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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 methods to silence genes in plant-parasitic cyst and root-knot nematodes. [ABSTRACT FROM AUTHOR]

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 procedures are generally applicable to any research program where the production of soluble recombinant protein in E. coli has proven difficult, or where there is a desire to evaluate E. coli thoroughly as a host before considering alternative hosts for heterologous expression. [ABSTRACT FROM AUTHOR]

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-invasive luciferase reporter assay and a simple method to detect activated Mitogen-Activated Protein Kinases (MAPKs) to identify and study, in a medium-throughput manner, new effectors suppressing early signal transduction events of MTI. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 asexual reproductive structures called sporangiophores in the compatible interaction between the Hpa isolate Noks1 and the Col-0 Arabidopsis accession. [ABSTRACT FROM AUTHOR]

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 identification of effector candidates, including OrthoMCL analysis to identify expanded gene families, are also described. [ABSTRACT FROM AUTHOR]

Although the discovery of the first oncogenic retrovirus was made almost 100 years ago, considerable insights into the specific host immunity involved in anti-tumor and anti-viral responses during acute and chronic infection with oncogenic retroviruses have been achieved only in the last few decades. Human T-cell lymphotropic virus 1 (HTLV-1) is the only known human oncogenic retrovirus, yet several animal retroviruses have provided knowledge of the mechanisms by which host immunity controls retroviral spread and the sequelae, such as tumor induction; as well as viral escape mechanisms, including mutation of virus-specific antigens and virus-induced immunosuppression of B-and T-cell responses. This chapter will cover advances in the understanding of the immune responses to HTLV-1 and its associated adult T-cell leukemia (ATL), after first examining the cell- and antibody-mediated responses to the AKR/Gross, Friend, and the LP-BM5 murine retroviruses and their tumors. [ABSTRACT FROM AUTHOR]

In 2005, a draft sequence of Magnaporthe oryzae isolate 70–15 genome was published (Dean et al. 2005). Complete rice genome sequence was also published in the same year (International Rice Genome Sequencing Project, 2005). As a result, study of the Magnaporthe-rice interaction has entered the ˵post-genomics″ era. The challenge now is how to make use of this large amount of information to improve our understanding of this interaction. By employing genomics information, we are trying to identify and characterize Magnaporthe effectors directly involved in molecular interactions between the pathogen and the host. In this paper, we present a short review of phytopathogen effectors and Magnaporthe avirulence factors. We then briefly describe our current approach to identify new effectors from Magnaporthe. [ABSTRACT FROM AUTHOR]

Several plant pathogenic oomycetes have been under investigation using modern molecular approaches. Genome sequencing and annotations are underway or near to completion for some of the species. Pathogen-associated molecular pattern molecules (PAMPs) and effector molecules perform inter- and intracellular tasks as adaptation factors and manipulators of the defence network. Hundreds of secreted putative effectors have been discovered and conserved molecular patterns such as RXLR and EER motifs have been identified and used for classifications. PAMPs and effectors are recognized directly or indirectly by the pattern recognition receptors at the cell surface including receptor-like kinases and receptor-like proteins, and/or by nucleotide binding site–leucine rich repeat proteins within the cytoplasm. The current knowledge of effectors, immune receptors and the defence network, will help us understand the `intricate genetic dance΄ between the oomycete pathogens and their hosts. This review concentrates on the recent findings in oomycete-plant interactions. [ABSTRACT FROM AUTHOR]

Recognition of non-self in plants is mediated by specialised receptors that upon pathogen perception trigger induction of host defence responses. Primary, or basal, defence is mainly triggered by trans-membrane receptors that recognise conserved molecules released by a variety of (unrelated) microbes. Pathogens can overcome these basal defences by the secretion of specific effectors. Subsequent recognition of these effectors by specialised receptors (called resistance proteins) triggers induction of a second layer of plant defence responses. These responses are qualitatively similar to primary defence responses; however, they are generally faster and stronger. Here we give an overview of the predicted (domain) structures of resistance proteins and their proposed mode of action as molecular switches of plant innate immunity. We also highlight recent advances revealing that some of these proteins act in the plant nucleus as transcriptional co-regulators and that crosstalk can occur between members of different resistance protein families. [ABSTRACT FROM AUTHOR]