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.Quantification of Plant Resistance to Isoprenoid Biosynthesis Inhibitors
Plants use two pathways for the production of the same universal isoprenoid precursors: the mevalonic acid (MVA) pathway and the methylerythritol 4-phosphate (MEP) pathway. Inhibitors of the MVA pathway prevent the activity of the shoot apical meristem and the development of true leaves in seedlings, whereas those inhibiting the MEP pathway show an additional bleaching phenotype. Here, we describe two methods to quantify plant resistance to inhibitors of the MVA pathway or the MEP pathway based on seedling establishment and photosynthetic pigment measurements. Although the methods are presented for Arabidopsis, they are va...
Source: Springer protocols feed by Plant Sciences - May 1, 2014 Category: Biology Source Type: news
Genomic DNA Library Preparation for Resistance Gene Enrichment and Sequencing (RenSeq) in Plants
Enrichment of genomic DNA for genes of interest prior to high-throughput sequencing offers an efficient and cost-effective approach to reduce genome complexity. Target enrichment typically yields higher read-depth for selected genes and is therefore suitable for determination of sequence polymorphisms and enables multiplexing of samples. Target enrichment also provides a means to annotate specific gene families within the sequenced organisms without the requirements for gene models. Here we describe enrichment procedures for NB-LRR-type plant resistance genes that can, for example, be used to establish the NB-LRR gene comp...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
A Bacterial Type III Secretion-Based Delivery System for Functional Assays of Fungal Effectors in Cereals
Large numbers of candidate effectors are being identified by genome sequencing of fungal pathogens and in planta expression studies. These effectors are both a boon and a curse for pathogens as they modulate the host cellular environment or suppress defense response to allow fungal growth as well as become targets of plant resistance (R) proteins. Recognition of a fungal effector by a plant R protein triggers a hypersensitive reaction (HR) leading to death of plant cells in and around the infection site, thus preventing further proliferation of the pathogen. Such HR induction has been used as an indicator of effector activ...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Protoplast Cell Death Assay to Study Magnaporthe oryzae AVR Gene Function in Rice
We describe a protocol for transient gene expression in rice protoplasts and its application to the study of Magnaporthe oryzae avirulence (AVR) gene function. In this assay the gene encoding the firefly luciferase protein is transfected into rice protoplasts by electroporation together with the candidate AVR genes. The luminescence can then be used to assess the viability of rice protoplasts. The hypersensitive response (HR) caused by the interaction between M. oryzae AVR and rice R genes can subsequently be monitored by recording the decrease in luminescence from the transfected cells. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
The Do’s and Don’ts of Effectoromics
Effectoromics, a high-throughput functional genomics approach that uses effectors to probe plant germplasm to detect R genes, has proven a potent contribution to modern resistance breeding. Advantages of effectoromics are summarized in four aspects: (1) accelerating R gene identification; (2) distinguishing functional redundancy; (3) detecting recognition specificity and (4) assisting in R gene deployment. In this manuscript, we provide suggestions as well as some reminders for applying effectoromics in the breeding process. The two routine functional assays that are widely used, agroinfiltration and agroinfection, are pre...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Functional Characterization of Nematode Effectors in Plants
Secreted effectors represent the molecular interface between the nematode and its host plant. Studies that aimed at deciphering molecular plant–nematode interactions are hampered by technical hurdles that prevent the generation of transgenic nematodes. However, RNA interference (RNAi) has proven to be a valuable tool to specifically knock-down nematode effector genes, both ex planta and in planta. Plant-mediated RNAi of nematode genes not only facilitates functional characterization of effectors but also lends itself to a novel control strategy against plant-parasitic nematodes. Here, we describe currently used metho...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Production of RXLR Effector Proteins for Structural Analysis by X-Ray Crystallography
Structural analysis of RXLR effector proteins from oomycete plant pathogens is an emerging area of research. These studies are aimed at understanding the molecular basis of how these proteins manipulate plant cells to promote infection and also to help define how they can lead to activation of the plant innate immune system. Here, we describe a medium-throughput procedure for cloning and expression testing oomycete RXLR proteins in Escherichia coli. We also describe methods for purification of soluble protein and crystallization, with the aim of determining three-dimensional structures by X-ray crystallography. The procedu...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
An Arabidopsis and Tomato Mesophyll Protoplast System for Fast Identification of Early MAMP-Triggered Immunity-Suppressing Effectors
Transient expression in plant mesophyll protoplasts allows rapid characterisation of gene functions in vivo in a simplified and synchronized manner without bias due to the use of bacteria-based gene or protein delivery systems. It offers the possibility to test whether microbial effectors can subvert early events of plant immune signaling that are activated upon recognition of Microbe-Associated Molecular Patterns (MAMPs), the so-called MAMP-triggered immunity (MTI). Here, we describe the isolation and transfection with effector genes of Arabidopsis thaliana and Solanum lycopersicum mesophyll protoplasts, the use of a non-...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
A Simple and Fast Protocol for the Protein Complex Immunoprecipitation (Co-IP) of Effector: Host Protein Complexes
Plant pathogens are responsible for enormous damage in natural and cultured ecosystems. One strategy most pathogenic organisms follow is the secretion of effector proteins that manipulate the host immune system to suppress defense responses. There is considerable interest in finding host targets of pathogen effectors as this helps to shape our understanding of how those proteins work in planta. The presented protocol describes a protein complex immunoprecipitation method aimed at verifying protein–protein interactions derived from protein complementation assays like Yeast-two-Hybrid. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
DIGE-ABPP by Click Chemistry: Pairwise Comparison of Serine Hydrolase Activities from the Apoplast of Infected Plants
Activity-based protein profiling (ABPP) is a targeted functional proteomics method that displays the active proteome by using small molecule probes that react covalently with the active sites of protein classes. Comparison of activity profiles from two different samples is not always easy, especially when using probes that generate too many signals. For accurate comparison of protein activities between two proteomes, we developed difference gel electrophoresis ABPP (DIGE-ABPP), which compares two fluorescently labeled proteomes in the same gel lane. This protocol describes the labeling of two proteomes with alkyne-labeled ...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Virus-Induced Gene Silencing and Agrobacterium tumefaciens-Mediated Transient Expression in Nicotiana tabacum
Virus-induced gene silencing (VIGS) is a rapid method for transient silencing of plant genes. In this chapter, we describe the methodology for Tobacco rattle virus (TRV)-based VIGS in Nicotiana tabacum. In combination with subsequent co-expression of the tomato immune receptor Ve1 and the corresponding Verticillium effector Ave1 through Agrobacterium tumefaciens-mediated transient transformation (agroinfiltration), we established a rapid system for assessing the requirement of candidate plant genes for Ve1-mediated immune signaling. (Source: Springer protocols feed by Plant Sciences)
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Simple Quantification of In Planta Fungal Biomass
An accurate assessment of the disease resistance status of plants to fungal pathogens is an essential requirement for the development of resistant crop plants. Many disease resistance phenotypes are partial rather than obvious immunity and are frequently scored using subjective qualitative estimates of pathogen development or plant disease symptoms. Here we report a method for the accurate comparison of total fungal biomass in plant tissues. This method, called the WAC assay, is based upon the specific binding of the plant lectin wheat germ agglutinin to fungal chitin. The assay is simple, high-throughput, and sensitive en...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
A Growth Quantification Assay for Hyaloperonospora arabidopsidis Isolates in Arabidopsis thaliana
There is a considerable interest in determining the role of individual oomycete effectors in promoting disease. Widely used strategies are based on manipulating effector-expression levels in the pathogen and by over-expressing particular effectors in the host by genetic transformation. In the case of the oomycete, Hyaloperonospora arabidopsidis (Hpa) genetic manipulation is not yet possible, so over-expression of predicted effectors in stably transformed Arabidopsis lines is used to investigate their capability for promoting virulence. Here, we describe a technique for quantifying pathogen growth based on the counting of a...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Leaf-Disc Assay Based on Transient Over-Expression in Nicotiana benthamiana to Allow Functional Screening of Candidate Effectors from Aphids
Aphids, like plant pathogens, are known to form close associations with their host. While probing and feeding, these insects deliver effectors inside the host, which are thought to be involved in suppression of host defenses and/or the release of nutrients. With increasing availability of aphid genome and transcriptome sequencing data, effectors can now be identified using bioinformatics- and proteomics-based approaches. The next step is then to apply functional assays relevant to plant–aphid interactions to identify effector activities. This chapter describes an effective and medium-throughput screen for the identif...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
Silencing of Aphid Genes by Feeding on Stable Transgenic Arabidopsis thaliana
Aphids are economically important pests that predominantly feed from the plant phloem. Genome, transcriptome, and proteome data are being generated for these insects, and predicted secreted proteins in aphid saliva have been identified. These secreted proteins are candidate effectors that may modulate plant processes and aid aphid colonization of plants. The next step is to develop post-genomics strategies to study the functions of identified aphid genes. One such strategy is to express aphid effector genes in planta to assess whether aphid effectors alter plant development and aphid survival and fecundity. A second strate...
Source: Springer protocols feed by Plant Sciences - March 19, 2014 Category: Biology Source Type: news
