Showing papers by "Jorunn I. B. Bos published in 2014"

Plant immunity in plant-aphid interactions.

Abstract: Aphids are economically important pests that cause extensive feeding damage and transmit viruses. While some species have a broad host range and cause damage to a variety of crops, others are restricted to only closely related plant species. While probing and feeding aphids secrete saliva, containing effectors, into their hosts to manipulate host cell processes and promote infestation. Aphid effector discovery studies pointed out parallels between infection and infestation strategies of plant pathogens and aphids. Interestingly, resistance to some aphid species is known to involve plant resistance proteins with a typical NB-LRR domain structure. Whether these resistance proteins indeed recognize aphid effectors to trigger ETI remains to be elucidated. In addition, it was recently shown that unknown aphid derived elicitors can initiate reactive oxygen species (ROS) production and callose deposition and that these responses were dependent on BAK1 (BRASSINOSTERIOD INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1) which is a key component of the plant immune system. In addition, BAK-1 contributes to non-host resistance to aphids pointing to another parallel between plant-pathogen and - aphid interactions. Understanding the role of plant immunity and non-host resistance to aphids is essential to generate durable and sustainable aphid control strategies. Although insect behavior plays a role in host selection and non-host resistance, an important observation is that aphids interact with non-host plants by probing the leaf surface, but are unable to feed or establish colonization. Therefore, we hypothesize that aphids interact with non-host plants at the molecular level, but are potentially not successful in suppressing plant defenses and/or releasing nutrients.

Single amino acid mutations in the potato immune receptor R3a expand response to Phytophthora effectors.

Abstract: Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NB-LRR or NLR) proteins to respond to invading pathogens and activate immune responses. How plant NB-LRR proteins respond to pathogens is poorly understood. We undertook a gain-of-function random mutagenesis screen of the potato NB-LRR immune receptor R3a to study how this protein responds to the effector protein AVR3a from the oomycete pathogen Phytophthora infestans. R3a response can be extended to the stealthy AVR3aEM isoform of the effector while retaining recognition of AVR3aKI. Each one of eight single amino acid mutations is sufficient to expand the R3a response to AVR3aEM and other AVR3a variants. These mutations occur across the R3a protein, from the N terminus to different regions of the LRR domain. Further characterization of these R3a mutants revealed that at least one of them was sensitized, exhibiting a stronger response than the wild-type R3a protein to AVR3aKI. Remarkably, the N336Y mutation, near the R3a nucleotide-binding pocket, conferred response to the effector protein PcAVR3a4 from the vegetable pathogen P. capsici. This work contributes to understanding how NB-LRR receptor specificity can be modulated. Together with knowledge of pathogen effector diversity, this strategy can be exploited to develop synthetic immune receptors.

Mp10 and Mp42 from the Aphid Species Myzus persicae Trigger Plant Defenses in Nicotiana benthamiana Through Different Activities

Abstract: Aphids are phloem-feeding insects that, like other plant parasites, deliver effectors inside their host to manipulate host responses. The Myzus persicae (green peach aphid) candidate effectors Mp10 and Mp42 were previously found to reduce aphid fecundity upon intracellular transient overexpression in Nicotiana benthamiana. We performed functional analyses of these proteins to investigate whether they activate defenses through similar activities. We employed a range of functional characterization experiments based on intracellular transient overexpression in N. benthamiana to determine the subcellular localization of Mp10 and Mp42 and investigate their role in activating plant defense signaling. Mp10 and Mp42 showed distinct subcellular localization in planta, suggesting that they target different host compartments. Also, Mp10 reduced the levels of Agrobacterium-mediated overexpression of proteins. This reduction was not due to an effect on Agrobacterium viability. Transient overexpression of Mp10 but not Mp42 activated jasmonic acid and salicylic acid signaling pathways and decreased susceptibility to the hemibiotrophic plant pathogen Phytophthora capsici. We found that two candidate effectors from the broad-host-range aphid M. persicae can trigger aphid defenses through different mechanisms. Importantly, we found that some (candidate) effectors such as Mp10 interfere with Agrobacterium-based overexpression assays, an important tool to study effector activity and function.

Recognition of an Avr3a Homologue Plays a Major Role in Mediating Nonhost Resistance to Phytophthora capsici in Nicotiana Species

Abstract: Nonhost resistance is a commonly occurring phenomenon wherein all accessions or cultivars of a plant species are resistant to all strains of a pathogen species and is likely the manifestation of multiple molecular mechanisms. Phytophthora capsici is a soil-borne oomycete that causes Phytophthora blight disease in many solanaceous and cucurbitaceous plants worldwide. Interest in P. capsici has increased considerably with the sequencing of its genome and its increasing occurrence in multiple crops. However, molecular interactions between P. capsici and both its hosts and its nonhosts are poorly defined. We show here that tobacco (Nicotiana tabacum) acts like a nonhost for P. capsici and responds to P. capsici infection with a hypersensitive response (HR). Furthermore, we have found that a P. capsici Avr3a-like gene (PcAvr3a1) encoding a putative RXLR effector protein produces a HR upon transient expression in tobacco and several other Nicotiana species. This HR response correlated with resistance in 19 of 23 Nicotiana species and accessions tested, and knock-down of PcAvr3a1 expression by host-induced gene silencing allowed infection of resistant tobacco. Our results suggest that many Nicotiana species have the capacity to recognize PcAvr3a1 via the products of endogenous disease resistance (R) genes and that this R gene-mediated response is a major component of nonhost resistance to P. capsici.

Plant-Pathogen Interactions

Abstract: 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 workfl ows, and shared with other Galaxy users, allowing them to easily perform the same analysis or protocol on their own data. We describe example Galaxy workfl ows for the identifi cation of candidate pathogen effector proteins. Our main example focuses on nematode plant pathogens where signal peptide and transmembrane prediction tools are used to identify predicted secreted proteins.

Phytophthora infestans RXLR-WY effector AVR3a associates with a Dynamin-Related Protein involved in endocytosis of a plant pattern recognition receptor

Abstract: Perception of pathogen associated molecular patterns (PAMPs) by cell surface localized pattern recognition receptors (PPRs), activates plant basal defense responses in a process known as PAMP/PRR–triggered immunity (PTI). In turn, pathogens deploy effector proteins that interfere with different steps in PTI signaling. However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that BAK1/SERK3, a regulatory receptor of several PRRs, contributes to basal immunity against the Irish potato famine pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated defense by binding the E3 ligase CMPG1. In contrast, AVR3aKI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and suppress INF1 cell death. Here we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3 dependent PTI responses using the plant PRR FLAGELLIN SENSING 2 (FLS2). We found that all tested variants of AVR3a, including AVR3aKI-Y147del, suppress early defense responses triggered by the bacterial flagellin-derived peptide flg22 and reduce internalization of activated FLS2 from the plasma membrane without disturbing its nonactivated localization. Consistent with this effect of AVR3a on FLS2 endocytosis, we discovered that AVR3a associates with the Dynamin-Related Protein DRP2, a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, DRP2 is required for ligand-induced FLS2 internalization but does not affect internalization of the growth receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1). Furthermore, overexpression of DRP2 suppressed accumulation of reactive oxygen species triggered by PAMP treatment. We conclude that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis and signaling. AVR3a is a multifunctional effector that can suppress BAK1/SERK3 mediated immunity through at least two different pathways.

Leaf-disc assay based on transient over-expression in Nicotiana benthamiana to allow functional screening of candidate effectors from aphids.

Abstract: 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 identification of effectors that affect aphid fecundity upon in planta over-expression. This assay will allow the identification of aphid effectors with a role in aphid virulence and can be adapted to other plant species amenable to agroinfiltration as well as to other assays based on transient expression, such as RNAi.

Plant-pathogen interactions : methods and protocols

Abstract: Part 1: Bioinformatics 1. Galaxy as a Platform for Identifying Candidate Pathogen Effectors Peter J. A. Cock and Leighton Pritchard 2. Bioinformatic analysis of expression data to identify effector candidates Adam J. Reid and John T. Jones 3. Two-dimensional data binning for the analysis of genome architecture in filamentous plant pathogens and other eukaryotes Diane G.O. Saunders, Joe Win, Sophien Kamoun, and Sylvain Raffaele 4. On The Statistics Of Identifying Candidate Pathogen Effectors Leighton Pritchard amd David Broadhurst Part 2: Cell Biology 5. High-throughput imaging of plant immune responses Martina Beck, Ji Zhou, Christine Faulkner, and Silke Robatzek 6. In vivo protein-protein interaction studies with BiFC: conditions, cautions and caveats Petra Boevink, Hazel McLellan, Tatyana Bukharova, Stefan Engelhardt, and Paul R.J. Birch 7. Particle bombardment-mediated transient expression to identify localization signals in plant disease resistance proteins and target sites for the proteolytic activity of pathogen effectors Daigo Takemoto and David Jones 8. Purification of fungal haustoria from infected plant tissue by flow cytometry Diana P. Garnica and John P. Rathjen Part 3: From assays of pathogen virulence to effector function 9. Functional Characterisation of Nematode Effectors in Plants Axel A. Elling and John T. Jones 10. Silencing of aphid genes by feeding on stable transgenic Arabidopsis thaliana Alexander D. Coleman, Marco Pitino, and Saskia A. Hogenhout 11. Leaf-disc assay based on transient over-expression in Nicotiana benthamiana to allow functional screening of candidate effectors from aphids Patricia A. Rodriguez, Saskia A. Hogenhout, and Jorunn I.B. Bos 12. A growth quantification assay for Hyaloperonospora arabidopsidis isolates in Arabidopsis thaliana Daniel F.A. Tome, Jens Steinbrenner, and Jim L. Beynon 13. Simple quantification of in planta fungal biomass Michael Ayliffe, Sambasivam K Periyannan, Angela Feechan, Ian Dry, Ulrike Schumann, Evans Lagudah, and Anthony Pryor 14. Virus-induced Gene Silencing and Agrobacterium Tumefaciens-Mediated Transient Expression in Nicotiana Tabacum Zhao Zhang and Bart P.H.J. Thomma 15. DIGE-ABPP by click chemistry: Pairwise comparison of serine hydrolase activities from the apoplast of infected plants Tram Ngoc Hong and Renier A.L. van der Hoorn 16. A Simple and Fast Protocol for Protein Complex Immunoprecipitation (Co-IP) of Effector-Host Protein Complexes Jens Steinbrenner, Matthew Eldridge, Daniel F.A. Tome, and Jim L. Beynon 17. An Arabidopsis and Tomato Mesophyll Protoplast System for fast Identification of early MAMP-triggered Immunity-Suppressing Effectors Malou Fraiture, Xiangzi Zheng, and Frederic Brunner 18. Production of RXLR effector proteins for structural analysis by X-ray crystallography Richard K. Hughes and Mark J. Banfield Part 4: Methods to identify Resistance genes and avirulence genes 19. The Do's and Don'ts of effectoromics Juan Du and Vivianne G.A.A. Vleeshouwers 20. Protoplast cell death assay to study Magnaporthe oryzae AVR gene function in rice Hiroyuki Kanzaki, Kentaro Yoshida, Hiromasa Saitoh, Muluneh Tamiru, and Ryohei Terauchi 21. A Bacterial Type III Secretion Based Delivery System for Functional Assays of Fungal Effectors in Cereals Narayana M. Upadhyaya, Jeffery G. Ellis, and Peter N. Dodds 22. Capture arrays to annotate resistance genes in plant genomes and to accelerate plant resistance gene discovery Florian Jupe, Xinwei Chen, Walter Verweij, Kamil Witek, Jonathan D.G. Jones, and Ingo Hein