Research interests

· Plant disease resistance: mechanisms and signal networks
· Plant natural products: biosynthesis, regulation and function

Rationale

Plants have evolved a battery of defence mechanisms that in aggregate provide protection against a wide range of potential viral, bacterial, fungal, and other pathogens encountered throughout the plant life cycle.  However, in the artificial setting of agriculture, disease, although the exception, can be costly and even devastating.  Crop diseases have played significant roles in human history, exemplified by the widespread starvation and mass emigration triggered by the failure of European potato crops in the mid-nineteenth century as a result of late blight.  Today, the use of pesticides, breeding for resistance, and integrated pest management provide important tools for reducing crop losses to pre-and post-harvest diseases.  However, agrochemicals are expensive, prohibitively so for many farmers in developing countries, and there are increasing concerns about environmental load from their intensive application.  Likewise, major disease resistance (R) genes are in many cases not durable, resistance breaking down within one or two seasons as a result of selection pressure on the pathogen population, and most breeding efforts now rely on combinations of minor resistance genes, to each of which gives partial protection.  For a number of important diseases, such as take-all of wheat, there is no effective genetic resistance.  Population growth, migration to cities, desertification, and climate change all now contribute to an urgent need to secure diversified food production against disease loss.

The major focus of my group is the dissection of signal mechanisms underlying the activation of inducible defences against pathogens and pests. New knowledge and understanding emerging from these studies will allow the development of novel approaches to enhance durable resistance of crops to pathogens and pests, thereby helping to secure the future supply of safe, nutritious food world-wide and reduce environmental load in diverse agricultural systems.

Programme

Background

Attempted infection by an avirulent pathogen elicits a battery of defences, many of which require transcriptional activation of functionally related sets of defence genes in spatially and temporal hierarchies. Activation of these protective mechanisms is often accompanied by the collapse of challenged plants cells in the so-called hypersensitive response (HR) which results in a restricted lesion clearly delimited from surrounding healthy tissue. In addition, immunity to subsequent attack by a broad range of normally virulent pathogens develops throughout the plant. There is great interest in this systemic acquired resistance (SAR) in relation to the nature of the mobile signals and as a key platform for developing novel approaches to enhance durable-broad, spectrum crop protection.

An early event in the HR is the generation of superoxide (O₂^-) and accumulation of hydrogen peroxide (H₂O₂) in an oxidative burst reminiscent of that producing reactive oxygen intermediates (ROIs) in activated macrophages. The oxidative burst drives peroxidase-mediated cross-linking of tyrosine-rich cell wall structural proteins which toughens the cell wall to slow pathogen ingress and spread prior to the activation of transcription-dependent defences such as the deployment of lytic enzymes and other antimicrobial proteins as well as the synthesis of protective natural products. Moreover, activation of the oxidative burst in the plant HR is part of a highly amplified and integrated signal system that also involves salicylic acid and perturbations in cytosolic calcium to trigger defence mechanisms and to mediate the establishment of systemic immunity underlying SAR. The oxidative burst is necessary but not sufficient to trigger host cell death and recent data indicate that nitric oxide (NO) co-operates with ROIs in the activation of hypersensitive cell death. In addition NO functions independently of ROIs in the induction of key sets of defence genes distinct from the cellular protectant genes activated by ROI signals.

Current work is focussed on the genetic dissection of the local and systemic signal networks mediated by ROI, NO and salicylic acid, and developing genetic approaches to the study of non-host resistance.

Genetic dissection of SAR

We have screened Agrobacterium tumefaciens transfer-DNA tagged lines of Arabidopsis thaliana for mutants specifically compromised in SAR. Defective induced resistance 1-1 (dir1-1) exhibits normal local resistance to avirulent Pseudomonas syringae but fails to develop SAR to virulent strains of this bacterium or to an oomycete pathogen. This novel mutant exhibits wild type defence responses at the site of the initial bacterial inoculation, but genes encoding pathogenesis-related proteins are not induced in uninoculated distant leaves, reflecting a defect in the production or transmission from the inoculated leaf of an essential mobile signal. DIR1 encodes a putative apoplastic lipid transfer protein and we propose that the DIR1 protein interacts with a lipid-derived molecule in systemic signalling. Current research is focussed on testing this hypothesis.

Activation tagging of stress signalling genes

Activation tagging allows the rapid cloning of tagged gain-of-function alleles and hence overcomes problems of redundant signal pathways or gene families.  From a screen of 7,000 activation tagged lines we identified the line Constitutive Disease Resistance 1, CDR1, which exhibits strong resistance to normally virulent strains of Pseudomonas syringae pv tomato and pv maculicola.  This resistance, which was accompanied by some stunting, reflects massive pre-activation in healthy leaves of the expression of several PR genes as well as rboh encoding the puative catalytic subunit of the respiratory burst oxidase.  Moreover, while the leaves of CDR1 plants show no visible lesion mimic phenotype, micro-burst and micro-HRs could be readily observed in leaves stained with diaminobenzidine and trypan blue respectively, in line with the proposed function of micro-bursts in the establishment of systemic immunity. CDR1 had substantially elevated levels of free and conjugated salicylic acid and crossing with nahG abolished all CDR1 phenotypes.

CDR1 encodes an apoplastic aspartate proteinase and ectopic expression this gene under the control of the 35S enhance or a desamethasone-inducible system confirmed that CDR1 entirely accounts for the observed phenotypes.  Moreover, CDR1 antisense lines are compromised for resistance to normally avirulent P. syringae and show enhanced disease susceptibility to virulent strains.

A second activation tagged allele, Constitutive Disease Suceptibility 1, CDS1, exhibits enhanced disease susceptibility to virulent P. syringae and is also compromised for localised resistance to isogenic avirulent strains.  Interestingly CDS1 appears to encode a second apoplastic aspartate proteinase and these emerging data suggest a model involving proteinase-mediated regulation of mobile peptide signal molecules in the modulation of disease resistance responses.

Current work is focussed on elucidation of the nature of these signals and further developing the activation approach to elucidate specific components of the signal network including salicylic acid- and jasmonic acid-mediated pathways and cross-talk between these pathways, as well as NO- and ROI-mediated pathways. This work uses new vectors that allow the rapid generation of secondary knockout alleles of the initial activation tagged alleles to determine loss-of-function as well as gain-of-function phenotypes.

Genetics of non-host resistance

While a reasonable experimental outline of the genetic requirements and biochemical events required to initiate and maintain race-specific defense responses exists, comparatively little is known concerning the genetics and biochemistry of non-host resistance.  We have recovered an antimicrobial activity from Arabidopsis thaliana extracts that is specifically toxic to non-host pathovars of Pseudomonas syringae while host pathovars are unaffected.  We have exploited this characteristic to screen a library from the host-pathogen Pseudomonas syringaepv. maculicola for genetic elements that confer resistance against this Arabidopsis-derived antimicrobial activity to non-host pathogens.   This has lead to the identification of a pathogenicity island in P.s. maculicola that codes for at least three genes.  The sax operon (for survival on Arabidopsis extract) appears to be under the control of a member of the AraC/XylS-family of transcriptional regulators (saxC).  The other functional components appear to be a putative Zn-binding-metalloenzyme of the beta-lactamase superfamily (saxA) and a putative member of the isochorismatase family (saxB).  Homologues of all of these gene products are known in other systems to be involved in recognizing and modifying small molecules in either detoxification or nutrient utilization.  Current experiments are focused on the biochemical identification of this antimicrobial activity, and genetic screens in Arabidopsis to isolate mutants with both altered non-host resistance in general and altered expression of this antimicrobial activity specifically.

Publications 

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