Publications from The Sainsbury Laboratory
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Mol Cell: Plant PRRs and the Activation of Innate Immune Signaling (2014)

Mol Cell: Plant PRRs and the Activation of Innate Immune Signaling (2014) | Publications from The Sainsbury Laboratory | Scoop.it

Despite being sessile organisms constantly exposed to potential pathogens and pests, plants are surprisingly resilient to infections. Plants can detect invaders via the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Plant PRRs are surface-localized receptor-like kinases, which comprise a ligand-binding ectodomain and an intracellular kinase domain, or receptor-like proteins, which do not exhibit any known intracellular signaling domain. In this review, we summarize recent discoveries that shed light on the molecular mechanisms underlying ligand perception and subsequent activation of plant PRRs. Notably, plant PRRs appear as central components of multiprotein complexes at the plasma membrane that contain additional transmembrane and cytosolic kinases required for the initiation and specificity of immune signaling. PRR complexes are under tight control by protein phosphatases, E3 ligases, and other regulatory proteins, illustrating the exquisite and complex regulation of these molecular machines whose proper activation underlines a crucial layer of plant immunity.


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Nature Biotech: News & Views: Genetics of a hardy crop (2017)

Nature Biotech: News & Views: Genetics of a hardy crop (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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New genomic resources for pearl millet are revealing how crops stand up to challenging environments.
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Plant J: A computational approach for inferring the cell wall properties that govern guard cell dynamics (2017)

Plant J: A computational approach for inferring the cell wall properties that govern guard cell dynamics (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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Guard cells dynamically adjust their shape in order to regulate photosynthetic gas exchange, respiration rates and defend against pathogen entry. Cell shape changes are determined by the interplay of cell wall material properties and turgor pressure. To investigate this relationship between turgor pressure, cell wall properties and cell shape, we focused on kidney-shaped stomata and developed a biomechanical model of a guard cell pair. Treating the cell wall as a composite of the pectin-rich cell wall matrix embedded with cellulose microfibrils, we show that strong, circumferentially oriented fibres are critical for opening. We find that the opening dynamics are dictated by the mechanical stress response of the cell wall matrix, and as the turgor rises, the pectinaceous matrix stiffens. We validate these predictions with stomatal opening experiments in selected Arabidopsis cell wall mutants. Thus, using a computational framework that combines a 3D biomechanical model with parameter optimization, we demonstrate how to exploit subtle shape changes to infer cell wall material properties. Our findings reveal that proper stomatal dynamics are built on two key properties of the cell wall, namely anisotropy in the form of hoop reinforcement and strain stiffening.
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Sci Rep: Comparative secretome analysis of Rhizoctonia solani isolates with different host ranges reveals unique secretomes and cell death inducing effectors (2017)

Sci Rep: Comparative secretome analysis of Rhizoctonia solani isolates with different host ranges reveals unique secretomes and cell death inducing effectors (2017) | Publications from The Sainsbury Laboratory | Scoop.it
Article
The Sainsbury Lab's insight:
Rhizoctonia solani is a fungal pathogen causing substantial damage to many of the worlds’ largest food crops including wheat, rice, maize and soybean. Despite impacting global food security, little is known about the pathogenicity mechanisms employed by R. solani. To enable prediction of effectors possessing either broad efficacy or host specificity, a combined secretome was constructed from a monocot specific isolate, a dicot specific isolate and broad host range isolate infecting both monocot and dicot hosts. Secretome analysis suggested R. solani employs largely different virulence mechanisms to well-studied pathogens, despite in many instances infecting the same host plants. Furthermore, the secretome of the broad host range AG8 isolate may be shaped by maintaining functions for saprophytic life stages while minimising opportunities for host plant recognition. Analysis of possible co-evolution with host plants and in-planta up-regulation in particular, aided identification of effectors including xylanase and inhibitor I9 domain containing proteins able to induce cell death in-planta. The inhibitor I9 domain was more abundant in the secretomes of a wide range of necrotising fungi relative to biotrophs. These findings provide novel targets for further dissection of the virulence mechanisms and potential avenues to control this under-characterised but important pathogen.
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Methods Mol Biol: Protein–Protein Interaction Assays with Effector–GFP Fusions in Nicotiana benthamiana (2017)

Methods Mol Biol: Protein–Protein Interaction Assays with Effector–GFP Fusions in Nicotiana benthamiana (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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Plant parasites secrete proteins known as effectors into host tissues to manipulate host cell structures and functions. One of the major goals in effector biology is to determine the host cell compartments and the protein complexes in which effectors accumulate. Here, we describe a five-step pipeline that we routinely use in our lab to achieve this goal, which consists of (1) Golden Gate assembly of pathogen effector–green fluorescent protein (GFP) fusions into binary vectors, (2) Agrobacterium-mediated heterologous protein expression in Nicotiana benthamiana leaf cells, (3) laser-scanning confocal microscopy assay, (4) anti-GFP coimmunoprecipitation–liquid chromatography–tandem mass spectrometry (coIP/MS) assay, and (5) anti-GFP western blotting. This pipeline is suitable for rapid, cost-effective, and medium-throughput screening of pathogen effectors in planta.
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bioRxiv: Dominant integration locus drives continuous diversification of plant immune receptors with exogenous domain fusions (2017)

bioRxiv: Dominant integration locus drives continuous diversification of plant immune receptors with exogenous domain fusions (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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The plant immune system is innate, encoded in the germline. Using it efficiently, plants are capable of recognizing a diverse range of rapidly evolving pathogens. A recently described phenomenon shows that plant immune receptors are able to recognize pathogen effectors through the acquisition of exogenous protein domains from other plant genes. We showed that plant immune receptors with integrated domains are distributed unevenly across their phylogeny in grasses. Using phylogenetic analysis, we uncovered a major integration clade, whose members underwent repeated independent integration events producing diverse fusions. This clade is ancestral in grasses with members often found on syntenic chromosomes. Analyses of these fusion events revealed that homologous receptors can be fused to diverse domains. Furthermore, we discovered a 43 amino acids long motif that was associated with this dominant integration clade and was located immediately upstream of the fusion site. Sequence analysis revealed that DNA transposition and/or ectopic recombination are the most likely mechanisms of NLR-ID formation. The identification of this subclass of plant immune receptors that is naturally adapted to new domain integration will inform biotechnological approaches for generating synthetic receptors with novel pathogen baits.
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Frontiers: Tyrosine-610 in the Receptor Kinase BAK1 Does Not Play a Major Role in Brassinosteroid Signaling or Innate Immunity (2017)

Frontiers: Tyrosine-610 in the Receptor Kinase BAK1 Does Not Play a Major Role in Brassinosteroid Signaling or Innate Immunity (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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The Sainsbury Lab's insight:
The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1) functions as a co-receptor with several receptor kinases including the brassinosteroid (BR) receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR), respectively, which are involved in immunity. BAK1 is a dual specificity protein kinase that can autophosphorylate on serine, threonine and tyrosine residues. It was previously reported that phosphorylation of Tyr-610 in the carboxy-terminal domain of BAK1 is required for its function in BR signaling and immunity. However, the functional role of Tyr-610 in vivo has recently come under scrutiny. Therefore, we have generated new BAK1 (Y610F) transgenic plants for functional studies. We first produced transgenic Arabidopsis lines expressing BAK1 (Y610F)-Flag in the homozygous bak1-4 bkk1-1 double null background. In a complementary approach, we expressed untagged BAK1 and BAK1 (Y610F) in the bak1-4 null mutant. Neither BAK1 (Y610F) transgenic line had any obvious growth phenotype when compared to wild-type BAK1 expressed in the same background. In addition, the BAK1 (Y610F)-Flag plants responded similarly to plants expressing BAK1-Flag in terms of brassinolide (BL) inhibition of root elongation, and there were only minor changes in gene expression between the two transgenic lines as monitored by microarray analysis and quantitative real-time PCR. In terms of plant immunity, there were no significant differences between plants expressing BAK1 (Y610F)-Flag and BAK1-Flag in the growth of the non-pathogenic hrpA- mutant of Pseudomonas syringae pv. tomato DC3000. Furthermore, untagged BAK1 (Y610F) transgenic plants were as responsive as plants expressing BAK1 (in the bak1-4 background) and wild-type Col-0 plants toward treatment with the EF-Tu- and flagellin-derived peptide epitopes elf18- and flg22, respectively, as measured by reactive oxygen species production, mitogen-activated protein kinase activation, and seedling growth inhibition. These new results do not support any involvement of Tyr-610 phosphorylation in either BR or immune signaling.
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bioRxiv: Heterologous expression of the immune receptor EFR in Medicago truncatula reduces pathogenic infection, but not rhizobial symbiosis (2017)

bioRxiv: Heterologous expression of the immune receptor EFR in Medicago truncatula reduces pathogenic infection, but not rhizobial symbiosis (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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The Sainsbury Lab's insight:
Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad-spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen-associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae-specific PRR ELONGATION FACTOR-THERMO UNSTABLE RECEPTOR (EFR) from Arabidopsis thaliana to the legume Medicago truncatula, conferring recognition of the bacterial EF-Tu protein. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF-Tu-derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFR-Medicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.
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Bakerlab Berkeley's curator insight, August 12, 3:08 PM
Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad-spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen-associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae-specific PRR ELONGATION FACTOR-THERMO UNSTABLE RECEPTOR (EFR) from Arabidopsis thaliana to the legume Medicago truncatula, conferring recognition of the bacterial EF-Tu protein. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF-Tu-derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFR-Medicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.
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BMC Genomics: Comparative analysis of targeted long read sequencing approaches for characterization of a plant’s immune receptor repertoire (2017)

BMC Genomics: Comparative analysis of targeted long read sequencing approaches for characterization of a plant’s immune receptor repertoire (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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Background
The Oxford Nanopore Technologies MinION™ sequencer is a small, portable, low cost device that is accessible to labs of all sizes and attractive for in-the-field sequencing experiments. Selective breeding of crops has led to a reduction in genetic diversity, and wild relatives are a key source of new genetic resistance to pathogens, usually via NLR immune receptor-encoding genes. Recent studies have demonstrated how crop NLR repertoires can be targeted for sequencing on Illumina or PacBio (RenSeq) and the specific gene conveying pathogen resistance identified.

Results
Sequence yields per MinION run are lower than Illumina, making targeted resequencing an efficient approach. While MinION generates long reads similar to PacBio it doesn’t generate the highly accurate multipass consensus reads, which presents downstream bioinformatics challenges. Here we demonstrate how MinION data can be used for RenSeq achieving similar results to the PacBio and how novel NLR gene fusions can be identified via a Nanopore RenSeq pipeline.

Conclusion
The described library preparation and bioinformatics methods should be applicable to other gene families or any targeted long DNA fragment nanopore sequencing project.
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Bakerlab Berkeley's curator insight, August 12, 3:09 PM
Background
The Oxford Nanopore Technologies MinION™ sequencer is a small, portable, low cost device that is accessible to labs of all sizes and attractive for in-the-field sequencing experiments. Selective breeding of crops has led to a reduction in genetic diversity, and wild relatives are a key source of new genetic resistance to pathogens, usually via NLR immune receptor-encoding genes. Recent studies have demonstrated how crop NLR repertoires can be targeted for sequencing on Illumina or PacBio (RenSeq) and the specific gene conveying pathogen resistance identified.

Results
Sequence yields per MinION run are lower than Illumina, making targeted resequencing an efficient approach. While MinION generates long reads similar to PacBio it doesn’t generate the highly accurate multipass consensus reads, which presents downstream bioinformatics challenges. Here we demonstrate how MinION data can be used for RenSeq achieving similar results to the PacBio and how novel NLR gene fusions can be identified via a Nanopore RenSeq pipeline.

Conclusion
The described library preparation and bioinformatics methods should be applicable to other gene families or any targeted long DNA fragment nanopore sequencing project.
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Plant J: A computational approach for inferring the cell wall properties that govern guard cell dynamics (2017)

Plant J: A computational approach for inferring the cell wall properties that govern guard cell dynamics (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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The Sainsbury Lab's insight:
Guard cells dynamically adjust their shape to regulate photosynthetic gas exchange, respiration rates and defend against pathogen entry. Cell shape changes are determined by the interplay of cell wall material properties and turgor pressure. To investigate this relationship between turgor pressure, cell wall properties and cell shape, we focused on kidney-shaped stomata and developed a biomechanical model of a guard cell pair. Treating the cell wall as a composite of the pectin-rich cell wall matrix embedded with cellulose microfibrils, we show that strong, circumferentially-oriented fibres are critical for opening. We find that opening dynamics are dictated by the mechanical stress-response of the cell wall matrix, and as the turgor rises, the pectinaceous matrix stiffens. We validate these predictions with stomatal opening experiments in selected Arabidopsis cell wall mutants. Thus, using a computational framework that combines a 3D biomechanical model with parameter optimisation we demonstrate how to exploit subtle shape changes to infer cell wall material properties. Our findings reveal that proper stomatal dynamics builds on two key properties of the cell wall, namely anisotropy in the form of hoop reinforcement and strain-stiffening.
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Curr Opin Plant Biol: Editorial overview: Biotic interactions: Inferring global implications for the molecular interface between plants and their biotic interactions across scales (2017)

Curr Opin Plant Biol: Editorial overview: Biotic interactions: Inferring global implications for the molecular interface between plants and their biotic interactions across scales (2017) | Publications from The Sainsbury Laboratory | Scoop.it
Curr Opin Plant Biol: 
The Sainsbury Lab's insight:
We now know that healthy plants are not free of microbes as postulated by Louis Pasteur, who is acknowledged as one of the founders of microbiology and the germ theory of disease. Discoveries made over the last four decades are conceptualized in the ‘zigzag model’ from Jones and Dangl, unifying that plant health is maintained by a two-tiered recognition system acting at the cell surface and in the cytosol, and the ability of pests and pathogens to cause disease based on their effector repertoires. Our exponentially growing knowledge regarding the molecular mechanisms that mediate the relationships between plants and other organisms has revealed many important questions, such as: how do plants recognize other organisms, why are plants not fully immune to some pests and pathogens, and how do plants tolerate and encourage infection with beneficial organisms. This has resulted in the discovery of universal principles of innate immunity that determine plant responses to all other organisms, whether symbiont or pathogen, at the stages of: 1) extracellular and intracellular recognition, 2) acute, transient and moderate, sustained responses, 3) encouragement or inhibition of colonisation, which are all subject to manipulation by effectors to promote the success of infection. Responses include the mobilization and production of ions, second messengers, hormones, transcripts, proteins and metabolites, and cellular transport.
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Ann Rev Phytopath: Function, Discovery, and Exploitation of Plant Pattern Recognition Receptors for Broad-Spectrum Disease Resistance (2017)

Ann Rev Phytopath: Function, Discovery, and Exploitation of Plant Pattern Recognition Receptors for Broad-Spectrum Disease Resistance (2017) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:
Plants are constantly exposed to would-be pathogens and pests, and thus have a sophisticated immune system to ward off these threats, which otherwise can have devastating ecological and economic consequences on ecosystems and agriculture. Plants employ receptor kinases (RKs) and receptor-like proteins (RLPs) as pattern recognition receptors (PRRs) to monitor their apoplastic environment and detect non-self and damaged-self patterns as signs of potential danger. Plant PRRs contribute to both basal and non-host resistances, and treatment with pathogen-/microbe-associated molecular patterns (PAMPs/MAMPs) or damage-associated molecular patterns (DAMPs) recognized by plant PRRs induces both local and systemic immunity. Here, we comprehensively review known PAMPs/DAMPs recognized by plants as well as the plant PRRs described to date. In particular, we describe the different methods that can be used to identify PAMPs/DAMPs and PRRs. Finally, we emphasize the emerging biotechnological potential use of PRRs to improve broad-spectrum, and potentially durable, disease resistance in crops.
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PLoS Genet: The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses (2017)

PLoS Genet: The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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The Sainsbury Lab's insight:
Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues.
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Frontiers: Insights into Host Cell Modulation and Induction of New Cells by the Corn Smut Ustilago maydis (2017)

Frontiers: Insights into Host Cell Modulation and Induction of New Cells by the Corn Smut Ustilago maydis (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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Many filamentous fungal pathogens induce drastic modulation of host cells causing abnormal infectious structures such as galls, or tumors that arise as a result of re-programming in the original developmental cell fate of a colonized host cell. Developmental consequences occur predominantly with biotrophic phytopathogens. This suggests that these host structures result as an outcome of efficient defense suppression and intimate fungal–host interaction to suit the pathogen’s needs for completion of its infection cycle. This mini-review mainly summarizes host cell re-programming that occurs in the Ustilago maydis – maize interaction, in which the pathogen deploys cell-type specific effector proteins with varying activities. The fungus senses the physiological status and identity of colonized host cells and re-directs the endogenous developmental program of its host. The disturbance of host cell physiology and cell fate leads to novel cell shapes, increased cell size, and/or the number of host cells. We particularly highlight the strategies of U. maydis to induce physiologically varied host organs to form the characteristic tumors in both vegetative and floral parts of maize.
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Front. Plant Sci: Enhanced Bacterial Wilt Resistance in Potato Through Expression of Arabidopsis EFR and Introgression of Quantitative Resistance from Solanum commersonii (2017)

Front. Plant Sci: Enhanced Bacterial Wilt Resistance in Potato Through Expression of Arabidopsis EFR and Introgression of Quantitative Resistance from Solanum commersonii (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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Bacterial wilt (BW) caused by Ralstonia solanacearum is responsible for substantial losses in cultivated potato (Solanum tuberosum) crops worldwide. Resistance genes have been identified in wild species; however, introduction of these through classical breeding has achieved only partial resistance, which has been linked to poor agronomic performance. The Arabidopsis thaliana (At) pattern recognition receptor elongation factor-Tu (EF-Tu) receptor (EFR) recognizes the bacterial pathogen-associated molecular pattern EF-Tu (and its derived peptide elf18) to confer anti-bacterial immunity. Previous work has shown that transfer of AtEFR into tomato confers increased resistance to R. solanacearum. Here, we evaluated whether the transgenic expression of AtEFR would similarly increase BW resistance in a commercial potato line (INIA Iporá), as well as in a breeding potato line (09509.6) in which quantitative resistance has been introgressed from the wild potato relative Solanum commersonii. Resistance to R. solanacearum was evaluated by damaged root inoculation under controlled conditions. Both INIA Iporá and 09509.6 potato lines expressing AtEFR showed greater resistance to R. solanacearum, with no detectable bacteria in tubers evaluated by multiplex-PCR and plate counting. Notably, AtEFR expression and the introgression of quantitative resistance from S. commersonii had a significant additive effect in 09509.6-AtEFR lines. These results show that the combination of heterologous expression of AtEFR with quantitative resistance introgressed from wild relatives is a promising strategy to develop BW resistance in potato.
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BMC Biol: Genome sequencing of the staple food crop white Guinea yam enables the development of a molecular marker for sex determination (2017)

BMC Biol: Genome sequencing of the staple food crop white Guinea yam enables the development of a molecular marker for sex determination (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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The Sainsbury Lab's insight:
Root and tuber crops are a major food source in tropical Africa. Among these crops are several species in the monocotyledonous genus Dioscorea collectively known as yam, a staple tuber crop that contributes enormously to the subsistence and socio-cultural lives of millions of people, principally in West and Central Africa. Yam cultivation is constrained by several factors, and yam can be considered a neglected “orphan” crop that would benefit from crop improvement efforts. However, the lack of genetic and genomic tools has impeded the improvement of this staple crop.
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Methods Mol Biol: MutRenSeq: A Method for Rapid Cloning of Plant Disease Resistance Genes (2017)

Methods Mol Biol: MutRenSeq: A Method for Rapid Cloning of Plant Disease Resistance Genes (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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The Sainsbury Lab's insight:
MutRenSeq is a method to clone disease resistance (R) genes in plants. Tips and detailed experimental protocols for the pipeline, including the complexity reduction by R gene targeted enrichment sequencing, and computational analysis based on comparative genomics are provided in this chapter.
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Plant Cell: The Arabidopsis Leucine-rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1 (2017)

Plant Cell: The Arabidopsis Leucine-rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1 (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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The Sainsbury Lab's insight:
BAK1 is a co-receptor and positive regulator of multiple ligand-binding leucine-rich-repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in Arabidopsis thaliana. BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand-binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand-binding receptors to prevent BAK1 receptor complex formation.
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Plant Physiol: PLC2 Regulates MAMP-Triggered Immunity by Modulating ROS Production in Arabidopsis (2017)

Plant Physiol: PLC2 Regulates MAMP-Triggered Immunity by Modulating ROS Production in Arabidopsis (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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The activation of phosphoinositide-specific phospholipase C (PI-PLC) is one of the earliest responses triggered by the recognition of several microbe-associated molecular patterns (MAMPs) in plants. The Arabidopsis PI-PLC gene family is composed of nine members. Previous studies suggested a role for PLC2 in MAMP-triggered immunity (MTI) as it is rapidly phosphorylated in vivo upon treatment with the bacterial MAMP flg22. Here we analyzed the role of PLC2 in plant immunity using an artificial microRNA to silence PLC2 expression in Arabidopsis. We found that PLC2-silenced plants are more susceptible to the type III secretion system-deficient bacterial strain Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC- (Pst DC3000 hrcC-) and to the non-adapted pea powdery mildew Erysiphe pisi. However, PLC2-silenced plants display normal susceptibility to virulent (Pst DC3000) and avirulent P. syringae strains (Pst DC3000 AvrRPM1), conserving typical hypersensitive response (HR) features. In response to flg22, PLC2-silenced plants maintain wild-type MAPK activation and PHI1, WRKY33 and FRK1 immune marker gene expression, but have reduced reactive oxygen species (ROS)-dependent responses such as callose deposition and stomatal closure. Accordingly, the generation of ROS upon flg22 treatment is compromised in the PLC2-defficient plants suggesting an effect of PLC2 in a branch of MTI and non-host resistance that involves early ROS-regulated processes. Consistently, PLC2 associates with the NADPH oxidase RBOHD, suggesting its potential regulation by PLC2.
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Sci Rep: Disruption of the plant-specific CFS 1 gene impairs autophagosome turnover and triggers EDS1-dependent cell death (2017)

Sci Rep: Disruption of the plant-specific CFS 1 gene impairs autophagosome turnover and triggers EDS1-dependent cell death (2017) | Publications from The Sainsbury Laboratory | Scoop.it
Article
The Sainsbury Lab's insight:
Cell death, autophagy and endosomal sorting contribute to many physiological, developmental and immunological processes in plants. They are mechanistically interconnected and interdependent, but the molecular basis of their mutual regulation has only begun to emerge in plants. Here, we describe the identification and molecular characterization of CELL DEATH RELATED ENDOSOMAL FYVE/SYLF PROTEIN 1 (CFS1). The CFS1 protein interacts with the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT I (ESCRT-I) component ELCH (ELC) and is localized at ESCRT-I-positive late endosomes likely through its PI3P and actin binding SH3YL1 Ysc84/Lsb4p Lsb3p plant FYVE (SYLF) domain. Mutant alleles of cfs1 exhibit auto-immune phenotypes including spontaneous lesions that show characteristics of hypersensitive response (HR). Autoimmunity in cfs1 is dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-mediated effector-triggered immunity (ETI) but independent from salicylic acid. Additionally, cfs1 mutants accumulate the autophagy markers ATG8 and NBR1 independently from EDS1. We hypothesize that CFS1 acts at the intersection of autophagosomes and endosomes and contributes to cellular homeostasis by mediating autophagosome turnover.
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bioRxiv: Lessons in effector and NLR biology of plant-microbe systems (2017)

bioRxiv: Lessons in effector and NLR biology of plant-microbe systems (2017) | Publications from The Sainsbury Laboratory | Scoop.it

A diversity of plant-associated organisms secrete effectors: proteins and metabolites that modulate plant physiology to favor host infection and colonization. However, effectors can also activate plant immune receptors, notably nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins, enabling plants to fight off invading organisms. This interplay between effectors, their host targets, and the matching immune receptors is shaped by intricate molecular mechanisms and exceptionally dynamic coevolution. In this article, we focus on three effectors, AVR-Pik, AVR-Pia, and AVR-Pii, from the rice blast fungus Magnaporthe oryzae (syn. Pyricularia oryzae), and their corresponding rice NLR immune receptors, Pik, Pia, and Pii, to highlight general concepts of plant-microbe interactions. We draw 12 lessons in effector and NLR biology that have emerged from studying these three little effectors and are broadly applicable to other plant-microbe systems.


Via Kamoun Lab @ TSL
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Traffic: An automated quantitative image analysis tool for the identification of microtubule patterns in plants (2017)

Traffic: An automated quantitative image analysis tool for the identification of microtubule patterns in plants (2017) | Publications from The Sainsbury Laboratory | Scoop.it
High throughput confocal imaging poses challenges in the computational image analysis of complex subcellular structures such as the microtubule cytoskeleton. Here, we developed CellArchitect, a
The Sainsbury Lab's insight:
High throughput confocal imaging poses challenges in the computational image analysis of complex subcellular structures such as the microtubule cytoskeleton. Here, we developed CellArchitect, an automated image analysis tool that quantifies changes to subcellular patterns illustrated by microtubule markers in plants. We screened microtubule-targeted herbicides and demonstrate that high throughput confocal imaging with integrated image analysis by CellArchitect can distinguish effects induced by the known herbicides indaziflam and trifluralin. The same platform was used to examine six other compounds with herbicidal activity, and at least three different effects induced by these compounds were profiled. We further show that CellArchitect can detect subcellular patterns tagged by actin and endoplasmic reticulum markers. Thus, the platform developed here can be used to automate image analysis of complex subcellular patterns for purposes such as herbicide discovery and mode of action characterisation. The capacity to use this tool to quantitatively characterise cellular responses lends itself to application across many areas of biology.
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PNAS: NLR network mediates immunity to diverse plant pathogens (2017)

PNAS: NLR network mediates immunity to diverse plant pathogens (2017) | Publications from The Sainsbury Laboratory | Scoop.it
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The Sainsbury Lab's insight:
Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept of NLR function is that “sensor” NLR proteins are paired with “helper” NLRs to mediate immune signaling. However, our fundamental knowledge of sensor/helper NLRs in plants remains limited. In this study, we discovered a complex NLR immune network in which helper NLRs in the NRC (NLR required for cell death) family are functionally redundant but display distinct specificities toward different sensor NLRs that confer immunity to oomycetes, bacteria, viruses, nematodes, and insects. The helper NLR NRC4 is required for the function of several sensor NLRs, including Rpi-blb2, Mi-1.2, and R1, whereas NRC2 and NRC3 are required for the function of the sensor NLR Prf. Interestingly, NRC2, NRC3, and NRC4 redundantly contribute to the immunity mediated by other sensor NLRs, including Rx, Bs2, R8, and Sw5. NRC family and NRC-dependent NLRs are phylogenetically related and cluster into a well-supported superclade. Using extensive phylogenetic analysis, we discovered that the NRC superclade probably emerged over 100 Mya from an NLR pair that diversified to constitute up to one-half of the NLRs of asterids. These findings reveal a complex genetic network of NLRs and point to a link between evolutionary history and the mechanism of immune signaling. We propose that this NLR network increases the robustness of immune signaling to counteract rapidly evolving plant pathogens.
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Plant & Cell Phys: Latest Advances in Plant Development and Environmental Response, the inaugural Cold Spring Harbor Asia Plant Biology Meeting in Japan (2017)

Plant & Cell Phys: Latest Advances in Plant Development and Environmental Response, the inaugural Cold Spring Harbor Asia Plant Biology Meeting in Japan (2017) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:
With stunning ocean views over Osaka Bay, Awaji Island played host to the first Cold Spring Harbor Asia Plant Biology meeting in Japan. The meeting, “Latest Advances in Plant Development and Environmental Response” (#CSHAPB), provided a platform to promote scientific communication and collaboration in the pan-pacific region. The event welcomed almost 200 scientists from around the world to showcase their cutting-edge research. Exemplary speakers from diverse research fields presented their latest discoveries, ranging from developmental mechanisms to host-pathogen interactions, environmental responses and stress memory. Here we seek to review the meeting and highlight some of the salient themes that emerged over the course of the three days.
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The Biochemist: Guarding the granary (2017)

The Biochemist: Guarding the granary (2017) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:
The introduction of microbial plant pathogens can cause tremendous damage to crops, food security, the economy and ecosystems in very little time. What do we know about these invaders? And what can be done to defend our crops and respond quickly to new threats?
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Lynne Reuber's curator insight, June 19, 12:02 PM
Emerging plant diseases have threatened food security throughout history and still do today.
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Methods in Mol Biol: Detection and Analyses of Endocytosis of Plant Receptor Kinases (2017)

Methods in Mol Biol: Detection and Analyses of Endocytosis of Plant Receptor Kinases (2017) | Publications from The Sainsbury Laboratory | Scoop.it
The Sainsbury Lab's insight:
Genetic dissection has led to a sophisticated understanding of receptor kinases in plant development and responses to abiotic and biotic stresses. Fluorescence confocal microscopy is essential to identify the (sub)cellular locations of resting and signaling receptor kinases that trigger molecular events in plant cells upon ligand perception. In this regard, the internalization of plasma membrane-localized FLAGELLIN SENSING 2 (FLS2) into endosomes induced by its ligand flg22, a peptide derived from bacterial flagellin, is a model system for studying activation status-dependent and endosomal receptor kinase trafficking routes and can be used in screens to identify pathogen effectors that target these trafficking routes for virulence promotion. In this chapter we describe approaches of visualizing fluorescently tagged FLS2, including protocols for flg22-induced endocytosis, instrument parameters, and image analysis. These approaches can be easily adapted for other receptor kinases, using the fast transient expression system in Nicotiana benthamiana for microscopic inspection.
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