Publications from The Sainsbury Laboratory
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Curr Opin Plant Biol: Should I stay or should I go? Traffic control for plant pattern recognition receptors (2015)

Curr Opin Plant Biol: Should I stay or should I go? Traffic control for plant pattern recognition receptors (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Plants employ cell surface-localised receptors to recognise potential invaders via perception of microbe-derived molecules. This is mediated by pattern recognition receptors (PRRs) that bind microbe-associated or damage-associated molecular patterns or perceive apoplastic effector proteins secreted by microorganisms. In either case, effective recognition and initiation of appropriate defence responses rely on a signalling competent pool of receptors at the cell surface. Maintenance of this pool of receptors at the plasma membrane is guaranteed by sorting of properly folded ligand-unbound and ligand-bound receptors via the secretory-endosomal network in an activation-dependent manner. Recent findings highlight that ligand-induced endocytosis is found across members of distinct PRR families suggesting a conserved mechanism by which PRRs and immunity is regulated.

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eLIFE: Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor (2015)

eLIFE: Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops.

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Plant J: Sequencing of 15,622 gene-bearing BACs clarifies the gene-dense regions of the barley genome (2015)

Plant J: Sequencing of 15,622 gene-bearing BACs clarifies the gene-dense regions of the barley genome (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6,278 BACs in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15,622 BACs representing the minimal tiling path of 72,052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low-recombination is particularly relevant.

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eLIFE: Lessons from Fraxinus, a crowd-sourced citizen science game in genomics (2015)

eLIFE: Lessons from Fraxinus, a crowd-sourced citizen science game in genomics (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

In 2013, in response to an epidemic of ash dieback disease in England the previous year, we launched a Facebook-based game called Fraxinus to enable non-scientists to contribute to genomics studies of the pathogen that causes the disease and the ash trees that are devastated by it. Over a period of 51 weeks players were able to match computational alignments of genetic sequences in 78% of cases, and to improve them in 15% of cases. We also found that most players were only transiently interested in the game, and that the majority of the work done was performed by a small group of dedicated players. Based on our experiences we have built a linear model for the length of time that contributors are likely to donate to a crowd-sourced citizen science project. This model could serve a guide for the design and implementation of future crowd-sourced citizen science initiatives.

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Mol Plant Path: High levels of cyclic-di-GMP in plant-associated Pseudomonas correlate with evasion of plant immunity (2015)

Mol Plant Path: High levels of cyclic-di-GMP in plant-associated Pseudomonas correlate with evasion of plant immunity (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

The plant innate immune system employs plasma membrane-localized receptors that specifically perceive pathogen/microbe-associated molecular patterns (PAMPs/MAMPs). This induces a defence response called pattern-triggered immunity (PTI) to fend off pathogen attack. Commensal bacteria are also exposed to potential immune recognition and must employ strategies to evade and/or suppress PTI to successfully colonize the plant. During plant infection the flagellum has an ambiguous role, acting as both a virulence factor and also as a potent immunogen due to the recognition of its main building block, flagellin, by the plant pattern-recognition receptors (PRRs), including FLAGELLIN SENSING2 (FLS2). Therefore, strict control of flagella synthesis is especially important for plant-associated bacteria. Here we show that cyclic-di-GMP, a central regulator of bacterial lifestyle, is involved in the evasion of PTI. Elevated cyclic-di-GMP levels in the pathogen Pseudomonas syringae pv. tomato (Pto) DC3000, the opportunist P. aeruginosa PAO1, and the commensal P. protegens Pf-5 inhibit flagellin synthesis and help the bacteria to evade FLS2-mediated signalling in Nicotiana benthamiana and Arabidopsis thaliana. Despite this, high cellular cyclic-di-GMP concentrations were shown to drastically reduce the virulence of Pto DC3000 during plant infection. We propose that this is due to reduced flagellar motility and/or additional pleiotropic effects of cyclic-di-GMP signalling on bacterial behaviour.

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Frontiers: Comparative genomic analysis of multiple strains of two unusual plant pathogens: Pseudomonas corrugata and Pseudomonas mediterranea (2015)

Frontiers: Comparative genomic analysis of multiple strains of two unusual plant pathogens: Pseudomonas corrugata and Pseudomonas mediterranea (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

The non-fluorescent pseudomonads, Pseudomonas corrugata (Pcor) and P. mediterranea (Pmed), are closely related species that cause pith necrosis, a disease of tomato that causes severe crop losses. However, they also show strong antagonistic effects against economically important pathogens, demonstrating their potential for utilization as biological control agents. In addition, their metabolic versatility makes them attractive for the production of commercial biomolecules and bioremediation. An extensive comparative genomics study is required to dissect the mechanisms that Pcor and Pmed employ to cause disease, prevent disease caused by other pathogens, and to mine their genomes for commercially significant chemical pathways. Here, we present the draft genomes of nine Pcor and Pmed strains from different geographical locations. This analysis covered significant genetic heterogeneity and allowed in-depth genomic comparison. All examined strains were able to trigger symptoms in tomato plants but not all induced a hypersensitive-like response in Nicotiana benthamiana. Genome-mining revealed the absence of a type III secretion system and of known type III effectors from all examined Pcor and Pmed strains. The lack of a type III secretion system appears to be unique among the plant pathogenic pseudomonads. Several gene clusters coding for type VI secretion system were detected in all genomes.

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Plant J: Rice Exo70 interacts with a fungal effector, AVR-Pii and is required for AVR-Pii-triggered immunity (2015)

Plant J: Rice Exo70 interacts with a fungal effector, AVR-Pii and is required for AVR-Pii-triggered immunity (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Vesicle trafficking including exocytosis pathway is intimately associated with host immunity against pathogens. However, we have still insufficiently known about how they contribute to immunity, and how pathogen factors affect them. In this study, we explored host interactors of Magnaporthe oryzae effector AVR-Pii. Gel filtration chromatography and co-immunoprecipitation assays identified a 150 kDa complex of proteins in the soluble fraction comprising AVR-Pii and OsExo70-F2 and OsExo70–F3, the two rice Exo70 proteins presumably involved in exocytosis. Simultaneous knockdown of OsExo70-F2/F3 totally abrogated Pii immune receptor-dependent resistance, but had no effect on Pia- and Pik-dependent resistance. Knockdown levels of OsExo70-F3 but not OsExo70-F2 correlated with reduction of Pii function suggesting that OsExo70-F3 is specifically involved in Pii-dependent resistance. In our current experimental conditions, overexpression of AVR-Pii or knockdown of OsExo70-F2 and -F3 genes in rice did not affect the virulence of compatible isolates of M. oryzae. AVR-Pii interaction with OsExo70-F3 seems to play a crucial role in effector triggered immunity by Pii, suggesting the role of OsExo70 as decoy or helper in Pii/AVR-Pii interactions.

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Plant Signal Behav: The calcium-dependent protein kinase CPK28 negatively regulates the BIK1-mediated PAMP-induced calcium burst (2015)

Plant Signal Behav: The calcium-dependent protein kinase CPK28 negatively regulates the BIK1-mediated PAMP-induced calcium burst (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Plants are protected from microbial infection by a robust immune system. Two of the earliest responses mediated by surface-localized immune receptors include an increase in cytosolic calcium (Ca2+) and a burst of apoplastic reactive oxygen species (ROS). The Arabidopsis plasma membrane-associated cytoplasmic kinase BIK1 is an immediate convergent substrate of multiple surface-localized immune receptors that is genetically required for the PAMP-induced Ca2+ burst and directly regulates ROS production catalyzed by the NADPH oxidase RBOHD. We recently demonstrated that Arabidopsis plants maintain an optimal level of BIK1 through a process of continuous degradation regulated by the Ca2+-dependent protein kinase CPK28. cpk28 mutants accumulate more BIK1 protein and display enhanced immune signaling, while plants over-expressing CPK28 accumulate less BIK1 protein and display impaired immune signaling. Here, we show that CPK28 additionally contributes to the PAMP-induced Ca2+ burst, supporting its role as a negative regulator of BIK1.

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Cell: A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors (2015)

Cell: A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a “decoy” domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain.

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bioRxiv: Rust fungal effectors mimic host transit peptides to translocate into chloroplasts (2015)

bioRxiv: Rust fungal effectors mimic host transit peptides to translocate into chloroplasts (2015) | Publications from The Sainsbury Laboratory | Scoop.it

Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane-rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified CTP1 (chloroplast-targeted protein 1), a candidate effector from the poplar leaf rust fungus Melampsora larici-populina that carries a predicted transit peptide and accumulates in chloroplasts. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts, and is cleaved after translocation. CTP1 is part of a Melampsora-specific family of polymorphic secreted proteins whose members translocate and are processed in chloroplasts in a N-terminal signal-dependent manner. Our findings reveal that fungi have evolved effector proteins that mimic plant-specific sorting signals to traffic within plant cells.


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Plant Cell Physiol: Regulation of the NADPH oxidase RBOHD during plant immunity (2015)

Plant Cell Physiol: Regulation of the NADPH oxidase RBOHD during plant immunity (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Pathogen recognition induces the production of reactive oxygen species (ROS) by NADPH oxidases in both plants and animals. ROS has direct anti-microbial properties, but also serve as signaling molecules to activate further immune outputs. However, ROS production has to be tightly controlled to avoid detrimental effects on host cells, but yet must be produced in the right amount, at the right place and at the right time upon pathogen perception. Plant NADPH oxidases belong to the respiratory burst oxidase homolog (RBOH) family, which contains 10 members in the model plant Arabidopsis thaliana. The perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) leads to a rapid, specific and strong production of ROS, which is dependent on RBOHD. RBOHD is mainly controlled by Ca2+ via direct binding to EF-hand motifs and phosphorylation by Ca2+-dependent protein kinases. Recent studies have, however, revealed a critical role for a Ca2+-independent regulation of RBOHD. The plasma membrane-associated cytoplasmic kinase BIK1, which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. Impairment of these phosphorylation events completely abolishes the function of RBOHD in immunity. These results suggest that RBOHD activity is tightly controlled by multilayered regulations. In this review, we summarize recent advances in our understanding of the regulatory mechanisms controlling RBOHD activation.

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Mol Cell Prot: Identification of regulatory and cargo proteins of endosomal and secretory pathways in Arabidopsis thaliana by proteomic dissection (2015)

Mol Cell Prot: Identification of regulatory and cargo proteins of endosomal and secretory pathways in Arabidopsis thaliana by proteomic dissection (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

The cell's endomembranes comprise an intricate, highly dynamic and well-organised system. In plants the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi Network (TGN), Early Endosomes (EE), secretory vesicles, Late Endosomes (LE), Multivesicular Bodies (MVB) and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localises to the Golgi, Clathrin Light Chain 2 (CLC2) labelling Clathrin-coated vesicles and pits and the Vesicle Associated Membrane Protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNARES and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA and recently defined complexes such as TPLATE. The sub-cellular localisation of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic data set to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localisation of proteins, identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system.

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BMC Research Notes: Broad application of a simple and affordable protocol for isolating plant RNA (2015)

BMC Research Notes: Broad application of a simple and affordable protocol for isolating plant RNA (2015) | Publications from The Sainsbury Laboratory | Scoop.it
Standard molecular biological methods involve the analysis of gene expression in living organisms under diverse environmental and developmental conditions. One of the most direct approaches to quantify gene expression is the isolation of RNA. Most techniques used to quantify gene expression require the isolation of RNA, usually from a large number of samples. While most published protocols, including those for commercial reagents, are either labour intensive, use hazardous chemicals and/or are costly, a previously published protocol for RNA isolation in Arabidopsis thaliana yields high amounts of good quality RNA in a simple, safe and inexpensive manner.
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New Phyt: An introduction to synthetic biology in plant systems: ERASynBio/OpenPlant summer school for early career researchers, September 2014 (2015)

New Phyt: An introduction to synthetic biology in plant systems: ERASynBio/OpenPlant summer school for early career researchers, September 2014 (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

European science policy is reflecting the increasing importance of synthetic biology as a tool to drive cutting-edge scientific developments. Significant strategic investment has been made, coordinated by the European Research Area Network for synthetic biology (ERASynBio), to ensure European synthetic biology research is coherent and world-leading. Strategies to achieve this include providing high-quality training for the next generation of synthetic biologists, and fostering international collaborations across a range of disciplines (ERASynBio, 2014). To realize these aims, ERASynBio has funded annual summer schools to bring together early career researchers from across ERASynBio partner countries for world-class synthetic biology training and networking. The second of these summer schools, which ran on 14–20 September 2014 at the John Innes Centre, Norwich, UK, was designed to provide the participants with ‘An introduction to synthetic biology in plant systems’ in conjunction with OpenPlant, a collaborative plant-focussed Synthetic Biology Research Centre linking the University of Cambridge, John Innes Centre and The Sainsbury Laboratory.

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Current Biology: Functional Divergence of Two Secreted Immune Proteases of Tomato (2015)

Current Biology: Functional Divergence of Two Secreted Immune Proteases of Tomato (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Rcr3 and Pip1 are paralogous secreted papain-like proteases of tomato. Both proteases are inhibited by Avr2 from the fungal pathogen Cladosporium fulvum, but only Rcr3 acts as a co-receptor for Avr2 recognition by the tomato Cf-2 immune receptor [ 1, 2, 3 and 4]. Here, we show that Pip1-depleted tomato plants are hyper-susceptible to fungal, bacterial, and oomycete plant pathogens, demonstrating that Pip1 is an important broad-range immune protease. By contrast, in the absence of Cf-2, Rcr3 depletion does not affect fungal and bacterial infection levels but causes increased susceptibility only to the oomycete pathogen Phytophthora infestans. Rcr3 and Pip1 reside on a genetic locus that evolved over 36 million years ago. These proteins differ in surface-exposed residues outside the substrate-binding groove, and Pip1 is 5- to 10-fold more abundant than Rcr3. We propose a model in which Rcr3 and Pip1 diverged functionally upon gene duplication, possibly driven by an arms race with pathogen-derived inhibitors or by coevolution with the Cf-2 immune receptor detecting inhibitors of Rcr3, but not of Pip1.

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Annual Review of Phytopathology: A Moving View: Subcellular Trafficking Processes in Pattern Recognition Receptor–Triggered Plant Immunity (2015)

Annual Review of Phytopathology: A Moving View: Subcellular Trafficking Processes in Pattern Recognition Receptor–Triggered Plant Immunity (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

A significant challenge for plants is to induce localized defense responses at sites of pathogen attack. Therefore, host subcellular trafficking processes enable accumulation and exchange of defense compounds, which contributes to the plant on-site defenses in response to pathogen perception. This review summarizes our current understanding of the transport processes that facilitate immunity, the significance of which is highlighted by pathogens reprogramming membrane trafficking through host cell translocated effectors. Prominent immune-related cargos of plant trafficking pathways are the pattern recognition receptors (PRRs), which must be present at the plasma membrane to sense microbes in the apoplast. We focus on the dynamic localization of the FLS2 receptor and discuss the pathways that regulate receptor transport within the cell and their link to FLS2-mediated immunity. One emerging theme is that ligand-induced late endocytic trafficking is conserved across different PRR protein families as well as across different plant species.

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Sridhar Ranganathan's curator insight, August 7, 2015 11:41 PM

A significant challenge for plants is to induce localized defense responses at sites of pathogen attack. Therefore, host subcellular trafficking processes enable accumulation and exchange of defense compounds, which contributes to the plant on-site defenses in response to pathogen perception. This review summarizes our current understanding of the transport processes that facilitate immunity, the significance of which is highlighted by pathogens reprogramming membrane trafficking through host cell translocated effectors. Prominent immune-related cargos of plant trafficking pathways are the pattern recognition receptors (PRRs), which must be present at the plasma membrane to sense microbes in the apoplast. We focus on the dynamic localization of the FLS2 receptor and discuss the pathways that regulate receptor transport within the cell and their link to FLS2-mediated immunity. One emerging theme is that ligand-induced late endocytic trafficking is conserved across different PRR protein families as well as across different plant species.

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IMA Fungus: Identification of a new order of root-colonising fungi in the Entorrhizomycota: Talbotiomycetales ord. nov. on eudicotyledons (2015)

The Sainsbury Lab's insight:

The recently described fungal phylum Entorrhizomycota was established solely for the genus Entorrhiza, species of which cause root-galls in Cyperaceae and Juncaceae. Talbotiomyces calosporus (incertae sedis) shares morphological characteristics and an ecological niche with species of Entorrhiza. We investigated the higher classification of T. calosporus to determine whether it belongs in Entorrhizomycota. Ribosomal DNA sequences showed Talbotiomyces to be a close relative of Entorrhiza and both taxa form a highly supported monophyletic group. Based on molecular phylogenetic analyses and in congruence with existing morphological and ecological data, Entorrhiza and Talbotiomyces represent a deep dichotomy within the Entorrhizomycota. While species of Entorrhiza are characterised by dolipores and occur on monocotyledons, members of Talbotiomyces are characterised by simple pores and are associated with eudicotyledons. This expands the host range of the recently described Entorrhizomycota from Poales to other angiosperms. Higher taxa, namely Talbotiomycetales ord. nov. and Talbotiomycetaceae fam. nov., are proposed here to accommodate Talbotiomyces. 

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Plant Cell: Phosphorylation of the Plant Immune Regulator RPM1-INTERACTING PROTEIN4 Enhances Plant Plasma Membrane H+-ATPase Activity and Inhibits Flagellin-Triggered Immune Responses in Arabidopsi...

Plant Cell: Phosphorylation of the Plant Immune Regulator RPM1-INTERACTING PROTEIN4 Enhances Plant Plasma Membrane H+-ATPase Activity and Inhibits Flagellin-Triggered Immune Responses in Arabidopsi... | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

The Pseudomonas syringae effector AvrB targets multiple host proteins during infection, including the plant immune regulator RPM1-INTERACTING PROTEIN4 (RIN4) and RPM1-INDUCED PROTEIN KINASE (RIPK). In the presence of AvrB, RIPK phosphorylates RIN4 at Thr-21, Ser-160, and Thr-166, leading to activation of the immune receptor RPM1. Here, we investigated the role of RIN4 phosphorylation in susceptible Arabidopsis thaliana genotypes. Using circular dichroism spectroscopy, we show that RIN4 is a disordered protein and phosphorylation affects protein flexibility. RIN4 T21D/S160D/ T166D phosphomimetic mutants exhibited enhanced disease susceptibility upon surface inoculation with P. syringae, wider stomatal apertures, and enhanced plasma membrane H+-ATPase activity. The plasma membrane H+-ATPase AHA1 is highly expressed in guard cells, and its activation can induce stomatal opening. The ripk knockout also exhibited a strong defect in pathogen-induced stomatal opening. The basal level of RIN4 Thr-166 phosphorylation decreased in response to immune perception of bacterial flagellin. RIN4 Thr166D lines exhibited reduced flagellin-triggered immune responses. Flagellin perception did not lower RIN4 Thr-166 phosphorylation in the presence of strong ectopic expression of AvrB. Taken together, these results indicate that the AvrB effector targets RIN4 in order to enhance pathogen entry on the leaf surface as well as dampen responses to conserved microbial features. 

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Frank Menke's curator insight, July 23, 2015 8:44 AM

The Pseudomonas syringae effector AvrB targets multiple host proteins during infection, including the plant immune regulator

RPM1-INTERACTING PROTEIN4 (RIN4) and RPM1-INDUCED PROTEIN KINASE (RIPK). In the presence of AvrB, RIPK

phosphorylates RIN4 at Thr-21, Ser-160, and Thr-166, leading to activation of the immune receptor RPM1. Here, we

investigated the role of RIN4 phosphorylation in susceptible Arabidopsis thaliana genotypes. Using circular dichroism

spectroscopy, we show that RIN4 is a disordered protein and phosphorylation affects protein flexibility. RIN4 T21D/S160D/

T166D phosphomimetic mutants exhibited enhanced disease susceptibility upon surface inoculation with P. syringae, wider

stomatal apertures, and enhanced plasma membrane H+-ATPase activity. The plasma membrane H+-ATPase AHA1 is highly

expressed in guard cells, and its activation can induce stomatal opening. The ripk knockout also exhibited a strong defect in

pathogen-induced stomatal opening. The basal level of RIN4 Thr-166 phosphorylation decreased in response to immune

perception of bacterial flagellin. RIN4 Thr166D lines exhibited reduced flagellin-triggered immune responses. Flagellin

perception did not lower RIN4 Thr-166 phosphorylation in the presence of strong ectopic expression of AvrB. Taken together,

these results indicate that the AvrB effector targets RIN4 in order to enhance pathogen entry on the leaf surface as well as

dampen responses to conserved microbial features.

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PLoS Genetics: Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress (2015)

PLoS Genetics: Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Receptor-like kinases (RLKs) are important regulators in signal transduction in plants. However, the large number of RLKs and their high sequence similarity has hampered the analysis of RLKs. One of the largest subgroups of RLKs, the cysteine-rich receptor-like kinases (CRKs), has been suggested to be involved in mediating the effects of reactive oxygen species (ROS). While ROS are recognized as important signalling elements with a large variety of roles in plants, their ligands and achievement of signalling specificity remain unknown. Using insertion mutants we analysed the roles of CRKs in plant development and stress responses and show that CRKs have important roles as mediators of signalling specificity during regulation of stomatal aperture. Our study shows that, despite their large number and high sequence conservation, individual CRKs have intriguingly distinct functions in different aspects of plant life. This makes the CRKs promising candidates for future studies of their biochemical function.

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New Phytologist: Standards for plant synthetic biology: a common syntax for exchange of DNA parts (2015)

New Phytologist: Standards for plant synthetic biology: a common syntax for exchange of DNA parts (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

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Nature Plants: Sweet! A naturally transgenic crop (2015)

Nature Plants: Sweet! A naturally transgenic crop (2015) | Publications from The Sainsbury Laboratory | Scoop.it
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One of the world’s most important staple crops, the sweet potato, is a naturally transgenic plant that was genetically modified thousands of years ago by a soil bacterium. This surprising discovery may influence the public view of GM crops. 

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bioRxiv: The Cf-4 receptor-like protein associates with the BAK1 receptor-like kinase to initiate receptor endocytosis and plant immunity (2015)

bioRxiv: The Cf-4 receptor-like protein associates with the BAK1 receptor-like kinase to initiate receptor endocytosis and plant immunity (2015) | Publications from The Sainsbury Laboratory | Scoop.it

The first layer of plant immunity is activated by cell surface receptor-like kinases (RLKs) and proteins (RLPs) that detect infectious pathogens. Constitutive interaction with the RLK SUPPRESSOR OF BIR1 (SOBIR1) contributes to RLP stability and kinase activity. As RLK activation requires transphosphorylation with a second associated RLK, it remains elusive how RLPs initiate downstream signaling. To address this, we investigated functioning of Cf RLPs that mediate immunity of tomato against Cladosporium fulvum. We employed live-cell imaging and co-immunoprecipitation in tomato and Nicotiana benthamiana to investigate the requirement of associated kinases for Cf activity and ligand-induced subcellular trafficking of Cf-4. Upon elicitation with the matching effector ligands Avr4 and Avr9, BRI1-ASSOCIATED KINASE 1 (BAK1) associates with Cf-4 and Cf-9. Furthermore, Cf-4 that interacts with SOBIR1 at the plasma membrane, is recruited to late endosomes after elicitation. Significantly, BAK1 is required for Avr4-triggered endocytosis, effector-triggered defenses in Cf-4 plants and resistance of tomato against C. fulvum. Our observations indicate that RLP-mediated immune signaling and endocytosis require ligand-induced recruitment of BAK1, reminiscent of BAK1 interaction and subcellular fate of the FLAGELLIN SENSING 2 RLK. This reveals that diverse classes of cell surface immune receptors share common requirements for signaling initiation and endocytosis.


Via Kamoun Lab @ TSL
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Methods in Mol Biol: Phosphopeptide Immuno-Affinity Enrichment to Enhance Detection of Tyrosine Phosphorylation in Plants (2015)

Methods in Mol Biol: Phosphopeptide Immuno-Affinity Enrichment to Enhance Detection of Tyrosine Phosphorylation in Plants (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Tyrosine (Tyr) phosphorylation plays an essential role in signaling in animal systems, but the relative contribution of Tyr phosphorylation to plant signal transduction has, until recently, remained an open question. One of the major issues hampering the analysis is the low abundance of Tyr phosphorylation and therefore underrepresentation in most mass spec-based proteomic studies. Here, we describe a working approach to selectively enrich Tyr-phosphorylated peptides from complex plant tissue samples. We describe a detailed protocol that is based on immuno-affinity enrichment step using an anti-phospho-tyrosine (pTyr)-specific antibody. This single enrichment strategy effectively enriches pTyr-containing peptides from complex total plant cell extracts, which can be measured by LC-MS/MS without further fractionation or enrichment.

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Mol Plant Path: SOBIR1 requires the GxxxG dimerization motif in its transmembrane domain to form constitutive complexes with receptor-like proteins (2015)

Mol Plant Path: SOBIR1 requires the GxxxG dimerization motif in its transmembrane domain to form constitutive complexes with receptor-like proteins (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

Receptor-like proteins (RLPs), forming an important group of transmembrane receptors in plants, play roles in development and immunity. RLPs contain extracellular leucine-rich repeats (LRRs) and, in contrast to receptor-like kinases (RLKs), lack a cytoplasmic kinase required for initiating downstream signalling. Recent studies revealed that the RLK SOBIR1/EVR (SUPPRESSOR OF BIR1-1/EVERSHED) specifically interacts with RLPs. SOBIR1 stabilizes RLPs and is required for their function. However, the mechanism by which SOBIR1 associates with RLPs and regulates RLP function remains unknown. The Cf immune receptors of tomato (Solanum lycopersicum), mediating resistance to the fungus Cladosporium fulvum, are RLPs that also interact with SOBIR1. Here, we show that both the LRR and kinase domain of SOBIR1 are dispensable for association with the RLP Cf-4, whereas the highly conserved GxxxGxxxG motif present in the transmembrane domain of SOBIR1 is essential for its interaction with Cf-4 and additional RLPs. Complementation assays in Nicotiana benthamiana, in which endogenous SOBIR1 levels were knocked-down by virus-induced gene silencing, showed that the LRR domain as well as kinase activity of SOBIR1 are required for the Cf-4/Avr4-triggered hypersensitive response (HR). In contrast, the LRRs and kinase activity of SOBIR1 are not required for facilitating Cf-4 accumulation. Together, these results suggest that in addition to being a stabilizing scaffold for RLPs, SOBIR1 is also required for initiating downstream signalling through its kinase domain.

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Mol Plant Microbe Interact: A recent expansion of the RXLR effector gene Avrblb2 is maintained in global populations of Phytophthora infestans indicating different contributions to virulence (2015)

Mol Plant Microbe Interact: A recent expansion of the RXLR effector gene Avrblb2 is maintained in global populations of Phytophthora infestans indicating different contributions to virulence (2015) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:

The introgression of disease resistance (R) genes encoding immunoreceptors with broad-spectrum recognition into cultivated potato appears to be the most promising approach to achieve sustainable management of late blight caused by the oomycete pathogen Phytophthora infestans. Rpi-blb2 from Solanum bulbocastanum, shows great potential for use in agriculture based on preliminary potato disease trials. Rpi-blb2 confers immunity by recognizing the P. infestans avirulence effector protein AVRblb2 after it is translocated inside the plant cell. This effector belongs to the RXLR class of effectors and is under strong positive selection. Structure-function analyses revealed a key polymorphic amino acid (position 69) in AVRblb2 effector that is critical for activation of Rpi-blb2. In this study, we reconstructed the evolutionary history of the Avrblb2 gene family and further characterized its genetic structure in worldwide populations. Our data indicates that Avrblb2 evolved as a single copy gene in a putative ancestral species of P. infestans and has recently expanded in the Phytophthora species that infect solanaceous hosts. As a consequence, at least four variants of AVRblb2 arose in P. infestans. One of these variants, with a Phe residue at position 69, evades recognition by the cognate resistance gene. Surprisingly, all Avrblb2 variants are maintained in pathogen populations. This suggests a potential benefit for the pathogen in preserving duplicated versions of AVRblb2 possibly because the variants may have different contributions to pathogen fitness in a diversified solanaceous host environment.

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