine whether a TF of interest is targeted to the 26S proteasome for degradation. The given method combines analyses of the effects of translational inhibition and the inhibition of proteasome activity. An important requirement for these experiments is to monitor in parallel the effects of translational and proteasomal inhibition on the abundance of the TF and (1) on ubiquitin, w... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991691 Sat, 02 Jul 2011 14:08:50 +0100 4991691 http://www.medworm.com/index.php?rid=4991690&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_11 Defining protein complexes is critical to virtually all aspects of cell biology because most cellular processes are regulated by stable or more dynamic protein interactions. Elucidation of the protein–protein interaction network around transcription factors is essential to fully understand their function and regulation. In the last decade, new technologies have emerged to study protein–protein interactions under near-physiological conditions. We have developed a high-throughput tandem affinity purification (TAP)/mass spectrometry (MS) platform for cell suspension cultures to analyze protein complexes in Arabidopsis thaliana. This streamlined platform follows an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of tra... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991690 Sat, 02 Jul 2011 14:08:49 +0100 4991690 http://www.medworm.com/index.php?rid=4991689&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_10 Protein–protein interactions are an important aspect of the gene regulation process. The expression of a gene in response to certain stimuli, within a specific cell type or at a particular developmental stage, involves a complex network of interactions between different regulatory proteins and the cis-regulatory elements present in the promoter of the gene. A number of methods have been developed to study protein–protein interactions in vitro and in vivo in plant cells, one of which is bimolecular fluorescence complementation (BiFC). BiFC is a relatively simple technique based upon the reconstitution of a fluorescent protein. The interacting protein complex can be visualized directly in a living plant cell when two non-fluorescent fragments, of an otherwise fluorescent protein,... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991689 Sat, 02 Jul 2011 14:08:49 +0100 4991689 http://www.medworm.com/index.php?rid=4991688&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_7 Microarray analysis is widely used to identify transcriptional changes associated with genetic perturbation or signaling events. Here we describe its application in the identification of plant transcription factor target genes with emphasis on the design of suitable DNA constructs for controlling TF activity, the experimental setup, the statistical analysis of the microarray data, and the validation of target genes. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991688 Sat, 02 Jul 2011 14:08:49 +0100 4991688 http://www.medworm.com/index.php?rid=4991687&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_6 Regulation of gene expression by transcription factors is a fundamental mechanism in essentially all aspects of cellular processes. Transient expression assay of a reporter plasmid containing a reporter gene driven by a promoter of interest and an effector plasmid expressing a transcription factor has been a powerful tool for analyzing transcription factors. Here we present a protocol for polyethylene glycol (PEG)-mediated transformation of Arabidopsis protoplasts. It details preparation of protoplasts from Arabidopsis suspension cultured cells or leaves of soil-grown Arabidopsis plants and subsequent PEG-mediated transformation with reporter and effector plasmids. This protocol can be completed within 24 h from protoplast preparation to reporter assay. As an example, analysis of the ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991687 Sat, 02 Jul 2011 14:08:49 +0100 4991687 http://www.medworm.com/index.php?rid=4991686&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_5 Chimeric REpressor gene Silencing Technology (CRES-T) is a useful tool for functional analysis of plant transcription factors. In this system, a chimeric repressor that is produced by fusion of a transcription factor to the plant-specific EAR-motif repression domain (SRDX) suppresses target genes of a transcription factor dominantly over the activity of endogenous and functionally redundant transcription factors. As a result, the transgenic plants that express a chimeric repressor exhibit phenotypes similar to loss-of-function of the alleles of the gene encoding the transcription factor. This system is simple and effective and can be used as a powerful tool not only for functional analysis of redundant transcription factors but also for the manipulation of plant traits by active suppressio... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991686 Sat, 02 Jul 2011 14:08:49 +0100 4991686 http://www.medworm.com/index.php?rid=4991685&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_4 Activation tagging is a powerful strategy to find new gene functions, especially from genes that are redundant or show lethal knock-out phenotypes. It has been applied using T-DNA or transposons. En/Spm-I/dSpm engineered transposons are efficient activation tags in Arabidopsis. An immobilized transposase source and an enhancer-bearing non-autonomous element are used in combination with positive and negative selectable markers to generate a population of single- or low-copy, stable insertions. This method describes the steps required for selection of parental lines, generation of a population of stable insertions, and gene identification. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991685 Sat, 02 Jul 2011 14:08:48 +0100 4991685 http://www.medworm.com/index.php?rid=4991684&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_3 The yeast one-hybrid (Y1H) system is a powerful tool for the identification and isolation of cDNAs of transcription factors using promoter segments or regulatory elements as baits. Here we propose an adaptation of the Y1H system for identification and cloning of transcription factors using Matchmaker (Clontech) Y2H cDNA libraries. The method is a modification of the standard one-hybrid screening protocol, utilising a mating step to introduce the library and reporter constructs into the same cell. This extends the compatibility of Matchmaker cDNA libraries from yeast two-hybrid screens to one-hybrid screens. Libraries were successfully prepared from wheat, barley and maize grain, spike, leaf and root tissues from plants subjected to several environmental stresses. Using this method, we have... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991684 Sat, 02 Jul 2011 14:08:48 +0100 4991684 http://www.medworm.com/index.php?rid=4991683&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_2 WRKY transcription factors are key regulators of numerous biological processes in plant growth and development, as well as plant responses to abiotic and biotic stresses. Research on biological functions of plant WRKY genes has focused in the past on model plant species or species with largely characterized transcriptomes. However, a variety of non-model plants, such as forest conifers, are essential as feed, biofuel, and wood or for sustainable ecosystems. Identification of WRKY genes in these non-model plants is equally important for understanding the evolutionary and function-adaptive processes of this transcription factor family. Because of limited genomic information, the rarity of regulatory gene mRNAs in transcriptomes, and the sequence divergence to model organism genes, identifica... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991683 Sat, 02 Jul 2011 14:08:48 +0100 4991683 http://www.medworm.com/index.php?rid=4991682&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-154-3_1 All major processes of life depend on differential gene expression, which is largely controlled by the activity of transcription factors (TFs). In plants many TFs are encoded by members of multigene families that expanded much more dramatically during land plant evolution than during the evolution of animals and fungi. Here we review typical features such as domain structure, DNA binding, and protein interactions of TFs from some families that have contributed to the development and evolution of plant-specific structures in especially important ways. Our survey includes the MADS-domain protein family involved in specifying meristem and organ identity; YABBY proteins controlling lamina outgrowth; TCP proteins controlling floral zygomorphy and apical dominance; and finally homeodomain protei... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4991682 Sat, 02 Jul 2011 14:08:48 +0100 4991682 http://www.medworm.com/index.php?rid=4682741&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_1 Plant tissue cultures are an efficient system to study cell wall biosynthesis in living cells in vivo. Tissue cultures also provide cells and culture medium where enzymes and cell wall polymers can easily be separated for further studies. Tissue cultures with tracheary element differentiation or extracellular lignin formation have provided useful information related to several aspects of xylem and lignin formation. In this chapter, methods for nutrient medium preparation, callus culture initiation, and its maintenance, as well as those for protoplast isolation and viability observation, are described. As a case study, we describe the establishment of a xylogenic culture of Zinnia elegans mesophyll cells. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682741 Tue, 29 Mar 2011 23:00:00 +0100 4682741 http://www.medworm.com/index.php?rid=4682740&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_20 This article summarizes the theory and practical aspects of measuring cell wall properties by four different extensometer techniques and how the results of these methods relate to the concept and ideal measurement of cell wall extensibility in the context of cell growth. These in vivo techniques are particularly useful for studies of the molecular basis of cell wall extension. Measurements of breaking strength, elastic compliance, and plastic compliance may be informative about changes in cell wall structure, whereas measurements of wall stress relaxation and creep are sensitive to both changes in wall structure and wall-loosening processes, such as those mediated by expansins and some lytic enzymes. A combination of methods is needed to obtain a broader view of cell wall behavior and prop... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682740 Tue, 29 Mar 2011 23:00:00 +0100 4682740 http://www.medworm.com/index.php?rid=4682739&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_2 Computerized molecular modeling continues to increase in capability and applicability to carbohydrates. This chapter covers nomenclature and conformational aspects of carbohydrates, perhaps of greater use to carbohydrate-inexperienced computational chemists. Its comments on various methods and studies might be of more use to computation-inexperienced carbohydrate chemists. New work on intrinsic variability of glucose, an overall theme, is described. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682739 Tue, 29 Mar 2011 23:00:00 +0100 4682739 http://www.medworm.com/index.php?rid=4682738&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_3 In today’s field of plant cell wall research, insights into the structure of wall components are obtained using many different techniques, ranging from spectroscopic and microscopic to chemical and biochemical. In this chapter, we describe one method: oligosaccharide mass profiling (OLIMP). Using OLIMP, we can harness the selective power of a specific wall hydrolase together with the speed and sensitivity of mass spectrometry to provide highly reproducible structural and compositional information about the wall molecule of interest. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682738 Tue, 29 Mar 2011 23:00:00 +0100 4682738 http://www.medworm.com/index.php?rid=4682737&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_4 HVPE is an excellent and often overlooked method for obtaining objective and meaningful information about cell-wall “building blocks” and their metabolic precursors. It provides not only a means of analysis of known compounds but also an insight into the charge and/or mass of any unfamiliar compounds that may be encountered. It can be used preparatively or analytically. It can achieve either “class separations” (e.g. delivering all hexose monophosphates into a single pool) or the resolution of different compounds within a given class (e.g. ADP-Glc from UDP-Glc; or GlcA from GalA). All information from HVPE about charge and mass can be obtained on minute traces of analytes, especially those that have been radiolabelled, e.g. by in-vivo feeding of a 3H- or 14C-labelle... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682737 Tue, 29 Mar 2011 23:00:00 +0100 4682737 http://www.medworm.com/index.php?rid=4682736&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_6 The importance of capillary zone electrophoresis (CZE) has been increasing in use for: structural analysis of plant cell walls, characterization of enzymes that degrade polysaccharides, and profiling of oligosaccharides to characterize cell wall mutants. CZE with laser-induced fluorescence detection provides high separation efficiencies, high speed analysis, with extremely small sample requirements. Here, we describe the instrumentation we use and the methods for attaching fluorescent labels to oligosaccharides so that they can be detected. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682736 Tue, 29 Mar 2011 23:00:00 +0100 4682736 http://www.medworm.com/index.php?rid=4682735&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_5 Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) relies on derivatization of the reducing ends of sugars with a fluorophore, followed by electrophoresis under optimized conditions in polyacrylamide gels. PACE is a sensitive and simple tool for studying polysaccharide structure or quantity and also has applications in the investigation of enzyme specificity. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682735 Tue, 29 Mar 2011 23:00:00 +0100 4682735 http://www.medworm.com/index.php?rid=4682734&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_8 Plant cells are surrounded by cell walls built largely from complex carbohydrates. The primary walls of growing plant cells consist of interdependent networks of three polysaccharide classes: cellulose, cross-linking glycans (also known as hemicelluloses), and pectins. Cellulose microfibrils are tethered together by cross-linking glycans, and this assembly forms the major load-bearing component of primary walls, which is infiltrated with pectic polymers. In the secondary walls of woody tissues, pectins are much reduced and walls are reinforced with the phenolic polymer lignin. Plant cell walls are essential for plant life and also have numerous industrial applications, ranging from wood to nutraceuticals. Enhancing our knowledge of cell wall biology and the effective use of cell wall mater... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682734 Tue, 29 Mar 2011 23:00:00 +0100 4682734 http://www.medworm.com/index.php?rid=4682733&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_7 Plant cell walls are diverse composites of complex polysaccharides. Molecular probes such as monoclonal antibodies (MABs) and carbohydrate-binding modules (CBMs) are important tools to detect and dissect cell wall structures in plant materials. We provide an account of methods that can be used to detect cell wall polysaccharide structures (epitopes) in plant materials and also describe treatments that can provide information on the masking of sets of polysaccharides that may prevent detection. These masking ­phenomena may indicate potential interactions between sets of cell wall polysaccharides, and methods to uncover them are an important aspect of cell wall immunocytochemistry. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682733 Tue, 29 Mar 2011 23:00:00 +0100 4682733 http://www.medworm.com/index.php?rid=4682732&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_9 High-resolution imaging of the membranous intermediates and cytoskeletal arrays involved in the assembly of a new cell wall during plant cytokinesis requires state-of-the-art electron microscopy techniques. The combination of cryofixation/freeze-substitution methods with electron tomography (ET) has revealed amazing structural details of this unique cellular process. This chapter deals with the main steps associated with these imaging techniques: selection of samples suitable for studying plant cytokinesis, sample preparation by high-pressure freezing/freeze substitution, and ET of plastic sections. In addition, immunogold approaches for the identification of proteins and polysaccharides during cell wall assembly are discussed. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682732 Tue, 29 Mar 2011 23:00:00 +0100 4682732 http://www.medworm.com/index.php?rid=4682731&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_11 Biosynthesis of pectin and hemicelluloses occurs in the Golgi apparatus and is thought to involve spatial regulations and complex formation of biosynthetic enzymes and proteins. We have demonstrated that a combination of heterologous expression of recombinant proteins tagged with fluorescent proteins and live cell imaging with confocal laser scanning microscopy (CLSM) allows efficient visualization of biosynthetic enzymes and proteins in subcellular compartments. We have also successfully utilized bimolecular fluorescence complementation (BiFC) for in situ visualization of protein–protein interactions of pectin biosynthetic enzymes and for the determination of their membrane topology in the Golgi apparatus. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682731 Tue, 29 Mar 2011 23:00:00 +0100 4682731 http://www.medworm.com/index.php?rid=4682730&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_10 Plant cells are delimited by a rigid cell wall that resists internal turgor pressure, but extends with a remarkable degree of control that allows the cell to grow and acquire specific shapes. Live cell fluorescence microscopy systems have allowed an amazing view into the complex and dynamic lives of individual proteins during cell morphogenesis. The current chapter will focus on methodology for live cell imaging of cellulose synthase (CESA) in Arabidopsis, which will also provide a launching pad to explore ones specific protein of interest. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682730 Tue, 29 Mar 2011 23:00:00 +0100 4682730 http://www.medworm.com/index.php?rid=4682729&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_12 Atomic force microscopy (AFM) can be used to obtain high-resolution images on a wide variety of materials. Unfortunately, plant cell wall material is typically too rough to be imaged as native tissue by AFM. Small tissue fragments can be produced through careful ball milling. These fragments can subsequently be imaged at high resolution in near native conditions showing the overall architecture and the arrangement of the individual cellulose fibrils. An overview of items that can cause practical difficulties is given, as is a description of common image artifacts. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682729 Tue, 29 Mar 2011 23:00:00 +0100 4682729 http://www.medworm.com/index.php?rid=4682728&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_13 Studies of the mobilities of polysaccharides or parts of polysaccharides in a cell-wall preparation may give clues about the molecular interactions among the polysaccharides in the cell wall and the relative locations of polysaccharides within the cell wall. A number of solid-state 13C NMR techniques have been developed that can be used to investigate different types of polysaccharide mobilities: rigid, semi-rigid, mobile, and highly mobile. In this chapter, techniques are described for obtaining spectra from primary cell-wall preparations using CP/MAS, proton-rotating frame, proton spin-spin, spin-echo relaxation spectra, and single-pulse excitation. We also describe how proton spin relaxation editing can be used to obtain subspectra for cell-wall polysaccharides of different mobilities. ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682728 Tue, 29 Mar 2011 23:00:00 +0100 4682728 http://www.medworm.com/index.php?rid=4682727&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_14 Gluconacetobacter xylinus synthesises cellulose in an analogous fashion to plants. Through fermentation of Ga. xylinus in media containing cell wall polysaccharides from the hemicellulose and/or pectin families, composites with cellulose can be produced. These serve as general models for the assembly, structure, and properties of plant cell walls. By studying structure/property relationships of cellulose composites, the effects of defined hemicellulose and/or pectin polysaccharide structures can be investigated. The macroscopic nature of the composites also allows composite mechanical properties to be characterised. The method for producing cellulose-based composites involves reviving and then culturing Ga. xylinus in the presence of desired hemicelluloses and/or pectins. Different conditi... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682727 Tue, 29 Mar 2011 23:00:00 +0100 4682727 http://www.medworm.com/index.php?rid=4682726&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_15 Structural proteins of the primary cell wall present unusual but interesting problems for structural biologists in particular and plant biologists in general. As structure is the key to function; then the biochemical isolation of these glycoproteins for further study is paramount. Here, we detail the “classical” method for isolating soluble extensin monomers by elution of monomeric precursors to network extensin from tissue cultures. We also outline an additional approach involving genetic engineering that can potentially yield the complete genomic range of extensins and other hydroxyproline-rich glycoprotein (HRGPs) currently underutilized for biotechnology. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682726 Tue, 29 Mar 2011 23:00:00 +0100 4682726 http://www.medworm.com/index.php?rid=4682725&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_16 Undoubtedly, the function of the plant cell wall in the control of cell growth far exceeds its mechanical role. The plant’s monitoring of cell wall function and integrity comprises a central checkpoint to integrate cues for survival and division, expansion and differentiation, as well as fluctuations in the biotic and abiotic environment (Somerville et al., Science 306:2206–2211, 2004). With their biochemical nature yet unknown, the identification of molecular constituents of cell wall performance, and integrity control initially depends on a combination of genetic and physiological approaches. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682725 Tue, 29 Mar 2011 23:00:00 +0100 4682725 http://www.medworm.com/index.php?rid=4682724&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_17 Arabinogalactan proteins are a diverse group of plant cell wall-associated proteoglycans. While structural and molecular genetic analyses have contributed to the emerging improved understanding of the wide-range of biological processes in which AGPs are implicated; the ability to detect, localise, and quantify them is fundamentally important. This chapter describes two commonly used methods, histological staining and radial gel diffusion, both of which utilise the ability of Yariv reagent to bind to AGPs. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682724 Tue, 29 Mar 2011 23:00:00 +0100 4682724 http://www.medworm.com/index.php?rid=4682723&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_18 Plant cell wall proteins play essential roles in many important biological processes, and yet there is still not a comprehensive catalogue of the cell wall proteome, or “secretome”. Here, we describe three procedures, including a yeast secretion trap (YST), Agrobacterium-mediated transient expression using a necrosis-inducing protein (NIP) and protein localization assay using a fluorescent protein, to identify and confirm the localization of cell wall proteins. The approaches are orthogonal and collectively provide a powerful suite of approaches to complement more commonly used strategies to isolate plant cell wall-associated proteins. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682723 Tue, 29 Mar 2011 23:00:00 +0100 4682723 http://www.medworm.com/index.php?rid=4682722&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-008-9_19 The moss Physcomitrella patens has become established as a model for investigating plant gene function due to the feasibility of gene targeting. The chemical composition of the P. patens cell wall is similar to that of vascular plants and phylogenetic analyses of glycosyltransferase sequences from the P. patens genome have identified genes that putatively encode cell wall biosynthetic enzymes, providing a basis for investigating the evolution of cell wall polysaccharides and the enzymes that synthesize them. The protocols described in this chapter provide methods for targeted gene knockout in P. patens, from constructing vectors and maintaining cultures to transforming protoplasts and analyzing the genotypes and phenotypes of the resulting transformed lines. (Source: Springer protocols fee... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4682722 Tue, 29 Mar 2011 23:00:00 +0100 4682722 http://www.medworm.com/index.php?rid=4605193&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_10 Redundancy between Pseudomonas syringae pv. tomato DC3000 virulence factors has made their characterization difficult. One method to circumvent redundancy for phenotypic characterization is to simultaneously delete all redundant factors through the generation of polymutant strains. Described here are methods by which single and polymutant strains of DC3000 can be generated through the use of the small mobilizable sucrose counter-selection vector pK18mobsacB, FRT-flanked antibiotic marker cassettes, and Flp recombination. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605193 Fri, 18 Mar 2011 03:34:44 +0100 4605193 http://www.medworm.com/index.php?rid=4605192&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_11 Genome sequencing combined with high-throughput functional analyses has proved vital in our quest to understand oomycete–plant interactions. With the identification of effector molecules from Phytophthora spp. we can now embark on dissecting the mechanisms by which effectors modulate host processes and thus ensure parasite fitness. One of the key limitations, however, is to genetically modify Phytophthora and assess gene function during parasitism. Here, we describe a straightforward protocol that allows rapid transformation of Phytophthora capsici, an emerging model in oomycete biology. P. capsici is a broad host range pathogen that can infect a wide variety of plants under lab conditions making it a suitable model for detailed studies on oomycete–host interactions. This proto... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605192 Fri, 18 Mar 2011 03:34:44 +0100 4605192 http://www.medworm.com/index.php?rid=4605191&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_13 The biolistic transient gene expression assay is a beneficial tool for studying gene function in vivo. However, biolistic transient assay systems have inherent pitfalls that often cause experimental inaccuracies such as poor transformation efficiency, which can be confused with biological phenomena. The double-barreled gene gun device is an inexpensive and highly effective attachment that enables statistically significant data to be obtained with one-tenth the number of experimental replicates compared to conventional biolistic assays. The principle behind the attachment is to perform two simultaneous bombardments with control and test DNA preparations onto the same leaf. The control bombardment measures the efficiency of the transformation while the ratio of the test bombardment to the co... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605191 Fri, 18 Mar 2011 03:34:43 +0100 4605191 http://www.medworm.com/index.php?rid=4605190&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_12 The oomycete pathogen Hyaloperonospora arabidopsidis is a natural pathogen of Arabidopsis thaliana and a laboratory model for (1) understanding how Arabidopsis responds to pathogen attack; (2) comparative and functional genomics of oomycetes; and (3) the molecular basis and evolution of obligate biotrophy. Here, we describe procedures for propagation and long-term storage of H. arabidopsidis, which address complications arising from its biotrophic lifestyle that precludes growth on synthetic media. We also describe four assays that provide information on different facets of the H. arabidopsidis–Arabidopsis interaction. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605190 Fri, 18 Mar 2011 03:34:43 +0100 4605190 http://www.medworm.com/index.php?rid=4605189&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_14 Pathogens such as bacteria, fungi, and oomycetes deliver diverse arrays of virulence or avirulence molecules, defined here as effectors, into the host cells. Effectors enable parasitic colonization by manipulating classes of biochemical, physiological, and morphological processes. An effective strategy to modulate host defense circuitry is to suppress their programmed cell death (PCD) response. Here, we describe a method for analyzing whether effectors function to suppress PCD in yeast. We use Bax and H2O2 to induce cell death and mimic some PCD features that naturally appear during the development of multicellular Saccharomyces cerevisiae colonies and assay whether plant pathogen effectors can inhibit the process. This technology provides an assay to test whether individual effectors can ... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605189 Fri, 18 Mar 2011 03:34:43 +0100 4605189 http://www.medworm.com/index.php?rid=4605188&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_15 This article describes a co-immunoprecipitation protocol aimed at identifying putative target complexes of the effectors by transiently overexpressing the tagged effectors in planta. The identification of the eluted protein complexes was achieved by LC-MS/MS mass spectrometry and peptide spectrum matching. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605188 Fri, 18 Mar 2011 03:34:43 +0100 4605188 http://www.medworm.com/index.php?rid=4605187&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_16 Assays to determine the role of pathogen effectors within an infected plant cell are yielding valuable information about which host processes are targeted to allow successful pathogen colonization. However, this does not necessarily inform on the cellular location of these interactions, or if these effector–virulence target interactions occur only in the presence of the pathogen. Here, we describe techniques to allow the subcellular localization of pathogen effectors inside infected plant cells or tissues, based largely on infiltration of plant tissue by Agrobacterium tumefaciens and its delivery of DNA encoding fluorescent protein-tagged effectors, and subsequent confocal microscopy. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605187 Fri, 18 Mar 2011 03:34:43 +0100 4605187 http://www.medworm.com/index.php?rid=4605186&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_17 The use of polyclonal antibodies enables the detection of proteins on a cellular and even subcellular level. Immunolocalization can be used on all pathosystems even if one or both partners of the interaction are unamenable to molecular tools like transformation. This chapter provides detailed information about how to obtain high quality antibodies, how to prepare samples, and finally how to detect the proteins. Methods for light and electron microscopy are presented. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605186 Fri, 18 Mar 2011 03:34:43 +0100 4605186 http://www.medworm.com/index.php?rid=4605185&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_18 Obligate plant-parasitic nematodes, such as cyst nematodes (Heterodera and Globodera spp.) and root-knot nematodes (Meloidogyne spp.), form specialized feeding cells in host plant roots. These feeding cells provide the sole source of nutrition for the growth and reproduction of the nematode to complete its life cycle. Feeding cell formation involves complex physiological and morphological changes to normal root cells and is accompanied by dramatic changes in plant gene expression. The distinct features of feeding cells suggest that their formation entails a unique gene expression profile, a better understanding of which will assist in building models to explain signaling pathways that modulate transcriptional changes in response to nematodes. Ultimately, this knowledge can be used to desig... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605185 Fri, 18 Mar 2011 03:34:42 +0100 4605185 http://www.medworm.com/index.php?rid=4605184&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_19 The molecular mechanisms that mediate the intimate interaction of an adapted obligate biotroph, such as the powdery mildew Golovinomyces orontii, on its host plant are spatially and temporally distinct. As G. orontii exclusively infects epidermal cells with a dominant host response occurring in the underlying mesophyll cells, we sought to develop a method to accurately and reproducibly perform global expression profiling on Arabidopsis thaliana leaf epidermal and mesophyll cells at the site of infection. Specific stages of G. orontii disease progression on Arabidopsis are visible by microscopy thus allowing distinct phases of the interaction to be studied. Tissue preparation, laser microdissection, and RNA isolation protocols that allow for temporally and spatially defined global expressio... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605184 Fri, 18 Mar 2011 03:34:42 +0100 4605184 http://www.medworm.com/index.php?rid=4605183&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_1 NB-LRR immune receptors in plants play dual roles as sentries and as activators of defense. The site in the cell where these activities take place can be different for different NB-LRRs. Furthermore, recognition and defense activation can occur in distinct subcellular compartments. Therefore, determining the subcellular localization of NB-LRRs is a key step toward understanding how they function. Recent advances in confocal microscopy enable high-resolution imaging of proteins in live cells. Agroinfiltration in the Nicotiana benthamiana model plant system is a convenient way of expressing proteins for localization studies. This chapter explains how to use N. benthamiana to transiently express NB-LRRs for confocal fluorescence microscopy. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605183 Fri, 18 Mar 2011 03:34:42 +0100 4605183 http://www.medworm.com/index.php?rid=4605182&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_20 Global expression profiling of RNA isolated from laser microdissected cells allows one to profile a specific set of cells allowing for enhanced sensitivity and for cell- or site-specific patterns of expression to emerge. In Chapter 19, we detail our optimized methods of tissue preparation, laser microdissection (LMD), and RNA isolation of cells at the site of Golovinomyces orontii infection of mature Arabidopsis leaves. Here, we describe (1) amplification of the RNA to obtain sufficient starting material for microarray analysis, (2) microarray hybridization and associated quality control assessments. As tissue preparation, LMD, and/or RNA amplification could impact mRNA quality, distribution, and/or microarray processing and output, it is important to include quality control assessments at... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605182 Fri, 18 Mar 2011 03:34:42 +0100 4605182 http://www.medworm.com/index.php?rid=4605181&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_21 Interactions between plant cells and microbial pathogens involve highly dynamic processes of cellular trafficking and reorganization. Substantial advances in imaging technologies, including the discovery and widespread use of fluorescent proteins as tags as well as advances in laser-based confocal microscopy have provided the first glimpses of the dynamic nature of the processes of defense and pathogenicity. Prior to the development of these techniques, high resolution imaging by electron microscopy gave only a static picture of these dynamic events and live cell imaging was significantly limited in resolution as well as the availability of relevant stains and markers. The incorporation of fluorescent protein fusions and laser-based confocal microscopy into studies of plant–microbe i... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605181 Fri, 18 Mar 2011 03:34:41 +0100 4605181 http://www.medworm.com/index.php?rid=4605180&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_2 Plant disease resistance (R) proteins confer protection against specific pathogens or pathogen isolates. R proteins function by recognizing pathogen-encoded avirulence (Avr) proteins and translating this recognition event into an initiation of downstream signaling pathways. Key to understanding this process is the study of the protein–protein interactions involving R proteins. Recognition and signaling mechanisms are mediated by both intramolecular interactions that take place between different domains of R proteins as well as intermolecular interactions between R proteins and additional plant proteins. These processes have been studied in part by using Agrobacterium-mediated transient expression of R protein fragments in Nicotiana benthamiana which allows for the rapid assessment of... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605180 Fri, 18 Mar 2011 03:34:41 +0100 4605180 http://www.medworm.com/index.php?rid=4605179&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_4 Protein complex purification represents a powerful approach to identify novel players in plant innate immunity. However, the identification of interacting protein partners within a natural context has been a challenge for researchers. In this chapter, we describe a method of immunoaffinity chromatography using purified, antibodies to isolate native protein complexes from wild-type tissue. We detail the antibody purification and immobilization steps in addition to the co-immunoprecipitation protocol. In addition, a method to prepare protein samples for mass spectroscopy analysis is described. This straightforward protocol has been used to isolate and identify novel components of Arabidopsis immunity-associated protein complexes. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605179 Fri, 18 Mar 2011 03:34:41 +0100 4605179 http://www.medworm.com/index.php?rid=4605178&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_3 Plant disease resistance (R) proteins confer strong resistance against pathogens by recognizing particular pathogen effectors. Identification of proteins associated with an R protein will provide insight into the mechanism of R protein function. Many R proteins are associated with the plasma membrane (PM) and expressed at low levels. Here, we describe a method to purify such low-abundance PM R protein ­complexes from Arabidopsis using a biotinylated affinity tag, called the HPB tag. We have successfully applied this method to identify candidate components of the RPS2 resistance protein complex(es). This method should also be applicable to purification of other low-abundance PM protein complexes. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605178 Fri, 18 Mar 2011 03:34:41 +0100 4605178 http://www.medworm.com/index.php?rid=4605177&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_5 The completion of the alfalfa, Arabidopsis, papaya, poplar, and rice genome sequences along with ongoing sequencing projects of various crop species, offers an excellent opportunity to study gene expression at the whole genome level and to unravel the complexity of gene networks underlying the reprogramming of plant defense toward pathogen challenge. Gene expression in eukaryotic cells is mainly controlled by regulatory elements that recruit transcription factors (TFs) to modulate transcriptional outputs. Therefore, methods allowing the identification of all cognate TF binding sites (TFBS) within the regulatory regions of target genes on a genome-wide basis are the next obvious step to elucidate the plant defense transcriptome. Chromatin immunoprecipitation (ChIP) is one such powerful tech... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605177 Fri, 18 Mar 2011 03:34:41 +0100 4605177 http://www.medworm.com/index.php?rid=4605176&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_7 Alternative splicing (AS) significantly contributes to transcriptome and proteome complexity. Transcriptome-wide studies concluded that approximately 22% of Arabidopsis and rice genes are subject to AS. Despite increasing recognition of AS in plants, little is known about the function of individual products of AS. In our studies of the Arabidopsis RPS4 resistance gene, which requires AS transcripts for function, the need to quantify AS transcripts became apparent. Because RPS4 expression levels are very low and the pattern of RPS4 splicing is complex, existing mRNA quantification methods were not adequate. We therefore developed a new method based on reverse transcription (RT) PCR amplification of all transcript variants with a common set of primers and separation of the PCR products by si... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605176 Fri, 18 Mar 2011 03:34:40 +0100 4605176 http://www.medworm.com/index.php?rid=4605175&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_6 Construction of transgenic plants is central to modern plant molecular genetics. Inducible systems permit spatial and temporal control of transgene expression. One commonly used inducible system relies on the use of dexamethasone to activate an endogenously expressed hybrid transcription factor, which positively regulates the expression of the gene of interest (Aoyama and Chua, Plant J 11:605–612, 1997). We have developed Arabidopsis plants using this inducible system to drive expression of a bacterial type III effector protein. The effector, AvrRpm1, elicits either strong cell death or weak cell death and chlorosis depending on the genetic background of the plant. Using these reagents, we examine several properties of the inducible system in Arabidopsis, including the timing of indu... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605175 Fri, 18 Mar 2011 03:34:40 +0100 4605175 http://www.medworm.com/index.php?rid=4605174&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_8 The haustorium is a distinguishing feature of biotrophic plant pathogens. Several highly diverged ­pathogen classes have independently evolved haustoria, suggesting that they represent an effective adaptation for growing within living plant tissue. Despite their clear importance in biotrophy, they have been difficult to study due to the close association of biotrophic pathogens with their host and the inability to produce haustoria in vitro. These drawbacks have been circumvented in the study of rust fungi by the development of a haustoria isolation technique. The strong binding of the lectin concanavalin A (ConA) to rust haustoria allows these structures to be purified from infected plant tissue by affinity chromatography on a ConA–Sepharose macrobead column. The isolation proce... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605174 Fri, 18 Mar 2011 03:34:39 +0100 4605174 http://www.medworm.com/index.php?rid=4605173&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61737-998-7_9 Identifying parasitism genes encoding proteins secreted from a plant-parasitic nematode’s esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Parasitism genes have been cloned by directly microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages of cyst or root-knot nematodes to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. cDNA clones are sequenced and deduced protein sequences with a signal peptide for secretion are identified for high-throughput in situ hybridization to confirm gland-specific expression. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=4605173 Fri, 18 Mar 2011 03:34:39 +0100 4605173 http://www.medworm.com/index.php?rid=3382161&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_9 In eukaryotes, RNA silencing encompasses a range of biochemical processes mediated by ∼20–25 nt small RNAs (smRNAs). This chapter describes northern blot hybridization techniques optimized for detection of such smRNAs, whether extracted from plant or animal tissues. The basic protocol is described, and control blots illustrate the detection specificity and sensitivity of this method using DNA oligonucleotide probes. Known endogenous smRNAs are analyzed in samples prepared from several model plant species, including Arabidopsis thaliana, Nicotiana benthamiana, Oryza sativa, Zea mays, and Physcomitrella patens, as well as the animals Drosophila melanogaster and Mus musculus. Finally, the usefulness of northern blotting in dissecting smRNA biogenesis is shown for the particular case... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=3382161 Fri, 19 Mar 2010 17:20:06 +0100 3382161 http://www.medworm.com/index.php?rid=3382160&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_8 Small RNA (sRNA) populations extracted from Arabidopsis plants submitted or not to biotic stress, were reverse-transcribed into cDNAs, and these were subsequently hybridized after labelling to a custom-made DNA tiling array covering Arabidopsis chromosome 4. We first designed a control experiment with eight cDNA clones corresponding to sequences located on chromosome 4 and obtained robust and specific hybridization signals. Furthermore, hybridization signals along chromosome 4 were in good agreement with sRNA abundance as previously determined by Massive Parallel Sequence Signature (MPSS) in the case of untreated plants, but differed substantially after stress treatment. These results demonstrate the utility of hybridization to DNA tiling arrays to detect major changes in small RNA populat... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=3382160 Fri, 19 Mar 2010 17:20:06 +0100 3382160 http://www.medworm.com/index.php?rid=3382159&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_7 Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of anonymous CCGG sequences in different fungi, plant and animal species. The main variation of this technique is based on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent cutter restriction enzyme. For each sample, AFLP analysis is performed using both EcoRI/HpaII and EcoRI/MspI digested samples. Comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) “Methylation-insensiti... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=3382159 Fri, 19 Mar 2010 17:20:06 +0100 3382159 http://www.medworm.com/index.php?rid=3382158&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_6 Restriction fragment length polymorphism (RFLP) is a difference in DNA sequences of organisms belonging to the same species. RFLPs are typically detected as DNA fragments of different lengths after digestion with various restriction endonucleases. The comparison of RFLPs allows investigators to analyze the frequency of occurrence of mutations, such as point mutations, deletions, insertions, and gross chromosomal rearrangements, in the progeny of stressed plants. The assay involves restriction enzyme digestion of DNA followed by hybridization of digested DNA using a radioactively or enzymatically labeled probe. Since DNA can be digested with methylation sensitive enzymes, the assay can also be used to analyze a methylation pattern of a particular locus. Here, we describe RFLP analysis using... Springer protocols feed by Plant Sciences news http://www.medworm.com/rss/comments.php?id=3382158 Fri, 19 Mar 2010 17:20:06 +0100 3382158