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Murray Grant

Publications and source records attributed to Murray Grant.

15 recordsLinked to original sources
PubMed2025

The Arabidopsis TIRome informs the design of artificial TIR (Toll/interleukin-1 receptor) domain proteins.

The TIR (Toll/interleukin-1 receptor) domain is an ancient protein module that functions in immune and cell death responses across the Tree of Life. TIR domains encoded by plants and prokaryotes function as enzymes to produce diverse small molecule immune signals. Plant genomes can encode hundreds of TIR-domain containing proteins-many of which confer important agricultural disease resistance as TIR-NLR (nucleotide-binding, leucine-rich repeat) immune receptors. Despite their importance, how natural variation influences TIR enzymatic output and immunity-associated cell death is largely unexplored. We assayed a complete collection of the TIR domains of Arabidopsis thaliana Col-0 (the "AtTIRome") to explore variation in TIR metabolite production and cell death signaling. Roughly half of the AtTIRome triggered cell death in transient assays. Artificial TIR proteins designed based on consensus sequences of the AtTIRome's cell death phenotypic classes revealed polymorphisms controlling variation in TIR cell death elicitation and metabolite production. Structure-function analyses of artificial TIRs revealed that natural variation in the "BB-loop", a flexible region overlying the catalytic pocket, determines differences in function across Arabidopsis TIR-containing proteins. We further demonstrate that artificial TIRs are functional on an NLR chassis and that BB-loop variation can tune the activity of a natural TIR-NLR protein. These findings shed light on the diversity of TIR outputs and reveal methods to design and engineer TIR-based immune receptors.

Arabidopsis↗
PubMed2007

Arabidopsis systemic immunity uses conserved defense signaling pathways and is mediated by jasmonates.

In the absence of adaptive immunity displayed by animals, plants respond locally to biotic challenge via inducible basal defense networks activated through recognition and response to conserved pathogen-associated molecular patterns. In addition, immunity can be induced in tissues remote from infection sites by systemic acquired resistance (SAR), initiated after gene-for-gene recognition between plant resistance proteins and microbial effectors. The nature of the mobile signal and remotely activated networks responsible for establishing SAR remain unclear. Salicylic acid (SA) participates in the local and systemic response, but SAR does not require long-distance translocation of SA. Here, we show that, despite the absence of pathogen-associated molecular pattern contact, systemically responding leaves rapidly activate a SAR transcriptional signature with strong similarity to local basal defense. We present several lines of evidence that suggest jasmonates are central to systemic defense, possibly acting as the initiating signal for classic SAR. Jasmonic acid (JA), but not SA, rapidly accumulates in phloem exudates of leaves challenged with an avirulent strain of Pseudomonas syringae. In systemically responding leaves, transcripts associated with jasmonate biosynthesis are up-regulated within 4 h, and JA increases transiently. SAR can be mimicked by foliar JA application and is abrogated in mutants impaired in jasmonate synthesis or response. We conclude that jasmonate signaling appears to mediate long-distance information transmission. Moreover, the systemic transcriptional response shares extraordinary overlap with local herbivory and wounding responses, indicating that jasmonates may be pivotal to an evolutionarily conserved signaling network that decodes multiple abiotic and biotic stress signals.

Arabidopsis↗
PubMed2006

Modifications to the Arabidopsis defense proteome occur prior to significant transcriptional change in response to inoculation with Pseudomonas syringae.

Alterations in the proteome of Arabidopsis (Arabidopsis thaliana) leaves during responses to challenge by Pseudomonas syringae pv tomato DC3000 were analyzed using two-dimensional gel electrophoresis. Protein changes characteristic of the establishment of disease, basal resistance, and resistance-gene-mediated resistance were examined by comparing responses to DC3000, a hrp mutant, and DC3000 expressing avrRpm1, respectively. The abundance of each protein identified was compared with that of selected transcripts obtained from comparable GeneChip experiments. We report changes in three subcellular fractions: total soluble protein, chloroplast enriched, and mitochondria enriched over four time points (1.5-6 h after inoculation). In total, 73 differential spots representing 52 unique proteins were successfully identified. Many of the changes in protein spot density occurred before significant transcriptional reprogramming was evident between treatments. The high proportion of proteins represented by more than one spot indicated that many of the changes to the proteome can be attributed to posttranscriptional modifications. Proteins found to show significant change after bacterial challenge are representative of two main functional groups: defense-related antioxidants and metabolic enzymes. Significant changes to photosystem II and to components of the mitochondrial permeability transition were also identified. Rapid communication between organelles and regulation of primary metabolism through redox-mediated signaling are supported by our data.

Arabidopsis↗
PubMed2006

Pseudomonas syringae effector AvrPtoB suppresses basal defence in Arabidopsis.

The virulence and avirulence activities of members of the Pseudomonas syringae HopAB family of effectors and AvrPto were examined in bean, tomato and Arabidopsis. Proteins were delivered by the RW60 strain of P. syringae pv. phaseolicola. RW60 causes a hypersensitive reaction (HR) in bean and tomato but is restricted without the HR in Arabidopsis. Dual avirulence and virulence functions in tomato and bean, respectively, were identified in virPphA homologues but only avrPtoB strongly enhanced virulence to Arabidopsis, overcoming basal defences operating against RW60. Virulence activity in both bean and Arabidopsis required regions of the C-terminus of the AvrPtoB protein, whereas elicitation of the rapid HR in tomato, with the matching Pto resistance gene, did not. The effect of AvrPtoB on Arabidopsis was accession-specific; most obvious in Wassilewskija (Ws-3), intermediate in Columbia and not detectable in Niedersenz (Nd-1) after inoculation with RW60 + avrPtoB. Analysis of crosses between Ws-3 and Nd-1 indicated co-segregation for the AvrPtoB virulence function with the absence of the Nd-1 FLS2 gene which mediates recognition of bacterial flagellin. In planta expression of AvrPtoB did not prevent the HR activated by P. syringae pv. tomato DC3000 + avrB, avrRpm1, avrRps4 or avrRpt2, but suppressed cell wall alterations, including callose deposition, characteristic of basal defence and was associated with reprogramming of the plant's transcriptional response. The success or failure of AvrPtoB in suppressing basal defences in Nd-1 depended on the timing of exposure of plant cells to the effector and the flagellin flg22 peptide.

Arabidopsis↗
PubMed2006

Systemic immunity.

Systemic acquired resistance (SAR) provides enhanced, long-lasting systemic immunity to secondary infection by a range of biotrophic, hemibiotrophic and necrotrophic pathogens that have diverse modes of infection. Considerable effort has focussed on the conserved central positive regulator of SAR, NON-EXPRESSOR OF PATHOGENESIS-RELATED1 (NPR1), and its control by changes in cellular redox potential. Recently, genetic and genomic approaches have highlighted a critical role for nucleocytoplasmic communication and protein secretion in establishing effective systemic immunity. Identification of the mobile signals and the mechanisms by which they are perceived in distal tissues remains challenging, but emerging evidence suggests that signal translocation uses lipid-derived (possibly jasmonate-based) signals and lipid-binding chaperones. Furthermore, the demonstration that autophagy interdicts and inactivates a systemic cell death signal adds further complexity to elucidating how mobile signals are decoded and transduced for effective immunity.

Gene Expression Regulation, Plant↗
PubMed2006

Analysis of the defence phosphoproteome of Arabidopsis thaliana using differential mass tagging.

Despite recent advances in proteomic technologies, quantitative analysis of the proteome remains a challenging task. Phosphorylation of proteins is central to signal transduction pathways and plays an important role in plant defence against pathogens, although the immediate targets of kinases remain elusive. Determining changes in the phosphoproteome during the defence response is a major goal in molecular plant pathology. In this first description of the novel mass tagging strategy (iTRAQ Applied Biosystems) applied to plant pathogen interactions, we describe early changes to the phosphoproteome of Arabidopsis thaliana during the defence response to Pseudomonas syringae pv. tomato DC3000. We identified five proteins which showed reproducible differences between a control and three different bacterial challenges, thus identifying proteins potentially phosphorylated as part of a plant basal defence response. Four of the five proteins a dehydrin, a putative p23 co-chaperone, heat shock protein 81 and a plastid-associated protein (PAP)/fibrillin, are known to be phosphorylated or have potential phosphorylation sites. One further protein, the large subunit of Rubisco, showed a significant difference between tissue undergoing the hypersensitive response and a basal defence response. We document the reproducibility, utility and problems associated with this approach.

Arabidopsis↗
PubMed2006

Type III effectors orchestrate a complex interplay between transcriptional networks to modify basal defence responses during pathogenesis and resistance.

To successfully infect a plant, bacterial pathogens inject a collection of Type III effector proteins (TTEs) directly into the plant cell that function to overcome basal defences and redirect host metabolism for nutrition and growth. We examined (i) the transcriptional dynamics of basal defence responses between Arabidopsis thaliana and Pseudomonas syringae and (ii) how basal defence is subsequently modulated by virulence factors during compatible interactions. A set of 96 genes displaying an early, sustained induction during basal defence was identified. These were also universally co-regulated following other bacterial basal resistance and non-host responses or following elicitor challenges. Eight hundred and eighty genes were conservatively identified as being modulated by TTEs within 12 h post-inoculation (hpi), 20% of which represented transcripts previously induced by the bacteria at 2 hpi. Significant over-representation of co-regulated transcripts encoding leucine rich repeat receptor proteins and protein phosphatases were, respectively, suppressed and induced 12 hpi. These data support a model in which the pathogen avoids detection through diminution of extracellular receptors and attenuation of kinase signalling pathways. Transcripts associated with several metabolic pathways, particularly plastid based primary carbon metabolism, pigment biosynthesis and aromatic amino acid metabolism, were significantly modified by the bacterial challenge at 12 hpi. Superimposed upon this basal response, virulence factors (most likely TTEs) targeted genes involved in phenylpropanoid biosynthesis, consistent with the abrogation of lignin deposition and other wall modifications likely to restrict the passage of nutrients and water to the invading bacteria. In contrast, some pathways associated with stress tolerance are transcriptionally induced at 12 hpi by TTEs.

Amino Acids, Aromatic↗
PubMed2005

RIN13 is a positive regulator of the plant disease resistance protein RPM1.

The RPM1 protein confers resistance to Pseudomonas syringae pv tomato DC3000 expressing either of the Type III effector proteins AvrRpm1 or AvrB. Here, we describe the isolation and functional characterization of RPM1 Interacting Protein 13 (RIN13), a resistance protein interactor shown to positively enhance resistance function. Ectopic expression of RIN13 (RIN13s) enhanced bacterial restriction mechanisms but paradoxically abolished the normally rapid hypersensitive response (HR) controlled by RPM1. In contrast with wild-type plants, leaves expressing RIN13s did not undergo electrolyte leakage or accumulate H2O2 after bacterial delivery of AvrRpm1. Overexpression of RIN13 also altered the transcription profile observed during a normal HR. By contrast, RIN13 knockout plants had the same ion leakage signatures and HR timing of wild-type plants in response to DC3000(avrRpm1) but failed to suppress bacterial growth. The modified phenotypes seen in the RIN13s/as plants were specific to recognition of AvrRpm1 or AvrB, and wild-type responses were observed after challenge with other incompatible pathogens or the virulent DC3000 isolate. Our results suggest that cell death is not necessary to confer resistance, and engineering enhanced resistance without activation of programmed cell death is a real possibility.

Amino Acid Sequence↗
PubMed2005

Characterisation of recombinant epithiospecifier protein and its over-expression in Arabidopsis thaliana.

Epithiospecifier protein (ESP) is a protein that catalyses formation of epithionitriles during glucosinolate hydrolysis. In vitro assays with a recombinant ESP showed that the formation of epithionitriles from alkenylglucosinolates is ESP and ferrous ion dependent. Nitrile formation in vitro however does not require ESP but only the presence of Fe(II) and myrosinase. Ectopic expression of ESP in Arabidopsis thaliana Col-5 under control of the strong viral CaMV 35S promoter altered the glucosinolate product profile from isothiocyanates towards the corresponding nitriles.

Amino Acid Sequence↗
PubMed2005

Biophoton imaging: a nondestructive method for assaying R gene responses.

Plant disease resistance (R) proteins of the nucleotide binding-leucine rich repeat class are responsible for pathogen recognition and activation of defense signaling networks leading to the hypersensitive response (HR). Genetically, R-protein signaling appears to be integrated through a limited set of common downstream components. However, the timing of development of visible HR is unique to individual R proteins. By utilizing the phenomena of ultraweak photon emission from leaves undergoing an incompatible interacttion, a powerful nondestructive and facile assay is described to compare timing of defense responses elicited by different R proteins. We demonstrate that ultraweak photon emission, or "biophoton generation," is demonstrated to be associated with hypersensitive cell death. Biophoton emission requires an intact R signaling network and increases in cytosolic calcium and nitric oxide, but elevated reactive oxygen species are not necessary. Importantly, the assay is robust and applicable to a range of incompatible interactions in various plant species. The ability to assay R responses nondestructively in real time and a chosen genetic background makes this technique amenable to subtle genetic dissection of plant defense responses.

Arabidopsis↗
PubMed2004

Specific changes in the Arabidopsis proteome in response to bacterial challenge: differentiating basal and R-gene mediated resistance.

Alterations in the proteome of Arabidopsis thaliana leaves during early responses to challenge by Pseudomonas syringae pv. tomato DC3000 (DC3000) were analysed using two-dimensional (2D) gel electrophoresis. Protein changes characteristic of the establishment of basal resistance and R-gene mediated resistance were examined by comparing responses to DC3000, a hrp mutant and DC3000 expressing avrRpm1 respectively. The abundance of selected transcripts was also analysed in GeneChip experiments. Here we present data from the soluble fraction of leaf protein, highlighting changes in two antioxidant enzyme groups; the glutathione S-transferases (GSTs F2, F6, F7 and F8) and peroxiredoxins (PrxA, B and IIE). Members of both enzyme groups showed signs of specific post-translational modifications, represented by multiple spots on gels. We suggest that oxidation of specific residues is responsible for some of the spot shifts. All forms of the GST proteins identified here increased following inoculation with bacteria. GSTF8 showed particularly dynamic responses to pathogen challenge, the corresponding transcript was significantly up-regulated by 2 h after inoculation, and the protein showed post-translational modifications specific to an incompatible interaction. Differential changes were observed with the peroxiredoxin proteins; PrxIIE and to a lesser extent PrxB, no change was observed with PrxA, but a truncated form PrxA-L was greatly reduced in abundance following bacterial challenges. Our data suggest that bacterial challenge generally induces Prxs and the antioxidants GSTs, however individual members of these families may be specifically modified dependent upon the virulence of the DC3000 strain and outcome of the interaction. Finally, proteomic and transcriptomic data derived from the same inoculation system are compared and the advantages offered by 2D gel analysis discussed in light of our results.

Arabidopsis↗
PubMed2004

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

Arabidopsis↗
PubMed2003

Expression profiling of the host response to bacterial infection: the transition from basal to induced defence responses in RPM1-mediated resistance.

Changes in transcription in leaves of Arabidopsis thaliana were characterised following challenge with strains of Pseudomonas syringae pv. tomato DC3000 allowing differentiation of basal resistance (hrpA mutants), gene-specific resistance (RPM1-specified interactions) and susceptibility (wild-type pathogen). In planta avirulence gene induction, changes in host [Ca2+]cyt and leaf collapse were used to delineate the transition from infection to induced resistance. The plant responds rapidly, dynamically and discriminately to infection by phytopathogenic bacteria. Within the first 2 h host transcriptional changes are common to all challenges indicating that Type III effector function does not contribute to early events in host transcriptome re-programming. The timing of induction for specific transcripts was reproducible, hierarchical and modulated at least in part through EDS1 function. R gene-specific transcripts were not observed until 3 h after inoculation. Intriguingly, the R gene-specific response proteins are expected to localise to diverse cellular addresses indicative of a global impact on cellular homeostasis. The altered transcriptional response rapidly manifests into initial symptoms of leaf collapse within 2 h, although establishment of the full macroscopic HR occurs significantly later.

Arabidopsis↗
PubMed2002

Differential expression of genes encoding Arabidopsis phospholipases after challenge with virulent or avirulent Pseudomonas isolates.

Phospholipase D (PLD; EC 3.1.4.4) has been linked to a number of cellular processes, including Tran membrane signaling and membrane degradation. Four PLD genes (alpha, beta, gamma1, and gamma2) have been cloned from Arabidopsis thalami. They encode isoforms with distinct regulatory and catalytic properties but little is known about their physiological roles. Using cDNA amplified fragment length polymorphism display and RNA blot analysis, we identified Arabidopsis PLDgamma1 and a gene encoding a lysophospholipase (EC 3.1.1.5), lysoPL1, to be differentially expressed during host response to virulent and avirulent pathogen challenge. Examination of the expression pattern of phospholipase genes induced in response to pathogen challenge was undertaken using the lysoPL1 and gene-specific probes corresponding to the PLD isoforms a, beta, and gamma1. Each mRNA class exhibited different temporal patterns of expression after infiltration of leaves with Pseudomonas syringae pv. tomato with or without avrRpm1. PLDalpha was rapidly induced and remained constitutively elevated regardless of treatment. PLDbeta was transiently induced upon pathogen challenge. However, mRNA for the lysoPL1 and PLDgamma1 genes showed enhanced and sustained elevation during an incompatible interaction, in both ndr1 and overexpressing NahG genetic backgrounds. Further evidence for differential engagement of these PLD mRNA during defense responses, other than gene-for-gene interactions, was demonstrated by their response to salicylic acid treatment or wounding. Our results indicate that genes encoding lysoPL1, PLDgamma1, and PLDbeta are induced during early responses to pathogen challenge and, additionally, PLDyl and lysoPL1 are specifically upregulated during gene-for-gene interactions, leading to the hypersensitive response. We discuss the possible role of these genes in plant-pathogen interactions.

Amino Acid Sequence↗