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. Sat, 20 Mar 2010 16:04:47 +0100 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 info 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 info 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 info 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 info http://www.medworm.com/rss/comments.php?id=3382158 Fri, 19 Mar 2010 17:20:06 +0100 3382158 http://www.medworm.com/index.php?rid=3382157&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_5 Epigenetic changes in the plant genome are associated with differential genome methylation, histone modifications, and the binding of various chromatin-binding factors. Methylation of cytosine residues is one of the most versatile mechanisms of epigenetic regulation. The analysis of DNA methylation can be performed in different ways. However, most of these procedures involve the extraction of chromatin from cells with further isolation and analysis of DNA. Modest success has been achieved in DNA methylation analysis in plant tissues in situ. Here, we present an in situ method for DNA methylation analysis, which has high sensitivity and good reproducibility. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382157 Fri, 19 Mar 2010 17:20:06 +0100 3382157 http://www.medworm.com/index.php?rid=3382156&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_4 Methylation is a reversible covalent chemical modification of DNA intended to regulate gene expression, genome stability, and chromatin structure. Although there are various methods of methylation analysis, most of them are either laborious or expensive, or both. Here, we describe a quick, inexpensive method for analysis of global genome methylation using a cytosine extension assay. The assay can be used for analysis of the total level of CpG, CNpG, and asymmetrical methylation in a given cell culture or in a plant tissue sample. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382156 Fri, 19 Mar 2010 17:20:06 +0100 3382156 http://www.medworm.com/index.php?rid=3382155&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_3 DNA methylation is a major mechanism for the reversible control of gene expression, chromatin structure, and genome stability. Methylation analysis at a given locus allows one to evaluate levels of chromatin packaging, gene expression, and even homologous recombination. We have shown that the combined bisulfite restriction analysis (COBRA) assay makes it possible to analyze methylation levels at a defined locus. The major steps are: bisulfite conversion of nonmethylate cytosines to uracils, locus-specific PCR amplification of converted DNA, restriction digestion, and analysis of restriction patterns on the gel. Due to the availability of various restriction enzymes that have cytosines in the restriction recognition sequence, the assay allows analysis of various cytosines, including those p... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382155 Fri, 19 Mar 2010 17:20:06 +0100 3382155 http://www.medworm.com/index.php?rid=3382154&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_2 Amplifying and sequencing DNA after bisulfite treatment of genomic DNA reveals the methylation state of cytosine residues at the highest resolution possible. However, a thorough analysis is required for statistical evaluation of methylation at all sites in each genomic region. Several software tools were developed to assist in quantitative evaluation of bisulfite sequencing data from complex methylation patterns occurring in plants. This chapter describes the application of Cytosine Methylation Analysis Tool for Everyone (CyMATE). From aligned sequences, CyMATE quantifies and illustrates general and pattern-specific methylation at CG, CHG, and CHH (H = A, C, or T) sites, both per sequence and per position. CyMATE is also able to perform a quality control of sequences and to detect redundan... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382154 Fri, 19 Mar 2010 17:20:06 +0100 3382154 http://www.medworm.com/index.php?rid=3382153&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_1 We describe the application of bisulfite sequencing for the analysis of DNA methylation at defined individual sequences of plant genomic DNA. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382153 Fri, 19 Mar 2010 17:20:06 +0100 3382153 http://www.medworm.com/index.php?rid=3382152&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_19 Epigenetic effects such as gene silencing and variable expression are unintended consequences of plant transformation, a problem that is present in the transformation of all plant species. There is not yet a reliable way to prevent epigenetic silencing; however, the probability of epigenetic effects may be reduced by choosing an appropriate method of transgene introduction into a plant cell. Most methods used in plant biotechnology, such as direct gene transfer and particle bombardment, result in the introduction of multiple DNA molecules and, as a consequence, multi-copy multi-locus insertion patterns. These multiple insertions may lead to variations in transgene expression, epigenetic silencing being the most extreme. In contrast, Agrobacterium-mediated plant transformation procedures ra... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382152 Fri, 19 Mar 2010 17:20:06 +0100 3382152 http://www.medworm.com/index.php?rid=3382151&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_18 Homologous recombination is a double-strand break repair mechanism operating in somatic cells and involved in meiotic crossovers in plants. It is responsible for the maintenance of genome stability and thus plays a crucial role in adaptation to stress. Recombination between homologous loci is believed to be regulated in part by epigenetic machinery such as methylation. Therefore, the recombination frequency at a specific locus can reflect the chromatin status. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382151 Fri, 19 Mar 2010 17:20:06 +0100 3382151 http://www.medworm.com/index.php?rid=3382150&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_17 DNA double strand breaks (DSBs) arise from spontaneous DNA damage due to metabolic activities or from direct and indirect damaging effects of stress. DSBs are also formed transiently during such processes as replication, transcription, and DNA repair. The level of DSBs positively correlates with the activities of homologous and nonhomologous DNA repair pathways, which in turn inversely correlate with methylation levels and chromatin structure. Thus, measurement of strand breaks can provide an informative picture of genome stability of a given cell. The use of random oligonucleotide-primed synthesis for the analysis of DSB levels is described. Applications of the assay for quantitative detection of 3′OH, 3′P, or DNA strand breaks at a cleavage site of the deoxyribose residue are... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382150 Fri, 19 Mar 2010 17:20:06 +0100 3382150 http://www.medworm.com/index.php?rid=3382149&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_16 Virus-induced gene silencing (VIGS) exploits endogenous plant antiviral defense mechanisms to posttranscriptionally silence the expression of targeted plant genes. VIGS is quick and relatively easy to perform and therefore serves as a powerful tool for high-throughput functional genomics in plants. Combined with the use of subtractive cDNA libraries for generating a collection of VIGS-ready cDNA inserts, VIGS can be utilized to screen a large number of genes to determine phenotypes resulting from the knockdown/knockout of gene function. Taking into account the optimal insert design for VIGS, we describe a methodology for producing VIGS-ready cDNA libraries enriched for inserts relevant to the biological process of interest. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382149 Fri, 19 Mar 2010 17:20:06 +0100 3382149 http://www.medworm.com/index.php?rid=3382148&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_15 Epigenetic gene regulation via histone modifications controls different processes ranging from embryonic development, vegetative development, floral induction, floral organ development, to pollen tube growth. The identification of an increasing number of epigenetically regulated processes was greatly advanced by genome-wide histone modification and chromatin-protein interaction surveys. However, genome-wide approaches are too global to access in detail a large number of histone modifications taking place at a single locus. Here we provide a robust Chromatin Immunoprecipitation (ChIP) protocol, allowing in vivo analyses of multiple chromatin modifications and binding of histone modifiers in different plant organs and tissues. This method is quantitative and provides a way to study the dynam... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382148 Fri, 19 Mar 2010 17:20:06 +0100 3382148 http://www.medworm.com/index.php?rid=3382147&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_14 Genome-wide analysis of histone modifications via ChIP-chip (chromatin immunoprecipitation followed by whole genome tiling array hybridization) may generate lists of up to several thousand potential target genes. In the case of the model organism Arabidopsis thaliana, several databases are available to alleviate further characterization and classification of genomic data sets. The term metaanalysis has been coined for this type of multidatabase comparison. In this chapter, we describe open source software and web tools that perform transcriptional and functional analysis of target genes. Sources of transcription data and clustering tools to subdivide genes according to their expression pattern are described. The user is guided through all necessary steps, including data download and format... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382147 Fri, 19 Mar 2010 17:20:05 +0100 3382147 http://www.medworm.com/index.php?rid=3382145&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_13 Genome-wide targets of chromatin-associated factors can be identified by a combination of chromatin-immunoprecipitation and oligonucleotide microarray hybridization. Genome-wide mircoarray data analysis represents a major challenge for the experimental biologist. This chapter introduces ChIPR, a package written in the R statistical programming language that facilitates the analysis of two-color microarrays from Roche-Nimblegen. The workflow of ChIPR is illustrated with sample data from Arabidopsis thaliana. However, ChIPR supports ChIP-chip data preprocessing, target identification, and cross-annotation of any species for which genome annotation data is available in GFF format. This chapter describes how to use ChIPR as a software tool without the requirement for programming skills in the ... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382145 Fri, 19 Mar 2010 17:20:05 +0100 3382145 http://www.medworm.com/index.php?rid=3382144&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_12 Chromatin immunoprecipitation in combination with DNA-microarray hybridization (ChIP-chip) allows the identification of chromatin regions that are associated with modified forms of histones on a genomic scale. The ChIP-chip workflow consists of the following steps: generation of biological material, in vivo formaldehyde-fixation of protein-DNA and protein-protein interactions, chromatin preparation and shearing, immunoprecipitation of chromatin with specific antibodies, fixation reversal and DNA purification, DNA amplification, microarray hybridization, and data analysis. In Part A of this chapter, we describe molecular methods of the experimental procedure employed to identify chromosomal regions of Arabidopsis thaliana associated with H3K27me3. In addition, some general information on th... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382144 Fri, 19 Mar 2010 17:20:05 +0100 3382144 http://www.medworm.com/index.php?rid=3382143&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_11 Small RNAs are key molecules in RNA silencing pathways that exert sequence-specific regulation of gene expression and chromatin modifications in many eukaryotes. In plants, endogenous small RNAs, including microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) play an important role in biological processes such as development and stress responses. In addition, viral genome-derived siRNAs are produced during viral infection, and they exhibit anti-viral defense by an RNA silencing pathway. These endogenous and exogenous small RNAs are mainly 21-24 nucleotides in length. Here, we describe a method to identify small RNA sequences from plant tissues. Small RNAs are purified by column fractionation and gel excision from total RNAs. These small RNAs are ligated at both termini to D... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382143 Fri, 19 Mar 2010 17:20:05 +0100 3382143 http://www.medworm.com/index.php?rid=3382142&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-646-7_10 Plant small RNAs are a class of 19- to 25-nucleotide (nt) RNA molecules that are essential for genome stability, development and differentiation, disease, cellular communication, signaling, and adaptive responses to biotic and abiotic stress. Small RNAs comprise two major RNA classes, short interfering RNAs (siRNAs) and microRNAs (miRNAs). Efficient and reliable detection and quantification of small RNA expression has become an essential step in understanding their roles in specific cells and tissues. Here we provide protocols for the detection of miRNAs by stem-loop RT-PCR. This method enables fast and reliable miRNA expression profiling from as little as 20 pg of total RNA extracted from plant tissue and is suitable for high-throughput miRNA expression analysis. In addition, this method ... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3382142 Fri, 19 Mar 2010 17:20:05 +0100 3382142 http://www.medworm.com/index.php?rid=3215813&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_8 For high frequency regeneration of Phalaenopsis, young leaf segments are cultured on gelrite-gelled ½ MS medium supplemented with 2% sucrose, 2.0 mg/L BA, 0.5 mg/L NAA, 10% coconut water (CW), 2 g/L peptone and 1 g/L activated charcoal. Cultures are incubated at 24 ± 2° C under fluorescence light 50 μmol/m2/s for 16 h photoperiod per day. The PLBs (protocorm like bodies) are induced within 12 weeks of culture and are subcultured to proliferate on the fresh nutrient culture medium for 8 weeks. The PLBs clumps are dissected and cultured on ½ MS medium containing 2% sucrose, 10% coconut water (CW), 2 g/L peptone, 150 mg/L l-glutamine and 1 g/L activated charcoal. The PLB sections elongate to form shoots and new PLBs are induced from the base within 8 weeks of cultu... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215813 Sun, 01 Mar 2009 00:00:00 +0100 3215813 http://www.medworm.com/index.php?rid=3215812&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_7 Poinsettia (Euphorbia pulcherrima) is one of the most popular ornamental pot plants. Conventional propagation is by cuttings, generally focused on a period prior to the most intensive time of sales. Rapid multiplication of elite clones, the production of pathogen-free plants and more rapid introduction of novel cultivars (cvs.) with desirable traits, represent important driving forces in the poinsettia industry. In recent years, different strategies have been adopted to micropropagate poinsettia, which could assist breeders to meet consumer demands. The development of reliable in vitro regeneration procedures is likely to play a crucial role in future production systems. Stem nodal explants cultured on semi-solid MS-based medium supplemented with benzylaminopurine (BAP) and naphthalene ace... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215812 Sun, 01 Mar 2009 00:00:00 +0100 3215812 http://www.medworm.com/index.php?rid=3215811&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_6 Bromeliads are tropical plants that are native to the Americas with a wide distribution in the rain forests, deserts and coastal areas. They are mostly epiphytes and terrestrial, diverse and important from the ecological point of view, they are found in microhabitats in strong interactions with fauna. Most of the ecosystems where they naturally occur are now endangered. Bromeliads are also one of the bases of the ornamental industry worldwide, being commercialized according to the features and colour of the foliage and flowers. This industry relies also on breeding programmes generating new hybrid bromeliads with improved bloom and foliage. Thus, advanced propagation systems based on micropropagation are valuable tools for both bromeliad germplasm conservation and for the mass clonal propa... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215811 Sun, 01 Mar 2009 00:00:00 +0100 3215811 http://www.medworm.com/index.php?rid=3215810&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_5 In this chapter, we describe multiplication of the superior and elite tree of Crataeva adansonii using plant tissue culture techniques. An ornamental and avenue tree, it is not available in abundance because of poor seed germination and seedling establishment. It reproduces in nature by root suckers, but that restricts its distribution to very limited areas. Efficient procedures are outlined for plant regeneration through direct shoot bud formation, indirect organogenesis, and somatic embryogenesis through callus formation. Different explants were utilized for separate pathways of regeneration. Murashige and Skoog’s (MS) medium containing 3 mg/L BA and 0.05–0.1 mg/L NAA is most effective in direct induction of axillary buds from nodal explants and shoot tips. Adventitious shoot... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215810 Sun, 01 Mar 2009 00:00:00 +0100 3215810 http://www.medworm.com/index.php?rid=3215809&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_4 Ranunculus asiaticus is an important ornamental species mainly cultivated in the countries surrounding the Mediterranean sea. So far, the multiplication of this plant has been mainly carried out by seed and tuberous root division; however, these systems present many drawbacks. Tissue culture is an attractive alternative for accelerated propagation of selected and indexed genotypes. In this chapter, we present a flow chart for the commercial production of Ranunculus clones by using in vitro axillary budding. Although the price of micropropagated plants is higher compared to traditional material (seedlings and tuberous roots from seed populations), we need to consider that micropropagation helps to supply growers with more performant and healthy genotypes, and a better production schedule ca... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215809 Sun, 01 Mar 2009 00:00:00 +0100 3215809 http://www.medworm.com/index.php?rid=3215808&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_3 For high frequency clonal propagation of Vanda teres, nodal segments are cultured on VW medium supplemented with 2% sucrose, 2 mg/L Kinetin, 0.5 mg/L NAA, 2 g/L peptone, 1 g/L activated charcoal and 2.2 g/L gelrite. The cultures are incubated at 24 ± 2° C under fluorescent light 50 μmol/m2/s for a 16 h photoperiod per day. The PLBs (protocorm like bodies) are induced within 12 weeks of culture and are subcultured to proliferate on the fresh nutrient culture medium for 8 weeks. The clumps of the PLBs are dissected and cultured on VW medium containing 2% sucrose, 15% coconut water (CW), 2 g/L peptone, 150 mg/L l-glutamine and 1 g/L activated charcoal. The PLB sections elongate to form shoots, and new PLBs are induced from the base within 8 weeks of culture. For plantlets format... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215808 Sun, 01 Mar 2009 00:00:00 +0100 3215808 http://www.medworm.com/index.php?rid=3215807&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_2 A protocol for micropropagation of begonia was established utilizing a thin cell layer (TCL) system. This system has been employed to produce several thousand shoots per sample. Explant size and position, and plant growth regulators (PGRs) contribute to the tissue morphogenesis. By optimizing the size of the tissue and applying an improved selection procedure, shoots were elongated in 8 weeks of culture, with an average number of 210 ± 9.7 shoots per segment. This system has facilitated a number of studies using TCL as a model for micropropagation and will enable the large-scale production of begonia. On an average, the best treatment would allow production of about 10,000 plantlets by the micropropagation of the axillary buds of one plant with five petioles, within a period of 8 mo... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215807 Sun, 01 Mar 2009 00:00:00 +0100 3215807 http://www.medworm.com/index.php?rid=3215806&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_26 Cyclamen (Cyclamen persicum) is an economically important ornamental pot plant with local use as cut flower as well. Traditionally, it is propagated via seeds, but interest is given in vegetative propagation of parental lines as well as superior single plants. Somatic embryogenesis is an efficient in vitro propagation method for many cyclamen cultivars. Starting from ovules of unpollinated flowers, callus is induced and propagated in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-(γ,γ-dimethylallylamino)purine (2iP). Transfer to hormone-free medium results in the differentiation of somatic embryos, which afterwards germinate on the same medium. These first culture stages take about 6–7 months and are carried out in complete darkness. Two to four months a... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215806 Sun, 01 Mar 2009 00:00:00 +0100 3215806 http://www.medworm.com/index.php?rid=3215805&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_25 Sunflower (Helianthus annuus L.) has been cultivated both as an oilseed and as an ornamental plant. Several protocols have been described for the micropropagation, direct plant regeneration by organogenesis being acceptable for this plant species. Besides a strong genotype dependency, the type and ontogenic stage of explants, environmental conditions of the culture, and media composition affect sunflower organogenesis. Several problems have hindered the ability to regenerate normal shoots; the most common being hyperhydricity and precocious flowering. This chapter describes a protocol for direct shoot regeneration from cotyledons developed and established in our laboratory, as well as the improvement regenerated shoot quality. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215805 Sun, 01 Mar 2009 00:00:00 +0100 3215805 http://www.medworm.com/index.php?rid=3215804&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_24 The Myrtle (Myrtus communis L.) is an evergreen shrub typical of the Mediterranean area; it is an interesting plant with multipurpose use. The ornamental use takes into account the production of green cut branches for indoor decoration and production of pot plants for gardening. In this species, there is a great variability in the natural germplasm around the Mediterranean coasts for type and size of fruit, plant architecture, leaf size and internode length. Selected genotypes have been successfully sterilized and cultured in vitro. The shoots were multiplied on MS (16) salts and vitamins, with 0.5 mg/L BA and 0.2 mg/L IAA. Clones showed variation of multiplication rate and rooting percentage. IAA or IBA at 0.5 mg/L increased the rooting percentage and noticed differences in root number an... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215804 Sun, 01 Mar 2009 00:00:00 +0100 3215804 http://www.medworm.com/index.php?rid=3215803&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_23 We describe here a new tulip micropropagation method based on the cyclic shoot multiplication in presence of the thidiazuron (TDZ), which enables the production of virus-free stock plants, speeds up breeding, and provides new genotypes for the market. In our novel protocol, cyclic shoot multiplication can be performed for 2–3 years by using TDZ instead of other cytokinins, as 6-benzylaminopurine (BAP) and N6-(-isopentyl)adenine (2iP). It makes possible to produce 500–2,000 microbulbs from one healthy plant. There are six main stages of tulip micropropagation. Stage 0 is the selection of true-to-type and virus-free plants, confirmed by ELISA. Fragments of flower stems isolated from bulbs are used as initial explants. Shoot multiplication is based on the regeneration of adventiti... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215803 Sun, 01 Mar 2009 00:00:00 +0100 3215803 http://www.medworm.com/index.php?rid=3215802&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_22 In this chapter, we describe a robust method for the micropropagation of Australian fan flower, Scaevola, a native plant increasingly being used in the ornamental horticulture industry. Shoot segments from different species of Scaevola can be successfully multiplied following this protocol. Multiple shoots can be obtained in hormone-free Murashige and Skoog medium. The regenerated shoot is rooted on hormone-free medium within 4–6 weeks. In vitro grown plantlets readily adapt to glasshouse conditions. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215802 Sun, 01 Mar 2009 00:00:00 +0100 3215802 http://www.medworm.com/index.php?rid=3215801&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_21 Procedures for somatic embryogenesis (SE) in in vitro culture of spring snowflake have been developed from different types of explants like scales and leaves isolated from bulbs, ovaries and fruits. Various plant growth regulators were tested including a cytokinin – benzyladenine (BA) and various concentrations of the exogenous auxins 3,6-dichloro-2-methoxybenzoic acid (Dicamba), 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (Picloram). Fruit explants, cultured on medium containing Picloram and BA, ensured the highest percentage of callusing and such calli were most efficient in inducing somatic embryos. The addition of abscisic acid (ABA) in combination with polyethylene glycol (PEG) stimulated somatic embryo maturation. Torpedo-stage embryos develo... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215801 Sun, 01 Mar 2009 00:00:00 +0100 3215801 http://www.medworm.com/index.php?rid=3215800&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_20 Marigold (Tagetes erecta) is an Asteraceous plant of industrial, ornamental and medicinal importance. Tagetes erecta species, popularly known as marigold, is grown as ornamental plant and is adapted to several agro climates. Inflorescences have been utilized as pigment source for food coloring, mainly of poultry skin and eggs. Lutein is the main pigment in marigold flowers. Some carotenoids are well known as provitamin A compounds. There are many reports on carotenoids and their effect on the prevention of certain ocular diseases, ischemic heart disease, strokes, photoprotection, immune response, aging and cancer. Marigold flowers are certainly a good source of carotenoids; they show very different pigmentation levels. This chapter describes the establishment of techniques for plant regene... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215800 Sun, 01 Mar 2009 00:00:00 +0100 3215800 http://www.medworm.com/index.php?rid=3215799&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_1 Tissue culture techniques are routinely used for mass propagation and the establishment of disease free stock material. Virtually all pot type Anthuriums available in the market today are produced by tissue culture. In this chapter, we describe an efficient protocol to obtain Anthurium andreanum cv Rubrun vitro plants through micropropagation and organogenesis. Seeds from plant spadixes were germinated on MS medium supplemented with 0.5 mg/L BA. Micro-cuttings from in vitro germinated seedlings were subcultured on MS medium containing 2 mg/L BA and 0.5 mg/L NAA. Four-week-old in vitro plants obtained from microcuttings, showed callus proliferation at the stem base. The development of shoots and plantlets was observed from callus tissue. We also describe a detailed method for the histologic... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215799 Sun, 01 Mar 2009 00:00:00 +0100 3215799 http://www.medworm.com/index.php?rid=3215798&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_19 Geraniums (Pelargonium spp.) are among the most popular bedding and pot plants (25 % of the French domestic market). On one hand, as vegetatively propagated plants, Pelargonium are submitted to pathogen pressure. On the other hand, innovation via interspecific hybridisation faces some difficulties. In this chapter, the two first protocols (from seeds and meristems) explain how in vitro plants free of virus could be obtained. The development of this technique is the long-term preservation of genetic resources via meristem cryopreservation. The third protocol describes propagation of Pelargonium with limited risks of variation. This technique also allows the constitution and the maintenance of a plant-stock from which explants can be taken for other studies. The two last protocols describe p... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215798 Sun, 01 Mar 2009 00:00:00 +0100 3215798 http://www.medworm.com/index.php?rid=3215797&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_18 Codiaeum variegatum (L) Blume cv. “Corazon de oro” and cv. “Norma” are successfully micropropagated when culture are initiated with explants taken from newly sprouted shoots. The establishment and multiplication steps are possible when 1 mg/L BA or 1 mg/L IAA and 3 mg/L 2iP are added to MS medium, according to the cultivar respectively selected. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215797 Sun, 01 Mar 2009 00:00:00 +0100 3215797 http://www.medworm.com/index.php?rid=3215796&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_17 Chrysanthemum flowers Chrysanthemum x grandiflorum (Ramat.) Kitam., are commercially significant worldwide as there are large number of cultivars for cut flowers, pot flowers, and garden flowers. Commercial in vitro multiplication of chrysanthemum is often based on stem nodal explants with lateral meristems. This chapter describes a protocol for in vitro propagation from stem nodal explants and by direct organogenesis from pedicel explants producing large number of true-to-type plantlets in 4–8 week on Murashige and Skoog (MS) based media. Also, true mutants with changed flower color are obtained without producing chimeras after gamma-irradiation in mutation breeding. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215796 Sun, 01 Mar 2009 00:00:00 +0100 3215796 http://www.medworm.com/index.php?rid=3215795&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_16 In vitro propagation of rose is an important tool for rapid multiplication and development of new cultivars with desirable traits. However, successful in vitro propagation requires an understanding of specific requirements and precise manipulation of various factors. Efficient protocols for different stages of micropropagation using apical buds or nodal segments are currently available. Recently, new challenges for refinements of protocols for high rate of shoot multiplication and development of cost effective methods has gained importance. Significance of the liquid static culture for shoot proliferation and root induction for rose has also gained prominence. Other distinct approaches of in vitro propagation include organogenesis and embryogenesis. These approaches are important for the s... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215795 Sun, 01 Mar 2009 00:00:00 +0100 3215795 http://www.medworm.com/index.php?rid=3215794&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_15 With more than 450 species, Passiflora is the most important genus of the family Passifloraceae. It comprises many species grown for their edible fruits, for their high ornamental value, and further for the therapeutic properties. With their striking exotic flowers, they are of particular interest for the floriculture market. With the aim of evaluating the in vitro propagation of an Italian ornamental hybrid, axillary tendrils of Passiflora “Guglielmo Betto” M. Vecchia (P. incarnata L. × P. tucumanensis L.) were sterilized and placed in vitro. Direct shoot regeneration was achieved from young tendrils cultivated on MS medium containing, either 4.43 μM 6-benzylaminopurine (BAP) and 11.41 μM indoleacetic acid (IAA), or 49.20 μM 6-γ-γ-dimethylallylaminop... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215794 Sun, 01 Mar 2009 00:00:00 +0100 3215794 http://www.medworm.com/index.php?rid=3215793&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_14 Methods for in vitro initiation and multiplication and general culture practices of Rhododendron are presented. Also acclimatization procedures are described. Protocols for callus, shoot, and root induction are described. Several protocols for breeding applications are highlighted in more detail. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215793 Sun, 01 Mar 2009 00:00:00 +0100 3215793 http://www.medworm.com/index.php?rid=3215792&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_13 Numerous shoots were directly regenerated from the leaf explants of Lysionotus pauciflorus on the MS medium containing 0.5–2 µM NAA with or without 1 µM BA. The calli were induced from the leaves on MS medium supplemented with 2 µM 2, 4-D. The calli proliferated about four times in fresh weight in the liquid medium of the same composition as the callus induction medium after 4 weeks of culture on a rotary shaker at 100 rpm. Shoots were induced from these calli on the regeneration medium amended with 32 µM BA or 0.5 µM zeatin. Regenerated shoots rooted easily on ½ MS medium without any plant growth regulators. Most of the regenerants from callus were diploid, whereas eight of 66 acclimatized plantlets were tetraploid determined by flow cytometric a... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215792 Sun, 01 Mar 2009 00:00:00 +0100 3215792 http://www.medworm.com/index.php?rid=3215791&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_12 The growing demand for flower extracts in perfume trade can primarily be met by increasing flower production and multiplying planting material. The major commercial aromatic flower yielding plants including Jasminum officinale L., a member of the Family Oleaceae have drawn the attention of a large section of the concerned sectors leading to a thrust upon developing advanced propagation technologies for these floral crops, in addition to conventional nature-dependent agro-techniques. This chapter describes concisely and critically, a protocol developed for in vitro propagation of Jasminum officinale by shoot regeneration from existing as well as newly developed adventitious axillary buds via proper phytohormonal stimulation. To start with nodal segments as explants, March–April is the... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215791 Sun, 01 Mar 2009 00:00:00 +0100 3215791 http://www.medworm.com/index.php?rid=3215790&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_11 Carnation (Dianthus caryophyllus L.) is one of the most popular ornamental plants worldwide and also among the most studied ones, mainly in cut flower postharvest physiology. Several protocols for the in vitro propagation of this species including nodal segment culture, somatic embryogenesis, and adventitious shoot induction are described in this chapter. The presence of hyperhydricity as an abnormality during micropropagation of carnation plants has also been the object of research for many years and different strategies to overcome this problem are also included in this study. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215790 Sun, 01 Mar 2009 00:00:00 +0100 3215790 http://www.medworm.com/index.php?rid=3215789&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-114-1_10 Enhancement of efficiency and efficacy of plant regeneration are primary goals of micropropagation. An efficient method with rapid and improved shoot regeneration of gladiolus based upon matrix supported liquid culture technique has been demonstrated. This technique in addition ensured the quality of the regenerated shoots by effectively alleviating the phenomenon of hyperhydricity. Efficacy in plant regeneration can be brought about by the ability to sort and objectively select plants or group of plants with a desired feature. A reliable, noninvasive, machine vision-neural network based sorting method for clustering photometric variants of regenerated shoots of gladiolus has also been described. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=3215789 Sun, 01 Mar 2009 00:00:00 +0100 3215789 http://www.medworm.com/index.php?rid=2875169&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-005-2_14 MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs associated with abiotic stress responses. Small RNA cloning and high-throughput deep sequencing methods provide expression profiles of not only known miRNAs, but also novel miRNAs. In this chapter, we describe the methods used to identify and characterize abiotic stress-associated miRNAs and their target genes. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2875169 Thu, 01 Jan 2009 00:00:00 +0100 2875169 http://www.medworm.com/index.php?rid=2875168&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-005-2_13 The methods described herein first highlight the strategies that were used to discover a biotic stress-associated miRNA. This involved (1) the selection of transcripts that were more abundant in transgenic plants expressing viral-derived suppressors of RNA silencing and transcripts that were repressed in wild-type seedlings treated with a biotic stress, (2) a 5′ RACE-derived assay to map miRNA target sites, and (3) a bioinformatic analysis to retrieve specific miRNA loci from the Arabidopsis genome. We then describe methods used to monitor (1) the levels of primary miRNA transcripts (pri-miRNAs)/mature miRNAs and (2) the transcriptional activity of miRNAs in response to a biotic stress and bacterial challenge. Furthermore, we present a strategy to identify additional biotic stress-re... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2875168 Thu, 01 Jan 2009 00:00:00 +0100 2875168 http://www.medworm.com/index.php?rid=2875167&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-005-2_12 Small RNAs play an important role in plant development, stress responses, and epigenetic regulation, primarily through their role in transcriptional and post-transcriptional silencing of specific target genes and loci. Most if not all plants utilize these small RNA signaling networks. We have developed a deep-sequencing based dataset of plant small RNAs, based on the hypothesis that comparisons among the complex pool of small RNAs from diverse plants will identify novel types of conserved, regulated, or species-specific molecules. A database containing upward of hundreds of millions of plant small RNA sequences is being created for comparative analyses. This small RNA database will allow the experimental characterization of the majority of the biologically important small RNAs for a range ... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2875167 Thu, 01 Jan 2009 00:00:00 +0100 2875167 http://www.medworm.com/index.php?rid=2875166&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-005-2_11 In this chapter, we present a brief overview of current knowledge about the promoters of plant microRNAs (miRNAs), and provide a step-by-step guide for predicting plant miRNA promoter elements using known transcription factor binding motifs. The approach to promoter element prediction is based on a carefully constructed collection of Positional Weight Matrices (PWMs) for known transcription factors (TFs) in Arabidopsis. A key concept of the method is to use scoring thresholds for potential binding sites that are appropriate to each individual transcription factor. While the procedure can be applied to search for Transcription Factor Binding Sites (TFBSs) in any pol-II promoter region, it is particularly practical for the case of plant miRNA promoters where upstream sequence regions and bin... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2875166 Thu, 01 Jan 2009 00:00:00 +0100 2875166 http://www.medworm.com/index.php?rid=2875165&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-005-2_10 After transcription, a large number of cellular RNAs employ modifications to increase their diversity and functional potential. Modifications can occur on the base, ribose, or both, and are important steps in the maturation of many RNAs. Our lab recently showed that plant microRNAs (miRNAs) possess a 2′-O-methyl group on the ribose of the 3′ terminal nucleotide, and that this methyl group is added after miRNA/miRNA* formation. One function of this modification is to protect miRNAs from 3′ terminal uridylation by an unknown enzymatic activity. It is possible that uridylation of miRNAs triggers their degradation. Here we describe a protocol to purify a specific miRNA in order to determine its molecular mass so that the presence of a modification can be inferred, an in vivo ... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2875165 Thu, 01 Jan 2009 00:00:00 +0100 2875165 http://www.medworm.com/index.php?rid=2601294&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_20 Systems biology is a comprehensive means of creating a complete understanding of how all components of an organism work together to maintain and procreate life. By quantitatively profiling one at a time, the effect of thousands and millions of genetic and environmental perturbations on the cell, systems biologists are attempting to recreate and measure the effect of the many different states that have been explored during the 3 billion years in which life has evolved. A key aspect of this work is the development of innovative new approaches to quantify changes in the transcriptome, proteome, and metabolome. In this chapter we provide a review and evaluation of several genomic profiling techniques used in plant systems biology as well as make recommendations for future progress in their use... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601294 Thu, 01 Jan 2009 00:00:00 +0100 2601294 http://www.medworm.com/index.php?rid=2601293&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_19 The recent development of analytic technologies allows fast analysis of metabolism in real time. Fluxomics aims to define the genes involved in regulation of flux through a metabolic or signaling pathway. Flux through a metabolic or signaling pathway is determined by the activity of its individual components; regulation can occur at many levels, including transcriptional, posttranslational, and allosteric levels. Currently two technologies are used to monitor fluxes. The first is pulse labeling of the organism with a tracer such as C13, followed by mass spectrometric analysis of the partitioning of label into different compounds. The second approach is based on the use of flux sensors, proteins that respond with a conformational change to ligand binding. Fluorescence resonance energy trans... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601293 Thu, 01 Jan 2009 00:00:00 +0100 2601293 http://www.medworm.com/index.php?rid=2601292&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_18 Chemical genomics (i.e., genomics-scale chemical genetics) approaches are based on the ability of low-molecular-mass molecules to modify biological processes. Such molecules are used to affect the activity of a protein or a pathway in a manner that is tunable and reversible. A major advantage of this approach compared to classical plant genetics is the fact that chemical genomics can address loss-of-function lethality and redundancy. Bioactive chemicals resulting from forward or reverse chemical screens can be useful in understanding and dissecting complex biological processes due to the essentially limitless variation in structure and activities inherent in chemical space. An important aspect of utilizing small molecules effectively is to characterize bioactive chemicals in detail includi... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601292 Thu, 01 Jan 2009 00:00:00 +0100 2601292 http://www.medworm.com/index.php?rid=2601291&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_17 Plants communicate with their surrounding ecosystems using a diverse array of volatile metabolites that are indicative of the physiological status of the emitter. A variety of systems have been adapted to capture, analyze, identify, and quantify airborne metabolites released by plants. Metabolomic experiments typically involve four steps: sample collection, preparation, product separation, and data analysis. To date, two different types of headspace sampling, static and dynamic, are widely used for volatile metabolome investigation. For static headspace analysis, solid-phase microextraction (SPME) is used to sample volatiles while push and pull as well as closed-loop stripping methods are used for dynamic headspace sampling. After collection, volatile blends are most efficiently and routin... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601291 Thu, 01 Jan 2009 00:00:00 +0100 2601291 http://www.medworm.com/index.php?rid=2601290&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_16 Morphological phenotypes due to mutations frequently provide key information about the biological function of the affected genes. This has long been true of the plant Arabidopsis thaliana, though phenotypes are known for only a minority of this model organism's approximately 25,000 genes. One common explanation for lack of phenotype in a given mutant is that a genetic redundancy masks the effect of the missing gene. Another possibility is that a phenotype escaped detection or manifests itself only in a certain unexamined condition. Addressing this potentially nettlesome alternative requires the development of more sophisticated tools for studying morphological development. Computer vision is a technical field that holds much promise in this regard. This chapter explains in general terms ho... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601290 Thu, 01 Jan 2009 00:00:00 +0100 2601290 http://www.medworm.com/index.php?rid=2601289&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_15 The shoot apical meristem (SAM) of higher plants represents a dynamic network of different cell types which exhibit distinct patterns of gene expression and cellular behaviors. The regulation of distinct patterns of gene expression and cellular behaviors is mediated by cell–cell communication networks. Live-imaging of spatiotemporal dynamics of cell–cell communication networks, gene expression patterns, and cellular behaviors is critical to deduce principles that underlie SAM growth and maintenance. In this chapter, we describe live-imaging methods, fluorescent reagents, and image processing protocols that have been developed to visualize the regulatory dynamics of SAM growth in Arabidopsis thaliana. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601289 Thu, 01 Jan 2009 00:00:00 +0100 2601289 http://www.medworm.com/index.php?rid=2601288&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_9 Advances in modern technologies have facilitated high-dimensional experiments (HDEs) that generate tremendous amounts of genomic, proteomic, and other “omic” data. HDEs involving whole-genome sequences and polymorphisms, expression levels of genes, protein abundance measurements, and combinations thereof have become a vanguard for new analytic approaches to the analysis of HDE data. Such situations demand creative approaches to the processes of statistical inference, estimation, prediction, classification, and study design. The novel and challenging biological questions asked from HDE data have resulted in many specialized analytic techniques being developed. This chapter discusses some of the unique statistical challenges facing investigators studying high-dimensional biology ... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601288 Thu, 01 Jan 2009 00:00:00 +0100 2601288 http://www.medworm.com/index.php?rid=2601287&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_14 Important patterns can be found in strings of characters such as nucleotides in a DNA sequence by examining the frequency of occurrence of specific character combinations or words. The abundance of words can reveal the presence of underlying trends governing the order of characters, even if the biological reasons for those trends remain mysterious. As an example of one way in which word frequencies have provided insight, we describe the IMEter, a word-based algorithm for analyzing introns and their effect on gene expression. The IMEter demonstrates that introns located near the beginning of genes are compositionally distinct from later introns and that these differences are closely related to the ability of some introns to increase gene expression. This word-based approach has proven more ... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601287 Thu, 01 Jan 2009 00:00:00 +0100 2601287 http://www.medworm.com/index.php?rid=2601286&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_13 Metabolic reactions and gene regulation are two primary processes of cells. In response to environmental changes cells often adjust the regulatory programs and shift the metabolic states. An integrative investigation and modeling of these two processes would improve our understanding of the cellular systems and may generate substantial impacts in medicine, agriculture, environmental protection, and energy. We review the studies of the various aspects of the crosstalk between metabolic reactions and gene regulation, including models, empirical evidence, and available databases. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601286 Thu, 01 Jan 2009 00:00:00 +0100 2601286 http://www.medworm.com/index.php?rid=2601285&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_12 Co-expression analysis allows experimenters to re-use archived expression microarray data to uncover previously unknown functional relationships between genes. An observation that a group of genes are co-expressed across diverse experimental conditions suggests they may play similar roles in the cell. Several thousand expression microarray experiments performed on samples from Arabidopsis thaliana have entered the public domain and it is now possible to use these data to investigate metabolic networks in plants. This chapter explains how to use a Web-based tool (CressExpress) to investigate co-expression of genes involved in metabolic pathways in Arabidopsis. Using CressExpress together with desktop visualization and analysis tools, one can easily identify clusters of genes that are co-exp... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601285 Thu, 01 Jan 2009 00:00:00 +0100 2601285 http://www.medworm.com/index.php?rid=2601284&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_11 Gene expression microarrays allow rapid and easy quantification of transcript accumulation for almost transcripts present in a genome. This technology has been utilized for diverse investigations from studying gene regulation in response to genetic or environmental fluctuation to global expression QTL (eQTL) analyses of natural variation. Typical analysis techniques focus on responses of individual genes in isolation of other genes. However, emerging evidence indicates that genes are organized into regulons, i.e., they respond as groups due to individual transcription factors binding multiple promoters, creating what is commonly called a network. We have developed a set of statistical approaches that allow researchers to test specific network hypothesis using a priori-defined gene networks... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601284 Thu, 01 Jan 2009 00:00:00 +0100 2601284 http://www.medworm.com/index.php?rid=2601283&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_10 A major aim of systems biology is the study of the inter-relationships found within and between large biological data sets. Here we describe one systems biology method, in which the tools of network analysis and discrete dynamic (Boolean) modeling are used to develop predictive models of cellular signaling in cases where detailed temporal and kinetic information regarding the propagation of the signal through the system is lacking. This approach is also applicable to data sets derived from some other types of biological systems, such as transcription factor-mediated regulation of gene expression during the control of developmental fate, or host defense responses following pathogen attack, and is equally applicable to plant and non-plant systems. The method also allows prediction of how eli... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601283 Thu, 01 Jan 2009 00:00:00 +0100 2601283 http://www.medworm.com/index.php?rid=2601282&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_8 Annotated genomes have provided a wealth of information about gene structure and gene catalogs in a wide range of species. Taking advantage of these developments, novel techniques have been implemented to investigate systematically diverse aspects of gene and protein functions underpinning biology processes. Here, we review functional genomics applications that require the mass production of cloned sequence repertoires, including ORFeomes and silencing tag collections. We discuss the techniques employed in large-scale cloning projects and we provide an up-to-date overview of the clone resources available for model plant species and of the current applications that may be scaled up for systematic plant gene studies. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601282 Thu, 01 Jan 2009 00:00:00 +0100 2601282 http://www.medworm.com/index.php?rid=2601281&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_7 To tackle the question of how chromatin organization is involved in global regulation of genome-related processes such as transcription, we have recently created a collection of 277 transposon-tagged Arabidopsis lines comprised of a single insert with a common luciferase reporter cassette and a LacO repeat array for visual tracking of the tagged region via fluorescent protein fusion technology. Using this collection of plants, one can begin to map transgene position effects as well as global epigenetic control in response to developmental or externally applied cues. In this chapter, we will outline the approach and methods for deploying this novel resource for the study of global gene control, using Arabidopsis as a convenient model system. (Source: Springer protocols feed by Plant Science... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601281 Thu, 01 Jan 2009 00:00:00 +0100 2601281 http://www.medworm.com/index.php?rid=2601280&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_6 Polyribosomes (polysomes) form as multiple ribosomes engage in translation on a single mRNA. This process is regulated for individual mRNAs by both development and the environment. To evaluate the translation state of an mRNA, ribosomal subunits, ribosomes, and polysomes can be isolated from detergent-treated cell extracts by high-speed differential centrifugation. These ribonucleoprotein complexes can be further purified by centrifugation through sucrose density gradients. By fractionation of the gradient the amount of an individual mRNA in a sub-population of polysomes can be quantitatively determined. Here, we describe methods for the isolation and quantification of polysome complexes from plant tissues. The mRNA obtained can be further analyzed by methods that evaluate polysomal mRNA a... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601280 Thu, 01 Jan 2009 00:00:00 +0100 2601280 http://www.medworm.com/index.php?rid=2601279&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_5 The genomics era has enabled scientists to more readily pose truly global questions regarding mutation, evolution, gene and genome structure, function, and regulation. Just as Sanger sequencing ushered in a paradigm shift that enabled the molecular basis of biological questions to be directly addressed, to an even greater degree, ultra-high-throughput DNA sequencing is poised to dramatically change the nature of biological research. New sequencing technologies have opened the door for novel questions to be addressed at the level of the entire genome in the areas of comparative genomics, systems biology, metagenomics, and genome biology. These new sequencing technologies provide a tremendous amount of DNA sequence data to be collected at an astounding pace, with reduced costs, effort, and t... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601279 Thu, 01 Jan 2009 00:00:00 +0100 2601279 http://www.medworm.com/index.php?rid=2601278&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_4 A series of large-scale Arabidopsis thaliana microarray expression experiments profiling genome-wide expression across different developmental stages, cell types, and environmental conditions have resulted in tremendous amounts of gene expression data. This gene expression is the output of complex transcriptional regulatory networks and provides a starting point for identifying the dominant transcriptional regulatory modules acting within the plant. Highly co-expressed groups of genes are likely to be regulated by similar transcription factors. Therefore, finding these co-expressed groups can reduce the dimensionality of complex expression data into a set of dominant transcriptional regulatory modules. Determining the biological significance of these patterns is an informatics challenge an... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601278 Thu, 01 Jan 2009 00:00:00 +0100 2601278 http://www.medworm.com/index.php?rid=2601277&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_3 DNA microarrays have become a mainstream tool in experimental plant biology. The constant improvements in the technological platforms have enabled the development of the tiling DNA microarrays that cover the whole genome, which in turn catalyzed the wide variety of creative applications of such microarrays in the areas as diverse as global studies of genetic variation, DNA-binding proteins, DNA methylation, and chromatin and transcriptome dynamics. This chapter attempts to summarize such applications as well as discusses some technical and strategic issues that are particular to the use of tiling microarrays. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601277 Thu, 01 Jan 2009 00:00:00 +0100 2601277 http://www.medworm.com/index.php?rid=2601276&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_2 RNA–protein interactions profoundly impact organismal development and function through their contributions to the basal gene expression machineries and their regulation of post-transcriptional processes. The repertoire of predicted RNA binding proteins (RBPs) in plants is particularly large, suggesting that the RNA–protein interactome in plants may be more complex and dynamic even than that in metazoa. To dissect RNA–protein interaction networks, it is necessary to identify the RNAs with which each RBP interacts and to determine how those interactions influence RNA fate and downstream processes. Identification of the native RNA ligands of RBPs remains a challenge, but several high-throughput methods for the analysis of RNAs that copurify with specific RBPs from cell extra... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601276 Thu, 01 Jan 2009 00:00:00 +0100 2601276 http://www.medworm.com/index.php?rid=2601275&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-563-7_1 Chromatin immunoprecipitation (ChIP) provides a versatile tool to investigate the in vivo location of DNA-binding proteins on genomic DNA. ChIP approaches are gaining significance in plants, in cases when entire genome sequences are available (e.g., Arabidopsis), for which several high-density oligo arrays have been or are being developed. Nevertheless, plant ChIP and ChIP-chip still present some technical challenges. Here, we describe general methods for ChIP and ChIP-chip, which have been successfully applied to maize and Arabidopsis. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2601275 Thu, 01 Jan 2009 00:00:00 +0100 2601275 http://www.medworm.com/index.php?rid=2715483&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-062-1_14 Many species of fungi have been shown to harbor double-stranded RNA (dsRNA) elements. A single fungal isolate of Rhizoctonia solani may have as many as five different dsRNA elements within them. The presence of specific dsRNA elements influence pathogenicity in host plants. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2715483 Mon, 01 Dec 2008 00:00:00 +0100 2715483 http://www.medworm.com/index.php?rid=2715482&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-062-1_13 The following chapter describes a PCR method for the identification of the raspberry root rot pathogen Phytophthora fragariae var. rubi. Furthermore, a nested PCR suitable for the detection of the pathogen in infected raspberry roots and validated against the “Duncan bait test” (EPPO Bull 35:87–91, 2005) is explained. Protocols for different DNA extraction methods are given which can be transferred to other fungal pathogens. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2715482 Mon, 01 Dec 2008 00:00:00 +0100 2715482 http://www.medworm.com/index.php?rid=2715481&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-062-1_12 Traditional methods for the isolation and identification of fungal spores can be time-consuming and laborious. DNA-based methods for fungal detection can be used to detect the spores of plant-pathogenic fungi. Air borne spores can be collected and identified by PCR allowing identification of the species. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2715481 Mon, 01 Dec 2008 00:00:00 +0100 2715481 http://www.medworm.com/index.php?rid=2715480&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-062-1_11 FISH is a widely used technique in many laboratories not only for cytogenetic studies, but also in other biological fields. It requires a combination of skills in molecular biology, cytogenetics, immunocytochemistry, microscopy and cellular imaging analysis. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2715480 Mon, 01 Dec 2008 00:00:00 +0100 2715480 http://www.medworm.com/index.php?rid=2715479&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-062-1_10 We describe a real-time PCR procedure that provides all requirements. This method is based on chromosomal genes rather than on the pEa29 plasmid and so can be used to measure isolates that have been cured of the plasmid. The method has been used very successfully in directly quantify whole E. amylovora cells, in a variety of tissues from the orchard environment. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2715479 Mon, 01 Dec 2008 00:00:00 +0100 2715479 http://www.medworm.com/index.php?rid=2145102&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_9 Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145102 Sun, 01 Jun 2008 04:00:00 +0100 2145102 http://www.medworm.com/index.php?rid=2145101&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_8 The moss Physcomitrella patens is a long-standing model for studying plant development, growth and cell differentiation in particular. Interest in this non-vascular plant arose following the discovery that homologous recombination is an efficient process. P. patens is, therefore, a tool of choice not only to study gene function but also for recombinant protein production. This system has many attributes that are advantageous for molecular farming: protein production in cell suspension, the possibility of generating targeted knockout mutants for glycoengineering and quantitative optimization for protein production. In terms of technical advances, P. patens is one of the most up-to-date plant expression systems and is a promising alternative to animal cell factories for the production of the... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145101 Sun, 01 Jun 2008 04:00:00 +0100 2145101 http://www.medworm.com/index.php?rid=2145100&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_7 Because of the wide use and high demand in medicine, monoclonal antibodies are among the main recombinant pharmaceuticals at present, although present limitations of the productive platforms for monoclonal antibodies are driving the improvement of the large-scale technologies and the development of alternative expression systems. This has drawn the attention on plants as expression system for monoclonal antibodies and related derivatives, owning the capacity of plants to properly express and process eukaryotic proteins with biological activity resembling that of the natural proteins. In this chapter, the procedures from the isolation of the monoclonal antibody genes to the biochemical and biological characterization of the plant-expressed monoclonal antibody are described. (Source: Springe... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145100 Sun, 01 Jun 2008 04:00:00 +0100 2145100 http://www.medworm.com/index.php?rid=2145099&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_6 Plants offer a number of attractive benefits over conventional mammalian or bacterial cell culture systems for the production of valuable pharmaceutical and industrial proteins. Currently, antibodies and their derived fragments represent the largest and most important group of biotechnological products in clinical trials. In particular, single-chain antibodies are an interesting class of biopharmaceuticals because they are able to overcome specific problems associated with full-length antibodies. Another valuable antibody format is the scFv-Fc: fusion of the Fc domain to a single-chain variable fragment restores antibody effector functions, allows purification, and mimicks, despite being a ‘single-gene’ product, the bivalent properties of a full-length IgG. Although many differ... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145099 Sun, 01 Jun 2008 04:00:00 +0100 2145099 http://www.medworm.com/index.php?rid=2145098&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_5 Among the many plant-based production systems that have been developed for pharmaceutical proteins, seeds have the useful advantage of accumulating proteins in a relatively small volume, and recom-binant proteins are very stable in dry seeds allowing long-term storage and facilitating distribution before processing. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145098 Sun, 01 Jun 2008 04:00:00 +0100 2145098 http://www.medworm.com/index.php?rid=2145097&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_4 Transgene product yield remains a key limitation in commercializing plant-derived pharmaceutical proteins. Although significant progress has been made in understanding the roles of promoters, enhancers, integration sites, codon usage, cryptic RNA sites, silencing, and product compartmentalization on product yield and quality, researchers still cannot reliably predict which proteins will be produced at high levels or what manipulations will guarantee enhanced productivity. We have optimized a simple transient expression system in Nicotiana benthamiana enabling rapid assessment of transgene potential for plant-based bioproduction. Briefly, intact Nicotiana benthamiana plants are vacuum-infiltrated with Agrobacterium tumefaciens cultures carrying the transgene of interest. After 48–96 h... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145097 Sun, 01 Jun 2008 04:00:00 +0100 2145097 http://www.medworm.com/index.php?rid=2145096&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_3 The improvements in agroinfiltration methods for plant-based transient expression now allow the production of significant amounts of recombinant proteins in a matter of days. While vacuum-based agroinfiltration has been brought to large scale to meet the cost, speed and surge capacity requirements for vaccine and therapeutic production, the more accessible and affordable syringe agroinfiltration procedure still represents a fast and high-yielding approach to recombinant protein production at lab scale. The procedure exemplified here has proven its reproducibility and high-yield capacity for the production of proteins with varying levels of complexity, including monoclonal antibodies. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145096 Sun, 01 Jun 2008 04:00:00 +0100 2145096 http://www.medworm.com/index.php?rid=2145095&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_20 The technology for plant-made pharmaceuticals (PMPs) has progressed significantly over the last few years, with the first commercial products for human use expected to reach the market by 2009 (see Note 1 ). As part of the ‘next generation’ of genetically modified (GM) crops, PMPs will be subject to additional biosafety considerations and are set to challenge the complex and overlapping regulations that currently govern GM plants, plant biologics (see Note 2 ) and ‘conventional’ pharmaceutical production. The areas of responsibility are being mapped out between the different regulatory agencies (Sparrow, P.A.C., Irwin, J., Dale, P., Twyman, R.M., and Ma, J.K.C. (2007) Pharma-Planta: Road testing the developing regulatory guidelines fo... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145095 Sun, 01 Jun 2008 04:00:00 +0100 2145095 http://www.medworm.com/index.php?rid=2145094&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_2 This chapter describes the use of Cowpea mosaic virus-based vectors for the production of foreign proteins such as antigens and antibodies in plants. The systems include vectors based on both full-length and deleted versions of RNA-2. In both cases, the modified RNA-2 is replicated by coinoculation with RNA-1. The constructs based on full-length RNA-2 retain the ability to spread systemically throughout an inoculated plant and the infection can be passaged. The vector based on a deleted version of RNA-2 can stably incorporate larger inserts but lacks the ability to move systemically. However, it has the added advantage of biocontainment. In both cases, vector constructs modified to contain a foreign gene of interest can be delivered by agroinfiltration to obtain transient expression of the... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145094 Sun, 01 Jun 2008 04:00:00 +0100 2145094 http://www.medworm.com/index.php?rid=2145093&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_19 Therapeutic proteins have an intrinsic potential to induce undesirable immune and allergic responses. The nature of the expression system and the control of the manufacturing process represent extrinsic factors that could modify this potential. Accordingly, regulatory agencies require sponsors to assess the risk of clinical immune and allergic responses that could be associated with the production of therapeutic proteins in transgenic plants. Since factors related to the clinical use of the product–including the immune status and genetic background of subjects–are also relevant, the risk assessment needs to balance the probability of a response induced by a plant-specific factor relative to the likely consequences for the specific product and therapeutic indication. (Source: Sp... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145093 Sun, 01 Jun 2008 04:00:00 +0100 2145093 http://www.medworm.com/index.php?rid=2145092&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_18 This chapter presents a general procedure for the on-chip detection and quantitation of low-molecular-weight recombinant proteins in transgenic plant crude extracts by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). A protocol is first described to detect the protein of interest in crude protein extracts of transgenic plant lines, by differential protein mapping against similar extracts from a control, nontransgenic line. A complementary protocol is then presented to generate a standard curve with the SELDI system, allowing the protein to be quantified in different transgenic lines. Overall, this procedure may be carried out within a few hours, without the need for prior purification or enrichment of the recombinant protein. (Source: Springer p... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145092 Sun, 01 Jun 2008 04:00:00 +0100 2145092 http://www.medworm.com/index.php?rid=2145091&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_17 In this chapter, we discuss and compare the different concepts and examples as well as present the basic protocols for applying intrabody-based approaches in plants for the investigation of cell functions and plant cell–pathogen interactions. The immunomodulation strategy, a molecular technique that allows to interfere with cellular metabolism, signal transduction pathways, or pathogen infectivity, is based on the ectopic expression of genes encoding specific recombinant antibodies. This needs basic prerequisites to be successfully applied as resources and techniques to isolate specific recombinant antibodies with sufficient binding parameters to bind and to block even low-concentrated targets or to compete successfully with substrates and ligands. Also techniques and constructs to e... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145091 Sun, 01 Jun 2008 04:00:00 +0100 2145091 http://www.medworm.com/index.php?rid=2145090&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_16 Transgenic plants are gaining increasing attention from the industry as a natural bioreactor for the production of industrial and chemical products. Optimization of transgene expression in plant cells holds the key to maximizing the potential of plants for producing proteins of commercial interest. This chapter is devoted to the description of the methods utilized for the generation of transgenic plants expressing a canine parvovirus vaccine peptide or virus-like particles from a rabbit calicivirus. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145090 Sun, 01 Jun 2008 04:00:00 +0100 2145090 http://www.medworm.com/index.php?rid=2145089&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_15 We describe a general approach for the use of recombinant protease inhibitors as stabilizing agents for clinically useful proteins extracted from transgenic plant tissues. A procedure is first described to assess the overall (in)stability of heterologous proteins in transgenic plant crude protein extracts. Step-by-step protocols are then presented for the choice and use of companion protease inhibitors inhibiting the host plant proteases during extraction. This strategy, that reproduces the protein-stabilizing effect of low-molecular-weight protease inhibitors often added to protein extraction media, does not require the exogenous addition of such expensive and often toxic compounds. It also presents the advantage of being intrinsically scalable to the amount of biomass processed. (Source:... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145089 Sun, 01 Jun 2008 04:00:00 +0100 2145089 http://www.medworm.com/index.php?rid=2145088&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_14 N-glycosylation is a maturation event necessary for the correct function, efficiency, and stability of a high number of biopharmaceuticals. This chapter presented here proposes various methods to determine whether, how, and where a plant pharmaceutical is N-glycosylated. These methods rely on blot detection with glycan-specific probes, specific deglycosylation of glycoproteins followed by mass spectrometry, N-glycan profile analysis, and glycopeptide identification by LC-MS. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145088 Sun, 01 Jun 2008 04:00:00 +0100 2145088 http://www.medworm.com/index.php?rid=2145087&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_13 Recombinant antibody therapeutics represent a significant success story in terms of clinical benefit delivered and revenue (profit) generated within the biopharmaceutical industry. Additionally, it is estimated that ̃30% of new drugs likely to be licensed during the next decade will be based on antibody products. High volume production with the maintenance of structural and functional fidelity of these large biological molecules results in high “cost of goods” that can limit their availability to patients, due to the strain it puts on national and private health budgets. The challenge in reducing cost of goods is that each antibody is unique, both in structure and function. Optimal clinical efficacy will require engineering of antibody genes to deliver products with enhanc... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145087 Sun, 01 Jun 2008 04:00:00 +0100 2145087 http://www.medworm.com/index.php?rid=2145086&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_12 Over the last decade, plant-based production of pharmaceuticals has made remarkable progress as the expression of a diverse set of proteins has been demonstrated in a range of plant crops. Although the commercial exploitation is still pending, today various plant-based expression technologies have reached significant milestones through clinical testing in humans. Each of the protein manufacturing platforms in plants has specific benefits and drawbacks. We have engaged in comparing some of these production systems with respect to their performance: protein yield and quality. Using a specific tester protein (aprotinin), it was shown that functional aprotinin can be manufactured in plants in substantial amounts, as illustrated in this chapter. (Source: Springer protocols feed by Plant Science... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145086 Sun, 01 Jun 2008 04:00:00 +0100 2145086 http://www.medworm.com/index.php?rid=2145085&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_11 We present a novel approach to produce recombinant proteins in plants based on the ability of γ zein-Zera domain to store recombinant proteins inside PBs. Zera domain fused to several proteins, including a enhanced cyan fluorescent protein (ECFP), calcitonin (Ct) and epidermal growth factor (EGF), were cloned into vectors for transient or stable transformation of tobacco plants. In tobacco leaves, we observed the formation of dense, ER-localized structures containing high concentrations of the respective target proteins. The intact synthetic organelles containing Zera fusions were readily isolated from cellular material using density-based separation methods. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145085 Sun, 01 Jun 2008 04:00:00 +0100 2145085 http://www.medworm.com/index.php?rid=2145084&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_10 Many vaccine antigens and biopharmaceutical proteins have been expressed at high levels via the chloroplast genome and their functionality has been evaluated using in vitro assays in cell cultures (i.e., macrophage lysis assay, inhibition of vesicular stomatitis virus-induced cytopathicity in baby hamster kidney cells, or inhibition of human HIV infection in TZM-BL cells) as well as protection after challenge with bacterial or viral pathogens or antitumor assays or delay the onset of insulitis in suitable animal models. Production of therapeutic proteins in chloroplasts eliminates the expensive fermentation technology. Moreover, oral delivery of chloroplast-derived therapeutic proteins eliminates expensive purification steps, cold storage, cold transportation, and delivery via sterile need... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145084 Sun, 01 Jun 2008 04:00:00 +0100 2145084 http://www.medworm.com/index.php?rid=2145083&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-407-0_1 Plants were the main source for human drugs until the beginning of the nineteenth century when plantderived pharmaceuticals were partly supplanted by drugs produced by the industrial methods of chemical synthesis. During the last decades of the twentieth century, genetic engineering has offered an alternative to chemical synthesis, using bacteria, yeasts and animal cells as factories for the production of therapeutic proteins. After a temporary decrease in interest, plants are rapidly moving back into human pharmacopoeia, with the recent development of plant-based recombinant protein production systems offering a safe and extremely cost-effective alternative to microbial and mammalian cell cultures. In this short review, we will illustrate that current improvements in plant expression syst... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=2145083 Sun, 01 Jun 2008 04:00:00 +0100 2145083 http://www.medworm.com/index.php?rid=1554951&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_31 This chapter describes techniques for in vivo imaging of fluorescent fusion proteins in living cells by confocal laser scanning microscopy (CLSM). Methods are provided for (i) producing the constructs for transient expression from plasmids or virus-based vectors, (ii) introduction of constructs to plant epidermal cells; (iii) imaging of the expressed proteins by CLSM and image processing, and (iv) studying the expression in the presence of agents that affect the integrity or function of cytoskeletal elements. Notes are provided to aid comprehension and indicate problems. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1554951 Tue, 01 Apr 2008 04:00:00 +0100 1554951 http://www.medworm.com/index.php?rid=1554950&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_29 Yeast two-hybrid systems are powerful tools to identify novel protein–protein interactions and have been extensively used to study viral protein interactions. The most commonly used systems are GAL4-based and LexA-based systems. Over the last decade, a range of modifications and improvements have been made to the original yeast two-hybrid system to expand the scope of molecular interaction assays and to eliminate false positives. Detailed protocols are provided for yeast strain storage, yeast transformation, yeast mating, preparation of growth and selection medium, quantitative reporter gene assays (α- and β-galactosidase liquid assays) and detection of fusion protein by Western blot. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1554950 Tue, 01 Apr 2008 04:00:00 +0100 1554950 http://www.medworm.com/index.php?rid=1554949&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_26 We report here on the use of a novel series of binary vectors for the transient or stable expression of autofluorescent protein fusions in plants. Use of these vectors in conjunction with advanced microscopy techniques such as fluorescent recovery after photobleaching and total internal fluorescence microscopy, has revealed novel insight into the membrane and protein dynamics of virus-infected cells. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1554949 Tue, 01 Apr 2008 04:00:00 +0100 1554949 http://www.medworm.com/index.php?rid=1554948&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_23 RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and animals. To counteract RNA silencing, viruses evolved silencing suppressors that interfere with siRNA guided RNA silencing pathway. We used the heterologous Drosophila in vitro embryo RNA to analyze the molecular mechanism of suppression of silencing suppressors. We found that different silencing suppressors inhibit the RNA silencing via binding to siRNAs. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, suppressors uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation. Here, we provide the protocol for the detailed analysis of p19 silencing suppressors of tombusviruses in ... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1554948 Tue, 01 Apr 2008 04:00:00 +0100 1554948 http://www.medworm.com/index.php?rid=1554947&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_22 Movement proteins (MPs) are virally encoded factors that mediate transport of viral nucleic acid between plant cells. Many MPs are able to move between cells themselves. This feature serves as the basis for evaluation of the transport activity of individual MPs. MPs are transiently expressed as a fusion to autofluo-rescent proteins such as green fluorescent protein (GFP) in individual epidermal cells of leaves by biolistic delivery. Expressing cells can be directly monitored for subcellular localization and cell-to-cell movement of the MP:GFP fusion protein into neighboring cells by confocal scanning microscopy. During the time frame of transient expression, numerous cells are evaluated at several time points, and the accumulated data are depicted in a graph termed “movement profile.... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1554947 Tue, 01 Apr 2008 04:00:00 +0100 1554947 http://www.medworm.com/index.php?rid=1554946&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_20 RNA–protein interactions control viral RNA replication, transcription, translation, and particle assembly. Progress toward understanding the functional significance of RNA–protein complexes in the viral life cycle is hindered by the lack of high resolution structural information. Challenges to acquiring structural data include RNA's inherent instability and conformational plasticity, coupled with the comparatively high cost of generating large quantities of RNA for biophysical experiments. The potential for successful structure determination is increased by conducting biochemical experiments that outline interacting domains and identify key residues. These approaches are aimed at defining and characterizing RNA and protein substrates that are suitable for high resolution struct... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1554946 Tue, 01 Apr 2008 04:00:00 +0100 1554946 http://www.medworm.com/index.php?rid=1554945&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_2 Coat proteins (CPs) of all plant viruses have an early function in disassembly of parental virus and a late function in assembly of progeny virus. Depending on the virus, however, CPs may play a role in many steps of the infection cycle in between these early and late functions. It has been shown that CPs can play a role in translation of viral RNA, targeting of the viral genome to its site of replication, cell-to-cell and/or systemic movement of the virus, symptomatology and virulence of the infection, activation of Rgene-mediated host defenses, suppression of RNA silencing, interference with suppression of RNA silencing, and determination of the specificity of virus transmission by vectors. These functions are reviewed in this chapter. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1554945 Tue, 01 Apr 2008 04:00:00 +0100 1554945 http://www.medworm.com/index.php?rid=1554944&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_19 Replication of the viral RNA genome performed by the viral replicase is the central process during the viral infection cycle (Nagy and Pogany, see earlier chapter four). Most RNA viruses assign one or more proteins translated from their own genomes for assembling the viral replicase complex, which consists of the viral RNA, viral proteins, and several subverted host proteins embedded in cellular membranes. Understanding the various biochemical activities of the replication proteins can lead to target identification for human intervention to control viral infections or the damage to the host cells. The replicase proteins of tomato bushy stunt virus (TBSV) are selected as model system to study the dynamics of interactions between viral replicase proteins using surface plasmon resonance (SPR)... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1554944 Tue, 01 Apr 2008 04:00:00 +0100 1554944 http://www.medworm.com/index.php?rid=1554943&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_17 Single-stranded RNA plant viruses not only code for viral proteins within their RNA genomes, they often maintain elaborate RNA secondary structures. These structures can be integral to a variety of viral processes, such as viral translation, genome replication, subgenomic mRNA transcription, and genome encapsidation. RNA secondary structures may function to recruit and bind trans-acting protein factors, or may become part of higher order tertiary RNA structures, which themselves may be functionally relevant. To fully understand such viral RNA elements and their mechanisms of action, it is necessary to first determine their secondary structures. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1554943 Tue, 01 Apr 2008 04:00:00 +0100 1554943 http://www.medworm.com/index.php?rid=1554942&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_16 During their infection in plants, viruses can form double stranded (ds) RNA structures. These dsRNAs can be recognized by plants as “aberrant” signals and short interfering RNA (siRNA) molecules of 19–25 nt will be produced with sequences derived from the viral source. Knowledge about antiviral siRNA profiles including siRNA size, distribution, polarity, etc. provides valuable insights to plant-virus interactions. In this chapter, we describe a simple method for cloning siRNA from virus-infected plants. This protocol includes isolation of small RNAs, their ligation to a pair of 5′ and 3′ adapters, RT-PCR/PCR amplification, and subsequent concatamerization before pGEM-T cloning and sequencing. Concatamers containing as many as 15 small RNA inserts can be produc... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1554942 Tue, 01 Apr 2008 04:00:00 +0100 1554942 http://www.medworm.com/index.php?rid=1554941&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_12 Viroids, as a consequence of not encoding any protein, are extremely dependent on their hosts. Replication of these minimal genomes, composed exclusively by a circular RNA of 246–401 nt, occurs in the nucleus (family Pospiviroidae) or in the chloroplast (family Avsunviroidae) by an RNA-based rolling-circle mechanism with three steps: (1) synthesis of longer-than-unit strands catalyzed by host DNA-dependent RNA polymerases recruited and redirected to transcribe RNA templates, (2) cleavage to unit-length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and (3) circularization through an RNA ligase or autocatalytically. This consistent but still fragmentary picture has emerged from a combination of studies with in vitro systems (analysis of RNA preparations from infec... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1554941 Tue, 01 Apr 2008 04:00:00 +0100 1554941 http://www.medworm.com/index.php?rid=1539238&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_38 We present here a brief overview of the recent applications of this method and a detailed protocol for agroinoculation and VIGS used in our laboratory. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539238 Tue, 01 Apr 2008 04:00:00 +0100 1539238 http://www.medworm.com/index.php?rid=1539237&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_43 Host factors are crucial determinants of viral pathogenicity. Identifying host factors and their contributions to virus infections may lead to the development of novel antiviral strategies. The recently developed virus-induced gene silencing (VIGS) approach offers a rapid means to knock down expression of a given gene in plants. VIGS can be used to determine biological function of candidate genes or to discover new genes that play a role in a given biological pathway. Here, we describe genome-wide Tobacco rattle virus (TRV)-based VIGS screening methods to identify host factors involved in viral pathogenicity. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539237 Tue, 01 Apr 2008 04:00:00 +0100 1539237 http://www.medworm.com/index.php?rid=1539236&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_11 The Begomovirus genus is the largest genus of the Geminiviridae family and comprises the whitefly transmitted geminiviruses that infect dicotyledonous plants. They can be either mono or bipartite. In this chapter, we describe the cloning of begomovirus replication modules and the subsequent functional characterization of geminivirus replication origins. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539236 Tue, 01 Apr 2008 04:00:00 +0100 1539236 http://www.medworm.com/index.php?rid=1539235&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_1 A variety of techniques have been used to examine plant viral genomes, the functions of virus-encoded proteins, plant responses induced by virus infection and plant–virus interactions. This overview considers these technologies and how they have been used to identify novel viral and plant proteins or genes involved in disease and resistance responses, as well as defense signaling. These approaches include analysis of spatial and temporal responses by plants to infection, and techniques that allow the expression of viral genes transiently or transgenically in planta, the expression of plant and foreign genes from virus vectors, the silencing of plants genes, imaging of live, infected cells, and the detection of interactions between viral proteins and plant gene products, both in plant... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539235 Tue, 01 Apr 2008 04:00:00 +0100 1539235 http://www.medworm.com/index.php?rid=1539234&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_14 The ability to combine nucleic acid hybridisation or immunospecific reactions with structural and ultrastructural analysis of virus-infected tissues has provided the opportunity to resolve the spatial details of infection with respect to the production of virus-specific products and the nature of the host response. These technologies may seem lengthy and complex but offer high rewards in terms of revealing the details of host—virus interactions not otherwise accessible. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539234 Tue, 01 Apr 2008 04:00:00 +0100 1539234 http://www.medworm.com/index.php?rid=1539233&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_5 RNA silencing suppressors, developed by plant viruses, are potent arms in the arm race between plant and invading viruses. In higher plants, these proteins efficiently inhibit RNA silencing, which has evolved to defend plants against viral infection in addition to regulation of gene expression for growth and development Virus-encoded RNA-silencing suppressors interfere with various steps of the different silencing pathways and the mechanisms of suppression are being progressively unraveled. Our better understanding of action of silencing suppressors at molecular level dramatically improved our basic knowledge about the intimate plant-virus interactions and also provided valuable tools to unravel the diversity, regulation, and evolution of RNA-silencing pathways. (Source: Springer protocols... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539233 Tue, 01 Apr 2008 04:00:00 +0100 1539233 http://www.medworm.com/index.php?rid=1539232&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_4 Identification of the roles of replication factors represents one of the major frontiers in current virus research. Among plant viruses, the positive-stranded (+) RNA viruses are the largest group and the most widespread. The central step in the infection cycles of (+) RNA viruses is RNA replication, which leads to rapid production of huge number of viral (+) RNA progeny in the infected plant cells. The RNA replication process is carried out by the virus-specific replicase complex consisting of viral RNA-dependent RNA polymerase, one or more auxiliary viral replication proteins, and host factors, which assemble in specialized membranous compartments in infected cells. Replication is followed by cell-to-cell and long- distance movement to invade the entire plant and/or encapsidation to faci... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539232 Tue, 01 Apr 2008 04:00:00 +0100 1539232 http://www.medworm.com/index.php?rid=1539231&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_15 Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) play crucial roles in establishing general host defense mechanisms against viral infections in plants and the development of disease symptoms. Understanding these fundamental processes requires the sensitive and specific detection of small RNA species. However, because of the small size of miRNAs and siRNAs, their detection is technically demanding. Here, we describe methods for robust and sensitive detection of small RNAs by Northern blot analysis and in situ hybridization. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539231 Tue, 01 Apr 2008 04:00:00 +0100 1539231 http://www.medworm.com/index.php?rid=1539230&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_30 Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to study the three-dimensional structure of proteins and nucleic acids at atomic resolution. Since the NMR data can be recorded in solution, conditions such as pH, salt concentration, and temperature can be adjusted so as to closely mimic the biomacromolecules natural milieu. In addition to structure determination, NMR applications can investigate time-dependent phenomena, such as dynamic features of the biomacromolecules, reaction kinetics, molecular recognition, or protein folding. The advent of higher magnetic field strengths, new technical developments, and the use of either uniform or selective isotopic labeling techniques, currently allows NMR users the opportunity to investigate the tertiary structure of biomacromolecu... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539230 Tue, 01 Apr 2008 04:00:00 +0100 1539230 http://www.medworm.com/index.php?rid=1539229&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_40 Since their conception in the late 1990s, microarray techniques have become a tool of choice for monitoring pangenomic gene expression. Although there are a large number of variations on the basic methodology the general approach remains standard and involves the comparison of a “test” RNA with a “control” RNA; in this case “healthy” and “virus-infected” plants. The protocol itself can be broken down into five main parts: RNA extraction, cDNA synthesis, hybridization, array scanning, and data analysis. The method presented is optimized for use with arrays based on glass slides spotted with cDNA, in this case 15,264 cDNAs from Solanum tuberosum. The labeling technique presented involves two steps: hybridization of cDNA produced using oligo-dT ... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539229 Tue, 01 Apr 2008 04:00:00 +0100 1539229 http://www.medworm.com/index.php?rid=1539228&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_18 Analysis of viral RNA encapsidation assay provides a rapid means of assaying which of the progeny RNA are competent for packaging into stable mature virions. Generally, a parallel analysis of total RNA and RNA obtained from purified virions is advisable for accurate interpretation of the results. In this, we describe a series of in vivo assays in which viral RNA encapsidation can be verified. These include whole plants inoculated either mechanically or by Agroinfiltration and protoplasts. The encapsidation assay described here is for an extensively studied plant RNA virus, brome mosaic virus, and can be reliably applied to other viral systems as well as with appropriate buffers. In principle, the encapsidation assay requires purification of virions from either symptomatic leaves or transfe... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539228 Tue, 01 Apr 2008 04:00:00 +0100 1539228 http://www.medworm.com/index.php?rid=1539227&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_21 Most plant viruses move between plant cells with the help of their movement proteins (MPs). MPs are multifunctional proteins, and one of their functions is almost invariably binding to nucleic acids. Presumably, the MP—nucleic acid interaction is directly involved in formation of nucleoprotein complexes that function as intermediates in the cell-to-cell transport of many plant viruses. Thus, when studying a viral MP, it is important to determine whether or not it binds nucleic acids, and to characterize the hallmark parameters of such binding, i.e., preference for single- or double-stranded nucleic acids and binding cooperativity and sequence specificity. Here, we present two major experimental approaches, native gel mobility shift assay and ultra violet (UV) light cross-linking, for... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539227 Tue, 01 Apr 2008 04:00:00 +0100 1539227 http://www.medworm.com/index.php?rid=1539226&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_33 Reverse genetic approach is widely used in virology as it makes possible direct identification of viral gene function and uses RNA genomes as vectors. Production of infectious cDNA clones is an essential step in developing a reverse genetic system for an RNA virus. Here, we present rapid method for generation of infectious cDNA clone for Turnip crinkle virus (TCV). The infectious cDNA clone could be used for production of in vitro transcripts with the T7 RNA polymerase which could be used for infection of plants or plant cell protoplasts. The procedure described here includes purification of TCV, viral RNA extraction, reverse transcription, PCR amplification of the full-length cDNA copy of TCV linked to a T7 RNA polymerase promoter, cloning into a plasmid vector, in vitro transcription, an... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539226 Tue, 01 Apr 2008 04:00:00 +0100 1539226 http://www.medworm.com/index.php?rid=1539225&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_25 Replication of the genome of positive-strand RNA plant viruses takes place in membrane-bound complexes that contain viral replicase proteins, viral RNA, and host proteins. Many viral replicase proteins play a crucial role in the assembly of replication complexes at intracellular membranes. They are integral membrane proteins that interact directly with the membranes and bring other proteins and the viral RNA to the complex via protein—protein or protein—RNA interactions. In this chapter, we describe subcellular fractionation methods that determine whether viral proteins are integral membrane proteins in planta. Differential centrifugation techniques are used to produce membrane-enriched fractions, which can then be analyzed for the presence of viral replicase proteins by immuno... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539225 Tue, 01 Apr 2008 04:00:00 +0100 1539225 http://www.medworm.com/index.php?rid=1539224&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_37 Apple latent spherical virus (ALSV) expressing green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP) was constructed and used to analyze the local and systemic movement of the virus in infected plants. In Chenopodium quinoa plants inoculated with GFP-ALSV, the infection foci first appeared as small fluorescent spots 2–3 days post inoculation (dpi). The GFP spots expanded as rings from 5 dpi, then fused to each other, and most fluorescence faded out at 10–12 dpi. In upper uninoculated leaves, GFP fluorescence was first observed 6–7 dpi on the basal area of mature leaves and on the entire area of young developing leaves. The appearance of fluorescent flecks on young leaves was first found on and near the class III and IV veins. ALSV labeled with two different flu... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539224 Tue, 01 Apr 2008 04:00:00 +0100 1539224 http://www.medworm.com/index.php?rid=1539223&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_32 The generation of infectious clones is routinely the first step for reverse genetic studies of RNA plant virus gene and sequence function. The procedure given here, details the creation of cDNA clones of tobacco mosaic virus, from which infectious transcripts can be generated in vitro with T7 RNA polymerase. The procedure describes methods for virion purification, viral RNA extraction, reverse transcription, PCR amplification of genomic cDNA fragments, generation of a full-length cDNA clone under the control of a T7 promoter, in vitro transcription, and infectivity testing. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539223 Tue, 01 Apr 2008 04:00:00 +0100 1539223 http://www.medworm.com/index.php?rid=1539222&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_42 Plant viruses encode movement proteins (MPs) which play important roles in spreading their infectious materials throughout host plants. This infection is facilitated by cell-to-cell trafficking of MPs through specialized channels termed plasmodesmata, which involves specific interactions between MPs and host factors. Recently, we have reported the identification of a host protein kinase named plasmodesmal-associated protein kinase (PAPK) which specifically phosphorylates a subset of noncell autonomous proteins in vitro, including MPs of Tobacco mosaic virus (TMV) and Bean dwarf mosaic virus (BDMV). Biochemical purification of PAPK was achieved by developing a method in which a series of liquid chroma-tographic separations of plasmodesmal-enriched subcellular fractions was coupled with phos... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539222 Tue, 01 Apr 2008 04:00:00 +0100 1539222 http://www.medworm.com/index.php?rid=1539221&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_24 Posttranslational modification of proteins is a key regulatory mechanism in a variety of cellular processes. This chapter outlines the concepts and methods used to investigate protein phosphorylation and its physiological relevance during plant virus infection. Rather than providing an exhaustive review of the experimental protocols for protein phosphorylation analysis, we focus on methods that can be used to study phosphorylation of viral proteins. We address the following points: how to determine that a viral protein of interest is phosphorylated; how to map the phosphorylation sites; how to identify the protein kinase(s) involved. Finally, we describe a number of useful strategies to evaluate the biological significance of phosphorylation. (Source: Springer protocols feed by Plant Scien... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539221 Tue, 01 Apr 2008 04:00:00 +0100 1539221 http://www.medworm.com/index.php?rid=1539220&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_10 The technique described was developed for the separation of begomovirus DNA. DNA products resulting from and during geminiviral replication are characterized by the application of strand-specific separation and identification by strand-specific DNA probing of Southern blots. The mapping of the initiation site of complementary-strand DNA synthesis, by this technique is also presented. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539220 Tue, 01 Apr 2008 04:00:00 +0100 1539220 http://www.medworm.com/index.php?rid=1539219&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_27 This chapter introduces an efficient and accurate site-directed mutagenesis protocol, which allows the color selection of mutants through the simultaneous activation or deactivation of the α-peptide of β-galactosidase. It uses doublestranded plasmid DNA as the mutational template. This protocol can efficiently create mutations of large inserts at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. Thus, constructs containing whole open-reading frames and whole viral genomes can be subjected to site-directed mutagenesis and used for subsequent functional studies. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539219 Tue, 01 Apr 2008 04:00:00 +0100 1539219 http://www.medworm.com/index.php?rid=1539218&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_9 Plant RNA viruses exploit nonorthodox strategies, such as the use of internal ribosomal entry sites (IRES), to express multiple genes from a single RNA species. IRES elements have been reported in tobacco etch virus (TEV), crucifer infecting tobamovirus (crTMV), hibiscus chlorotic ringspot virus (HCRSV), and many other animal and plant RNA viruses. In this chapter, the methodology used to identify and characterize a plant virus IRES element, including construction of a translation reporter vector for testing the IRES activity, testing the IRES activity in coupled in vitro transcription and translation systems and mammalian cells analysis of RNA stability, and sucrose gradient analysis and polysome profiling, is presented. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539218 Tue, 01 Apr 2008 04:00:00 +0100 1539218 http://www.medworm.com/index.php?rid=1539217&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_13 The interaction between viral polymerases and their cognate RNAs is vital to regulate the timing and abundance of viral replication products. Despite this, only minimal detailed information is available for the interaction between viral polymerases and cognate RNAs. We study the biochemical interactions using two viral polymerases that could serve as models for other plus-strand RNA viruses: the replicase from the tripartite brome mosaic virus (BMV), and the recombinant RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV). Replicase binding sites in the BMV RNAs were mapped using a template competition assay. The minimal length of RNA required for RNA binding by the HCV RdRp was determined using fluorescence spectroscopy. Lastly, regions of the HCV RdRp that contact the RNA wer... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539217 Tue, 01 Apr 2008 04:00:00 +0100 1539217 http://www.medworm.com/index.php?rid=1539216&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_3 Plant viruses spread from the initially infected cells to the rest of the plant in several distinct stages. First, the virus (in the form of virions or nucleic acid protein complexes) moves intracellularly from the sites of replication to plasmodesmata (PD, plant-specific intercellular membranous channels), the virus then transverses the PD to spread intercellularly (cell-to-cell movement). Long-distance movement of virus occurs through phloem sieve tubes. The processes of plant virus movement are controlled by specific viral movement proteins (MPs). No extensive sequence similarity has been found in MPs belonging to different plant virus taxonomic groups. Moreover, different MPs were shown to use different pathways and mechanisms for virus transport. Some viral transport systems require a... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539216 Tue, 01 Apr 2008 04:00:00 +0100 1539216 http://www.medworm.com/index.php?rid=1539215&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_34 To characterize a virus at the molecular and biological levels, it is necessary to produce an infectious clone. For most of the Geminiviridae, cloning of the genome is relatively easy because of their small genomes and the presence of the virus double-stranded (replicative) DNA form in infected plants. Indeed, the presence of conserved sequences between species in the genera Begomovirus, Curtovirus, and Topocuvirus allows the PCR amplification of most genomes using degenerate “universal” primers. Unlike the other genera, no universal primers are reported that are suitable for all mastreviruses and alternative, more time-consuming methods must be used. (Source: Springer protocols feed by Plant Sciences) Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539215 Tue, 01 Apr 2008 04:00:00 +0100 1539215 http://www.medworm.com/index.php?rid=1539214&cid=s_37130_62_f&fid=37130&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-102-4_28 Maize streak virus (MSV) genome has four open reading frames. C1 and C2 encoded by the complementary sense are required for virus replication, while V1 and V2 encoded by virion sense are required for infectivity. V1 encodes movement protein (MP), while V2 encodes coat protein (CP). Deletion or mutation of MSV CP does not prevent virus replication in single cells or protoplasts but leads to a loss of infectivity in the inoculated plant suggesting that MSV CP is required for virus movement. Towards understanding the role of MSV CP and MP in virus movement, the interaction of MSV CP and MP with viral DNA was investigated using the South-western assay. Wild type and truncated MSV CPs and MP were expressed in E. coli and the expressed CPs and MP were used to investigate interaction with single-... Springer protocols feed by Plant Sciences info http://www.medworm.com/rss/comments.php?id=1539214 Tue, 01 Apr 2008 04:00:00 +0100 1539214