Springer protocols feed by Plant Sciences 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Purification of Fungal Haustoria from Infected Plant Tissue by Flow Cytometry
A hallmark of biotrophy in many fungal plant pathogens is the formation of a specialized pathogenic structure called the haustorium from infectious hyphae. This is the major parasitic structure, where nutrients are taken up from the host and pathogenicity factors are exported to the host tissue. Obligate biotrophic fungi can typically be cultured in vivo only to a limited extent and do not produce haustoria under these conditions. This has hampered the application of classic molecular biology techniques to haustoria-forming pathogens. The lectin Concanavalin A (Con A), which binds specifically to sugars present on the exte...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Particle Bombardment-Mediated Transient Expression to Identify Localization Signals in Plant Disease Resistance Proteins and Target Sites for the Proteolytic Activity of Pathogen Effectors
Plant pathogens, including fungi, oomycetes, bacteria, aphids, and nematodes, produce a variety of effector proteins to counter plant disease resistance mechanisms. After delivery into the cytosol of the plant cell, effectors may target proteins localized to different compartments within the plant cell. Plants, in turn, have evolved disease resistance (R) proteins to recognize the action of effectors. Elucidation of the subcellular localization of pathogen effectors, the plant proteins they target, and plant disease resistance proteins is essential to fully understand their interactions during pathogen challenge. In recent...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
In Vivo Protein–Protein Interaction Studies with BiFC: Conditions, Cautions, and Caveats
Bimolecular fluorescence complementation (BiFC), performed with suitable controls and the right conditions, can be a straightforward and simple method to assess protein–protein interactions accessible to anyone with basic confocal microscopy skills. It is of course not without its own potential pitfalls and requires specific controls. Here we describe its use to study the interactions between pathogen effector proteins and host proteins inside plant cells. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
High-Throughput Imaging of Plant Immune Responses
Fluorescence confocal microscopy has emerged in the past decade as an important method for studying the cellular changes associated with plant–microbe interactions. One such change is the internalization into endosomes of the cell surface receptor FLAGELLIN SENSING 2 (FLS2) upon activation by its ligand, bacterial flagellin (flg22). Quantification of endosomes containing FLS2 can thus be used as a direct readout of immune response activation at the cellular level. High-throughput imaging of cellular events is routinely applied in chemical screening for pharmaceutical drug discovery, and we have adapted this system fo...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
On the Statistics of Identifying Candidate Pathogen Effectors
High-throughput sequencing is an increasingly accessible tool for cataloging gene complements of plant pathogens and their hosts. It has had great impact in plant pathology, enabling rapid acquisition of data for a wide range of pathogens and hosts, leading to the selection of novel candidate effector proteins, and/or associated host targets (Bart et al., Proc Nat Acad Sci U S A doi:10.1073/pnas.1208003109, 2012; Agbor and McCormick, Cell Microbiol 13:1858–1869, 2011; Fabro et al., PLoS Pathog 7:e1002348, 2011; Kim et al., Mol Plant Pathol 2:715–730, 2011; Kimbrel et al., Mol Plant Pathol 12:580–594, 2011...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Two-Dimensional Data Binning for the Analysis of Genome Architecture in Filamentous Plant Pathogens and Other Eukaryotes
Genome architecture often reflects an organism’s lifestyle and can therefore provide insights into gene function, regulation, and adaptation. In several lineages of plant pathogenic fungi and oomycetes, characteristic repeat-rich and gene-sparse regions harbor pathogenicity-related genes such as effectors. In these pathogens, analysis of genome architecture has assisted the mining for novel candidate effector genes and investigations into patterns of gene regulation and evolution at the whole genome level. Here we describe a two-dimensional data binning method in R with a heatmap-style graphical output to facilitate ...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Bioinformatic Analysis of Expression Data to Identify Effector Candidates
Pathogens produce effectors that manipulate the host to the benefit of the pathogen. These effectors are often secreted proteins that are upregulated during the early phases of infection. These properties can be used to identify candidate effectors from genomes and transcriptomes of pathogens. Here we describe commonly used bioinformatic approaches that (1) allow identification of genes encoding predicted secreted proteins within a genome and (2) allow the identification of genes encoding predicted secreted proteins that are upregulated at important stages of the life cycle. Other approaches for bioinformatic identificatio...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Galaxy as a Platform for Identifying Candidate Pathogen Effectors
The Galaxy web platform provides an integrated system for its users to run multiple computational tools, linking their output in order to perform sophisticated analysis without requiring any programming or installation of software beyond a modern web-browser. Analyses can be saved as reusable workflows, and shared with other Galaxy users, allowing them to easily perform the same analysis or protocol on their own data. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Integration and Expression of gfp in the Plastid of Medicago sativa L.
Here we describe a protocol of alfalfa (Medicago sativa L.) plastid transformation by which gfp, a gene encoding the green fluorescent protein (GFP), is inserted into plastid genome via particle bombardment and homoplastomic plant is obtained. Plastid engineering is likely to make a significant contribution to the genetic improvement of this crop and the production of vaccines and therapeutic proteins. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news
Plastid Transformation in Sugar Beet: Beta vulgaris
We describe here a method to obtain transplastomic sugar beet plants trough biolistic transformation. The availability of sugar beet transplastomic plants should avoid the risk of gene flow between these cultivated genetic modified sugar beet plants and the wild-type plants or relative wild species. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news
Plastid Transformation in Cabbage (Brassica oleracea L. var. capitata L.) by the Biolistic Process
Cabbage (Brassica oleracea L. var. capitata L.) is one of the most important vegetable crops grown worldwide. Scientists are using biotechnology in addition to traditional breeding methods to develop new cabbage varieties with desirable traits. Recent biotechnological advances in chloroplast transformation technology have opened new avenues for crop improvement. In 2007, we developed a stable plastid transformation system for cabbage and reported the successful transformation of the cry1Ab gene into the cabbage chloroplast genome. This chapter describes the methods for cabbage transformation using biolistic procedures. The...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news
Plastid Transformation in Soybean
The biotechnological potential of plastid genetic engineering has been illustrated in a limited number of higher plant species. We have developed a reproducible method to generate plastid transformants in soybean (Glycine max), a crop of major agronomic importance. The transformation vectors are delivered to embryogenic cultures by the particle gun method and selection performed using the aadA antibiotic resistance gene. Homoplasmy is established rapidly in the selected events without the need for further selection or regeneration cycles, and genes of interest can be expressed at a high level in green tissues. This is a si...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news
Plastid Transformation in Lettuce (Lactuca sativa L.) by Biolistic DNA Delivery
The interest in producing pharmaceutical proteins in a nontoxic plant host has led to the development of an approach to express such proteins in transplastomic lettuce (Lactuca sativa L.). A number of therapeutic proteins and vaccine antigen candidates have been stably integrated into the lettuce plastid genome using biolistic DNA delivery. High levels of accumulation and retention of biological activity suggest that lettuce may provide an ideal platform for the production of biopharmaceuticals. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news
Plastid Transformation in Lettuce (Lactuca sativa L.) by Polyethylene Glycol Treatment of Protoplasts
A detailed protocol for PEG-mediated plastid transformation of Lactuca sativa cv. Flora, using leaf protoplasts, is described. Successful plastid transformation using protoplasts requires a large number of viable cells, high plating densities, and an efficient regeneration system. Transformation was achieved using a vector that targets genes to the trnI/trnA intergenic region of the lettuce plastid genome. The aadA gene, encoding an adenylyltransferase enzyme that confers spectinomycin resistance, was used as a selectable marker. With the current method, the expected transformation frequency is 1–2 spectinomycin-resi...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news
Plastid Transformation in Eggplant
Eggplant (Solanum melongena L.) is an important vegetable crop of tropical and temperate regions of the world. Here we describe a procedure for eggplant plastid transformation, which involves preparation of explants, biolistic delivery of plastid transformation vector into green stem segments, selection procedure, and identification of the transplastomic plants. Shoot buds appear from cut ends of the stem explants following 5–6 weeks of spectinomycin selection after bombardment with the plastid transformation vector containing aadA gene as selectable marker. Transplastomic lines are obtained after the regenerated sho...
Source: Springer protocols feed by Plant Sciences - March 6, 2014 Category: Biology Source Type: news
