Award details

Genetic dissection of basal resistance to microbial colonisation in Arabidopsis

Reference	BB/D523135/1
Principal Investigator / Supervisor	Professor John Mansfield
Co-Investigators / Co-Supervisors	Dr Marta de Torres Zabala, Professor Murray Grant
Institution	Imperial College London
Department	Life Sciences
Funding type	Research
Value (£)	362,939
Status	Completed
Type	Research Grant
Start date	01/10/2005
End date	31/05/2009
Duration	44 months

Abstract

The proposal addresses the basal resistance (BR) of Arabidopsis to strains of the bean pathogen Pseudomonas syringae pv. Phaseolicola (Psph). We have developed the Psph/Arabidopsis interaction as a model to examine the activity of effector proteins from pvs. of P. syringae. We have mainly used the plasmid-cured strain RW60 of race 7 which activates BR in Arabidopsis and fails to cause symptoms or multiply. The effector AvrPtoB was found to promote virulence recognised by lesion formation and bacterial multiplication when expressed in Psph strain RW60 but only in certain accessions. In particular Niederzenz (ND-0) remained resistant and asymptomatic whereas Wassilewskja (Ws-3) was susceptible. Basal resistance in Nd to RW60+avrPtoB was inherited as a single dominant gene which we mapped to chromosome 5 and identified as the flagelin receptor encoding gene FLS2. Screening the mapping population with additional Psph strains has revealed that a second gene (designated BR1) is required with FLS2 to confer basal resistance to the race 6 strain 1448A. One target of the proposed research is the cloning of genes identified to control the very effective BR found in Nd. We now have a population from a ND x Ws cross segregating for BR1 in a homozygous FLS2 background and this will be used for map based cloning of BR1. As an alternative approach we will identify other BR genes by mutagenesis. We have an EMS mutagenised population of Nd which will be screened by inoculation with 1448A and RW60. The use of the two onocula will allow the identification of not only mutations in BR1 and also the Nd allele of FLS2, but importantly also in other components that contribute to basal resistance. A secondary screen with RW60+avrPtoB should allow the differentiation of pathways to activation of BR that are attacked by AvrPtoB. Map-based cloning of the mutated genes will be achieved from F2 populations of crosses with Nd wild type plants. The second and integrated target of the project is todetermine the mode of action of AvrTtoB in Arabidopsis and why it fails to overcome fully the FLS2-based systems of BR. We have already identified seven proteins from a yeast two hybrid screen (using AvrPtoB as bait to an Arabidopsis cDNA library) which interact in vitro. As a second approach to identify AvrPtoB/protein complexes we propose to use protein pull down methods to recover interactions from plant cells using both HA-tagged AvrPtoB and anti-AvrPtoB antisera we raised in rabbit. We have confirmed that N termina HA-tagged protein retains virulence activity in Arabidopsis following delivery by Psph. Interacting proteins will be recovered from leaves inoculated with RW60+avrPtoB-HA or from transgenic plants expressing AvrPtoB under the control of a dexamethosone inducible promoter. We have established that in planta expression of AvrPtoB causes symptom development and promotes bacterial multiplication. Identification of interacting proteins will be achieved using our LC-Qtrap MS system. Complexes will be dissociated in denaturing conditions, digested with trypsin and resultant peptides separated and sequenced by 1 or 2D nano LC/MS. Working with Arabidopsis we are able to use reverse genetics to assess the roles in BR of the genes and proteins identified. Insertion lines with mutations in the genes encoding putative interactors already recovered from yeast two hybrid screens, those to be identified from the hunt for AvrPtoB containing complexes and BR gene cloning experiments, are available from different resource centres and will be examined for modified basal defence responses. By the examination of reactions of wild-type and flagellin minus strains of RW60 with or without avrPtoB, and also wild type race 6 1448A we will be able to assess the contribution of interacting proteins to the different routes to basal resistance. Lesion development and bacterial multiplication will both be measured.

Summary

Like humans and animals, plants have the ability to defend themselves against colonisation by the microbes (pathogens) such as fungi and bacteria that cause disease. One form of resistance is the type that operates against fungi and bacteria whether they are pathogens of other plants or saprophytes, which are only able to feed on dead material. This widespread and effective form of defence is called basal resistance and is more like the process of innate immunity that operates in animals. One aim of this project is to find out which plant genes encode the proteins that are required for basal defence. Such proteins might be sensors (or receptors) which detect the presence of invading microbes and activate resistance mechanisms. One such receptor called FLS2, is known to detect a small fragment of flagellin, a protein which is part of the bacterial flagellum which propels bacteria through the films of water that you find on plant surfaces. We will use Arabidopsis thaliana for the research. It is termed a model plant because it is very easy to use for genetics and in particular the cloning of genes for resistance. It is closely related to plants in the Brassica family such as cabbage and oilseed rape. We have found clear differences between the basal resistance of two varieties of Arabidopsis called Niederzenz (Nd) and Wassilewskja (Ws) and have shown by crossing the two varieties that the resistance is inherited as a single gene. This gene is not FLS2 but seems to work with FLS2 to restrict certain strains of the bacterium Pseudomonas syringae. By the analysis of progeny from the cross between Nd and Ws we will be able to map, i.e. locate the position of the gene on one of the five Arabidopsis chromosomes and eventually clone the new gene we call BR1 for Basal Resistance 1. Once we know the DNA sequence of BR1 we will be able to predict the nature and properties of the protein that it encodes and this should indicate how it might function in partnership with FLS2. Additional BR genes will be identified by screening a population of Nd plants that has been treated with a compound (EMS) which causes very small, usually single base, changes to DNA. Mutants will be identified by their response to different strains of P. syringae. As with BR1 we will be able to clone the additional genes for further experiments. The evolution of the pathogens that cause disease in animals and plants seems to have been brought about by their production of effector proteins which damage certain parts of their hosts defences. We have found that one such effector named AvrPtoB can partially suppress basal resistance in Arabidopsis. It is most successful if plants do not have a functional FLS2 system of basal defence but may target a second subsidiary mechanism either at the point of perception of the invading microbe or downstream of the recognition event as part of the generation of antibacterial conditions. We will identify the plant proteins which are targets of AvrPtoB by searching for interactors that bind to the effector protein. We will be able to isolate the interaction complexes through a system which uses yeast cells to identify interactors and also by the direct recovery of protein mixtures using antibodies to AvrPtoB. The proteins within the complexes will be identified through the separation of the individual components and the measurement of their molecular weight using a mass spectrometer. The role in basal resistance of the genes and proteins we will isolate can be tested in Arabidopsis because of the availability of plants with single gene mutations. Thus we can examine responses in plants with a knock out in our gene of interest. Through this project we should be able to understand how the long lasting basal resistance operates at the molecular level. We hope to find new defence processes that we will be able to use to develop sustainable disease control strategies which reduce spraying with harmful pesticides.

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Crop Science, Microbiology, 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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