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Dissecting the apoplastic oxidative burst in Arabidopsis

Reference	BB/E021166/1
Principal Investigator / Supervisor	Professor Godfrey Bolwell
Co-Investigators / Co-Supervisors
Institution	Royal Holloway, Univ of London
Department	Biological Sciences
Funding type	Research
Value (£)	336,096
Status	Completed
Type	Research Grant
Start date	01/01/2008
End date	31/12/2010
Duration	36 months

Abstract

Two main mechanisms for the generation of reactive oxygen species (ROS) in plants have been proposed, one of which involves apoplastic peroxidase and the other plasma membrane-associated NADPH oxidases. Recent studies have relegated the role of NADPH oxidases to involvement in R-gene resistance and not basal resistance. The overall aim of the proposed project therefore is to establish the contribution of peroxidases to ROS accumulation in Arabidopsis as a key component of basal resistance. A key resource will be an existing homozygous transgenic Arabidopsis line which is hyper-susceptible to a variety of pathogens and was constructed antisense expression of a cDNA encoding FBP1, the oxidative burst peroxidase from French bean. This transgenic line in conjunction with full-genome microarray analysis confirmed by RT-PCR was used to identify two tandemly arranged Arabidopsis FBP1 orthologues as the targets for down-regulation with At3g49120 (AtPCb) playing the more important role. To confirm this we will use proteomic analysis to identify that AtPCb is down-regulated in FBP1 anti-sense plants. To confirm the role of AtPCb in rOS generation in basal resistance, and we will construct additional AtPCb knockdowns using RNAi and anti-sense techniques, and if they become available, mutants from public libraries and we will test these various transgenic and mutants plants for pathogen susceptibility and for their ability to mount an oxidative burst using biochemical, molecular and imaging techniques. In comparison with the French bean system, we will use metabolomic profiling to identify the substrate, known to be anionic, in apoplastic fluid, which is secreted or transported as a consequence of elicitation in both suspension cultured cells and plants. The link between peroxidase, substrate provision and ROS generation and ion-fluxes will be studied by the effect of modulators and in AtPCb knock-outs on the appearance of substrate and product by metabolic profiling.

Summary

Over twenty per cent of the world's crops are lost to disease every year. In addition, quality, safety and nutrition are compromised in infected crops that make it to the market. Crop protection has therefore been a vital factor since the dawn of agriculture. However many basic processes in natural plant defence are still unknown to this day. Progress has been made in identifying genes for resistance to pests and the processes that bring defence about but some mechanisms are still not understood. Plants cannot run away and continually face the threat from pests and disease-causing organisms (pathogens) landing upon them. They have devised a number of defences to allow them to defend themselves against such challenges. They make a number of chemical compounds which are characteristic of each type of plant that can ward off pests by being obnoxious to their senses (antifeedants) or can kill microbial pathogens by interfering with their metabolism so that they die(phytoalexins). Plants do not have an immune system in which specialised cells recognise and engulf and kill pathogens by literally exposing them to 'bleach' as in human cells in the body (macrophages). However they can produce similar 'bleaching' compounds such as hydrogen peroxide which can potentially be lethal to microbes. In order to do this the plant cell uses oxygen in a sudden burst and converts it to hydrogen peroxide at the site of attack. This 'oxidative burst' can be demonstrated in all sorts of interactions between different plants and microbes and is an important part of the defence armory of the plants. However despite its importance as such a widespread mechanism we cannot yet be certain of how it occurs. A number of ways in which plants carry out these reactions have been proposed, some internal to the cells some external. What is certain is that microscopic techniques can stain the hydrogen peroxide at the site of the attack and invariably it can be seen located in the wall that surroundsall plant cells. Therefore some of the mechanism resides here. There are a number of ways to explore this. The hydrogen peroxide must come from an enzyme. A big question is which one? Many enzymes can produce this compound. However there are a number of enzymes that could do this job that are located outside the cell, including one type, peroxidase. If peroxidases are responsible they must be linked to other processes that occur immediately after the pathogen is recognised. Peroxidases normally use hydrogen peroxide so how can they turn around and produce it? The answer lies in changes in the environment surrounding them in the wall and these are precisely what happens when cells respond to pathogens! Molecules from the pathogen that is trying to feed on the plant cell are recognised and signals at sent to various components in the cell. One component is movement of charged molecules so that the inside of the cell becomes more acid and the outside more alkaline. This allows the peroxidase to function as a hydrogen peroxide producer. To do this it needs something to work on and we know this is passed outside but we don't know what it is yet. So we need to show that mutant plants that lack this special peroxidase cannot make hydrogen peroxide and will therefore die when infected. We need to confirm we have identified the actual one as there are seventy three in the model plant, Arabidopsis. Then we need to identify the substrate which we will do by finding out exactly when it is moved outside and then we will analyse what is tranferred out and find out which one can produce hydrogen peroxide in the presence of the special peroxidase. Who knows when we have proved all this we might use this information to engineer or breed better disease resistance in plants!

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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